Downregulation of Microvascular Endothelial Type B Endothelin Receptor Is a Central Vascular Mechanism in Hypertensive Pregnancy Marc Q. Mazzuca, Wei Li, Ossama M. Reslan, Peng Yu, Karina M. Mata and Raouf A. Khalil Hypertension. 2014;64:632-643; originally published online June 9, 2014; doi: 10.1161/HYPERTENSIONAHA.114.03315 Hypertension is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 2014 American Heart Association, Inc. All rights reserved. Print ISSN: 0194-911X. Online ISSN: 1524-4563
The online version of this article, along with updated information and services, is located on the World Wide Web at: http://hyper.ahajournals.org/content/64/3/632
Data Supplement (unedited) at: http://hyper.ahajournals.org/content/suppl/2014/06/09/HYPERTENSIONAHA.114.03315.DC1.html
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Hypertension can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. Further information about this process is available in the Permissions and Rights Question and Answer document. Reprints: Information about reprints can be found online at: http://www.lww.com/reprints Subscriptions: Information about subscribing to Hypertension is online at: http://hyper.ahajournals.org//subscriptions/
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Preeclampsia Downregulation of Microvascular Endothelial Type B Endothelin Receptor Is a Central Vascular Mechanism in Hypertensive Pregnancy Marc Q. Mazzuca, Wei Li, Ossama M. Reslan, Peng Yu, Karina M. Mata, Raouf A. Khalil See Editorial Commentary, pp 461–462 Abstract—Preeclampsia is a pregnancy-related disorder characterized by hypertension with an unclear mechanism. Studies have shown endothelial dysfunction and increased endothelin-1 (ET-1) levels in hypertensive pregnancy (HTN-Preg). ET-1 activates endothelin receptor type-A in vascular smooth muscle to induce vasoconstriction, but the role of vasodilator endothelial endothelin receptor type-B (ETBR) in the changes in blood pressure (BP) and vascular function in HTN-Preg is unclear. To test whether downregulation of endothelial ETBR expression/activity plays a role in HTN-Preg, BP was measured in normal pregnancy (Norm-Preg) rats and rat model of HTN-Preg produced by reduction of uteroplacental perfusion pressure (RUPP), and mesenteric microvessels were isolated for measuring diameter, [Ca2+]i, and endothelin receptor type-A and ETBR levels. BP, ET-1– and potassium chloride–induced vasoconstriction, and [Ca2+]i were greater in RUPP than in NormPreg rats. Endothelium removal or microvessel treatment with ETBR antagonist BQ-788 enhanced ET-1 vasoconstriction and [Ca2+]i in Norm-Preg, but not RUPP, suggesting reduced vasodilator ETBR in HTN-Preg. The ET-1+endothelin receptor type-A antagonist BQ-123 and the ETBR agonists sarafotoxin 6c and IRL-1620 caused less vasorelaxation and nitrate/ nitrite production in RUPP than in Norm-Preg. The nitric oxide synthase inhibitor Nω-nitro-L-arginine methyl ester reduced sarafotoxin 6c– and IRL-1620–induced relaxation in Norm-Preg but not in RUPP, supporting that ETBR-mediated nitric oxide pathway is compromised in RUPP. Reverse transcription polymerase chain reaction, Western blots, and immunohistochemistry revealed reduced endothelial ETBR expression in RUPP. Infusion of BQ-788 increased BP in Norm-Preg, and infusion of IRL-1620 reduced BP and ET-1 vasoconstriction and [Ca2+]i and enhanced ETBR-mediated vasorelaxation in RUPP. Thus, downregulation of microvascular vasodilator ETBR is a central mechanism in HTN-Preg, and increasing ETBR activity could be a target in managing preeclampsia. (Hypertension. 2014;64:632-643.) Online Data Supplement
•
Key Words: calcium
■
endothelins
N
■
ormal pregnancy (Norm-Preg) is associated with increased plasma volume and cardiac output, decreased vascular resistance, and slight or no change in blood pressure (BP).1 Preeclampsia is a major disorder affecting ≈5% to 8% of pregnancies in the United States and 8 million pregnancies worldwide.2 Preeclampsia is manifested as maternal hypertension3,4 and, if untreated, could lead to eclampsia with severe hypertension and seizures. Preeclampsia may be associated with intrauterine growth restriction and could lead to in utero programming of cardiovascular, renal, and metabolic disorders and adult-onset hypertension and diabetes mellitus.5 Although preeclampsia poses serious consequences to the health of mother and fetus, the mechanisms involved are unclear. Placental ischemia could be an initiating event4 leading to release of various bioactive factors in the maternal circulation including cytokines, anti-angiogenic factors, angiotensin receptor type-1 agonistic auto-antibody,
hypertension
■
nitric oxide
■
pre-eclampsia
reactive oxygen species, and hypoxia-inducible factor.6–8 These circulating factors are thought to cause generalized endotheliosis in the peripheral, glomerular, and cerebral vessels leading to some of the hallmarks of preeclampsia/eclampsia, namely hypertension, proteinuria, and seizures, respectively9; however, the central endothelial cell target involved is unclear. One of the major factors released by endothelium is endothelin-1 (ET-1). ET-1 is a potent vasoconstrictor in some forms of hypertension including hypertensive pregnancy (HTNPreg).3,10 Also, plasma ET-1 levels are increased in preeclamptic women.3,11 Because of the difficulty to perform mechanistic studies in pregnant women, we and others have utilized animal models of HTN-Preg such as the rat model of placental ischemia produced by reduction of uteroplacental perfusion pressure (RUPP).6,8,12 RUPP rats mimic some of the features of preeclampsia in women, including hypertension, endothelial
Received February 4, 2014; first decision February 21, 2014; revision accepted April 14, 2014. From the Division of Vascular and Endovascular Surgery, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA. The online-only Data Supplement is available with this article at http://hyper.ahajournals.org/lookup/suppl/doi:10.1161/HYPERTENSIONAHA. 114.03315/-/DC1. Correspondence to Raouf A. Khalil, Division of Vascular Surgery, Brigham and Women’s Hospital, 75 Francis St, Boston, MA 02115. E-mail raouf_ [email protected] © 2014 American Heart Association, Inc. Hypertension is available at http://hyper.ahajournals.org
DOI: 10.1161/HYPERTENSIONAHA.114.03315
Downloaded from http://hyper.ahajournals.org/ 632 at University of Utah on October 13, 2014
Mazzuca et al Downregulated Vasodilator ETBR in Hypertensive Pregnancy 633 dysfunction, and increased vascular reactivity.3,4,12 RUPP rats also exhibit increased expression of preproET mRNA in the renal medulla and cortex.13 ET-1 activates endothelin receptor type-A (ETAR) and type-B (ETBR). ETAR is mainly expressed in vascular smooth muscle (VSM) to induce vasoconstriction, whereas ETBR is predominately expressed in endothelial cells to induce the release of vasodilator substances,14,15 and also plays a role in ET-1 clearance.16 Although a role of ET-1 and its vasoconstrictor ETAR in modulation of cardiovascular-renal function during HTN-Preg has been suggested,3,17–19 the role of vasodilator endothelial ETBR during Norm-Preg and HTNPreg, and the post-ETBR mediators involved are less clear. We have recently shown that ET-1–induced vasoconstriction is reduced and that ETBR expression and ETBR-mediated vasodilation are enhanced in microvessels of Norm-Preg rats.20 The present study was designed to test the hypothesis that downregulation of endothelial ETBR is an important mechanism in HTN-Preg. We used mesenteric microvessels from RUPP and Norm-Preg rats to determine whether (1) the increased BP in HTN-Preg reflects increased ET-1–induced constriction and decreased ETBR-mediated relaxation via the nitric oxide (NO), prostacyclin, or endothelium-derived hyperpolarizing factor (EDHF) pathway, (2) HTN-Preg is associated with reduced endothelial ETBR expression/activity, and (3) downregulation of ETBR causes HTN-Preg, while enhancing ETBR activity reverses the increase in BP, promotes vasodilation, and reduces vasoconstriction in HTN-Preg. Because changes in [Ca2+]i are major determinants of VSM contraction/relaxation,20 particularly during Norm-Preg and HTN-Preg,21 the effects of ETBR modulation on microvascular [Ca2+]i were also measured.
Methods See details in Methods in the online-only Data Supplement.
Animals Time-pregnant (day 13) Sprague–Dawley rats were either shamoperated (Norm-Preg) or underwent surgical RUPP by placing a silver clip (0.203 mm ID) on the lower abdominal aorta and 2 clips (0.1 mm ID) on uterine branches of the ovarian artery.8,12 To test the role of ETBR in HTN-Preg, other pregnant rats were infused with the ETBR antagonist BQ-788 100 μg/kg per day subcutaneously for 5 days using osmotic minipump. Also, some RUPP rats were infused with the ETBR agonist IRL-1620 100 μg/kg per day subcutaneously for 5 days. All experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the guidelines of Harvard Medical Area Standing Committee on Animals.
Blood Pressure On day 19 of gestation, BP was measured via a PE-50 carotid arterial catheter connected to a pressure transducer.
Tissue Preparation Rats were euthanized by CO2 inhalation, the uterus was excised, and the litter size and individual pup weight were recorded. Third-order mesenteric arteries (≤300 μm OD) were dissected for measuring microvascular function, nitrate/nitrite production, and Western blots. The thoracic aorta was used for measuring nitrate/nitrite production and reverse transcription polymerase chain reaction (RT-PCR).
Measurement of Microvessel Diameter and [Ca2+]i
Mesenteric microvessels were mounted between 2 glass cannulae (Living Systems Instrumentation, Burlington, VT), pressurized under 60
mm Hg,20,21 and the vessel diameter was measured using automatic edgedetection system. For measurement of [Ca2+]i, microvessels were incubated in Krebs solution containing fura-2/AM (5 μmol/L) for 1 hour.20,21
Nitrate/Nitrite (NOx) Production Endothelium-intact aortic and mesenteric artery segments were treated with acetylcholine (ACh, 10–6 mol/L), BQ-123+ET-1, sarafotoxin 6c (S6c), or IRL-1620 (10–7 mol/L) for 30 minutes and the incubation solution was assayed for the stable end product of NO, NO2− using Griess reagent.12
Reverse Transcription Polymerase Chain Reaction Total RNA was extracted from thoracic aorta using RNeasy Fibrous Tissue Mini Kit (QIAGEN, Valencia, CA), 1 μg total RNA was used for reverse transcription to synthesize single-strand cDNA, and 2 μL of cDNA dilution (1:5 for preproET, ETAR and ETBR, and 1:25 for α-actin) was applied to 20 μL RT-PCR reaction. Gene expression was measured using quantitative RT-PCR system (Mx4000, Stratagene, La Jolla, CA), published oligonucleotide primers for preproET, ETAR, and ETBR (Integrated DNA Technologies, Coralville, IA), and iQ-SYBR Green Supermix (Bio-Rad, Hercules, CA). Relative mRNA expression was calculated by the comparative ΔΔCT method, with α-actin as internal control.22
Western Blots Microvessels were homogenized and proteins were separated by electrophoresis and transferred to nitrocellulose membranes. Membranes were probed with polyclonal anti-ETAR (sc-33536) or anti-ETBR antibody (sc-33538; 1:1000, Santa Cruz Biotechnology, Dallas, TX) at 4°C overnight. Immunoreactive bands were visualized using the ECL detection kit (GE Healthcare Bio-Sciences) and the intensity was normalized to β-actin.20
Immunohistochemisty Transverse 6-μm-thick cryosections of mesenteric artery were labeled with polyclonal ETAR and ETBR antibodies (1:100). Vessel images were acquired on a Nikon microscope and analyzed using ImageJ (National Institutes of Health). The total wall thickness, relative thickness of the intima, media and adventitia, and the amount of ETAR and ETBR in the whole vessel wall and in each layer were measured.
Statistical Analysis Experiments were conducted on vessels from n=4 to 12 rats per group. Data were presented as means±SEM. Concentration-dependent responses were fitted to sigmoidal curves using the least squares method and pD2 (−log EC50) was calculated using Graphpad Prism 5.01. Data were analyzed using repeated measures ANOVA and Bonferroni post hoc test for multiple comparisons or Student t test for comparison of 2 means. Differences were statistically significant when P
The online version of this article, along with updated information and services, is located on the World Wide Web at: http://hyper.ahajournals.org/content/64/3/632
Data Supplement (unedited) at: http://hyper.ahajournals.org/content/suppl/2014/06/09/HYPERTENSIONAHA.114.03315.DC1.html
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Hypertension can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. Further information about this process is available in the Permissions and Rights Question and Answer document. Reprints: Information about reprints can be found online at: http://www.lww.com/reprints Subscriptions: Information about subscribing to Hypertension is online at: http://hyper.ahajournals.org//subscriptions/
Downloaded from http://hyper.ahajournals.org/ at University of Utah on October 13, 2014
Preeclampsia Downregulation of Microvascular Endothelial Type B Endothelin Receptor Is a Central Vascular Mechanism in Hypertensive Pregnancy Marc Q. Mazzuca, Wei Li, Ossama M. Reslan, Peng Yu, Karina M. Mata, Raouf A. Khalil See Editorial Commentary, pp 461–462 Abstract—Preeclampsia is a pregnancy-related disorder characterized by hypertension with an unclear mechanism. Studies have shown endothelial dysfunction and increased endothelin-1 (ET-1) levels in hypertensive pregnancy (HTN-Preg). ET-1 activates endothelin receptor type-A in vascular smooth muscle to induce vasoconstriction, but the role of vasodilator endothelial endothelin receptor type-B (ETBR) in the changes in blood pressure (BP) and vascular function in HTN-Preg is unclear. To test whether downregulation of endothelial ETBR expression/activity plays a role in HTN-Preg, BP was measured in normal pregnancy (Norm-Preg) rats and rat model of HTN-Preg produced by reduction of uteroplacental perfusion pressure (RUPP), and mesenteric microvessels were isolated for measuring diameter, [Ca2+]i, and endothelin receptor type-A and ETBR levels. BP, ET-1– and potassium chloride–induced vasoconstriction, and [Ca2+]i were greater in RUPP than in NormPreg rats. Endothelium removal or microvessel treatment with ETBR antagonist BQ-788 enhanced ET-1 vasoconstriction and [Ca2+]i in Norm-Preg, but not RUPP, suggesting reduced vasodilator ETBR in HTN-Preg. The ET-1+endothelin receptor type-A antagonist BQ-123 and the ETBR agonists sarafotoxin 6c and IRL-1620 caused less vasorelaxation and nitrate/ nitrite production in RUPP than in Norm-Preg. The nitric oxide synthase inhibitor Nω-nitro-L-arginine methyl ester reduced sarafotoxin 6c– and IRL-1620–induced relaxation in Norm-Preg but not in RUPP, supporting that ETBR-mediated nitric oxide pathway is compromised in RUPP. Reverse transcription polymerase chain reaction, Western blots, and immunohistochemistry revealed reduced endothelial ETBR expression in RUPP. Infusion of BQ-788 increased BP in Norm-Preg, and infusion of IRL-1620 reduced BP and ET-1 vasoconstriction and [Ca2+]i and enhanced ETBR-mediated vasorelaxation in RUPP. Thus, downregulation of microvascular vasodilator ETBR is a central mechanism in HTN-Preg, and increasing ETBR activity could be a target in managing preeclampsia. (Hypertension. 2014;64:632-643.) Online Data Supplement
•
Key Words: calcium
■
endothelins
N
■
ormal pregnancy (Norm-Preg) is associated with increased plasma volume and cardiac output, decreased vascular resistance, and slight or no change in blood pressure (BP).1 Preeclampsia is a major disorder affecting ≈5% to 8% of pregnancies in the United States and 8 million pregnancies worldwide.2 Preeclampsia is manifested as maternal hypertension3,4 and, if untreated, could lead to eclampsia with severe hypertension and seizures. Preeclampsia may be associated with intrauterine growth restriction and could lead to in utero programming of cardiovascular, renal, and metabolic disorders and adult-onset hypertension and diabetes mellitus.5 Although preeclampsia poses serious consequences to the health of mother and fetus, the mechanisms involved are unclear. Placental ischemia could be an initiating event4 leading to release of various bioactive factors in the maternal circulation including cytokines, anti-angiogenic factors, angiotensin receptor type-1 agonistic auto-antibody,
hypertension
■
nitric oxide
■
pre-eclampsia
reactive oxygen species, and hypoxia-inducible factor.6–8 These circulating factors are thought to cause generalized endotheliosis in the peripheral, glomerular, and cerebral vessels leading to some of the hallmarks of preeclampsia/eclampsia, namely hypertension, proteinuria, and seizures, respectively9; however, the central endothelial cell target involved is unclear. One of the major factors released by endothelium is endothelin-1 (ET-1). ET-1 is a potent vasoconstrictor in some forms of hypertension including hypertensive pregnancy (HTNPreg).3,10 Also, plasma ET-1 levels are increased in preeclamptic women.3,11 Because of the difficulty to perform mechanistic studies in pregnant women, we and others have utilized animal models of HTN-Preg such as the rat model of placental ischemia produced by reduction of uteroplacental perfusion pressure (RUPP).6,8,12 RUPP rats mimic some of the features of preeclampsia in women, including hypertension, endothelial
Received February 4, 2014; first decision February 21, 2014; revision accepted April 14, 2014. From the Division of Vascular and Endovascular Surgery, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA. The online-only Data Supplement is available with this article at http://hyper.ahajournals.org/lookup/suppl/doi:10.1161/HYPERTENSIONAHA. 114.03315/-/DC1. Correspondence to Raouf A. Khalil, Division of Vascular Surgery, Brigham and Women’s Hospital, 75 Francis St, Boston, MA 02115. E-mail raouf_ [email protected] © 2014 American Heart Association, Inc. Hypertension is available at http://hyper.ahajournals.org
DOI: 10.1161/HYPERTENSIONAHA.114.03315
Downloaded from http://hyper.ahajournals.org/ 632 at University of Utah on October 13, 2014
Mazzuca et al Downregulated Vasodilator ETBR in Hypertensive Pregnancy 633 dysfunction, and increased vascular reactivity.3,4,12 RUPP rats also exhibit increased expression of preproET mRNA in the renal medulla and cortex.13 ET-1 activates endothelin receptor type-A (ETAR) and type-B (ETBR). ETAR is mainly expressed in vascular smooth muscle (VSM) to induce vasoconstriction, whereas ETBR is predominately expressed in endothelial cells to induce the release of vasodilator substances,14,15 and also plays a role in ET-1 clearance.16 Although a role of ET-1 and its vasoconstrictor ETAR in modulation of cardiovascular-renal function during HTN-Preg has been suggested,3,17–19 the role of vasodilator endothelial ETBR during Norm-Preg and HTNPreg, and the post-ETBR mediators involved are less clear. We have recently shown that ET-1–induced vasoconstriction is reduced and that ETBR expression and ETBR-mediated vasodilation are enhanced in microvessels of Norm-Preg rats.20 The present study was designed to test the hypothesis that downregulation of endothelial ETBR is an important mechanism in HTN-Preg. We used mesenteric microvessels from RUPP and Norm-Preg rats to determine whether (1) the increased BP in HTN-Preg reflects increased ET-1–induced constriction and decreased ETBR-mediated relaxation via the nitric oxide (NO), prostacyclin, or endothelium-derived hyperpolarizing factor (EDHF) pathway, (2) HTN-Preg is associated with reduced endothelial ETBR expression/activity, and (3) downregulation of ETBR causes HTN-Preg, while enhancing ETBR activity reverses the increase in BP, promotes vasodilation, and reduces vasoconstriction in HTN-Preg. Because changes in [Ca2+]i are major determinants of VSM contraction/relaxation,20 particularly during Norm-Preg and HTN-Preg,21 the effects of ETBR modulation on microvascular [Ca2+]i were also measured.
Methods See details in Methods in the online-only Data Supplement.
Animals Time-pregnant (day 13) Sprague–Dawley rats were either shamoperated (Norm-Preg) or underwent surgical RUPP by placing a silver clip (0.203 mm ID) on the lower abdominal aorta and 2 clips (0.1 mm ID) on uterine branches of the ovarian artery.8,12 To test the role of ETBR in HTN-Preg, other pregnant rats were infused with the ETBR antagonist BQ-788 100 μg/kg per day subcutaneously for 5 days using osmotic minipump. Also, some RUPP rats were infused with the ETBR agonist IRL-1620 100 μg/kg per day subcutaneously for 5 days. All experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the guidelines of Harvard Medical Area Standing Committee on Animals.
Blood Pressure On day 19 of gestation, BP was measured via a PE-50 carotid arterial catheter connected to a pressure transducer.
Tissue Preparation Rats were euthanized by CO2 inhalation, the uterus was excised, and the litter size and individual pup weight were recorded. Third-order mesenteric arteries (≤300 μm OD) were dissected for measuring microvascular function, nitrate/nitrite production, and Western blots. The thoracic aorta was used for measuring nitrate/nitrite production and reverse transcription polymerase chain reaction (RT-PCR).
Measurement of Microvessel Diameter and [Ca2+]i
Mesenteric microvessels were mounted between 2 glass cannulae (Living Systems Instrumentation, Burlington, VT), pressurized under 60
mm Hg,20,21 and the vessel diameter was measured using automatic edgedetection system. For measurement of [Ca2+]i, microvessels were incubated in Krebs solution containing fura-2/AM (5 μmol/L) for 1 hour.20,21
Nitrate/Nitrite (NOx) Production Endothelium-intact aortic and mesenteric artery segments were treated with acetylcholine (ACh, 10–6 mol/L), BQ-123+ET-1, sarafotoxin 6c (S6c), or IRL-1620 (10–7 mol/L) for 30 minutes and the incubation solution was assayed for the stable end product of NO, NO2− using Griess reagent.12
Reverse Transcription Polymerase Chain Reaction Total RNA was extracted from thoracic aorta using RNeasy Fibrous Tissue Mini Kit (QIAGEN, Valencia, CA), 1 μg total RNA was used for reverse transcription to synthesize single-strand cDNA, and 2 μL of cDNA dilution (1:5 for preproET, ETAR and ETBR, and 1:25 for α-actin) was applied to 20 μL RT-PCR reaction. Gene expression was measured using quantitative RT-PCR system (Mx4000, Stratagene, La Jolla, CA), published oligonucleotide primers for preproET, ETAR, and ETBR (Integrated DNA Technologies, Coralville, IA), and iQ-SYBR Green Supermix (Bio-Rad, Hercules, CA). Relative mRNA expression was calculated by the comparative ΔΔCT method, with α-actin as internal control.22
Western Blots Microvessels were homogenized and proteins were separated by electrophoresis and transferred to nitrocellulose membranes. Membranes were probed with polyclonal anti-ETAR (sc-33536) or anti-ETBR antibody (sc-33538; 1:1000, Santa Cruz Biotechnology, Dallas, TX) at 4°C overnight. Immunoreactive bands were visualized using the ECL detection kit (GE Healthcare Bio-Sciences) and the intensity was normalized to β-actin.20
Immunohistochemisty Transverse 6-μm-thick cryosections of mesenteric artery were labeled with polyclonal ETAR and ETBR antibodies (1:100). Vessel images were acquired on a Nikon microscope and analyzed using ImageJ (National Institutes of Health). The total wall thickness, relative thickness of the intima, media and adventitia, and the amount of ETAR and ETBR in the whole vessel wall and in each layer were measured.
Statistical Analysis Experiments were conducted on vessels from n=4 to 12 rats per group. Data were presented as means±SEM. Concentration-dependent responses were fitted to sigmoidal curves using the least squares method and pD2 (−log EC50) was calculated using Graphpad Prism 5.01. Data were analyzed using repeated measures ANOVA and Bonferroni post hoc test for multiple comparisons or Student t test for comparison of 2 means. Differences were statistically significant when P
