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Vascular reactivity in cardiopulmonary bypassSamarska, Iryna

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Vascular reactivity in

cardiopulmonary bypass

Iryna Samarska

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Financial support by the Groningen University Institute for Drug Exploration (GUIDE) and the University of Groningen for the publication of this thesis is gratefully acknowledged.

Samarska, I.V. Vascular reactivity in Cardiopulmonary bypass Proefschrift Groningen ISBN: 978-90-367-4789-9 ISBN: 978-90-367-4788-2

© Copyright 2011 I.V.Samarska All rights are reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, without permission of the author. Cover: Iryna Samarska. Source: Servier Medical Arts. Lay-out: Iryna Samarska Printed by Ipskamp Drukkers, Enschede

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RIJKSUNIVERSITEIT GRONINGEN !!!!

Vascular reactivity in cardiopulmonary bypass

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Proefschrift

ter verkrijging van het doctoraat in de Medische Wetenschappen

aan de Rijksuniversiteit Groningen op gezag van de

Rector Magnificus, dr. E.Sterken, in het openbaar te verdedigen op

woensdag 6 april 2011 om 11.00 uur

door

Iryna Samarska geboren op 8 januari 1979

te Kiev, Oekraïne

Promotores: Prof. dr. R.H. Henning Prof. dr. M.M.R.F. Struys Copromotores: Dr. J.H. Buikema Dr. A.H. Epema Beoordelingscommissie: Prof. dr. P. Wouters Prof. dr. G. Molema Prof. dr. J.G.R. de Mey

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Paranimfen: Magdalena Mazagova

Roelien Meijering

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Table of contents

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Chapter 1 Introduction and the aims of the thesis

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Chapter 2 Changes in vasomotor function of small coronary and mesenteric artery during 5-day follow-up after cardiopulmonary bypass in the rat. Characterization of endothelial mediators and contractility Submitted

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Chapter 3 Changes of vascular reactivity of rat thoracic aorta during prolonged recovery following cardiopulmonary bypass Manuscript in preparation

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Chapter 4 Microarray analysis of gene expression profiles in the kidney demonstrates a local inflammatory response induced by cardiopulmonary bypass in rat Submitted

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Chapter 5 Protracted pulmonary inflammation during long-term recovery following cardiopulmonary bypass in the rat Manuscript in preparation

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Chapter 6 Adjunct nitrous oxide normalizes vascular reactivity changes after hemorrhagic shock in mice under isoflurane anesthesia Anesthesiology 2009 Sep;111(3):600-8

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Chapter 7 S1P1 receptor modulation improves systemic vascular dysfunction after CPB in the rat independent of depletion of lymphocyte: a comparison between FTY720 and SEW2871 Manuscript in preparation

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Chapter 8 Summary and future perspectives

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Supplements Supplemental digital content to the Chapter 4 Editorial Views. Sanders RD, Maze M. Does correcting the numbers improve long-term outcome? Anesthesiology 2009 Sep;111(3):475-7

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Nederlandse Samenvatting

Ukrainian Summary

Acknowledgments

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Chapter 1

Introduction and the aims of the thesis

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Clinical aspects of cardiopulmonary bypass Cardiopulmonary bypass (CPB) is a widely used technique in cardiothoracic

surgery, including procedures such as coronary artery bypass grafting, valvular heart surgery, repair of aortic aneurysms, surgical treatment of congenital heart defects, heart-, lung or heart-lung transplant, and pulmonary thrombectomiy or tromboendarterectomy. The first successful open-heart surgery with extracorporeal circulation on a patient was performed in 1953, by John Gibbon in Philadelphia (USA) and consisted of an atrial septal defect repair. Surgery with CPB has now become a standard procedure.1 The principal components of the cardiopulmonary circuit include tubing, a pump, an oxygenator and cannulae. They enable clinicians’ to reach the main goal of CPB, namely temporarily taking over the function of heart and lung (fig. 1).2

However, CPB is also associated with serious postoperative clinical complications, including dysfunction of vital organs such as heart, kidney, lung, and liver.3;4 Several clinical syndromes are related to CPB, including increased pulmonary vascular resistance with postoperative pulmonary insult,5;6 pulmonary dysfunction,5;6 myocardial ischemia and atrial fibrillation,7 postoperative renal function deterioration,8-

11 renal ischemic injury up to dialysis-dependent renal failure,3;4;12;13 intestinal ischemia,12 stroke, neurocognitive dysfunction and neurologic complications.14. The post-CPB incidence of these syndromes and associated conditions is substantial, and associated with a high morbidity and mortality rate or prolonged hospital stay.15;16. Moreover, a transient organ dysfunction may be observed in patients with preoperatively normal function.10;11

Renal complications Acute kidney injury is thought to represent the most frequently occurring

adverse effect of CPB, present in up to 30% of the patients after cardiac surgery.17 Because of reduced clearance, decreased renal function results in metabolic disturbances with metabolic acidosis and hyperkalemia, which in turn affect function in other organs. Kidney injury post-CPB varies from mild renal dysfunction up to full blown renal failure requiring dialysis.18 The etiology of the CPB associated renal injury is multifactorial and includes perioperative renal hypoperfusion with possible ischemia-reperfusion injury, presence of nephrotoxins and microembolism.15;16;19. Moreover, activation of neutrophils, monocytes, endothelial cells and the complement system resulting from this systemic inflammatatory response, further adds to glomerular and tubular disturbances.18 In addition, postoperative renal dysfunction negatively affects long-term survival after cardiac surgery.8-11;18

Pulmonary complications Another severe complication of CPB is a postoperative lung dysfunction with

interstitial pulmonary edema and subsequent abnormal gas exchange. Clinical data demonstrated that approximately 25% of the patients following open heart surgery exhibited signs of pulmonary impairment for at least one week thereafter.20-22 Several CPB-related pathophysiological processes were described in the lung, namely, increased vascular permeability, water content, vascular resistance, neutrophils

Introduction and the aim of the thesis 11

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sequestration, arterial-alveolar gradient, decrease in pulmonary compliance, alveolar-capillary injury, rise in shunt fraction, and likelihood of atelectasis and pneumonia.15;16;23 The induction of such critical complications can be explained by the specifics of the lung perfusion during CPB, since lungs are deprived of the majority of their normal blood supply in this period. Thus extracorporeal circulation causes serious pulmonary injury with vascular injury and edema, atelectasis, collapse of non-ventilated lungs, and massive or submassive pulmonary embolism.17;24

CPB-related hypotension Although the phenomenon of CPB-induced hypotension has been addressed

during the last decade, the exact underlying mechanisms are still unknown. In this context, platelet-mediated serotonin release is thought to be one of the most prominent explanations as extracorporeal circulation, contact of blood with artificial surfaces and the roller pump induces platelet activation and triggers release of serotonin.25 Serotonin may act as a vasodilator through activation of the most sensitive and widespread 5-HT2B receptors, mediating enhanced NO-release.25 Prolonged anesthesia, which is common in surgery employing CPB, may have an additional impact on hemodynamic state during the operation. Volatile anesthetics have been shown to modulate vascular smooth muscle tone and depress the cardiovascular system. The systemic hypotensive effect of isoflurane reported previously26-29 has been ascribed to a direct inhibitory effect on vascular endothelial and/or smooth muscle cells, involving a decrease in myofilament calcium-sensitivity, intracellular calcium concentration and voltage-gated calcium influx.30 Lower arterial pressure under isoflurane anesthesia may also be explained by isoflurane-induced activation of ATP-sensitive potassium channels of vascular smooth muscle cells causing cellular membrane hyperpolarization and inhibition of calcium influx.29-32 Moreover, Pypendop et al. (2003) showed that addition of 70% nitrous oxide to isoflurane anesthesia improved arterial pressure and central venous pressure, but the mechanism of this effect was not investigated.31;33 Pathogenesis of the CPB-associated complications

Despite recent progress, the pathological mechanisms involved in CPB-associated complications, being complex and multifactorial, still are largely unknown. CPB circuit and CPB surgical procedure consist of different elements, each of them can be accounted as a contributory factor for the CPB-related complications.34 The main cause of the systemic inflammatory response is thought to be contact of blood with the artificial plastic surfaces of the circuit.34-37 The priming solutions of the CPB circuit induce a drop of hematocrit and colloid oncotic pressure that leads to decreased oxygen delivery and formation of tissue edema.34;38 The usage of the roller pump is responsible for blood cell damage, hemolysis and complement activation.34;39 Thus, CPB is associated with a wide range of the etiological factors that evoke different compensatory and/or pathological processes. Systemic inflammatory response syndrome, ischemia-reperfusion injury, hemodynamic abnormalities, and vascular dysfunction are thought to play a major role.

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CPB-related pathophysiological processes Surgical trauma, contact of blood with artificial surfaces of the extracorporeal circuit and ischemia-reperfusion injury due to insufficient perfusion cause stimulation of local and systemic cellular and humoral defense mechanisms.6;15;16;40-42 Particularly blood interaction with the extracorporeal circuit and air is thought to activate factor XII and the alternative complement pathway that in turn activate cells of the host defense system and stimulate coagulation, fibrinolysis and the kallikrein pathway.15;16;34 Activated white blood cells release cytokines, free oxygen radicals, and other mediators cause activation of the nuclear factor kB and cell adhesion molecules, and as a result initiate SIRS.6;15;16;20

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!Particularly, CPB is associated with release of inflammatory cytokines tumor

necrosis factor-! (TNF-!), interleukin-6 and 8, increased plasma neutrophils elastase level, but also increased anti-inflammatory cytokine interleukin-10 levels.15;16 The systemic inflammatory events may manifest itself as a decline of systemic vascular resistance, systemic hypotension, increased heart rate and cardiac output followed by myocardial failure and increased vascular permeability.34;43 Changes in systemic vascular resistance and arterial blood pressure may cause substantial hemodynamic alterations that lead to redistribution of the blood to the vital organs (heart and brain), decreased supply of other organs, and to microcirculatory disturbances herein. Such processes in the intestine may initiate release of endotoxins in the blood circulation, which activate both classical and alternative complement pathways.34 Activated leukocytes, through release of free radicals, inflammatory cytokines and proteolytic

Figure 1. The principal scheme of the cardiopulmonary circuit. The CPB circuit includes a venous reservoir, a pump, an oxygenator, and a heat exchanger. The venous blood is removed from the body to the venous reservoir. The roller pump propels it through the system. The membrane oxygenator enriches the blood with oxygen and removes carbon dioxide. Then oxygenated blood is returned to the body. The figure was produced using Servier Medical Art.


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enzymes, evoke endothelial abnormalities and tissue destruction.20;38;43 Upregulation of tumor necrosis factor-!, IL-6, IL-8, and vascular endothelial growth factor (VEGF) results in increased vascular permeability and attenuation of endothelial cell integrity.44 Capillary leak (due to increased vascular permeability) with decreased plasma colloid osmotic pressure (due to hemodilution) lead to accumulation of the fluid in the interstitial spaces and tissue edema, that diminish tissue perfusion, nutrients and oxygen delivery, and state the stage for organ dysfunction.38 Moreover, inflammatory events may cause rigidness of the erythrocytes, decrease their deformability, and affect their ability to flow in microcirculatory beds, which also participate in disturbances of tissue perfusion and oxygen delivery.34;38

Therefore, CPB is associated with hemodynamic abnormalities (centralisation of the circulation, decreased systemic vascular resistance and systemic hypotension), that result in ischemia-reperfusion injury, oxidative stress, and evoke local pathophysiological processes.32;44 Augmented local release of nitrous oxide, thromboxane A2 and products generated by inducible cyclooxygenase are thought also to affect vascular reactivity. Oxidative stress after ischemia-reperfusion injury may itself cause endothelial cells dysfunction. Moreover, formation of the peroxynitrite radicals, because of interaction of superoxide anion with NO, has an additional role in the free-radical induced injury.32;44

To summarise, systemic inflammatory events and ischemia-reperfusion injury trigger the development of systemic and/or local vascular dysfunction. Hemodynamic disorders and microcirculatory abnormalities, which initiate secondary tissue edema and tissue hypoperfusion, become the basis of the multiple organ dysfunction syndrome, and the serious co-morbidity and – mortality associated herewith.3;34;38;45;46

Changes in vascular motor function Vascular endothelium and vasculature represent an important aspect/feature

of CPB-related complications since major organ pathological conditions result from impaired vascular function.47 Alteration of cerebral blood flow with impairment of the mechanisms of cerebral vascular auto-regulation is thought to be the main cause of the CPB-mediated brain injury and early neuropsychological dysfunction.48-51 Impairment of the lung vascular function is believed to cause the pulmonary dysfunction of the CPB.24;52 CPB with deep hypothermic circulatory arrest is related to major renal and pulmonary artery dysfunction.53;54 Endothelial dysfunction was shown to be one of the major contributors to post-CPB intestinal complications.55 CPB is also associated with increased numbers of circulating endothelial cells, and studies suggested this to be a marker of the systemic endothelial dysfunction and injury after cardiac surgery with CPB.56;57

CPB is associated with both impairment of the endothelial function and the alteration of the myogenic tone. Short-term CPB (30 minutes) was found to be associated with loss in acetylcholine-induced vasodilatation in preparations of the middle cerebral artery.48 Short-term CPB with 90 minutes recovery period resulted in aggravated pulmonary vascular resistance with hyperreactivity to serotonin.58 In mesenteric arteries, 90 minutes of CPB followed by 6 hours recovery period induced

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endothelial dysfunction and increased vascular reactivity to !1-adrenoceptor agonists.59 Heart failure was shown to aggravate mesenteric artery endothelial and smooth muscle dysfunction after CPB due to increased oxidative stress.60 CPB was also shown to decrease acetylcholine-mediated relaxation in pulmonary resistances artery and seemed to have no effect on contractile reactivity of these arteries to norepinephrine, vasopressin, and the thromboxane A2.52 In another study pulmonary endothelial dysfunction with decreased relaxant response to acetylcholine was shown after fourth day of the post-operative period after CPB.61

The origin of the CPB-related endothelial dysfunction involves inflammatory mechanisms with neutrophils-mediated endothelial injury, while other processes also may contribute herein. Endothelial activation after CPB with increased circulatory levels of the soluble endothelial adhesion molecules (E-selectin and ICAM-1) was even confirmed after 48h of the postoperative period.62 The degradation of the endothelial and cardiomyocytes adherens junctions were shown to mediate CPB-related increased vascular permeability and cardiomyocyte dysfunction.63 Moreover, apoptosis of endothelial cells might represent another possible mechanism of the CPB-mediated vascular dysfunction and aggravated capillary permeability.56;57

CPB has also been shown to affect peripheral vasomotor tone. Decreased myogenic tone has been reported in CPB-related injury of skeletal microvascular beds.64-67 Inhibition of the mitogen-activated protein kinases was suggested to cause decreased coronary myogenic tone after CPB with cardioplegia.68 The activation of the large conductance calcium-activated potassium channels (BKCa) was shown to be the main cause of the reduced myogenic tone of the skeletal muscles arterioles after CPB in humans.64 Altered alpha-adrenergic receptors and protein kinase C-mediated contractions were observed in skeletal muscle microvessels.67 Together, these alteration in vascular tone and reduced peripheral vascular resistance are thought to be responsible for a systemic hypotensive response, which can be observed up to one week post-operatively.44 The suggested mechanisms involved in decreased intrinsic tone include increased circulating levels of vasoactive substances, increased expression of iNOS, adrenergic receptor desensitization and uncoupling from the second messenger system.43

In the majority of these studies, vascular responsiveness was evaluated in one vessel type and at one time point post-CPB only, mostly after a relatively short-term recovery period.43;48;58;64-67;69 It should be noted, however, that organ dysfunction post-CPB has been shown to influence not only in-hospital mortality and morbidity but also mid-term and long-term survival.8-11;19;64;65 However, data on alterations in vasoresponsiveness during the postoperative period that might account for the late effects of CPB are lacking.

Treatment and/or prevention of CPB-associated complications

Several anti-inflammatory pharmacological agents, including nonsteroid anti-inflammatory agents, corticosteroids, aprotinin, antioxidants, complement inhibitors and phosphodiesterase inhibitors have been proposed to inhibit the CPB-related inflammatory processes and vascular dysfunction.41;42;45 Though, none of them entirely


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prevented the adverse pathological and clinical outcomes that are associated with extracorporeal circulation. While a single dose of dexamethasone after the induction of anesthesia has been shown to reduce IL-6 related to CPB, it had no effect on clinical outcome.70 However, dexamethasone decreased the concentration of the circulating cytokines in plasma and had anti-inflammatory properties.71;72 Several clinical studies also showed that dexamethasone had no effect on perioperative abdominal organ damage.73 Conversely, recent data suggest that this compound offers a pulmonary protective effect.19 Another way to reduce the inflammatory response after CPB was to remove activated leukocytes from the circulating blood by leukocyte-depleting filters, but several clinical studies showed controversial data regarding its efficacy.19;74

Alternative therapeutic agents for the prevention of CPB-related complications are still under investigations. One of the possible interventions to limit CPB associated complication is the use of sphingosine-1 phosphate receptors antagonists. Fingolimod (FTY720) is a non-selective sphingosine 1-phosphate (S1P) receptor modulator that acts as an immunosuppressive agent. It was demonstrated to be effective in treatment of the autoimmune disorders (multiple sclerosis, autoimmune neuritis, autoimmune glomerulonephritis).75-79 and organ transplant.80 The main mechanism of its immunomodulatory action concerns activation of the S1P-receptors and is associated with inhibition of the egress of lymphocytes from the secondary lymphoid organs to the peripheral blood.81 Moreover, FTY720 has been shown to prevent or ameliorate ischemia-reperfusion injury,82-87 enhance endothelial barrier and decrease vascular permeability,75;88;89 and improve vascular function through modulation of the S1P1,3,4,5 receptors.90;91 Thus, taking into account the above described pharmacological properties, FTY720 might be a promising therapeutic agent to prevent CPB-related vascular dysfunction.

Aims of this thesis

Despite recent progress, there are major questions to be answered concerning the effects of CPB. First, with respect to changes in vascular function following CPB, it should be noted that previously mainly short-term changes (approximately 24 hours period) have been evaluated, while a substantial amount of critical post-operative events take place between the second and fifth day of the recovery period. Also, the association between vascular functionality and inflammatory events has not been addressed sufficiently. Finally, the possibilities of pre-operative and/or intraoperative intervention in vascular reactivity, as an approach to prevent the CPB-related complications, were not formerly evaluated. Thus, the principle aim of this thesis is to investigate the pathophysiology and novel therapeutic options with respect to CPB-related complications in a rat model of CPB.

The first part of this thesis investigates the changes in vascular function evoked by CPB. To this end, vasomotor responses were obtained in different types of vessels during a clinically relevant postoperative recovery period (up to 5 days) in rat. In addition, the relationship between endothelial activation and vascular responsiveness was studied in rat aorta throughout the whole recovery period.

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In the second part of this thesis, the pathophysiology of CPB related inflammation was further characterized by measurement of the expression of inflammatory markers in lung and kidney during the entire recovery period by PCR, Western blot and micro-array analysis in our rat model of CPB.

In the last part of the thesis, the effects of drugs are explored. Since anesthetics influence vascular tone, they may add to the CPB-related hypotension. Thus, the impact of different types of anesthesia was studied in a model of extreme hypotension in mice (hemorrhagic shock). Finally, the effect of the novel immunosuppressive drugs on CPB induced changes in vasomotor response was measured to define its therapeutic potential to prevent the CPB-related complications. To this end, rats were pretreated with selective and non-selective agonists of the S1P-receptors and contractile and relaxant vascular reactivity was assessed.

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55. Andrasi TB, Buhmann V, Soos P, Juhasz-Nagy A, Szabo G: Mesenteric complications after hypothermic cardiopulmonary bypass with cardiac arrest: underlying mechanisms. Artif.Organs 2002; 26: 943-6

56. Schmid FX, Vudattu N, Floerchinger B, Hilker M, Eissner G, Hoenicka M, Holler E, Birnbaum DE: Endothelial apoptosis and circulating endothelial cells after bypass grafting with and without cardiopulmonary bypass. Eur.J.Cardiothorac.Surg. 2006; 29: 496-500

57. Schmid FX, Floerchinger B, Vudattu NK, Eissner G, Haubitz M, Holler E, Andreesen R, Birnbaum DE: Direct evidence of endothelial injury during cardiopulmonary bypass by demonstration of circulating endothelial cells. Perfusion 2006; 21: 133-7

58. Sato K, Li J, Metais C, Bianchi C, Sellke F: Increased pulmonary vascular contraction to serotonin after cardiopulmonary bypass: role of cyclooxygenase. J.Surg.Res. 2000; 90: 138-43

59. Doguet F, Litzler PY, Tamion F, Richard V, Hellot MF, Thuillez C, Tabley A, Bouchart F, Bessou JP: Changes in mesenteric vascular reactivity and inflammatory response after cardiopulmonary bypass in a rat model. Ann.Thorac.Surg. 2004; 77: 2130-7

60. Andrasi TB, Bielik H, Blazovics A, Zima E, Vago H, Szabo G, Juhasz-Nagy A: Mesenteric vascular dysfunction after cardiopulmonary bypass with cardiac arrest is aggravated by coexistent heart failure. Shock 2005; 23: 324-9

61. Nyhan D, Gaine S, Hales M, Zanaboni P, Simon BA, Berkowitz D, Flavahan N: Pulmonary vascular endothelial responses are differentially modulated after cardiopulmonary bypass. J.Cardiovasc.Pharmacol. 1999; 34: 518-25

62. Galea J, Rebuck N, Finn A, Manche A, Moat N: Expression of soluble endothelial adhesion molecules in clinical cardiopulmonary bypass. Perfusion 1998; 13: 314-21


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63. Bianchi C, Araujo EG, Sato K, Sellke FW: Biochemical and structural evidence for pig myocardium adherens junction disruption by cardiopulmonary bypass. Circulation 2001; 104: I319-I324

64. Feng J, Liu Y, Khabbaz KR, Sodha NR, Osipov RM, Hagberg R, Alper SL, Sellke FW: Large conductance calcium-activated potassium channels contribute to the reduced myogenic tone of peripheral microvasculature after cardiopulmonary bypass. J.Surg.Res. 2009; 157: 123-8

65. Feng J, Chu LM, Robich MP, Clements RT, Khabbaz KR, Hagberg R, Liu Y, Osipov RM, Sellke FW: Effects of cardiopulmonary bypass on endothelin-1-induced contraction and signaling in human skeletal muscle microcirculation. Circulation 2010; 122: S150-S155

66. Stamler A, Wang SY, Aguirre DE, Johnson RG, Sellke FW: Cardiopulmonary bypass alters vasomotor regulation of the skeletal muscle microcirculation. Ann.Thorac.Surg. 1997; 64: 460-5

67. Wang SY, Stamler A, Li J, Johnson RG, Sellke FW: Decreased myogenic reactivity in skeletal muscle arterioles after hypothermic cardiopulmonary bypass. J.Surg.Res. 1997; 69: 40-4

68. Khan TA, Bianchi C, Ruel M, Voisine P, Li J, Liddicoat JR, Sellke FW: Mitogen-activated protein kinase inhibition and cardioplegia-cardiopulmonary bypass reduce coronary myogenic tone. Circulation 2003; 108 Suppl 1: II348-II353

69. Putnam EA, Manners JM: Vascular resistance during cardiopulmonary bypass. Its effect on cardiac performance in the immediate post-bypass period. Anaesthesia 1983; 38: 635-43

70. Sobieski MA, Graham JD, Pappas PS, Tatooles AJ, Slaughter MS: Reducing the effects of the systemic inflammatory response to cardiopulmonary bypass: can single dose steroids blunt systemic inflammatory response syndrome? ASAIO J. 2008; 54: 203-6

71. Bronicki RA, Backer CL, Baden HP, Mavroudis C, Crawford SE, Green TP: Dexamethasone reduces the inflammatory response to cardiopulmonary bypass in children. Ann.Thorac.Surg. 2000; 69: 1490-5

72. El A, Sr., Rosseel PM, De Lange JJ, Groeneveld AB, Van SR, Van Wijk EM, Scheffer GJ: Dexamethasone decreases the pro- to anti-inflammatory cytokine ratio during cardiac surgery. Br.J.Anaesth. 2002; 88: 496-501

73. Morariu AM, Loef BG, Aarts LP, Rietman GW, Rakhorst G, van OW, Epema AH: Dexamethasone: benefit and prejudice for patients undergoing on-pump coronary artery bypass grafting: a study on myocardial, pulmonary, renal, intestinal, and hepatic injury. Chest 2005; 128: 2677-87

74. Boodram S, Evans E: Use of leukocyte-depleting filters during cardiac surgery with cardiopulmonary bypass: a review. J.Extra.Corpor.Technol. 2008; 40: 27-42

75. Brinkmann V, Cyster JG, Hla T: FTY720: sphingosine 1-phosphate receptor-1 in the control of lymphocyte egress and endothelial barrier function. Am.J.Transplant. 2004; 4: 1019-25

76. Brinkmann V: FTY720 (fingolimod) in Multiple Sclerosis: therapeutic effects in the immune and the central nervous system. Br.J.Pharmacol. 2009; 158: 1173-82

77. Chun J, Hartung HP: Mechanism of Action of Oral Fingolimod (FTY720) in Multiple Sclerosis. Clin.Neuropharmacol. 2010;

78. Zhang Z, Schluesener HJ: FTY720: a most promising immunosuppressant modulating immune cell functions. Mini.Rev.Med.Chem. 2007; 7: 845-50

79. Zhang Z, Zhang ZY, Fauser U, Schluesener HJ: FTY720 ameliorates experimental autoimmune neuritis by inhibition of lymphocyte and monocyte infiltration into peripheral nerves. Exp.Neurol. 2008; 210: 681-90

80. Martini S, Peters H, Bohler T, Budde K: Current perspectives on FTY720. Expert.Opin.Investig.Drugs 2007; 16: 505-18

81. Mullershausen F, Zecri F, Cetin C, Billich A, Guerini D, Seuwen K: Persistent signaling induced by FTY720-phosphate is mediated by internalized S1P1 receptors. Nat.Chem.Biol. 2009; 5: 428-34

82. Delbridge MS, Shrestha BM, Raftery AT, El Nahas AM, Haylor JL: Reduction of ischemia-reperfusion injury in the rat kidney by FTY720, a synthetic derivative of sphingosine. Transplantation 2007; 84: 187-95

83. Egom EE, Ke Y, Musa H, Mohamed TM, Wang T, Cartwright E, Solaro RJ, Lei M: FTY720 prevents ischemia/reperfusion injury-associated arrhythmias in an ex vivo rat heart model via activation of Pak1/Akt signaling. J.Mol.Cell Cardiol. 2010; 48: 406-14

84. Hasegawa Y, Suzuki H, Sozen T, Rolland W, Zhang JH: Activation of sphingosine 1-phosphate receptor-1 by FTY720 is neuroprotective after ischemic stroke in rats. Stroke 2010; 41: 368-74

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85. Kaudel CP, Schmiddem U, Frink M, Bergmann S, Pape HC, Krettek C, Klempnauer J, Winkler M: FTY720 for treatment of ischemia-reperfusion injury following complete renal ischemia in C57/BL6 mice. Transplant.Proc. 2006; 38: 679-81

86. Kaudel CP, Frink M, van GM, Schmiddem U, Probst C, Bergmann S, Krettek C, Klempnauer J, Winkler M: FTY720 application following isolated warm liver ischemia improves long-term survival and organ protection in a mouse model. Transplant.Proc. 2007; 39: 493-8

87. Lee KD, Chow WN, Sato-Bigbee C, Graf MR, Graham RS, Colello RJ, Young HF, Mathern BE: FTY720 reduces inflammation and promotes functional recovery after spinal cord injury. J.Neurotrauma 2009; 26: 2335-44

88. Camp SM, Bittman R, Chiang ET, Moreno-Vinasco L, Mirzapoiazova T, Sammani S, Lu X, Sun C, Harbeck M, Roe M, Natarajan V, Garcia JG, Dudek SM: Synthetic analogs of FTY720 [2-amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol] differentially regulate pulmonary vascular permeability in vivo and in vitro. J.Pharmacol.Exp.Ther. 2009; 331: 54-64

89. Dudek SM, Camp SM, Chiang ET, Singleton PA, Usatyuk PV, Zhao Y, Natarajan V, Garcia JG: Pulmonary endothelial cell barrier enhancement by FTY720 does not require the S1P1 receptor. Cell Signal. 2007; 19: 1754-64

90. Sarai K, Shikata K, Shikata Y, Omori K, Watanabe N, Sasaki M, Nishishita S, Wada J, Goda N, Kataoka N, Makino H: Endothelial barrier protection by FTY720 under hyperglycemic condition: involvement of focal adhesion kinase, small GTPases, and adherens junction proteins. Am.J.Physiol Cell Physiol 2009; 297: C945-C954

91. Tolle M, Levkau B, Keul P, Brinkmann V, Giebing G, Schonfelder G, Schafers M, von Wnuck LK, Jankowski J, Jankowski V, Chun J, Zidek W, van der GM: Immunomodulator FTY720 Induces eNOS-dependent arterial vasodilatation via the lysophospholipid receptor S1P3. Circ.Res. 2005; 96: 913-20

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Chapter 2

Changes in vasomotor function of small coronary and mesenteric artery during 5-day follow-up after

cardiopulmonary bypass in the rat. Characterization of endothelial mediators and contractility

Iryna V. Samarska, Hendrik Buikema, Hubert Mungroop,

Martin C. Houwertjes, Fellery de Lange, Yumei Wang, Anne H. Epema, Robert H. Henning

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Abstract Background: Changes in vascular function may represent key elements of hemodynamic instability and organ injury after cardiopulmonary bypass (CPB). We investigated vascular contraction and endothelium dependent relaxation of small coronary and mesenteric arteries in a rat model of CPB with a follow-up period of 5 days. Materials and Methods: Male Wistar rats (n=78) were anesthetized with isoflurane (2.5 %) and fentanyl/midazolam during CPB. Animals were assigned to sham or CPB with normothermic extracorporeal circulation for 60 min at a flow of 120 mL kg-1 min-1. After recovery for 60 min to 5 days, constriction to phenylephrine (mesenteric artery) and serotonin (coronary artery), and relaxation to acetylcholine (both arteries) were assessed. The expression of nitrotyrosine was assessed by western blot. Results: Sham and CPB decreased the sensitivity to constricting agents at 1 day of recovery compared to control both in mesenteric (-log EC50; sham: 5.9±0.3, CPB: 5.9±0.1, control: 6.3±0.2) and in coronary arteries (-log EC50; sham: 5.8±0.3, CPB: 5.8±0.6, and control: 6.5±0.3). Moreover, CPB progressively decreased total ACh-mediated relaxation, the reduction becoming maximal at 2 days of recovery (AUC; sham: 248.8±48.8, CPB: 100.9±73.0), mainly due to decreased EDHF contribution. In addition, coronary artery of CPB animals displayed a biphasic change characterized by initial increased relaxation due to EDHF, followed by an inhibition of NO mediated relaxation. Nitrotyrosine content of mesenteric artery was about 8-fold higher at 2 days of recovery in CPB group compared to Sham. Conclusions: In Sham animals, vasoconstrictor responses were primarily inhibited, demonstrating profound changes in vascular function following a relative mild procedure of anesthesia and cannulation. In addition, CPB inhibited endothelial-mediated vasorelaxation related to changes in EDHF and NO. Notably, vascular responses after CPB were not normalized at 5 days after the procedure. The observed changes may contribute to hemodynamic instability and organ injury during a protracted period after CPB. Introduction

Cardiopulmonary bypass (CPB) enables complex cardiac surgical procedures. CPB however, is still associated with increased morbidity and mortality1 due to damage of various organ and systems including heart, kidney, lung, brain and gut.2-9 While the mechanisms initiating organ injury are complex and not fully understood, CPB induced activation of systemic inflammation and ischemia reperfusion injury are considered major factors. Previous research showed that this results in the impairment of vascular function immediately following CPB, consisting of both endothelial and smooth muscle dysfunction of small arteries.10-19 These changes affect organ blood supply contributing to the development of secondary tissue edema and tissue hypo-perfusion, setting the stage for multiple organ dysfunction. Thus, changes in vasoreactivity represent a pivotal component of the development of organ dysfunction following CPB. 20-22

Vascular reactivity of the small vessels 23

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Characteristic differences exist in the regulation of vascular tone in small artery beds of different organs. In general, vascular tone is controlled by neurohumoral factors acting through specific receptor pathways in the endothelium and smooth muscles, as well as by local hemodynamic factors including shear stress and intraluminal pressure.23;24 Further, vasodilation is governed mainly by the endothelium through the production of various components, including nitric oxide (NO), endothelium dependent hyperpolarizing factor (EDHF) and prostaglandins.25;26 The contribution of each component is dependent on the vascular bed studied, i.e. mesenteric artery is mainly dependent on EDHF, whereas coronary artery represents a mixed type dependent on NO and EDHF.27;28

Clinical and experimental data suggest that the CPB induced initial systemic inflammatory response (SIRS) weans off within 24 h following surgery.29 In contrast, organ injury following CPB is observed not only in the immediate post-operative period (in the ICU),30 but during a protracted time frame following surgery.31, 32 Thus, we hypothesized that acute CPB injury results in protracted dysfunction of vasomotor control. Therefore, our objective was to evaluate vascular reactivity of different small arteries in an experimental rat model of CPB. Thus, we measured changes in vascular contractility as well as endothelial relaxation function in mesenteric and coronary arteries, including assessment of the contribution of different endothelial dilative components. Further, vascular reactivity was evaluated during a clinically relevant post-operative period. Therefore, time-dependent changes in vascular reactivity were examined from 60 min up to 5 days of recovery.

Material and Methods

The experimental protocol was approved by the Institutional Animal Care and Use Committee of the University of Groningen. This study was performed in n=78 adult male Wistar rats with body weights of 507.4 ± 31.3 g (Harlan, Zeist, The Netherlands) housed under standard conditions with free access to food (standard rat chow; Hope Farms, Woerden, The Netherlands) and drinking water throughout the study period.

Experimental groups

Experimental animals were randomly allocated to one of four experimental groups with different duration of the recovery period after the procedure, being 60 min and 1, 2 and 5 days. Each group was subdivided in two sub-groups: CPB and Sham. CPB animals were subjected to the full experimental protocol described below, including anesthesia, cannulation, extracorporeal circulation, weaning and the corresponding recovery period. Sham animals followed the same procedure except for the extracorporeal circulation, but maintained cannulated and mechanically ventilated. In order to evaluate the normal vascular reactivity in rats without invasive interventions, an additional untreated control group was examined. Animals in this group were sacrificed under brief isoflurane anesthesia (2.5 %) only.

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Experimental protocol The experimental protocol consisted of the following parts: anesthesia, preparation, CPB, a weaning and a recovery period (see fig. 1). Anesthesia was induced with 2.5 % isoflurane in O2/air (1:1) before intubation and mechanical ventilation (Amsterdam Infant Ventilator; HoekLoos, Amsterdam, The Netherlands). Tidal volume was set to achieve normocapnia (verified by capnography 35-42 mmHg, and arterial blood gas analysis), with O2/air (1:2) at a ventilation rate of 50 min-1 (0.5 s inspiration time). Rectal temperature was kept at 37.0 ± 0.5 °C, using an electrical heating pad. The left femoral artery was cannulated (26-gauge catheter) for continuous blood pressure monitoring. The mean arterial pressure (MAP) was kept between 70 and 100 mm Hg by adjusting the isoflurane concentration as necessary (typically between 2.0-2.5%). Immediately before insertion of the arterial line, 250 IU kg-1 heparin was administered. The left carotid artery was cannulated for arterial inflow using a 22-gauge catheter. A multi-orifice 4.5 French cannula (Desilets-Hoffmann catheter, Cook Son, The Netherlands) was advanced into the right heart using the right common jugular vein for access. The tail vein was cannulated for the administration of intravenous anesthetics, heparin, and protamine sulfate.

Cardiopulmonary bypass Subsequently, CPB was initiated for 60 min. The set-up consisted of a glass

venous reservoir, a peristaltic pump (Pericor® SF70, Verder, Haan, Germany), a rat membrane oxygenator (M.Humbs, Valley, Germany) and a glass counter-flow heat exchanger with built-in bubble trap. The oxygenator carried a sterile, disposable three-layer artificial diffusion membrane, made from hollow polypropylene fibres (Jostra AG, Hirrlingen, Germany). All components were connected with polyethylene tubing (1.6 mm inner diameter). The venous reservoir and heat exchanger were sterilized prior to use. The circuit was primed with 15 ml of haes 60 mg ml-1 solution (Voluven®, Fresenius Kabi, Bad Homburg, Germany). No donor blood was used. Animals were additionally heparinized (250 IU kg-1) after the start of ECC. During CPB, rats were anesthetized with intravenous fentanyl (10 "g kg-1), atracurium (0.5 mg kg-1), and

Figure 1. Scheme of the experimental protocol in Sham and CPB groups regarding the duration of recovery period. The experimental protocol consisted of the following parts: anesthesia, preparation, extracorporeal circulation, a weaning and a recovery period (see methods). Abbreviations: CPB=cardiopulmonary bypass.

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midazolam (2 mg kg-1) and blood oxygen saturation was monitored continuously by a pulse oxymeter. Targeted CBP flow was 120 mL kg-1 min-1. During CPB ventilation was stopped and oxygenator gas flow was maintained at 800 ml min–1 of O2:air mixture (1:4). Samples for blood gas analysis (0.1 "l) were drawn at four time-points: at the end of preparation period (15 min before start of CPB), twice during the CPB (at 15 and 45 min), and at 45 min after the end of CPB.

Weaning from CPB and recovery At the end, CPB flow was gradually decreased and mechanical ventilation was

initiated. Protamine (150 IU kg-1 i.v.) was administered to neutralize heparin and cannules were removed and wounds sutured. Animals were kept ventilated under isoflurane anesthesia (0.8-1.0%) for 1 h to stabilize, followed by extubation. The total duration of the recovery period lasted 1 h, and 1, 2 and 5 days after the end of the CPB. Sacrification was performed under brief isoflurane anesthesia (2.5 %).

Vascular reactivity At sacrification, all mesenteric loops and the heart were removed and placed in

cold physiological saline solution. Several segments of the third branch of the mesenteric superior artery and the interseptal coronary artery were dissected, prepared as ring vessel preparations (1.8-2.0 mm in length) and mounted on two 40 µm stainless wires connected to force transducers in individual organ bath chambers for isometric tension recordings in a wire-myograph (Danish Myo Technology A/S, Aarhus, Denmark). The baths contained 6 ml of Kreb’s solution warmed to 37°C and bubbled continuously with 95%O2/5%CO2 at pH 7.4. Vessels were subjected to a standardized normalization procedure [33] and left to equilibrate for 40 min until they were at a steady baseline. In brief, the vessels were distended stepwise until effective pressure exceeded 100 mmHg (13.3 kPa). The internal circumference, IC100, was found from the Laplace’s equation and the experiments were performed at the IC1=0.9*IC100. Vascular segments were primed and checked for viability by two consecutive exposures to potassium chloride (60 mM).

The experimental protocol consisted of evaluation of contractile responses to phenylephrine (PE; 10 nM to 100 "M; mesenteric arteries) or serotonin (10 nM to 100 "M; coronary arteries). Endothelium-dependent relaxation to acetylcholine (ACh; 10 nM to 300 "M) was assessed in rings precontracted with PE (mesenteric artery) or serotonin (coronary artery). To assess the contribution of different endothelial mediators, ACh-induced relaxations were studied in the absence and presence of L-NMMA (0.1 mM; an inhibitor of NO-synthase) and/or indomethacine (1 "M; an inhibitor of cyclooxygenase) administered 20 min before application of phenylephrine.34 It has been shown previously that the remaining ACh induced relaxation in presence of both cyclooxygenase and NO-synthase inhibitors was fully dependent on EDHF.35 Following the final concentration of ACh, a maximal concentration of the NO-donor sodium nitroprusside (SNP; 0.1 mM) was applied to assess maximal endothelium-independent relaxation.

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Western blot The methods used were described previously.36;37 Briefly, after grinding, the

frozen mesenteric beds were placed in 300 "l of boiling 2% SDS followed by pounding by a polytron (Kinematica AG Littau, Switzerland). Then, samples were centrifuged (4000 rpm, 1 min) and boiled (95oC) for 5 min. After a second centrifugation (13000 rpm, 3 min), supernatant was collected and used for measurements. Protein concentration was determined by Bio-Rad Dc Protein Assay (Bio-Rad, Hercules, CA). Twenty µg of total protein from each sample was separated on 10% Tris-Glycine-SDS-polyacrylamide gel and transferred to polyvinylidene difluoride (PVDF) membranes. Anti-nitrotyrosine antibody (ALEXIS Biochemicals, Lausen, Switzerland) and anti-GAPDH (Sigma, St. Louis, MO) were used as primary antibodies. Horseradish peroxidase-linked rabbit anti-mouse antibody (Sigma, St. Louis, MO) was applied as a secondary antibody. GAPDH served as a housekeeping protein.

Drugs The composition of Krebs solution was as follows (mM): 120.4 NaCl, 5.9 KCl,

2.5 CaCl2, 1.2 MgSO4, 25.0 NaHCO3, 1.2 NaH2PO4, 11.5 glucose; these chemicals were obtained from Merck (Darmstadt, Germany). The following drugs were used: acetylcholine chloride (ACh), phenylephrine (PE), serotonin, indomethacine, SNP, NG-methyl-L-arginine (L-NMMA). Stock solution (10 mM) for indomethacine was prepared in 96% ethanol. All other drugs were dissolved in deionized water. L-NMMA was purchased from MP Biomedicals (Illkirch, France). All other compounds were purchased from Sigma (St. Louis, MO). The concentrations presented in the concentration-curve responses are expressed as a final molar concentration in the bath.

Data analysis Data are given as mean ± SD. Contractile responses to PE and serotonin are

presented in mN and relaxations to ACh and SNP were calculated as a percentage of pre-constriction. For each concentration-response curve the following parameters were determined: (1) the concentration at which half-maximal response was reached (EC50), (2) the maximal response (Emax) and (3) the Area Under the Curve (AUC) in arbitrary units (AU) (SigmaPlot 11, Systat Software, San Jose, CA, USA). The difference in AUC of concentration-response curves to ACh in the absence and presence of inhibitor(s) was used to quantify the contribution of different endothelial relaxing components.38 Differences were evaluated using Student’s t-test, one-way ANOVA, repeated measurements ANOVA combined with post-hoc Bonferroni test or with t-test (SPSS 16.0, Chicago, IL, USA). Differences were considered significant at P<0.05 (2-tailed). The relationship between different relaxant pathways and total acetylcholine-mediated relaxation was evaluated with regression analysis and Spearman correlation.


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Results Hemodynamics and blood gas analysis At baseline, all parameters measured were similar and in the normal range.

During CPB, MAP was significantly lower compared with Sham (fig. 2). Blood gas analysis showed a decreased pH, bicarbonate (HCO-

3), and Base excess and increased levels of pCO2 and pO2 during CPB (fig. 2), indicating mild acidosis. Hematocrit was significantly decreased in CPB groups as a consequence of hemodilution with the priming solution. Intraoperative hyperglycemia was observed in both Sham and CPB groups during the first 2 hours. After weaning from CPB, all parameters, including MAP, returned to normal values and were similar in both Sham and CPB, except for the hematocrit (fig. 2).

Figure 2. Data on blood gas analysis and mean arterial pressure (MAP, femoral artery) and glucose concentration in Sham and CPB groups during the experimental protocol. Abbreviations: CPB= cardiopulmonary bypass. * indicates P<0.05, independent t-test. !

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Contraction studies in isolated arteries Full concentration-response curves to phenylephrine and serotonin were

constructed in mesenteric (fig. 3, left panel) and coronary arteries (fig. 3, right panel), respectively. EC50-values and Emax derived from these curves are presented in table 1. As compared to mesenteric arteries of control rats, concentration–response curves to PE in Sham rats were shifted to the right (fig. 3A) and statistical significance was reached at 1h, 2 days and 5 days of recovery (table 1). In contrast, Emax of PE was significantly decreased in Sham rats at 1 day of recovery, while normalizing again at longer periods of recovery (table 1). The time pattern of the changes in overall contractility to PE is shown in figure 3C as AUC for concentration-response curves to PE. In Sham rats, overall contractility was reduced most prominently at 1 day of recovery, followed by normalization at the 5th post-procedure day. Contractile reactivity in CPB treated animals followed the same pattern (fig. 3B, table 1). Collectively, our data show that changes in contractility of mesenteric arteries were due to the anesthetic and cannulation procedure.

Concentration–response curves to serotonin in coronary artery of Sham rats (fig. 3D) were significantly shifted to the right after short-term recovery compared to control (i.e. at 1 h and 1 day; table 1). In contrast, Emax for Serotonin in Sham was increased when studied after longer periods of recovery (i.e. at 2 days and 5 days), but not at short-term (table 1). Collectively, AUC of concentration-response curves show a tendency for a reduction in overall contractility in coronary arteries of Sham rats after acute recovery, followed by normalization towards the 5th post-procedural day, but variation was too large to reach statistical significance (fig. 3F). Contractile reactivity in the group of CPB rats largely followed the same pattern (fig. 3E). Comparison of

Table 1. Contractile response to phenylephrine in mesenteric arteries and to serotonin in coronary artery in Sham and CPB groups

Mesenteric artery Recovery Time

-Log EC50 Emax, µµM Control Sham CPB Control Sham CPB

-6.3 ±0.2

11.3±3.1

1hour -6.1±0.5 -6.0±0.4 10.5±3.6 10.8±2.8 1 day -5.9±0.3* -5.9±0.1* 5.8±2.7 * 6.9±3.6* 2 days -5.9±0.2* -6.2±0.3 10.4±3.1 9.7±5.9 5 days -6.2±0.3 -6.2±0.5 10.0±2.3 10.0±5.2

Coronary artery Recovery Time

-Log EC50 Emax, µµM Control Sham CPB Control Sham CPB

-6.5 ±0.3

1.4±1.0

1 hour -5.6±0.1* -5.7±0.3* 1.5±0.6 1.7±0.9 1 day -5.8±0.3* -5.8±0.6* 1.3±0.6 1.7±1.3 2 days -6.2±0.4 -6.4±0.5 3.4±1.9 1.4±0.8 5 days -5.8±0.3# -5.7±0.2* 2.7±0.9 2.4±1.8 Data are given as mean±SD. Abbreviations: CPB, cardiopulmonary bypass; Emax, the maximal response; -Log EC50, negative logarithm of the concentration at which half-maximal response was reached (EC50).*-P<0.05 vs Control, one-way ANOVA with post-hoc Bonferroni test #- P<0.05 vs Control, t-test


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individual time points of Sham and CPB to controls showed a significant difference in EC50 values at almost all time points in both groups (table 1). These data indicate that the protracted changes in contractility in coronary arteries were also due to the anesthetic and cannulation procedure rather than ECC. Collectively, the above findings demonstrate that CPB-related surgical procedures and/or extended anesthesia induced extensive and protracted changes in the contractility of mesenteric and coronary arteries, without significant additional influence of CPB.

Relaxation studies in isolated arties Endothelium-dependent relaxation was assessed by construction of full

concentration-response curves to acetylcholine (ACh), shown in the figure in the Supplement Digital Content 1 for mesenteric and coronary arteries of normal control rats. The major endothelial dilative pathways were investigated by blocking the action of two components: vasoactive prostaglandins (PGs) and nitric oxide (NO) with indomethacin and additional pre-incubation with L-NMMA. The difference between AUCs of concentration-response curves in the absence and presence of inhibitors was used to calculate contribution of PGs, NO and endothelial derived hyperpolarizing factor (EDHF; Supplement Digital Content 1). Endothelium-independent relaxation was unaffected in both mesenteric and coronary artery since the relaxant response to sodium nitroprusside was similar in all groups (see table, Supplement Digital Content 2).

Preconstriction levels to phenylephrine were not significantly different among groups (see table, Supplement digital data 3, which is a table containing all preconstriction levels to phenylephrine). The changes in the AUC of total ACh-mediated relaxation and contributions of the endothelial dilative mediators in mesenteric artery were plotted against time of recovery (fig. 4). In Sham rats, total ACh-induced relaxation progressively increased during the recovery period to become even significantly larger as compared normal control rats (fig. 4A). In contrast, total ACh-mediated relaxation progressively decreased in CPB rats, the reduction becoming maximal at 2 days recovery (fig. 4A). Interestingly, the observed changes in total ACh-induced relaxation were similar to changes in EDHF contribution (fig. 4C), while contribution of NO and PGs did not change (fig. 4B, D). By the 5th day of recovery, AUC of total ACh-induced relaxation and the EDHF contribution had normalized again. Finally, EC50 and Emax of individual time points of Sham and CPB were compared to control by Student t-test (See table in Supplemental Digital Content 4), that showed significant changes in some point up to 5 days of recovery. The above findings indicate that the surgical procedure under general anesthesia induced a temporal increase in endothelium-dependent relaxation of mesenteric arteries via mobilization of EDHF, which is attenuated after CPB.

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Preconstriction levels of coronary artery to serotonin were not significantly different among groups (See table in Supplement Digital Content 3). Total relaxation of ACh mediated dilatation was unaltered in Sham compared to control rats (fig. 5 A,C). Regarding its components, the NO-contribution to ACh-induced relaxation was profoundly decreased at 1 h in Sham rats and to a lesser extent in later time points (fig.

Figure 3. Vascular contractility of rat mesenteric and coronary arteries at different periods of recovery (1h, 1day, 2 or 5 days) in Sham groups (A,B and D,F, respectively) and following subjection of the animals to CPB-related procedures (A,C and E,F, respectively). Contractile responses are given as full CR-curves (A,B,D,E) and the AUC (C). Data are mean ± SD (n=6-9). * indicates P<0.05 CPB vs Control, one way ANOVA with post-hoc Bonferroni test. # indicates P<0.05 Sham vs Control, repeated measurements ANOVA combined with post-hoc Bonferroni test. Abbreviations: PE= phenylephrine; CPB= cardiopulmonary bypass; SE= serotonine.

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5D). However, decreased NO contribution was compensated by the increase in the contribution of dilatory PGs (fig. 5B).

Following CPB a more complex pattern was observed, resulting in an enhanced total relaxation to ACh at 1 h of recovery, followed by a larger inhibition of relaxation at 2 days (fig. 5A). Enhanced relaxation was mainly due to a significant increase in EDHF (fig. 5C), whereas inhibition at 2 days seems caused by a persistent decrease of NO contribution (fig. 5D). Collectively, the above findings suggest that the unchanged relaxation observed following cannulation procedures and general anesthesia results from a balanced decrease in NO and increase in PGs. In addition, CPB mainly resulted in the acute mobilization of EDHF, thus enhancing relaxation at 90 min of recovery.

Western blotting Reactive oxygen species (ROS) have been implicated to impair EDHF

mediated relaxation of mesenteric artery.39;40 As CPB is associated with enhanced inflammatory response and oxidative stress [41], production of ROS was studied at 2 days of recovery, when relaxation in mesenteric artery was greatly enhanced in CPB compared to Sham (fig. 4). ROS production in mesenteric artery was quantified by measurement of nitrotyrosine content.42. Nitrotyrosine content was about 8-fold higher at 2 days of recovery in CPB group in comparison to Sham (fig. 6). Thus, these data suggest that CPB induced excess production of ROS at 2 days of recovery inhibiting the EDHF component of relaxation. !!

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Figure 4. Total acetylcholine (ACh-)-induced relaxation (A) - and the contribution of (B) prostaglandins (PG’s), (C) NO, and (D) EDHF hereto, all provided as AUC - in rat mesenteric arteries at different periods of recovery (1h, 1day, 2 or 5 days) following subjection of the animals to CPB-related procedures. Data are mean ± SD (n=5-8). * indicates P<0.05 CPB vs Control, t-test; # indicates P<0.05 Sham vs Control, one-way ANOVA with following Bonferroni test; # indicates P<0.05 CPB vs Sham, t-test. Abbreviations: CPB= cardiopulmonary bypass.

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Figure 5. Total acetylcholine (ACh-)-induced relaxation (A) - and the contribution of (B) prostaglandins (PG’s), (C) NO, and (D) EDHF hereto, all provided as AUC - in rat coronary arteries at different periods of recovery (1h, 1day, 2 or 5 days) following subjection of the animals to CPB-related procedures. Data are mean ± SD (n=5-8). *indicates P<0.05 Sham vs CPB, one-way ANOVA with following Bonferroni test; # indicates P<0.05 Sham vs Control, one-way ANOVA with following Bonferroni test; # indicates P<0.05 CPB vs Sham, one-way ANOVA with following Bonferroni test. Abbreviations: CPB=cardiopulmonary bypass.

Figure 6. Nitrotyrosine expression in mesenteric beds after 48h recovery period. representative blot (A) in Sham (n=3) and CPB (n=4) and Relative expressions patterns (B) in Sham and CPB samples. All data is normalized to expression level of GAPDH and present in arbitrary units. *P<0.05 CPB 2 days vs Sham 2 days, independent t-test.


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Discussion Changes in the vascular responsiveness of small arteries after cardiopulmonary bypass were studied during 5 days of recovery. First, our data show that CPB affects the vasomotor response of isolated arteries during a protracted time period, as vascular function following CPB did not normalize during 5 days of follow-up. Second, Sham and CPB induce complex changes in vasomotor response of small arteries following CPB in rat, which differ between vascular beds. Sham operation primarily induced changes in vasoconstrictor responses, with additional changes in vasorelaxation caused by CPB.

Sham operation caused a temporal depression in contractility of mesenteric and coronary artery around day 1 of recovery. In addition, sham operation caused an increased relaxant responsiveness of mesenteric artery after 2 days of recovery. In contrast, CPB strongly inhibited the vascular relaxation of mesenteric artery at 2 days recovery caused by decreased EDHF contribution.

Coronary artery of CPB animals displayed a biphasic change characterized by initial increased relaxation due to EDHF, followed by an inhibition of NO mediated relaxation. Collectively, these data demonstrate the induction of profound changes in vascular function following a relative mild procedure of anesthesia and cannulation. Specific changes induced by CPB consist of a change in endothelial-mediated relaxation, mainly related to its EDHF component. Furthermore, since the relaxant response to sodium nitroprusside was similar in all groups, alterations in relaxation seem due to changes at the level of the endothelial cell rather than caused by altered vascular smooth muscle responsiveness. Finally, the inhibited EDHF response in mesenteric artery of CPB animals was accompanied by the upregulation of nitrotyrosine, suggesting a role for an inflammatory response and ROS. Vasoresponsiveness after CPB

Vascular reactivity after CPB has been investigated previously in various vascular beds43-49 although follow-up is limited to a few hours after CPB. In rat mesenteric artery, 90 min of CPB with 2.5 h recovery increased the contractile response to phenylephrine and evoked endothelial dysfunction.50 CPB at 280C body temperature with aortic cross-clamping and cardioplegic arrest in dog caused depressed endothelial mediated mesenteric vasodilation immediately following CPB.51 The differences from our results, in which vasorelaxation was unaltered shortly following CPB, may be explained by differences from our model, i.e. a longer duration of CPB, cardioplegic arrest, hypothermia and the use of donor blood. Data on vascular reactivity of coronary artery following CPB are limited. In particular, a decreased myogenic vascular tone has been reported in pig coronary and human atrial arteries.52-

54 However, data on changes in receptor mediated vasomotor responses following CPB are lacking, precluding comparison of our results to others.

Sham induced changes in vascular reactivity One of the main findings of our study constitutes of changes in vascular

reactivity following Sham procedure with a delay of 1 to 2 days following the procedure. Moreover, Sham induced changes appear more prominent in mesenteric artery. The impaired vascular function observed in isolated mesenteric artery favors vasodilation,

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which in vivo may relate to a low flow state and splanchnicus hypoperfusion with increased incidence of gastrointestinal complications after major surgery.55

The underlying mechanism of changes in vascular reactivity in Sham remains unknown. In our study, a main candidate for the induction of these changes is isoflurane, but limited data are available. Volatile anesthetics, including isoflurane, are well known for their vasorelaxing effect.56 Additionally, volatile anesthetics have been shown to inhibit AngII-mediated contraction.57 Studies employing administration of isoflurane to isolated arterial segments indicate that inhibition of contraction by isoflurane, if any, is limited to a short time interval of the recovery (up to 25 min).58;59 Given the time course of our experiments, including the 40 min equilibration time in the absence of isoflurane, it seems unlikely that recovery from prolonged isoflurane administration accounts for inhibited contractility of isolated arteries following the Sham procedure.

A second cause of the Sham-mediated changes in vascular reactivity might be an inflammatory response evoked by the procedure. Indeed, a Sham procedure of CPB in the rat results in a substantial increased plasma concentration of TNF-alpha up to 6 hours post-procedure,60 a cytokine inducing endothelial dysfunction through various mechanisms (reviewed in Zhang et al., 2009). 61

Collectively our data suggest that the relatively mild procedure of anesthesia and cannulation have a long-lasting impact on vasoresponsiveness of small arteries and warrant further studies to its mechanism and the contribution of specific anesthetics.

CPB induced changes in vascular reactivity The observation that CPB selectively affects endothelium dependent relaxation

constitutes a second main finding of our study. Most likely, endothelial dysfunction is caused by the substantially aggravated systemic inflammation initiated by CPB,62-64 as increased plasma TNF-alpha concentrations relate to impaired endothelial mediated relaxation in rat.65 Further, impaired hemodynamics during CPB may add to vascular dysfunction, as demonstrated in a hypovolemic shock model in mice.66 Additionally, the observed changes in vascular reactivity might originate from metabolic disturbances during CPB. Slight changes in pH, HCO-

3, and BE during CPB were observed and likely represent mild metabolic acidosis, which normalized after weaning from the CPB.

Our data imply that CPB affects the relaxation of mesenteric artery predominantly. This may be caused by the mesenteric artery being dependent on a single component of relaxation (EDHF), compared to coronary artery, which is balanced between NO, EDHF and prostaglandins. Thus, the compensatory capacity of the coronary vascular bed seems intrinsically superior over mesenteric artery. Possibly, the limited capacity of adjustment of mesenteric function relates to gastrointestinal complications after CPB, which is one of the serious complications with high mortality rate.67

In summary, our study demonstrates that vascular responsiveness of small vessels was significantly altered during the entire follow-up period of 5 days following CPB in the rat. Not only CPB, but also Sham procedure causes protracted and


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substantial alteration of vascular function. Changes in contractile and relaxation function of small arteries following CPB may lead to impaired control of vasomotor response, possibly related to hemodynamic instability and organ injury during the recovery period. Reference List 1. Loef BG, Epema AH, Smilde TD, Henning RH, Ebels T, Navis G, Stegeman CA: Immediate

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27. Gschwend S, Pinto-Sietsma SJ, Buikema H, Pinto YM, van Gilst WH, Schulz A, de ZD, Kreutz R: Impaired coronary endothelial function in a rat model of spontaneous albuminuria. Kidney Int. 2002; 62: 181-91

28. Gschwend S, Henning RH, de ZD, Buikema H: Coronary myogenic constriction antagonizes EDHF-mediated dilation: role of KCa channels. Hypertension 2003; 41: 912-8

29. Morariu AM, Loef BG, Aarts LP, Rietman GW, Rakhorst G, van OW, Epema AH: Dexamethasone: benefit and prejudice for patients undergoing on-pump coronary artery bypass grafting: a study on myocardial, pulmonary, renal, intestinal, and hepatic injury. Chest 2005; 128: 2677-87

30. Loef BG, Epema AH, Navis G, Ebels T, van OW, Henning RH: Off-pump coronary revascularization attenuates transient renal damage compared with on-pump coronary revascularization. Chest 2002; 121: 1190-4

31. Loef BG, Epema AH, Smilde TD, Henning RH, Ebels T, Navis G, Stegeman CA: Immediate postoperative renal function deterioration in cardiac surgical patients predicts in-hospital mortality and long-term survival. J.Am.Soc.Nephrol. 2005; 16: 195-200

32. Loef BG, Epema AH, Navis G, Ebels T, Stegeman CA: Postoperative renal dysfunction and preoperative left ventricular dysfunction predispose patients to increased long-term mortality after coronary artery bypass graft surgery. Br.J.Anaesth. 2009; 102: 749-55

33. Xu Y, Henning RH, Lipsic E, van BA, van Gilst WH, Buikema H: Acetylcholine stimulated dilatation and stretch induced myogenic constriction in mesenteric artery of rats with chronic heart failure. Eur.J.Heart Fail. 2007; 9: 144-51



36. Darblade B, Pendaries C, Krust A, Dupont S, Fouque MJ, Rami J, Chambon P, Bayard F, Arnal JF: Estradiol alters nitric oxide production in the mouse aorta through the alpha-, but not beta-, estrogen receptor. Circ.Res. 2002; 90: 413-9

37. Samarska IV, van MM, Buikema H, Houwertjes MC, Wulfert FM, Molema G, Epema AH, Henning RH: Adjunct nitrous oxide normalizes vascular reactivity changes after hemorrhagic shock in mice under isoflurane anesthesia. Anesthesiology 2009; 111: 600-8


39. Dal-Ros S, Oswald-Mammosser M, Pestrikova T, Schott C, Boehm N, Bronner C, Chataigneau T, Geny B, Schini-Kerth VB: Losartan prevents portal hypertension-induced, redox-mediated endothelial dysfunction in the mesenteric artery in rats. Gastroenterology 2010; 138: 1574-84

40. Inkster ME, Cotter MA, Cameron NE: Effects of trientine, a metal chelator, on defective endothelium-dependent relaxation in the mesenteric vasculature of diabetic rats. Free Radic.Res. 2002; 36: 1091-9

41. Deblier I, Sadowska AM, Janssens A, Rodrigus I, DeBacker WA: Markers of inflammation and oxidative stress in patients undergoing CABG with CPB with and without ventilation of the lungs: a pilot study. Interact.Cardiovasc.Thorac.Surg. 2006; 5: 387-91


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45. Mazer CD, Briet F, Blight KR, Stewart DJ, Robb M, Wang Z, Harrington AM, Mak W, Li X, Hare GM: Increased cerebral and renal endothelial nitric oxide synthase gene expression after cardiopulmonary bypass in the rat. J.Thorac.Cardiovasc.Surg. 2007; 133: 13-20


47. Modine T, Azzaoui R, Fayad G, Lacroix D, Bordet R, Warembourg H, Gourlay T: A recovery model of minimally invasive cardiopulmonary bypass in the rat. Perfusion 2006; 21: 87-92








55. Giglio MT, Marucci M, Testini M, Brienza N: Goal-directed haemodynamic therapy and gastrointestinal complications in major surgery: a meta-analysis of randomized controlled trials. Br.J.Anaesth. 2009; 103: 637-46

56. Kokita N, Stekiel TA, Yamazaki M, Bosnjak ZJ, Kampine JP, Stekiel WJ: Potassium channel-mediated hyperpolarization of mesenteric vascular smooth muscle by isoflurane. Anesthesiology 1999; 90: 779-88

57. Ishikawa A, Ogawa K, Tokinaga Y, Uematsu N, Mizumoto K, Hatano Y: The mechanism behind the inhibitory effect of isoflurane on angiotensin II-induced vascular contraction is different from that of sevoflurane. Anesth.Analg. 2007; 105: 97-102

58. Izumi K, Akata T, Takahashi S: Role of endothelium in the action of isoflurane on vascular smooth muscle of isolated mesenteric resistance arteries. Anesthesiology 2001; 95: 990-8

59. Akata T, Kanna T, Yoshino J, Takahashi S: Mechanisms of direct inhibitory action of isoflurane on vascular smooth muscle of mesenteric resistance arteries. Anesthesiology 2003; 99: 666-77


61. Zhang H, Park Y, Wu J, Chen X, Lee S, Yang J, Dellsperger KC, Zhang C: Role of TNF-alpha in vascular dysfunction. Clin.Sci.(Lond) 2009; 116: 219-30

62. Larmann J, Theilmeier G: Inflammatory response to cardiac surgery: cardiopulmonary bypass versus non-cardiopulmonary bypass surgery. Best.Pract.Res.Clin.Anaesthesiol. 2004; 18: 425-38

63. Sistino JJ, Acsell JR: Systemic inflammatory response syndrome (SIRS) following emergency cardiopulmonary bypass: a case report and literature review. J.Extra.Corpor.Technol. 1999; 31: 37-43

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A

-Log [ACh]-8 -7 -6 -5 -4

rela

xatio

n, %

-120

-100

-80

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-Log [ACh]-8 -7 -6 -5 -4

rela

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250 Mesenteric artery Coronary artery

Mesenteric artery Coronary artery

64. Markewitz A, Lante W, Franke A, Marohl K, Kuhlmann WD, Weinhold C: Alterations of cell-mediated immunity following cardiac operations: clinical implications and open questions. Shock 2001; 16 Suppl 1: 10-5


66. Samarska IV, van MM, Buikema H, Houwertjes MC, Wulfert FM, Molema G, Epema AH, Henning RH: Adjunct nitrous oxide normalizes vascular reactivity changes after hemorrhagic shock in mice under isoflurane anesthesia. Anesthesiology 2009; 111: 600-8

67. McSweeney ME, Garwood S, Levin J, Marino MR, Wang SX, Kardatzke D, Mangano DT, Wolman RL: Adverse gastrointestinal complications after cardiopulmonary bypass: can outcome be predicted from preoperative risk factors? Anesth.Analg. 2004; 98: 1610-7, table

Supplement Digital Content 1. Acetylcholine (ACh-)induced relaxation in the absence and presence of 10 "M indomethacin (INDO; i.e. to inhibit cyclooxygenase-derived prostaglandins (PG’s)) only or with 100 "M L-NMMA (i.e. to inhibit NO production) additionally present, in arteries of normal control rats that underwent short anesthesia (for sacrifice) only. Full CR-curves are given for mesenteric (A) and coronary (B) arteries, as well as the individual contribution of different endothelial mediators hereto in both artery types (C), presented as the AUC (for further explanation and description, see text). Data are mean ± SD (n=5). Abbreviations: CPB, cardiopulmonary bypass.

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Supplement Digital Content 3 Table. Preconstriction level to phenylephrine of mesenteric and coronary arteries in different experimental groups Groups Preconstriction level, mN

Phenylephrine in Mesenteric artery

Serotonin in Coronary artery

Control 10.9±3.5 2.3±0.93

Sham 1h 10.8±5.9 1.7±0.7

CPB 1h 9.7±3.7 1.4±0.9

Sham 1day 7.3±4.0 1.3±0.6

CPB 1day 6.6±3.1 1.3±0.9

Sham 2 days 9.2±3.7 2.2±2.0

CPB 2days 10.5±4.9 1.5±0.9

Sham 5 days 10.0±3.8 2.7±1.0

CPB 5 days 8.8±5.7 2.7±1.9

Data are given as mean±SD. Abbreviations: CPB, cardiopulmonary bypass.

Supplement Digital Content 2 Table. Relaxant response to Sodium Nitroprusside of rat mesenteric and coronary arteries at different periods of recovery (1h, 1day, 2 and 5 days) in Sham and CPB groups Groups Response to Sodium Nitroprusside, %

Mesenteric artery Coronary artery Control 93.9±7.5 116.8±17.5

Sham 1h 95.2±12.6 99.1±16.5

CPB 1h 97.6±1.4 100.7±12.7

Sham 1day 95.7±2.8 110.5±13.5

CPB 1day 98.5±4.2 150.2±59.6

Sham 2days 94.6±9.0 119.7±29.9

CPB 2days 92.3±29.4 164.5±55.5

Sham 5days 92.4±9.8 107.3±10.6

CPB 5days 84.2±21.5 112.0±15.6

Data are given as mean±SD. Abbreviations: CPB=cardiopulmonary bypass.

Supplement Digital Content 4 Table. Characteristics of the ACh-mediated relaxation in coronary and mesenteric vessels during prolong recovery period. Comparisons were done with t-test vs Control group

Groups Coronary artery Mesenteric artery

Emax, % of the relaxation -log EC50 Emax, % of the relaxation -log EC50

Control 54.8±18.6 -6.6±0.2 58.6±16.3 -7.2±0.4

Sham 1h 58.0±8.3 -6.3±0.4 70.8±12.6* -6.3±0.6*

Sham 1d 51.5±22.6 -6.6±0.7 64.7±21.3 -7.1±0.4

Sham 2d 53.8±21.6 -6.4±0.3 70.7±25.5 -7.1±0.3

Sham 5d 39.9±9.3** -6.0±0.2** 47.9±33.5 -7.1±0.2

CPB 1h 72.55±11.0# -6.6±0.3 81.0±13.0* -6.9±0.7

CPB 1d 54.6±26.5 -6.3±0.7 66.8±38.2 -6.8±0.8

CPB 2d 41.9±21.3 -5.9±0.4** 51.1±32.8 -6.5±0.6*

CPB 5d 53.3±21.5 -6.4±0.3 49.54±25.3 -6.9±0.5

*P<0.05, t-test versus Control; **P<0.001, t-test versus Control; # P=0.051, t-test versus Control

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Chapter 3

Changes of vascular reactivity of rat thoracic

aorta during prolonged recovery following cardiopulmonary bypass

Iryna V. Samarska, Anouk Oldenburger, Hendrik Buikema, Hubert Mungroop Martin C. Houwertjes, Michel M.R.F. Struys,

Anne H. Epema, Robert H. Henning

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Abstract Background: Changes in vascular reactivity may represent key elements of pathogenesis of cardiopulmonary bypass (CPB). The relationship between endothelial activation and the endothelial dependent vasomotor control following cardiopulmonary bypass has not been addressed. The aims of the present study were to examine vascular reactivity of rat thoracic aorta after CPB within a follow up from 60 min to 5 days and correlate the observed changes in vasoresponsiveness with the level of induction of markers of the endothelial activation. Materials and Methods: Male Wistar rats (n=78) were anesthetized with isoflurane (2,5-3%) and fentanyl/midazolam during CPB. Animals were assigned to sham or CPB with normothermic extracorporeal circulation (ECC) for 60 min at a flow of 100-110 mL kg-1 min-1. After recovery for 60 min to 5 days, constriction to phenylephrine (PE) and potassium chloride (KCl) and relaxation to acetylcholine (Ach) in thoracic aorta were assessed. The expression of E-selectin, VCAM-1, ICAM-1, TNF-!, HO-1 and TGF-! was assessed by PCR and of COX-2, p22phox and TxA2 receptors by western blot. Results: In both Sham and CPB the sensitivity to phenylephrine and potassium chloride were similarly and significantly decreased at day 5 of recovery compared to control (for PE AUC value in au: 322.0±152.7, 435.9±132.8 and 1200±245; Emax KCl in uM: 153.9±15.2, 183.6±10.9 and 331.4±71.3, respectively) At day 5 of recovery, EDHF-mediated relaxation to acetylcholine was significantly decreased in both Sham and CPB as compared to control, while total Ach-mediated relaxation was unchanged. Expressions of all molecular markers were upregulated in CPB group after 1hour recovery period. Conclusions: Extracorporeal circulation induced endothelial cell activation after short-term recovery period (1 hour). Sham-related procedures (surgical intervention and/or prolonged anesthesia) affected vasoresponsiveness by attenuating vascular contractility and elevation of EDHF-mediated reactivity after 5 days of recovery period. However, extracorporeal circulation did not additionally influence vascular reactivity. Hence, endothelial cell activation did not translate/result into impaired endothelium-dependent relaxation in rat aorta. Introduction

Cardiopulmonary bypass (CPB) represents a widely used technique in thoracic surgery enabling various surgical procedures such as coronary artery bypass grafting, valve surgery, heart-lung transplantation and pulmonary surgical interventions. CPB is associated with a systemic inflammatory response syndrome (SIRS), which is triggered by multiple factors such as surgical trauma, contact of blood with artificial surfaces of the extracorporeal circuit, and ischemia-reperfusion injury.1;2 The abovementioned processes may lead to the activation of the endothelium with accompanying up-regulation of the cell adhesions molecules.3-7 Induction of the soluble E-selectin, which mediates adhesion of the neutrophils to the endothelium, has been shown after cardiac surgery previously, and is thought to represent a marker of the neutrophil-evoked endothelial injury.3;8;9 CPB has been documented to change vascular reactivity in

Vascular reactivity and endothelial activation 43

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mesenteric, cerebral, coronary, and skeletal muscle vessels.10-17 CPB caused endothelial dysfunction of the mesenteric artery after 6 hours of recovery,12 in cerebral artery directly after extracorporeal circulation,13 and reduced myogenic reactivity of the coronary and skeletal muscles arterioles.13;16;17 However, the relationship between endothelial activation and worsening of endothelial dependent vasomotor control following cardiopulmonary bypass has not been addressed.

The aims of the present study were 1) to examine vascular reactivity of rat thoracic aorta after CPB with a follow up from 60 min to 5 days and 2) correlate the observed changes in vasoresponsiveness with the level of induction of markers of the endothelial activation. Material and Methods

The experimental protocol was approved by the Institutional Animal Care and Use Committee of the University of Groningen. This study was performed in n=78 adult male Wistar rats (507.4±31.3 g; Harlan, Zeist, The Netherlands) housed under standard conditions with free access to food (standard rat chow; Hope Farms, Woerden, The Netherlands) and drinking water throughout the study.

Experimental groups

Animals were randomly divided in four experimental groups with different duration of the recovery period after the procedure: (1) an 1hour group where animals were allowed to recover for 60 min after the end of the extracorporeal circulation, (2) a 1day group in which recovery lasted one day, (3) a 2days group that was allowed to recover for 2 days; and (4) a 5days group in which animals were allowed to recover for 5 days. Each group was subdivided in two sub-groups: CPB and Sham. CPB animals were subjected to the full experimental protocol described below, including anesthesia, cannulation, extracorporeal circulation, weaning and the corresponding recovery period. Sham animals followed the same procedure except for the extracorporeal circulation, but maintained cannulated and mechanically ventilated. In order to evaluate the normal vascular reactivity in rats without invasive interventions, an additional untreated Control group was introduced. Animals in Control group were sacrificed under isoflurane anesthesia (2.5-3%) only.

Experimental protocol The experimental protocol consisted of the following parts: anesthesia,

preparation, extracorporeal circulation, weaning with a recovery period (see fig. 1). Anesthesia was induced (2-3% isoflurane in O2/air (1:1)) before the trachea was intubated and the lungs mechanically ventilated (Amsterdam Infant Ventilator; HoekLoos, Amsterdam, The Netherlands). Tidal volume was set to achieve normocapnia (verified by capnography and arterial blood gas analysis), with O2/air (0.5:1) at a respiration frequency of 50 min-1 (0.5 s inspiration time). During the preparation period, the left femoral artery was cannulated (26-gauge catheter) for blood pressure monitoring. Immediately before insertion of the arterial line, 250 IU kg-1 heparin was administered. The carotid artery was canulated for arterial inflow using a

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22-gauge catheter; a multi-orfice 4.5 French cannula (adapted from a Desilets-Hoffmann catheter, Cook Son, The Netherlands) was advanced into the right heart using the common jugular vein for access. The tail artery was canulated for the administration of intravenous anesthetics, heparin, and protamine sulfat.

Extracorporeal circulation In CPB groups extracorporeal circulation (ECC) was initiated with a duration of

60 min. The set-up consisted of a glass venous reservoir, a peristaltic pump (Pericor® SF70), a neonatal membrane oxygenator (0140GM, Polystan, Vaerlose, Denmark) and a glass counter-flow heat exchanger with built-in bubble trap. All components were connected with polyethylene tubing (1.6mm inner diameter). The circuit was primed with 15 ml of Haes 60mg ml -1 solution (Voluven®, Fresenius Kabi, Bad Homburg, BRD)). Animals were also heparinised (250 IU) after start of ECC. During period of extracorporeal circulation, rats were anesthetized with intravenous fentanyl (125 "g kg-

1), atracurium (0.5mg kg-1), and midazolam (2 mg kg-1). During experimental procedure blood and venous saturation was continuously monitored. Targeted CBP flow was 100-110 mL kg-1 min-1, corresponding to 60-70% of normal cardiacoutput. Samples for the blood gas analysis (0.1"l) were withdrawn at four time-points: at the end of the preparation period (15 min before start of ECC), twice during the ECC period (15 min after the start of it and 15 min before the its ending), and after ECC (45 min after the end of ECC).

Weaning and recovery During the weaning period, ECC was terminated and mechanical ventilation

was initiated. Protamine (150 IU kg-1 i.v.) was administered in order to neutralize heparin after which the animals were decannulated and the wounds sutured. Following extubation during the recovery period thereafter, animals were first kept under isoflurane anesthesia (0.8-1.0%) for another 60 min in order to stabilize. The duration of the recovery period varied from 1 till 120 hours after the end of the extracorporeal circulation according the experimental design. Sacrification was performed in the end of assigned protocol under isoflurane anesthesia (2.5-3%) by exsanguinations due to taking blood and organs for the analysis.

Figure 1. Scheme of the experimental protocol in Sham and CPB groups regarding the duration of recovery period. The experimental protocol consisted of the following parts: anesthesia, preparation, extracorporeal circulation, a weaning and a recovery period (see methods). Abbreviations: ECC, extracorporeal circulation.

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!

Vascular reactivity Immediately following sacrifice, thoracic aorta was removed and placed in cold

physiological saline solution, cleaned from connective tissue and cut into 8 rings of 2-2.5 mm, which were placed in the individual organ bathes for isotonic tension recording. The baths contained 15ml of Kreb’s solution at 37°C and bubbled continuously with 95%O2/5%CO2 at pH 7.4. Rings were allowed to equilibrate for 40 min to reach a steady baseline. Vascular segments were primed and checked for viability by two consecutive exposures to potassium chloride (60 mM).

The experimental protocol consisted of evaluation of contractile responses to phenylephrine (PE; 10 nM to 100 "M) and endothelium-dependent relaxation to acetylcholine (ACh; 10 nM to 300 "M) in rings precontracted with PE. To assess the contribution of different endothelial mediators, ACh-induced relaxation was additionally studied in the absence and presence of L-NMMA (0.1 mM; an inhibitor of NO-synthase) and/or indomethacine (1 "M; an inhibitor of cyclooxygenase) administered 20 min before application of PE [18]. Prostaglandin-mediated relaxation was determined as a difference between total Ach-mediated relaxation and response to Ach in the presence of indomethacin. NO-mediated contribution was evaluated as the portion of acetylcholine induced relaxation in presence of indomethacin additionally sensitive to eNOS-inhibition by L-NMMA. By exclusion, the rest relaxation was accounted to be EDHF-mediated [19]. To quantify the contribution of the above endothelial mediators, the area between the concentration-response curve in the absence and presence of one or more inhibitors was taken. Following the final concentration of ACh, a maximal concentration of the NO-donor sodium nitroprusside (SNP; 0.1 mM) was applied to assess total endothelium-independent relaxation [20]. Mainly two types of the impacts were evaluated: Sham-related affects (namely, anesthesia and cannulation) and influence of the extracorporeal circulation (ECC) on vascular reactivity.

Western blotting

The methods used were described previously.21 Briefly, after grinding, the frozen abdominal aortas were placed in 300 "l of boiling 2% SDS followed by pounding by a polytron (Kinematica AG Littau, Switzerland). Then, samples were centrifuged (4000 rpm, 1 min) and heated (95 oC) for 5 min. After a second centrifugation (13000 rpm, 3 min), supernatant was collected and used for measurements. Protein concentration was determined by Bio-Rad Protein Assay (Bio-Rad, Hercules, CA). Forty µg of total protein from each sample was separated on 4-20% Precise Protein Gels (Pierce, Rockford, IL) and transferred to nitrocellulose membranes. Anti-cyclooxygenase-2 antibody (BD Bioscience Pharmingen, San Diego, CA), p22 phox (BD Bioscience Pharmingen, San Diego, CA), thromboxane A2 (BD Bioscience Pharmingen, San Diego, CA), and anti-$-actin (Sigma, St. Louis, MO) were used as a primary antibody. Horseradish peroxidase-linked rabbit anti-mouse antibody was applied as a secondary antibody. The blots were analyzed using Super Signal assay (Pierce, Rockford, IL). $-actin served as a housekeeping protein. Experimental levels are expressed as ratios to $-actin protein levels.

46 Chapter 3

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Real time PCR Total RNA was extracted from abdominal aorta using Trizol Reagent

(Invitrogen, Carlsbad, CA, USA) with subsequent quality control by ethidium bromide staining in 1% agarose gel. DNase treatment of the RNA samples prior to RT-PCR was performed with RQ1 RNase-Free DNase (Promega, Madison, WI, USA). First-Strand cDNA Synthesis from RNA (1"g) was performed by using Reverse Transcription reagents (Promega, Madison, WI, USA). The expressions of E-selectin, VCAM-1, ICAM-1, TNF!, HO-1 and TGF$ 1 (table 1) were analyzed by real-time PCR with Absolute QPCR SYBR Green reagents (Molecular Probes, Leiden, Netherlands) and CFX384 Real-Time PCR Detection System (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The PCR protocol consisted of 15 min at 95ºC, followed by 40 cycles with heating to 95ºC and cooling to 60ºC for 1 minute. The amplification product was evaluated by melting curves analysis and agarose gel (1.5%) electrophoresis. Sequence-specific primers (table 1) were obtained from Biolegio (Nijmegen, the Netherlands). Cycle threshold (CT) values for genes analyzed were normalized to their CT value of GAPDH.

Drugs The ionic millimolar composition of Krebs solution was as follows: 120.4 NaCl,

5.9 KCl, 2.5 CaCl2, 1.2 MgSO4, 25.0 NaHCO3, 1.2 NaH2PO4, 11.5 glucose; these chemicals were obtained from Merck (Darmstadt, Germany). The following drugs were used: acetylcholine chloride (ACh), phenylephrine (PE), indomethacine, SNP, NG-methyl-L-arginine (L-NMMA). Stock solution (10 mmol/L) for indomethacine was prepared in 96% ethanol. All other drugs were dissolved in deionized water. L-NMMA was purchased from MP Biomedicals (Illkirch, France). All other compounds were purchased from Sigma (St. Louis, Missouri, USA). The concentrations presented in the concentration-curve responses are expressed as a final molar concentration in the bath. Data analysis

Data are given as mean±SD and n refers to the number of animals in a corresponding group. Contractile responses to phenylephrine (PE) and potassium chloride (KCl) are presented in "m displacement and relaxations to ACh and SNP were calculated as a percentage of pre-constriction. For each concentration-response curve

Table 1. Primer pairs for RT-PCR!

Gene Forward Reverse

VCAM-1 TAAGTTACACAGCAGTCAAATGGA CACATACATAAATGCCGGAATCTT

ICAM-1 CTGCCACCATCACTGTGT CTGACCTCGGAGACATTCTT

TGF!1 AGAGCTGCGCCTGCAGAG GAAGCCGGTTACCAAGGT

HO-1 GTGCACATCCGTGCAGAGAA GAAGGCCATGTCCTGCTCTA

TNF" CACGCTCTTCTGTCTACTGA GTACCACCAGTTGGTTGTCT

E-selectin CCATTCGGCCTCTTCAAGCTA TGCAGCTCACAGAGCCATTC

GAPDH AAGGTCGGTGTCAACGGATTT CAATGTCCACTTTGTCACAAGAGAA


!

the following parameters were determined: 1) the Area Under the Curve (AUC) in arbitrary units (AU) (Sigma, Systat Software, Inc, Germany), 2) the maximal response (Emax) and 3) the concentration at which half-maximal response was reached (EC50). The difference in AUC for concentration-response curves to ACh in the absence or presence of an inhibitor was used as an estimate for the contribution of different endothelial mediators, as described previously [18;19]. Differences were evaluated using one-way ANOVA or repeated measurements ANOVA combined with post-hoc Bonferroni test or with t-test where applicable. Differences were considered significant at P<0.05 (2-tailed). Results

Hemodynamics and blood gas analysis during the protocol During the procedure, hemodynamic measurements (MAP) and blood gas

analysis were performed in order to monitor the animals. At baseline all parameters were in the normal range. During CPB, MAP was significantly lower compared with Sham (65.4 ± 13.9 and 105.3 ± 4.0 mmHg, respectively). During weaning, MAP of CPB animals returned to baseline levels (75.5 ± 25.1 mmHg).

Blood gas analysis was performed before start of ECC, twice during ECC and after weaning (table 2). CPB decreased pH, bicarbonate ion (HCO-

3), and Base excesses and increased levels of pCO2 and pO2, which may represent moderate respiratory acidosis. Hematocrit was significantly decreased in CPB groups as a consequence of hemodilution with priming solution. Intraoperative hyperglycemia was observed in both SHAM and CPB groups during the first 2 hours.

Contractile responses in aorta In order to evaluate contractile properties of rat thoracic aorta, maximal

response to potassium chloride (KCl; fig.2) and full concentration-response curves to phenylephrine (PE; fig.3) were constructed. In both Sham and CPB, maximal contractile responses to KCl were significantly decreased at 5 days of recovery, both compared to controls and to shorter periods of recovery. After 1hour of the recovery period Sham-related procedures (anesthesia and cannulation) decrease contractile response to PE, while ECC counteract those changes in vascular reactivity. Similarly at 5 days of recovery, concentration-response curves for PE showed a right shift and decreased Emax, and hence a decrease in AUC (fig. 3, panel A: IV, table 3).

Thus, the above findings suggest that the Sham-related surgical procedures (extended anesthesia and cannulation) induced the depression in thoracic aorta contractility after prolonged recovery, without significant additional influence of extracorporeal circulation.

48 Chapter 3

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Tabl

e 2.

Dat

a on

blo

od g

ases

ana

lysi

s, g

luco

se c

once

ntra

tion

and

mea

n ar

teri

al p

ress

ure

(M

AP

, fem

oral

art

ery)

in S

ham

and

CP

B g

roup

s du

ring

the

expe

rim

enta

l pro

toco

l P

aram

eter

s S

HA

M

CP

B

Bef

ore

EC

C

15 m

in E

CC

45

min

EC

C

Aft

er E

CC

B

efor

e E

CC

15

min

EC

C

45 m

in E

CC

A

fter

EC

C

MA

P, m

mH

g 10

2.4±

32.8

10

9.8±

12.9

10

3.5±

22.4

92

.0±1

8.2

84.1

±11.

6 57

.9*±

12.9

81

.4*±

25.6

79

.5±2

5.1

pH

7.43

±0.0

5 7.

45±0

.05

7.46

±0.0

7 7.

41±0

.06

7.42

±0.0

5 7.

38 *

±0.0

7 7.

32 *

±0.0

8 7.

37±0

.05

pCO

2, k

Pa

4.74

±1.0

4.

51±0

.8

4.68

±1.0

5.

15±0

.9

4.77

±0.8

5.

07*±

1.1

5.72

*±1.

0 5.

22±0

.8

pO2,

kP

a 17

.4±4

.7

16.6

±3.9

17

.7±4

.2

16.9

±4.6

19

.6±7

.0

34.1

*±17

.1

35.8

*±18

.3

19.4

±10.

2

HC

O3,

mm

ol/l

22.9

±1.5

23

.3±1

.5

24.7

±1.5

24

.4±2

.2

22.6

±2.3

21

.5±3

.6

21.6

*±2.

4 22

.4*±

1.7

BE

, mm

ol/l

-0

.4±1

.0

0.5±

1.2

1.8±

1.5

0.5±

1.4

-0.6

±1.7

-1

.9*±

2.7

-3.6

*±3.

1 -2

.04±

1.9

Hct

c 0.

4±0.

02

0.

4±0.

03

0.

4±0.

03

0.

4±0.

03

0.4±

0.02

0.3*

±0.0

5

0.3*

±0.0

2

0.3*

±0.0

3

Glu

cose

, mm

ol/l

20.6

±5.4

15.2

±5.4

8.4±

4.3

6.

6±1.

0

22.9

±4.1

20.5

±5.1

11.6

±4.4

6.9±

3.0

Abb

revi

atio

ns: H

ctc,

hem

atoc

rit; C

PB

, car

diop

ulm

onar

y by

pass

. *

deno

tes

P<0

.05,

Sha

m v

s C

PB

, ind

epen

dent

t-te

st

!Ta

ble

3. P

aram

eter

s of

the

Phe

nyle

phri

ne-m

edia

ted

cont

ract

ion

Gro

ups

EC

50, u

Mol

/L

Hill

slo

pe

Max

imal

res

pons

e, u

M

Con

trol

2.

7±0.

5

0.9±

0.1

488.

9±13

.8

Sha

m1h

our

33.8

±4.2

**

0.7±

0.09

42

2.8±

23.5

* C

PB

1 h

our

6.8±

2.0

1.3±

0.6

402.

8±22

.9*

Sha

m 1

day

8.1±

2.7

1.2±

0.6

415.

6±31

.0

CP

B 1

day

6.5±

3.9

1.07

±0.7

39

7.5±

45.5

S

ham

2da

ys

6.4±

1.7

1.4±

0.5

427.

0±19

.7*

CP

B 2

days

5.

1±1.

1 1.

2±0.

3 42

7.3±

14.9

* S

ham

5da

ys

12.6

±3.1

* 1.

03±0

.3

210.

2±13

.0*

CP

B 5

days

18

.1±7

.6

0.76

±0.3

26

4.9±

26.6

* *-

P<0

.05,

vs

Con

trol,

t-tes

t; **

- P<0

.001

, vs

Con

trol,

t-tes

t


!

Relaxant response in aorta Aortic rings were additionally studied for endothelium-dependent responses to

ACh. Data are present as parameters of the concentration-response curves (table 4) and contribution of different relaxant pathways (fig. 4). Relaxant reactivity of thoracic aorta of the control rats were characterized by major contribution of NO in total acetylcholine mediated relaxation counteracted by contractile prostaglandins (fig. 4). Neither extracorporeal circulation, nor the duration of recovery period affected total acetylcholine-mediated relaxation. In contrast, various changes were observed in the contribution of PG-, NO- and EDHF to overall relaxation. Both in Sham and in CPB groups, NO-contribution was non-significantly reduced after 5 days of recovery period. Significantly increased contribution of EDHF-mediated pathways was found at 1day and 5 days time-points of the recovery period. Further, the “amount” of contractile prostaglandins seemed to be augmented during 1-2 days of the recovery period with normalization by the 5th day. Collectively, the findings showed that Sham-related procedures (prolonged anesthetic intervention and/or cannulation) increased the

Table 4. Parameters of the Acetylcholine-mediated relaxation Groups EC50, Mol/L Hill slope Maximal response, % Control 5.3±0.8*10-8 1.5±0.2 36.4±0.5 Sham1hour 4.7±6.2*10-6 * 0.3±0.2 * 66.07±11.0 * CPB 1 hour 17.7±1.2*10-8 * 0.8±0.03 * 42.7±0.4 * Sham 1day 9.8±2.02*10-8 1.5±0.4 38.05±1.4 CPB 1day 10.5±3.3*10-8 1.1±0.3 32.5±1.5 Sham 2days 7.6±1.02*10-8 1.5±0.3 45.9±1.1 * CPB 2days 7.3±2.5*10-8 1.3±0.5 50.6±2.3 * Sham 5days 30.3±9.4*10-8 * 0.6±0.1 * 60.6±2.4 * CPB 5days 47.7±9.3*10-8 * 0.5±0.1 * 52.7±1.7 * *-P<0.05, vs Control, t-test

Figure 2. Vascular contractility to potassium chloride of rat thoracic aorta at different periods of recovery (1 hour, 1day, 2 days, 5 days) in Sham groups and following subjection of the animals to CPB-related procedures. Contractile responses are given as Emax. Data are mean ± SD (n=6-9). * indicates P<0.05 vs Control, one way ANOVA with post-hoc Bonferroni test. Abbreviations: KCl=, potassium chloride; CPB=, cardiopulmonary bypass.

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contribution of EDHF-pathway in total acetylcholine-mediated relaxation to maintain the overall relaxation response to ACh. However, extracorporeal circulation did not add any influence herein. !

Figure 3. Vascular contractility to phenylephrine of rat thoracic aorta at different periods of recovery (1 hour, 1day, 2 days, 5 days) in Sham groups and following subjection of the animals to CPB-related procedures. Contractile responses are given as full CR-curves (A) and the AUC (B). Data are mean ± SD (n=6-9). * indicates P<0.05 vs Control, one way ANOVA with post-hoc Bonferroni test. # indicates P<0.05 vs Control, repeated measurements ANOVA combined with post-hoc Bonferroni test. Abbreviations: PE=phenylephrine; CPB=cardiopulmonary bypass.


!

Protein expression Because of the elevated contribution of contractile prostaglandins in ACh-induced responses in almost all groups we additionally assessed protein expression of COX-2 - hence, the isoform of inducible COX, might be responsible for the synthesis of contractile prostanoids- and of TxA2 receptors - hence, implied to be involved in cyclooxygenase-dependent vascular contraction [22] – in aortic segments. p22phox protein, a NAD(P)H oxidase subunit associated with increased ROS production and oxidative stress in vascular endothelium [23] , was also assessed. Both COX-2 and TxA2 receptor protein as well as p22phox were similarly expressed in all groups regardless of type of experimental protocol (fig.5, for TxA2 receptors and p22phox – data not shown). However, regression analysis revealed that the degree of relaxation-inhibition by (indomethacine-sensitive) COX-derived prostaglandins was proportionally correlated to COX-2 protein expression (Pearson correlation coefficient=-0.50, n=22, P=0.01), but not the TxA2 receptor or p22phox. These findings suggest the (overall)

Figure 4. Total acetylcholine (ACh-)-induced relaxation (A) - and the contribution of (B) prostaglandins (PG’s), (C) NO, and (D) EDHF hereto, all provided as AUC - in rat thoracic aorta at different periods of recovery (1h, 1day, 2days or 5days) following subjection of the animals to CPB -related procedures. Data are mean ± SD (n=5-8). * indicates P<0.05 vs control, one-way ANOVA with following Bonferroni test. # indicates P<0.05 vs control Mann-Whitney Rank Sum Test. $ indicates P<0.05 vs control; # independent t-test. Abbreviations: CPB= cardiopulmonary bypass.

52 Chapter 3

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outcome for endothelial function in vascular reactivity in the present study to an important extent was determined by the level of vascular COX-2 protein expression.

Expression of markers of endothelial activation, anti-inflammatory marker, and hypoxia

To investigate changes in endothelial cells related to an inflammatory response, we the evaluated expression of the adhesion factors E-selectin, VCAM-1, ICAM-1 and of TNF-" in abdominal aorta of a subset of animals. HO-1, an inducible enzyme in response oxidative stress and hypoxia and TGF-!, which might modulate the cell adhesion molecules expression, were also evaluated. No expression of these 6 factors was detected in control animals and in Sham and CPB animals beyond 60 min of recovery (fig. 6). In contrast, a consistent upregulation of all factors tested was found in all tested CPB animals at 1 hour recovery period. In contrast, sham animals at 1 h of recovery showed consistently no increase in expression (VCAM-1 and ICAM-1) or only in 1 out of 4 animals (fig. 6). Discussion

The present work studied the changes in vascular responsiveness of thoracic aorta after cardiopulmonary bypass during 5 days of recovery in relation to endothelial cell activation. The main finding of this study is that endothelial cell activation did not result into impaired endothelium-dependent relaxation to ACh in rat aorta. We found that CPB induced endothelial cell activation only after a short-term recovery period (1 hour), as evidenced by a consistently increased expression of 6 markers studied. Secondly, changed vasoresponsiveness, characterized by attenuated vascular contractility and elevation of EDHF-mediated relaxation, was found only after 5 days of recovery. Moreover, these vasomotor changes were to a similar extent present in both CPB and Sham, indicating that they were mainly the result of the accompanying

Figure 5. COX-2 expression in thoracic aorta in all groups (at least 4 per group). Representative blot (A), relative expressions patterns (B) and relation between individual Pr’s-mediated contribution and COX-2 expression; Data were evaluated by regression analysis and Pearson correlation. Abbreviations: CPB= cardiopulmonary bypass.


!

procedures (cannulation procedures and/or prolonged anesthesia), but not of the extracorporeal circulation. Collectively, these data indicate that endothelial cell activation after extracorporeal circulation was not directly related to altered vascular properties of rat thoracic aorta.

Hemodynamics and blood gas analysis during the protocol Hemodynamic data showed that extracorporeal circulation significantly

decreased MAP to around 60-80 mmHg in CBP group, which corresponds to data in previously published studies of CBP in rat.12; 13 Although the phenomenon of CPB-induced hypotension has been addressed previously during last decade, the exact

Figure 6. Relative mRNA expression of E-selectin (A), VCAM-1 (B), ICAM-1 (C), TNF-! (D), HO-1 (E) and TGF-$ (F) in thoracic aorta. Abbreviations: CPB= cardiopulmonary bypass.

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54 Chapter 3

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underlying mechanisms are still unknown. In this context, platelet-mediated serotonin release is thought to be one of the most prominent explanations as extracorporeal circulation, contact of blood with artificial surfaces and the roller pump induces platelet activation and triggers release of serotonin. Serotonin acts as vasodilator through activation of the most sensitive and widespread 5-HT2B receptors mediating NO-release.24 Several other changes in parameters (pH, HCO-

3, and BE) indicate moderate metabolic acidosis in CPB animals. Since all levels returned to the normal range by the end of CPB, it is unlikely that this affected vascular reactivity. Previously severe acidosis has been shown to increase circulating TNF, IL-6, and IL-10 in sever septic shock.25 In our experimental setup only mild acidosis occurred, that turned to basal level after the end of extracorporeal circulation, hence it might doubtful modulate to some extend expression of the inflammatory cytokines. Intraoperative hyperglycemia was observed in both Sham and CPB groups during first 2 hours, indicating that this represents a reaction to the surgical intervention.

Vascular reactivity The present work showed an alteration of rat aorta vasoreactivity only after a

prolonged recovery period of 5 days in both Sham and CPB animals, demonstrating these changes to be caused by cannulation and/or prolonged anesthesia. Contractile response to PE and KCl at day 5 were decreased to a similar extent. Its decrease may be related to increased basal release of endothelium-mediated relaxing factors, particularly EDHF, which was found in the same time point of the 5th day of the recovery period. Previously, the role of the endothelium, in phenylephrine- and potassium chloride-mediated contraction has been shown, while mainly the role of COX, 20-HETE and NO have been investigated.25-28 Alternatively, inhibition of the contractile vascular reactivity may be mediated by decreased Ca2+ sensitivity, as both receptor dependent (PE) and independent (KCl) pathways seem affected. Possibly, cannulation and/or prolonged anesthesia inhibit contractile vascular reactivity through modulation of the MLC kinase and/or MLC phosphatase activity.

The main change in relaxant properties constitutes of an increase in the contribution of EDHF to maintain total endothelial dependent relaxation at 1day and 5 days of recovery in both Sham and CPB. Increased EDHF may serve a compensatory role in response to the depletion of the principal relaxant mediator in aorta, NO, as shown in several studies.29-31

Also our data showed that relaxant reactivity of thoracic aorta are characterized by involvement of contractile prostaglandins. Moreover the amount of contractile prostaglandins seemed to be augmented during first 2 days of the recovery period with normalization by the 5th day. Both types of cyclooxygenases (COX-1 and COX-2) can be involved in production of arachidonic acid contracting metabolites. COX-1 is accounted to be constitutively expressed in endothelium, while COX-2 is inducible form. In our study occurrence of contractile prostaglandins correlated with expression of COX-2. That corresponds to previously published data.22;32 Achieved results did not allow us to draw exact conclusion about the changes in COX-2 expression due to large inter-individual variability. However, correlation analysis


!

showed relation between COX-2 expression and the value of the contractile prostaglandin’s participation in the total relaxation.

Endothelial activation and endothelial dysfunction To substantiate endothelial cell activation, we quantified the expression of

adhesion molecules, pro-inflammatory factors and oxidative stress by qPCR and western blot. Substantial upregulation of all these factors was found consistently in CPB and occasionally in Sham only after 60 min of recovery from CPB. Thus, we found ECC activation of E-selectin, which is a main marker of the endothelium activation in response to inflammatory cytokine (TNF-! and IL-1$)33 and the cell adhesions molecules of the immunoglobulin superfamily, ICAM-1 and VCAM-1, that serve as ligands for leukocyte integrins.34 In addition, CPB induced the upregulation of TNF- ! and TGF-$, which might modulate expression of adhesion molecules, provide a chemotactic gradient for leukocytes and other cells participating in an inflammatory response34-36 and HO-1, an enzyme induced in response to oxidative stress and hypoxia, with the same time-course. As a consequence, it is unlikely that endothelial activation, which is mainly present in the CPB group, accounts for vascular changes as these are present in both CPB and Sham to a similar extent. Nevertheless a direct relationship between the both has been suggested before in mouse thoracic aorta in diet-induced obesity model.34;37 Differences in pathophysiology of these two models (acute surgical intervention in our study and chronic disease state in later) do not allow us to make a comparison between two studies. Circulating cell adhesion molecules (E-selectin, VCAM-1, ICAM-1) have been shown to correlate with the development of the nephropathy, retinopathy, and cardiovascular disease in both type 1 and type 2 diabetes and with the degree of atherosclerosis and albuminuria in type-2 diabetic patients.18-21 Impaired acetylcholine-dependent vasodilation was recently associated with increased levels of the plasma soluble E-selectin and P-selectin in essential hypertension.38 However, in abovementioned studies E-selectin was measured in plasma (and therefore has unknown origin), while we evaluated its expression directly in rat abdominal aorta.

In conclusion, our study demonstrates CPB to induce an immediate and short-term endothelial activation in rat aorta. In addition, vasoresponsiveness is altered at the long run due to prolonged cannulation and/or anesthesia, without any additional effect of extracorporeal circulation. Achieved data suppose better understanding of the pathophysiological processes underlying CPB and possible therapeutical approaches of its complications. Reference List 1. Levy JH, Tanaka KA: Inflammatory response to cardiopulmonary bypass. Ann.Thorac.Surg. 2003;

75: S715-S720 2. Rinder C: Cellular inflammatory response and clinical outcome in cardiac surgery.

Curr.Opin.Anaesthesiol. 2006; 19: 65-8

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3. Galea J, Rebuck N, Finn A, Manche A, Moat N: Expression of soluble endothelial adhesion molecules in clinical cardiopulmonary bypass. Perfusion 1998; 13: 314-21

4. John AE, Galea J, Francis SE, Holt CM, Finn A: Interleukin-8 mRNA expression in circulating leucocytes during cardiopulmonary bypass. Perfusion 1998; 13: 409-17

5. Onorati F, Rubino AS, Nucera S, Foti D, Sica V, Santini F, Gulletta E, Renzulli A: Off-pump coronary artery bypass surgery versus standard linear or pulsatile cardiopulmonary bypass: endothelial activation and inflammatory response. Eur.J.Cardiothorac.Surg. 2009;

6. Onorati F, Santarpino G, Tangredi G, Palmieri G, Rubino AS, Foti D, Gulletta E, Renzulli A: Intra-aortic balloon pump induced pulsatile perfusion reduces endothelial activation and inflammatory response following cardiopulmonary bypass. Eur.J.Cardiothorac.Surg. 2009; 35: 1012-9

7. Paparella D, Yau TM, Young E: Cardiopulmonary bypass induced inflammation: pathophysiology and treatment. An update. Eur.J.Cardiothorac.Surg. 2002; 21: 232-44

8. Kilbridge PM, Mayer JE, Newburger JW, Hickey PR, Walsh AZ, Neufeld EJ: Induction of intercellular adhesion molecule-1 and E-selectin mRNA in heart and skeletal muscle of pediatric patients undergoing cardiopulmonary bypass. J.Thorac.Cardiovasc.Surg. 1994; 107: 1183-92

9. Williams HJ, Rebuck N, Elliott MJ, Finn A: Changes in leucocyte counts and soluble intercellular adhesion molecule-1 and E-selectin during cardiopulmonary bypass in children. Perfusion 1998; 13: 322-7










19. Gschwend S, Buikema H, Henning RH, Pinto YM, de ZD, van Gilst WH: Endothelial dysfunction and infarct-size relate to impaired EDHF response in rat experimental chronic heart failure. Eur.J.Heart Fail. 2003; 5: 147-54

20. Westendorp B, Schoemaker RG, van Gilst WH, Buikema H: Improvement of EDHF by chronic ACE inhibition declines rapidly after withdrawal in rats with myocardial infarction. J.Cardiovasc.Pharmacol. 2005; 46: 766-72

21. Darblade B, Pendaries C, Krust A, Dupont S, Fouque MJ, Rami J, Chambon P, Bayard F, Arnal JF: Estradiol alters nitric oxide production in the mouse aorta through the alpha-, but not beta-, estrogen receptor. Circ.Res. 2002; 90: 413-9

22. Feletou M, Verbeuren TJ, Vanhoutte PM: Endothelium-dependent contractions in SHR: a tale of prostanoid TP and IP receptors. Br.J.Pharmacol. 2009; 156: 563-74

23. Siekmeier R, Grammer T, Marz W: Roles of oxidants, nitric oxide, and asymmetric dimethylarginine in endothelial function. J.Cardiovasc.Pharmacol.Ther. 2008; 13: 279-97

24. Borgdorff P, Fekkes D, Tangelder GJ: Hypotension caused by extracorporeal circulation: serotonin from pump-activated platelets triggers nitric oxide release. Circulation 2002; 106: 2588-93


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25. Kellum JA, Song M, Venkataraman R: Effects of hyperchloremic acidosis on arterial pressure and circulating inflammatory molecules in experimental sepsis. Chest 2004; 125: 243-8

26. Aloamaka CP, Ezimokhai M, Morrison J: The role of endothelium in phenylephrine- and potassium-induced contractions of the rat aorta during pregnancy. Res.Exp.Med.(Berl) 1993; 193: 407-17

27. Chabot F, Mestiri H, Sabry S, l'Ava-Santucci J, Lockhart A, nh-Xuan AT: Role of NO in the pulmonary artery hyporeactivity to phenylephrine in experimental biliary cirrhosis. Eur.Respir.J. 1996; 9: 560-4

28. Guo Z, Su W, Allen S, Pang H, Daugherty A, Smart E, Gong MC: COX-2 up-regulation and vascular smooth muscle contractile hyperreactivity in spontaneous diabetic db/db mice. Cardiovasc.Res. 2005; 67: 723-35

29. Ekse S, Clapp LH, Revhaug A, Ytrebo LM: Endothelium-derived hyperpolarization factor (EDHF) is up-regulated in a pig model of acute liver failure. Scand.J.Gastroenterol. 2007; 42: 356-65

30. Katz SD, Krum H: Acetylcholine-mediated vasodilation in the forearm circulation of patients with heart failure: indirect evidence for the role of endothelium-derived hyperpolarizing factor. Am.J.Cardiol. 2001; 87: 1089-92

31. Luksha L, Agewall S, Kublickiene K: Endothelium-derived hyperpolarizing factor in vascular physiology and cardiovascular disease. Atherosclerosis 2009; 202: 330-44

32. Wong SL, Leung FP, Lau CW, Au CL, Yung LM, Yao X, Chen ZY, Vanhoutte PM, Gollasch M, Huang Y: Cyclooxygenase-2-derived prostaglandin F2alpha mediates endothelium-dependent contractions in the aortae of hamsters with increased impact during aging. Circ.Res. 2009; 104: 228-35

33. Ulbrich H, Eriksson EE, Lindbom L: Leukocyte and endothelial cell adhesion molecules as targets for therapeutic interventions in inflammatory disease. Trends Pharmacol.Sci. 2003; 24: 640-7


35. Sanjabi S, Zenewicz LA, Kamanaka M, Flavell RA: Anti-inflammatory and pro-inflammatory roles of TGF-beta, IL-10, and IL-22 in immunity and autoimmunity. Curr.Opin.Pharmacol. 2009; 9: 447-53

36. Li MO, Wan YY, Sanjabi S, Robertson AK, Flavell RA: Transforming growth factor-beta regulation of immune responses. Annu.Rev.Immunol. 2006; 24: 99-146

37. Kim F, Pham M, Maloney E, Rizzo NO, Morton GJ, Wisse BE, Kirk EA, Chait A, Schwartz MW: Vascular inflammation, insulin resistance, and reduced nitric oxide production precede the onset of peripheral insulin resistance. Arterioscler.Thromb.Vasc.Biol. 2008; 28: 1982-8

38. de la SA, Larrousse M: Endothelial dysfunction is associated with increased levels of biomarkers in essential hypertension. J.Hum.Hypertens. 2009;

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Chapter 4

Microarray analysis of gene expression profiles in the kidney demonstrates a local inflammatory response

induced by cardiopulmonary bypass in rat

Hjalmar R. Bouma *; Iryna V. Samarska *; H. Maria Schenk; Marry Duin; Martin C. Houwertjes; Berthus G. Loef; Hubert E. Mungroop;

Michel M.R.F. Struys; Anne H. Epema; Robert H. Henning • these authors contributed equally

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Abstract Background: Cardiopulmonary bypass (CPB) is a commonly used technique in cardiac surgery. However, CPB is associated with acute renal dysfunction. Also, a temporary peri-operative decrease of renal function negatively influences long-term survival. To obtain an insight into the pathogenesis of renal dysfunction following CPB, we performed a microarray analysis of kidney gene expression in rat. Methods: Rats underwent CPB or Sham procedure and were sacrificed at 60 min, 1 and 5 days following the procedure. Microarray analysis was used to determine expression changes in the kidney following CPB and Sham. Results: Expression of 421 genes was significantly altered in CPB as compared to Sham, of which 407 genes in the acute phase (60 min) following CPB. Gene-ontology analysis revealed 28 of these genes being involved in inflammatory responses, with major activation of mitogen activated protein-kinase (MAP-kinase) signaling pathways. Potent inducers identified constitute of the interleukin-6 cytokine family that consists of interleukin-6 (IL6) and oncostatin M (OSM), which signal through the gp130-cytokine receptor. Downstream signaling leads to production of chemokines, adhesion molecules and molecules involved in coagulative pathways. Production of chemokines and adhesion molecules stimulate early influx of monocytes/macrophages, neutrophils and lymphocytes, thereby augmenting the renal inflammatory response. Conclusions: CPB induces an acute and local inflammatory response in the kidney, which might contribute to the induction of renal injury. The signaling pathways identified by microarray analysis of renal gene expression may represent pharmacological targets to limit renal injury following CPB. Introduction

Despite advances in anesthetic and surgical management, cardiac surgery with cardiopulmonary bypass (CPB) is still associated with increased morbidity and mortality. A serious and frequent complication of CPB is renal dysfunction, affecting up to 30% of patients.1-3 Importantly, this incidence has not changed in the last few decades. Serious renal dysfunction requiring renal replacement therapy has a high mortality rate, up to 80%.3 In addition, recent studies demonstrate that small changes in renal function across a broad spectrum of medical and surgical conditions resulted in increased in-hospital morbidity and mortality, and impaired long-term survival.4-6 For example, patients with a temporarily decline of renal function postoperatively, defined as a >25% increase of serum creatinine from baseline, have a substantially increased long-term mortality rate at 13 years of follow-up.3

The etiology of CPB-induced renal dysfunction is considered multifactorial involving hemodynamic changes, an inflammatory response and nephrotoxins.7 Although several contributing factors have been put forward, the most prominent being systemic inflammatory response syndrome (SIRS) and ischemia/reperfusion injury,8 the exact molecular mechanism of renal injury following CPB is still unexplored. To identify signaling pathways involved in the etiology of kidney injury following CPB, we

Gene expression profile in the kidney 61

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investigated the genome-wide expression profile in the kidney during 5 days of follow-up of rats that had undergone CPB. Material and Methods

Animals The experimental protocol was approved by the Animal Ethics Committee of

the University of Groningen. This study was performed in n = 46 adult male Wistar rats with body weights of 510.4±31.0 g (Harlan, Zeist, The Netherlands) housed under standard conditions with free access to food (standard rat chow; Hope Farms, Woerden, The Netherlands) and drinking water throughout the study period.

Experimental groups Experimental animals were randomly allocated to one of four experimental

groups with different duration of the recovery period after the procedure, being 60 min and 1 and 5 days. Each group was subdivided in two sub-groups: CPB and Sham. CPB animals were subjected to the full experimental protocol described below, including anesthesia, cannulation, extracorporeal circulation, weaning and the corresponding recovery period. Sham animals followed the same procedure except for the extracorporeal circulation, but maintained cannulated and mechanically ventilated. In order to evaluate the normal vascular reactivity in rats without invasive interventions, an additional untreated control group was examined. Animals in this group were sacrificed under brief isoflurane anesthesia (2.5 %) only.


preparation, extracorporeal circulation, a weaning and a recovery period (fig. 1). Anesthesia was induced with 2.5 % isoflurane in O2/air (1:1) before intubation and mechanical ventilation (Amsterdam Infant Ventilator; HoekLoos, Amsterdam, The Netherlands). Tidal volume was set to achieve normocapnia (verified by capnography 35-42 mmHg, and arterial blood gas analysis), with O2/air (1:2) at a ventilation rate of 50 min-1 (0.5 s inspiration time). Rectal temperature was kept at 37.0 ± 0.5 °C, using an electrical heating pad. The left femoral artery was cannulated (26-gauge catheter) for continuous blood pressure monitoring. The mean arterial pressure (MAP) was kept between 70 and 100 mm Hg by adjusting the isoflurane concentration as necessary (typically between 2.0-2.5%). Immediately before insertion of the arterial line, 250 IU kg-1 heparin was administered. The left carotid artery was cannulated for arterial inflow using a 22-gauge catheter. A multi-orifice 4.5 French cannula (Desilets-Hoffmann catheter, Cook Son, The Netherlands) was advanced into the right heart using the right common jugular vein for access. The tail vein was cannulated for the administration of intravenous anesthetics, heparin, and protamine sulfate.

Extracorporeal circulation Subsequently, extracorporeal circulation was initiated in the CPB group for 60

min. The set-up consisted of a glass venous reservoir, a peristaltic pump (Pericor® SF70, Verder, Haan, Germany), a rat membrane oxygenator (M.Humbs, Valley, Germany). The oxygenator carried a sterile, disposable three-layer artificial diffusion

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membrane, made from hollow polypropylene fibres (Jostra AG, Hirrlingen, Germany) and a glass counter-flow heat exchanger with built-in bubble trap. All components were connected with polyethylene tubing (1.6 mm inner diameter). The venous reservoir and heat exchanger were sterilized prior to use. The circuit was primed with 15 ml of haes 60 mg ml-1 solution (Voluven®, Fresenius Kabi, Bad Homburg, Germany). No donor blood was used. Animals were additionally heparinized (250 IU kg-1) after the start of ECC. During CPB, rats were anesthetized with intravenous fentanyl (10 "g kg-1), atracurium (0.5 mg kg-1), and midazolam (2 mg kg-1) and blood oxygen saturation was monitored continuously by a pulse oxymeter. Targeted CBP flow was 120 mL kg-1 min-1. During CPB ventilation was stopped and oxygenator gas flow was maintained at 800 ml min–1 of O2:air mixture (1:4). Samples for blood gas analysis (0.1 "l) were drawn at four time-points: at the end of preparation period (15 min before start of CPB), twice during the CPB (at 15 and 45 min), and at 45 min after the end of CPB.

Weaning and recovery At the end of extracorporal circulation, CPB flow was gradually decreased and

mechanical ventilation was initiated. Protamine (150 IU kg-1 i.v.) was administered to neutralize heparin and cannules were removed and wounds sutured. Animals were kept ventilated under isoflurane anesthesia (0.8-1.0%) for 1 h to stabilize, followed by extubation. The total duration of the recovery period lasted 1 h, and 1, 2 and 5 days after the end of the CPB. Sacrification was performed under brief isoflurane anesthesia (2.5 %). Sacrification was performed in the end of the assigned protocol under isoflurane anesthesia (2.5-3%). Samples were snap-frozen in liquid nitrogen and stored at -80°C until further analysis.

Microarray RNA isolation was performed according to the manufacturer’s instructions

(RNA Isolation Kit, Bioké, The Netherlands). Preceding hybridization, RNA-quality was checked on a BioAnalyzer 2088 (Agilent, USA). In each group (n = 5-8), RNA-samples of two animals were pooled and randomized over the different arrays as shown in a table (Supplemental Digital Content 1, which is a table listing the randomization of samples on arrays). Microarrays were performed on RatRef-12 Expression Beadchips (Illumina, USA) according to the manufacturer’s protocol. After hybridization, iScan (Illumina, USA) was used to scan the arrays, and Genomestudio 2009.1 (Illumina, USA) was used to subtract background noise and normalize intensity values (quantile method). Next, Genespring GX 11.0 (Agilent, USA) was used to analyze these normalized values.

Statistics Differences in blood gas parameters between groups over time were

compared using a GLM Repeated Measures analysis. Prior to analysis of the microarray expression profiles, probes were filtered on normalized expression values from 20th to 100th percentile present in at least 50% of the arrays in any of the groups. Next, significant differences were calculated using a One-Way ANOVA with post-hoc


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Tukey HSD corrected for multiple comparisons using Benjamini Hochberg FDR in Genespring GX 11.0 (Agilent, USA) to compare CPB to Sham (per time-point; P < 0.05).

Gene ontology analysis Gene ontology analysis is a statistical method that demonstrates which sets of

genes that are grouped to cellular component, biological process or molecular function (called “ontologies”) are significantly different expressed by the experiment. Of all genes being significantly expressed, the fold change versus pre-operative control was calculated and gene ontology analysis for biological processes was performed (cut-off P < 0.10).

Results Hemodynamic and blood gas analysis At baseline, all hemodynamic and blood gas parameters were similar and in

the normal range. During CPB, the mean arterial pressure (MAP) was significantly decreased compared to Sham treated animals. In addition, blood gas analysis demonstrated lower values for pH, bicarbonate (HCO3

-) and hematocrit (HCT), while arterial oxygen and carbon dioxide pressure (PaO2 and PaCO2) were increased as compared to Sham (P < 0.01; fig. 2).

Transcriptional alterations induced by CPB Microarray analysis demonstrated a substantial effect of CPB on genome-wide expression profiles in the kidney (fig. 3). Statistical analysis revealed 421 significant differences in expression of gene transcripts between CPB and Sham treated groups (P<0.05; Supplemental Digital Content 1, table, which is a table listing all genes different in expression). While 407 of these genes were different in the acute phase (60 min) between CPB and Sham, only ten and four genes were different at day 1 and 5 following the procedure, respectively. Of the genes that were differentially affected by CPB and Sham, 183 genes were downregulated in both, 158 genes were upregulated in both, while 80 genes were oppositely affected by Sham and CPB.

Figure 1. Time-line of the experimental procedure. Shown in this scheme are the induction of anesthesia using volatile anesthetics (2-3% isofluorane in O2/air), followed by preparation of the animals for extracorporeal circulation (±40 minutes). Animals are ventilated during the entire procedure except during extracorporeal circulation (60 minutes) when animals are anesthetized using intravenous anesthesia (fentanyl, atracurium, midazolam). After the weaning period (±15 minutes), animals are kept under very light anesthesia (0.8-1% isofluorane in O2/air) during the first hour of recovery in order to stabilize.

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Gene ontology analysis Gene ontology (GO) analysis was used to investigate classes of genes that

are involved in the same biological process among the differences between Sham and CPB across ~340,000 described assocations in the GO-database (cut-off P<0.10; tables 2–4). Herewith, gene ontology analysis provides initial information about signaling pathways that might be affected by CPB. Genes involved in cytokine biosynthetic processes and cytokine mediated signaling pathways (table 1; 11 genes), genes involved in defense and immune response (table 2; 17 genes), and genes involved in regulation of cellular metabolic processes (table 3; 23 genes) represented ontologies that were significantly affected by CPB.

Figure 3. Genomewide expression profiles following CPB (A) and Sham-surgery (B). Profile plots demonstrate the fold-change of gene expression for all genes with significant expression in the kidney as compared to healthy control for CPB (A) and Sham (B) during the acute phase (60 min), 1 and 5 days following the procedure. Changes are more profound following CPB and occur mainly in the acute phase.

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Figure 2. Perioperative hemodynamic values. Figure shows perioperative hemodynamic values of the different groups. MAP: mean arterial blood pressure; PaCO2: arterial carbon dioxide pressure; PaO2: arterial oxygen pressure; cHCO3-: bicarbonate; HCT: hematocrit. Blood gas analysis was performed on arterial blood samples drawn at different time-points during the experiment. Shown in the figures are mean ± standard deviation (SD). P=0.05, t-test

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Gene ontologies (biological processes) that were significantly altered in

expression by CPB were limited to the acute phase following surgery: no differences existed after 24 hours (tables 2 – 4). In the acute phase, profound (> 10-fold) upregulation occured of interleukin-6 (IL6; 283-fold), IL-1$ (19-fold), oncostatin M receptor (OSMR; 17-fold), IL-10 (14-fold) (table 1), chemokine (C-X-C-motif) ligand 1 and 2 (CXCL1/2; 238- and 241-fold, respectively);P-selectin (Selp; 160-fold), CXCL10 (60-fold), FBJ osteosarcoma oncogene (FOS; 33-fold), E-selectin (Sele; 30-fold), chemokine (C-C-motif) ligand 2 (CCL2; 19-fold), peptidoglycan recognition protein 1 (PGLYRP1; 19-fold), CCL7 (13-fold) (table 2), early growth response 1 (EGR1; 20-fold), myelocytomatosis oncogene (MYC; 14-fold), zinc finger protein 36 (ZFP36; 11-fold) and runt related transcription factor 1 (RUNX1; 10-fold) (table 3). No genes were profoundly downregulated by CPB as compared to Sham. Of these 17 genes that are profoundly affected by CPB as compared to Sham, 13 were involved in the regulation of immune responses (tables 2 and 3) while the minority of affected genes was associated with other (non-immunological) processes (table 3).

Table 1. Genes involved in cytokine biosynthetic process and cytokine mediated signaling pathways GO: cytokine biosynthetic process and cytokine mediated signaling pathways

Acute day 1 day 5

Entrez ID

Symbol Definition CPB Sham p CPB Sham p CPB Sham p

24498 IL6 interleukin 6 283.4 0.8 .00 1.5 1.3 ns 0.4 2.0 ns 24494 IL1B interleukin 1 $ 19.4 2.6 .00 1.1 0.9 ns. 1.1 1.1 ns 310132 OSMR oncostatin M

receptor 17.4 4.1 .00 1.4 1.9 ns 1.6 1.7 ns

25325 IL10 interleukin 10 14.2 1.0 .00 1.0 1.0 ns 1.0 1.4 ns 24253 CEBPB CCAAT/enhancer

binding protein (C/EBP), $

9.3 2.3 .00 1.2 1.4 ns 1.2 1.2 ns

114203 SH2B2 SH2B adaptor protein 2

9.3 1.6 .00 0.5 1.3 ns 1.4 1.1 ns

24508 IRF1 interferon regulatory factor 1

6.2 1.5 .00 1.0 1.2 ns 1.1 0.9 ns

58954 KLF6 Kruppel-like factor 6

5.5 1.9 .00 1.2 1.3 ns 1.0 1.0 ns

310553 TLR2 toll-like receptor 2 3.4 1.1 .00 0.8 0.7 ns 0.9 0.8 ns 25625 TNFRSF1A tumor necrosis

factor receptor superfamily, member 1a

2.8 1.4 .00 0.9 1.3 ns 0.9 1.0 ns

301059 MYD88 myeloid differentiation primary response gene 88

2.2 1.0 .01 1.2

1.2 ns 1.2 1.0 ns

Table shows genes involved in cytokine biosynthetic processes and cytokine mediated signaling pathways, as determined by gene ontology analysis (cut-off P< 0.10) performed on all significant differences between CPB and Sham (ANOVA and post-hoc Tukey HSD; p < 0.05). Shown in the table is the fold-change compared to healthy control for CPB and Sham during the acute phase (60 minutes), 1 and 5 days following the procedure, as well as the p-value between CPB and Sham (ANOVA and post-hoc Tukey


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Discussion

Our analysis reveals 421 genes in rat kidney to be affected by CPB. The majority of changes was detected at a short interval after CPB, and normalized by 1 day following CPB. Geneontology analysis was performed to calculate which gene sets were significantly affected by CPB and demonstrated that these comprise mainly genes involved in immune responses (acute phase response) and metabolic processes (tables 2 – 4). The gp-130-cytokine receptor mediated signal transduction

Table 2. Genes involved in defense/immune response other than cytokine biosynthesis and signaling GO: defense/immune response other than cytokine biosynthesis and signaling

Acute day 1 day 5

Entrez ID


114105 CXCL2 chemokine (C-X-Cmotif) ligand 2

241.3 2.5 .00 1.4 2.4 ns 2.9 0.7 ns

81503 CXCL1 chemokine (C-X-Cmotif) ligand 1

237.6 3.1 .00 1.1 1.1 ns 0.6 1.0 ns

25651 SELP selectin P 160.2 1.0 .01 0.5 0.9 ns 1.4 0.9 ns 245920 CXCL10 chemokine (C-

X-Cmotif) ligand 10

59.9 7.3 .00 1.0 1.9 ns 1.3 1.3 ns

314322 FOS FBJ osteosarcoma oncogene

33.3 1.3 .01 0.4 1.2 ns 1.6 1.6 ns

25544 SELE selectin E 29.7 1.5 .02 1.4 0.9 ns 1.9 1.2 ns

24770 CCL2 chemokine (C-Cmotif) ligand 2

19.3 2.1 .00 0.5 0.7 ns 0.9 0.5 ns

84387 PGLYRP1 peptidoglycan recognition protein 1

19.3 1.0 .01 0.7 1.6 ns 0.7 1.7 ns

287561 CCL7 chemokine (C-Cmotif) ligand 7

13.4 1.9 .00 0.8 0.9 ns 0.9 0.7 ns

25369 ADORA2A adenosine A2a receptor

6.1 2.1 .00 1.5 1.0 ns 1.4 1.2 ns

29187 CD69 CD69 molecule 4.9 1.1 .00 0.7 1.0 ns 0.4 0.6 ns 117029 CCR5 chemokine (C-

Cmotif) receptor 5

4.1 1.0 .01 1.3 1.2 ns 1.2 1.2 ns

60350 CD14 CD14 molecule 3.6 1.5 .00 1.2 1.0 ns 1.0 0.9 ns 171164 GBP2 guanylate

binding protein 2 3.3 0.8 .01 1.2 1.1 ns 1.1 0.7 ns

117540 PLSCR1 phospholipid scramblase 1

2.8 0.7 .01 1.1 1.3 ns 1.1 1.1 ns

246208 IFI47 Interferon-% inducible protein 47

2.6 1.0 .00 1.1 0.8 ns 0.8 0.8 ns

24165 ADA adenosine deaminase

0.4 1.5 .00 0.7 0.6 ns 0.7 0.7 ns

Table shows genes involved in defense/immune response other than cytokine biosynthesis and cytokine mediated signaling (which are shown in table 1), as determined by gene ontology analysis (cut-off P< 0.10) performed on all significant differences between CPB and Sham (ANOVA and post-hoc Tukey HSD; P < 0.05). Shown in the table is the fold-change compared to healthy control for CPB and Sham during the acute phase (60 minutes), 1 and 5 days following the procedure, as well as the p-value between CPB and Sham (ANOVA and post-hoc Tukey HSD; P < 0.05); a fold-change of 1.0 represents equal expression as observed in healthy control rats.

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that can be activated by (a.o.) IL6 and OSM was significantly upregulated following CPB. IL6 and the pathways that can be activated by IL6 are major players in the induction of the acute phase response. Downstream signaling leads to expression of chemokines, selectins and adhesion molecules in the kidney (fig. 4). Ultimately, this inflammatory response leads to influx of leukocytes (neutrophils, monocytes/macrophages and lymphocytes), and activation of coagulation cascades that lead to augmentation of the inflammatory response. Thus, CPB induces an acute phase response in the kidney that leads to the induction of a local inflammatory response with influx of leukocytes, which may play a role in the pathogenesis of renal dysfunction post-operatively.9;10

Although the etiology of renal dysfunction following CPB is complex, the design of our experiment allowed us to focus on the effect of the extracorporeal circulation on renal inflammation. Inflammation induced by CPB is postulated to be due to contact of leukocytes with the artificial surfaces of the machine, endotoxin released from the inflamed intestine, ischemia/reperfusion, hypothermia and surgical trauma during the procedure.9-12 In our model, hypothermia and surgical trauma were ruled out as factors influencing inflammation a priori, since animals were all kept normothermic during the procedure and the Sham procedure was included as a control for surgical trauma. Thus, our data suggests that the induction of a profound acute inflammatory response following CPB is primarily due to factors related to the use of extracorporal circulation.

Signal transduction pathways involved in the acute phase response induced by

CPB Interleukins play important signaling roles in inflammatory responses. In the

kidney, interleukin-1$ (IL1$) and IL6 are increased in expression in the acute phase (60 minutes) following CPB (table 1). IL1$ is 19-fold upregulated in CPB, while IL6 was almost 300-fold upregulated in CPB. Expression of IL6 might be induced by IL1$ via activation of MAP-kinases (ERK1/2 and p38),13 but also by Oncostatin M (OSM) that plays an important role in the acute phase response in the kidney.14 Both OSM and IL6 can activate the gp130-cytokine-receptor leading to the induction of expression of IL6, fibrinogen beta (FGB), OSMR and Serpine peptidase inhibitor 1 (Serpine 1)14 (fig. 4). Binding of IL6 to the gp130-receptor stimulates homodimerization that in turn leads to activation of downstream signalling pathways. Also heterodimerization of gp130 with OSMR, which shares extensive functional similarities with the gp130-cytokine receptor, leads to activation of downstream signal transduction pathways.15 In our analysis, OSMR was upregulated more than 17-fold by CPB (table 1). Transcription of IL6 stimulates a positive feedback mechanism, while negative feedback in the pathway is supplied by SOCS-3,16 which is upregulated more than 100-fold in the acute phase (60 minutes) following CPB. It has been shown that plasma levels of IL6 correlate with the presence of renal injury following CPB.17 Induction of expression of upstream activators and downstream signaling targets is consistent with activation of the gp130-cytokine receptor signal transduction pathway.


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Interleukin-6 (gp130-cytokine receptor) mediated signal transduction In the kidney, IL6 signals through the glycoprotein-130 (gp130)-cytokine-

receptor, downstream Janus Kinase/signal transducer and activator of transcription (JAK/STAT), and mitogen activated protein kinase (MAP-kinase) signal transduction pathways.16 Activation of this MAPK pathways is confirmed by our microarray, as the downstream transcription factors of MAPK-signaling Myc, Jun, Fos, and ATF-4 were upregulated by CPB (table 3 and Supplemental Digital Content 1, table), suggesting major involvement of MAPK-signaling in the kidney following CPB. Activation of MAP-kinases might occur via growth arrest and DNA-damage-inducible alpha and beta (GADD45A and –B) that are both upregulated in the acute phase (60 minutes) following CPB (Supplemental Digital Content 1, table) and can induce activation of mitogen activated protein kinase kinase kinase (MAP3K4). By activation of p38 and JNK, MAP3K4 can induce apoptosis of the cells.18 Another upstream activator of p38, dual specificity mitogen activated protein kinase kinase 6 (MAP2K6), was downregulated twice as much by CPB as by Sham (Supplemental Digital Content 1, table). Although MAP2K6 has been linked to p38, it does not cause activation of ERK.19 Downregulation of MAP2K6 might therefore be a counterregulatory pathway limiting p38-activation. This might shift the balance in MAPK-activation towards the ‘pro-survival’ MAP-kinase ERK.16 Our data are consistent with previous observations on gene expression changes in cardiac tissue following CPB, in which upregulation of Fos, Myc and Jun in patients20 and of mitogen activated protein kinase 1 (MAPK1) and mitogen activated protein kinase 3 (MAPK3) in rat.21 MAPK-pathways can be activated by several stimuli, including ischemia/reperfusion and circulating cytokines.16;22;23 Activation of different MAPK-signaling pathways determines cellular faith in terms of survival or apoptosis of the cells.24 MAP-kinases play an important role in signal transduction in inflammation by regulating the transcription of cytokines and/or growth factors16;22;23 (fig. 4). Taken together, our data suggest that a main pathway of local inflammation induction in kidney consists of activation of the gp130-cytokine receptor, activating downstream MAP-kinase signal transduction pathways that play a pivotal role in cell cycle regulation but also regulate the expression of cytokines.

Chemokines, selectins and adhesion molecules stimulate cellular influx into

the kidney Expression of chemoattractive cytokines (chemokines) can stimulate influx of

leukocytes into the kidney, thereby exaggerating the inflammatory response. In our analysis, C-X-C motif chemokine ligand 1 (CXCL1), CXCL2, C-C motif chemokine ligand 2 (CCL2), CCL7, and CXCL10 were grossly upregulated in the acute phase following CPB (table 2). While CXCL1 and CXCL2 stimulate attraction of neutrophils 25;26, CCL2 and CCL7 promote influx of monocytes/macrophages27;28 and CXCL10 stimulates influx of activated (i.e. CD69+-cells) T-lymphocytes.29 Local production of chemokines will stimulate influx of leukocytes into the kidney. Leukocyte influx is facilitated by the upregulation of E- and P-selectin, VCAM1 and ICAM1 in the acute phase (60 minutes) following CPB (table 2 and Supplemental Digital Content 1, table).

Table 3. Genes involved in regulation of cellular metabolic process (without direct immune function)

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GO: regulation of cellular metabolic process (without direct immune function)

acute day 1 day 5

Entrez ID


24330 EGR1 early growth response 1

19.7 2.6 .00 0.5 1.2 ns 1.6 1.3 ns

24577 MYC myelocytomatosis oncogene

13.6 3.3 .00 1.2 1.8 ns 1.1 1.2 ns

79426 ZFP36 zinc finger protein 36

10.7 1.2 .00 1.2 1.2 ns 1.1 1.1 ns

50662 RUNX1 runt related transcription factor 1

10.1 2.2 .01 1.1 1.1 ns 2.0 1.3 ns

24517 JUNB jun B proto-oncogene

8.1 1.6 .00 0.9 1.4 ns 1.3 1.1 ns

59329 SNF1LK SNF1-like kinase 6.0 0.9 .01 1.1 1.2 ns 1.6 1.3 ns 304663 LYL1 lymphoblastic

leukemia derived sequence 1

0.2 1.0 .00 1.2 0.8 ns 1.3 1.0 ns

309452 NFKB2 nuclear factor of kappa light polypeptide gene enhancer in B-cells 2, p49/p100

4.6 1.3 .00 1.1 1.1 ns 1.0 1.1 ns

309175 CDC42EP2 CDC42 effector protein (Rho GTPase binding) 2

3.7 1.5 .01 1.2 1.2 ns 1.5 1.1 ns

63839 FHL2 four and a half LIM domains 2

0.3 0.7 .01 1.1 0.8 ns 0.9 0.9 ns

114121 CCNL1 cyclin L1 3.4 1.7 .00 1.3 1.1 ns 1.4 1.4 ns 306330 KLF2 Kruppel-like factor 2 0.3 0.6 .00 1.3 1.1 ns 0.9 1.1 ns 79431 BHLHE40 basic helix-loop-

helix family, member e40

3.3 1.7 .00 1.2 1.0 ns 1.1 1.0 ns

246760 MAFK v-maf musculoaponeurotic fibrosarcoma oncogene homolog K (avian)

2.9 1.7 .00 1.0 1.3 ns 1.1 1.2 ns

79255 ATF4 activating transcription factor 4 (tax-responsive enhancer element B67)

2.9 1.7 .00 0.9 1.1 ns 0.9 0.9 ns

114090 EGR2 early growth response 2

2.8 1.0 .00 1.0 0.9 ns 0.8 0.9 ns

302937 ANKS3 ankyrin repeat and sterile ! motif domain containing 3

0.4 0.8 .01 1.1 0.8 ns 0.8 0.8 ns

24516 JUN Jun oncogene 2.6 1.2 .00 0.8 1.0 ns 0.9 0.9 ns 29344 ZFP36L1 zinc finger protein

36, C3H type-like 1 2.3 1.0 .03 0.9 1.3 ns 1.0 1.1 ns

29618 BTG1 B-cell translocation gene 1, anti-proliferative

2.3 1.2 .00 1.0 1.0 ns 1.0 1.0 ns

311807 BMYC brain expressed myelocytomatosis oncogene

0.4 0.7 .00 0.9 0.9 ns 1.1 0.9 ns

288912 MRI1 methylthioribose-1-phosphate isomerase homolog

0.5 0.9 .01 0.9 0.8 ns 1.0 1.0 ns

286988 PNRC1 proline-rich nuclear receptor coactivator 1

2.1 1.2 .00 0.8 0.8 ns 0.8 0.8 ns

Table shows genes involved in regulation of cellular metabolic process (without direct immune function; these are shown in table 1 and 3), as determined by gene ontology analysis (cut-off P< 0.10) performed on all significant differences between CPB and Sham (ANOVA and post-hoc Tukey HSD; P < 0.05). Shown in the table is the fold-change compared to healthy control for CPB and Sham during the acute phase (60 minutes), 1 and 5 days following the procedure, as well as the p-value between CPB and Sham (ANOVA and post-hoc Tukey HSD; P < 0.05); a fold-change of 1.0 represents equal expression as observed in healthy control rats.


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Increased expression of adhesion molecules has also been found in cardiac tissue of rats and in renal tissue from humans undergoing CPB.21;30 Taken together, our analysis provides evidence that local production of chemokines and increased expression of selectins and adhesion molecules following CPB stimulate cellular influx into the kidney, thereby amplifying the inflammatory response.

Neutrophil and monocyte influx Clinically, the number of circulating monocytes31 and neutrophils32 is increased

following surgery. Activation and influx of neutrophils and monocytes correlates with renal injury following CPB.31-34 The expression of CD14 (a marker of monocytes/macrophages) was significantly higher in the kidney following CPB (table 2). Activation of macrophages induces expression of CCL2, CCL7,35 CXCL1 and CXCL2,36;37 which is induced by CPB (table 2). While CCL2 and CCL7 provide a

Figure 4. Summary of major signal transduction pathways involved in renal injury following cardiopulmonary bypass. This figure shows schematically how activation of the interleukin-1-receptor (IL1R), gp130-cytokine receptor (interleukin-6-receptor or oncostatin-receptor; IL6R or OSMR) lead to activation of the mitogen activated protein kinases (MAP-kinases) extracellular signal-regulated kinase (ERK), Jun-kinase (JNK) and p38. Activation of JNK and p38 might result in apoptosis of the cell. Downstream signal transduction from the gp130-cytokine receptor can result in transcription of genes involved in the acute phase response in the kidney (fibrogen-$; FGB, OSMR; oncostatin M receptor, and Serpine 1), but also induces expression and activation of suppressor of cytokine signaling 3 (SOCS3) that provides a negative feedback loop by inhibiting the receptor. Expression of C-X-C chemokine ligand 1 and -2 (CXCL1 and -2) can be induced by downstream signal transduction of the IL1R in which MyD88 and the MAP-kinases ERK and p38 play important signaling roles. Further, activation of MAP-kinases is enhanced by growth arrest and DNA-damage inducible gene (GADD45) and MAP-kinase kinase kinase 4 (MAP3K4) and lead via activation of transcription factors (Myc, Fos, Jun, ATF2 and ATF4) to transcription of several genes that are involved in the inflammatory response and might contribute to the development of acute kidney injury following cardiopulmonary bypass (E-selectin, C-C motif chemokine ligand 2; CCL2, tumor necrosis factor alpha; TNF!, heme oxygenase; HMOX, and IL6).

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chemotactic signal for monocytes,27;28 CXCL1 and CXCL2 stimulate early recruitment of activated neutrophils into the tissue.36;37 Activated neutrophils increase their expression of CCRL2, which is a receptor for circulating chemokines and thus augments their sensitivity to chemokines.38 Expression of CCRL2 is significantly higher than Sham in the acute phase (60 minutes) following CPB (table 2); a finding that might represent influx of activated neutrophils into the kidney. CXCL1 and CXCL2 can be upregulated by ischemia/reperfusion,39 but also IL1$ is able to upregulate expression of CXCL1 and CXCL2 in which MyD88 and the MAP-kinases ERK1/2 and p38 play important signaling roles13;37 (fig. 4). Both the expression of IL1$ and MyD88 are increased in the acute phase (60 minutes) following CPB (table 1). Since CCL2, CCL7, CXCL1 and CXCL2 are mainly produced by macrophages; our array data support the view that the early activation of (resident) macrophages induces the production of chemokines, in turn causing an influx of monocytes/macrophages and neutrophils into the kidney.

Lymphocyte influx The number of circulating T-helper (Th-) cells, B-cells and natural killer cells

(NK-cells) is reduced following CPB.40;41 However, CPB induces the activation of both NK-cells40 and T-lymphocytes (i.e. CD69+-cells)42 in peripheral blood. Activated T-lymphocytes increase their expression of CD69.43 In our microarray, expression of CD69 increased about four fold in the acute phase (60 minutes) following CPB and thus likely represents an influx of activated T-cells in the kidney. Further, one of the chemokine receptors mainly expressed on T-cells and monocytes/macrophages, the C-C motif Chemokine Receptor 5 (CCR5),44 is upregulated significantly by CPB (table 2). A factor that plays a key role in the migration of lymphocytes from peripheral lymphoid organs is Sphingosine-1-phosphate (S1P).45 The expression of Sphk was more than eight fold increased by CPB, while this was only slightly upregulated following Sham (Supplemental Digital Content 1, table). We speculate that local production of S1P in the kidney stimulates influx of lymphocytes into the kidney. Further, influx of lymphocytes into the kidney is promoted by CXCL10.29 In our analysis, expression of CXCL10 was induced about 60-fold by CPB. Taken together, CPB induces activation of lymphocytes, while production of S1P and CXCL10 in the kidney causes influx of these activated cells.

Heme oxygenase 1 Heme oxygenase 1 (HMOX1) is the rate-limiting enzyme in the catabolism of

heme to form biliverdin, which is subsequently cleaved to free iron and carbon monoxide (CO).46 HMOX1 (Supplemental Digital Content 1, table) is upregulated almost 100-fold during the acute phase (60 minutes) following CPB, being ten times as much as following Sham. Ischemia induces expression of HMOX1, which can be mediated by activating transcription factor 4 (ATF4).47;48 Further, IL10 can induce expression of HMOX1 via p38 (fig. 4).49 Expression of ATF4 is increased almost three fold, while expression of IL10 was increased about 14-fold in the acute phase (60 minutes) following CPB (tables 4 and Supplemental Digital Content 1, table).


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HMOX1/CO are believed to regulate several (repair) processes to limit tissue injury: they stimulate the production of anti-inflammatory IL10 over pro-inflammatory IL6,50;51 induce the formation of FoxP3+ regulatory T-lymphocytes,50 and stimulate re-endothelialization after vascular injury by mobilizing progenitor cells.52 Induction of HMOX1 in the kidney seems to reflect direct cell injury as well as an adaptive response towards a cytoprotective state to limit renal injury, thereby protecting the kidneys from further ischemic or toxic damage.53

Characterization of the renal inflammatory response following CPB The sequence of events following CPB can be divided in events on a

molecular level (e.g. intracellular signaling pathways; summarized in fig. 4) as well as on a cellular level (e.g. influx of leukocytes). Taken together, our data suggest early activation of (resident) macrophages in the kidney following CPB that leads to local production of chemokines, thereby attracting other leukocytes into the kidney. Whereas production of CCL2 and CCL7 stimulates influx of monocytes, influx of neutrophils is promoted by CXCL1 and CXCL2, and production of CXCL10 and S1P stimulates lymphocyte infiltration. Upon activation of circulating cells, the expression of ligands for adhesion molecules is increased (CD11b, CCRL2 on neutrophils and CCR5 on monocytes and lymphocytes) that can bind the adhesion molecules expressed on endothelial cells on kidney vasculature (E-selectin, ICAM1, VCAM1) and facilitate transmigration of cells into the tissue. Signaling between leukocytes is mainly mediated by (chemoattractive) cytokines, while intercellular signaling can occur after these cytokines bind their receptors and activate the downstream signaling pathways. An important signaling pathway identified by our microarray is the gp130-cytokinereceptor mediated signaling pathway. This receptor can be activated by IL6 or OSM (grossly increased in our array) that leads via activation of MAP-kinase pathways to expression of adhesion molecules, (chemoattractive) cytokines and other molecules involved in the acute phase response.

Identification of pharmacological targets Resident macrophages might play a more important role than infiltrating cells

from the circulation in initiating the inflammatory response in the kidney. Our micoarray analysis shows early upregulation of chemokines that are mainly produced by (resident) macrophages rather than neutrophils or lymphocytes and stimulate influx of both monocytes/macrophages, neutrophils and lymphocytes. Since the application of leukocyte depleting filters during CPB is only partially effective in reducing the inflammatory response54;55 and does not affect clinical outcome following surgery,56 one may speculate that influx of circulating leukocytes into organs is of minor importance in the initiation of the local inflammatory response in organs following CPB. Conditional ablation of resident macrophages, an experimental technique that can be used to deplete the kidney of resident macrophages, may provide valuable data regarding the role of resident cells.57 Inhibition of signaling pathways, either directed at leukocytes (e.g. resident macrophages) or endothelial cells in the kidney, might reduce the inflammatory response in the kidney. As described above, signaling through the

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gp130-cytokine-receptor following activation of OSM or IL6 and downstream activation of MAP-kinases represents an important target in renal inflammation following CPB. Reducing the presence of the receptor-ligand, antagonizing the receptor or intervening in its signal transduction might limit renal inflammation following CPB. Identification of pharmacological agents that successfully reduce renal injury following CPB does not only provide more insight into the etiology of CPB-induced renal dysfunction and its influence on mortality, but might also improve survival following surgery.

Conclusion

Changes in gene expression of kidney occur early following CPB and represent mainly genes involved in inflammatory responses. The principle activation route consists of release of IL6 or OSM by resident macrophages, which in turn activate gp130-cytokine mediated signaling and downstream MAP-kinase pathways. Intervening with receptor activation or downstream signaling may represent novel therapeutic strategies to limit CPB-induced renal injury.

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45. Schwab SR, Cyster JG: Finding a way out: lymphocyte egress from lymphoid organs. Nat.Immunol. 2007; 8: 1295-301

46. Guenegou A, Leynaert B, Benessiano J, Pin I, Demoly P, Neukirch F, Boczkowski J, Aubier M: Association of lung function decline with the heme oxygenase-1 gene promoter microsatellite polymorphism in a general population sample. Results from the European Community Respiratory Health Survey (ECRHS), France. J.Med.Genet. 2006; 43: e43

47. He CH, Gong P, Hu B, Stewart D, Choi ME, Choi AM, Alam J: Identification of activating transcription factor 4 (ATF4) as an Nrf2-interacting protein. Implication for heme oxygenase-1 gene regulation. J.Biol.Chem. 2001; 276: 20858-65

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50. Xia ZW, Xu LQ, Zhong WW, Wei JJ, Li NL, Shao J, Li YZ, Yu SC, Zhang ZL: Heme oxygenase-1 attenuates ovalbumin-induced airway inflammation by up-regulation of foxp3 T-regulatory cells, interleukin-10, and membrane-bound transforming growth factor- 1. Am.J.Pathol. 2007; 171: 1904-14

51. Kamimoto M, Mizuno S, Nakamura T: Reciprocal regulation of IL-6 and IL-10 balance by HGF via recruitment of heme oxygenase-1 in macrophages for attenuation of liver injury in a mouse model of endotoxemia. Int.J.Mol.Med. 2009; 24: 161-70

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54. Warren O, Wallace S, Massey R, Tunnicliffe C, Alexiou C, Powell J, Meisuria N, Darzi A, Athanasiou T: Does systemic leukocyte filtration affect perioperative hemorrhage in cardiac surgery? A systematic review and meta-analysis. ASAIO J. 2007; 53: 514-21


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55. Lim HK, Anderson J, Leong JY, Pepe S, Salamonsen RF, Rosenfeldt FL: What is the role of leukocyte depletion in cardiac surgery? Heart Lung Circ. 2007; 16: 243-53

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57. Duffield JS, Tipping PG, Kipari T, Cailhier JF, Clay S, Lang R, Bonventre JV, Hughes J: Conditional ablation of macrophages halts progression of crescentic glomerulonephritis. Am.J.Pathol. 2005; 167: 1207-19

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Supplemental Digital Content 1 Microarray annotations Group Microarray

number Rat numbers

Control 48716040004_I 77 + 78

Control 48716040004_L 79 + 80

Control 48716040005_E 81 + 82

Acute Sham 48716040004_D 18 + 19

Acute Sham 48716040005_C 20 + 21

Acute Sham 48716040005_G 16 + 17

1 day Sham 48716040004_E 42 + 61

1 day Sham 48716040005_H 34 + 36

1 day Sham 48716040005_J 39 + 41

1 day Sham 48716040005_K 37 + 38

5 day Sham 48716040004_B 70 + 73

5 day Sham 48716040004_H 68 + 69

5 day Sham 48716040005_D 74

Acute CPB 48716040004_A 12 + 14

Acute CPB 48716040004_K 15 + 43

Acute CPB 48716040005_B 5 + 7

Acute CPB 48716040005_L 9 + 10

1 day CPB 48716040004_C 32 + 35

1 day CPB 48716040004_F 25 + 26

1 day CPB 48716040004_G 27 + 31

1 day CPB 48716040004_J 63 + 64

5 day CPB 48716040005_A 2 + 67

5 day CPB 48716040005_F 71 + 72

5 day CPB 48716040005_I 75

Table shows annotation of microarray samples. In each group (n = 5 – 8 animals), RNA-samples were pooled to obtain n = 3 – 4 microarray samples. Samples were included in the different arrays as shown.

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Chapter 5

Protracted pulmonary inflammation during long-term recovery following

cardiopulmonary bypass in the rat

Iryna V. Samarska, Pieter A. van der Vorm, Hjalmar Bouma, Hendrik Buikema, Martin C. Houwertjes, Michel M.R.F. Struys,


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Abstract Background: Cardiopulmonary bypass (CPB) is associated with a multiple organ dysfunction syndrome involving disturbances in lung function. Pulmonary inflammation represents a main cause of postoperative lung dysfunction. The present study was intended to evaluate the time-dependent changes in markers of the pulmonary inflammation during a clinically relevant post-operative period. Materials and Methods: Male Wistar rats (n=78) were anesthetized with isoflurane (2,5-3%) and fentanyl/midazolam during CPB. Femoral and carotid artery and the right heart were cannulated and animals were randomly assigned to sham or CPB with normothermic extracorporeal circulation (ECC) for 60 min at a flow of 100-110 mL kg-1 min-1. Different markers of the endothelial activation, oxidative stress, pro- and anti-inflammatory cytokines were evaluated within 5 days recovery period by RT-PCR. Infiltration of the lung tissue with macrophages was evaluated by immunohistochemistry with anti-ED-1 staining. Results: After one hour of recovery, CPB rats showed increased expression of HO-1, E-selectin, ICAM-1, VCAM-1, IL6, IL-1beta (as compared with Control, p<0.05). At the second day of the recovery, CPB rats showed upregulation of SOD-1, TGFbeta-1 and IL1beta, which was higher in CPB group through the entire 5 days following CPB The amount of macrophages in lung tissue was relatively unchanged throughout the whole recovery period. Activity of neutrophil elastase in plasma was increased in CPB group after 1 hour of recovery (p<0.05). Conclusion: CPB evokes production of IL-6 and IL-1beta by residential macrophages of lung, which in turn activate neutrophils and endothelium. Activated neutrophils release neutrophil elastase, which participates in degradation of extra-cellular matrix components and contribute to acute lung tissue injury. Increased expression of TGFbeta1 is supposed to have immunosuppressive effects with modulatory influence on T-cell response. The prolonged upregulation of IL-1beta, TGFbeta1 and SOD-1 after 5 days of recovery period suggest ongoing inflammatory process in lungs that may contribute to post-CPB lung complications. Introduction

Cardiopulmonary bypass (CPB) plays an important role in thoracic surgery, including procedures such as coronary artery bypass grafting, valve surgery, heart-lung transplantation and pulmonary surgical interventions. However, CPB is also associated with the initiation of a systemic inflammatory response, which may affect function of vital organs including heart, kidney, lung, and liver.1-3 One of the severe clinical consequences of CPB includes postoperative lung dysfunction with interstitial pulmonary edema and subsequent abnormal gas exchange. Clinical data suggest that approximately 25% of the patients following an open-heart surgery exhibited signs of the pulmonary impairment during at least one week after. 4, 5 Lung damage following CPB may be explained by the specific character of during CPB, since lungs are more or less excluded from the circulation in this period. It is thought that the extracorporeal

Protracted inflammation in the lungs 81

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circulation causes the lung vascular injury, atelectasis, collapse of non-ventilated lungs, and massive or submassive pulmonary embolism.4, 5, 6 On the other hand, the CPB-related systemic inflammatory response plays a prominent role as well. Several triggers (surgical technique, effects of the anesthetic technique, extracorporeal circulation, metabolic changes, hypothermia, temporary cardiac dysfunction, contact of the blood with artificial surfaces, ischemia-reperfusion injury) may lead to the activation of the humoral and cellular immune system with subsequent release of the various mediators and factors that in turn cause multiple organ dysfunction syndrome. Several studies investigated lung injury after CPB and pulmonary inflammation was found to be the main cause.2, 7-10 The present study was intended to evaluate the time-dependent changes in markers of the pulmonary inflammation during a clinically relevant post-operative period (from 60 min up to 5 days of recovery). Material and Methods

The experimental protocol was approved by the Animal Ethics Committee of the University of Groningen. This study was performed in n=78 adult male Wistar rats with body weights of 507.4±31.3 g (Harlan, Zeist, The Netherlands) housed under standard conditions with free access to food (standard rat chow; Hope Farms, Woerden, The Netherlands) and drinking water throughout the study.

Experimental groups Experimental animals were randomly allocated to one of four experimental

groups with different duration of the recovery period after the procedure, being 60 min and 1, 2 and 5 days. Each group was subdivided in two sub-groups: CPB and Sham. CPB animals were subjected to the full experimental protocol described below, including anesthesia, cannulation, extracorporeal circulation, weaning and the corresponding recovery period. Sham animals followed the same procedure except for the extracorporeal circulation, but maintained cannulated and mechanically ventilated. In order to evaluate the normal vascular reactivity in rats without invasive interventions, an additional untreated control group was examined. Animals in this group were sacrificed under brief isoflurane anesthesia (2.5-3%) only.

Experimental protocol The experimental protocol includes following parts: anesthesia, preparation,

extracorporeal circulation, a weaning and a recovery period. Anesthesia was induced with 2-3% isoflurane in O2/air (1:1) before intubation and mechanical ventilation (Amsterdam Infant Ventilator; HoekLoos, Amsterdam, The Netherlands). Tidal volume was set to achieve normocapnia (verified by capnography and arterial blood gas analysis), with O2/air (1:2) at a ventilation rate of 50 min-1 (0.5 s inspiration time). Rectal temperature was kept at 37.5±0.5 °C, using an electrical heating pad. The left femoral artery was cannulated (26-gauge catheter) for blood pressure monitoring. The mean arterial pressure was kept between 70 and 100 mm Hg by adjusting the isoflurane concentration as necessary (typically between 2.0-2.5%). Immediately before insertion of the arterial line, 250 IU kg-1 heparin was administered. The left

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carotid artery was cannulated for arterial inflow using a 22-gauge catheter. A multi-orifice 4.5 French cannula (adapted from a Desilets-Hoffmann catheter, Cook Son, The Netherlands) was advanced into the right heart using the right common jugular vein for access. The tail vein was cannulated for the administration of intravenous anesthetics, heparin, and protamine sulfate.

Extracorporeal circulation Subsequently, extracorporeal circulation (ECC) was initiated in the CPB group

for 60 min. The set-up consisted of a glass venous reservoir, a peristaltic pump (Pericor® SF70), a neonatal membrane oxygenator (0140GM, Polystan, Vaerlose, Denmark) and a glass counter-flow heat exchanger with built-in bubble trap. All components were connected with polyethylene tubing (1.6 mm inner diameter). The venous reservoir and heat exchanger were sterilized prior to use. The circuit was primed with 15 ml of Haes 60 mg ml -1 solution (Voluven®, Fresenius Kabi, Bad Homburg, Germany). Animals were additionally heparinised (250 IU kg-1) after the start of ECC. During EEC, rats were anesthetized with intravenous fentanyl (125 "g kg-1), atracurium (0.5 mg kg-1), and midazolam (2 mg kg-1). During CBP, blood oxygen saturation was monitored continuously by a pulse oximeter. Targeted CBP flow was 100-110 mL kg-1 min-1, corresponding to 60-70% of normal cardiac output. Samples for blood gas analysis (0.1 "l) were drawn at four time-points: at the end of preparation period (15 min before start of ECC), twice during the ECC period (15 min after the start of it and 15 min before the its ending), and at 45 min after the end of ECC.


was initiated. Protamine (150 IU kg-1 i.v.) was administered in order to neutralize heparin after which cannules were removed and the wounds sutured. Following extubation, animals were first kept under isoflurane anesthesia (0.8-1.0%) for the first h of recovery in order to stabilize. The duration of the recovery period of various groups lasted 1 h, 1, 2 and 5 days after the end of the extracorporeal circulation. Sacrification was performed in the end of assigned protocol under isoflurane anesthesia (2.5-3%).

Real time PCR Total RNA was extracted from the lung using NucleoSpin RNAII (Macherey-

Nagel, Duren, Germany). First-Strand cDNA Synthesis from RNA (1ug) was performed by using Reverse Transcription reagents (Promega, Madison, WI, USA). The expression of SOD1, p22, HO-1, ICAM-1, E-selectin, VCAM-1, TNF-alpha, TGF beta 1, IL-6, IL1beta, FOXP3 (table 1) were analyzed by real-time PCR with Absolute QPCR SYBR Green reagents (Molecular Probes, Leiden, Netherlands) and CFX384 Real-Time PCR Detection System (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The PCR protocol consisted of 15 min at 95ºC, followed by 40 cycles with heating to 95ºC and cooling to 60ºC for 1 minute. The amplification product was evaluated by melting curves analysis and agarose gel (1,5%) electrophoresis. Sequence-specific primers


!

(table 1) were obtained from Biolegio (Nijmegen, the Netherlands). Cycle threshold (CT) values for genes analyzed were normalized to their CT value of GAPDH and presented as delta delta Ct (2-&&Ct).

Neutrophil elastase activity Neutrophil elastase activity was measured in plasma by using its specific

substrate N-methoxysuccinyl-Ala- Ala-Pro-Val p-nitroanilide.11 Briefly, 100ul plasma was incubated with 0.1M Tris–HCl buffer containing 0.5MNaCl and 1mM substrate at the temperature 37oC during 24 hours. This results in the formation of the product p-nitroanilide that was measured by spectrophotometry (Bio-Rad Laboratories, USA) at 405nm.

Immunohistochemistry The infiltration of the lung tissue with macrophages was evaluated by

immunohistochemistry. Fresh frozen lung sections were incubated with a monoclonal primary IgG antibody specific for the monocyte/macrophage antigen, ED-1 (Serotec, Oxford, UK). The immunostaining was quantitated by counting the number of ED-1 positive cells in 10 fields under dimensions 400x.

Data analysis Data are given as mean±SD and n refers to the number of animals in a

corresponding group. If the data passed normality test, then differences were evaluated using one-way ANOVA followed by Bonferroni test or with t-test (SPSS, Chicago, IL, USA). Unless the normality test was passed, data were analyzed by Kruskal-Wallis One Way Analysis of Variance on Ranks followed by Turkey test or Mann-Whitney Rank Sum Test (SigmaStat for SigmaPlot version 11, Systat Software Inc, San Jose, California). Differences were considered significant at P<0.05 (2-tailed).

Table 1. Primer pairs for RT-PCR

Gene Forward Reverse SOD1 AACATGGCGGTCCAGCGGAT GCCAAGCGGCTTCCAGCATT

p22 TGTTGCAGGAGTGCTCATCTGTCT AGGACAGCCCGGACGTAGTAATTT

HO-1 GTGCACATCCGTGCAGAGAA GAAGGCCATGTCCTGCTCTA

E-selectin CCATTCGGCCTCTTCAAGCTA TGCAGCTCACAGAGCCATTC

ICAM-1 CTGCCACCATCACTGTGT CTGACCTCGGAGACATTCTT

VCAM-1 TAAGTTACACAGCAGTCAAATGGA CACATACATAAATGCCGGAATCTT

IL-6 CCAGAAGACCAGAGCAGATT CACACTAGCAGGTCGTCATC

IL1beta CTGTGGCAGCTACCTATGTC CACACTAGCAGGTCGTCATC

TNF" CACGCTCTTCTGTCTACTGA GTACCACCAGTTGGTTGTCT

TGF beta 1 AGAGCTGCGCCTGCAGAG GAAGCCGGTTACCAAGGT

FOXP3 GCACAAGTGCTTTGTGCGAGT TGTCTGTGGTTGCAGACGTTGT

GAPDH AAGGTCGGTGTCAACGGATTT CAATGTCCACTTTGTCACAAGAGAA

84 Chapter 5

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Results Hemodynamics and blood gas analysis during the protocol During the procedure, hemodynamic measurements (MAP) and blood gas

analysis were performed in order to monitor the animals. At baseline all parameters were in the normal range. Blood gas analysis was performed before start of ECC, twice during ECC and after weaning. During ECC, mean arterial blood pressure was significantly lower in the CPB groups compared with Sham (table 2). Also in the CPB group, a decreased pH, bicarbonate (HCO-

3), and Base excesses and increased levels of pCO2 and pO2 were found, which may represent moderate respiratory acidosis. Hematocrit was significantly decreased in CPB groups as a consequence of hemodilution with the priming solution. Intraoperative hyperglycemia was observed in both Sham and CPB groups during the first 2 hours. In the post-ECC period all parameters in CPB, including mean arterial blood pressure, returned to normal ranges and were similar in both Sham and CPB, except for hematocrit (table 2).

Expression of markers of the oxidative stress CPB is known to be associated with ischemia-reperfusion injury, hypoxia,

oxidative stress and increased ROS-production. Therefore the expression of SOD-1, p22 phox, HO-1 was evaluated by RT-PCR during long-term recovery (fig. 1). HO-1 was significantly increased in CPB after 1hour of recovery as compared with Control (P<0.05, t-test). Expression of SOD-1 was increased in CPB group at the second day of recovery and remained upregulated at 5th day of the postoperative period without a tendency for normalization. Expression of p22phox showe a tendency to increase in both CPB and Sham compared to Controls.

Thus, CPB evoked expression of HO-1, a marker of hypoxia injury, during short-term recovery and upregulated SOD-1, a marker of the oxidative stress, during later phases of the postoperative period.

Expression of markers of endothelial activation In order to evaluate the activation of the pulmonary endothelium, the relative

expression of E-selectin, ICAM-1, and VCAM-1 were studied by RT-PCR (fig.2). All three parameters were significantly upregulated in CPB after 1 hour of recovery as compared with Sham and/or Control. Interestingly, after 1 day of recovery, the values of all three markers returned to the baseline levels, even in the CPB group. Thus, CPB evoked an immediate endothelial activation which normalized within 24 h.


!

Tabl

e 2.

Dat

a on

blo

od g

ases

ana

lysi

s, g

luco

se c

once

ntra

tion

and

mea

n ar

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and

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roup

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C

15 m

in E

CC

45

min

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er E

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B

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e E

CC

15

min

EC

C

45 m

in E

CC

A

fter

EC

C

MA

P, m

mH

g 10

2.4±

32.8

10

9.8±

12.9

10

3.5±

22.4

92

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8.2

84.1

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6 56

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12.9

81

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25.6

80

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pH

7.43

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45±0

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7.46

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7 7.

41±0

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7.42

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40*±

0.07

7.

32 *

±0.0

8 7.

37±0

.05

pCO

2, k

Pa

4.74

±1.0

4.

51±0

.8

4.68

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15±0

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4.77

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1*±1

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5.72

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pO2,

kP

a 17

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16.6

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17

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17.0

±5.1

19

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.0

34.1

*±17

.1

36.0

*±18

.3

20.0

±10.

0

HC

O3,

mm

ol/l

23.0

±1.6

23

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.5

24.7

±1.5

24

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22.6

±2.3

21

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21.6

*±2.

4 22

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1.7

BE

, mm

ol/l

-0

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1.8±

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, mm

ol/l

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atio

ns: H

ctc,

hem

atoc

rit; C

PB

, car

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P<0

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, ind

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t-te

st

86 Chapter 5

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Figure 1. Relative expression of markers of the oxidative stress and hypoxia (HO-1, SOD-1, p22phox) in Sham, CPB and Control groups within 5 days recovery period. * P<0.05, vs Control, one way ANOVA, Bonferroni test.

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Figure 2. Relative expression of markers of the endothelial activation (E-selectin, ICAM-1, VCAM-1) in Sham, CPB and Control groups within 5 days recovery period. * P<0.05, vs Control, Kruskal-Wallis One Way Analysis of Variance on Ranks, Turkey test; ** P<0.05, vs Control, One Way ANOVA, Bonferroni test; # P<0.05, vs 2days Sham, One way ANOVA, Bonferroni test

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Neutrophil elastase activity CPB caused a marked increase in

the activity of neutrophil elastase in plasma after 1 hour recovery as compared with corresponding Sham group and Control (fig.3). After 1 day of recovery, neutrophil elastase acivity returned to baseline values. Thus, CPB induced an immediate increase in neutrophil elastase activity in plasma.

Expression of the pro and anti-inflammatory markers IL1beta, IL6 and TNF alpha expression were studied in lung tissue as pro-

inflammatory markers (fig. 4). mRNA levels of IL6 and IL1beta were significantly increased in CPB 1hour group as compared with Control and Sham. At 1 day of recovery, expression of IL6 was normalized, while IL 1beta remained upregulated in CPB groups up to 5th day of the recovery period. TNF alpha did not show pronounced changes in its expression and was even down-regulated at the earliest time point of recovery (1hour).

TGF beta 1 was evaluated as anti-inflammatory factor, as it is considered to be upregulated in SIRS.12 CPB induced a biphasic pattern in the expression of TGF beta: it was down regulated during the 1st day of the recovery period, followed by an upregulation ate the 5th day of the recovery period.

Together, these data show that CPB causes a profound upregulation of pro- and anti-inflammatory cytokines during recovery. During short-term recovery, CPB increased the expression of the pro-inflammatory cytokines IL-6 and IL-1beta and decreased the expression of anti-inflammatory cytokine TGF-beta. Expression of IL-6 was normalized at 1th day of the recovery period, while IL-1beta remained up-regulated during long-term recovery. Expression of TGF-beta was increased above normal value at 5th day of the recovery period.

Infiltration of lung with macrophages ED-1 staining was performed in order to evaluate the infiltration of lung tissue

with macrophages (fig. 6). Throughout the whole recovery period number of ED-1 positive cells remained almost on the same level in all groups. Thus, the amount of macrophages seems to be unaffected by the experimental procedure or CPB during 5 days recovery.

Figure 3. Neutrophil elastase activity in plasma in Sham, CPB and Control groups within 5 days recovery period. # P=0.051, vs Sham 1h, Mann-Whitney Rank Sum Test. * P<0.05, vs Control, Anova one way, Dunnet C test

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Figure 4. Relative expression of pro-inflammatory markers (Il-1beta, IL-6, TNF alpha) in Sham, CPB and Control groups within 5 days recovery period. * P<0.001, vs Control, Kruskal-Wallis One Way Analysis of Variance on Ranks, Turkey test. ** P<0.05, vs Control One way Anova with Bonferroni test # p<0.05, vs Sham 5days One way Anova with Bonferroni test.

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90 Chapter 5

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Figure 5. Relative expression of anti-inflammatory marker TGF beta 1 in Sham, CPB and Control groups within 5 days recovery period. * P<0.05, vs Control, One Way ANOVA, Bonferroni test. ** P<0.05, vs Control, Kruskal-Wallis One Way Analysis of Variance on Ranks, Turkey test

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Figure 6. Infiltration of the lung with macrophages. ED-1 staining was performed in order to evaluate the infiltration of lung tissue with macrophages in Sham, CPB, and Control groups (resolution 400x). A: ED-1 staining in lung of Control group; B: negative control for anti-ED-1 antibody; C: Number of macrophages in the lung in all groups within the whole recovery period.

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Discussion The present study evaluates the time-dependent changes in markers of

pulmonary inflammation during a clinically relevant post-operative period (from 60 min up to 5 days of recovery). Several parameters were evaluated, including markers of hypoxia and oxidative stress (HO-1, SOD1, p22phox), markers of the endothelial activation (E-selectin, ICAM-1, and VCAM-1), pro- and anti-inflammatory markers (IL1 beta, IL-6, TNF alpha, and TGF beta), and activity of neutrophil elastase. Results show that CPB promoted endothelial activation and activation of neutrophils during short-term recovery period (1hour) and caused profound upregulation of specific pro- and anti-inflammatory cytokines during short and long-term recovery period.

Hypoxia and oxidative stress after CPB. CPB is known to be associated with hypoxia and oxidative stress injury, which

may contribute to acute lung injury after cardiac surgery.13, 14 In our study, CPB evoked significant upregulation of the hypoxia injury marker HO-1 during the first hour of recovery and a trend towards upregulation of the small NAD(P)H oxidase subunit p22phox. Similar results were shown previously in pigs after 2 hours CPB.13, 14 It is thought that ischemia-reperfusion injury may activate alveolar residential macrophages initiating NADPH-mediated ROS-production, which in turn cause alveolar tissue damage, and release of the proinflammatory cytokines.14 Interestingly, expression of antioxidant enzyme SOD1, that neutralizes superoxide radicals in cells, was increased only after the second day of recovery and remained upregulated during the entire recovery period without normalization. Such maintained activity of SOD1 in the CPB groups suggests ongoing pathological processes in lung tissue, probably due to increased inflammation, since acute CPB-mediated ischemia-reperfusion injury occurs immediately after the operation.15, 16

Endothelial and neutrophils activation. Our data show that CPB caused upregulation of E-selectin, which is a main

marker of the endothelium activation in response to inflammatory cytokine (TNF-! and IL-1$).17 Also CPB upregulated the cell adhesion molecules of the immunoglobulin superfamily, ICAM-1 and VCAM-1, that serve as ligands for leukocyte integrins.18-21 Further, CPB increased the plasma concentration of neutrophil elastase, which is a serine proteinase from the azurophilic granules of neutrophils, which is associated with acute lung injury.21 This enzyme is involved in several biological processes, such as degradation of extra-cellular matrix components and the modulation of the activity of some pro-inflammatory agents (TNF alpha, IL6, IL8) and anti-inflammatory mediators (TGF beta, TGF alpha, EGF).22

CPB is known to activate neutrophils and endothelial cells during acute phase reaction.23 The specific character of the lung functionality during CPB is represent extraordinary aspect since lungs are more or less excluded from the circulation in this period.3, 4 Clinical data suggest that approximately 25% of the patients following open heart surgery exhibited signs of the pulmonary impairment during at least one week after,3 while experimental studies in humans on endothelial activation marker

92 Chapter 5

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expression covered mainly short term recovery period. Thus, elevated levels of circulating ICAM-1, VCAM-1, and E-selectin were shown during short term24 and 24h post-operative period.25 Our data showed that CPB caused endothelial activation during 1hour post-operative period with complete normalization till 1st day of recovery period. Similar pattern was found in our study for increased plasma level of neutrophil elastase as a marker of the neutrophils activation.

Pro and anti--inflammatory cytokines. The present study shows that CPB causes up-regulation of the pro-

inflammatory cytokines IL1beta and IL6 in lung tissue already after a short-term recovery period. Interestingly, CPB evoked short-lasting expression of IL6, which was completely normalized at 1day of recovery, while IL-1beta remained up-regulated during long-term recovery period, suggesting ongoing inflammation of the lung. CPB did not cause pronounced changes in TNF alpha expression. It is known that in lung TNF-alpha and IL-1beta are produced by resident or infiltrated macrophages in response to infective and non-infective agents and initiates the activation of neutrophils and cell adhesion molecules.26 IL-6 is a cytokine with various physiological function and several cells are responsible for its production, such as monocytes/macrophages, endotheliocytes, smooth muscle cells, fibroblasts.19, 26 Rapid up-regulation of IL-6 and IL-1beta, which was found in our study, might be as a result of its release from alveolar macrophages, which are abundantly distributed in lung tissue. The latter is supported by unchanged amounts of ED-1 positive cells (macrophages) in lung tissue during the whole recovery period, as found in this study.

Previously, short-time upregulation of these cytokines was shown after cardiac surgery and CPB 23, 27, 28 in different animal29 model of CPB and in human studies. We also evaluated long-term postoperative period. Interestingly, CPB evoked short-lasting expression of IL6, which was completely normalized up to 1day of recovery. IL-1beta remained up-regulated during long-term recovery, suggesting ongoing pulmonary inflammation.12, 30

TGF beta 1, which was evaluated as an anti-inflammatory factor, was found upregulated in SIRS.12, 31 TGF beta 1 has been shown to have a wide range of the immunosuppressive and immunomodulatory activity, inhibit superoxide anions production, and decrease the release of TNF-alpha.12, 31-33 In our study, CPB evoked an initial downregulation of TGF-beta during 1st day of the recovery period and a subsequent upregulation at the 5th day of recovery.

In summary, we suggest the following scheme of the CPB-mediated changes in lung: heart-lung machine-related triggers (i.e. ischemia-reperfusion injury, hypoxia, contact of blood with artificial surfaces or others) evoke the production of IL-6 and IL-1beta by residential macrophages of lung, which in turn activate neutrophils and endothelium. Activated neutrophils release neutrophil elastase, which participates in the degradation of extra-cellular matrix components and evoke acute lung tissue injury. At the same time, CPB increases expression of TGFbeta1, which has several immunosuppressive effects with a negative influence on T-cell response and may contribute to the CPB-evoked immunological disturbances. The prolonged upregulation


!

of IL-1beta, TGFbeta1 and SOD-1 after 5 days of recovery suggests ongoing pulmonary inflammation that may contribute to post-CPB lung complications.

Reference list 1. Salis S, Mazzanti VV, Merli G, Salvi L, Tedesco CC, Veglia F, Sisillo E: Cardiopulmonary bypass

duration is an independent predictor of morbidity and mortality after cardiac surgery. J Cardiothorac Vasc Anesth 2008; 22: 814-22

2. Yoshizumi K, Ishino K, Ugaki S, Ebishima H, Kotani Y, Kasahara S, Sano S: Effect of a miniaturized cardiopulmonary bypass system on the inflammatory response and cardiac function in neonatal piglets. Artif Organs 2009; 33: 941-6

3. Apostolakis EE, Koletsis EN, Baikoussis NG, Siminelakis SN, Papadopoulos GS: Strategies to prevent intraoperative lung injury during cardiopulmonary bypass. J Cardiothorac Surg 2010; 5: 1

4. Apostolakis E, Filos KS, Koletsis E, Dougenis D: Lung dysfunction following cardiopulmonary bypass. J Card Surg 2010; 25: 47-55

5. Sadaba JR, Greco E, Alvarez LA, Pulitani I, Juaristi A, Goiti JJ: The surgical option in the management of acute pulmonary embolism. J Card Surg 2008; 23: 729-32

6. Groeneveld AB, Jansen EK, Verheij J: Mechanisms of pulmonary dysfunction after on-pump and off-pump cardiac surgery: a prospective cohort study. J Cardiothorac Surg 2007; 2: 11

7. Goebel U, Siepe M, Mecklenburg A, Doenst T, Beyersdorf F, Loop T, Schlensak C: Reduced pulmonary inflammatory response during cardiopulmonary bypass: effects of combined pulmonary perfusion and carbon monoxide inhalation. Eur J Cardiothorac Surg 2008; 34: 1165-72

8. Risnes I, Wagner K, Ueland T, Mollnes T, Aukrust P, Svennevig J: Interleukin-6 may predict survival in extracorporeal membrane oxygenation treatment. Perfusion 2008; 23: 173-8

9. Yasser MA, Elmistekawy E, El-Serogy H: Effects of dexamethasone on pulmonary and renal functions in patients undergoing CABG with cardiopulmonary bypass. Semin Cardiothorac Vasc Anesth 2009; 13: 231-7

10. Zheng JH, Gao BT, Jiang ZM, Yu XQ, Xu ZW: Evaluation of Early Macrophage Activation and NF-kappaB Activity in Pulmonary Injury Caused by Deep Hypothermia Circulatory Arrest: An Experimental Study. Pediatr Cardiol 2009

11. Hagio T, Kishikawa K, Kawabata K, Tasaka S, Hashimoto S, Hasegawa N, Ishizaka A: Inhibition of neutrophil elastase reduces lung injury and bacterial count in hamsters. Pulm Pharmacol Ther 2008; 21: 884-91

12. Torre D, Tambini R, Aristodemo S, Gavazzeni G, Goglio A, Cantamessa C, Pugliese A, Biondi G: Anti-inflammatory response of IL-4, IL-10 and TGF-beta in patients with systemic inflammatory response syndrome. Mediators Inflamm 2000; 9: 193-5

13. Dodd-o JM, Welsh LE, Salazar JD, Walinsky PL, Peck EA, Shake JG, Caparrelli DJ, Ziegelstein RC, Zweier JL, Baumgartner WA, Pearse DB: Effect of NADPH oxidase inhibition on cardiopulmonary bypass-induced lung injury. Am J Physiol Heart Circ Physiol 2004; 287: H927-36

14. Shimoyama T, Tabuchi N, Kojima K, Akamatsu H, Arai H, Tanaka H, Sunamori M: Aprotinin attenuated ischemia-reperfusion injury in an isolated rat lung model after 18-hours preservation. Eur J Cardiothorac Surg 2005; 28: 581-7

15. Mumby S, Block R, Petros AJ, Gutteridge JM: Hydrogen peroxide and catalase are inversely related in adult patients undergoing cardiopulmonary bypass: implications for antioxidant protection. Redox Rep 1999; 4: 49-52

16. Davies SW, Duffy JP, Wickens DG, Underwood SM, Hill A, Alladine MF, Feneck RO, Dormandy TL, Walesby RK: Time-course of free radical activity during coronary artery operations with cardiopulmonary bypass. J Thorac Cardiovasc Surg 1993; 105: 979-87

17. Vallely MP, Bannon PG, Bayfield MS, Hughes CF, Kritharides L: Endothelial activation after coronary artery bypass surgery: comparison between on-pump and off-pump techniques. Heart Lung Circ 2010; 19: 445-52


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19. Asimakopoulos G, Taylor KM: Effects of cardiopulmonary bypass on leukocyte and endothelial adhesion molecules. Ann Thorac Surg 1998; 66: 2135-44

20. Elangbam CS, Qualls CW,Jr, Dahlgren RR: Cell adhesion molecules--update. Vet Pathol 1997; 34: 61-73

21. Moraes TJ, Zurawska JH, Downey GP: Neutrophil granule contents in the pathogenesis of lung injury. Curr Opin Hematol 2006; 13: 21-7

22. Lungarella G, Cavarra E, Lucattelli M, Martorana PA: The dual role of neutrophil elastase in lung destruction and repair. Int J Biochem Cell Biol 2008; 40: 1287-96

23. Paparella D, Yau TM, Young E: Cardiopulmonary bypass induced inflammation: pathophysiology and treatment. An update. Eur J Cardiothorac Surg 2002; 21: 232-44

24. Kalawski R, Bugajski P, Smielecki J, Wysocki H, Olszewski R, More R, Sheridan DJ, Siminiak T: Soluble adhesion molecules in reperfusion during coronary bypass grafting. Eur J Cardiothorac Surg 1998; 14: 290-5

25. Vallely MP, Bannon PG, Bayfield MS, Hughes CF, Kritharides L: Endothelial activation after coronary artery bypass surgery: comparison between on-pump and off-pump techniques. Heart Lung Circ 2010; 19: 445-52

26. Bhatia M, Moochhala S: Role of inflammatory mediators in the pathophysiology of acute respiratory distress syndrome. J Pathol 2004; 202: 145-56

27. Ng CS, Wan S, Yim AP, Arifi AA: Pulmonary dysfunction after cardiac surgery. Chest 2002; 121: 1269-77

28. Gilliland HE, Armstrong MA, McMurray TJ: Tumour necrosis factor as predictor for pulmonary dysfunction after cardiac surgery. Lancet 1998; 352: 1281-2

29. Bernard F, Romano A, Granel B: Regulatory T cells and systemic autoimmune diseases: systemic lupus erythematosus, rheumatoid arthritis, primary Sjogren's syndrome. Rev Med Interne 2010; 31: 116-27

30. Weber A, Wasiliew P, Kracht M: Interleukin-1beta (IL-1beta) processing pathway. Sci Signal 2010; 3: cm2

31. Sablotzki A, Welters I, Lehmann N, Menges T, Gorlach G, Dehne M, Hempelmann G: Plasma levels of immunoinhibitory cytokines interleukin-10 and transforming growth factor-beta in patients undergoing coronary artery bypass grafting. Eur J Cardiothorac Surg 1997; 11: 763-8


33. Sanjabi S, Zenewicz LA, Kamanaka M, Flavell RA: Anti-inflammatory and pro-inflammatory roles of TGF-beta, IL-10, and IL-22 in immunity and autoimmunity. Curr Opin Pharmacol 2009; 9: 447-5

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Chapter 6

Adjunct nitrous oxide normalizes vascular reactivity changes after hemorrhagic shock in mice

under isoflurane anesthesia1

Iryna V. Samarska, Matijs van Meurs, Hendrik Buikema, Martin C. Houwertjes, Francis M. Wulfert, Grietje Molema,


Anesthesiology 2009; 111:600 –8

1-This article is accompanied by an editorial in Anesthesiology: Does correcting the numbers improve long-term outcome? Robert D. Sanders, Mervyn Maze See supplement of this thesis

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Abstract Background: Hemorrhagic shock is associated with changes in vascular responsiveness that may lead to organ dysfunction and ultimately multiple organ dysfunction syndrome. Volatile anesthetics interfere with vasoresponsiveness, which may contribute to organ hypoperfusion. In this study, we examined the influence of adjunct nitrous oxide on the vascular responsiveness after short-term hemorrhagic shock under isoflurane anesthesia. Methods: Spontaneously breathing mice (n=31, 27.6 ± 0.31g) were anesthetized with isoflurane (1.4%) or with isoflurane (1.4%) and adjunct nitrous oxide (66%). Both groups were divided in Sham, Shock and Resuscitated groups. Vascular reactivity to phenylephrine and acetylcholine and expression of cyclooxygenases were studied in aorta. Results: In isoflurane-anesthetized groups, the contractile response to phenylephrine was increased in Shock as compared with the Sham and Resuscitated groups (Emax=3.2±0.4, 1.2±0.4 and 2.5±0.5 mN, respectively). Adjunct nitrous oxide increased phenylephrine contraction to a similar level in all three groups. In Sham-isoflurane group, acetylcholine caused a biphasic response: an initial relaxation followed by a contractile response sensitive to cyclooxygenases inhibition by indomethacine. The contractile response was abrogated in isoflurane-anesthetized groups that underwent shock. In all groups, adjunct nitrous oxide preserved the contractile phase. Shock induced a downregulation of cyclooxygenases-1, which was normalized by adjunct nitrous oxide. Conclusion: Adjunct nitrous oxide attenuates shock-induced changes in vascular reactivity and cyclooxygenases expression of mice under isoflurane anesthesia. This implies that vascular reactive properties during anesthesia in hemorrhagic shock conditions may be influenced by the choice of anesthetics. Introduction

Changed vascular reactivity in hemorrhagic shock (HS) may represent an important factor in the development of a multiple organ dysfunction syndrome. The mechanisms underlying this syndrome are not fully understood. However, early changes in vascular responsiveness may be of significance, as arterial hypotension and vascular leakage have been recognized as hallmarks of the post-shock state.1-3 Indeed, hypotension has been associated with altered vascular reactivity to different modulators of vascular tone.1 Several mechanisms have been proposed to cause post-shock vascular hyporeactivity including systemic and local release of nitric oxide, decreased plasma levels of vasopressin and changes in properties of vascular smooth muscle cells.2-5

Patients with hemorrhagic shock frequently receive anesthesia to facilitate diagnostic and therapeutic procedures. Many of the agents employed interfere with vasoresponsiveness and/or influence the therapeutic effectiveness of vasoactive drugs, and the development and outcome of hemorrhagic shock. Volatile anesthetics may evoke peripheral vasodilatation, myocardial depression, and lower sympathetic nervous system activity, subsequently leading to a decrease in blood pressure.6, 7

Anesthesia and vascular reactivity 97

!

Moreover, these agents may influence systemic secretion of vasoconstrictors such as vasopressin, angiotensin, and endothelin, further contributing to a reduction in blood pressure and deterioration of organ microcirculation.8, 9

Nitrous oxide, still widely used as an adjunct in various anesthetic techniques, has also been implied to influence hemodynamics. Adjunct nitrous oxide to sevoflurane (at 1.5 Minimum Alveolar Concentration [MAC]) or isoflurane (at 1.45 MAC) stabilizes several hemodynamic parameters of regional and systemic flow.10-12 The mechanisms through which nitrous oxide offsets the hemodynamic effects of volatile anesthetics are still not fully understood,13 although venoconstictor effects of nitrous oxide and sympathetic stimulation have been proposed as a possible explanation.12

The purpose of this study was to determine the change in vascular responsiveness obtained from mice after short-term hemorrhagic shock under isoflurane anesthesia, and to investigate the effects of adjunct nitrous oxide administration on the observed changes.

Materials and Methods

The experimental protocol was approved by the Institutional Animal Care and Use Committee of the University of Groningen (Groningen, the Netherlands). This study was performed in 31 adult mice (27.6±0.31g; Harlan, Zeist, The Netherlands) housed under standard conditions with free access to food (standard rat chow) and drinking water throughout the study.

Experimental animals were assigned to two groups: A) a group of mice that were anesthetized with isoflurane (ISO,1.4%) in oxygen/air mixture (O2:33%), and B) a group of mice that were anesthetized with isoflurane (1.4%) and adjunct nitrous oxide (N2O, 66%) in oxygen (33%). Each group was divided in three experimental sub-groups (n = 5-6 per group). Sham animals were anesthetized and cannulated but not hemorrhaged, and kept under anesthesia for 90 min before being killed. Shock animals underwent anesthesia and hemorrhage and were sacrificed after 90 min of hemorrhagic shock. Resuscitated animals underwent anesthesia with hemorrhagic shock for 90 minutes, followed by resuscitation with 6% hydroxyethyl starch 130/0.4 (Voluven; Fresenius-Kabi, Bad Homburg, Germany). The resuscitation volume was twice the estimated volume of the blood withdrawn to induce hemorrhagic shock. Resuscitated mice were sacrificed 24 hours after induction of shock. All animals were breathing spontaneously throughout the protocol. A fixed-blood pressure model of hemorrhagic shock was used as described previously.14 Briefly, after induction of anesthesia the left femoral artery was cannulated with polyethylene tubing (internal diameter of 0.28 mm and an external diameter of 0.61 mm). Hemorrhagic shock was induced by blood withdrawal until mean arterial pressure (MAP) reached 30 mmHg; this blood pressure reduction was reached in approximately 11 minutes. An initial 0.5 ml of blood was withdrawn followed by additional portions of about 0.1 ml mean arterial pressure reached 30 mmHg. Total amount of blood withdrawn was estimated from the number of portions taken from the animal. Blood was collected in a heparinized 1-mL syringe to prevent clotting. Hypotension at 30 mmHg was maintained during 90 min with a continuous monitoring

98 Chapter 6

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of MAP. Small increments of blood were infused or withdrawn during the shock period to maintain MAP at 30 mmHg. In both Shock-groups, blood gas analysis was measured twice: just after femoral cannulation (t=0 min) and after the shock period immediately before killing (t=90 min). In both Resuscitated groups, blood gas analysis was performed just after femoral cannulation (t=0 min) and immediately before killing (t=24 hours). In Sham-groups, blood gas analysis was performed only immediately before sacrification (90 min) to avoid potential activation of endothelium and modification of vascular reactivity by withdrawal of blood at t=0. After killing, freshly isolated thoracic aorta was collected for in vitro vasomotor studies. Abdominal aorta was removed and snap-frozen in liquid nitrogen without pretreatment with any drug, and stored at -80oC until analysis.

Vasomotor responses After removal, the descending thoracic aorta was immediately placed into cold

physiological saline. Freshly isolated thoracic aortic rings (1.5 - 2 mm in length) were mounted on two 200µm stainless wires in the individual myograph baths wire myograph (Danish Myo Technology A/S, Aarhus, Denmark), containing 6ml of Kreb’s solution, warmed to 37°C and bubbled continuously with 95%O2/5%CO2 to maintain pH at 7.4. Length of aortic strips was assessed by microscopy. Aortic rings were equilibrated for 40 min until they were at a steady baseline. Then, rings were subjected twice to stimulation with potassium chloride (60 mM) in order to obtain reproducible contractile responses. Contraction was measured by obtaining concentration-response curves to phenylephrine (10 nM-100 "M). Endothelium-dependent relaxation was measured by obtaining concentration-response curves to acetylcholine (10 nM-300 "M) in rings precontracted with phenylephrine. The influence of vasoactive prostanoids on the response to acetylcholine was examined by incubating rings with the non-specific cyclooxygenase inhibitor indomethacine (10 "M) administered 20 min before application of phenylephrine. In the end of each experimental protocol, endothelium-independent relaxation was measured by applying the nitric oxide-donor sodium nitroprusside (0.1 mM).

Western blotting The methods used were described previously.15, 16 Briefly, after grinding, the

frozen aortas were placed in 300 "l of boiling 2% SDS followed by pounding by a polytron (Kinematica AG Littau, Switzerland). Then, samples were centrifuged (4000 rpm, 1 min) and boiled (95oC) for 5 min. After a second centrifugation (13000 rpm, 3 min), supernatant was collected and used for measurements. Protein concentration was determined by Bio-Rad Protein Assay (Bio-Rad, Hercules, CA). Forty µg of total protein from each sample was separated on 4-20% Precise Protein Gels (Pierce, Rockford, IL) and transferred to nitrocellulose membranes. Anti-cyclooxygenase 1 antibody (ALEXIS Biochemicals, Lausen, Switzerland), anti-cyclooxygenase-2 antibody (BD Bioscience Pharmingen, San Diego, CA) and anti-$-actin (Sigma, St. Louis, MO) were used as a primary antibodies. Horseradish peroxidase-linked rabbit anti-mouse antibody was applied as a secondary antibody. Macrophage+IFNg/LPS (BD


!

Bioscience Pharmingen) was used as a positive control for cyclooxygenase-2. The blots were analyzed using Super Signal assay (Pierce, Rockford, IL). $-actin served as a housekeeping protein. Cyclooxygenase-1 and cyclooxygenase-2 levels are expressed as ratios to $-actin protein levels.

Drugs Krebs solution was prepared freshly and of the following composition (mM):

120.4 NaCl, 5.9 KCl, 2.5 CaCl2, 1.2 MgSO4, 25.0 NaHCO3, 1.2 NaH2PO4, 11.5 glucose; these chemicals were obtained from Merck (Darmstadt, Germany). The following drugs were used: Acetylcholine chloride, phenylephrine, indomethacine, sodium nitroprusside, NG-Monomethyl-L-arginine acetate salt. Stock solution (10 mM) for indomethacine, was prepared in 96% ethanol. All other drugs were dissolved in deionized water. NG-Monomethyl-L-arginine acetate salt was purchased form MP Biomedicals (Illkirch, France). All other compounds were purchased from Sigma. The concentrations presented in the concentration-curve responses are expressed as a final molar concentration in the organ baths.

Statistical analysis Data are present as mean ± SD and n refers to the number of animals in

corresponding group. The contractile response to phenylephrine was expressed in mN and relaxant responses to acetylcholine and sodium nitroprusside are expressed as a percentage of the preconstriction obtained with phenylephrine (0.1 mM). Concentration- response curves to phenylephrine and acetylcholine were characterized by Area Under the Curve, the maximal response (Emax), and the negative logarithm of the molar concentration that caused half-maximal response (EC50). Concentration-response curves of individual rings were plotted with SigmaPlot version 10.0 (Systat Software Inc, San Jose, CA). EC50 was calculated by Four Parameter Logistic Curve (SigmaPlot) for each ring. Statistical analysis was done with SPSS 16.0.2 for Windows (SPSS Inc., Chicago, IL). Differences between concentration-response curves were evaluated by Two-way Repeated-Measures ANOVA followed by Bonferroni test. Comparison of single parameters among multiple groups was done by One-Way ANOVA followed by Bonferroni test. Levene's test was used to confirm the homogeneity of variance. Independent sample t-test was used to compare the means of two groups where applicable. All tests were two-tailed and differences were considered significant at P<0.05. Results

Hemodynamic changes and routine lab parameters To monitor the animals during the procedure, hemodynamic measurements

and blood gas analyses were performed. After instrumentation at the start of the experimental protocol, hemodynamic parameters were comparable between the 3 experimental subgroups for a given type of anaesthesia. In mice undergoing blood withdrawal, shock was confirmed by a significant drop in blood pressure; MAP varied between 26 and 36 mmHg during the 90 min shock period followed by a near

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normalization to baseline values in resuscitated animals (fig. 1). The main difference between both types of anesthesia was that in Sham isoflurane animals MAP was slightly lower (~ 10 mmHg throughout the protocol) compared to Sham isoflurane/nitrous oxide animals receiving adjunct nitrous oxide (P<0.05). Blood gases, pCO2, Base excess, HCO3

-, SaO2, Hb, and Hct did not significantly differ between both Sham groups at all time points measured (table 1). The pH was increased in Shock isoflurane group compared to Sham isoflurane. Adjunct nitrous oxide resulted in a similar pH in Sham isoflurane/nitrous oxide and Shock isoflurane/nitrous oxide groups (table 1). During shock, pO2 was slightly increased in animals anesthetised with isoflurane only as compared to group with adjunct nitrous oxide (table 1). Other blood gas parameters were similar in both Shock groups.

Figure 1. Mean arterial pressure (MAP, femoral artery) of Sham, Shock and Resuscitated groups during the experimental procedure in animals anesthetized with isoflurane (ISO, panel A), or anesthesia with isoflurane and adjunct nitrous oxide (ISO + N2O, panel B). * denotes significantly different curves between Sham ISO and Sham ISO+N2O (P<0.05). ** P<0.05 Sham vs corresponding Shock and Resuscitated groups

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!

Tabl

e 1.

Art

eria

l blo

od g

as d

ata

at th

e di

ffer

ent t

ime

poin

ts o

f the

exp

erim

enta

l pro

toco

l

Par

amet

ers

Isof

lura

ne

Isof

lura

ne+N

itrou

s ox

ide

SH

AM

gro

up

Sho

ck g

roup

R

esus

cita

ted

grou

p S

HA

M g

roup

S

hock

gro

up

Res

usci

tate

d gr

oup

Num

ber o

f ani

mal

s 5

6 5

5 5

5 w

eigh

t, g

26.9

±2.5

27

.7±3

.3

26.2

±1.7

26

.6±3

.0

28.9

±0.9

29

.0±3

.2

pH

0 m

in

7.

32±0

.03

7.29

±0.0

4

7.37

±0.0

8 7.

34±0

.05

90 m

in

7.34

±0.0

1$ 7.

42±0

.03*

7.35

±0.0

5 7.

33±0

.01

24

h

7.40

±0.0

6

7.

37±0

.07

pCO

2, kP

a

0

min

4.5±

0.6

4.9±

0.4

3.

9±0.

4 4.

6±0.

5 90

min

3.

5±0.

8 2.

9±0.

7

3.4±

0.1

3.4±

0.9

24

h

2.8±

0.4

3.5±

0.1

pO2,

kPa

0

min

30.3

±1.4

*

31.9

±0.9

#

25.3

±1.7

25

.2±1

.9

90 m

in

33.1

±0.6

27

.8±1

.7

22

.7±9

.2

30.3

±3.3

24 h

27

.3±5

.0

39.2

±13.

7 B

E, m

mol

/l

0 m

in

-8

.3±1

.2

-8.3

±1.6

-7.3

±3.8

-6

.6±2

.2

90 m

in

-10.

5±2.

4 -9

.4±2

.2

-1

0.3±

2.5

-11.

5±4.

2

24 h

-1

1.9±

2.4

-9. 5

±3.0

H

CO

- 3, m

mol

/l

0 m

in

16

.6±1

.4

17.3

±0.1

16.4

±2.7

18

.0±1

.5

90 m

in

13.6

±2.7

13

.8±2

.6

13

.6±1

.6

13.0

±3.9

24 h

12

.1±1

.7

14.6

±2.1

S

aO2 ,

%

0 m

in

10

0.0±

0 10

0.0±

0

99.9

±0.2

10

0.0±

0 90

min

10

0.0±

0 10

0.0±

0

99.7

±0.5

10

0.0±

0

24 h

99

.9±0

.1

100.

0±0

Hb,

mm

ol/l

0 m

in

7.

9±0.

4 7.

9±0.

4

8.4±

0.3

8.6±

0.2

90 m

in

6.9±

0.9

4.6±

0.6

7.

3±0.

6 5.

6±1.

3

24 h

3.

1±0.

3

3.

6±0.

4 H

ct

0 m

in

39

.4±2

.1

39.5

±1.8

41.3

±1.5

42

.4±1

.1

90 m

in

34.7

±4.5

23

.0±2

.6

35

.9±2

.5

28.1

±6.4

24 h

15

.9±1

.3

18.4

±2.1

D

ata

on t

he v

alue

of

pH,

pCO

2, pO

2, B

E,

HC

O-3

, S

aO2,

Hb,

Hct

in g

roup

s of

mic

e an

esth

etiz

ed w

ith I

soflu

rane

sol

e an

d Is

oflu

rane

with

adj

unct

Nitr

ous

oxid

e. p

CO

3 ,

the

parth

ial a

rteria

l pre

ssur

e of

car

bon

diox

ide;

pO

2, th

e pa

rtial

arte

rial p

ress

ure

of o

xyge

n;

HC

O-3

, bi

carb

onat

e; B

E, B

ase

exce

ss; H

b, H

emog

lobi

n; H

ct, h

emat

ocrit

; SaO

2, th

e ox

ygen

sat

urat

ion.

*- P

<0.0

5 S

hock

Iso

flura

ne v

s S

hock

Is

oflu

rane

+Nitr

ous

oxid

e; $ -

P<0

.05

Sha

m I

soflu

rane

vs

Sho

ck I

soflu

rane

; #-

P<0

.05

Res

usci

tate

d Is

oflu

rane

vs

Res

usci

tate

d Is

oflu

rane

+Nitr

ous

oxid

e

102 Chapter 6

!

Tabl

e 2.

The

wid

th o

f aor

ta ri

ngs

in a

ll gr

oups

G

roup

s Is

oflu

rane

Is

oflu

rane

/Nitr

ous

oxid

e S

ham

1.

8 ±

0.3

1.7

± 0.

1

Sho

ck

1.7

± 0.

2 1.

8 ±

0.2

Res

usci

tate

d

1.7

± 0.

1 1.

7 ±

0.1

All

data

are

pre

sent

ed a

s m

ean

± S

D in

mm

.

Tabl

e 3.

Para

met

ers

of P

heny

leph

rine-

med

iate

d C

ontr

actio

n du

ring

Diff

eren

t Pha

ses

of E

xper

imen

tal S

hock

afte

r A

naes

thes

ia w

ith e

ither

Isof

lura

ne o

r Is

oflu

rane

with

Adj

unct

N

itrou

s O

xide

G

roup

s Is

oflu

rane

Is

oflu

rane

/Nitr

ous

oxid

e -lo

gEC

50

AU

C

Emax

, mN

-lo

gEC

50

AU

C

Emax

, mN

S

ham

-6

.9 ±

0.2

2.

1 ±

1.6

1.5

± 0.

41

-7.0

± 0

.1

4.2

± 1.

0*

2.19

± 0

.31

Sho

ck

-7.0

± 0

.3

7.

8 ±

2.8!

3

.24

± 0.

43!

-6

.9 ±

0.2

6.

2 ±

1.8

3.17

± 0

.44

Res

usci

tate

d -7

.0 ±

0.4

4.

9 ±

1.8

2.28

± 0

.54

-6.8

± 0

.3

3.8

± 1.

2 2.

1 ±

0.41

A

ll da

ta a

re p

rese

nted

as

mea

n ±

SD

; AU

C=a

rea

unde

r the

cur

ve; E

max

=max

imal

con

tract

ile re

spon

se; I

SO

=iso

flura

ne; N

2O=n

itrou

s ox

ide.

* P

=0.0

48, S

ham

-ISO

vs

Sha

m-IS

O/N

2O, t

wo-

taile

d t-t

est; !

P<0

.05,

Sha

m-IS

O v

s S

hock

-ISO

, one

-way

AN

OV

A fo

llow

ed b

y B

onfe

rron

i tes

t.


!

Contractile reactivity Experiments in vitro were performed on freshly isolated aorta rings of similar

length (P=0.1) in all groups (table 2).Contractile responses of isolated aortic rings were obtained by constructing concentration-response curves to phenylephrine. The contractile response to phenylephrine in the Sham isoflurane/nitrous oxide group was enhanced (P < 0.05) as compared with the Sham isoflurane group (fig. 2). This enhancement was characterized by an increased Emax (P < 0.05) and an unchanged EC50 (P=0.2, table 3). The contractile response to phenylephrine in the Shock isoflurane and Resuscitated isoflurane groups was also enhanced as compared with the Sham isoflurane group (P < 0.05; fig. 2A). This enhancement was also characterized by an increased Emax (P < 0.05) and an unchanged EC50 (P=0.7, table 3). However, the contractile response to phenylephrine was identical between the Shock isoflurane and Shock isoflurane/nitrous oxide groups and between Resuscitated isoflurane and Resuscitated isoflurane/nitrous oxide groups (fig. 2); i.e. no significant differences were observed in the Emax and EC50 values between the corresponding two groups (table 3). These findings suggest that both shock and nitrous oxide increase contractility in a nonadditive manner.

Relaxation responses to acetylcholine Administration of acetylcholine to aortic rings from mice subjected to sham

conditions caused a biphasic response, characterized by a relaxation, which was

Figure 2. Contractile reactivity of aortic rings to phenylephrine of mice anesthetized with isoflurane (panel A) and isoflurane with adjunct nitrous oxide (panel B) in Sham, Shock and Resuscitated groups. Inserts show the responsiveness to phenylephrine presented as maximal response (Emax) in mN. ISO=isoflurane; N2O= Nitrous oxide. #P<0.05 Sham vs Shock Isoflurane. *P<0.05 Sham vs Shock Isoflurane

104 Chapter 6

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maximal at 0.3 "M, followed by a contractile response at concentrations ' 0.3 "M (Sham groups in fig. 3, A and B). In Shock isoflurane and Resuscitated isoflurane mice, the contractile phase and hence the biphasic pattern in the concentration-response curve was fully suppressed (fig. 3A). In contrast, adjunct nitrous oxide preserved the biphasic pattern also after conditions of shock and resuscitation (fig. 3B). The latter is also reflected by the profound increase in acetylcholine concentration at which maximal relaxation was seen in Shock and Resuscitation groups that received anesthesia with isoflurane only (at 10 "M), as compared to groups with adjunct nitrous oxide (at 1 "M).

The biphasic pattern in the acetylcholine concentration-response curve was also abolished in the presence of indomethacine, an inhibitor of cyclooxygenase (fig. 3, C and D). Consequently, under these conditions concentration-response curves for acetylcholine did not differ between groups. These findings imply the involvement of contractile prostaglandins in the upward stroke at higher concentrations of acetylcholine. Collectively, these findings indicate that the biphasic pattern of the

Figure 3. Acetylcholine induced relaxation of aortic rings from Sham, Shock and Resuscitated groups anesthetized with isoflurane (A and C) or isoflurane with adjunct nitrous oxide (B and D), and in the absence of indomethacine (A and B) or presence of indomethacine (C and D). Concentration-response curves of Sham groups (closed circle), Shock groups (open circles) and Resuscitated groups (triangular down) are expressed as percentage of preconstriction value to phenylephrine (0.1mM) and are present as mean±SD. INDO=indomethacine; ISO= isoflurane; N2O= nitrous oxide.


!

response to acetylcholine after conditions of shock and resuscitation was maintained by adjunct nitrous oxide.

While groups differed in anesthetic regimes and the levels of phenylephrine-precontraction, the relaxant response to sodium nitroprusside was similar in all groups (table 4), suggesting that acetylcholine responses represent changes in endothelial cell function rather than altered vascular smooth muscle response to nitric oxide.

Vascular cyclooxygenase-1 and cyclooxygenase-2 expression Since short-term hemorrhagic shock differentially affected relaxation

responses to acetylcholine with both modes of anesthesia, we subsequently investigated vascular protein expression of cyclooxygenase-1 and cyclooxygenase-2 in segments collected from the unstimulated abdominal aorta. In mice anaesthetized with isoflurane only, vascular cyclooxygenase-1 expression decreased significantly after shock, followed by a recovery during resuscitation (fig. 4, A,B and C). In contrast, cyclooxygenase-1 expression was preserved in groups receiving adjunct nitrous oxide. A similar pattern was observed for vascular cyclooxygenase-2 expression, but changes did not reach statistical significance (fig. 4, A and B). Collectively, these findings suggest that adjunct nitrous oxide mitigates the shock-induced decrease in the expression of cyclooxygenases, particularly cyclooxygenase-1.

Discussion

We investigated the effects of nitrous oxide adjunct to anesthesia with isoflurane on vascular reactivity in an experimental model of shock in mice. Our data show that hemorrhagic shock in animals that received isoflurane during the procedure induced profound changes in the subsequent vascular reactivity of isolated thoracic rings, consisting of an increased contraction to phenylephrine and a loss of cyclooxygenase mediated contraction to acetylcholine. These functional changes were accompanied by a decrease in the vascular expression of cyclooxygenase-1. Secondly, adjunct nitrous oxide augmented phenylephrine-induced contractility of Sham mice, but no further increase was observed when these animals underwent shock. In addition, adjunct nitrous oxide protected from the loss of contractile response to acetylcholine during shock and preserved the vascular expression of cyclooxygenase-1. Collectively, these findings suggest that vascular reactivity during different phases of shock may be preserved when adjunct nitrous oxide is employed,

Table 4. Maximal relaxant response to sodium nitroprusside in all groups Maximal relaxation to SNP (%)

Isoflurane Isoflurane/Nitrous oxide Sham Shock Resuscitated Sham Shock Resuscitated

101.2±21.7 99.4±8.1 101.5±1.8 101.2±1.3 103.4±7.1 98.0±11.3 Data are expressed as a percentage of phenylephrine-mediated precontraction and were calculated from concentration response curves at 0.1 mM of sodium nitroprusside. No differences were found between groups (one-way ANOVA). SNP, sodium nitroprusside.

106 Chapter 6

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which may involve (amongst others) a preservation of vascular cyclooxygenase-expression.

In this study, we investigated the effect of different anesthetic techniques applied during hemorrhagic shock in vivo on the vascular properties of aortic tissue that was harvested subsequently and examined in the absence of anesthetics. To our knowledge, the influence of anesthetics on vascular reactivity has not been explored in this way previously. Importantly, the changes observed in vascular reactivity in the shock group under isoflurane anesthesia imply that this procedure induces protracted changes in the vascular bed. Indeed, such view is in keeping with the observed downregulation of cyclooxygenase-1 in this group.

Systemic hypotension after isoflurane anesthesia has been reported previously,4, 12, 17 and its general depressant effects on hemodynamics are quite

Figure 4. Expressions level of COX enzymes in aortic tissue from Sham, Shock and Resuscitated groups in groups anesthetized with isoflurane (ISO), or anesthesia with isoflurane and adjunct nitrous oxide (ISO+N2O). Expression of COX-1 (A) and COX-2 (B) was assessed in Sham (n=5), Shock (n=5) and Resuscitated (n=5) groups. Data are normalized to the expression level of $-actin and presented in arbitrary units. Representative expressions pattern in Sham (n=5), Shock (n=5), Resuscitated (n=5) groups anesthetized with sole isoflurane with isoflurane and adjunct nitrous oxide (C). The difference between groups was borderline significant with One-Way ANOVA (P=0.06). However, independent t-test showed a significant reduction of COX-1 expression in Shock ISO versus Sham ISO (*P<0.05).


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notorious indeed.10 It may represent an important unwanted side effect of anesthesia, particularly during conditions of shock in which tissue perfusion already is at threat. Investigation of the underlying mechanisms was not within the scope of this study, but previous studies reported the involvement of decrements in processes such as myofilament calcium-sensitivity, intracellular calcium concentration, or voltage-gated calcium influx,18-24 all of which might have contributed to decreased contractility after anesthesia. In the present study, decreased arterial pressure was observed together with decreased constriction to phenylephrine in isolated aortic ring preparation obtained from Sham mice anaesthetized with isoflurane, as compared to those receiving adjunct nitrous oxide. Recently, Pypendop et al showed that addition of 70% nitrous oxide to isoflurane anesthesia results in improved arterial and central venous pressures.12 In addition, a study in patients under sevoflurane anesthesia suggests that adjunct nitrous oxide normalizes hemodynamic parameters of regional and systemic flow.11 These findings suggest that hemodynamics may be better preserved during adjunct anesthesia with nitrous oxide compared to monoanesthesia with isoflurane. It should also be noted, however, that shock increased constriction to phenylephrine. It thus appears that contractile mechanisms become mobilized to maintain perfusion pressure during shock and overrule the effect of anesthesia. In keeping with that, contractility to phenylephrine was not further increased after shock with adjunct nitrous oxide, indicating that the effect of nitrous oxide was not additive.

Vascular responsiveness was also investigated by studying endothelial-mediated responses. As reported previously,25 administration of acetylcholine caused relaxation at low concentrations and contraction at higher concentrations thus generating a biphasic concentration-response curve. Preincubation with indomethacine fully abolished the normal upward stroke in the concentration-response curve, thus indicating the involvement of contractile prostaglandins derived from cyclooxygenase herein. Interestingly, the upward stroke in the concentration-response curve was also lost in aorta preparations of shock-mice anesthetized with isoflurane, but not in those receiving adjunct nitrous oxide. Such findings suggest that shock may alter the production of contractile prostaglandins, which may be preserved during anesthesia with adjunct nitrous oxide. In mouse aorta, endothelial denudation abolishes the acetylcholine-mediated contractile responses,25 implicating that contractile cyclooxygenases metabolites are derived from endothelial cells. In turn, this would implicate that the decreased cyclooxygenase-1 expression as observed following shock is caused by downregulation of the enzyme in endothelial cells. Previous studies suggest both isoforms of cyclooxygenase to be expressed in aorta endothelial and/or vascular smooth muscle cells,26, 27 and that both cyclooxygenase-1 and cyclooxygenase-2 may be involved in production of contractile prostaglandins. At present, there are few data on the influence of isoflurane on cyclooxygenase-protein expression. It has been reported that isoflurane produces cyclooxygenase-2 mediated anesthetic preconditioning, but it did not affect cyclooxygenase-1 and cyclooxygenase-2 protein expression,28, 29 Taken together, our results indicate that the loss of acetylcholine-induced contractile function in Shock-mice anesthetized solely with isoflurane, is due to decreased endothelial production of contractile prostaglandins

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caused by downregulation of endothelial cyclooxygenase-1. Moreover, adjunct nitrous oxide counteracts these changes most likely due to a preservation of normal endothelial function.

Alternatively, nitrous oxide-evoked hyperhomocysteinemia may offer an explanation for enhanced vascular reactivity to phenylephrine in Sham animals, because of an increase in asymmetric dimethylarginine, an endogenous inhibitor of nitric oxide synthase.30 Asymmetric dimethylarginine has been reported to increase arteriolar basal tone31 and participate in the maintenance of vasospasm.32 Previously, nitrous oxide anesthesia has been shown to enhance homocysteine concentration and impairment of endothelial function.33 However, acetylcholine evoked relaxation of groups with and without nitrous oxide was similar in the presence of indomethacine, i.e. upon blockade of prostaglandin synthesis. Consequently, nitric oxide mediated relaxation does not differ between both groups, making the involvement of hyperhomocysteinemia unlikely.

Furthermore, nitrous oxide was reported to stimulate the sympathetic nervous system, which induces systemic and local vasoconstriction. This effect possibly explains the increased MAP during anesthesia with nitrous oxide.34 Whether this phenomenon also explains the normalization of phenylephrine-mediated contraction in resuscitated animals from the nitrous oxide group remains to be established. Aortic rings were studied in the absence of anesthetics in the organ bath. Our study clearly demonstrates vasomotor changes and decreased expression of cyclooxygenase-1 in the post-shock period, which were prevented by the use of nitrous oxide during the shock period. In view of the absence of volatile anesthetics and nitrous oxide in the organ baths, these results likely reflect vascular changes present in the immediate post-shock period, e.g. at the post anesthesia care unit or intensive care unit. However, it is unknown how these results obtained in isolated arteries correspond to vasoreactivity in in vivo conditions. Further, isoflurane and isoflurane/nitrous oxide groups differed in depth of anesthesia, since equivalent concentrations of isoflurane were used. However, as the study aimed to evaluate the effect of adjunct nitrous oxide, we chose to use the same isoflurane concentration in all experimental groups. Also, because the blood volume withdrawn for the induction of hemorrhagic shock was not assessed, we cannot comment on possible intra-operative differences in circulating volume. Finally, as the number of experiments was about six, small differences in calculated parameters of acetylcholine and phenylephrine-mediated responsiveness, or of cyclooxygenase expression may have been unnoticed. Nowadays, despite intensive scientific discussion on the influence of adjunct nitrous oxide to volatile anesthesia,33-38 only few experimental data regarding this question are available. The present study shows positive effects of adjunct nitrous oxide on vasoresponsiveness after short-term hemorrhagic shock in mice, since this anesthetic agent normalized vascular reactivity after as observed under isoflurane anesthesia.

In summary, the findings of this study show that vascular reactivity after hemorrhagic shock is affected by the choice of general anesthesia. Short-term hemorrhagic shock under isoflurane anesthesia increased phenylephrine-mediated


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contractile response and suppressed the acetylcholine-evoked contractile effect in thoracic mouse aorta, while adjunct nitrous oxide may attenuate changes in vascular reactivity caused by hemorrhagic shock and/or subsequent resuscitation. The results of this study also indicate that cyclooxygenase proteins participate in shock-dependent changes of vasoresponsiveness to acetylcholine. Our data suggest that the choice of the anesthetic regimen during emergency surgery for hemorrhagic shock may influence post-surgery vascular reactivity. !!Reference List 1. Keel M, Trentz O: Pathophysiology of polytrauma. Injury 2005; 36: 691-709 2. Landry DW, Oliver JA: The pathogenesis of vasodilatory shock. N Engl J Med 2001; 345: 588-95 3. Musser JB, Bentley TB, Griffith S, Sharma P, Karaian JE, Mongan PD: Hemorrhagic shock in swine:

Nitric oxide and potassium sensitive adenosine triphosphate channel activation. Anesthesiology 2004; 101: 399-408

4. Baumert JH, Hecker KE, Hein M, Reyle-Hahn SM, Horn NA, Rossaint R: Haemodynamic effects of haemorrhage during xenon anaesthesia in pigs. Br J Anaesth 2005; 94: 727-32

5. Xu J, Liu L: The role of calcium desensitization in vascular hyporeactivity and its regulation after hemorrhagic shock in the rat. Shock 2005; 23: 576-81

6. Park WK, Kim MH, Ahn DS, Chae JE, Jee YS, Chung N, Lynch C 3rd: Myocardial depressant effects of desflurane: Mechanical and electrophysiologic actions in vitro. Anesthesiology 2007; 106: 956-66

7. Akata T: General anesthetics and vascular smooth muscle: direct actions of general anesthetics on cellular mechanisms regulating vascular tone. Anesthesiology 2007; 106: 365-91

8. Akata T, Kanna T, Yoshino J, Takahashi S: Mechanisms of direct inhibitory action of isoflurane on vascular smooth muscle of mesenteric resistance arteries. Anesthesiology 2003; 99: 666-77

9. Izumi K, Akata T, Takahashi S: Role of endothelium in the action of isoflurane on vascular smooth muscle of isolated mesenteric resistance arteries. Anesthesiology 2001; 95: 990-8

10. Hopkins PM: Nitrous oxide: A unique drug of continuing importance for anaesthesia. Best Pract Res Clin Anaesthesiol 2005; 19: 381-9

11. Kaisti KK, Langsjo JW, Aalto S, Oikonen V, Sipila H, Teras M, Hinkka S, Metsahonkala L, Scheinin H: Effects of sevoflurane, propofol, and adjunct nitrous oxide on regional cerebral blood flow, oxygen consumption, and blood volume in humans. Anesthesiology 2003; 99: 603-13

12. Pypendop BH, Ilkiw JE, Imai A, Bolich JA: Hemodynamic effects of nitrous oxide in isoflurane-anesthetized cats. Am J Vet Res 2003; 64: 273-8

13. Sanders RD, Weimann J, Maze M. Biologic effects of nitrous oxide: A mechanistic and toxicologic review. Anesthesiology 2008;109:707-22.

14. van Meurs M, Wulfert FM, Knol AJ, De Haes A, Houwertjes M, Aarts LP, Molema G: Early organ-specific endothelial activation during hemorrhagic shock and resuscitation. Shock 2008; 29: 291-9

15. Darblade B, Pendaries C, Krust A, Dupont S, Fouque MJ, Rami J, Chambon P, Bayard F, Arnal JF: Estradiol alters nitric oxide production in the mouse aorta through the alpha-, but not beta-, estrogen receptor. Circ Res 2002; 90: 413-9

16. Potenza MA, Botrugno OA, De Salvia MA, Lerro G, Nacci C, Marasciulo FL, Andriantsitohaina R, Mitolo-Chieppa D: Endothelial COX-1 and -2 differentially affect reactivity of MVB in portal hypertensive rats. Am J Physiol Gastrointest Liver Physiol 2002; 283: G587-94

17. Conzen PF, Vollmar B, Habazettl H, Frink EJ, Peter K, Messmer K: Systemic and regional hemodynamics of isoflurane and sevoflurane in rats. Anesth Analg 1992; 74: 79-88

18. Akata T: Cellular and molecular mechanisms regulating vascular tone. Part 2: regulatory mechanisms modulating Ca2+ mobilization and/or myofilament Ca2+ sensitivity in vascular smooth muscle cells. J Anesth 2007; 21: 232-42

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19. Stekiel TA, Contney SJ, Kokita N, Bosnjak ZJ, Kampine JP, Stekiel WJ: Mechanisms of isoflurane-mediated hyperpolarization of vascular smooth muscle in chronically hypertensive and normotensive conditions. Anesthesiology 2001; 94: 496-506

20. Stekiel TA, Kokita N, Yamazaki M, Bosnjak ZJ, Kampine JP, Stekiel WJ: Effect of isoflurane on in situ vascular smooth muscle transmembrane potential in spontaneous hypertension. Anesthesiology 1999; 91: 207-14

21. Kokita N, Stekiel TA, Yamazaki M, Bosnjak ZJ, Kampine JP, Stekiel WJ: Potassium channel-mediated hyperpolarization of mesenteric vascular smooth muscle by isoflurane. Anesthesiology 1999; 90: 779-88

22. Li T, Liu L, Xu J, Yang G, Ming J: Changes of Rho kinase activity after hemorrhagic shock and its role in shock-induced biphasic response of vascular reactivity and calcium sensitivity. Shock 2006; 26: 504-9

23. Li T, Liu L, Liu J, Ming J, Xu J, Yang G, Zhang Y: Mechanisms of Rho kinase regulation of vascular reactivity following hemorrhagic shock in rats. Shock 2008; 29: 65-70

24. Liu LM, Ward JA, Dubick MA: Hemorrhage-induced vascular hyporeactivity to norepinephrine in select vasculatures of rats and the roles of nitric oxide and endothelin. Shock 2003; 19: 208-14

25. Zhou Y, Varadharaj S, Zhao X, Parinandi N, Flavahan NA, Zweier JL: Acetylcholine causes endothelium-dependent contraction of mouse arteries. Am J Physiol Heart Circ Physiol 2005; 289: H1027-32

26. Li M, Kuo L, Stallone JN: Estrogen potentiates constrictor prostanoid function in female rat aorta by upregulation of cyclooxygenase-2 and thromboxane pathway expression. Am J Physiol Heart Circ Physiol 2008; 294:H2444-55

27. Kawka DW, Ouellet M, Hetu PO, Singer II, Riendeau D: Double-label expression studies of prostacyclin synthase, thromboxane synthase and COX isoforms in normal aortic endothelium. Biochim Biophys Acta 2007; 1771: 45-54

28. Alcindor D, Krolikowski JG, Pagel PS, Warltier DC, Kersten JR: Cyclooxygenase-2 mediates ischemic, anesthetic, and pharmacologic preconditioning in vivo. Anesthesiology 2004; 100: 547-54

29. Tanaka K, Ludwig LM, Krolikowski JG, Alcindor D, Pratt PF, Kersten JR, Pagel PS, Warltier DC: Isoflurane produces delayed preconditioning against myocardial ischemia and reperfusion injury: role of cyclooxygenase-2. Anesthesiology 2004; 100: 525-31

30. Dayal S, Lentz SR: ADMA and hyperhomocysteinemia. Vasc Med 2005; 10:S27-33. 31. Toth J, Racz A, Kaminski PM, Wolin MS, Bagi Z, Koller A: Asymmetrical Dimethylarginine inhibits

shear stress-induced nitric oxide release and dilation and elicits superoxide-mediated increase in arteriolar tone. Hypertension 2007; 49:563-8

32. Pluta RM, Oldfield EH: Analysis of nitric oxide in cerebral vasospasm after aneursymal bleeding. Rev Recent Clin Trials 2007; 2:59-67.

33. Myles PS, Chan MT, Kaye DM, McIlroy DR, Lau CW, Symons JA, Chen S: Effect of nitrous oxide anesthesia on plasma homocysteine and endothelial function. Anesthesiology 2008;109:657-63

34. Jahn UR, Berendes E: Nitrous oxide--an outdated anaesthetic. Best Pract Res Clin Anaesthesiol 2005; 19: 391-7

35. Nagele P, Zeugswetter B, Wiener C, Burger H, Hüpfl M, Mittlböck M, Födinger M: Influence of methylenetetrahydrofolate reductase gene polymorphisms on homocysteine concentrations after nitrous oxide anesthesia. Anesthesiology 2008;109:36-43

36. Myles PS, Leslie K, Chan MT, Forbes A, Paech MJ, Peyton P, Silbert BS, Pascoe E; ENIGMA Trial Group: Avoidance of nitrous oxide for patients undergoing major surgery: a randomized controlled trial. Anesthesiology 2007;107:221-31

37. Preckel B, Bolten J: Pharmacology of modern volatile anaesthetics. Best Pract Res Clin Anaesthesiol 2005; 19: 331-48

38. Mashour GA, Forman SA, Campagna JA: Mechanisms of general anesthesia: From molecules to mind. Best Pract Res Clin Anaesthesiol 2005; 19: 349-64

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Chapter 7

S1P receptor modulation improves systemic vascular dysfunction after CPB in the rat independent of depletion

of lymphocytes: a comparison between FTY720 and SEW2871

Iryna V. Samarska, Hjalmar Bouma, Hendrik Buikema, Martin C. Houwertjes, Anne H. Epema, Robert H. Henning

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Abstract Background: Cardiopulmonary bypass (CPB) is associated with a systemic inflammatory response syndrome and disturbances in endothelial function of systemic arteries. FTY720, a non-selective agonist for sphingosine 1-phosphate (S1P) receptors, evokes lymphopenia by sequestration of lymphocytes from circulation to secondary lymphoid organs. SEW2871 is a selective agonist of S1P1 receptors. We investigated whether FTY720 and SEW2871 improve vascular reactivity after CPB in the rat. Materials and Methods: Experiments were done in male Wistar rats (n=48). Anesthesia-induction consisted of isoflurane (2.5-3%), followed by fentanyl and midazolam during CPB. After catheterization of the left femoral and carotid artery and the right heart, normothermic extracorporeal circulation was instituted for 60 min. Following 1 day of recovery, constriction to phenylephrine (PE) and serotonin (SE) and relaxation to acetylcholine of small mesenteric artery segments was assessed in a wired myograph system. Relaxation was expressed as % of preconstriction and analyzed as the area under the concentration-response curve (AUC, arbitrary units). Results: Contractile responses were inhibited after both CPB and SHAM experimental procedures. In mesenteric artery, FTY720 normalized SE- and PE-mediated vascular reactivity after CPB. FTY720 also increased total relaxation to acetylcholine as compared with untreated CPB groups (AUC: 256.8±43.9 and 168.2± 28.4; respectively). SEW2871 produced vascular effects comparable with FTY720. Conclusions: FTY720 and SEW2871 improve vascular function in mesenteric after CPB. This pharmacological effect was mediated mainly through S1P1 receptors and did not require lymphopenia. S1P1 receptor agonism may provide a promising therapeutic intervention to prevent CPB-related vascular dysfunction. Introduction

Cardiopulmonary bypass (CPB) is a widely used technique invaluable to thoracic surgery. CPB is however also associated with several side-effects such as induction of a systemic inflammatory response syndrome, a multiple organ dysfunction syndrome and ischemia-reperfusion injury. Two main mechanisms of CPB-related complications have been investigated during last decade: systemic inflammatory events and vascular dysfunction with secondary tissue edema and/or tissue hypoperfusion, setting the stage for multiple organ dysfunction.1 In a previous study, we characterized the vascular changes in small systemic arteries during a 5 day follow-up post-CPB in rat. That was found that overall contractility was inhibited by experimental procedures most prominently at 1 day of the recovery period, suggesting CPB-related surgical procedures and/or extended anesthesia induce extensive and protracted changes in the contractility of small vessels.

Several anti-inflammatory pharmacological agents, including corticosteroids, aprotinin, antioxidants, complement inhibitors and phosphodiesterase inhibitors have been proposed to inhibit the CPB-related inflammatory processes and vascular

S1P receptors’ agonists and vascular reactivity 113

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dysfunction. However, none of them satisfactorily prevented pathological and clinical outcomes that are associated with extracorporeal circulation.2

FTY720 (Fingolimod) is a non-selective sphingosine 1-phosphate (S1P) receptor modulator and acts as an immunosuppressive agent. Treatment with FTY720 thus far has been studied for autoimmune disorders (multiple sclerosis, autoimmune neuritis, autoimmune glomerulonephritis)3-6 and in the setting of transplantation.7 One main mechanisms of its therapeutic activity is the induction of lymphopenia, through inhibition of the egress of lymphocytes from the secondary lymphoid organs to the peripheral blood. In addition, FTY720 exerts pharmacological effects, including vascular effects, via activation of different S1P receptors.8 FTY720 has been shown to prevent or decrease ischemia-reperfusion injury,9-15 enhance endothelial barrier and decrease vascular permeability,16-19 and improve vascular function through modulation of the type 1, 3, 4 and 5 S1P receptors.16;18;20

From the above we hypothesized that treatment with FTY720 may be a promising pharmacological strategy to prevent vascular dysfunction in CPB. To address this, we investigated the effect of FTY720 on systemic vascular dysfunction in a rat model of experimental CPB. Rats were pretreated with FTY720 and subjected to CPB-related anaesthetical and surgical procedures with or without extracorporeal circulation. 24-Hours following recovery, small mesenteric arteries were removed and studied for contractility and endothelial relaxant reactivity. Comparisons were made to control rats that underwent short anesthesia only. Additional comparisons were made to rats treated with the selective S1P1 receptor agonist SEW2871 to further detail the type of S1P receptor involved. Material and Methods

The experimental protocol was approved by the Animal Ethics Committee of the University of Groningen. This study was performed in n=48 adult male Wistar rats with body weights of 509.3±32.3 g (Harlan, Zeist, The Netherlands) housed under standard conditions with free access to food (standard rat chow; Hope Farms, Woerden, The Netherlands) and drinking water throughout the study.

Experimental groups Experimental animals were randomly allocated to one of three experimental

groups with different treatment: untreated, FTY720-pretreated and SEW2871-pretreated. Each group was subdivided in three sub-groups: Control, Sham and CPB. CPB animals were subjected to the full experimental protocol described below, including anesthesia, cannulation, extracorporeal circulation, weaning and the recovery period. Sham animals followed the same procedure except for the extracorporeal circulation, but maintained cannulated and mechanically ventilated. The duration of the recovery period in Sham and CPB groups was 24 hours, in the end of which animals were sacrificed. Control groups were employed in order to evaluate the normal vascular reactivity in rats without invasive interventions. Animals in this group were sacrificed under brief isoflurane anesthesia (2.5-3%) only.

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preparation, extracorporeal circulation, a weaning and a recovery period (see fig. 1). Three hour before the beginning of the protocol animals were injected with FTY720 (0.5mg/kg), SEW2871 (0.5mg/kg) or vehicle. Anesthesia was induced with 2-3% isoflurane in O2/air (1:1) before intubation and mechanical ventilation (Amsterdam Infant Ventilator; HoekLoos, Amsterdam, The Netherlands). Tidal volume was set to achieve normocapnia (verified by capnography and arterial blood gas analysis), with O2/air (1:2) at a ventilation rate of 50 min-1 (0.5 s inspiration time). Rectal temperature was kept at 37.5±0.5 °C, using an electrical heating pad. The left femoral artery was cannulated (26-gauge catheter) for blood pressure monitoring. The mean arterial pressure was kept between 70 and 100 mm Hg by adjusting the isoflurane concentration as necessary (typically between 2.0-2.5%). Immediately before insertion of the arterial line, 250 IU kg-1 heparin was administered. The left carotid artery was cannulated for arterial inflow using a 22-gauge catheter. A multi-orifice 4.5 French cannula (adapted from a Desilets-Hoffmann catheter, Cook Son, The Netherlands) was advanced into the right heart using the right common jugular vein for access. The tail vein was cannulated for the administration of intravenous anesthetics, heparin, and protamine sulfate.

Extracorporeal circulation Subsequently, extracorporeal circulation (ECC) was initiated in the CPB group

for 60 min. The set-up consisted of a glass venous reservoir, a peristaltic pump (Pericor® SF70), a neonatal membrane oxygenator (0140GM, Polystan, Vaerlose, Denmark) and a glass counter-flow heat exchanger with built-in bubble trap. All components were connected with polyethylene tubing (1.6 mm inner diameter). The venous reservoir and heat exchanger were sterilized prior to use. The circuit was primed with 15 ml of haes 60 mg ml -1 solution (Voluven®, Fresenius Kabi, Bad Homburg, Germany). Animals were additionally heparinized (250 IU kg-1) after the start of ECC. During EEC, rats were anesthetized with intravenous fentanyl (125 "g kg-1), atracurium (0.5 mg kg-1), and midazolam (2 mg kg-1). During CBP, blood oxygen

Figure 1. Scheme of the experimental protocol in Sham and CPB groups regarding the duration of recovery period. The experimental protocol consisted of the following parts: anesthesia, preparation, extracorporeal circulation, a weaning and a recovery period (see methods). Abbreviations: ECC, extracorporeal circulation

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saturation was monitored continuously by a pulse oximeter. Targeted CBP flow was 100-110 mL kg-1 min-1, corresponding to 60-70% of normal cardiac output.


was initiated. Protamine (150 IU kg-1 i.v.) was administered in order to neutralize heparin after which cannulas were removed and the wounds sutured. Following extubation, animals were first kept under isoflurane anesthesia (0.8-1.0%) for the first hour of recovery in order to stabilize. The duration of the recovery period of various groups lasted 24 hours after the end of the extracorporeal circulation. Sacrifice was performed in the end of the recovery period under isoflurane anesthesia (2.5-3%).

Blood gas and cell analysis Samples for blood gas analysis (0.1 "l) were drawn at four time-points: at the

end of preparation period (15 min before start of ECC), twice during the ECC period (15 min after the start of ECC and 15 min before its ending), and at 45 min after the end of ECC. During the procedure, the amount of neutrophils, lymphocytes and monocytes was measured 4 times: just prior to injecting the compound (FTY720, SEW2871 or vehicle), 15 min after before the start of the ECC, 15 min after the end of the ECC, and at sacrification (24h after ECC). Blood gas analysis and cell count were performed at the Central laboratory of the University Medical Centre Groningen. MAP was monitored continuously during the entire protocol.

Vascular reactivity studies At sacrifice, all mesenteric loops were removed and placed into a cold

physiological saline solution. Several segments of third branch of the mesenteric superior artery were dissected, prepared as ring vessels preparations (1.8-2.0 mm in length) and mounted on two 40 µm stainless wires connected to force transducers in individual organ bath chambers for isometric tension recordings in a wire-myograph (Danish Myo Technology A/S, Denmark). The baths contained 6 ml of Kreb’s solution warmed to 37°C and bubbled continuously with 95%O2/5%CO2 at pH 7.4. Vessels were subjected to the standard normalization procedure21 and left to equilibrate for 40 min until they were at a steady baseline. In brief, the vessels were distended stepwise until effective pressure exceeded 100 mmHg (13.3 kPa). The internal circumference, IC100, was found from the Laplace’s equation and the experiments were performed at the IC1=0.9*IC100. Vascular segments were primed and checked for viability by two consecutive exposures to potassium chloride (60 mM).

The experimental protocol consisted of evaluation of contractile responses to potassium chloride (Emax to 60 mM) as a G-protein coupled receptor (GPCR)-independent contractile agent, and phenylephrine (PE; 10 nM to 100 "M) as a GPCR-dependent contractile agent. Endothelium-dependent relaxation to acetylcholine (ACh; 10 nM to 300 "M) was assessed in rings pre-constricted with PE. To assess the contribution of different endothelial mediators, ACh-induced relaxations were studied in the absence and presence of L-NMMA (0.1 mM; an inhibitor of NO-synthase) and/or

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indomethacine (1 "M; an inhibitor of cyclooxygenase) administered 20 min before application of PE [22]. Following the final concentration of ACh, a high concentration of the NO-donor sodium nitroprusside (SNP; 0.1 mM) was applied to assess maximal endothelium-independent relaxation.

Drugs The composition of Kreb’s solution was as follows (mM): 120.4 NaCl, 5.9 KCl,

2.5 CaCl2, 1.2 MgSO4, 25.0 NaHCO3, 1.2 NaH2PO4, 11.5 glucose; these chemicals were obtained from Merck (Darmstadt, Germany). The following drugs were used: acetylcholine chloride (ACh), phenylephrine (PE), indomethacine, SNP, NG-methyl-L-arginine (L-NMMA All other drugs were dissolved in deionized water. L-NMMA was purchased from MP Biomedicals (Illkirch, France). All other compounds were purchased from Sigma (St. Louis, Missouri, USA). The concentrations presented in the concentration-curve responses are expressed as a final molar concentration in the bath.

Data analysis Data are given as mean±SD and n refers to the number of animals in a

corresponding group. Contractile responses to KCl and PE are presented in mN and relaxations to ACh and SNP were calculated as a percentage of pre-constriction. For each concentration-response curve the following parameters were determined: 1) the Area Under the Curve (AUC) in arbitrary units (AU) (SigmaPlot, Systat Software, San Jose, CA, USA); 2) the maximal response (Emax); 3) the concentration at which half-maximal response was reached (EC50).Tthe difference in AUC for concentration-response curves to ACh in the absence or presence of an inhibitor was used as an estimate the contribution of different endothelial mediators [23]. The remainder of ACh induced relaxation resistant to the inhibition of both cyclooxygenase and NO-synthase was taken as an estimate of EDHF, as showed previously [24]. Differences were evaluated using one-way ANOVA combined with post-hoc Bonferroni test or with t-test (SPSS, Chicago, IL, USA). When data were not normally distributed, then nonparametric tests were used. Differences were considered significant at P<0.05 (2-tailed). The relationship between different relaxant pathways and total acetylcholine-mediated relaxation was evaluated with regression analysis and Spearman correlation. Results

Hemodynamics and blood gas analysis during the procedure Blood gas analysis was performed before start of ECC, twice during ECC and

after weaning. In untreated Sham and CPB groups at baseline all parameters were in the normal range. During ECC both in the untreated and treated CPB groups, a decreased pH, bicarbonate (HCO-

3), and Base excesses (BE) and increased levels of pCO2 were found, which may represent moderate respiratory acidosis (table 1). Hematocrit was significantly decreased in CPB groups as a consequence of hemodilution with the priming solution. In the post-ECC period, all parameters in CPB


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groups returned to normal ranges and were similar for Sham and CPB groups, except for hematocrit (table 1).

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%&!untreated rats, baseline MAP was similar between the groups. After onset of ECC, MAP dropped and remained significantly decreased until re-institution of normal circulation during which it recovered (fig. 2). In contrast, in pre-treated rats (all groups), MAP was significantly reduced already at baseline before the onset of ECC, and remained lower throughout the whole experimental procedure (fig. 2).!

Influence of FTY-720 and SEW-2871 on the amount of neutrophils, lymphocytes and monocytes in peripheral blood

Data on the amount of neutrophils, lymphocytes and monocytes are present in table 2. Since hemodilution can influence the results in CPB groups, all values are normalized to Hct value. At the start of recovery, the number of circulating neutrophils transiently increased in both Sham and CPB rats (table 2). Treatments did not affect the number of circulating neutrophils.

The amount of circulating lymphocytes did not significantly change in Sham rats, but was reduced in CPB rats at the end of the 24h recovery period (table 2). Treatment with FTY720 (0.5mg/kg i.p.) induced a pronounced lymphopenia, which was present already before onset of Sham/ECC (~4 hours after injection) and lasted until animals were sacrificed 24h thereafter. SEW2871 (0.5mg/kg) on the other hand, did not induce lymphopenia. Finally, the amount of monocytes did not significantly differ between groups throughout the study period. Thus, FTY720 caused pronounced lymphopenia, while SEW2871 did not show such effect.

Figure 2. Data on Mean arterial pressure (MAP, femoral artery) in Sham and CPB groups during the experimental protocol. Abbreviations: CPB, cardiopulmonary bypass. * indicates P<0.05, t-test; # indicates P<0.05, repeated measurements Anova, Bonferonni test !

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Alterations in vascular reactivity after CPB and the effect of treatments Differences in vascular contractility at the end of the 1-day study period were

observed particularly in Control versus Sham/CPB-groups, with no obvious additional effect of CPB compared to Sham. I.e. in mesenteric arteries both GPCR-independent and –dependent responses to KCl and PE, respectively, were significantly decreased in Sham/CPB as compared to Control (see fig. 3). Both treatments significantly increased and fully normalized responses to KCl - in Sham as well as in CPB rats - but seemed less effective in restoring contractility to PE. It should be noted, however, that both treatments also reduced GPCR-dependent responses to PE in control rats, while leaving GPCR-independent contraction to KCl unaffected (fig. 3).

Relaxant reactivity to ACh was similar in untreated control and Sham/CPB rats, thus suggesting that (systemic) endothelial function in this vessel type at this time-point post-CPB was (still) intact. Interestingly, both treatments enhanced total relaxation to ACh in all groups, including in control rats. Augmented endothelium-dependent reactivity was most pronounced in CPB rats treated with FTY720 and SEW2871, particularly due to increased contribution of EDHF in ACh-induced relaxation (fig. 4).

Thus, both treatments had tendency to enhance contractile and relaxant reactivity in mesenteric artery that became more pronounced in CPB groups.


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7 7.

41±0

.06

7.4±

0.06

7.

4±0.

07

7.4±

0.07

7.

36±0

.07

7.4±

0.06

7.

4±0.

05

7.37

±0.0

8 7.

35±0

.03

pCO 2

, kPa

4.

74±1

.0

4.51

±0.8

4.

68±1

.0

5.15

±0.9

4.

8±0.

8 4.

6±0.

8 5.

0±0.

8 5.

6±1.

1 4.

7±0.

9 4.

7±0.

8 5.

7±1.

3 5.

9±0.

7

HCO 3

,mm

ol/l

22.9

±1.5

23

.3±1

.5

24.7

±1.5

24

.4±2

.2

22.1

±0.7

22

.3±1

.2

23.8

±1.3

23

.8±1

.2

21.5

±2.0

21

.5±1

.7

23.9

±1.6

23

.9±1

.4

BE,m

mol

/l -0

.4±1

.0

0.5±

1.2

1.8±

1.5

0.5±

1.4

-1.8

±0.9

-0

.84±

1.3

0.05

±0.7

-1

.4±1

.1

-2.1

±1.6

-1

.9±1

.2

-0.7

±1.9

-1

.2±1

.1

Hctc

0.

4±0.

02

0.4±

0.03

0.

4±0.

03

0.4±

0.03

0.

39±0

.03

0.34

±0.0

3 0.

34±0

.06

0.34

±0.0

7 0.

4±0.

01

0.4±

0.06

0.

4±0.

02

0.4±

0.03

Gluc

ose,m

mol

/l 20

.6±5

.4

15.2

±5.4

8.

4±4.

3 6.

6±1.

0 19

.6±2

.4

16.4

±3.0

6.

9±1.

2 6.

9±0.

7 22

.7±3

.1

19.2

±4.4

7.

8±1.

4 8.

3±1.

2

CPB

untre

ated

CP

B FT

Y720

CP

B

SEW

2871

Be

fore

ECC

15

min

ECC

45

min

ECC

Af

ter E

CC

Befo

re E

CC

15 m

in E

CC

45 m

in E

CC

Afte

r ECC

Be

fore

ECC

15

min

ECC

45

min

ECC

Af

ter E

CC

pH

7.42

±0.0

5 7.

4*±0

.07

7.32

*±0.

08

7.

37±0

.05

7.

37±0

.07

7.

3#±0

.02

7.

3#±0

.04

7.33

±0.0

3 7.

35±0

.03

7.

3±0.

07

7.

3±0.

03

7.4±

0.08

pCO 2

, kPa

4.

77±0

.8

5.07

*±1.

1

5.72

*±1.

0

5.22

±0.8

5.0±

0.9

6.2#

±0.4

5

6.9#

±0.8

8 5.

8±0.

4 5.

4±0.

6 5.

7±0.

7 6.

6±0.

54

5.6±

1.2

HCO 3

,mm

ol/l

22.6

±2.3

21

.5±3

.6

21

.6*±

2.4

22.4

*±1.

7

21.3

±0.7

21.9

±0.5

6

22.3

±1.6

6

22.3

±1.5

22

.5±1

.0

21

.7±0

.9

23

.7±2

.5

23.7

±1.6

BE,m

mol

/l -0

.6±1

.7

-1.9

*±2.

7

-3.6

*±3.

1

-2.0

4±1.

9 -3

.2#±

0.7

-3

.6#±

0.5

-4

.2#±

1.7

-3.0

#±1.

4 -2

.3±0

.9

-3.2

±1.9

-2

.3±2

.1

1±2.

1

Hctc

0.

4±0.

02

0.3*

±0.0

5

0.3*

±0.0

2

0.3*

±0.0

3 0.

38±0

.03

0.2#

±0.0

2 0.

2#±0

.02

0.33

±0.0

2 0.

04±0

.07

0.22

±0.0

2 0.

28±0

.02

0.38

&±0.

02

Gluc

ose,m

mol

/l 22

.9±4

.1

20.5

±5.1

11

.6±4

.4

6.

9±3.

0

23.4

±1.9

22.0

±1.3

13.0

±2.3

7.

4±1.

4 24

.5±7

.4

23

.5±8

.5

18

.5±1

0.0

12.1

5&±3

.9

Ab

brev

iation

: CPB

, car

diopu

lmon

ary b

ypas

s. *P

<0.0

5, vs

Sha

m u

ntre

ated

, t-te

st; #

P<0

.05,

vs S

ham

FTY

720,

t-te

st; &

P<0

.05,

vs S

ham

SEW

2871

, t-te

st.

120 Chapter 7

!

Tabl

e 2.

Am

ount

of n

eutr

ophi

ls, l

ymph

ocyt

es a

nd m

onoc

ytes

in b

lood

of t

he e

xper

imen

tal a

nim

als

in a

ll gr

oups

. Va

lues

wer

e no

rmal

ized

to H

ct (1

0-9/l)

Sham

unt

reat

ed

CPB

unt

reat

ed

base

line

star

t EC

C

end

ECC

24

h re

cove

ry

base

line

star

t EC

C

end

ECC

24

h re

cove

ry

Neu

troph

ils

1.7±

0.4

1.7±

0.9

6.5a

±2.4

3.

1±1.

4 2.

4±1.

2 6.

5±2.

6 10

.3±5

.7a

6.6±

0.8

Lym

phoc

ytes

21

.1±6

.4

14.7

±2.6

18

.7±4

.7

15.0

±1.8

16

.1±7

.6

31.3

±16.

0 34

.9±5

.3

9.8±

3.6

b

Mon

ocyt

es

0.6±

0.5

0.3±

0.2

0.4±

0.5

0.75

±0.3

1.

2±0.

8 2.

3±2.

4 1.

8±1.

3 2.

3±2.

0

Sh

am F

TY 7

20

CPB

FTY

720

base

line

star

t EC

C

end

ECC

24

h re

cove

ry

base

line

star

t EC

C

end

ECC

24

h re

cove

ry

Neu

troph

ils

1.2±

0.1

4.2±

2.7

7.1±

0.9

c

4.6±

0.8

2.0±

1.5

7.8±

2.7

3.0

±0.7

5.

8±2.

9

Lym

phoc

ytes

23

.3±5

.4

5.9±

1.4

d 5.

1±1.

1 d

1.3

±0.7

d

19.0

±3.2

8.

3±2.

6 e

6.2±

3.8

e 2.

0±1.

0 e

Mon

ocyt

es

1.0±

0.3

0.6±

0.5

1.5±

0.6

0.9±

0.7

0.7±

0.4

0.7±

0.4

1.0±

0.6

0.7±

0.3

Sh

am S

EW28

71

CPB

SEW

2871

ba

selin

e st

art E

CC

en

d EC

C

24 h

reco

very

ba

selin

e st

art E

CC

en

d EC

C

24 h

reco

very

N

eutro

phils

2.

2±1.

4 2.

3±0.

9 9.

5±2.

2 f

5.2±

1.5

2.3±

1.7

4.8±

2.3

5.7±

1.7

6.3±

2.5

Lym

phoc

ytes

18

.8±2

.7

18.2

±7.5

23

.0±0

.65

12.8

±6.6

g

23.4

±3.2

40

.6±1

4.5

32.0

±7.

7 11

.4±5

.8 i

M

onoc

ytes

0.

7±0.

3 0.

5±0.

27

0.2±

0.1

0.5±

0.37

0.

5 ±0

.3

0.4

±0.2

1.

4±0.

9 0.

8 ±0

.4

Abb

revi

atio

n: C

PB=c

ardi

opul

mon

ary

bypa

ss;

base

line,

15

min

afte

r in

ject

ion

of t

he e

xper

imen

tal

com

poun

d of

the

veh

icle

; st

art

ECC

, 15

min

bef

ore

extra

corp

orea

l circ

ulat

ion;

end

EC

C, 1

5 m

in a

fter e

xtra

corp

orea

l circ

ulat

ion;

24h

reco

very

, sac

rific

atio

n, 2

4h la

ter t

he e

nd o

f the

ext

raco

rpor

eal c

ircul

atio

n. a

- P<

0.05

, vs

Sham

bas

elin

e, t-

test

; b -

P<0.

05, u

ntre

ated

CPB

24h

vs

all o

ther

CPB

unt

reat

ed g

roup

s, o

ne w

ay A

NO

VA, B

onfe

rroni

test

; c -

P<0.

05, v

s Sh

am

FTY7

20 b

asel

ine,

one

way

AN

OVA

on

rank

s, D

unn'

s M

etho

d; d

- P

<0.0

5, v

s Sh

am F

TY72

0 ba

selin

e, o

ne w

ay A

NO

VA, B

onfe

rroni

test

;e -

P<0

.05,

vs

CPB

FT

Y720

bas

elin

e, o

ne w

ay A

NO

VA, B

onfe

rroni

test

;f - P

<0.0

5, v

s Sh

am S

EW28

71 b

asel

ine,

one

way

AN

OVA

, Bon

ferro

ni te

st; g

- P<

0.05

, vs

Sham

SEW

2871

en

d EC

C, o

ne w

ay A

NO

VA, B

onfe

rroni

test

; i -

P<0.

05, C

PB S

EW28

71 2

4h v

s al

l oth

er C

PB28

71 g

roup

s, o

ne w

ay A

NO

VA, B

onfe

rroni

test


!

Discussion

The present work studied the acute pre-treatment effects of FTY720 and SEW2871, two different S1P receptor agonists, on systemic vascular reactivity in an experimental rat model of cardiopulmonary bypass. Isolated mesenteric artery preparations were studied 1-day post-CPB, hence at which time point contractility in untreated animals was generally depressed. The major finding of this study is that both FTY720 and SEW2871 both restored vascular contractility and augmented endothelial relaxant reactivity after CPB. Both compounds also evoked hypotension, yet differentially decreased the amount of lymphocytes in the peripheral blood. The observed effects are discussed with respect to the anti-inflammatory activity of S1P agonists and the modulation of the S1P receptors. It is concluded that the observed systemic vascular treatment effects were independent from lymphopenia but rather involved modulation of vascular S1P1 receptors.

Impaired contractility of mesenteric arteries to PE and KCl in the present study confirmed the development of systemic vascular dysfunction post-CPB in this model.

Figure 3. Contractile reactivity to phenylephrine and potassium chloride in all groups. A: maximal response to single dose of potassium chloride, mN; B: AUC values of the contractile responses to phenylephrine, mN. *- P<0.05, vs Control untreated, one way ANOVA, Bonferroni test; **- P<0.05, vs CPB untreated, one way ANOVA, Bonferroni test; #- P<0.05, vs Control, t-test; + -P<0.05 vs Sham untreated, one way ANOVA Bonferroni test

!

122 Chapter 7

!

Since both GPCR-dependent (PE) and –independent responses (KCl) were attenuated, this indicated a general depression of contractility. A Ca2+-desensitization process developing post-CPB might be implied, but further mechanistic investigation or qualification hereof was beyond the scope of the present study. Instead, because vascular dysfunction is believed to contribute to multiple organ dysfunction syndrome and related complications post-CPB, and therefore considered a relevant target of therapy, depressed mesenteric contractility was taken as an index (marker) to evaluate the potential of S1P receptor agonist treatment to modulate systemic vascular dysfunction in CPB.

Figure 4. Relaxant reactivity of mesenteric vessels to Acetylcholine in Control (A), Sham (B), and CPB (C) groups. Data are present as AUC values. ** - P=0.001 vs Control untreated, one way ANOVA, Bonferroni test. * P=0.003, vs CPB untreated, one way ANOVA, Bonferroni test.# P<0.05, vs CPB untreated, one way ANOVA, Bonferroni test.


!

Although S1P is of importance in the entire human body, it is a major regulator of vascular and immune systems. In this respect, the immunomodulatory effects of S1P agonists have been associated with the inhibition after S1P receptor activation of the egress of lymphocytes from secondary lymphoid organs to peripheral blood.8 Consistent herewith, FTY720 at the dose 0.5mg/kg in the present study induced a profound decrease in the amount of circulating lymphocytes (hence, but not in neutrophils or monocytes). Moreover, FTY720-induced lymphopenia was present at the time of onset of ECC (approximately 4 hour after the injection) and lasted for (at least) 24 hours thereafter (at which time-point vascular reactivity was assessed). In contrast, levels of circulating lymphocytes in animals treated SEW2871 at the same dose (0.5mg/kg) did not differ from (sham or CPB) rats that remained untreated. FTY720 is a potent non-selective S1P receptors agonist that causes the internalization of the receptors with their following degradation.25 SEW2871 is a highly selective S1P1 receptors agonist, that in contrast to FTY720 are capable only to internalize receptors, posses less efficacy and unable to downregulate S1P1 receptors.26;27

Despite the differential effects of FTY720 and SEW2871 on circulating lymphocytes in the present study, both compounds shared a similar treatment effect on vascular reactivity and blood pressure (see below). This suggests that the vascular treatment effects occurred independently from lymphopenia but rather involved (direct) modulation of vascular S1P receptors. Second to the above, therefore, S1P receptor agonists may exert direct vascular effects that may help to maintain vascular function. The five types of S1P receptors, S1P1-5, are widely distributed in different organs and tissues with S1P1-3 being the main receptors in the cardiovascular system. They are functionally involved in various processes, such as vascular permeability, endothelial proliferation, angiogenesis, and regulation of vascular tone and heart rate.19;28;29 The S1P receptor is a G-protein coupled receptor (GPCR), the type of G-protein coupled to it providing the diversity in downstream signaling pathways and responses.30 S1P receptors are not homogeneously expressed in the endothelial and smooth muscle cells derived from different vascular beds.29;30 Although it is generally considered that in endothelium the S1P1-receptor type is most abundantly expressed,30 both S1P1, -2 and -3 receptors participate in the maintenance of endothelial integrity and hence, the vascular barrier. Activation of S1P1 type receptor increases the endothelial integrity and thus decreases the vascular permeability, as found in studies with S1P1 agonists.17;19;30 Activation of S1P2 and S1P3 receptors on the other hand, leads to an increased vascular permeability. Activation of S1P2 and S1P3 receptors on smooth muscle cells causes vasoconstriction through activation of the phospholipase C, activation of myosine light chain kinase, and/or Rho-associated kinase-dependent inhibition of myosine light chain phosphatase.29-31 Several studies also showed the involvement of S1P1 receptors in regulation of contractile reactivity.32

In our study, impaired contractility was restored in Sham and CPB-rats pre-treated with FTY720 or SEW2871. This improvement mainly concerned the GRCP-independent responses to KCl, but was less eminent for GRCP-dependent responses to PE (except in SEW2871-treated CPB-rats). Of note, both treatments reduced

124 Chapter 7

!

contractility to PE, but not that to KCl, in control rats. Possibly, stimulation of the S1P receptor, which is also a G-protein coupled receptor, leads to a downregulation/desensitzation of G-protein signalling in general. This could explain the decreased response to PE, but not KCl, in treated control rats as well as in treated Sham and CPB rats.

Endothelium-dependent relaxation to ACh in untreated Sham and CPB rats was comparable to that in untreated controls. This indicates that endothelial function was still intact 1-day post-CPB but does not rule out attenuation of endothelial function in other vascular beds and/or at later stages post-CPB.chapter 2 Interestingly, FTY720 and SEW2871 both enhanced total relaxation to ACh in all groups, including in control rats. It has been shown previously that S1P1 and S1P3 receptors on endothelial cells may evoke the release of NO through activation of eNOS.12-15 On the other hand, NO is not the major mediator of endothelium-dependent relaxation in mesenteric artery. Augmented endothelium-dependent reactivity was most pronounced in CPB rats treated with FTY720 and SEW2871, due to an increased contribution of EDHF. Although the effect of S1P agonists on NO production has been described before, the effect of S1P-receptor activation in EDHF has not been reported previously. S1P was shown to activate large-conductance Ca(2+)-activated K(+) channel, but in G-protein independent manner.32 Thus, this effect requires further elucidation but that was not an aim of the present study. Nevertheless, because both experimental compounds exerted their effect in comparable values, mainly S1P1 receptor involvement may be implied in above vascular effects.

Finally, the results of our study showed that both experimental compounds decreased mean arterial blood pressure during the whole experimental procedure in both Sham and CPB-operated groups. Previously it has been shown that FTY720 increased blood pressure in human.29;32; 33 Probably, simultaneous application of S1P agonists and prolonged anesthetical intervention might produce hypotensive effects since both can modulate open probability of the potassium channels34;35 that are involved in the regulation of the heart rate and vascular tone. S1P1 receptors participate in the regulation of both relaxant and contractile reactivity of vascular beds that serve as balance of concurrent activity. The final effect depends on the most sensitive and most expressed pathway.31;32

In conclusion, our study for the first times demonstrated that agonists of the S1P receptors enhanced systemic vascular reactivity after CPB mainly due to modulation of the S1P1 subtype. However, this effect does not depend on the lymphopenia. This finding can initiate further research for the evaluation of such therapeutical agents in the prevention and/or treatment of the CPB-related complications. Reference List

1. Hirleman E, Larson DF: Cardiopulmonary bypass and edema: physiology and pathophysiology.

Perfusion 2008; 23: 311-22


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2. Apostolakis EE, Koletsis EN, Baikoussis NG, Siminelakis SN, Papadopoulos GS: Strategies to prevent intraoperative lung injury during cardiopulmonary bypass. J.Cardiothorac.Surg. 2010; 5: 1

3. Chun J, Hartung HP: Mechanism of Action of Oral Fingolimod (FTY720) in Multiple Sclerosis. Clin.Neuropharmacol. 2010;

4. Brinkmann V: FTY720 (fingolimod) in Multiple Sclerosis: therapeutic effects in the immune and the central nervous system. Br.J.Pharmacol. 2009; 158: 1173-82

5. Zhang Z, Zhang ZY, Fauser U, Schluesener HJ: FTY720 ameliorates experimental autoimmune neuritis by inhibition of lymphocyte and monocyte infiltration into peripheral nerves. Exp.Neurol. 2008; 210: 681-90

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9. Hasegawa Y, Suzuki H, Sozen T, Rolland W, Zhang JH: Activation of sphingosine 1-phosphate receptor-1 by FTY720 is neuroprotective after ischemic stroke in rats. Stroke 2010; 41: 368-74

10. Egom EE, Ke Y, Musa H, Mohamed TM, Wang T, Cartwright E, Solaro RJ, Lei M: FTY720 prevents ischemia/reperfusion injury-associated arrhythmias in an ex vivo rat heart model via activation of Pak1/Akt signaling. J.Mol.Cell Cardiol. 2010; 48: 406-14

11. Lee KD, Chow WN, Sato-Bigbee C, Graf MR, Graham RS, Colello RJ, Young HF, Mathern BE: FTY720 reduces inflammation and promotes functional recovery after spinal cord injury. J.Neurotrauma 2009; 26: 2335-44

12. Delbridge MS, Shrestha BM, Raftery AT, El Nahas AM, Haylor JL: Reduction of ischemia-reperfusion injury in the rat kidney by FTY720, a synthetic derivative of sphingosine. Transplantation 2007; 84: 187-95

13. Kaudel CP, Frink M, Schmiddem U, Probst C, Bergmann S, Krettek C, Klempnauer J, van GM, Winkler M: FTY720 for treatment of ischemia-reperfusion injury following complete renal ischemia; impact on long-term survival and T-lymphocyte tissue infiltration. Transplant.Proc. 2007; 39: 499-502

14. Kaudel CP, Frink M, van GM, Schmiddem U, Probst C, Bergmann S, Krettek C, Klempnauer J, Winkler M: FTY720 application following isolated warm liver ischemia improves long-term survival and organ protection in a mouse model. Transplant.Proc. 2007; 39: 493-8

15. Kaudel CP, Schmiddem U, Frink M, Bergmann S, Pape HC, Krettek C, Klempnauer J, Winkler M: FTY720 for treatment of ischemia-reperfusion injury following complete renal ischemia in C57/BL6 mice. Transplant.Proc. 2006; 38: 679-81

16. Sarai K, Shikata K, Shikata Y, Omori K, Watanabe N, Sasaki M, Nishishita S, Wada J, Goda N, Kataoka N, Makino H: Endothelial barrier protection by FTY720 under hyperglycemic condition: involvement of focal adhesion kinase, small GTPases, and adherens junction proteins. Am.J.Physiol Cell Physiol 2009; 297: C945-C954

17. Camp SM, Bittman R, Chiang ET, Moreno-Vinasco L, Mirzapoiazova T, Sammani S, Lu X, Sun C, Harbeck M, Roe M, Natarajan V, Garcia JG, Dudek SM: Synthetic analogs of FTY720 [2-amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol] differentially regulate pulmonary vascular permeability in vivo and in vitro. J.Pharmacol.Exp.Ther. 2009; 331: 54-64

18. Dudek SM, Camp SM, Chiang ET, Singleton PA, Usatyuk PV, Zhao Y, Natarajan V, Garcia JG: Pulmonary endothelial cell barrier enhancement by FTY720 does not require the S1P1 receptor. Cell Signal. 2007; 19: 1754-64

19. Brinkmann V, Cyster JG, Hla T: FTY720: sphingosine 1-phosphate receptor-1 in the control of lymphocyte egress and endothelial barrier function. Am.J.Transplant. 2004; 4: 1019-25

20. Tolle M, Levkau B, Keul P, Brinkmann V, Giebing G, Schonfelder G, Schafers M, von Wnuck LK, Jankowski J, Jankowski V, Chun J, Zidek W, van der GM: Immunomodulator FTY720 Induces eNOS-dependent arterial vasodilatation via the lysophospholipid receptor S1P3. Circ.Res. 2005; 96: 913-20

21. Xu Y, Henning RH, Lipsic E, van BA, van Gilst WH, Buikema H: Acetylcholine stimulated dilatation and stretch induced myogenic constriction in mesenteric artery of rats with chronic heart failure. Eur.J.Heart Fail. 2007; 9: 144-51

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25. Graler MH, Goetzl EJ: The immunosuppressant FTY720 down-regulates sphingosine 1-phosphate G-protein-coupled receptors. FASEB J. 2004; 18: 551-3

26. Graler MH: Targeting sphingosine 1-phosphate (S1P) levels and S1P receptor functions for therapeutic immune interventions. Cell Physiol Biochem. 2010; 26: 79-86

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28. Brinkmann V, Baumruker T: Pulmonary and vascular pharmacology of sphingosine 1-phosphate. Curr.Opin.Pharmacol. 2006; 6: 244-50

29. Brinkmann V: Sphingosine 1-phosphate receptors in health and disease: mechanistic insights from gene deletion studies and reverse pharmacology. Pharmacol.Ther. 2007; 115: 84-105

30. Lucke S, Levkau B: Endothelial Functions of Sphingosine-1-phosphate. Cell Physiol Biochem. 2010; 26: 87-96

31. Yatomi Y: Sphingosine 1-phosphate in vascular biology: possible therapeutic strategies to control vascular diseases. Curr.Pharm.Des 2006; 12: 575-87

32. Igarashi J, Michel T: Sphingosine-1-phosphate and modulation of vascular tone. Cardiovasc.Res. 2009; 82: 212-20

33. Comi G, O'Connor P, Montalban X, Antel J, Radue EW, Karlsson G, Pohlmann H, Aradhye S, Kappos L: Phase II study of oral fingolimod (FTY720) in multiple sclerosis: 3-year results. Mult.Scler. 2010; 16: 197-207

34. Agnew NM, Pennefather SH, Russell GN: Isoflurane and coronary heart disease. Anaesthesia 2002; 57: 338-47

35. Koyrakh L, Roman MI, Brinkmann V, Wickman K: The heart rate decrease caused by acute FTY720 administration is mediated by the G protein-gated potassium channel I. Am.J.Transplant. 2005; 5: 529-36

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Chapter 8

Summary and future perspectives

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Cardiopulmonary bypass (CPB) is a widely used technique invaluable to thoracic surgery. However, CPB also associates with an (temporal) organ dysfunction and an increased incidence of postoperative clinical complications, not only in-hospital but also at the mid- and long-term thereafter.1 Changes in vascular function of macro- and microcirculatory beds represent a pivotal component in the development of organ dysfunction and are thought to result from systemic inflammation and ischemia-reperfusion injury following CPB.2 Hence, documenting the time-course and mechanisms of altered vascular reactivity post-CPB as well as its interrelationship with mediators of reperfusion injury and inflammation may provide better and/or new rationales for pharmacotherapeutic interventions to improve morbidity- and mortality outcome after CPB. The studies in the present thesis intended to explore this and help to fill the gap in the data on the role of altered vascular function in the pathogenesis of CPB. To this end, the main focus was put on (1) alterations in vascular reactivity after CPB in relation to (2) inflammatory processes induced in vital organs, and (3) the effect of pharmacological modulation of S1P-receptors as a therapeutic intervention herein. Employing a rat model of experimental CPB, vascular reactivity in the second and third chapter of this thesis was assessed at different time-points post-operatively during a clinically relevant recovery period (up to 5 days). Because anesthesia is part of CPB, but may have its own impact on vascular reactivity, this was further explored in the forth chapter in which the effect of different types of general anesthesia on vascular reactivity was tested in a mice model of hemorrhagic shock. In chapters five and six, the inflammatory response of vital organs (kidney and lung) to CPB was studied. Finally, the effect of pretreatment with two different S1P-receptor agonists on altered vascular reactivity after CPB was studied in chapter seven. Alterations in vascular reactivity after CPB

Influence of CPB-related anesthetic and surgical procedures Apart from extracorporeal circulation (ECC), CPB consists of intensive

anesthetic and surgical procedures, which by themselves represent a profound challenge to the subject’s condition. In chapters 2 and 3, time-course analysis of vascular reactivity was assessed (in vitro) in preparations of 3 different vessel types obtained from rats subjected to CPB-related procedures with or without ECC (i.e. rats from CPB and sham groups) and sacrificed at different time points (up to 5 days) after recovery hereof. Compared to animals studied after short anesthesia only (i.e. control rats), small mesenteric and coronary artery (but not aorta) contractility was depressed in sham rats the first day after recovery after which it normalized again. This early inhibition of small artery contractility concerned both receptor-dependent (phenylephrine or serotonin) and -independent (KCl) pathways. Such finding suggests a temporal decrease in general Ca2+ sensitivity post-CPB, possibly through modulation of the MLC kinase and/or MLC phosphatase activity.3

Volatile anesthetics are well known for their vasorelaxing effects4 and constitute a main candidate for the induction of changes in vascular contractility. In chapter 4, the effects of N2O adjunct to anesthesia with isoflurane (ISO) on vascular reactivity was investigated in a mouse model of hemorrhagic shock. Both arterial

Summary and future perspectives 129

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pressure and aortic contractility to phenylephrine were decreased in sham mice anaesthetized with ISO only, as compared to those receiving adjunct N2O. Systemic hypotension after ISO has been reported previously5-7 and may represent an unwanted side-effect of anesthesia. Volatile anesthetics including ISO have also been shown to inhibit contraction to angiotensin II, amongst others.4, 8 Collectively, the findings in chapter 6 suggest that vascular contractility during different phases of shock was preserved when adjunct N2O was employed. Moreover, changes in COX expression may represent the underlying mechanism of those effects of N2O. The results of this study fundamentally demonstrate that vascular contractility was modulated by the choice of general anesthesia. This may demand for a rational choice of intra-operative anesthesia to maintain vascular function post-operatively and avoid clinical complications after surgical intervention, including in CPB.

CPB-related anesthetic and surgical procedures not only affected vascular smooth cell function but also endothelial cell function. Additionally, as described in chapter 2, endothelium-dependent relaxation in small mesenteric arteries was significantly enhanced in sham rats at 2 days after recovery. Together with the above-mentioned findings, the results indicate that anesthetic and invasive procedures in CPB may induce profound changes in vascular function, even in the absence of ECC. The results suggest a pattern of temporal changes early post-CPB (i.e. 24-48h) both at the level of smooth muscle as well as endothelial cell function in small arteries. In contrast, however, aorta contractility and endothelium-dependent relaxation in sham rats remained relatively unaffected after early post-CPB (but increased EDHF in EF after 5 days). This seems to highlight that different vessel types may become differentially affected and underlines the differences along the vascular tree in susceptibility for alterations in vascular function following CPB.

Additional effects of extracorporeal circulation On top of the described effects of CPB-related anesthetic and surgical

procedures, ECC attenuated vascular relaxation in small mesenteric and coronary arteries (chapter2). This impairment was most outspoken at 2 days of recovery and involved a decrement of various different mediators of endothelial relaxation function. In mesenteric arteries, particularly, the contribution of EDHF was reduced, while in coronary arteries all endothelial mediators appeared suppressed by ECC.

The observation that ECC selectively affected (small artery) endothelial function constitutes an important finding. Others have found increased plasma TNF-! concentrations correlates to impaired endothelium dependent relaxation in rats after CPB9 and implied that (systemic) endothelial dysfunction results from the aggravated systemic inflammatory response that may be elicited by CPB.10; 11 ; 12 In chapter 3, we examined vascular expression of E-selectin, ICAM-1, and VCAM-1 and evaluated the correlation between these markers of endothelial cell activation and endothelial relaxation in rat aorta. Hence, E-selectin is a main marker of the endothelium activation in response to inflammatory cytokine (TNF-! and IL-1$)13 and the cell adhesion molecules of the immunoglobulin superfamily, ICAM-1 and VCAM-1, serve as ligands for leukocyte integrins.1 Substantial up-regulation of all these factors was found consistently in CPB and occasionally in Sham but only after 60 min of recovery from

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CPB. CPB also induced the up-regulation of TNF-! and TGF-$, which may modulate the expression of adhesion molecules, provide a chemotactic gradient for leukocytes and other cells participating in an inflammatory response,14 and increase expression of HO-1, an enzyme induced in response to oxidative stress and hypoxia. Again, however, these increases occurred only very acutely after CPB. Because aortic endothelium-dependent relaxation at that time-point of recovery did not differ between sham and CPB rats, it seems unlikely that endothelial relaxation (dys-)function was related with endothelial cell activation (which was present mainly in the CPB group). Alternatively, it seems that transient aortic inflammation acutely following CPB may have induced processes that lead to alterations in aortic reactivity at the long-term.

To summarise, vascular reactivity after CPB was greatly affected in different vessel types during long-term recovery period without trends for normalization. Sham-related procedures (anesthesia and surgery) seemed to evoke an initial decrease in general vascular contractility, whereas extracorporeal circulation (as a major constitute of CPB) additionally attenuated endothelial relaxant function (particularly in small vessels). Although ECC also prompted for a rapid systemic inflammatory response, endothelial cell activation alone did not explain the selective CPB-mediated impairment in endothelial function (see also fig. 1, A and B).

Changes in inflammatory processes in vital organs (kidney and lung) A high rate of postoperative morbidity in CPB is being associated with

(temporal) renal and pulmonary organ dysfunction. The systemic inflammatory response syndrome in CPB is thought to play a major role herein. In chapters 4 and 5, we evaluated the time-dependent changes in inflammatory marker expression after CPB in the lung and the kidney.

The observed expression patterns of different pro- and anti-inflammatory cytokines, endothelial activation markers, and markers of neutrophil activation indicated that CPB evoked an acute inflammatory response in both organs. Early activation of macrophages and profound release of the inflammatory cytokines (TNF-!, IL-6, IL-1$ and others) during the first hours of recovery has been shown previously both in lung15-18 and kidney.1, 19, 20 In our studies, macrophages infiltration in the lung was relatively high and remained elevated over time post-CPB in all groups, including in controls rats. However, expression of inflammatory cytokines was not increased in the latter. Such findings seem to dispute for a role solely of infiltrating macrophages in increased inflammatory cytokine production. Instead, macrophages already resident in organs may be importantly involved in the rapid up-regulation of inflammatory cytokines. Literature reporting on a poor correlation between inflammatory mRNA expression in organs with cytokine concentrations in the plasma may further support the importance of inflammatory processes locally in the kidney and the lung.21

Despite early similarities, the expressional patterns at later stages post-CPB differed between lung and kidney in our studies. In the kidney, all up-regulated markers were normalized by day 5 of the recovery period. In contrast, pulmonary expression of Il-1beta and TGFbeta1 remained increased during recovery, indicating the continuation of inflammatory processes in the lung. Such situation may be explained by the specific


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character of the lung functionality during CPB. Lungs are not only subjected to CPB-evoked release of cytokines initiating a prominent inflammatory response in this organ, but they are also more or less excluded from the circulation during the extracorporeal circulation period22, 23 that aggravates CPB-related pathological consequences.

To summarize, the inflammatory processes in kidney and lung in our study appeared early after CPB. Whereas all up-regulated markers in kidney normalised in due course thereafter, inflammatory processes in lung seem were maintained for at least 5 days thereafter with little trend for the normalization (fig.1C) Pharmacological modulation of S1P-receptors as a therapeutic intervention in CPB

Systemic inflammation has been implied to underlie changes in vascular function of macro- and microcirculatory beds. Altered vascular function is believed to represent a pivotal component in the development of organ dysfunction and clinical complications hereof.22, 23 Therefore, interventions to maintain vascular function in CPB may be aimed both at modulation of systemic inflammation and/or direct modulation of vascular function. Because sphingosine 1-phosphate (S1P) is a main regulator of immune as well as vascular systems, pharmacological modulation of S1P-receptors was considered a potential therapeutic intervention in CPB.24

Employing our experimental rat model of CPB, the acute pre-treatment effects of FTY720 and SEW2871, two different S1P-receptor agonists, on (systemic) vascular reactivity was investigated in chapter seven. Isolated mesenteric artery preparations were studied 1-day post-CPB, hence, at which time point contractility in untreated animals was generally depressed (see chapter 2). FTY720 and SEW2871 both restored contractility and augmented endothelial relaxant reactivity after CPB. Both treatments also evoked hypotension, yet differentially affected the amount of lymphocytes in the peripheral blood. I.e. circulating lymphocytes were decreased after FTY720, a non-selective S1P receptor modulator, but not after SEW2871, a selective agonist of the S1P1-receptor (see also fig. 2). Because both compounds shared a similar treatment effect on vascular reactivity and blood pressure, this suggests that the vascular treatment effects occurred independent from lymphopenia and inhibition of systemic inflammation but rather involved (direct) modulation of vascular S1P receptors.

The five types of S1P receptors, S1P1-5, are widely distributed in different organs and tissues with S1P1-3 being the main receptors in the cardiovascular system.25 S1P1 receptors participate in the regulation of both relaxant and contractile reactivity of vascular beds that serve as balance of concurrent activity.26, 27 Given the fact that the observed systemic vascular effects were also obtained with the selective agonist SEW2871, involvement of the vascular S1P1-receptor subtype herein is strongly implied. The findings of these studies may encourage further research for the evaluation of such therapeutic agents in the prevention and/or treatment of the CPB-related complications.

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Implications The studies in the first part of this thesis demonstrated that vascular reactivity

post-CPB was profoundly influenced in a vessel-type specific manner by CPB-related procedures (anesthesia and surgical intervention), and ECC in addition hereof. Per se anesthetic and surgical procedures in CPB evoked a pattern of temporal changes early post-CPB both at the level of smooth muscle (decreased contractility) as well as endothelial cell function (enhanced relaxation) in small (coronary and mesenteric) arteries. As depicted in figure 1A, this is in contrast to large conductance artery reactivity which was relatively unaffected early post-CPB but rather changed at the longer term (i.e. increased EDHF in endothelial function). ECC as a major constitute of CPB attenuated endothelial relaxant function in small arteries, but not in large vessel. The findings suggest differential sensitivity along the vascular tree for alterations in vascular function following CPB, and imply increased susceptibility of small arteries for impairment of endothelial function by ECC. These pathophysiological events may explain observations made in the clinic. The disturbances in microcirculatory beds of different organs can initiate secondary tissue edema and tissue hypoperfusion, thus founding a multiple organ dysfunction syndrome. Hemodynamic and microcirculatory abnormalities were shown to set a stage for the mesenteric complications,28, 29 acute lung injury and acute respiratory distress syndrome,15, 30 acute kidney injury 31, 32 after CPB.

As depicted in figure 1B, ECC also initiated a rapid systemic inflammatory response and vascular endothelial cell activation in the aorta, however, without a momentary impairment in the endothelial relaxation. The latter challenges the immediate translation of vascular inflammation into altered vascular reactivity. Rather, inflammation may act as an trigger of subsequent alterations in the expression or activity of components involved in vasomotor regulation, subsequently altering vascular reactivity after an initial lag time [2]. If so, the lag time may leave time for pharmacological modulation of vascular function to prevent microcirculatory disturbances.

Studies in the second part of this thesis showed that while ECC evoked an acute inflammatory processes in all organs studied, organs differed with respect of the duration of the response. As depicted in figure 1C, up-regulated markers in vascular and renal tissue normalised in due course, while the inflammatory processes in lung was maintained for at least 5 days post-CPB. The difference may be explained by the specifics of the lung perfusion during CPB, since lungs are deprived of the majority of their normal blood supply in this period, additionally to the exposition to all CPB-related pathogenetic factors that affect functionality of all organs.1, 20 Maintenance of the inflammatory processes in lung during long-term recovery can set the stage for the frequently occurring acute pulmonary complication after CPB, since approximately 25% of the patients following open heart surgery exhibited signs of pulmonary impairment for at least one week thereafter.1, 22, 23


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Figure 1. Schematic representation of the changes in vascular reactivity and inflammatory processes following cardiopulmonary bypass (CPB). (A) CPB evoked a differential pattern of temporal changes early post-CPB (i.e. 24-48h) in small (mesenteric and coronary) arteries vs. “long-term” changes in large conductance artery (aorta); (B) Rapid systemic inflammation and aorta endothelial cell activation after extracorporeal circulation (ECC) were not paralleled by acute changes in aorta endothelial relaxation function, but rather by changes at the “long-term”; (C) Up-regulated inflammatory markers in kidney and aorta normalised in due course after ECC, but were maintained elevated in the lung for at least 5 days after CPB.

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Figure 2. Effect of S1P-receptor agonism on circulating lymphocytes and vascular reactivity following CPB. (A) The amount of circulating lymphocytes was reduced after pre-treatment with the non-selective S1P-receptor agonist FTY720, but not with the selective S1P1-receptor agonist SEW2871. In contrast, both compounds similarly enhanced vascular (B) contractility and (C) endothelial relaxation function. This suggests that the enhanced vascular reactivity following pre-treatment was mediated via modulation vascular S1P1-receptors, independently from lymphopenia. * P<0.05, vs untreated group, one way ANOVA with Bonferroni test. # P<0.05, vs untreated group, t-test


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Studies in the last part of this thesis demonstrated that pre-treatment with an S1P-receptor agonist restored vascular reactivity post-CPB both in the absence and presence of concurrent reduction in circulating lymphocytes. As depicted in figure 2, lymphopenia was induced with FTY720 only, but not with SEW2871. However, both compounds shared a similar effect on vascular reactivity and blood pressure. The results imply positive vascular treatment effects independent from lymphopenia via direct modulation of vascular S1P1-receptors. Therefore agonists of S1P receptors can represent a new approach for the improvement of the functionality of microcirculatory beds and preventing of the organ dysfunction syndrome due to enhancing of the tissue perfusion. In conclusion, there are several major points disclosed to the research described in this thesis. First, CPB evokes profound abnormalities, which include different physiological processes; herein among others changes in vascular reactivity play a prominent role. Secondly, key components of physiology are not fully normalized after a prolonged recovery period, suggesting continuance of the pathological process. Thirdly, normalization of the microcirculation following CPB may be accomplished by modulation of S1P receptors. Despite recent progress in the understanding of the pathophysiological consequences of CPB, the remaining unsolved CPB-related problems point at a range of directions for future research. First, further investigation of the relative contribution of different factors to CPB pathogenesis is warranted. The contribution of the different components of the CPB-circuit to CPB-related complications needs exploration, as changes of the technical design of the CPB-circuit or the surgical protocol may represent a possible way for the prevention and/or treatment of the CPB-mediated medical problems.23, 33 Secondly, as shown in this thesis, anesthetic interventions may have profound impact on the physiology of vascular beds, however, our current knowledge in this respect is very limited. Thus, the choice of the optimal inhalation/intravenous anesthetics prevent multiple organ dysfunction syndrome, by improving hemodynamics, organ microcirculation and tissue perfusion.chapter 2, 3, and 6 of this

thesis; 34, 35 In addition, the influence of preoperatively existing chronic disease, such diabetes, heart, renal, and pulmonary disease, may influence the pathophysiology of CPB.36, 37. Further research in this direction provides better understanding of the underlying mechanisms and possible selective therapeutical modulations. Finally, evaluation of the efficacy of different therapeutic interventions to limit CPB-related complications remains an actual research topic. Further investigation of the S1P receptor modulation may be promising in prevention and/or treatment of the CPB-related complications. Reference List 1. Asimakopoulos G: Systemic inflammation and cardiac surgery: an update. Perfusion 2001; 16: 353

60 2. Verrier ED, Morgan EN: Endothelial response to cardiopulmonary bypass surgery. Ann Thorac Surg

1998; 66: S17,9; discussion S25-8

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3. Savineau JP, Marthan R: Modulation of the calcium sensitivity of the smooth muscle contractile apparatus: molecular mechanisms, pharmacological and pathophysiological implications. Fundam Clin Pharmacol 1997; 11: 289-99

4. Preckel B, Bolten J: Pharmacology of modern volatile anaesthetics. Best Pract Res Clin Anaesthesiol 2005; 19: 331-48

5. Baumert JH, Hecker KE, Hein M, Reyle-Hahn SM, Horn NA, Rossaint R: Haemodynamic effects of haemorrhage during xenon anaesthesia in pigs. Br J Anaesth 2005; 94: 727-32

6. Pypendop BH, Ilkiw JE, Imai A, Bolich JA: Hemodynamic effects of nitrous oxide in isoflurane-anesthetized cats. Am J Vet Res 2003; 64: 273-8

7. Conzen PF, Vollmar B, Habazettl H, Frink EJ, Peter K, Messmer K: Systemic and regional hemodynamics of isoflurane and sevoflurane in rats. Anesth Analg 1992; 74: 79-88

8. Qi F, Ogawa K, Tokinaga Y, Uematsu N, Minonishi T, Hatano Y: Volatile anesthetics inhibit angiotensin II-induced vascular contraction by modulating myosin light chain phosphatase inhibiting protein, CPI-17 and regulatory subunit, MYPT1 phosphorylation. Anesth Analg 2009; 109: 412-7

9. Doguet F, Litzler PY, Tamion F, Richard V, Hellot MF, Thuillez C, Tabley A, Bouchart F, Bessou JP: Changes in mesenteric vascular reactivity and inflammatory response after cardiopulmonary bypass in a rat model. Ann Thorac Surg 2004; 77: 2130,7; author reply 2137

10. Larmann J, Theilmeier G: Inflammatory response to cardiac surgery: cardiopulmonary bypass versus non-cardiopulmonary bypass surgery. Best Pract Res Clin Anaesthesiol 2004; 18: 425-38

11. Sistino JJ, Acsell JR: Systemic inflammatory response syndrome (SIRS) following emergency cardiopulmonary bypass: a case report and literature review. J Extra Corpor Technol 1999; 31: 37-43


13. Ulbrich H, Eriksson EE, Lindbom L: Leukocyte and endothelial cell adhesion molecules as targets for therapeutic interventions in inflammatory disease. Trends Pharmacol Sci 2003; 24: 640-7

14. Sanjabi S, Zenewicz LA, Kamanaka M, Flavell RA: Anti-inflammatory and pro-inflammatory roles of TGF-beta, IL-10, and IL-22 in immunity and autoimmunity. Curr Opin Pharmacol 2009; 9: 447-53

15. Fujii M, Miyagi Y, Bessho R, Nitta T, Ochi M, Shimizu K: Effect of a neutrophil elastase inhibitor on acute lung injury after cardiopulmonary bypass. Interact Cardiovasc Thorac Surg 2010; 10: 859-62

16. Zheng JH, Gao BT, Jiang ZM, Yu XQ, Xu ZW: Evaluation of Early Macrophage Activation and NF-kappaB Activity in Pulmonary Injury Caused by Deep Hypothermia Circulatory Arrest: An Experimental Study. Pediatr Cardiol 2009



19. Gueret G, Lion F, Guriec N, Arvieux J, Dovergne A, Guennegan C, Bezon E, Baron R, Carre JL, Arvieux C: Acute renal dysfunction after cardiac surgery with cardiopulmonary bypass is associated with plasmatic IL6 increase. Cytokine 2009; 45: 92-8

20. Asimakopoulos G, Taylor KM: Effects of cardiopulmonary bypass on leukocyte and endothelial adhesion molecules. Ann Thorac Surg 1998; 66: 2135-44

21. Brix-Christensen V, Vestergaard C, Chew M, Johnsen CK, Andersen SK, Dreyer K, Hjortdal VE, Ravn HB, Tonnesen E: Plasma cytokines do not reflect expression of pro- and anti-inflammatory cytokine mRNA at organ level after cardiopulmonary bypass in neonatal pigs. Acta Anaesthesiol Scand 2003; 47: 525-31



24. Huwiler A, Pfeilschifter J: New players on the center stage: sphingosine 1-phosphate and its receptors as drug targets. Biochem Pharmacol 2008; 75: 1893-900

25. Brinkmann V, Baumruker T: Pulmonary and vascular pharmacology of sphingosine 1-phosphate. Curr Opin Pharmacol 2006; 6: 244-50


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26. Graler MH: Targeting sphingosine 1-phosphate (S1P) levels and S1P receptor functions for therapeutic immune interventions. Cell Physiol Biochem 2010; 26: 79-86

27. Lucke S, Levkau B: Endothelial functions of sphingosine-1-phosphate. Cell Physiol Biochem 2010; 26: 87-96

28. Abboud B, Daher R, Boujaoude J: Acute mesenteric ischemia after cardio-pulmonary bypass surgery. World J Gastroenterol 2008; 14: 5361-70

29. Andrasi TB, Buhmann V, Soos P, Juhasz-Nagy A, Szabo G: Mesenteric complications after hypothermic cardiopulmonary bypass with cardiac arrest: underlying mechanisms. Artif Organs 2002; 26: 943-6

30. Aghdaii N, Faritous SZ, Yazdanian F, Zade HR: Acute respiratory distress syndrome: rapid and significant response to volume-controlled inverse ratio ventilation--a case report. Middle East J Anesthesiol 2009; 20: 457-60

31. Picca S, Ricci Z, Picardo S: Acute kidney injury in an infant after cardiopulmonary bypass. Semin Nephrol 2008; 28: 470-6

32. Abu-Omar Y, Ratnatunga C: Cardiopulmonary bypass and renal injury. Perfusion 2006; 21: 209-13 33. Su XW, Undar A: Brain protection during pediatric cardiopulmonary bypass. Artif Organs 2010; 34:

E91-102 34. Piriou V, Mantz J, Goldfarb G, Kitakaze M, Chiari P, Paquin S, Cornu C, Lecharny JB, Aussage P,

Vicaut E, Pons A, Lehot JJ: Sevoflurane preconditioning at 1 MAC only provides limited protection in patients undergoing coronary artery bypass surgery: a randomized bi-centre trial. Br J Anaesth 2007; 99: 624-31

35. Pouzet B, Lecharny JB, Dehoux M, Paquin S, Kitakaze M, Mantz J, Menasche P: Is there a place for preconditioning during cardiac operations in humans? Ann Thorac Surg 2002; 73: 843-8

36. Ji Q, Mei Y, Wang X, Feng J, Cai J, Sun Y: Impact of diabetes mellitus on old patients undergoing coronary artery bypass grafting. Int Heart J 2009; 50: 693-700

37. Guo HW, Chang Q, Xu JP, Song YH, Sun HS, Hu SS: Coronary artery bypass grafting for Kawasaki disease. Chin Med J (Engl) 2010; 123: 1533-6

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ns

0.7

1.7

ns

3163

27

AR

ID5A

A

T ric

h in

tera

ctiv

e do

mai

n 5A

(Mrf1

like

) 17

.9

1.7

.00

0.6

4.2

ns

2.3

1.7

ns

3101

32

OS

MR

on

cost

atin

M re

cept

or

17.4

4.

1 .0

0 1.

4 1.

9 ns

1.

6 1.

7 ns

29

9206

B

ATF

ba

sic

leuc

ine

zipp

er tr

ansc

riptio

n fa

ctor

, ATF

-like

16

.8

1.7

.00

1.1

1.2

ns

0.9

1.1

ns

7897

1 B

IRC

3 ba

culo

vira

l IA

P re

peat

-con

tain

ing

3 16

.3

2.1

.00

0.9

1.0

ns

1.1

1.0

ns

3652

45

SA

A4

seru

m a

myl

oid

A4,

con

stitu

tive

16.1

1.

3 .0

1 2.

1 9.

5 ns

1.

7 1.

3 ns

30

9527

C

H25

H

chol

este

rol 2

5-hy

drox

ylas

e 16

.1

2.7

.00

1.0

1.1

ns

1.0

0.8

ns

3023

78

LPA

R4

lyso

phos

phat

idic

aci

d re

cept

or 4

0.

1 0.

5 .0

1 0.

4 0.

6 ns

1.

2 0.

6 ns

29

619

BTG

2 B

TG fa

mily

, mem

ber 2

15

.1

1.8

.00

0.8

1.0

ns

1.1

0.9

ns

2879

74

KLH

L6

kelc

h-lik

e 6

0.1

0.9

.00

0.8

0.9

ns

0.9

0.9

ns

2568

5 IG

FBP

1 in

sulin

-like

gro

wth

fact

or b

indi

ng p

rote

in 1

14

.3

3.0

.00

1.2

1.7

ns

0.8

1.1

ns

2532

5 IL

10

inte

rleuk

in 1

0 14

.2

1.0

.00

1.0

1.0

ns

1.0

1.4

ns

5885

3 N

R4A

3 nu

clea

r rec

epto

r sub

fam

ily 4

, gro

up A

, mem

ber 3

14

.2

1.2

.00

1.0

1.3

ns

1.1

0.7

ns

2457

7 M

YC

m

yelo

cyto

mat

osis

onc

ogen

e 13

.6

3.3

.00

1.2

1.8

ns

1.1

1.2

ns

3628

48

PR

AM

1 P

ML-

RA

RA

regu

late

d ad

apto

r mol

ecul

e 1

13.5

1.

7 .0

2 2.

9 0.

6 ns

3.

1 1.

0 ns

Supplements 141

!

2875

61

CC

L7

chem

okin

e (C

-C m

otif)

liga

nd 7

13

.4

1.9

.00

0.8

0.9

ns

0.9

0.7

ns

1170

22

IL1R

2 in

terle

ukin

1 re

cept

or, t

ype

II 11

.7

1.1

.00

0.9

1.2

ns

0.3

0.4

ns

2956

71

RG

D13

0889

0 si

mila

r to

RIK

EN

cD

NA

493

0506

L13

0.1

0.6

.02

0.9

1.1

ns

1.1

1.0

ns

3063

51

GD

F1

grow

th d

iffer

entia

tion

fact

or 1

0.

1 0.

8 .0

1 1.

2 1.

0 ns

1.

5 1.

8 ns

69

0672

LO

C69

0672

si

mila

r to

Dis

cs la

rge

hom

olog

5 (P

lace

nta

and

pros

tate

DLG

) (D

iscs

larg

e pr

otei

n P

-dlg

) 0.

1 0.

7 .0

4 0.

4 0.

8 ns

0.

9 0.

4 ns

2924

61

RA

ET1

L re

tinoi

c ac

id e

arly

tran

scrip

t 1L

10.8

1.

0 .0

0 1.

0 4.

2 ns

1.

2 1.

0 ns

79

426

ZFP

36

zinc

fing

er p

rote

in 3

6 10

.7

1.2

.00

1.2

1.2

ns

1.1

1.1

ns

3628

61

SLC

41A

2 so

lute

car

rier f

amily

41,

mem

ber 2

10

.4

1.1

.02

1.6

1.4

ns

1.9

1.0

ns

3088

94

RG

D13

0984

7 si

mila

r to

pept

idyl

glyc

ine

alph

a-am

idat

ing

mon

ooxy

gena

se C

OO

H-te

rmin

al

inte

ract

or; p

eptid

ylgl

ycin

e al

pha-

amid

atin

g m

onoo

xyge

nase

CO

OH

-term

inal

in

tera

ctor

pro

tein

-1

0.1

0.8

.00

0.5

0.8

ns

1.1

1.0

ns

8531

1 P

LA1A

ph

osph

olip

ase

A1

mem

ber A

10

.2

1.8

.00

4.0

2.4

ns

1.2

1.1

ns

5066

2 R

UN

X1

runt

rela

ted

trans

crip

tion

fact

or 1

10

.1

2.2

.01

1.1

1.1

ns

2.0

1.3

ns

2543

3 H

BE

GF

hepa

rin-b

indi

ng E

GF-

like

grow

th fa

ctor

9.

8 1.

1 .0

1 0.

4 0.

6 ns

0.

7 0.

4 ns

11

6596

M

AP

3K8

mito

gen-

activ

ated

pro

tein

kin

ase

kina

se k

inas

e 8

9.5

1.1

.00

1.0

1.1

ns

0.9

0.9

ns

2869

29

CY

P3A

23/3

A1

cyto

chro

me

P45

0, fa

mily

3, s

ubfa

mily

a, p

olyp

eptid

e 23

/pol

ypep

tide

1 9.

4 1.

5 .0

2 1.

0 5.

6 ns

1.

0 1.

0 ns

25

066

PV

R

polio

viru

s re

cept

or

9.3

2.0

.01

1.0

1.4

ns

1.0

0.8

ns

2425

3 C

EB

PB

C

CA

AT/

enha

ncer

bin

ding

pro

tein

(C/E

BP

), be

ta

9.3

2.3

.00

1.2

1.4

ns

1.2

1.2

ns

1142

03

SH

2B2

SH

2B a

dapt

or p

rote

in 2

9.

3 1.

6 .0

0 0.

5 1.

3 ns

1.

4 1.

1 ns

31

5711

S

EM

A7A

se

ma

dom

ain,

imm

unog

lobu

lin d

omai

n (Ig

), an

d G

PI m

embr

ane

anch

or,

(sem

apho

rin) 7

A

9.2

2.5

.00

1.2

0.9

ns

1.0

1.0

ns

4449

83

AP

OLD

1 ap

olip

opro

tein

L d

omai

n co

ntai

ning

1

8.9

1.7

.00

1.1

1.4

ns

1.1

1.1

ns

2546

4 IC

AM

1 in

terc

ellu

lar a

dhes

ion

mol

ecul

e 1

8.9

2.0

.00

1.1

1.2

ns

0.9

1.0

ns

4986

82

RG

D15

6632

5 si

mila

r to

regu

lato

r of s

ex-li

mita

tion

cand

idat

e 16

0.

1 0.

9 .0

0 1.

7 1.

2 ns

1.

0 1.

3 ns

17

0897

S

PH

K1

sphi

ngos

ine

kina

se 1

8.

5 1.

4 .0

1 0.

8 1.

1 ns

1.

2 1.

1 ns

31

6241

N

FKB

IE

nucl

ear f

acto

r of k

appa

ligh

t pol

ypep

tide

gene

enh

ance

r in

B-c

ells

inhi

bito

r, ep

silo

n 4.

2 1.

0 n

s 0.

1 0.

8 0.

00

0.6

0.5

ns

5003

00

LOC

5003

00

sim

ilar t

o hy

poth

etic

al p

rote

in M

GC

6835

8.

3 0.

4 .0

0 1.

5 1.

1 ns

0.

8 1.

0 ns

29

527

PTG

S2

pros

tagl

andi

n-en

dope

roxi

de s

ynth

ase

2 8.

2 0.

8 .0

0 1.

0 0.

7 ns

0.

8 0.

8 ns

28

8529

G

JC3

gap

junc

tion

prot

ein,

gam

ma

3 8.

1 1.

9 .0

2 1.

2 1.

5 ns

1.

0 1.

0 ns

24

517

JUN

B

jun

B p

roto

-onc

ogen

e 8.

1 1.

6 .0

0 0.

9 1.

4 ns

1.

3 1.

1 ns

83

518

AP

LNR

ap

elin

rece

ptor

0.

1 0.

5 .0

0 0.

7 0.

8 ns

1.

0 1.

0 ns

28

7422

P

ER

1 pe

riod

hom

olog

1

7.8

1.6

.00

4.0

3.8

ns

2.6

3.0

ns

2898

01

UP

P1

urid

ine

phos

phor

ylas

e 1

7.7

3.2

.00

1.3

1.4

ns

0.9

0.9

ns

3676

20

MG

C11

6197

si

mila

r to

RIK

EN

cD

NA

170

0001

E04

0.

1 0.

6 .0

2 0.

7 0.

9 ns

1.

2 0.

8 ns

25

531

RA

B3A

R

AB

3A, m

embe

r RA

S o

ncog

ene

fam

ily

0.1

0.7

.01

1.1

1.2

ns

1.2

1.2

ns

1706

37

SA

MS

N1

SA

M d

omai

n, S

H3

dom

ain

and

nucl

ear l

ocal

izat

ion

sign

als,

1

7.5

1.2

.00

1.8

1.1

ns

1.0

0.9

ns

2536

1 V

CA

M1

vasc

ular

cel

l adh

esio

n m

olec

ule

1 7.

0 1.

4 .0

0 1.

1 1.

1 ns

1.

0 1.

0 ns

79

252

AD

AM

TS1

AD

AM

met

allo

pept

idas

e w

ith th

rom

bosp

ondi

n ty

pe 1

mot

if, 1

6.

8 1.

5 .0

2 1.

2 1.

7 ns

1.

2 1.

1 ns

84

586

FGL2

fib

rinog

en-li

ke 2

6.

7 2.

1 .0

0 1.

1 1.

0 ns

0.

7 0.

9 ns

29

194

CH

KA

ch

olin

e ki

nase

alp

ha

6.6

3.3

.00

1.1

1.1

ns

1.3

1.2

ns

4977

57

GU

CY

1A3

guan

ylat

e cy

clas

e 1,

sol

uble

, alp

ha 3

0.

2 0.

6 .0

0 1.

0 0.

9 ns

1.

0 0.

9 ns


!

3084

08

GP

R4

G p

rote

in-c

oupl

ed re

cept

or 4

6.

4 1.

6 .0

0 1.

5 1.

4 ns

1.

3 1.

2 ns

29

9626

G

AD

D45

B

grow

th a

rres

t and

DN

A-d

amag

e-in

duci

ble,

bet

a 6.

4 1.

2 .0

0 0.

8 0.

7 ns

0.

9 1.

1 ns

31

2309

ZN

F775

zi

nc fi

nger

pro

tein

775

0.

2 0.

7 .0

1 1.

2 0.

9 ns

1.

2 1.

4 ns

24

508

IRF1

in

terfe

ron

regu

lato

ry fa

ctor

1

6.2

1.5

.00

1.0

1.2

ns

1.1

0.9

ns

3108

77

TIFA

TR

AF-

inte

ract

ing

prot

ein

with

fork

head

-ass

ocia

ted

dom

ain

6.2

1.0

.00

1.0

0.9

ns

0.8

0.8

ns

1165

10

TIM

P1

TIM

P m

etal

lope

ptid

ase

inhi

bito

r 1

6.1

1.6

.02

2.7

1.7

ns

1.1

1.1

ns

2536

9 A

DO

RA

2A

aden

osin

e A

2a re

cept

or

6.1

2.1

.00

1.5

1.0

ns

1.4

1.2

ns

3146

19

SB

NO

2 st

raw

berr

y no

tch

hom

olog

2

6.1

1.4

.02

1.1

1.2

ns

1.2

1.2

ns

2924

4 G

CH

1 G

TP c

yclo

hydr

olas

e 1

6.1

0.7

.00

0.9

0.6

ns

0.5

0.5

ns

2549

3 N

FKB

IA

nucl

ear f

acto

r of k

appa

ligh

t pol

ypep

tide

gene

enh

ance

r in

B-c

ells

inhi

bito

r, al

pha

6.0

1.4

.00

1.2

1.2

ns

1.1

1.0

ns

3080

97

TAG

AP

T-

cell

activ

atio

n G

TPas

e ac

tivat

ing

prot

ein

6.0

1.0

.03

1.3

1.3

ns

1.0

1.0

ns

5932

9 S

NF1

LK

SN

F1-li

ke k

inas

e 6.

0 0.

9 .0

1 1.

1 1.

2 ns

1.

6 1.

3 ns

30

8451

IT

PK

C

inos

itol 1

,4,5

-tris

phos

phat

e 3-

kina

se C

5.

9 1.

1 .0

0 0.

9 0.

9 ns

1.

0 1.

1 ns

49

9839

R

GD

1564

664

sim

ilar t

o LO

C38

7763

pro

tein

5.

9 1.

3 .0

0 0.

7 0.

7 ns

0.

7 0.

7 ns

29

3538

H

MX

2 H

6 fa

mily

hom

eobo

x 2

0.2

1.2

.02

0.7

0.9

ns

0.6

0.9

ns

7896

5 C

SF1

co

lony

stim

ulat

ing

fact

or 1

(mac

roph

age)

5.

6 1.

3 .0

0 0.

9 1.

0 ns

1.

0 0.

9 ns

58

954

KLF

6 K

rupp

el-li

ke fa

ctor

6

5.5

1.9

.00

1.2

1.3

ns

1.0

1.0

ns

3096

96

BC

R

brea

kpoi

nt c

lust

er re

gion

5.

5 1.

1 .0

0 1.

0 1.

0 ns

1.

2 1.

2 ns

31

3806

R

NF1

9B

ring

finge

r pro

tein

19B

5.

4 1.

1 .0

0 1.

0 1.

0 ns

1.

3 0.

9 ns

64

195

MG

L1

mac

roph

age

gala

ctos

e N

-ace

tyl-g

alac

tosa

min

e sp

ecifi

c le

ctin

1

5.3

1.7

.00

1.8

1.2

ns

1.6

1.2

ns

3035

19

KR

T25

kera

tin 2

5 1.

0 1.

0 ns

3.

1 1.

0 ns

5.

2 1.

5 0.

02

3623

36

FAM

180A

fa

mily

with

seq

uenc

e si

mila

rity

180,

mem

ber A

0.

2 1.

0 .0

0 2.

1 1.

4 ns

1.

3 1.

4 ns

49

8276

LO

C49

8276

Fc

gam

ma

rece

ptor

II b

eta

5.1

1.7

.00

1.3

0.9

ns

1.5

1.1

ns

1710

71

PP

P1R

15A

pr

otei

n ph

osph

atas

e 1,

regu

lato

ry (i

nhib

itor)

sub

unit

15A

4.

9 1.

3 .0

0 0.

8 0.

9 ns

0.

9 1.

0 ns

29

187

CD

69

Cd6

9 m

olec

ule

4.9

1.1

.00

0.7

1.0

ns

0.4

0.6

ns

4943

44

IER

2 im

med

iate

ear

ly re

spon

se 2

4.

7 1.

3 .0

0 0.

8 0.

9 ns

1.

0 0.

9 ns

30

4663

LY

L1

lym

phob

last

ic le

ukem

ia d

eriv

ed s

eque

nce

1 0.

2 1.

0 .0

0 1.

2 0.

8 ns

1.

3 1.

0 ns

30

9452

N

FKB

2 nu

clea

r fac

tor o

f kap

pa li

ght p

olyp

eptid

e ge

ne e

nhan

cer i

n B

-cel

ls 2

, p4

9/p1

00

4.6

1.3

.00

1.1

1.1

ns

1.0

1.1

ns

5006

23

RG

D15

6370

1 si

mila

r to

BC

0682

81 p

rote

in

0.2

0.7

.00

0.9

0.9

ns

1.1

0.7

ns

2433

4 E

NO

2 en

olas

e 2,

gam

ma,

neu

rona

l 4.

6 1.

6 .0

0 1.

2 1.

2 ns

1.

6 1.

2 ns

36

2491

R

IPK

2 re

cept

or-in

tera

ctin

g se

rine-

thre

onin

e ki

nase

2

4.6

1.4

.00

1.3

1.2

ns

1.3

1.2

ns

4052

97

OLR

114

olfa

ctor

y re

cept

or 1

14

1.3

1.0

ns

1.0

1.0

ns

4.4

1.0

0.04

25

426

CY

P1B

1 cy

toch

rom

e P

450,

fam

ily 1

, sub

fam

ily b

, pol

ypep

tide

1 4.

4 1.

4 .0

0 0.

7 0.

8 ns

0.

5 0.

5 ns

30

7351

TU

BB

6 tu

bulin

, bet

a 6

4.4

1.5

.02

1.3

1.3

ns

1.1

0.8

ns

2491

4 LO

X

lysy

l oxi

dase

4.

3 1.

4 .0

0 1.

0 1.

0 ns

1.

0 0.

9 ns

29

7902

G

EM

G

TP b

indi

ng p

rote

in (g

ene

over

expr

esse

d in

ske

leta

l mus

cle)

4.

3 1.

7 .0

0 1.

6 1.

4 ns

0.

8 0.

9 ns

31

6019

C

CR

L2

chem

okin

e (C

-C m

otif)

rece

ptor

-like

2

4.3

0.9

.00

0.7

0.5

ns

0.5

0.9

ns

2909

11

RG

D15

6480

7 si

mila

r to

zinc

fing

er p

rote

in, s

ubfa

mily

1A

, 5

1.4

1.1

ns

4.3

1.0

0.01

1.

2 1.

0 ns

28

8239

D

SC

R6

Dow

n sy

ndro

me

criti

cal r

egio

n ho

mol

og 6

0.

2 0.

8 .0

0 0.

9 1.

0 ns

0.

9 1.

0 ns

17

1099

R

AS

D2

RA

SD

fam

ily, m

embe

r 2

0.2

0.9

.03

1.0

0.9

ns

0.9

1.0

ns

1408

60

SLC

O2B

1 so

lute

car

rier o

rgan

ic a

nion

tran

spor

ter f

amily

, mem

ber 2

b1

0.2

0.6

.00

1.1

0.9

ns

1.2

1.2

ns

Supplements 143

!

1170

29

CC

R5

chem

okin

e (C

-C m

otif)

rece

ptor

5

4.1

1.0

.01

1.3

1.2

ns

1.2

1.2

ns

4981

79

RG

D15

6348

2 si

mila

r to

hypo

thet

ical

pro

tein

FLJ

3866

3 0.

2 0.

6 .0

0 0.

9 0.

8 ns

1.

0 0.

9 ns

50

2018

R

GD

1563

728

sim

ilar t

o co

iled-

coil-

helix

-coi

led-

coil-

helix

dom

ain

cont

aini

ng 2

1.

2 1.

0 ns

4.

0 1.

0 0.

04

1.0

1.0

ns

3037

98

AR

VC

F ar

mad

illo

repe

at g

ene

dele

ted

in v

elo-

card

io-fa

cial

syn

drom

e 0.

3 0.

6 .0

0 0.

8 0.

7 ns

1.

0 1.

0 ns

36

3140

R

AS

SF1

R

as a

ssoc

iatio

n (R

alG

DS

/AF-

6) d

omai

n m

embe

r 1

4.0

1.5

.00

0.9

1.0

ns

0.8

1.0

ns

2940

07

PP

RC

1 pe

roxi

som

e pr

olife

rato

r-ac

tivat

ed re

cept

or g

amm

a, c

oact

ivat

or-r

elat

ed 1

4.

0 1.

8 .0

0 0.

9 1.

2 ns

1.

1 1.

2 ns

29

4235

IE

R3

imm

edia

te e

arly

resp

onse

3

3.9

1.0

.00

0.9

1.1

ns

1.2

1.1

ns

5026

57

RG

D15

6356

5 si

mila

r to

cyto

chro

me

c ox

idas

e su

buni

t IV

0.

7 0.

9 ns

0.

7 0.

7 ns

3.

9 0.

7 0.

00

3137

22

SP

SB

1 sp

lA/ry

anod

ine

rece

ptor

dom

ain

and

SO

CS

box

con

tain

ing

1 3.

9 1.

7 .0

0 1.

6 1.

5 ns

1.

5 1.

3 ns

49

7886

C

1QTN

F2

C1q

and

tum

or n

ecro

sis

fact

or re

late

d pr

otei

n 2

0.3

0.8

.00

0.9

1.0

ns

1.1

1.0

ns

2542

9 C

YP

7B1

cyto

chro

me

P45

0, fa

mily

7, s

ubfa

mily

b, p

olyp

eptid

e 1

3.8

0.8

.00

0.9

1.0

ns

1.0

0.9

ns

2882

57

DO

NS

ON

do

wns

tream

nei

ghbo

r of S

ON

3.

8 1.

3 .0

0 1.

5 1.

7 ns

1.

4 1.

4 ns

11

4856

D

US

P1

dual

spe

cific

ity p

hosp

hata

se 1

3.

8 1.

5 .0

0 0.

8 1.

0 ns

1.

1 0.

9 ns

30

7811

S

LC7A

6 so

lute

car

rier f

amily

7 (c

atio

nic

amin

o ac

id tr

ansp

orte

r, y+

sys

tem

), m

embe

r 6

3.7

1.3

.00

1.0

1.0

ns

1.0

0.8

ns

3091

75

CD

C42

EP

2 C

DC

42 e

ffect

or p

rote

in (R

ho G

TPas

e bi

ndin

g) 2

3.

7 1.

5 .0

1 1.

2 1.

2 ns

1.

5 1.

1 ns

24

423

GS

TM1

glut

athi

one

S-tr

ansf

eras

e m

u 1

1.5

0.9

ns

3.6

1.1

0.00

0.

9 0.

7 ns

60

350

CD

14

CD

14 m

olec

ule

3.6

1.5

.00

1.2

1.0

ns

1.0

0.9

ns

2991

45

RH

OJ

ras

hom

olog

gen

e fa

mily

, mem

ber J

3.

6 1.

5 .0

0 0.

9 0.

8 ns

1.

1 0.

9 ns

28

9443

LR

RC

8C

leuc

ine

rich

repe

at c

onta

inin

g 8

fam

ily, m

embe

r C

3.6

1.0

.04

1.0

0.9

ns

0.6

0.8

ns

2511

2 G

AD

D45

A

grow

th a

rres

t and

DN

A-d

amag

e-in

duci

ble,

alp

ha

3.6

1.3

.00

0.8

1.0

ns

1.0

1.0

ns

2417

3 A

DR

A1B

ad

rene

rgic

, alp

ha-1

B-,

rece

ptor

0.

3 0.

6 .0

0 0.

9 1.

0 ns

0.

8 1.

0 ns

29

1908

TO

X3

TOX

hig

h m

obili

ty g

roup

box

fam

ily m

embe

r 3

0.3

0.6

.01

0.7

0.9

ns

0.7

0.8

ns

3105

53

TLR

2 to

ll-lik

e re

cept

or 2

3.

4 1.

1 .0

0 0.

8 0.

7 ns

0.

9 0.

8 ns

36

4206

P

LEK

pl

ecks

trin

3.4

1.4

.00

1.3

1.1

ns

1.1

1.0

ns

6383

9 FH

L2

four

and

a h

alf L

IM d

omai

ns 2

0.

3 0.

7 .0

1 1.

1 0.

8 ns

0.

9 0.

9 ns

28

7129

TM

EM

204

trans

mem

bran

e pr

otei

n 20

4 0.

3 0.

8 .0

0 1.

1 1.

0 ns

1.

0 1.

1 ns

29

7508

N

AM

PT

nico

tinam

ide

phos

phor

ibos

yltra

nsfe

rase

3.

4 1.

3 .0

1 1.

2 1.

6 ns

1.

1 1.

2 ns

11

4121

C

CN

L1

cycl

in L

1 3.

4 1.

7 .0

0 1.

3 1.

1 ns

1.

4 1.

4 ns

36

1993

R

GD

1562

059

sim

ilar t

o R

IKE

N c

DN

A 1

1100

38F2

1 0.

3 0.

6 .0

1 0.

6 0.

7 ns

0.

7 1.

0 ns

30

6330

K

LF2

Kru

ppel

-like

fact

or 2

0.

3 0.

6 .0

0 1.

3 1.

1 ns

0.

9 1.

1 ns

25

7634

LH

X1

LIM

hom

eobo

x 1

0.3

0.8

.00

0.8

0.9

ns

0.9

1.0

ns

8152

0 M

AR

CK

SL1

M

AR

CK

S-li

ke 1

3.

3 1.

3 .0

1 1.

0 0.

8 ns

1.

5 0.

8 ns

84

398

CD

93

CD

93 m

olec

ule

3.3

1.2

.00

0.7

0.7

ns

1.0

0.9

ns

1711

64

GB

P2

guan

ylat

e bi

ndin

g pr

otei

n 2

3.3

0.8

.01

1.2

1.1

ns

1.1

0.7

ns

7943

1 B

HLH

E40

ba

sic

helix

-loop

-hel

ix fa

mily

, mem

ber e

40

3.3

1.7

.00

1.2

1.0

ns

1.1

1.0

ns

3054

23

C1Q

TNF7

C

1q a

nd tu

mor

nec

rosi

s fa

ctor

rela

ted

prot

ein

7 0.

3 0.

8 .0

0 1.

3 1.

1 ns

1.

1 1.

3 ns

29

3023

K

LHL2

5 ke

lch-

like

25

0.3

1.0

.04

1.1

0.6

ns

0.6

0.8

ns

7923

7 H

HE

X

hem

atop

oiet

ical

ly e

xpre

ssed

hom

eobo

x 0.

3 1.

2 .0

0 1.

0 1.

0 ns

1.

1 1.

0 ns

36

2564

B

EN

D5

BE

N d

omai

n co

ntai

ning

5

0.3

0.7

.00

0.7

0.8

ns

1.1

1.2

ns

3049

54

UA

P1L

2 U

DP

-N-a

ctey

lglu

cosa

min

e py

roph

osph

oryl

ase

1-lik

e 2

3.1

1.3

.00

0.7

1.0

ns

1.1

0.9

ns

3135

95

RG

D15

6307

2 si

mila

r to

hypo

thet

ical

pro

tein

FLJ

3898

4 0.

3 1.

1 .0

0 1.

2 1.

2 ns

1.

4 1.

2 ns

36

2703

W

DR

43

WD

repe

at d

omai

n 43

3.

1 1.

8 .0

0 1.

0 1.

2 ns

1.

0 1.

0 ns


!

3142

43

WD

R89

W

D re

peat

dom

ain

89

0.3

0.7

.00

0.9

0.9

ns

1.0

1.0

ns

2520

2 G

UC

Y1B

3 gu

anyl

ate

cycl

ase

1, s

olub

le, b

eta

3 0.

3 0.

7 .0

2 1.

0 0.

9 ns

0.

9 0.

8 ns

30

1416

C

OQ

10B

co

enzy

me

Q10

hom

olog

B

3.1

1.5

.00

1.0

1.2

ns

1.0

0.9

ns

2953

23

TRIM

45

tripa

rtite

mot

if-co

ntai

ning

45

0.3

0.6

.00

1.1

1.0

ns

1.2

1.0

ns

2469

7 P

TPN

1 pr

otei

n ty

rosi

ne p

hosp

hata

se, n

on-r

ecep

tor t

ype

1 3.

0 1.

3 .0

0 1.

0 1.

0 ns

1.

1 1.

2 ns

14

0942

D

DIT

4 D

NA

-dam

age-

indu

cibl

e tra

nscr

ipt 4

3.

0 1.

0 .0

4 1.

9 1.

2 ns

1.

1 1.

2 ns

31

3436

ZC

CH

C12

zi

nc fi

nger

, CC

HC

dom

ain

cont

aini

ng 1

2 3.

0 1.

3 .0

0 0.

6 1.

1 ns

1.

2 1.

3 ns

11

4495

M

AP

2K6

mito

gen-

activ

ated

pro

tein

kin

ase

kina

se 6

0.

3 0.

6 .0

0 1.

1 1.

1 ns

1.

0 1.

0 ns

29

9411

LO

C29

9411

si

mila

r to

Ig h

eavy

cha

in V

regi

on M

OP

C 2

1 pr

ecur

sor

1.0

1.3

ns

1.0

1.0

ns

3.0

1.3

0.00

65

161

LITA

F lip

opol

ysac

char

ide-

indu

ced

TNF

fact

or

3.0

1.5

.00

1.2

1.3

ns

1.1

1.1

ns

8458

3 R

GS

2 re

gula

tor o

f G-p

rote

in s

igna

ling

2 3.

0 1.

1 .0

0 1.

2 1.

3 ns

1.

1 1.

1 ns

24

6760

M

AFK

v-

maf

mus

culo

apon

euro

tic fi

bros

arco

ma

onco

gene

hom

olog

K (a

vian

) 2.

9 1.

7 .0

0 1.

0 1.

3 ns

1.

1 1.

2 ns

79

255

ATF

4 ac

tivat

ing

trans

crip

tion

fact

or 4

(tax

-res

pons

ive

enha

ncer

ele

men

t B67

) 2.

9 1.

7 .0

0 0.

9 1.

1 ns

0.

9 0.

9 ns

24

5963

E

GFL

7 E

GF-

like-

dom

ain,

mul

tiple

7

0.3

1.2

.01

0.8

0.9

ns

1.0

1.0

ns

3005

19

ES

AM

en

doth

elia

l cel

l adh

esio

n m

olec

ule

2.9

1.5

.00

1.0

1.0

ns

1.3

1.1

ns

3641

36

AA

SD

H

amin

oadi

pate

-sem

iald

ehyd

e de

hydr

ogen

ase

0.4

0.9

.00

1.3

0.9

ns

1.1

1.3

ns

3081

13

CN

KS

R3

Cnk

sr fa

mily

mem

ber 3

2.

8 1.

3 .0

1 0.

8 0.

9 ns

1.

1 1.

1 ns

11

7540

P

LSC

R1

phos

phol

ipid

scr

ambl

ase

1 2.

8 0.

7 .0

1 1.

1 1.

3 ns

1.

1 1.

1 ns

83

626

UG

CG

U

DP

-glu

cose

cer

amid

e gl

ucos

yltra

nsfe

rase

2.

8 0.

9 .0

0 0.

8 1.

0 ns

1.

0 1.

0 ns

25

472

KC

NJ8

po

tass

ium

inw

ardl

y-re

ctify

ing

chan

nel,

subf

amily

J, m

embe

r 8

2.8

1.1

.01

1.9

1.1

ns

1.2

1.1

ns

1140

90

EG

R2

early

gro

wth

resp

onse

2

2.8

1.0

.00

1.0

0.9

ns

0.8

0.9

ns

2562

5 TN

FRS

F1A

tu

mor

nec

rosi

s fa

ctor

rece

ptor

sup

erfa

mily

, mem

ber 1

a 2.

8 1.

4 .0

0 0.

9 1.

3 ns

0.

9 1.

0 ns

25

655

GJA

4 ga

p ju

nctio

n pr

otei

n, a

lpha

4

2.8

1.0

.03

1.1

0.8

ns

1.1

1.1

ns

2930

8 A

RL4

A

AD

P-r

ibos

ylat

ion

fact

or-li

ke 4

A

2.8

1.8

.00

1.2

1.3

ns

1.0

1.0

ns

1165

01

SLC

9A3R

2 so

lute

car

rier f

amily

9 (s

odiu

m/h

ydro

gen

exch

ange

r), m

embe

r 3 re

gula

tor 2

0.

4 0.

8 .0

1 1.

1 1.

0 ns

1.

0 1.

2 ns

17

0929

B

CL2

A1D

B

-cel

l leu

kem

ia/ly

mph

oma

2 re

late

d pr

otei

n A

1d

2.7

1.3

.00

1.0

1.0

ns

1.0

0.9

ns

3047

35

TME

M17

7 tra

nsm

embr

ane

prot

ein

177

0.4

0.9

.02

1.0

1.0

ns

1.0

1.0

ns

3063

38

AB

HD

8 ab

hydr

olas

e do

mai

n co

ntai

ning

8

0.4

0.8

.00

0.9

0.8

ns

0.8

0.8

ns

2906

48

PG

PE

P1

pyro

glut

amyl

-pep

tidas

e I

0.4

1.1

.01

1.0

1.0

ns

0.9

1.0

ns

3608

78

KLH

DC

9 ke

lch

dom

ain

cont

aini

ng 9

0.

4 0.

7 .0

0 0.

9 0.

9 ns

1.

1 1.

1 ns

30

2937

A

NK

S3

anky

rin re

peat

and

ste

rile

alph

a m

otif

dom

ain

cont

aini

ng 3

0.

4 0.

8 .0

1 1.

1 0.

8 ns

0.

8 0.

8 ns

50

672

ED

NR

B

endo

thel

in re

cept

or ty

pe B

2.

6 1.

8 .0

0 1.

0 1.

1 ns

1.

1 1.

3 ns

29

3511

ZN

F688

zi

nc fi

nger

pro

tein

688

0.

4 0.

7 .0

0 0.

8 0.

8 ns

0.

8 0.

9 ns

24

6311

P

DP

2 py

ruva

te d

ehyd

roge

nase

pho

spha

tase

isoe

nzym

e 2

0.4

0.8

.04

0.8

0.8

ns

0.9

0.9

ns

2936

69

CD

248

CD

248

mol

ecul

e, e

ndos

ialin

0.

4 0.

9 .0

0 1.

3 1.

1 ns

0.

9 1.

0 ns

24

516

JUN

Ju

n on

coge

ne

2.6

1.2

.00

0.8

1.0

ns

0.9

0.9

ns

2462

08

IFI4

7 in

terfe

ron

gam

ma

indu

cibl

e pr

otei

n 47

2.

6 1.

0 .0

0 1.

1 0.

8 ns

0.

8 0.

8 ns

31

4228

S

NA

PC

1 sm

all n

ucle

ar R

NA

act

ivat

ing

com

plex

, pol

ypep

tide

1 2.

6 1.

2 .0

0 0.

8 1.

1 ns

0.

9 1.

0 ns

49

7832

TR

PC

1 tra

nsie

nt re

cept

or p

oten

tial c

atio

n ch

anne

l, su

bfam

ily C

, mem

ber 1

2.

5 1.

1 .0

1 1.

0 0.

9 ns

1.

1 1.

2 ns

60

341

CA

MK

K1

calc

ium

/cal

mod

ulin

-dep

ende

nt p

rote

in k

inas

e ki

nase

1, a

lpha

0.

4 1.

0 .0

0 1.

1 1.

1 ns

1.

0 1.

1 ns

30

2642

S

AT1

sp

erm

idin

e/sp

erm

ine

N1-

acet

yl tr

ansf

eras

e 1

2.5

1.6

.00

1.1

1.3

ns

0.9

1.0

ns

4983

86

RG

D15

6104

2 si

mila

r to

RIK

EN

cD

NA

573

0509

K17

gen

e 0.

4 0.

8 .0

0 1.

1 1.

1 ns

1.

1 1.

1 ns

24

165

AD

A

aden

osin

e de

amin

ase

0.4

1.5

.00

0.7

0.6

ns

0.7

0.7

ns

Supplements 145

!

3141

26

BA

Z1A

br

omod

omai

n ad

jace

nt to

zin

c fin

ger d

omai

n, 1

A

2.5

1.4

.00

1.1

1.1

ns

0.9

0.9

ns

2945

18

SE

SN

1 se

strin

1

0.4

0.8

.00

0.9

0.9

ns

0.9

0.9

ns

3127

28

TSP

AN

9 te

trasp

anin

9

0.4

0.8

.01

1.0

0.7

ns

0.8

0.9

ns

3132

62

PA

PP

A

preg

nanc

y-as

soci

ated

pla

sma

prot

ein

A

2.5

1.1

.00

0.8

1.0

ns

1.1

1.1

ns

3612

26

CD

83

CD

83 m

olec

ule

2.5

1.2

.00

0.9

0.9

ns

0.7

0.9

ns

3151

19

MFN

G

MFN

G O

-fuco

sylp

eptid

e 3-

beta

-N-a

cety

lglu

cosa

min

yltra

nsfe

rase

0.

4 0.

8 .0

0 1.

5 1.

1 ns

1.

1 1.

0 ns

68

3385

LO

C68

8922

si

mila

r to

Insu

lin-in

duce

d ge

ne 1

pro

tein

(IN

SIG

-1) (

Insu

lin-in

duce

d gr

owth

re

spon

se p

rote

in C

L-6)

(Im

med

iate

-ear

ly p

rote

in C

L-6)

2.

5 1.

1 .0

1 1.

1 0.

8 ns

0.

8 0.

7 ns

3098

28

TSP

YL4

TS

PY

-like

4

0.4

0.7

.01

0.8

1.0

ns

1.0

1.0

ns

6502

6 R

WD

D3

RW

D d

omai

n co

ntai

ning

3

0.4

0.8

.00

1.0

0.9

ns

1.0

1.1

ns

3608

72

RC

SD

1 R

CS

D d

omai

n co

ntai

ning

1

0.4

0.7

.01

0.9

0.7

ns

0.9

0.9

ns

4982

72

UA

P1

UD

P-N

-act

eylg

luco

sam

ine

pyro

phos

phor

ylas

e 1

2.4

1.0

.01

0.9

0.8

ns

0.9

0.8

ns

2569

2 P

LAT

plas

min

ogen

act

ivat

or, t

issu

e 2.

4 1.

6 .0

0 0.

8 1.

0 ns

1.

1 0.

9 ns

36

2762

D

CA

F4

DD

B1

and

CU

L4 a

ssoc

iate

d fa

ctor

4

0.4

0.9

.01

0.8

0.9

ns

1.1

0.9

ns

2478

3 S

LC9A

2 so

lute

car

rier f

amily

9 (s

odiu

m/h

ydro

gen

exch

ange

r), m

embe

r 2

0.4

0.7

.01

0.7

1.0

ns

1.0

0.9

ns

6812

52

LOC

6812

52

sim

ilar t

o M

yris

toyl

ated

ala

nine

-ric

h C

-kin

ase

subs

trate

(MA

RC

KS

) (P

rote

in

kina

se C

sub

stra

te 8

0 kD

a pr

otei

n)

2.4

0.5

.02

1.0

1.0

ns

1.1

0.9

ns

2934

4 ZF

P36

L1

zinc

fing

er p

rote

in 3

6, C

3H ty

pe-li

ke 1

2.

3 1.

0 .0

3 0.

9 1.

3 ns

1.

0 1.

1 ns

30

1227

TR

EM

2 tri

gger

ing

rece

ptor

exp

ress

ed o

n m

yelo

id c

ells

2

0.7

0.6

ns

2.3

0.9

0.00

1.

7 1.

3 ns

29

618

BTG

1 B

-cel

l tra

nslo

catio

n ge

ne 1

, ant

i-pro

lifer

ativ

e 2.

3 1.

2 .0

0 1.

0 1.

0 ns

1.

0 1.

0 ns

31

1807

B

MY

C

brai

n ex

pres

sed

mye

locy

tom

atos

is o

ncog

ene

0.4

0.7

.00

0.9

0.9

ns

1.1

0.9

ns

3072

35

RG

D13

1191

0 si

mila

r to

hypo

thet

ical

p38

pro

tein

0.

4 0.

9 .0

4 0.

7 0.

7 ns

0.

8 0.

9 ns

36

5571

TY

SN

D1

tryps

in d

omai

n co

ntai

ning

1

0.4

0.8

.01

1.0

0.9

ns

1.0

1.0

ns

8533

2 P

RK

CD

BP

pr

otei

n ki

nase

C, d

elta

bin

ding

pro

tein

0.

4 0.

9 .0

0 1.

1 0.

8 ns

0.

9 0.

8 ns

30

4346

M

BLA

C1

met

allo

-bet

a-la

ctam

ase

dom

ain

cont

aini

ng 1

0.

4 0.

9 .0

1 0.

9 0.

8 ns

0.

9 0.

8 ns

50

0150

R

GD

1559

612

sim

ilar t

o tig

ger t

rans

posa

ble

elem

ent d

eriv

ed 2

0.

4 0.

8 .0

0 0.

8 0.

8 ns

0.

8 1.

0 ns

30

8336

ZN

F580

zi

nc fi

nger

pro

tein

580

0.

8 0.

5 ns

0.

4 1.

1 0.

04

1.0

0.9

ns

3099

18

ELL

2 el

onga

tion

fact

or R

NA

pol

ymer

ase

II 2

2.2

1.2

.00

1.0

1.1

ns

1.2

1.1

ns

5896

6 R

AM

P2

rece

ptor

(G p

rote

in-c

oupl

ed) a

ctiv

ity m

odify

ing

prot

ein

2 0.

5 1.

1 .0

0 1.

4 1.

1 ns

1.

1 1.

0 ns

63

997

SLC

29A

1 so

lute

car

rier f

amily

29

(nuc

leos

ide

trans

porte

rs),

mem

ber 1

0.

5 0.

8 .0

5 0.

8 0.

8 ns

0.

8 0.

9 ns

30

1059

M

YD

88

mye

loid

diff

eren

tiatio

n pr

imar

y re

spon

se g

ene

88

2.2

1.0

.01

1.2

1.2

ns

1.2

1.0

ns

3032

00

MA

P2K

3 m

itoge

n ac

tivat

ed p

rote

in k

inas

e ki

nase

3

2.2

1.3

.02

1.1

1.2

ns

1.0

1.1

ns

2889

12

MR

I1

met

hylth

iorib

ose-

1-ph

osph

ate

isom

eras

e ho

mol

og

0.5

0.9

.01

0.9

0.8

ns

1.0

1.0

ns

2871

48

RP

US

D1

RN

A p

seud

ourid

ylat

e sy

ntha

se d

omai

n 1

0.5

0.8

.01

0.8

0.8

ns

0.9

1.0

ns

2869

88

PN

RC

1 pr

olin

e-ric

h nu

clea

r rec

epto

r coa

ctiv

ator

1

2.1

1.2

.00

0.8

0.8

ns

0.8

0.8

ns

4992

14

CH

CH

D8

coile

d-co

il-he

lix-c

oile

d-co

il-he

lix d

omai

n 8

0.5

0.9

.03

1.1

1.0

ns

0.8

0.9

ns

2961

19

DTW

D1

DTW

dom

ain

cont

aini

ng 1

0.

5 0.

8 .0

0 0.

9 1.

0 ns

1.

1 1.

1 ns

30

7907

R

GD

1304

884

sim

ilar t

o R

IKE

N c

DN

A 6

4305

48M

08

0.5

1.0

.00

1.1

1.1

ns

1.1

1.1

ns

3135

60

CTP

S

CTP

syn

thas

e 2.

1 1.

0 .0

1 0.

8 0.

8 ns

0.

8 0.

8 ns

49

9630

R

GD

1565

059

sim

ilar t

o hy

poth

etic

al p

rote

in E

1303

11K

13

0.5

0.9

.00

1.3

1.1

ns

1.1

1.2

ns

3065

87

TCTA

T-

cell

leuk

emia

tran

sloc

atio

n al

tere

d ge

ne

0.5

0.8

.00

1.0

0.9

ns

0.8

0.9

ns

8438

6 S

LPI

secr

etor

y le

ukoc

yte

pept

idas

e in

hibi

tor

2.1

0.8

.01

1.1

0.7

ns

0.8

0.7

ns

6037

3 N

OP

58

nucl

eola

r pro

tein

NO

P58

2.

1 1.

2 .0

0 0.

8 1.

3 ns

1.

0 1.

0 ns


!

2553

7 R

OC

K2

Rho

-ass

ocia

ted

coile

d-co

il co

ntai

ning

pro

tein

kin

ase

2 2.

1 1.

3 .0

0 1.

1 1.

6 ns

1.

4 1.

6 ns

28

7063

N

MR

AL1

N

mrA

-like

fam

ily d

omai

n co

ntai

ning

1

0.5

0.7

.00

0.8

0.8

ns

0.9

1.1

ns

3108

11

PA

LMD

pa

lmde

lphi

n 0.

5 1.

0 .0

1 0.

9 1.

0 ns

1.

0 0.

9 ns

30

9109

TM

EM

80

trans

mem

bran

e pr

otei

n 80

0.

5 0.

9 .0

1 0.

9 0.

9 ns

0.

9 1.

0 ns

29

9207

R

GD

1310

769

sim

ilar t

o H

SP

C28

8 0.

5 0.

9 .0

0 1.

1 1.

1 ns

1.

1 1.

1 ns

30

5351

K

LHL5

ke

lch-

like

5

0.5

0.9

.00

1.0

0.9

ns

0.9

0.9

ns

3035

77

AC

BD

4 ac

yl-C

oenz

yme

A b

indi

ng d

omai

n co

ntai

ning

4

0.5

0.9

.04

0.9

0.8

ns

1.0

1.0

ns

5001

26

HO

XA

9 ho

meo

box

A9

0.5

0.9

.00

1.0

1.0

ns

0.9

1.0

ns

2919

75

RG

D13

0735

7 si

mila

r to

hypo

thet

ical

pro

tein

DK

FZp4

34A

1319

0.

5 0.

9 .0

0 1.

2 1.

1 ns

1.

1 1.

3 ns

29

1661

TM

CO

6 tra

nsm

embr

ane

and

coile

d-co

il do

mai

ns 6

0.

5 1.

0 .0

1 1.

3 1.

2 ns

0.

9 1.

3 ns

49

9085

P

ELI

1

pelli

no 1

2.

0 1.

3 .0

0 1.

0 1.

3 ns

1.

1 1.

1 ns

25

675

HM

GC

R

3-hy

drox

y-3-

met

hylg

luta

ryl-C

oenz

yme

A re

duct

ase

2.0

0.9

.00

1.0

0.9

ns

0.9

0.9

ns

3145

84

ZFP

563

zinc

fing

er p

rote

in 5

63

0.5

0.8

.00

1.1

1.1

ns

0.9

1.0

ns

3636

76

ZPB

P2

zona

pel

luci

da b

indi

ng p

rote

in 2

0.

5 0.

7 .0

0 1.

1 1.

0 ns

1.

2 1.

1 ns

58

840

MA

PK

6 m

itoge

n-ac

tivat

ed p

rote

in k

inas

e 6

1.9

1.1

.00

0.8

0.9

ns

1.1

1.0

ns

3036

12

PO

LG2

poly

mer

ase

(DN

A d

irect

ed),

gam

ma

2, a

cces

sory

sub

unit

0.5

1.1

.00

1.2

1.1

ns

1.0

1.1

ns

3604

61

RG

D13

0582

3 si

mila

r to

RIK

EN

cD

NA

061

0037

P05

0.

5 0.

9 .0

1 0.

9 1.

0 ns

1.

1 0.

9 ns

28

7427

TR

AP

PC

1 tra

ffick

ing

prot

ein

parti

cle

com

plex

1

0.5

0.9

.02

1.0

0.8

ns

0.8

0.9

ns

8434

8 C

XC

R7

chem

okin

e (C

-X-C

mot

if) re

cept

or 7

1.

9 0.

9 .0

0 0.

8 0.

8 ns

0.

8 0.

8 ns

26

6773

ZF

P70

9 zi

nc fi

nger

pro

tein

709

0.

5 1.

1 .0

0 1.

3 1.

1 ns

1.

3 1.

5 ns

28

2845

R

NF3

4 rin

g fin

ger p

rote

in 3

4 0.

5 0.

8 .0

0 1.

1 1.

1 ns

0.

9 1.

0 ns

29

6565

R

GD

1306

215

sim

ilar t

o hy

poth

etic

al p

rote

in M

GC

3683

1 0.

5 0.

9 .0

0 0.

9 0.

8 ns

0.

8 0.

7 ns

29

9639

A

NK

RD

24

anky

rin re

peat

dom

ain

24

0.5

0.9

.04

1.0

0.9

ns

0.8

1.0

ns

3606

47

ICA

M2

inte

rcel

lula

r adh

esio

n m

olec

ule

2 0.

5 1.

0 .0

0 1.

1 1.

0 ns

1.

1 1.

1 ns

36

0754

ZF

P35

8 zi

nc fi

nger

pro

tein

358

0.

5 1.

1 .0

0 1.

0 1.

0 ns

1.

0 1.

0 ns

49

7782

A

BC

B1A

A

TP-b

indi

ng c

asse

tte, s

ub-fa

mily

B (M

DR

/TA

P),

mem

ber 1

A

1.4

0.7

ns

0.5

1.0

0.00

1.

3 1.

0 ns

28

8651

G

TF2H

3 ge

nera

l tra

nscr

iptio

n fa

ctor

IIH

, pol

ypep

tide

3 0.

5 1.

0 .0

1 1.

0 1.

1 ns

0.

9 0.

9 ns

29

534

PX

MP

3 pe

roxi

som

al m

embr

ane

prot

ein

3 0.

5 0.

9 .0

5 0.

9 0.

9 ns

0.

9 0.

8 ns

49

7745

LS

S

lano

ster

ol s

ynth

ase

(2,3

-oxi

dosq

uale

ne-la

nost

erol

cyc

lase

) 1.

8 1.

0 .0

2 0.

9 1.

0 ns

1.

0 1.

0 ns

31

1723

S

OX

18

SR

Y (s

ex d

eter

min

ing

regi

on Y

)-bo

x 18

0.

5 1.

7 .0

1 1.

2 0.

9 ns

1.

1 1.

0 ns

83

614

PIA

S3

prot

ein

inhi

bito

r of a

ctiv

ated

STA

T, 3

0.

6 0.

8 .0

1 0.

9 0.

9 ns

1.

0 0.

9 ns

28

9150

C

EN

PL

cent

rom

ere

prot

ein

L 0.

6 0.

8 .0

0 1.

1 1.

0 ns

0.

9 0.

9 ns

30

9446

H

PS

6 H

erm

ansk

y-P

udla

k sy

ndro

me

6 0.

6 1.

2 .0

0 1.

0 1.

0 ns

1.

0 0.

9 ns

84

019

NA

E1

NE

DD

8 ac

tivat

ing

enzy

me

E1

subu

nit 1

0.

6 0.

9 .0

0 1.

0 0.

9 ns

1.

0 1.

0 ns

64

134

XY

LT2

xylo

syltr

ansf

eras

e II

0.6

0.8

.00

0.9

1.0

ns

1.0

1.1

ns

2976

27

MG

C94

282

sim

ilar t

o 59

3041

6I19

Rik

pro

tein

0.

6 0.

9 .0

0 1.

0 1.

1 ns

1.

1 1.

1 ns

36

5314

LI

PT2

lip

oyl(o

ctan

oyl)

trans

fera

se 2

(put

ativ

e)

0.6

1.0

.01

1.0

0.9

ns

0.9

0.9

ns

2882

33

WR

B

trypt

opha

n ric

h ba

sic

prot

ein

0.6

0.9

.01

1.0

0.9

ns

0.9

0.9

ns

3111

85

KB

TBD

4 ke

lch

repe

at a

nd B

TB (P

OZ)

dom

ain

cont

aini

ng 4

0.

6 1.

0 .0

2 0.

9 0.

9 ns

0.

9 0.

9 ns

36

2576

R

GD

1305

347

sim

ilar t

o R

IKE

N c

DN

A 2

6105

28J1

1 0.

6 0.

8 .0

0 0.

9 1.

0 ns

1.

0 1.

1 ns

36

2667

TH

AP

3 TH

AP

dom

ain

cont

aini

ng, a

popt

osis

ass

. pro

tein

3

0.6

0.9

.01

1.0

1.0

ns

1.0

1.1

ns

2998

28

XR

CC

6BP

1 X

RC

C6

bind

ing

prot

ein

1 0.

6 0.

8 .0

1 1.

0 0.

8 ns

0.

9 1.

0 ns

36

3022

ZF

P42

6L2

zinc

fing

er p

rote

in 4

26-li

ke 2

0.

6 1.

0 .0

1 1.

1 1.

2 ns

1.

0 1.

0 ns

Supplements 147

!

2537

4 A

LAD

am

inol

evul

inat

e, d

elta

-, de

hydr

atas

e 0.

6 1.

0 .0

1 1.

0 0.

9 ns

1.

0 1.

1 ns

30

7395

A

BLI

M3

actin

bin

ding

LIM

pro

tein

fam

ily, m

embe

r 3

0.7

0.7

ns

0.6

0.9

0.02

0.

9 0.

9 ns

29

1927

O

RC

6L

orig

in re

cogn

ition

com

plex

, sub

unit

6 lik

e 0.

6 0.

9 .0

0 1.

1 1.

1 ns

0.

9 0.

9 ns

36

2425

ZF

P63

7 zi

nc fi

nger

pro

tein

637

0.

6 0.

9 .0

2 1.

0 1.

0 ns

0.

9 1.

0 ns

30

4396

LO

C30

4396

si

mila

r to

hypo

thet

ical

pro

tein

DK

FZp4

34K

1815

0.

6 1.

0 .0

1 1.

0 1.

0 ns

1.

1 1.

0 ns

30

5341

R

HO

H

ras

hom

olog

gen

e fa

mily

, mem

ber H

1.

7 0.

9 .0

4 0.

8 1.

0 ns

1.

1 1.

1 ns

36

7874

R

GD

1563

579

sim

ilar t

o 60

S ri

boso

mal

pro

tein

L29

(P23

) 0.

6 1.

1 .0

3 0.

9 0.

9 ns

0.

7 0.

9 ns

28

9468

FA

M17

5A

fam

ily w

ith s

eque

nce

sim

ilarit

y 17

5, m

embe

r A

0.6

1.0

.00

1.0

1.0

ns

1.1

1.1

ns

3614

53

DE

AD

C1

deam

inas

e do

mai

n co

ntai

ning

1

0.6

1.1

.01

1.2

1.3

ns

0.9

1.0

ns

5006

27

RG

D15

6280

1 si

mila

r to

RN

A, U

tran

spor

ter 1

0.

6 0.

8 .0

0 0.

9 1.

0 ns

1.

0 1.

0 ns

36

2849

M

AR

CH

2 m

embr

ane-

asso

ciat

ed ri

ng fi

nger

(C3H

C4)

2

0.6

1.0

.02

1.0

0.9

ns

1.0

1.0

ns

2954

58

BD

H2

3-hy

drox

ybut

yrat

e de

hydr

ogen

ase,

type

2

0.6

0.9

.01

1.2

0.9

ns

0.9

1.0

ns

3089

93

SE

PH

S2

sele

noph

osph

ate

synt

heta

se 2

0.

6 0.

8 .0

0 1.

0 0.

8 ns

0.

9 0.

9 ns

49

9561

FA

M17

3B

fam

ily w

ith s

eque

nce

sim

ilarit

y 17

3, m

embe

r B

0.6

1.1

.02

1.3

1.0

ns

0.8

1.1

ns

2980

75

NC

BP

1 nu

clea

r cap

bin

ding

pro

tein

sub

unit

1, 8

0kD

a 0.

6 0.

9 .0

4 1.

0 1.

0 ns

0.

8 1.

0 ns

68

6765

LO

C68

6765

si

mila

r to

CG

1428

6-P

A

0.6

0.9

.01

1.0

0.9

ns

0.9

0.9

ns

1714

11

TME

M17

6B

trans

mem

bran

e pr

otei

n 17

6B

0.6

0.8

.00

1.2

1.0

ns

1.0

0.9

ns

5004

11

RG

D15

6494

0 si

mila

r to

RIK

EN

cD

NA

300

0004

N20

0.

6 1.

1 .0

0 1.

1 1.

3 ns

1.

0 1.

2 ns

49

9900

LO

C49

9900

si

mila

r to

Zinc

fing

er p

rote

in 1

33

0.6

1.1

.02

1.0

1.1

ns

0.9

1.0

ns

3119

52

GA

LNT1

1 U

DP

-N-a

cety

l-alp

ha-D

-gal

acto

sam

ine:

pol

ypep

tide

N-

acet

ylga

lact

osam

inyl

trans

fera

se 1

1 (G

alN

Ac-

T11)

0.

6 1.

0 .0

5 1.

2 1.

0 ns

1.

0 1.

1 ns

8448

0 B

NIP

3 B

CL2

/ade

novi

rus

E1B

inte

ract

ing

prot

ein

3 1.

7 1.

2 .0

0 1.

1 1.

0 ns

0.

9 1.

0 ns

50

0533

D

MA

P1

DN

A m

ethy

ltran

sfer

ase

1-as

soci

ated

pro

tein

1

0.6

0.8

.00

0.9

0.9

ns

0.9

0.9

ns

2558

6 A

LPL

alka

line

phos

phat

ase,

live

r/bon

e/ki

dney

0.

6 0.

8 .0

0 1.

0 0.

9 ns

0.

8 0.

8 ns

31

4788

R

GD

1307

947

sim

ilar t

o R

IKE

N c

DN

A C

4300

08C

19

0.6

0.9

.01

0.9

0.9

ns

0.9

0.9

ns

6422

6 M

LST8

M

TOR

ass

ocia

ted

prot

ein,

LS

T8 h

omol

og

0.6

0.9

.00

0.9

0.9

ns

1.0

1.1

ns

6451

3 P

AW

R

PR

KC

, apo

ptos

is, W

T1, r

egul

ator

1.

7 1.

1 .0

0 1.

0 1.

0 ns

1.

0 1.

0 ns

30

2890

C

PP

ED

1 ca

lcin

eurin

-like

pho

spho

este

rase

dom

ain

cont

aini

ng 1

0.

6 0.

8 .0

0 0.

9 0.

8 ns

0.

9 0.

9 ns

29

6758

A

RM

C10

ar

mad

illo

repe

at c

onta

inin

g 10

0.

6 0.

9 .0

0 0.

9 0.

9 ns

0.

9 1.

0 ns

36

2793

R

GD

1307

315

LOC

3627

93

0.6

1.0

.00

1.0

1.1

ns

1.0

1.1

ns

6828

93

LYR

M2

LYR

mot

if co

ntai

ning

2

0.6

0.8

.00

1.0

1.0

ns

0.9

0.9

ns

3615

42

U2A

F1L4

U

2 sm

all n

ucle

ar R

NA

aux

iliar

y fa

ctor

1-li

ke 4

0.

6 0.

9 .0

2 1.

0 0.

9 ns

0.

9 0.

9 ns

36

2789

ZF

YV

E21

zi

nc fi

nger

, FY

VE

dom

ain

cont

aini

ng 2

1 0.

6 0.

9 .0

0 1.

1 1.

0 ns

1.

0 1.

0 ns

31

6626

H

ES

6 ha

iry a

nd e

nhan

cer o

f spl

it 6

0.

6 0.

9 .0

0 1.

4 1.

1 ns

1.

2 1.

1 ns

28

7456

M

ED

11

med

iato

r com

plex

sub

unit

11

0.6

0.9

.01

1.1

1.0

ns

1.1

1.0

ns

3613

01

RG

D13

1057

1 si

mila

r to

hypo

thet

ical

pro

tein

0.

6 0.

8 .0

1 0.

7 0.

8 ns

0.

7 0.

7 ns

31

6008

C

CD

C51

co

iled-

coil

dom

ain

cont

aini

ng 5

1 0.

6 1.

0 .0

5 0.

9 1.

0 ns

1.

0 0.

9 ns

28

7383

B

9D1

B9

prot

ein

dom

ain

1 0.

6 0.

9 .0

0 0.

9 1.

1 ns

0.

9 0.

9 ns

84

427

GR

B7

grow

th fa

ctor

rece

ptor

bou

nd p

rote

in 7

0.

6 1.

2 .0

0 1.

0 1.

2 ns

1.

1 1.

3 ns

31

1902

R

BM

18

RN

A b

indi

ng m

otif

prot

ein

18

1.6

1.0

.01

0.8

0.9

ns

0.9

0.8

ns

6787

41

LOC

6787

41

sim

ilar t

o Zi

nc fi

nger

CC

CH

-type

dom

ain

cont

aini

ng p

rote

in 6

0.

6 1.

0 .0

1 1.

1 1.

0 ns

1.

0 1.

0 ns

64

364

PLL

P

plas

ma

mem

bran

e pr

oteo

lipid

(pla

smol

ipin

) 0.

6 0.

9 .0

3 1.

0 1.

1 ns

0.

9 0.

9 ns

36

0894

R

GD

1310

587

sim

ilar t

o hy

poth

etic

al p

rote

in F

LJ14

146

0.6

0.9

.00

1.1

1.1

ns

1.0

1.0

ns


!

3634

42

FUN

DC

1 FU

N14

dom

ain

cont

aini

ng 1

0.

6 0.

9 .0

3 0.

9 0.

9 ns

1.

0 0.

9 ns

36

1197

R

GD

1561

537

sim

ilar t

o pu

tativ

e re

pair

and

reco

mbi

natio

n he

licas

e R

AD

26L

0.6

0.9

.05

0.8

0.7

ns

0.7

0.7

ns

2903

22

INTS

9 in

tegr

ator

com

plex

sub

unit

9 0.

6 0.

9 .0

1 1.

0 0.

9 ns

1.

0 1.

0 ns

24

706

RA

RB

re

tinoi

c ac

id re

cept

or, b

eta

0.6

1.0

.04

0.9

0.8

ns

1.0

0.8

ns

3159

23

LYS

MD

3 Ly

sM, p

utat

ive

pept

idog

lyca

n-bi

ndin

g, d

omai

n co

ntai

ning

3

1.6

1.1

.01

0.9

1.2

ns

1.2

1.2

ns

3606

39

DC

AK

D

deph

osph

o-C

oA k

inas

e do

mai

n co

ntai

ning

0.

6 0.

9 .0

0 0.

9 1.

0 ns

1.

0 1.

0 ns

24

5974

C

OM

MD

5 C

OM

M d

omai

n co

ntai

ning

5

0.6

1.0

.00

1.0

0.9

ns

1.1

1.0

ns

3616

50

HIR

IP3

HIR

A in

tera

ctin

g pr

otei

n 3

0.6

1.0

.02

1.0

1.0

ns

1.0

1.0

ns

1711

59

ZHX

1 zi

nc fi

nger

s an

d ho

meo

boxe

s 1

0.6

0.8

.01

0.9

0.9

ns

0.9

0.9

ns

3651

79

ZNF5

24

zinc

fing

er p

rote

in 5

24

0.6

1.0

.00

1.1

1.1

ns

1.0

1.1

ns

3622

27

DTD

1 D

-tyro

syl-t

RN

A d

eacy

lase

1 h

omol

og

0.6

0.9

.01

0.9

0.9

ns

1.0

1.0

ns

2885

57

MO

SP

D3

mot

ile s

perm

dom

ain

cont

aini

ng 3

0.

6 1.

0 .0

0 1.

1 1.

0 ns

1.

0 1.

1 ns

30

4579

U

SP

30

ubiq

uitin

spe

cific

pep

tidas

e 30

0.

6 0.

9 .0

1 0.

9 1.

0 ns

1.

0 0.

9 ns

49

8957

K

LHL3

6 ke

lch-

like

36

0.6

1.0

.02

1.1

1.0

ns

1.0

1.1

ns

2424

4 C

ALM

3 ca

lmod

ulin

3

0.6

1.0

.00

1.3

1.0

ns

1.1

1.0

ns

2877

03

EIF

1 eu

kary

otic

tran

slat

ion

initi

atio

n fa

ctor

1

1.5

1.2

.00

1.0

1.1

ns

1.0

1.0

ns

4982

79

FCE

R1G

Fc

frag

men

t of I

gE, h

igh

affin

ity I,

rece

ptor

for;

gam

ma

poly

pept

ide

1.5

1.2

ns

1.5

1.0

0.00

1.

1 1.

0 ns

28

7436

E

IF4A

1 eu

kary

otic

tran

slat

ion

initi

atio

n fa

ctor

4A

1 1.

5 1.

1 .0

0 0.

9 1.

0 ns

1.

0 1.

0 ns

36

2304

O

RC

5L

orig

in re

cogn

ition

com

plex

, sub

unit

5-lik

e 0.

7 0.

8 .0

0 1.

0 1.

0 ns

1.

1 1.

0 ns

25

671

SM

AD

1 S

MA

D fa

mily

mem

ber 1

1.

5 1.

0 .0

0 0.

9 0.

8 ns

0.

8 0.

9 ns

25

670

IL15

in

terle

ukin

15

0.7

1.0

.00

1.3

1.3

ns

1.3

1.3

ns

2936

20

PTD

SS

2 ph

osph

atid

ylse

rine

synt

hase

2

0.7

0.9

.01

1.1

0.9

ns

0.9

0.9

ns

3061

82

IPO

5 im

porti

n 5

1.5

1.1

.02

0.9

1.1

ns

1.1

1.0

ns

4997

55

TME

M14

1 tra

nsm

embr

ane

prot

ein

141

0.7

0.9

.00

1.0

0.9

ns

1.0

1.0

ns

3008

49

FBX

O9

f-box

pro

tein

9

0.7

1.1

.05

0.9

1.0

ns

1.1

1.0

ns

3680

42

BTA

F1

BTA

F1 R

NA

pol

ymer

ase

II, B

-TFI

ID tr

ansc

riptio

n fa

ctor

-ass

ocia

ted,

(Mot

1 ho

mol

og, S

. cer

evis

iae)

1.

5 1.

2 .0

0 0.

8 1.

0 ns

0.

9 0.

9 ns

3014

32

PP

IL3

pept

idyl

prol

yl is

omer

ase

(cyc

loph

ilin)

-like

3

0.7

1.0

.04

0.9

1.0

ns

0.9

0.9

ns

2906

32

MR

PL3

4 m

itoch

ondr

ial r

ibos

omal

pro

tein

L34

0.

7 0.

9 .0

0 0.

9 1.

0 ns

1.

0 1.

0 ns

29

7432

A

BTB

1 an

kyrin

repe

at &

BTB

(PO

Z) d

omai

n co

ntai

ning

1

0.7

1.1

.00

1.1

1.2

ns

1.4

1.4

ns

3636

03

CN

OT8

C

CR

4-N

OT

trans

crip

tion

com

plex

, sub

unit

8 0.

7 1.

0 .0

0 0.

9 1.

0 ns

1.

0 1.

0 ns

36

1767

P

OLL

po

lym

eras

e (D

NA

dire

cted

), la

mbd

a 0.

7 1.

0 .0

0 1.

1 1.

1 ns

1.

2 1.

3 ns

36

2757

R

DH

11

retin

ol d

ehyd

roge

nase

11

(all-

trans

/9-c

is/1

1-ci

s)

0.7

0.9

.02

1.0

0.8

ns

0.9

0.9

ns

2996

12

RG

D13

5912

7 si

mila

r to

RIK

EN

cD

NA

231

0011

J03

0.7

1.0

.01

0.9

0.9

ns

1.0

0.9

ns

5009

65

RG

D15

6269

0 si

mila

r to

L-la

ctat

e de

hydr

ogen

ase

A c

hain

(LD

H-A

) 1.

5 1.

0 .0

3 1.

1 1.

1 ns

1.

2 1.

0 ns

17

0842

TO

B1

trans

duce

r of E

rbB

-2.1

0.

7 1.

0 .0

2 1.

1 1.

0 ns

1.

0 0.

9 ns

50

0899

M

FSD

3 m

ajor

faci

litat

or s

uper

fam

ily d

omai

n co

ntai

ning

3

0.7

0.9

.00

1.2

1.1

ns

1.2

1.2

ns

3152

87

AS

B8

anky

rin re

peat

SO

CS

box

-con

tain

ing

prot

ein

8 0.

7 0.

9 .0

0 0.

9 1.

1 ns

1.

0 1.

0 ns

29

4283

R

GL2

ra

l gua

nine

nuc

l. di

ssoc

iatio

n st

imul

ator

-like

2

0.7

1.1

.01

1.2

1.1

ns

1.2

1.1

ns

6877

99

LOC

6894

08

sim

ilar t

o H

2A h

isto

ne fa

mily

, mem

ber V

-1

0.7

0.9

.03

1.0

1.0

ns

0.9

1.0

ns

2950

50

EX

OS

C8

exos

ome

com

pone

nt 8

0.

7 1.

0 .0

0 1.

1 1.

1 ns

1.

1 1.

1 ns

36

2414

TA

DA

3L

trans

crip

tiona

l ada

ptor

3 (N

GG

1 ho

mol

og, y

east

)-lik

e 0.

7 1.

0 .0

3 1.

0 0.

9 ns

1.

0 1.

1 ns

58

918

CA

SP

9 ca

spas

e 9,

apo

ptos

is-r

elat

ed c

yste

ine

pept

idas

e 0.

7 1.

0 .0

4 1.

0 1.

0 ns

1.

1 1.

0 ns

Supplements 149

!

1409

08

CD

K5

cycl

in-d

epen

dent

kin

ase

5 0.

7 1.

0 .0

1 1.

0 1.

0 ns

1.

0 1.

0 ns

36

2484

P

LEK

HF2

pl

ecks

trin

hom

olog

y do

mai

n co

ntai

ning

, fam

ily F

(with

FY

VE

dom

ain)

m

embe

r 2

0.7

1.1

.03

0.9

0.9

ns

0.8

1.0

ns

9420

1 C

DK

4 cy

clin

-dep

ende

nt k

inas

e 4

0.7

1.1

.02

1.2

0.9

ns

1.0

0.9

ns

5026

03

SFR

S11

sp

licin

g fa

ctor

, arg

inin

e/se

rine-

rich

11

1.4

1.0

.03

1.0

1.0

ns

1.2

1.1

ns

2889

24

MO

RG

1 m

itoge

n-ac

tivat

ed p

rote

in k

inas

e or

gani

zer 1

0.

7 1.

0 .0

3 0.

9 1.

0 ns

1.

1 1.

0 ns

17

1565

S

TAM

BP

S

tam

bin

ding

pro

tein

0.

7 1.

0 .0

3 1.

1 1.

0 ns

1.

0 1.

1 ns

29

6851

P

ON

2 pa

raox

onas

e 2

0.7

0.9

.00

1.1

1.0

ns

1.0

0.9

ns

2916

70

CX

XC

5 C

XX

C fi

nger

5

0.7

1.0

.02

1.0

1.1

ns

1.0

1.0

ns

4989

09

CO

Q9

coen

zym

e Q

9 ho

mol

og

0.7

0.9

.03

1.0

0.9

ns

0.9

0.9

ns

3067

34

RM

I1

RM

I1, R

ecQ

med

iate

d ge

nom

e in

stab

ility

1, h

omol

og

0.7

1.0

.02

1.1

1.0

ns

1.1

1.2

ns

5672

5 C

RIP

T cy

stei

ne-r

ich

PD

Z-bi

ndin

g pr

otei

n 0.

7 1.

0 .0

2 0.

9 0.

9 ns

0.

9 1.

0 ns

28

8621

M

RP

S17

m

itoch

ondr

ial r

ibos

omal

pro

tein

S17

0.

7 0.

9 .0

1 1.

0 1.

1 ns

0.

9 1.

0 ns

29

5357

H

BX

IP

hepa

titis

B v

irus

x in

tera

ctin

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!

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Supplement

Does correcting the numbers improve long-term outcome? This Editorial View accompanies the following article: Samarska IV, van Meurs M, Buikema H, Houwertjes MC, Wulfert FM, Molema G, Epema AH, Henning RH: Adjunct nitrous oxide normalizes vascular reactivity changes after hemorrhagic shock in mice under isoflurane anesthesia.

Robert D. Sanders, B.Sc., M.B.B.S., F.R.C.A.,* Mervyn Maze, M.B., Ch.B., F.R.C.P., F.R.C.A., F.Med.Sci. *Department of Anaesthetics,

Pain Medicine, and Intensive Care, Imperial College London, United Kingdom. [email protected]


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Advances in the understanding of anesthetic pharmacology and perioperative physiology, coupled with improved patient monitoring, have significantly contributed to improvements in quality of care and perioperative outcome.1–3 In this issue of Anesthesiology, Samarska et al. describe preclinical research that addresses the anesthetic modulatory effects on the physiologic adaptation to hemorrhagic shock; their data have led them to the conclusion that nitrous oxide promotes hemodynamic stability.4 In their studies, mice were exposed to anesthesia, either isoflurane (1.4%) in oxygen (33%) or isoflurane (1.4%) plus nitrous oxide (66%) in oxygen (33%), and underwent a sham procedure, hemorrhagic shock, or shock plus fluid resuscitation, during which time hemodynamic measurements were obtained. Thereafter, vascular responsiveness was assessed ex vivo in aortic rings. Isoflurane treatment attenuated the maximal aortic contractile responses to phenylephrine, corroborating earlier reports with volatile anesthetics.5 Shock, with or without resuscitation, mitigated the isoflurane-induced attenuation of phenylephrine responses, although the biphasic pattern of relaxation and then contraction with acetylcholine was altered. The ex vivo effects induced by in vivo isoflurane exposure were mitigated when supplemented with nitrous oxide. However, in the shock state the addition of nitrous oxide induced acidosis when compared with isoflurane, and further physiologic differences (such as oxygenation) confounds clear interpretation of the experimental findings. Even though animals were at different depths of anesthesia under these conditions, the authors attribute the pharmacologic properties of nitrous oxide for “normalizing” vasoreactivity, and speculate that nitrous oxide may induce increased perioperative hemodynamic stability. Samarska et al. also observed that nitrous oxide exposure was associated with a higher mean arterial blood pressure in the sham-treated animals despite the increased depth of anesthesia;4 this finding corroborates previous studies demonstrating the vasoconstrictive properties of nitrous oxide.6 Yet recent clinical studies have reported minor difference in blood pressure when comparing nitrous oxide or air as the carrier gas.6–9 Thus it is highly speculative that the modest increments (of the order of 10 mmHg) reported by Samarska et al. and others will exert a positive long-term clinical benefit. ! Placing Physiologic Normalization into Clinical Context

It is not possible to ascribe a benefit to the “normalization” achieved by the administration of nitrous oxide because there was no “nonanesthetized” control group, no correlation with postoperative hemodynamic changes, and no assessment of long-term outcome. Nonetheless, the authors’ interest in improving hemodynamic stability in the postoperative period is commendable, especially as there is a tendency to view improvement as simply an intraoperative endpoint within anesthesiology. Rather our discipline needs to focus on endpoints of anesthetic management that are important by virtue of the fact that patient outcome is affected. In this manner, physiologic variables should not be used as surrogate markers for long-term outcomes unless their association is tightly correlated.

Supplements 153

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When Can Modifying Intraoperative Numbers Improve Long-term Outcome? Identification of patient comorbidities is critical to understanding risk

stratification of vulnerable patients and, therefore, their level of care. In addition, anesthesiologists need to determine the modifiable risk factors occurring in the perioperative period that may be manipulated to improve outcomes. A recent analysis started this process by evaluating the importance of intraoperative physiologic variables to determine long-term cardiovascular outcomes and death.10 Data from this large cohort study identified higher-risk patients as having two or more comorbidities: Age, obesity, emergency surgery, previous cardiac intervention, congestive cardiac failure, cerebrovascular disease, and hypertension. Patients with two or more risk factors who had an adverse cardiac event were more likely to have had intraoperative hypotension (mean arterial pressure less than 50 mmHg or decreased by 40%, lasting at least 10 min) among other modifiable risks. Similar to previous data for vascular surgical patients,11 those with three or more risk factors who sustained an adverse cardiac event were also more likely to have endured intraoperative tachycardia. Unfortunately the study was underpowered to ascertain an independent effect of the hemodynamic variables on adverse cardiac events; adequately powered studies are required to further investigate these findings and that of physiologic changes in the postoperative period,11 to determine long-term patient outcomes. Clearly it is important to “correct the numbers” intraoperatively, but how? Perhaps if tachycardia predisposes patients with three or more cardiovascular risk factors to adverse cardiac events, then these are the subjects who are most likely to benefit from perioperative $-blockade.11,

12 Exposing patients with fewer risk factors may merely increase their chance of hypotension and thus increase their cardiac and stroke risk.13 These findings also question the clinical significance of nitrous oxide induced improvement in intraoperative mean arterial blood pressure, suggesting that this effect will be too modest alone (approximately 10 mmHg) to alter cardiac risk. Whether postoperative hemodynamic parameters are improved after nitrous oxide exposure remains unknown.

Long-term Anesthetic Effects: Nitrous Oxide Case Study

The use of nitrous oxide for the maintenance of anesthesia exemplifies the need to focus on long-term outcomes. While the purported increased hemodynamic stability (“correcting the numbers”) with nitrous oxide has been regarded as good for cardiac risk, other factors may mitigate this benefit; for example, halogenated volatile anesthetics consistently demonstrate superior organ-protective effects as compared with nitrous oxide or intravenous agents in experimental studies.14–16 This may translate into improved tolerance to lower perfusion pressure or reduced oxygen supply with an anesthetic technique that is based solely on halogenated volatile anesthesia. Thus the addition of nitrous oxide, while “sparing” the volatile, may reduce this potential benefit accrued from the volatile anesthetic gas. Similarly, individual anesthetic effects on cellular metabolism could also be important.17 Nitrous oxide may also influence cardiac risk by increasing homocysteine levels.18, 19 Raised


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homocysteine levels predispose to higher cardiac risk in the community20 and in cardiac surgical patients21 via endothelial dysfunction and possible effects on coagulation.19 Putatively related to this increased perioperative myocardial ischemia, increased homocysteine levels have been noted with nitrous oxide-based anesthesia (however, long-term follow-up of these patients has not been conducted).18 To further ignite debate, nitrous oxide administration was recently associated with an increased number of delayed ischemic neurologic events in a post hoc subgroup analysis of the intraoperative hypothermia for aneurysm surgery trial.8 Again this may be secondary to raised homocysteine levels in the nitrous oxide group (although these were not measured). Critically though, the long-term outcomes between the nitrous oxide and no nitrous oxide groups were not different. Therefore, the use of nitrous oxide to improve hemodynamic stability based on the assumption that it will alter long-term patient outcomes may be flawed. It is therefore timely that the ENIGMA-II trial protocol has recently been published.22 ENIGMA-II is designed to ask whether nitrous oxide predisposes to adverse cardiac events based on its ability to modify homocysteine levels.

The trial has a solid scientific foundation,19, 22, 23 with proof of principal demonstrated in smaller clinical trials.18, 23 ENIGMA-II will be a 7,000-patient study designed to evaluate whether avoidance of nitrous oxide administration is associated with a 25% decrease risk of cardiac events or death (! =0.05; $=0.1). Of course it is possible that the study may find that the higher intraoperative mean arterial blood pressure induced by nitrous oxide may improve outcomes. Whatever the results of ENIGMA II, the critical approach here is to focus on long-term patient outcomes; outcomes that matter to the patient. Impact of Long-term Outcome Studies

Long-term outcomes studies are needed to define the optimal anesthetic management for the more than 234 million patients who undergo surgery each year.24 Going beyond the results that are based on cohorts of “average” patients, anesthesiologists will need to further personalize care for the individual patients, using careful clinical phenotyping that will be guided in the future by biomarkers that evolve from postgenomic research endeavors. Both our specialty and the welfare of our patients will benefit from rigorous translation of the evidence from well-conducted clinical research into practice. Our discipline’s research program has to focus on long-term outcomes to improve endpoints that both matter to the patient and improve the efficiency of healthcare resource use. Defining how to “correct the numbers” is a critical part of this approach; we need studies to define how these values should be modified. It is more than likely that anesthesiologists can continue to improve long-term patient outcomes, but we need the studies to demonstrate how.

Reference list 1. Holland R: Anaesthetic mortality in New South Wales. Br J Anaesth 1987; 59:834–41

Supplements 155

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2. Lagasse RS: The right stuff: Veterans Affairs National Surgical Quality Improvement Project. Anesth Analg 2008; 107:1772–4

3. Mayfield JB: The impact of intraoperative monitoring on patient safety. Anesthesiol Clin 2006; 24:407–17

4. Samarska IV, van Meurs M, Buikema H, Houwertjes MC, Wulfert FM, Molema I, Epema AH, Hennings RH: Adjunct nitrous oxide normalizes vascular reactivity changes after hemorrhagic shock in mice under isoflurane anesthesia. Anesthesiology 2009; 111:600–8

5. Spiss CK, Smith CM, Tsujimoto G, Hoffman BB, Maze M: Prolonged hyporesponsiveness of vascular smooth muscle contraction after halothane anesthesia in rabbits. Anesth Analg 1985; 64:1–6

6. Inada T, Inada K, Kawachi S, Takubo K, Tai M, Yasugi H: Haemodynamic comparison of sevoflurane and isoflurane anaesthesia in surgical patients. Can J Anaesth 1997; 44:140–5

7. Fleischmann E, Lenhardt R, Kurz A, Herbst F, Fu¨lesdi B, Greif R, Sessler DI, Akc¸a O; Outcomes Research Group: Nitrous oxide and risk of surgical wound infection: A randomised trial. Lancet 2005; 366:1101–7

8. Pasternak JJ, McGregor DG, Lanier WL, Schroeder DR, Rusy DA, Hindman B, Clarke W, Torner J, Todd MM; IHAST Investigators: Effect of nitrous oxide use on long-term neurologic and neuropsychological outcome in patients who received temporary proximal artery occlusion during cerebral aneurysm clipping surgery. Anesthesiology 2009; 110:563–73

9. McKinney MS, Fee JP: Cardiovascular effects of 50% nitrous oxide in older adult patients anaesthetized with isoflurane or halothane. Br J Anaesth 1998; 80:169–73

10. Kheterpal S, O’Reilly M, Englesbe MJ, Rosenberg AL, Shanks AM, Zhang L, Rothman ED, Campbell DA, Tremper KK: Preoperative and intraoperative predictors of cardiac adverse events after general, vascular, and urological surgery. Anesthesiology 2009; 110:58–66

11. Feringa HH, Bax JJ, Boersma E, Kertai MD, Meij SH, Galal W, Schouten O, Thomson IR, Klootwijk P, van Sambeek MR, Klein J, Poldermans D: High-dose beta-blockers and tight heart rate control reduce myocardial ischemia and troponin T release in vascular surgery patients. Circulation 2006; 114:I344–9

12. Beattie WS, Wijeysundera DN, Karkouti K, McCluskey S, Tait G: Does tight heart rate control improve beta-blocker efficacy? An updated analysis of the noncardiac surgical randomized trials. Anesth Analg 2008; 106:1039–48

13. POISE Study Group; Devereaux PJ, Yang H, Yusuf S, Guyatt G, Leslie K, Villar JC, Xavier D, Chrolavicius S, Greenspan L, Pogue J, Pais P, Liu L, Xu S, Ma´laga G, Avezum A, Chan M, Montori VM, Jacka M, Choi P: Effects of extendedrelease metoprolol succinate in patients undergoing non-cardiac surgery (POISE trial): A randomised controlled trial. Lancet 2008; 371:1839–47

14. De Hert SG, Van der Linden PJ, Cromheecke S, Meeus R, Nelis A, Van Reeth V, ten Broecke PW, De Blier IG, Stockman BA, Rodrigus IE: Cardioprotective properties of sevoflurane in patients undergoing coronary surgery with cardiopulmonary bypass are related to the modalities of its administration. Anesthesiology 2004; 101:299–310

15. Sanders RD, Ma D, Maze M: Anaesthesia induced neuroprotection. Best Pract Res Clin Anaesthesiol 2005; 19:461–74

16. Weber NC, Toma O, Awan S, Fra¨ssdorf J, Preckel B, Schlack W: Effects of nitrous oxide on the rat heart in vivo: Another inhalational anesthetic that preconditions the heart? Anesthesiology 2005; 103:1174–82

17. Kaisti KK, La°ngsjo¨ JW, Aalto S, Oikonen V, Sipila¨ H, Tera¨s M, Hinkka S, Metsa¨honkala L, Scheinin H: Effects of sevoflurane, propofol, and adjunct nitrous oxide on regional cerebral blood flow, oxygen consumption, and blood volume in humans. Anesthesiology 2003; 99:603–13

18. Badner NH, Beattie WS, Freeman D, Spence JD: Nitrous oxide-induced increased homocysteine concentrations are associated with increased postoperative myocardial ischemia in patients undergoing carotid endarterectomy. Anesth Analg 2000; 91:1073–9

19. Myles PS, Chan MT, Kaye DM, McIlroy DR, Lau CW, Symons JA, Chen S: Effect of nitrous oxide anesthesia on plasma homocysteine and endothelial function. Anesthesiology 2008; 109:657–63

20. Nygard O, Nordrehaug JE, Refsum H, Ueland PM, Farstad M, Vollset SE: Plasma homocysteine levels and mortality in patients with coronary artery disease. N Engl J Med 1997; 337:230–6

21. Ranucci M, Ballotta A, Frigiola A, Boncilli A, Brozzi S, Costa E, Mehta RH: Pre-operative homocysteine levels and morbidity and mortality following cardiac surgery. Eur Heart J 2009; 30:995–1004


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22. Myles PS, Leslie K, Peyton P, Paech M, Forbes A, Chan MT, Sessler D, Devereaux PJ, Silbert BS, Jamrozik K, Beattie S, Badner N, Tomlinson J, Wallace S; ANZCA Trials Group: Nitrous oxide and perioperative cardiac morbidity (ENIGMA-II) Trial: Rationale and design. Am Heart J 2009; 157:488–94

23. Sanders RD, Weimann J, Maze M: Biologic effects of nitrous oxide: A mechanistic and toxicologic review. Anesthesiology 2008; 109:707–22

24. Weiser TG, Regenbogen SE, Thompson KD, Haynes AB, Lipsitz SR, Berry WR, Gawande AA: An estimation of the global volume of surgery: A modeling strategy based on available data. Lancet 2008; 372:139–44

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Samenvatting in het Nederlands

Cardiopulmonaire bypass (CPB) is een techniek waarbij een hart-longmachine de functie van het hart als circulatiepomp overneemt, waardoor openhartchirurgie mogelijk wordt gemaakt. CPB is daarmee van grote waarde voor de thoraxchirurgie gebleken. Het is echter ook een techniek waarbij er sprake is van een verhoogd optreden van postoperatieve klinische complicaties, zowel tijdens verblijf in het ziekenhuis als op midden en lange termijn daarna. CPB gaat gepaard met activering van ontstekingsprocessen en een meer of mindere mate van (tijdelijke) achteruitgang in de werking van verschillende organen. Het goed functioneren van organen is mede afhankelijk van het “gedrag” van bloedvaten (vaatfunctie) in deze organen en daarbuiten. Dit proefschrift richt zich op het beschrijven van het verloop van veranderingen in vaatfunctie na CPB in relatie tot de ontstekingsprocessen die hierbij optreden. Daarbij werd onderzocht of beïnvloeding van de vaatfunctie een mogelijk aangrijpingspunt van farmacotherapeutische interventie is (behandeling met geneesmiddelen).

In hoofdstuk 1 worden de huidige opvattingen besproken over de pathofysiologische veranderingen en de klinische gevolgen van CPB. Het is bekend dat het chirurgisch trauma, het contact van bloed met kunstmatige oppervlakken van de hart-long machine, en de afwijkende doorbloeding van organen tijdens CPB en het herstel van de normale doorbloeding na CPB (=ischemie/reperfusie) allen cellulaire en humorale afweermechanismen activeren. Deze processen spelen zowel op lokaal niveau in de organen als in de systemische circulatie. Ontstekingsprocessen en ischemie-reperfusie schade leidt tot de ontwikkeling van vaatdisfunctie. Hierbij speelt verhoogde productie van zuurstof radicalen (oxidatieve stress) - zoals bij ischemie-reperfusie - een belangrijke rol. Oxidatieve stress draagt bij aan het ontstaan van endotheeldisfunctie, d.w.z. het niet goed meer functioneren van de cellaag die de binnenbekleding van de vaatwand vormt en normaliter belangrijke processen reguleert t.b.v. van een gezonde structuur en functie van bloedvaten. Als gevolg van bovengenoemde veranderingen treden afwijkingen op van de bloedsomloop in grote en kleine bloedvaten, die – samen met weefseloedeem en secundaire hypoperfusie - aan de basis liggen van het zogenaamde meervoudige orgaan disfunctie syndroom in CPB.

Vervolgens is in hoofdstukken 2 en 3 eerst de veranderde reactiviteit van bloedvaten na CPB het onderwerp van onderzoek. Deze werd bestudeerd in een experimenteel model van CPB in de rat waarbij drie verschillende type bloedvaten (kleine coronair en mesenteriaal arteriën, en de aorta) werden bemeten op verschillende tijdstippen na CPB gedurende een klinische relevante periode van 5 dagen. Tijdens deze periode na CPB trad er een verandering in vaatreactiviteit op die kan worden omschreven als een algehele verschuiving naar minder contractie/meer (endotheel-) relaxatie, met daarbij een opvallend verschil in het tijdsverloop voor verschillende bloedvaten. De patronen in figuur 1A suggereren dat veranderingen in vaatfunctie in kleine bloedvaten op korte termijn na CPB (1-2 dagen) optreden en tijdelijk van aard zijn, terwijl veranderingen in grote bloedvaten pas later opkomen (5

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dagen na CPB) en mogelijk een meer blijvend karakter hebben. Deze veranderingen in vaatfunctie zijn grotendeels toe te schrijven aan chirurgische procedures bij CPB, zoals anesthesie en cannulaties.

Het overnemen van de bloedsomloop door de hartlongmachine (extracorporele circulatie [ECC]) lijkt hieraan nog specifiek een eigen effect toe te voegen m.b.t. de endotheelfunctie. Zo bleek de toename in endotheel-afhankelijke relaxatie in kleine mesenteriaal arteriën 2 dagen na CPB teniet te zijn gedaan door ECC. Dit effect viel samen met een verhoogd niveau van nitrotyrosine in dit vasculaire bed, wat aangeeft dat de oxidatieve stress verhoogd was bij ECC. Verder is opmerkelijk dat het effect van ECC op endotheelfunctie selectief aanwezig was bij kleine maar niet bij grote arteriën, hetgeen verder onderstreept hoe verschillende vaatbedden op eigen verschillende wijze reageren op CPB. Om veranderde vaatfunctie verder te onderzoeken in relatie tot ontstekingsreacties in CPB werd in hoofdstuk 3 de expressie van markers en mediatoren van ontsteking en endotheelcel-activatie in de aorta vaatwand bepaald. Het patroon in figuur 1B suggereert dat CPB gepaard gaat met acute ontstekingsreacties in de aorta die daarna echter ook snel weer normaliseren. Dit in tegenstelling tot de veranderingen in reactiviteit in de aorta, die zoals eerder besproken pas op latere tijdstippen (5 dagen) na CPB optraden. Het lijkt er dus mogelijk op dat tijdelijke ontsteking van de vaatwand acuut na CPB bepaalde processen in gang zet die zich pas op langere termijn vertalen naar functionele verandering in reactiviteit. Of dit ook het geval is in kleine bloedvaten, of dat hier ontstekingsreacties en veranderde vaatfunctie hand in hand gaan (en gelijktijdig optreden) zou nog verder onderzocht moeten worden.

In de hoofdstukken 4 en 5 van dit proefschrift werd vervolgens getracht de pathofysiologie van CPB-gerelateerde ontstekingsprocessen verder te karakteriseren. Hiertoe werd in het zelfde model als boven de expressie van ontstekingsmediatoren in longen en de nieren bepaald m.b.v. PCR, Western blot en micro-array analyse technieken. De expressiepatronen die daarbij naar voren komen van verschillende pro-en anti-inflammatoire cytokines, en van markers van endotheelcel- en neutrofiel-activatie, suggereren dat CPB een acute ontstekingsreactie opwekt in beide organen. Micro-array analyse van nierweefsel liet zien dat CPB hier de expressie beïnvloedde van 421 genen die voornamelijk betrokken zijn bij de regulatie van de afweermechanismen.

Daar waar acute en heftige ontstekingsreacties in nier en aorta van voorbijgaande aard waren en volledig normaliseerden op langere termijn na CPB, bleef de expressie van de markers van oxidatieve stress, endotheelcel- en neutrofiel-activatie, en van pro- en anti-inflammatoire markers in longweefsel substantieel verhoogd gedurende veel langere tijd. De verlengde verhoging in longweefsel van markers, zoals IL-1$, TGF-$1 en SOD-1 tot wel 5 dagen van herstel na CPB, suggereert een doorlopende ontstekingreactie in longweefsel, welke bij zou kunnen dragen aan longcomplicaties zoals waargenomen na hartchirurgie. CPB is ook een situatie waarin (tijdelijke) hypotensieve condities kunnen ontstaan, waarbij er op dat moment een lagere bloeddruk “heerst” dan eigenlijk wenselijk als het

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gaat om adequate orgaandoorbloeding. Immers, te weinig bloeddruk leidt tot te weinig bloeddoorstroming en daarmee tot orgaan-ischemie, -schade en –functieverlies (net als extreem het geval is in hemodynamische shock). De boven beschreven bevindingen in dit proefschrift over veranderde vaatfunctie in CPB – met een shift naar minder contractie/meer (endotheel-) relaxatie – passen bij dit beeld en geven een functionele verklaring. Omdat anesthesie – als onderdeel van chirurgische procedures bij CPB - zelf ook van invloed kan zijn op de vaattonus is in hoofdstuk 6 een klein uitstapje gemaakt naar het effect van verschillende vormen van anesthesie op reactiviteit van bloedvaten in een muis model van extreme hypotensie (hemorragische shock). De resultaten bevestigen in eerste instantie dat een gangbaar anestheticum zoals isofluraan, ingrijpende effecten heeft op reactiviteit van bloedvaten (- en daarmee orgaanfunctie). Tevens blijkt dat een geselecteerde combinatie met andere anesthetica (in dit geval distikstofoxide, N2O ofwel lachgas) deze beïnvloeding dramatisch kan beperken. Het fundamentele punt wat deze studie naar voren brengt is dat post-operatieve vaatreactiviteit kan worden beïnvloed/gestuurd door rationeel gebruik van intra-operatieve anesthesie, en daarmee wellicht ook de klinische gevolgen na een chirurgische ingreep.

Tot slot wordt in dit proefschrift de aandacht gericht op mogelijke therapeutische interventies (=ontwikkeling van geneesmiddelen) voor de preventie en/of behandeling van de complicaties die gerelateerd zijn aan CPB. De strategie die hierbij werd getest was om systemische immuunreacties die veranderde vaatreactiviteit zouden kunnen opwekken te onderdrukken en tegelijkertijd om vaatreactiviteit direct te beïnvloeden d.m.v. modulatie van de zogenaamde S1P-receptor, die bij beide processen betrokken is. In hoofdstuk 7 werden ratten kort voorafgaand aan CPB behandeld met FTY720, een niet-selectieve agonist (=stimulerende stof) die aangrijpt op verschillende subtypes S1P-receptoren, of met SEW2871, een selectieve agonist van de S1P1-subtype-receptor. In figuur 2 is een patroon te zien waarin beide stoffen eenzelfde effect hadden op vaatreactiveit maar een verschillend effect op de systemische immuunrespons. D.w.z. beide stoffen versterkten contractiele en relaxerende reactiviteit na CPB (figuur 2B en 2C), maar alleen FTY720 beïnvloede het aantal lymfocyten in het bloed (figuur 2A). Deze resultaten suggereren dat beide S1P-receptor agonisten de vaatreactiviteit na CPB direct versterkten door modulatie van de S1P1-receptoren op de endotheelcellen en gladde spiercellen in de vaatwand.

Samengevat, het onderzoek in dit proefschrift onderstreept dat zowel de chirurgische procedures als het overnemen van lichaamsfuncties door de hart-long machine bij CPB verschillende fysiologische processen sterk verandert. Het optreden van ontstekingsreacties en vaatfunctie zijn hiervan belangrijke uitingen. Verder blijkt dat belangrijke componenten zelfs tijdens een langere herstelperiode niet volledig normaliseren, hetgeen suggereert dat sommige processen langdurig verstoord zijn. Deze langdurige verstoring vormt wellicht een belangrijk onderdeel van de klinische complicaties die zowel op korte als langere termijn na CPB zijn beschreven. Om dit te vermijden is directe beïnvloeding van vaatreactiviteit m.b.v. S1P1 receptor agonisten voorafgaand aan CPB een potentieel interessante optie.


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!Figure 1. Schematic representation of the changes in vascular reactivity and inflammatory processes following cardiopulmonary bypass (CPB). (A) CPB evoked a differential pattern of temporal changes early post-CPB (i.e. 24-48h) in small (mesenteric and coronary) arteries vs. “long-term” changes in large conductance artery (aorta); (B) Rapid systemic inflammation and aorta endothelial cell activation after extracorporeal circulation (ECC) were not paralleled by acute changes in aorta endothelial relaxation function, but rather by changes at the “long-term”; (C) Up-regulated inflammatory markers in kidney and aorta normalised in due course after ECC, but were maintained elevated in the lung for at least 5 days after CPB.

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Figure 2. Effect of S1P-receptor agonism on circulating lymphocytes and vascular reactivity following CPB. (A) The amount of circulating lymphocytes was reduced after pre-treatment with the non-selective S1P-receptor agonist FTY720, but not with the selective S1P1-receptor agonist SEW2871. In contrast, both compounds similarly enhanced vascular (B) contractility and (C) endothelial relaxation function. This suggests that the enhanced vascular reactivity following pre-treatment was mediated via modulation vascular S1P1-receptors, independently from lymphopenia. * P<0.05, vs untreated group, one way ANOVA with Bonferroni test. # P<0.05, vs untreated group, t-test


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166 Ukrainian summary

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Acknowledgments

It is my pleasure to say that I enjoy working with all people from the Departments of Clinical Pharmacology and Anesthesiology whom I have been lucky to meet during these years. I will always keep the nice memories of this time in my hart.

First of all I would like to express my gratitude to my promotor Prof. Dr. Robert H. Henning, whose expertise, knowledge, assistance, fast work are always a template for his PhD-students. Dear Robert, it is always a pleasure to work with you because of your ability to encourage people, to make a nice work atmosphere, and, of course, to generate an enormous amount of the research ideas.

Prof. Dr. Michel M.R.F. Struys, my second promotor, thank you for your suggestions to improve our papers, the scientific support and for the constructive feedback.

Dr. Hendrik Buikema, my copromotor, thank you for your ideas, the excellent talent to present data and to write perfect articles, and for your work. I appreciate very much your supervision and learned a lot from you.

Dr. Anne H. Epema, my copromotor, thank you for your medical expertise in the developing of the experimental model, for sharing his knowledge and experience in the CPB research, and for the supervision and the encouragement.

During the past years I was lucky to work with Martin C. Houwertjes, who improved to the highest degree the experimental setup, performed the surgery perfectly, provided technical support of all experiments, and has done a lot of great work. Dear Martin, many thanks for your professionalism and for the excellent human qualities.

I would like to thank the members of the reading committee, Prof. Dr. Patrick Wouters, Prof. Dr. Ingrid Molema and Prof. Dr. Jozef G.R de Mey, for the revision and critical evaluation of this thesis.

Many nice people made my work in the Department enjoyable and convenient. AIO’s, postdoc’s, and the staff: Maggie, Roelien, Peter, Diana, Hjalmar, Talaei, Madhi, Yan, Sjoerd, Nadir, Hiddo, Maria, Bernadet, Hisko, Irma, Femke, Azuwerus, Marry, Maaike, Jan, Linda, Maria, Anouk, Pieter. Thank you all for the fun and support during theses years. Special thanks to the supporting staff of the Department: Ardy Kuperus, Alexandra Doeglas, Wessel Sloof, Adriaan van Doorn, Marja van der Ende. I appreciate very much everything you have done for me.

I would like to show appreciation to the academic staff of the Department of Clinical Pharmacology for the support and for the constructive, interesting meetings: Prof. Dr. Dick de Zeeuw, Dr. Bianca Brundel. Dr. Leo Deelman, Dr. Petra Denig, Dr. Richard van Dokkum, Dr. Pieter de Graeff, Prof. Dr. Floor Haaijer-Ruskamp,

168 Acknoledgments

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Dr. Frank Holtkamp, Dr. Frouke Hut, Dr. Cees de Langen, Dr. Peter Mol, Dr. Margie Monster-Simons, Dr. Itte de Waard.

I was also glad to collaborate and communicate with Matijs van Meurs, Francis M. Wulfert, Dr. Willem van Oeveren, Hubert Mungroop, Dr. Fellery de Lange, Yumei Wang.

Further, the work of the GUIDE team, Prof. Dr. Han Moshage, Riekje Banus, Maaike Bansema, Mathilde Pekelaer, and many others, is greatly appreciated.

I would like also to thank my friends Anna, Katya, Jullia, Yana, Natasha, Olya, Vladimir, Zhenya, Katya, Sasha, Zanna, Sergei, and many others for the friendship and support. It was a pleasure for me to meet you.

It is a pleasure to thank you, my paranimfen Maggie and Roelien, for your generous help and assistance.

Last but not at least, I would like to express my enormous gratitude to my family; my parents Tetiana and Vasyl, my husband Volodymyr, my brother Oleksii and my sister-in-law Ella, my small nephew Stella, a parents-in-law Georgii and Maria, also Iryna and Oleg, uncle Victor, aunt Ellena, cousins Maria, Iwan, Michail, and of course my small daughter Katja for the support during these years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170 Notes

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university of groningen vascular reactivity in ... · cardiopulmonary bypass (cpb) is a widely used...

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