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48-1

VASCULAR PHYSIOLOGYAshraf A. Sabe • David G. Harrison • Frank W. Sellke

Myocardial and pulmonary perfusion is regulated by a complex array of influences intrinsic and extrinsic to the vasculature. Surgical decisions are generally based on the anatomy of large arteries, where the presence of obstruc-tive lesions and vasomotor state of these vessels can affect myocardial, pulmonary, or other organ perfusion. Under normal circumstances, the microcirculation actually plays a more significant role in the regulation of blood flow. A basic understanding of vascular tone and vasomotor regulation, and the influence of various disease states, is necessary to optimize patient care. Over the past four decades, a great deal has been learned, and this has pro-moted the understanding of blood vessel regulation and organ perfusion in healthy and in diseased states.

The blood vessel wall is organized in three layers: the intima, media, and adventitia. The innermost, or intimal, layer is made of endothelial cells. Initially, the endothe-lium was thought to serve mainly as a barrier to the dif-fusion of macromolecules, but it is now known that the endothelium plays a pivotal role in vascular function, regulation of vascular tone, and control of local blood flow.1 The medial layer surrounds this intimal endothelial layer, and it is composed of a variable number of smooth muscle cells. Smooth muscle cells also control vascular tone via humoral vasoactive factors, neural mediators, or local paracrine factors (Fig. 48-1). The outermost, adven-titial, layer surrounds these vascular smooth muscle cells and provides structural integrity to the blood vessel, par-ticularly larger arteries.

The classification of microvessels based on structural characteristics is rather arbitrary, and there is a lack of uniformity in the definitions of microvascular seg-ments such as small arteries, arterioles, and venules.

Furthermore, the transition between these segments is gradual, and there is no clear demarcation between them. In general, microvessels are defined as vessels less than 300 µm in internal diameter. Capillaries are the smallest blood vessels, defined as vessels whose walls are com-posed of only endothelial tubes. The microvessels through which blood flows toward capillaries are arterial microves-sels, and those that drain from capillaries are venous microvessels.2 Arterial microvessels usually have three coats: (1) a thin tunica intima; (2) a relatively thick tunica media, composed of one to several layers of smooth muscle cells disposed circumferentially; and (3) tunica adventitia, composed of fibrous elements and fibroblasts. Venous microvessels collect the blood from capillaries and have thinner vascular walls than arterial microvessels do. Venules (50 µm in diameter) do not possess smooth muscle cell layers. Smaller venules are composed of only endothelial cells and pericytes. Venules are highly perme-able and have an important role in nutrient exchange.

The regulation of myocardial perfusion depends on many intrinsic and extrinsic factors that might be affected by atherosclerotic lesions. In the coronary circulation, it has been shown that vasomotor regulation of vessels, in addition to the actual anatomy, plays an important role in coronary perfusion and operative decision making. Blood flow is also largely dependent on the resistance generated by the microcirculation. Although early studies on vasomotor regulation consisted of indirect assess-ments using measurements of flow and calculations of resistance, recent investigations into the properties of the intact coronary circulation have yielded much informa-tion, as have modern methods of analysis for interpreta-tion of physiologic data.3-6

C H A P T E R 4 8

VASCULAR RESISTANCE

REGULATION OF VASCULAR TONEIntrinsic and Extrinsic Vasomotor ControlRole of the EndotheliumRole of Metabolism and AutoregulationFlow-Induced DilationNeurohumoral Influence on MicrocirculationIntrinsic Myogenic ToneImpact of Extravascular and Humoral Forces

on the MicrocirculationRole of Venules in Vascular Resistance

CHAPTER OUTLINE

ENDOTHELIAL FACTORS IN VASCULAR GROWTH AND RESPONSE TO INJURY

EFFECT OF DISEASE STATES ON CORONARY CIRCULATION

PULMONARY VASCULAR PHYSIOLOGY

IMPROVING MYOCARDIAL PERFUSIONAngiogenesisChallenges and Future Directions

SUMMARY

ISBN: 978-0-323-24126-7; PII: B978-0-323-24126-7.00048-X; Author: Sellke & del Nido & Swanson; 00048ISBN: 978-0-323-24126-7; PII: B978-0-323-24126-7.00048-X; Author: Sellke & del Nido & Swanson; 00048

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48-2 SECTION 2 ADULT CARDIAC SURGERY

The microcirculation possesses unique features that allow it to respond to the dynamic changes in nutrient requirements and to interact with surrounding tissue. It is important to note that although the various vascular beds in the body possess many similarities, there are also subtle differences. This chapter will discuss regulation of vascular tone, with an emphasis on coronary and pulmo-nary circulation, and will review the physiologic and molecular basis of recent advances in ischemic cardiac disease.

VASCULAR RESISTANCE

An understanding of vascular resistance is important, because these resistance vessels cause pressure losses and are responsible for regulation of perfusion. Initially, it was thought that the precapillary arterioles were respon-sible for vascular resistance, with little resistance involve-ment by the vessels larger than 25 to 50 µm in diameter. However, subsequent work revealed that over half of total vascular resistance is caused by vessels with diameters larger than 100 µm, even including vessels up to 300 µm.7,8 Contrary to previous belief, the venous circu-lation, under conditions of vasodilation, may account for up to 30% of vascular resistance. Figure 48-2 shows that after vasodilation with dipyridamole, larger arteries and veins assume a greater role in resistance.8,9 Similarly, is-chemia results in a significant redistribution of vascular

FIGURE 48-1 ■ Regulation of vascular tone by factors released from the endothelium, activated platelets and leukocytes, neuronally released factors, and circulating substances. Ace, Angiotensin-converting enzyme; Ach, acetylcholine; ADP, adenosine diphosphate; Ang, angiotensin; cGMP, cyclic guanosine monophosphate; EDHF, endothelium-derived hyperpolarizing factor; ET, endothelin; 5HT, 5-hydroxytryptamine (serotonin); NE, norepinephrine; NO, nitric oxide; NOS, nitric oxide synthase; PGI2, prostaglandin I2.

Aggregating platelets

Shearstress

Ang IAng II

Arginine

ET-1Ace

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Ach

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*Endothelial receptors

Muscarinic5-HT�-adrenergicBradykininPurinergic

Ca++

VY

HistaminergicThrombinSubstance PProstaglandin E2Endothelin

5HT — ADP- Thromboxane

FIGURE 48-2 ■ Intravascular pressures in the coronary microcir-culation under basal conditions and during vasodilation with dipyridamole. The distribution of vascular resistance is not static. Instead, the size of the vessels regulating vascular tone depends on the tone of the vasculature. (Adapted from Chilian WM, Layne SM, Klausner EC, et al: Redistribution of coronary microvascular resistance produced by dipyridamole. Am J Physiol 256:H383–H390, 1989.)

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48 VASCULAR PHYSIOLOGY 48-3

vasomotor tone is critically involved in organ perfusion. Vascular responses to endogenous substances are sum-marized in Figure 48-4. All these factors have an espe-cially significant role in the setting of microvascular tone.2

Based on in vitro observations, Jones and colleagues12 found that larger arterioles are more sensitive to shear stress than myogenic factors, whereas small microvessels are more sensitive to metabolic factors. They organized arterial microvessels into three microdomains as gov-erned by distinct forms of regulation based on vessel size: (1) small arterioles (<50 µm), most sensitive to metabolic mediators; (2) intermediate arterioles (50 to 80 µm), most sensitive to myogenic mechanisms; and (3) large arteri-oles (80 to 150 µm), most sensitive to flow-induced dila-tion. Undoubtedly, there is overlap among these three components; however, this model provides a framework for understanding the regulation of microvessels.

Jones and colleagues12 hypothesized that the longitu-dinal disposition of these three microdomains enables integrated adjustment of flow conductance in the face of various influences, such as increased metabolism or a reduction in perfusion pressure. For example, dilation of small arterioles by augmented metabolism produces a decrease in luminal pressure in upstream microvessels, leading to the dilation of intermediate arterioles by decreasing the myogenic tone. These microvascular dila-tions could also produce an increase in shear stress and result in enhanced flow-induced dilation in large arteri-oles. As a result, all sizes, or domains, of arterial microves-sels dilate in response to the metabolic stimulation. The marked longitudinal heterogeneity of microvascular responses may be at least partly explained by this micro-domain hypothesis.

resistance9; this reveals that the distribution of vascular resistance is dynamic and is also largely dependent on vascular tone.

The redistribution of microvascular resistance can change the myogenic tone in each microvascular segment because the luminal pressure in a certain vascular segment is determined by the systemic pressure and the upstream distribution of vascular resistance. For example, when resistance is shifted upstream by the dilation of small arterioles, the luminal pressure in the upstream microves-sels decreases, resulting in myogenic dilation. Changes in the venular pressure caused by the resistance redistribu-tion can also affect the capacity for substance exchange and lead to edema formation.

In the coronary circulation, pressure losses also occur as vessels course from the epicardium through the myocardium10; this is accentuated further in the setting of cardiac hypertrophy. Such a phenomenon is particu-larly relevant clinically, as it in part explains the propen-sity of the subendocardium to develop ischemia in hypertrophied hearts (Fig. 48-3). The hypertrophied pathologic state causes a decrease in the perfusion pres-sure of the subendocardium, predisposing it to ischemia and infarction.11

REGULATION OF VASCULAR TONE

Intrinsic and Extrinsic Vasomotor ControlVasomotor tone is influenced by the intrinsic properties of the vessel wall, by local innervation, and by substances from surrounding parenchymal tissue. This regulation of

FIGURE 48-3 ■ Transmural losses of coronary perfusion pressure in normal and hypertrophied hearts. Pressures were measured using micropuncture–servo null techniques in hearts perfused via the left main coronary artery at 100 mm Hg. (Adapted from Fujii M, Nuno DW, Lamping KG, et al: Effect of hypertension and hypertrophy on coronary microvascular pressure. Circ Res 71:120–126, 1992.)

Aortic pressure = 100 mm Hg

Normal left ventricle Hypertrophied left ventricle

65 mm Hg

57 mm Hg

52 mm Hg40 mm Hg


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FIGURE 48-5 ■ Endothelial cells have both metabolic and synthetic functions. Through the secretion of a large variety of mediators, these cells are able to influence cellular function throughout the body. LDL, Low-density lipoprotein; MHC, major histocompatibility complex. (Adapted from Galley HF, Webster NR: Physiology of the endothelium. Br J Anaesth 93:105–113, 2004.)

Antithrombotic factorsProstacyclinThrombomodulinAntithrombinPlasminogen activatorHeparin

Matrix productsFibronectinLamininCollagenProteoglycansProteases

Procoagulant factorsvon Willebrand factorThromboxane A2ThromboplastinFactor VPlatelet activating factorPlasminogen activator inhibitor

Vasodilator factors

ENDOTHELIAL CELL

Nitric oxideProstacyclin

Inflammatory mediatorsInterleukins 1, 6, 8LeukotrienesMHC II

Lipid metabolismLDL-receptorLipoprotein lipase

Growth factorsInsulin like growth factorTransforming growth factorColony stimulating factor

Vasoconstricting factorsAngiotensin converting enzymeThromboxane A2LeukotrienesFree radicalsEndothelin

FIGURE 48-4 ■ Factors that influence microvascular tone. ADP, Adenosine diphosphate; BK, Ca2+-activated K+; CGRP, calcitonin gene-related peptide; ET, endothelin; 5HT, 5-hydroxytryptamine (serotonin); NPY, neuropeptide Y; TXA2, thromboxane A2. (Adapted from Komaru T, Kanatsuka H, Shirato K: Coronary microcirculation: physiology and pharmacology. Pharmacol Ther 86:217–261, 2000.)

Neural factors• Sympathetic (norepinephrine, NPY, etc.)• Parasympathetic (acetylcholine, etc.)• Nonadrenergic noncholinergic (CGRP, substance P, etc.)

Substances releasedfrom vascular wall

• Angiotensin II• Vasopressin• ET• BKetc.

Metabolic factors(myocardium)

Circulating hormones• Angiotensin II• Vasopressin• Sex hormones• Growth factorsetc.

Thrombus-related substances• 5-HT• TXA2

• ADP• Thrombin, etc.

Myogenic factorShear stress

Role of the EndotheliumThe endothelium plays a pivotal role in vasomotor tone regulation. Many substances affect tone via endothelium-mediated mechanisms. Endothelial cells also release several substances that affect coronary

resistance, including vasodilators such as nitric oxide (NO•), prostaglandins, and vasoconstrictors including angiotensin converting enzyme endothelin, and reactive oxygen species (summarized in Fig. 48-5). As the major regulatory molecule, NO• is produced by a constitutively expressed enzyme known as endothelial nitric oxide synthase


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exposure to cytokines.18-20 In the coronary circulation, the release of NO• confers a state of basal vasodilation; therefore, administration of NO• synthase antagonists increases resting coronary resistance. However, when substances such as acetylcholine and bradykinin are administered, coronary microvessels of all sizes dilate, resulting in decreased coronary resistance. Endothelial NO• production is affected by a variety of mechanisms in many disease states. The signal transduction pathways through which NO• acts are summarized in Figure 48-6. It is likely that the most important pathway involves activation of soluble guanylate cyclase, which catalyzes the formation of cGMP from guanidine triphosphate. The cGMP serves as an allosteric regulator of the enzyme cGMP-dependent protein kinase, which phosphorylates contractile proteins and ion channels, decreasing intra-cellular calcium and the sensitivity of contractile proteins to intracellular calcium. The binding of NO• to cyto-chrome oxidase in the mitochondria alters oxygen con-sumption and in turn can affect oxygen demand. Similarly, receptors for atrial natriuretic peptide and brain natri-uretic peptide are also the particulate forms of guanylate cyclases, and these signal vasodilation via similar path-ways. NO• is also released in response to sodium nitro-prusside and organic nitrates.

(eNOS or NOS-3). NO• is formed as a result of a series of electron transfers from reduced nicotinamide adenine dinucleotide phosphate (i.e., NADPH) to flavin adenine dinucleotide (FAD) and flavin mononucle-otide (FMN) on the reductase domain, and electron transfer to a prosthetic heme group in the oxygenase domain. When heme reduction occurs, arginine is catalyzed to citrulline and NO•. The NO• formed dif-fuses to underlying vascular smooth muscle, where its actions include stimulation of soluble guanylate cyclase, increasing the level of cyclic guanosine monophosphate (cGMP) and prompting vasodilation via activation of cGMP-dependent protein kinase.13 Although binding of calcium and calmodulin is a prerequisite for activity of eNOS, other events, such as phosphorylation,14 mem-brane binding,15 binding of eNOS with heat-shock protein 90, and association with the integral membrane protein caveolin16 can also modulate NOS activity. In addition, NO• can undergo reactions with thiol-containing compounds to form biologically active nitroso molecules.17

Although eNOS is expressed constitutively, it under-goes important gene expression regulation by factors such as shear stress, endothelial cell growth, hypoxia, exposure to oxidized low-density lipoprotein, and

FIGURE 48-6 ■ Signal transduction pathways for the vascular responses to agonists. Nitric oxide (NO), prostaglandin I2 (PGI2), and endothelium-derived hyperpolarizing factor (EDHF) are important for the cross-talk between the endothelium and the vascular smooth muscle. AC, Adenylyl cyclase; COX, cyclooxygenase; Cyt P450, cytochrome P450; DG, diacylglycerol; eNOS, endothelial nitric oxide synthase; Gi, Gi-protein; Gq, Gq protein; PCS, prostacyclin synthase; PI, phosphatidylinositol; PIP, phosphatidylinositol 4-phosphate; PIP2, phosphatidylinositol 4,5-bisphosphate; PKA, protein kinase A; PLA2, phospholipase A2; PLC, phospholipase C; R, receptors; TK, tyrosine kinase; VGCC, voltage-gated Ca2+ channel. (Adapted from Komaru T, Kanatsuka H, Shirato K: Coronary micro-circulation: physiology and pharmacology. Pharmacol Ther 86:217–261, 2000.)

Arachidonic acid

Proteinphosphorylation

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PLC-β

PLC-β

PLA2Gi

Gi

PKC

GsAC Gs

G i

Gi/q

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K+ Channel

Hyperpolarization

cAMP

cGMP GTP

ATP

PKAVGCC inhibition

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of endothelial cells with vascular smooth muscle cells and the intermediates involved is outlined in Figure 48-7.

Role of Metabolism and AutoregulationThe ability of a vascular bed to adjust its tone to maintain a constant flow during changes in perfusion pressure is termed autoregulation.22 This process is most effective in the coronary circulation when pressure is between 40 and 160 mm Hg. The subendocardium and subepicardium differ in the range of pressures over which autoregulation can be observed. In the subendocardium flow begins to decrease at pressures of less than 60 to 70 mm Hg, whereas in the subepicardium these decreases in flow occur at significantly lower pressures.23 Clinically, sys-temic arterial hypertension affects the range over which autoregulation occurs in the subendocardium, such that flow begins to decline at even higher pressures. Such a change in subendocardial perfusion pressure in the setting of hypertrophic myocardium increases the likelihood of subendocardial ischemia. Patients with systemic hyper-tension may have an increased lower limit of autoregula-tion and may be more likely to suffer brain ischemia during surgery, or other times, even at pressures suffi-cient to sustain adequate perfusion in patients with normal blood pressure.

The predominant changes in vasomotor tone, con-cerning both autoregulation and metabolic vascular regu-lation, occur in vessels smaller than 100 µm in diameter. The rate of oxygen consumption in the myocardium is closely related to myocardial perfusion via coronary microvascular tone. The ability of the myocardium to

Although NO• is the major regulator of vascular tone, there are other factors that modulate endothelium-dependent vascular tone in coronary, pulmonary, and peripheral circulations. Endothelium-derived hyperpo-larizing factor (EDHF) is an example. The endothelium-dependent hyperpolarization of vascular smooth muscle is mediated by the opening of a calcium-dependent potas-sium channel, leading to K+ exit from the cell. When the vascular smooth muscle is hyperpolarized, voltage-sensitive calcium channels are closed, leading to a reduc-tion in intracellular calcium. The role of the various EDHFs probably varies depending on the vessel size, the species, and the vascular bed under consideration. It is likely that several EDHFs exist, but current evidence suggests that hydrogen peroxide and epoxyeicosatrienoic acid, a cytochrome p450 metabolite of arachidonic acid, play major roles. Hydrogen peroxide is formed by the mitochondria in response to shear stress and acetylcho-line. The hydrogen peroxide not only opens large con-ductance potassium channels; it also activates protein kinase G, causing oxidation and dimerization.21 Prosta-glandin synthesis by the endothelium also modulates tone in the microcirculation. The predominant prostaglandin produced by endothelial cells is prostacyclin. There is substantial interaction between NO•, EDHF, and pros-tacyclin. A major stimulus for release of these factors is shear stress, or the tangential force of fluid as it flows over the endothelium, resulting in flow-dependent vasodila-tion. Interestingly, the importance of NO• seems to decline and the role of the EDHF increases as blood vessels decrease in size. In addition, the production of EDHF may increase when NO• is low. The interaction

FIGURE 48-7 ■ Role of the increase in cytosolic calcium concentration in the release of endothelium-derived relaxing factors (EDRF). Endothelial receptor activa-tion induces an influx of calcium into the cytoplasm of the endothelial cell. After inter-action with calmodulin, NO-synthase and cyclooxygenase are activated, leading to the release of endothelium-derived hyperpolar-izing factor (EDHF). NO causes relaxation by activating the formation of cyclic GMP (cGMP) from GTP. EDHF causes hyperpolar-ization and relaxation by opening K+ chan-nels. Prostacyclin (PGI2) causes relaxation by activating adenylate cyclase (AC), which leads to the formation of cyclic AMP (cAMP). Any increase in cytosolic calcium (including that induced by the calcium ionophore A23187) causes the release of relaxing factors. When agonists activate the endothe-lial cells, an increase in inositol phosphate may contribute to the increase in cytoplas-mic Ca2+ by releasing it from the sarcoplas-mic reticulum (SR). (Adapted with permission from Vanhoutte PM, Boulanger CM, Vidal M, et al: Endothelium-derived mediators and the renin-angiotensin system. In Robertson JIS, Nicholls MG, editors: The renin-angiotensin system, London, 1993, Gower Medical.)

Ca2�-Calmodulin

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Adenylatecyclase

Cyclo-oxygenase

Smoothmuscle cell

Endothelialcell

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Relaxation

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have been shown to be involved, at least in porcine coro-nary arterioles.27,28 The exact mechanism of regulation can depend on age, vessel size, and the vascular bed under consideration. A possible flow-induced arteriolar dilation mechanism is summarized in Figure 48-8.

Autoregulation is mediated by the actions of several factors such as NO•, EDHF, and adenosine. Removal of a particular factor does not prevent autoregulation, because the other factors seem to take over its function. Adenosine and hydrogen peroxide also cause hyperpolar-ization of vascular smooth muscle. From the work of Duncker and colleagues,22 and others, we now know that several factors work together to influence metabolic reg-ulation and autoregulation, leading to adequate regula-tion of coronary vascular tone despite interruption of any particular pathway.22

Neurohumoral Influence on MicrocirculationThe coronary arterial system is densely innervated with sympathetic and parasympathetic nerves.29 Neurotrans-mitters released from these, and a wide variety of humoral substances, significantly affect the microvascular tone. Neurohumoral factors affect coronary microvascular tone, and together with myogenic, flow-induced, and local metabolic controls, they participate in determining the coronary vascular resistance necessary for oxygen and nutrition supply to the myocardium (see Fig. 48-4).

extract additional oxygen to meet increased demand is limited because myocardial oxygen extraction is already near its maximal threshold under resting conditions. To account for this limitation, coronary flow rises in response to increased myocardial oxygen requirements.

Flow-Induced DilationFlow-induced dilation is a ubiquitous phenomenon of blood vessels in various organs of animals, including humans.2,24,25 Flow-induced dilation plays important physiologic roles by: (1) protecting the vessel wall against friction induced injury, (2) preventing vascular steal phe-nomenon by dilating upstream vessels when there is focal hyperemia, (3) reducing the heterogeneity of flow distri-bution, and (4) buffering the pressure distribution in response to rapid pressure changes.

The mechanism by which endothelial cells sense flow has been a focus of substantial investigation. Studies by Tzima and colleagues26 have defined a mechanosensory complex composed of VE cadherin, the platelet endothe-lial cell adhesion molecule (PECAM-1), and the vascular endothelial growth factor receptor-2 (VEGFR-2) that is critical in this process. Shear seems to stimulate PECAM-1 and VE-cadherin, which in turn transactivate VEGFR2, leading to downstream signaling events. In contrast to the myogenic response, the endothelium is required for flow-induced dilation. Regulation of flow-mediated vasodila-tion is controversial, and both NO• and prostaglandins

FIGURE 48-8 ■ Possible signal transduction pathway for the flow-induced arteriolar dilation, which is mediated by mechanotransduc-tion via actin stress fibers, and the subsequent activation of the focal adhesion kinase and endothelial nitric oxide synthase (eNOS) phosphorylation. cGMP, Cyclic guanosine monophosphate; FAK, focal adhesion kinase; GTP, guanosine triphosphate; NO, nitric oxide; sGC, soluble guanylyl cyclase. (Adapted from Komaru T, Kanatsuka H, Shirato K: Coronary microcirculation: physiology and phar-macology. Pharmacol Ther 86:217–261, 2000.)

Actin stress fiber

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Extracellular matrixFocal adhesion site

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such as these play a critical role in determining the basal tone and in maintaining the intraluminal pressure of the downstream exchange vessels within a physio-logic level.41,42 In the myocardium, myogenic responses to increases in pressure are greater in subepicardial microvessels than in vessels from the subendocardium. In addition, myogenic tone can be reduced during inflammatory states when increased expression of induc-ible nitric oxide synthase (iNOS) causes altered myocar-dial perfusion. Increases in myogenic tone, which occur during stretch of vascular smooth muscle, are associated with an increase in inositol 1,4,5-trisphosphate, presum-ably because of activation of phospholipase C.39,43,44 In addition, the myogenic tone mediator 20-HETE pro-duces constriction of vascular smooth muscle by promot-ing Ca2+-activated K+ (BK) channel inhibition. This induces depolarization and increases levels of [Ca2+]I. This effect is most likely caused by activation of L-type Ca2+ channels or the activation of protein kinase C and inhibition of the Na/K-ATPase.45 Other mediators prob-ably involved in the myogenic tone response include mitogen-activated protein kinases and Rho protein. The downstream mediator Rho kinase can modulate myo-genic tone by regulation of the actin cytoskeleton. One potential therapeutic agent for treatment of hypertension and coronary spasm is the use of Rho kinase inhibitors. A summary of possible mechanisms of myogenic tone is shown in Figure 48-9.

Impact of Extravascular and Humoral Forces on the MicrocirculationIn the setting of pathologic processes such as ischemia leading to decreased tissue compliance or increased tissue edema, extravascular forces have an especially important role. For example, collateral perfusion is particularly sen-sitive to changes in heart rate (more frequent extravascu-lar compression) and ventricular diameter (stretch).6,46 The coronary circulation is particularly unique in that it is exposed to a large number of extravascular forces pro-duced by contraction of adjacent myocardium and intra-ventricular pressures. Of relevance to the concept of extravascular forces is the idea that these might collapse coronary vessels under certain circumstances. Of note, flow through the epicardial coronary arteries halted when aortic pressure fell to levels ranging from 25 to 50 mm Hg, raising the possibility that extravascular forces might be sufficiently high to collapse vessels when intraluminal pressures declined to values below this critical value.47 Flow in the coronary microcirculation continued even when the arterial driving pressure was minimally higher than coronary venous pressure. Based on modeling and various experimental interventions, it has been deter-mined that the decrease of antegrade blood flow in larger upstream vessels is associated with continued forward flow in microvessels and that this is due to capacitance in the coronary circulation.48 Kanatsuka and colleagues49 used a floating microscope to visualize epicardial capil-laries and were able to show that red cells continued to flow, even after perfusion had stopped in the more proxi-mal vessels.49 Using this approach, they showed that the pressure at which flow stops in the epicardial coronary

The role of the autonomic sympathetic and parasym-pathetic nervous systems is important in regulation of coronary perfusion. In vivo, the vascular response to sym-pathetic stimulation is mediated by both α-adrenergic and β-adrenergic receptors. In the coronary circulation, the predominant receptor subtype is the β-adrenergic receptor.30 For example, direct sympathetic nerve stimu-lation evokes coronary vasodilation and an increase in coronary flow. If β-adrenergic antagonists are adminis-tered, a transient vasoconstriction can be observed.30 When coronary microvessels are studied in vitro, α-adrenergic stimulation has minimal contractile effects. When selective α2-adrenergic stimulation is applied using pharmacologic stimuli, there is rather potent vaso-dilation of all sizes of coronary microvessels, predomi-nantly the result of the release of endothelium-derived NO•. β-Adrenergic stimulation produces a potent relax-ation of all coronary arteries, but especially of small-resistance vessels.30 In addition, β2-adrenergic receptor subtype predominates in vessels smaller than 10 µm in diameter in in vitro studies, whereas a mixed β1- and β2-adrenergic receptor population controls vascular resis-tance in in vivo studies.30 On the other hand, larger coronary vessels are regulated by a mixed β1- and β2-adrenoceptor subtype population. Activation of choliner-gic receptors by either vagal stimulation or the infusion of acetylcholine produces uniform vasodilation of coro-nary vessels.31 This vasodilation is predominantly medi-ated by endothelium-derived NO•, although release of EDHF32 and release of prostaglandin substances may contribute.33 The coronary flow increase by vagal stimu-lation could be blunted by a metabolically mediated flow decrease caused by a decrease in the heart rate and myo-cardial contractility.34

Other neurotransmitters that act on the coronary cir-culation include neuropeptide Y, which is mainly released with norepinephrine as a co-transmitter from sympa-thetic postganglionic nerve terminals upon intense sym-pathetic activation.2,35 Intracoronary application of neuropeptide Y markedly decreases coronary flow, pro-ducing myocardial ischemia without large coronary artery constriction.36,37 These results point to its potent and specific constrictor effects on coronary microvessels. Substance P, a potent vasodilator whose effect is depen-dent on the endothelium, is contained in perivascular nerve fibers and sensory ganglia.38

Intrinsic Myogenic ToneMyogenic contraction is observed when applying luminal pressure to microvessels, which causes development of intrinsic vascular tone, as shown by elevated wall tension or a decrease in vessel diameter. The microcirculation possesses this intrinsic myogenic tone response, which also contributes to maintaining basal vascular tone and autoregulation.39 Recent evidence has shown that in the microcirculation, fenestrations in the internal elastic lamina allow communications between the endothelium and vascular smooth muscle. Lowering pressure activates endothelial TRPV4 channels in this microdomain, pro-moting endothelial cell calcium sparklets, vascular smooth muscle hyperpolarization, and vasodilatation.40 Responses


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Role of Venules in Vascular ResistanceControl of vasomotor regulation differs between the venous and arterial microcirculations, and certain reac-tions to pathologic stimuli occur preferentially on one side of the capillary bed. Therefore, consideration of the venous circulation apart from the arterial circulation is needed. Venules have considerable importance under conditions of vascular dilation, such as during exercise, metabolic stress, or reperfusion after myocardial isch-emia.9 The venous circulation can influence myocardial stiffness and relaxation properties of the heart. Veins also respond differently to agonists and neuronal stimulation compared with arteries in the same vascular bed.33,52 Venules are also the initiating site of neutrophil adher-ence and transmigration, whereas arterioles seldom man-ifest these initial changes in the inflammatory response.53 Ischemia-reperfusion has been determined to cause endothelial dysfunction in veins but, under similar condi-tions, arterioles appear to be more susceptible to a reduc-tion in endothelium-dependent relaxation than are coronary venules, despite the fact that leukocytes prefer-entially adhere to venular rather than to arterial endothe-lial cells.54 In addition, complement fragment C5a causes

microvessels was only a few millimeters of mercury higher than right atrial pressure.49 In addition, when ven-tricular diastolic pressure is high, vessels deeper in the subendocardium might be made to collapse by pressure transmitted from the ventricular chamber. In contrast, coronary epicardial vessels do not close at any pressure. It therefore seems likely that the concept of “critical closing pressure” is not applicable to all vessels in the coronary circulation.

The response of the coronary microcirculation to humoral agents differs with vessel size and location. Endothelin-1 produces vasoconstriction when adminis-tered to the adventitial surfaces of coronary microvessels. The degree of constriction produced by endothelin-1 is inversely related to the size of the vessels. In contrast, when endothelin-1 is administered intra-arterially, vaso-dilation occurs, presumably via release of NO•.31,33 Sero-tonin constricts vessels less than 100 µm in diameter, whereas it causes vasodilation of smaller arteries.50 Vaso-pressin, on the other hand, produces greater constriction of microvessels less than 100 µm in diameter than it produces in larger microvessels.51,52 In the larger epi-cardial coronary arteries, vasopressin predominantly causes vasodilation.

FIGURE 48-9 ■ Schematic illustrations forthe possible mechanisms of (A) the myo-genic constriction and (B) its compensatory mechanisms. DG, Diacylglycerol; 20-HETE, 20-hydroxyeicosatetraenoic acid; IP3, inositol 1,4,5-trisphosphate; PKC, protein kinase C; PLA2, phospholipase A2; PLC, phospholipase C. (Adapted from Komaru T, Kanatsuka H, Shirato K: Coronary microcirculation: physiology and pharmacology. Pharmacol Ther 86:217–261, 2000.)

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promotes vascular growth, because while endothelial cells are proliferating to form new blood vessels, the high levels of NO• promote tube formation. Similarly, in response to the denudation injury, proliferating endothe-lial cells increase NO• production during the growth period to compensate for the lack of endothelial cells in the denuded area while also decreasing platelet adhesion and vascular smooth muscle proliferation in that same area. Moreover, endothelial progenitor cells (EPCs) from the bone marrow have a role in repair of denuded vessels as well as angiogenesis. Although not completely eluci-dated, circulating EPCs seem to vary in quantity from one patient to the next. Common risk factors such as diabetes and hypercholesterolemia decrease these cells, whereas lipid-lowering drugs such as 3-hydroxy-3- methylglutaryl-coenzyme A (HMG-CoA) reductase inhi-bitors increase them.

EFFECT OF DISEASE STATES ON CORONARY CIRCULATION

Coronary microvascular homeostasis can be adversely af-fected in disease states by altering vascular diameter, quantity, or response to humoral factors. The contribu-tion of the endothelium to vascular regulation is particu-larly vulnerable to pathologies such as atherosclerosis, hyperlipidemia, diabetes, and the aging process. This mechanism is highlighted in Figure 48-11. The mecha-nisms underlying these abnormal endothelium-dependent responses are most likely multifactorial. Factors re-sponsible include abnormalities of G-protein signaling,

neutrophil adherence in venules but not in arterioles, suggesting that different mechanisms mediate neutrophil-endothelial adherence in the two vessel types.55

ENDOTHELIAL FACTORS IN VASCULAR GROWTH AND RESPONSE TO INJURY

It is important to identify the role of NO• and NO•-related factors in vascular development (Fig. 48-10). Nitric oxide inhibits vascular smooth muscle prolifera-tion via apoptosis. Animal models have shown that treat-ment with L-nitroarginine methyl ester, an inhibitor of NO• formation, markedly increases neointimal develop-ment after vascular injury.56 Local transfection with the eNOS cDNA also reduces the intimal proliferation that follows balloon injury.57 The vascular response to injury is enhanced in mice deficient in eNOS.58,59 Thus, NO• and cGMP-elevating agents inhibit the growth of fibro-blasts and vascular smooth muscle. This effect of NO• on vascular smooth muscle growth is mediated by cGMP and can be mimicked by cGMP analogs such as atrial natriuretic factor.59,60

NO• plays an important role in the process of angio-genesis, and endothelial cells do not seem to be sensitive to the growth-inhibitory effects of NO•. In fact, vascular endothelial growth factor (VEGF)-1 actions during angiogenesis are mediated by NO•. Endothelial cells in the proliferative phase have a sixfold increase in eNOS expression compared with confluent ones, and eNOS knockout mice have little VEGF activity.19 During the vascular injury response, this feed-forward condition

FIGURE 48-10 ■ Schematic representation of endothelium and vascular smooth muscle, demonstrating the multifaceted roles of nitric oxide released from the endothelium in the modulation of vascular function, structure, and the response to injury. cGMP, Cyclic guanidine monophosphate; PGI2, prostacyclin.

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role in the development of clinical symptoms despite normal coronary anatomy.

Impaired endothelium-dependent vasodilation has also been linked to increased cardiovascular events. The loss of NO• in cardiovascular disease not only leads to a decrease in vasodilation; it also predisposes to atheroscle-rotic lesion formation and vascular smooth muscle pro-liferation. NO• also has antioxidant properties and prevents adhesion molecule expression by endothelial cells. An example of relevance to the clinical setting is the endothelial changes in the coronary microcirculation after cardioplegic arrest and cardiopulmonary bypass during cardiac surgery.67 In this setting, endothelial dysfunction persists for some time after cardiopulmonary bypass, and it normalizes thereafter. This result has important clinical implications, because it is common for patients undergoing coronary artery bypass grafting, with seemingly complete coronary revascularization, to exhibit signs of myocardial ischemia during the hours after surgery—most likely caused by endothelial dysfunction.

Collateral vessels in the coronary circulation are par-ticularly important in coronary disease. These allow for normal resting perfusion to a region of the myocardium that is served by an occluded vessel, albeit at a lower perfusion pressure; however, the coronary arterioles nourished by collaterals develop markedly abnormal vas-cular reactivity—for example, impaired endothelium-dependent vascular relaxations and enhanced constrictions to vasopressin.51 Possible mechanisms of this impaired microvascular endothelium-dependent relaxation in the collateral-dependent region can involve changes in shear stress, pulsatile flow in the collateral-dependent micro-vasculature, or intracellular calcium levels. Such changes can cause disturbance in microvascular tone during a disease state.18

Clinically, patients suffering from ventricular hyper-trophy often complain of angina-like symptoms. Animal and human studies have demonstrated that cardiac hypertrophy causes a reduction in the maximal capacity of the coronary circulation to dilate in response to either reactive hyperemia or pharmacologic stimuli.5,64,68 One possible cause for this abnormal response may be a mismatch between the elevated myocardial mass and the relatively reduced coronary microcirculation. Peak flow normalized to myocardial mass may be reduced because of this relative paucity of coronary arterioles, because as the myocardium hypertrophies, the coronary resistance circulation might not increase enough to keep pace with the larger muscle mass. Another possible mechanism of impaired vasodilator responses may be explained by endothelial dysfunction, because many of the diseases associated with myocardial hypertrophy are also associated with a loss of endothelial NO• production.

In normal hearts, there is a linear relationship between the diameter of an epicardial coronary artery and the mass of myocardium perfused. Interestingly, epicardial coronary arteries do not enlarge to the same extent as the myocardium hypertrophies, so that for any diameter coronary artery, the amount of myocardium perfused is increased twofold. This phenomenon is particularly

FIGURE 48-11 ■ Reduced production and bioreactivity of endothelium-derived nitric oxide (NO•) in the setting of athero-sclerosis, diabetes, and many other pathologic conditions. Asymmetric dimethylarginine (ADMA) acts as an antagonist of I-arginine. Superoxide and other oxygen free radicals may inter-fere with NO• availability in conditions of increased oxidant stress. The peroxynitrite radical (OONO•) can inhibit tetrahydro-biopterin (BH4), a cofactor for nitric oxide synthase (NOS). LOO•, Peroxyl radical.

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resulting in reduced activation of eNOS in response to endothelial cell receptor activation; an alteration of levels of the critical cofactor for eNOS tetrahydrobiopterin; and an overproduction of asymmetric dimethylarginine, which acts as an antagonist for the eNOS substrate L-arginine. It has been shown that oxidative stress (via in-creased production of vascular superoxide O2−•) is particularly increased in the presence of common risk factors. Such an increase in oxidative stress will cause a reduction in endothelium-dependent vasodilation.

It is accepted that diseases that affect endothelium-dependent vascular dilation affect the coronary microcirculation as well as the larger vessels. Endothelium-dependent vasodilatation acetylcholine and bradykinin is dramatically impaired in coronary microvessels from monkeys fed a high-cholesterol diet for 18 months, and sometimes paradoxic constrictions can be observed.61 Similar findings have been made in other animal models. Subsequent studies performed using in vivo techniques showed that vasoconstriction caused by serotonin and ergonovine (both known to be modulated by the endothe-lium) is markedly enhanced in the coronary microcircula-tion of hypercholesterolemic monkeys.62 These findings are impressive because the coronary microcirculation is spared from the development of overt atherosclerosis. Therefore, in the setting of a risk factor for atherosclerosis, endothelial dysfunction occurs, leading to abnormal vascular responses. In humans with hypercholesterolemia, diminished flow responses to acetylcholine are restored by cholesterol lowering.63 Similar observations have been made either in humans or in experimental models of hyper-tension,64 ischemia-reperfusion,55,65 and diabetes.66 It has been suggested that this endothelial dysfunction plays a


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Hypoxia is a potent vasoconstrictive stimulus in these patients.74

Although the exact mechanism of HPV remains unclear, many believe that it is related to the inhibition of calcium channels in the pulmonary vascular smooth muscle that leads to contraction.75-78 This hypothesis is supported by increasing evidence that an increase in cyto-solic calcium appears to be an important factor in HPV development.77,79 Hypoxia has also been shown to promote membrane depolarization.79 Alternatively, HPV might also be related to the inhibition or secretion of an unknown endogenous mediator that results in vasocon-striction.71 Pulmonary venous hypertension causes pul-monary endothelial dysfunction characterized by reduced bioavailability of NO• and increased formation of vaso-constrictors such as endothelin 1 and thromboxane A2.80,81 Pulmonary venous hypertension may therefore increase pulmonary vasoconstriction and remodeling and cause an increase in pulmonary vascular resistance.82,83 Lung vas-cular responses may further increase pulmonary arterial pressure in congestive heart failure and augment the risk for right ventricular failure.

Aside from oxygen, agents used as potential therapies for HPV have include inhaled NO•, prostaglandins, endothelin receptor antagonists, protein kinase C inhibi-tors, and potassium channel activators (Fig. 48-13). Currently, advanced therapies are more likely to include combination treatments, and often incorporate endothelin receptor antagonists or cyclic GMP phospho-diesterase-5 inhibitors.84 Inhaled vasodilators are thought to circumvent potentially deleterious systemic side effects by acting predominantly on the pulmonary circulation (e.g., epoprostenol or the phosphodiesterase-3 inhibitor milrinone).85,86

relevant in the presence of a coronary stenosis, where a small lesion that is otherwise considered minimal becomes flow limiting in a hypertrophic state.

PULMONARY VASCULAR PHYSIOLOGY

The pulmonary vascular response has unique character-istics that distinguish it from other vascular beds. The normal pulmonary circulation is a low-pressure, low-resistance circuit with little or no resting tone. In contrast to the systemic circulation, where neural and humoral mechanisms predominate, the pulmonary circulation is under the control of both active factors that affect vascu-lar smooth muscle tone (e.g., autonomic nerves, humoral factors, gasses), and passive factors (e.g., cardiac output, left atrial pressure, airway pressure).69

In a normal state, unlike systemic arteries, pulmonary arteries have a much thinner smooth muscle layer, which is consistent with a low-pressure system. Small pulmo-nary arteries are the main effectors of pulmonary vascular resistance, such as hypoxic pulmonary vasoconstriction. In addition, the pulmonary capillary bed and the systemic capillary bed respond differently. Pulmonary veins are similar in structure to pulmonary arteries, but have less smooth muscle and may be regulated differently. Con-striction of pulmonary arteries results in elevated pulmo-nary artery pressure, which increases the pressure on the right side of the heart, whereas constriction of pulmonary veins increases pulmonary capillary pressure, and this could result in pulmonary edema. With disease, the structure of pulmonary vessels can change significantly. With a chronic increase in pulmonary vascular pressure, there is a structural remodeling, with fibrosis, particularly in the intimal layer, and increased size of the smooth muscle layer, which results in a marked alteration in control mechanisms.69

A unique characteristic of the pulmonary circulation is its response to hypoxia. Pulmonary arteries contract when oxygen tension is acutely decreased (Fig. 48-12), unlike systemic vessels, which dilate in response to hypoxia. This phenomenon, called hypoxia-induced pulmo-nary vasoconstriction (HPV) is an important mechanism that aids in matching ventilation with perfusion by direct-ing blood flow from poorly ventilated regions of the lung to areas with normal or relatively high ventilation.70 Although acute HPV benefits gas exchange and maxi-mizes oxygenation of venous blood in the pulmonary artery, sustained HPV or chronic exposure to hypoxia is a major cause of the elevated pulmonary vascular resis-tance and pulmonary arterial pressure in patients with pulmonary arterial hypertension associated with hypoxic cardiopulmonary diseases.71 Chronic vasoconstriction leads to vascular remodeling, pulmonary hypertension, and possibly cor pulmonale. Patients with chronic obstructive pulmonary disease, for example, are usually hypoxemic and may fall into this category. Similarly, pul-monary vasoconstriction presents a challenge in pediatric patients with congenital heart disease, as they are particu-larly susceptible to developing pulmonary hypertensive crises after cardiac interventions.72 Pulmonary vascular resistance is often increased after lung transplantation.73

FIGURE 48-12 ■ Actual tracing of isolated pulmonary artery response to hypoxia. Isolated rat pulmonary artery, supported between steel wires in a 37° C organ bath containing modified Henseleit solution and connected to a force transducer, was contracted with phenylephrine (10−7 M) and gassed with 95% oxygen, 5% carbon dioxide (hypoxia). The resulting tension tracing exhibits relaxation preceding a transient vascular con-traction. (Adapted from Tsai BM, Wang M, Turrentine MW, et al: Hypoxic pulmonary vasoconstriction in cardiothoracic surgery: basic mechanisms to potential therapies. Ann Thorac Surg 78:360–368, 2004.)

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gene therapy may enhance endothelium-dependent relax-ation, in addition to improving other aspects of cardiac performance. Several animal studies have demonstrated that therapeutic angiogenic interventions, in the setting of chronic ischemia, are associated with an improvement in myocardial perfusion and endothelium-dependent vasodilation in the area supplied by collaterals.87-89 These studies used growth factors such as VEGF, fibroblast growth factor (FGF)-1, or FGF-2 placed in the perivas-cular area. Possible mechanisms through which these factors act include FGF-2– and VEGF-induced release of NO•, which improves collateral perfusion and decreases tissue ischemia.90 In addition, it has been shown that during periods of chronic ischemia there occurs an upreg-ulation of FGF-2 and VEGF receptors. This finding is consistent with results showing that after administration of growth factors, endothelium-dependent relaxation occurs in the collateral-dependent region, but not in myo-cardium perfused via the original vessels.91 In addition, these growth factors can stimulate the release of bone marrow–derived endothelial progenitor cells that promote collateral growth and endothelial function at the treated sites. Unfortunately, clinical trials to date have not dem-onstrated much benefit with growth factor and gene therapy in patients. Similarly, in clinical trials cell therapy has largely failed to enhance collateral formation to provide sufficient perfusion in the setting of myocardial ischemia. A number of clinical studies have been per-formed in which patients received bone marrow–derived mononuclear cells or endothelial progenitor cells by infu-sion several days after myocardial infarction. Other clini-cal trials have similarly found clinically insignificant benefits or no benefit to cell therapy.92 These studies dem-onstrate that cell therapy is safe in the short and long term. Differences in study outcomes are likely related to patient selection, comorbidities, and method of delivery.

Challenges and Future DirectionsMultiple factors may explain the failure to reproduce the multiple effective preclinical trials with growth factors, genes or cell therapy on vascular regeneration. Patients with end-stage CAD often have significant comorbid conditions, including diabetes and hypercholesterolemia, resulting in aberrant vascular signaling and endothelial dysfunction.93 These conditions result in hostile local environments with increased oxidative stress and increased expression of anti-angiogenic proteins.94 Technical issues may contribute to failure in growth factor therapy, includ-ing incomplete or nonsustained drug delivery. Similarly, cell-based therapies are fraught with biological and tech-nical failures. Issues include technical failure during injection resulting in “washout,” or “leak out” of cells from injection sites in the heart, lack of homing and incorporation of cells, and cell death.95 Potential approaches to improve homing and survival of the cells include heat shock treatment and incorporation of bioactive matrices. Another effort to improve functional-ity is the coadministration of growth factors with cell-based therapy. Gene-based therapies may hold the greatest promise for improving angiogenesis and myo-cardial perfusion, yet are the least studied and most

IMPROVING MYOCARDIAL PERFUSION

Despite improvement in catheter-based interventions and the use of coronary artery bypass grafting, a number of patients present with such diffuse coronary artery disease (CAD) that these interventions result in incom-plete or failed revascularization. These patients have poor clinical outcomes associated with decreased survival and decreased angina free survival. We will briefly discuss alternative and theoretical approaches to improve col-lateral formation for patients suffering from CAD not amenable to current interventions. These therapies include growth factor therapy, gene therapy, cell therapy, and adjuvant drug treatment.

It is important to note that collateral formation result-ing from the generic term angiogenesis actually encom-passes three distinct modes of vessel formation with differential regulation and developmental processes. These modes of vessel formation include “true angiogen-esis,” arteriogenesis, and vasculogenesis. True angiogen-esis is the growth of vessels from pre-existing vessels; arteriogenesis is the growth of large muscular arteries from existing vessels; and vasculogenesis is de novo blood vessel formation from progenitor cells. These three enti-ties have differential regulatory and developmental pro-cesses. Of these processes, arteriogenesis most effectively improves collateral dependent myocardial perfusion.

AngiogenesisIt has also been found that treatment of collateral-dependent vessels with angiogenic growth factors and

FIGURE 48-13 ■ Therapeutic strategies for blocking hypoxic pul-monary vasoconstriction. (A) Inhaled nitric oxide (NO) and (B) prostacyclin (PGI2) activate guanylate cyclase (GC) and adenyl-ate cyclase (AC), respectively, to cause vasodilation. (C) Protein kinase C (PKC) inhibitors prevent protein kinase C–mediated vasoconstriction. (D) Endothelin receptor (ETA, ETB) antagonists prevent endothelin-1 (ET-1) from binding to ETA, thus inhibiting vasoconstriction. (E) Potassium channel activation prevents calcium-dependent vasoconstriction. ATP, Adenosine triphos-phate; cAMP, adenosine 3′,5′-cyclic monophosphate; cGMP, guanidine 3′,5′-cyclic monophosphate; GTP, guanosine triphos-phate; Kv, voltage-gated potassium channel. (Adapted from Tsai BM, Wang M, Turrentine MW, et al: Hypoxic pulmonary vasocon-striction in cardiothoracic surgery: basic mechanisms to potential therapies. Ann Thorac Surg 78:360–368, 2004.)

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8. Chilian WM, Eastham CL, Marcus ML: Microvascular distribu-tion of coronary vascular resistance in beating left ventricle. Am J Physiol 251:H779–H788, 1986.

9. Chilian WM, Layne SM, Klausner EC, et al: Redistribution of coronary microvascular resistance produced by dipyridamole. Am J Physiol 256:H383–H390, 1989.

10. Chilian WM: Microvascular pressures and resistances in the left ventricular subepicardium and subendocardium. Circ Res 69:561–570, 1991.

11. Fujii M, Nuno DW, Lamping KG, et al: Effect of hypertension and hypertrophy on coronary microvascular pressure. Circ Res 71:120–126, 1992.

12. Jones CJ, Kuo L, Davis MJ, et al: Regulation of coronary blood flow: coordination of heterogeneous control mechanisms in vas-cular microdomains. Cardiovasc Res 29:585–596, 1995.

13. Murad F: Cyclic guanosine monophosphate as a mediator of vaso-dilation. J Clin Invest 78:1–5, 1986.

14. Corson MA, James NL, Latta SE, et al: Phosphorylation of endo-thelial nitric oxide synthase in response to fluid shear stress. Circ Res 79:984–991, 1996.

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16. Michel JB, Feron O, Sacks D, et al: Reciprocal regulation of endothelial nitric-oxide synthase by Ca2+-calmodulin and caveo-lin. J Biol Chem 272:15583–15586, 1997.

17. Myers PR, Minor RL, Jr, Guerra R, Jr, et al: Vasorelaxant proper-ties of the endothelium-derived relaxing factor more closely resemble S-nitrosocysteine than nitric oxide. Nature 345:161–163, 1990.

18. Uematsu M, Ohara Y, Navas JP, et al: Regulation of endothelial cell nitric oxide synthase mRNA expression by shear stress. Am J Physiol 269:C1371–C1378, 1995.

19. Arnal JF, Yamin J, Dockery S, et al: Regulation of endothelial nitric oxide synthase mRNA, protein, and activity during cell growth. Am J Physiol 267:C1381–C1388, 1994.

20. McQuillan LP, Leung GK, Marsden PA, et al: Hypoxia inhibits expression of eNOS via transcriptional and posttranscriptional mechanisms. Am J Physiol 267:H1921–H1927, 1994.

21. Zhang DX, Borbouse L, Gebremedhin D, et al: H2O2-induced dilation in human coronary arterioles: role of protein kinase G dimerization and large-conductance Ca2+-activated K+ channel activation. Circ Res 110:471–480, 2012.

22. Duncker DJ, van Zon NS, Ishibashi Y, et al: Role of K+ ATP channels and adenosine in the regulation of coronary blood flow during exercise with normal and restricted coronary blood flow. J Clin Invest 97:996–1009, 1996.

23. Boatwright RB, Downey HF, Bashour FA, et al: Transmural varia-tion in autoregulation of coronary blood flow in hyperperfused canine myocardium. Circ Res 47:599–609, 1980.

24. Pohl U, Holtz J, Busse R, et al: Crucial role of endothelium in the vasodilator response to increased flow in vivo. Hypertension 8:37–44, 1986.

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poorly understood. Gene-based therapies result in over-expression of survival proteins, use of genetically engi-neered bone marrow cells, and embryonic stem cells.95-97 In addition, investigators have also searched for adjuvant drugs to improve regenerative therapies. Commonly pre-scribed drugs like statins, cyclo-oxygenase inhibitors, and metformin, as well as naturally occurring substances like resveratrol have demonstrated a number of pleotropic effects in preclinical and clinical studies.98-104 The direct and indirect effects of these and other drugs on hyper-glycemia, hypercholesterolemia, and oxidative stress can act synergistically with other therapies to improve col-lateral formation.98-104 Despite the overall lack of clinical efficacy to date, the future of regenerative treatments holds much promise. Improvements in treating patient comorbidities along with continued advances in under-standing the basic science of angiogenesis, pharmacology, genetic engineering, and more procedure based interven-tions will be required to demonstrate successful clinical application of regenerative therapies.

SUMMARY

This chapter provided an overview of some of the newer concepts regarding physiologic and pathophysiologic control of vascular tone. Properties of peripheral vessels cannot be extrapolated to the coronary or pulmonary circulation. Similarly, properties of one size or class of coronary microvasculature cannot be generalized. Cer-tainly, the technology used in recent studies is dramati-cally changed from that of older ones. Although we attempted to focus on studies that directly examined the microvasculature using the newer technology (in vitro preparations or in situ observations), it was also impor-tant to present classical studies of the intact circulation performed in intact animals or isolated hearts. Newer research questions have necessitated the use of more basic techniques, including cell culture and molecular biological approaches. A more recent development has been the ability to make many in vivo hemodynamic measurements in human subjects in the catheterization laboratory, thus obviating the need and the expenses of flow studies in large animals. As the field of vascular biology grows, we will continue to validate our observa-tions in the intact circulation of our patients and to trans-late this basic science to the clinical setting.

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