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Endothelium-Dependent Relaxation and Angiotensin II Sensitivity in ExperimentalPreeclampsiavan der Graaf, Anne Marijn; Wiegman, Marjon J.; Plosch, Torsten; Zeeman, Gerda G.; vanBuiten, Azuwerus; Henning, Robert; Buikema, Jan; Faas, MariaPublished in:PLoS ONE


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Publication date:2013

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Citation for published version (APA):van der Graaf, A. M., Wiegman, M. J., Plosch, T., Zeeman, G. G., van Buiten, A., Henning, R. H., ... Faas,M. M. (2013). Endothelium-Dependent Relaxation and Angiotensin II Sensitivity in ExperimentalPreeclampsia. PLoS ONE, 8(11), [e79884].

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Endothelium-Dependent Relaxation and Angiotensin IISensitivity in Experimental PreeclampsiaAnne Marijn van der Graaf1,4*, Marjon J. Wiegman1, Torsten Plsch2, Gerda G. Zeeman1, Azuwerus vanBuiten3, Robert H. Henning3, Hendrik Buikema3, Marijke M. Faas4

1 Department of Obstetrics and Gynecology, University of Groningen, University Medical Centre Groningen, Groningen, The Netherlands, 2 Center for Liver,Digestive and Metabolic Diseases, Laboratory of Pediatrics, University of Groningen, University Medical Centre Groningen, Groningen, The Netherlands,3 Department of Clinical Pharmacology, University of Groningen, University Medical Centre Groningen, Groningen, The Netherlands, 4 Division of MedicalBiology, Department of Pathology and Medical Biology, University of Groningen, University Medical Centre Groningen, Groningen, The Netherlands


Objective: We investigated endothelial dysfunction and the role of angiotensin (Ang)-II type I (AT1-R) and type II(AT2-R) receptor in the changes in the Ang-II sensitivity in experimental preeclampsia in the rat.Methods: Aortic rings were isolated from low dose lipopolysaccharide (LPS) infused pregnant rats (experimentalpreeclampsia; n=9), saline-infused pregnant rats (n=8), and saline (n=8) and LPS (n=8) infused non-pregnant rats.Endothelium-dependent acetylcholine--mediated relaxation was studied in phenylephrine-preconstricted aortic ringsin the presence of vehicle, NG-nitro-L-arginine methyl ester and/or indomethacin. To evaluate the role for AT1-R andAT2-R in Ang-II sensitivity, full concentration response curves were obtained for Ang-II in the presence of losartan orPD123319. mRNA expression of the AT1-R and AT2-R, eNOS and iNOS, COX1 and COX2 in aorta were evaluatedusing real-time RT-PCR.Results: The role of vasodilator prostaglandins in the aorta was increased and the role of endothelium-derivedhyperpolarizing factor and response of the AT1-R and AT2-R to Ang-II was decreased in pregnant saline infused ratsas compared with non-pregnant rats. These changes were not observed during preeclampsia.Conclusion: Pregnancy induced adaptations in endothelial function, which were not observed in the rat model forpreeclampsia. This role of lack of pregnancy induced endothelial adaptation in the pathophysiology of experimentalpreeclampsia needs further investigation.

Citation: van der Graaf AM, Wiegman MJ, Plsch T, Zeeman GG, van Buiten A, et al. (2013) Endothelium-Dependent Relaxation and Angiotensin IISensitivity in Experimental Preeclampsia. PLoS ONE 8(11): e79884. doi:10.1371/journal.pone.0079884

Editor: Ana Claudia Zenclussen, Otto-von-Guericke University Magdeburg, GermanyReceived June 7, 2013; Accepted September 26, 2013; Published November 6, 2013Copyright: 2013 van der Graaf et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, whichpermits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No current externalfunding was received for this study.

Competing interests: The authors have declared that no competing interests exist.* E-mail:


Preeclampsia is a pregnancy specific syndrome, clinicallycharacterized by the presence of hypertension, associated withproteinuria in the second half of pregnancy [1]. Preeclampsiacomplicates about 5% of pregnancies and is a leading cause ofmaternal and perinatal mortality [1]. The etiology ofpreeclampsia remains unknown, but appears to be related tothe presence of the placenta [2]. In preeclamptic patientsphysiological remodelling of the uterine spiral arteries isdiminished, resulting in decreased placental perfusion [3].Several mechanisms have been implicated in thepathophysiology of preeclampsia, including activation ofinflammatory cells [4], endothelial cell activation and vascular

dysfunction [5], as well as changes in the renin-angiotensin-aldosterone system (RAAS) [6].

During normal pregnancy, vascular function changesdramatically; increased endothelium dependent vascularrelaxation as well as increased flow mediated dilation can beobserved [7]. Together this may result in a decrease in bloodpressure (mainly in the second trimester) and a decrease inperipheral vascular resistance [8]. By production of vasoactivefactors, endothelial cells are important mediators of vasculartone [9]. Changes in the production of these vasoactive factorsmay therefore account for the pregnancy-related changes invascular relaxation. Indeed, the production of endothelialprostacyclin, nitric oxide (NO) as well as the unidentifiedendothelium-derived hyperpolarizing factor (EDHF) has beenshown to be increased during pregnancy [1013]. In contrast to

PLOS ONE | 1 November 2013 | Volume 8 | Issue 11 | e79884

normal pregnancy, vascular relaxation is reduced inpreeclampsia [8]. The endothelial cell dysfunction inpreeclampsia appears to be associated with an impairedregulation and secretion of vasodilating factors, such as NO,prostacyclin production or EDHF [12,14,15].

In addition, the RAAS may also be involved in the changes invascular dysfunction in preeclampsia. While normal pregnancyis associated with a decreased sensitivity to the vasoconstrictorangiotensin II (Ang-II) [16], preeclampsia is associated with anincreased response to Ang-II as compared to normalpregnancy [17]. During preeclampsia, the increased Ang-IIsensitivity may even develop before the clinical manifestationof the disease [18,19]. Ang-II exerts its effects via tworeceptors. Binding of Ang-II to the Ang-II Type I receptor (AT1-R) causes contraction [17]. The other Ang-II receptor is theType II receptor (AT2-R). The function of this receptor is lesswell understood. There is however, increasing evidence thatthe AT2-R may exert an inhibitory influence on AT1-Rmediated stimulation [20]. It is largely unknown if and howthese receptors are involved in the changes in Ang-II sensitivityduring normal pregnancy and preeclampsia. However, it seemslikely that the AT1-R is involved in the pathophysiology ofpreeclampsia, since in both rat and mice it has beendemonstrated that treatment with AT1-R blockers inhibited thedevelopment of clinical signs in models of preeclampsia[21,22]. Unfortunately, treatment with AT1-R blockers iscontraindicated during pregnancy [23].

In the present study, we evaluated endothelial functionduring pregnancy and experimental preeclampsia in the rat, bystudying the role of the vasoactive factors in endothelialfunction as well as the role of the AT1-R and AT2-R in the Ang-II sensitivity. We used the well-established model forpreeclampsia, i.e. the low-dose lipopolysaccharide (LPS)infused pregnant rat [24]. This model is characterized byhypertension and proteinuria and has been used as a model forpreeclampsia for many years and was used in many studies[21,2527], including a recent study by Wang et al.[28].

Materials and Methods

AnimalsExperiments were conducted in accordance with the National

Institutes of Health Guide for the Care and Use of LaboratoryAnimals and approved by the Committee for Animal EthicalExperiments of the University of Groningen (applicationnumber: DEC-5516A).

Female Wistar outbred rats (Harlan Inc, Horst, theNetherlands) were kept in a 12 hour light-dark cycle andconstant room temperature, with food and water available adlib in the home cages. Until selection for experiments vaginalsmears were taken daily. Rats were rendered pregnant byhousing them on pro-oestrus with fertile males for one night.Day 0 of pregnancy was documented by the presence ofspermatozoa in the vaginal smear. In cyclic and pregnant rats,the latter ones on day 0 of pregnancy, a cannula was insertedinto th


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