Uteroplacental insufficiency temporally exacerbates salt-induced hypertension associated with a reduced natriuretic response in male rat offspring.

KEY POINTS: Low weight at birth increases the risk of developing chronic diseases in adulthood A diet that is high in salt is known to elevate blood pressure, which is a major risk factor for cardiovascular and kidney diseases The present study demonstrates that growth restricted male rats have a heightened sensitivity to high dietary salt, in the context of raised systolic blood pressure, reduced urinary sodium excretion and stiffer mesenteric resistance vessels Other salt-induced effects, such as kidney hyperfiltration, albuminuria and glomerular damage, were not exacerbated by being born small The present study demonstrates that male offspring born small have an increased cardiovascular susceptibility to high dietary salt, such that that minimizing salt intake is probably of particular benefit to this at-risk population ABSTRACT: Intrauterine growth restriction increases the risk of developing chronic diseases in adulthood. Lifestyle factors, such as poor dietary choices, may elevate this risk. We determined whether being born small increases the sensitivity to a dietary salt challenge, in the context of hypertension, kidney disease and arterial stiffness. Bilateral uterine vessel ligation or sham surgery (offspring termed Restricted and Control, respectively) was performed on 18-day pregnant Wistar Kyoto rats. Male offspring were allocated to receive a diet high in salt (8% sodium chloride) or remain on standard rat chow (0.52% sodium chloride) from 20 to 26 weeks of age for 6 weeks. Systolic blood pressure (tail-cuff), renal function (24 h urine excretions) and vascular stiffness (pressure myography) were assessed. Restricted males were born 15% lighter than Controls and remained smaller throughout the study. Salt-induced hypertension was exacerbated in Restricted offspring, reaching a peak systolic pressure of ∼175 mmHg earlier than normal weight counterparts. The natriuretic response to high dietary salt in Restricted animals was less than in Controls and may explain the early rise in arterial pressure. Growth restricted males allocated to a high salt diet also had increased passive arterial stiffness of mesenteric resistance arteries. Other aspects of renal function, including salt-induced hyperfiltration, albuminuria and glomerular damage, were not exacerbated by uteroplacental insufficiency. The present study demonstrates that male offspring exposed to uteroplacental insufficiency and born small have an increased sensitivity to salt-induced hypertension and arterial remodelling.

Angiotensin receptor blockade in juvenile male rat offspring: Implications for long-term cardio-renal health.

Inhibition of the renin-angiotensin system in early postnatal life is a potential therapeutic approach to prevent long-term cardiovascular and kidney diseases in individuals born small. We determined the long-term effects of juvenile losartan treatment on cardiovascular and kidney function in control male rat offspring and those exposed to uteroplacental insufficiency and born small. Bilateral uterine vessel ligation (Restricted) or sham (Control) surgery was performed in late gestation in Wistar Kyoto rats. At weaning, male offspring were randomly assigned to receive losartan in their drinking water or drinking water alone from 5 to 8 weeks of age, and followed to 26 weeks of age. Systolic blood pressure and kidney function were assessed throughout the study. Pressure myography was used to assess passive mechanical wall properties in mesenteric and femoral arteries from 26-week-old offspring. Losartan treatment for three weeks lowered systolic blood pressure in both Control and Restricted groups but this difference was not sustained after the cessation of treatment. Losartan, irrespective of birth weight, mildly increased renal tubulointerstitial fibrosis when assessed at 26 weeks of age. Mesenteric artery stiffness was increased by the early losartan treatment, and was associated with increased collagen and decreased elastin content. Losartan also exerted long-term increases in fat mass and decreases in skeletal muscle mass. In this study, untreated Restricted offspring did not develop hypertension, vascular dysfunction or kidney changes as anticipated. Regardless, we demonstrate that short-term losartan treatment in the juvenile period negatively affects postnatal growth, and kidney and vascular parameters in adulthood, irrespective of birth weight. The long-term effects of early-life losartan treatment warrant further consideration in settings where the potential benefits may outweigh the risks; i.e. when programmed adulthood diseases are apparent and in childhood cardiovascular and kidney diseases.

Increased vascular and uteroplacental matrix metalloproteinase-1 and -7 levels and collagen type I deposition in hypertension in pregnancy: role of TNF-α.

Preeclampsia is a pregnancy-related disorder manifested as maternal hypertension in pregnancy (HTN-Preg) and fetal growth restriction. Placental ischemia could be an initiating event that leads to abnormal vascular and uteroplacental remodeling in HTN-Preg; however, the molecular targets and intermediary mechanisms involved are unclear. We tested the hypothesis that placental ischemia could target vascular and uteroplacental matrix metalloproteinases (MMPs) through an inflammatory cytokine-mediated mechanism. MMP levels and distribution were measured in the aorta, uterus, and placenta of normal pregnant (Preg) rats and pregnant rats with reduced uterine perfusion pressure (RUPP). Maternal blood pressure was higher and the litter size and pup weight were lower in RUPP compared with Preg rats. Gelatin zymography showed prominent uterine MMP-2 and MMP-9 activity that was dependent on the amount of loaded protein. At saturating protein loading, both gelatin and casein zymography revealed two additional bands corresponding to MMP-1 and MMP-7 that were greater in the aorta, uterus, and placenta of RUPP compared with Preg rats. Western blots and immunohistochemistry confirmed increased MMP-1 and MMP-7 in the aorta, uterus, and placenta of RUPP versus Preg rats. The levels of MMP-1 and MMP-7 substrate collagen type I were greater in tissues of RUPP compared with Preg rats. In organ culture, TNF-α increased MMP-1 and MMP-7 in the aorta, uterus, and placenta of Preg rats, and a TNF-α antagonist prevented the increases in MMPs in tissues of RUPP rats. Thus, placental ischemia, possibly through TNF-α, increases vascular and uteroplacental MMP-1 and MMP-7, which, in turn, alter collagen deposition and cause inadequate tissue remodeling in HTN-Preg. Cytokine antagonists may reverse the increase in MMP-1 and MMP-7 expression/activity and, in turn, restore proper vascular and uteroplacental remodeling in HTN-Preg and preeclampsia.NEW & NOTEWORTHY The molecular mechanisms of preeclampsia are unclear, making it difficult to predict, prevent, or manage the pregnancy-associated disorder. This study showed that placental ischemia, possibly through the release of TNF-α, causes increases in the levels of matrix metalloproteinase (MMP)-1 and MMP-7, which could alter collagen deposition and cause inadequate uteroplacental and vascular remodeling in hypertension in pregnancy. The data suggest that targeting MMP-1 and MMP-7 and their upstream modulators, such as TNF-α, could provide a new approach in the management of hypertension in pregnancy and preeclampsia.

Estrogen receptor subtypes mediate distinct microvascular dilation and reduction in [Ca2+]I in mesenteric microvessels of female rat.

Estrogen interacts with estrogen receptors (ERs) to induce vasodilation, but the ER subtype and post-ER relaxation pathways are unclear. We tested if ER subtypes mediate distinct vasodilator and intracellular free Ca(2+) concentration ([Ca(2+)]i) responses via specific relaxation pathways in the endothelium and vascular smooth muscle (VSM). Pressurized mesenteric microvessels from female Sprague-Dawley rats were loaded with fura-2, and the changes in diameter and [Ca(2+)]i in response to 17ß-estradiol (E2) (all ERs), PPT (4,4',4''-[4-propyl-(1H)-pyrazole-1,3,5-triyl]-tris-phenol) (ERα), diarylpropionitrile (DPN) (ERß), and G1 [(±)-1-[(3aR*,4S*,9bS*)-4-(6-bromo-1,3-benzodioxol-5-yl)-3a,4,5,9b-tetrahydro:3H-cyclopenta(c)quinolin-8-yl]-ethanon] (GPR30) were measured. In microvessels preconstricted with phenylephrine, ER agonists caused relaxation and decrease in [Ca(2+)]i that were with E2 = PPT > DPN > G1, suggesting that E2-induced vasodilation involves ERα > ERß > GPR30. Acetylcholine caused vasodilation and decreased [Ca(2+)]i, which were abolished by endothelium removal or treatment with the nitric oxide synthase blocker Nω-nitro-l-arginine methyl ester (L-NAME) and the K(+) channel blockers tetraethylammonium (nonspecific) or apamin (small conductance Ca(2+)-activated K(+) channel) plus TRAM-34 (1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole) (intermediate conductance Ca(2+)-activated K(+) channel), suggesting endothelium-derived hyperpolarizing factor-dependent activation of KCa channels. E2-, PPT-, DPN-, and G1-induced vasodilation and decreased [Ca(2+)]i were not blocked by L-NAME, TEA, apamin plus TRAM-34, iberiotoxin (large conductance Ca(2+)- and voltage-activated K(+) channel), 4-aminopyridine (voltage-dependent K(+) channel), glibenclamide (ATP-sensitive K(+) channel), or endothelium removal, suggesting an endothelium- and K(+) channel-independent mechanism. In endothelium-denuded vessels preconstricted with phenylephrine, high KCl, or the Ca(2+) channel activator Bay K 8644 (1,4-dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)phenyl]-3-pyridinecarboxylic acid methyl ester), ER agonist-induced relaxation and decreased [Ca(2+)]i were with E2 = PPT > DPN > G1 and not inhibited by the guanylate cyclase inhibitor ODQ [1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one], and showed a similar relationship between decreased [Ca(2+)]i and vasorelaxation, supporting direct effects on Ca(2+) entry in VSM. Immunohistochemistry revealed ERα, ERß, and GPR30 mainly in the vessel media and VSM. Thus, in mesenteric microvessels, ER subtypes mediate distinct vasodilation and decreased [Ca(2+)]i (ERα > ERß > GPR30) through endothelium- and K(+) channel-independent inhibition of Ca(2+) entry mechanisms of VSM contraction.

Adaptive regulation of endothelin receptor type-A and type-B in vascular smooth muscle cells during pregnancy in rats.

Normal pregnancy is associated with systemic vasodilation and decreased vascular contraction, partly due to increased release of endothelium-derived vasodilator substances. Endothelin-1 (ET-1) is an endothelium-derived vasoconstrictor acting via endothelin receptor type A (ETA R) and possibly type B (ETB R) in vascular smooth muscle cells (VSMCs), with additional vasodilator effects via endothelial ETB R. However, the role of ET-1 receptor subtypes in the regulation of vascular function during pregnancy is unclear. We investigated whether the decreased vascular contraction during pregnancy reflects changes in the expression/activity of ETAR and ETBR. Contraction was measured in single aortic VSMCs isolated from virgin, mid-pregnant (mid-Preg, day 12), and late-Preg (day 19) Sprague-Dawley rats, and the mRNA expression, protein amount, tissue and cellular distribution of ETAR and ETBR were examined using RT-PCR, Western blots, immunohistochemistry, and immunofluorescence. Phenylephrine (Phe, 10(-5) M), KCl (51 mM), and ET-1 (10(-6) M) caused VSMC contraction that was in late-Preg < mid-Preg and virgin rats. In VSMCs treated with ETB R antagonist BQ788, ET-1 caused significant contraction that was still in late-Preg < mid-Preg and virgin rats. In VSMCs treated with the ETAR antagonist BQ123, ET-1 caused a small contraction; and the ETBR agonists IRL-1620 and sarafotoxin 6c (S6c) caused similar contraction that was in late-Preg < mid-Preg and virgin rats. RT-PCR revealed similar ETAR, but greater ETBR mRNA expression in pregnant versus virgin rats. Western blots revealed similar ETAR, and greater protein amount of ETBR in endothelium-intact vessels, but reduced ETBR in endothelium-denuded vessels of pregnant versus virgin rats. Immunohistochemistry revealed prominent ETBR staining in the intima, but reduced ETAR and ETBR in the aortic media of pregnant rats. Immunofluorescence signal for ETAR and ETBR was less in VSMCs of pregnant versus virgin rats. The pregnancy-associated decrease in ETAR- and ETBR-mediated VSMC contraction appears to involve downregulation of ETAR and ETBR expression/activity in VSM, and may play a role in the adaptive vasodilation during pregnancy.

Increased uterine arterial tone, stiffness and remodeling with augmented matrix metalloproteinase-1 and -7 in uteroplacental ischemia-induced hypertensive pregnancy.

Preeclampsia is a pregnancy-related disorder manifested as hypertensive pregnancy (HTN-Preg) and often fetal growth restriction (FGR), but the mechanisms involved are unclear. We have reported enhanced reactivity of systemic vessels in HTN-Preg rats, but the critical changes in the uterine circulation are less clear. We tested whether HTN-Preg involves localized aberrations in uterine arterial tone, stiffness and remodeling by matrix metalloproteinases (MMPs). Blood pressure (BP) and litter size were recorded in normal pregnant (Preg) rats and Preg rats with reduced uteroplacental perfusion pressure (RUPP). Isolated uterine arteries were placed in a pressure myograph for measuring intrinsic and extrinsic tone and arterial stiffness. Arteries were bathed in normal Krebs solution (2.5 mM Ca2+), Ca2+-free (2 mM EGTA) Krebs, treated with sodium nitroprusside (SNP), or endothelium denuded, then pressurized at 10 mmHg steps from 10 to 110 mmHg, and the % change in diameter was analyzed to measure total (active + passive), active Ca2+-dependent myogenic, passive, and endothelium-dependent tone, respectively. BP was higher and the litter size and pup weight were reduced in RUPP vs Preg rats. In normal Krebs, increasing intraluminal pressure caused smaller increments in diameter in arteries of RUPP vs Preg rats, suggesting greater total vascular tone. Arterial incubation in Ca2+-free Krebs, treatment with SNP or endothelium-removal abolished the differences in vascular tone, and subtraction of each of these components from total vascular tone revealed significant active Ca2+-dependent myogenic, passive, and endothelium-dependent tone, respectively, in RUPP vs Preg rats. The total and passive strain-stress curves were shifted leftward in arteries of RUPP vs Preg rats, indicating increased uterine arterial stiffness. Arterial sections showed decreased lumen/total and increased wall/total area, and immunohistochemistry revealed greater MMP-1 and MMP-7 staining particularly in the media, suggesting uterine arterial remodeling by MMPs in RUPP vs Preg rats. The increased uterine arterial active myogenic, passive, and endothelium-dependent tone, arterial stiffness and remodeling by MMPs would further reduce uterine blood flow and exacerbate uteroplacental ischemia, FGR and HTN-Preg.

Increased Ca2+-dependent intrinsic tone and arterial stiffness in mesenteric microvessels of hypertensive pregnant rats.

Preeclampsia is a pregnancy-related hypertensive disorder (HTN-Preg) with unclear mechanisms. We have shown increased vascular reactivity to extrinsic vasoconstrictors in HTN-Preg rats. Here, we test whether microvascular intrinsic tone and arterial stiffness could contribute to HTN-Preg, and examined the underlying cellular mechanisms. On gestational day 19, BP was recorded in normal pregnant (Preg) rats and Preg rats with reduced uterine perfusion pressure (RUPP), and mesenteric microvessels were mounted on a pressure myograph for measurement of intrinsic tone, simultaneous changes in [Ca2+]i (fura-2 340/380 ratio), and arterial stiffness. Arteries were incubated in Ca2+-containing and 0 Ca2+ (2 mM EGTA) Krebs, pressurized at 10 to 110 mmHg in 10 mmHg increments, and the % change in vessel diameter from initial diameter at 10 mmHg was analyzed for measurement of total (active + passive) intrinsic tone and passive intrinsic response, respectively. The passive response was then subtracted from the total intrinsic tone to determine the active myogenic tone. The strain-stress relationship was also constructed as a measure of arterial stiffness. BP was higher in RUPP vs Preg rats. In Ca2+-containing Krebs, increases in intraluminal pressure caused smaller increases in diameter and greater increases in [Ca2+]i in microvessels of RUPP vs Preg rats, suggesting increased Ca2+-dependent myogenic tone. In 0 Ca2+ Krebs, increases in pressure also caused less increases in diameter in microvessels of RUPP vs Preg rats, but with no changes in [Ca2+]i, suggesting changes in the structure and mechanics of the arterial wall. The total and passive strain-stress relationship was shifted to the left in microvessels of RUPP vs Preg rats, suggesting increased arterial wall stiffness. Histology and immunohistochemistry showed greater vascular wall thickness and collagen-I staining in RUPP vs Preg rats, supporting changes in the wall architecture and structural proteins. The increased active myogenic tone and underlying increases in Ca2+ signaling as well as the increased passive intrinsic response, arterial stiffness and collagen-I in the mesenteric microvessels could play a role in the regulation of blood flow to the splanchnic region and the increased vascular resistance and BP in HTN-Preg.

Uteroplacental insufficiency programmes vascular dysfunction in non-pregnant rats: compensatory adaptations in pregnancy.

Intrauterine growth restriction is a risk factor for cardiovascular disease in adulthood. We have previously shown that intrauterine growth restriction caused by uteroplacental insufficiency programmes uterine vascular dysfunction and increased arterial stiffness in adult female rat offspring. The aim of this study was to investigate vascular adaptations in growth restricted female offspring when they in turn become pregnant. Uteroplacental insufficiency was induced in WKY rats by bilateral uterine vessel ligation (Restricted) or sham surgery (Control) on day 18 of pregnancy. F0 pregnant females delivered naturally at term. F1 Control and Restricted offspring were mated at 4 months of age and studied on day 20 of pregnancy. Age-matched non-pregnant F1 Control and Restricted females were also studied. Wire and pressure myography were used to test endothelial and smooth muscle function, and passive mechanical wall properties, respectively, in uterine, mesenteric, renal and femoral arteries of all four groups. Collagen and elastin fibres were quantified using polarized light microscopy and qRT-PCR. F1 Restricted females were born 10­15% lighter than Controls (P <0.05). Non-pregnant Restricted females had increased uterine and renal artery stiffness compared with Controls (P <0.05), but this difference was abolished at day 20 of pregnancy. Vascular smooth muscle and endothelial function were preserved in all arteries of non-pregnant and pregnant Restricted rats. Collagen and elastin content were unaltered in uterine arteries of Restricted females. Growth restricted females develop compensatory vascular changes during late pregnancy, such that region-specific vascular deficits observed in the non-pregnant state did not persist in late pregnancy.

Cardio-renal and metabolic adaptations during pregnancy in female rats born small: implications for maternal health and second generation fetal growth.

Intrauterine growth restriction caused by uteroplacental insufficiency increases risk of cardiovascular and metabolic disease in offspring. Cardio-renal and metabolic responses to pregnancy are critical determinants of immediate and long-term maternal health. However, no studies to date have investigated the renal and metabolic adaptations in growth restricted offspring when they in turn become pregnant. We hypothesised that the physiological challenge of pregnancy in growth restricted females exacerbates disease outcome and compromises next generation fetal growth. Uteroplacental insufficiency was induced by bilateral uterine vessel ligation (Restricted) or sham surgery (Control) on day 18 of gestation in WKY rats and F1 female offspring birth and postnatal body weights were recorded. F1 Control and Restricted females were mated at 4 months and blood pressure, renal and metabolic parameters were measured in late pregnancy and F2 fetal and placental weights recorded. Age-matched non-pregnant Control and Restricted F1 females were also studied. F1 Restricted females were born 10-15% lighter than Controls. Basal insulin secretion and pancreatic ß-cell mass were reduced in non-pregnant Restricted females but restored in pregnancy. Pregnant Restricted females, however, showed impaired glucose tolerance and compensatory glomerular hypertrophy, with a nephron deficit but normal renal function and blood pressure. F2 fetuses from Restricted mothers exposed to physiological measures during pregnancy were lighter than Controls highlighting additive adverse effects when mothers born small experience stress during pregnancy. Female rats born small exhibit mostly normal cardio-renal adaptations but altered glucose control during late pregnancy making them vulnerable to lifestyle challenges.

Uteroplacental insufficiency programs regional vascular dysfunction and alters arterial stiffness in female offspring.

Intrauterine growth restriction caused by uteroplacental insufficiency increases the risk of cardiovascular disease in adulthood. Vascular mechanisms in female offspring are poorly understood. The aim of this study was to investigate the effects of uteroplacental insufficiency on blood pressure, vascular reactivity and arterial stiffness in four vascular beds in female offspring born growth restricted. Uteroplacental insufficiency was induced on day 18 of gestation in Wistar Kyoto rats by bilateral uterine vessel ligation (Restricted) or sham surgery (Controls). Wire and pressure myography were used to test endothelial and smooth muscle function, and passive mechanical wall properties, respectively, in uterine, mesenteric, renal and femoral arteries of 18-month-old female offspring. Collagen and elastin fibres were quantified using circular crossed-polarized light microscopy and quantitative real time polymerase chain reaction. Restricted female offspring were born 10-15% smaller. Restricted females were normotensive, had plasma triglycerides 2-fold elevated and had uterine endothelial dysfunction, attributed to a 23% reduction in the maximal relaxation produced by endothelium-derived hyperpolarizing factor. Uterine artery stiffness was increased, with an augmented proportion of thick and decreased proportion of thin collagen fibres. Vascular reactivity and mechanical wall properties were preserved in mesenteric, renal and femoral arteries in growth restricted females. Female offspring born growth restricted have selective uterine artery endothelial dysfunction and increased wall stiffness. The preserved vascular function in other arteries may explain the lack of hypertension in these females. The uterine artery specific dysfunction has potential implications for impaired pregnancy adaptations and a compromised intrauterine environment of the next generation.

Uteroplacental insufficiency causes a nephron deficit, modest renal insufficiency but no hypertension with ageing in female rats.

In rats, uteroplacental insufficiency induced by uterine vessel ligation restricts fetal growth and impairs mammary development compromising postnatal growth. In male offspring, this results in a nephron deficit and hypertension which can be reversed by improving lactation and postnatal growth. Here, growth, blood pressure and nephron endowment in female offspring from mothers which underwent bilateral uterine vessel ligation (Restricted) on day 18 of pregnancy were examined. Sham surgery (Control) and a reduced litter group (Reduced at birth to 5, equivalent to Restricted group) were used as controls. Offspring (Control, Reduced, Restricted) were cross-fostered on postnatal day 1 onto a Control (normal lactation) or Restricted (impaired lactation) mother. Restricted-on-Restricted offspring were born small but were of similar weight to Control-on-Control by postnatal day 35. Blood pressure was not different between groups at 8, 12 or 20 weeks of age. Glomerular number was reduced in Restricted-on-Restricted offspring at 6 months without glomerular hypertrophy. Cross-fostering a Restricted pup onto a Control dam resulted in a glomerular number intermediate between Control-on-Control and Restricted-on-Restricted. Blood pressure, along with renal function, morphology and mRNA expression, was examined in Control-on-Control and Restricted-on-Restricted females at 18 months. Restricted-on-Restricted offspring did not become hypertensive but developed glomerular hypertrophy by 18 months. They had elevated plasma creatinine and alterations in renal mRNA expression of transforming growth factor-beta(1), collagen IV (alpha1) and matrix matelloproteinase-9. This suggests that perinatally growth restricted female offspring may be susceptible to onset of renal injury and renal insufficiency with ageing in the absence of concomitant hypertension.

Interference of doxycycline pretreatment in a model of abdominal aortic aneurysms.

BACKGROUND: Abdominal aortic aneurysm (AAA) is characterized by chronic inflammation and degradation of the extracellular matrix, mediated by matrix metalloproteinases (MMPs). Doxycycline has been reported to control the progression of AAA by regulation of MMP. We hypothesized that doxycycline pretreatment in a rat model of AAA would cause reduction in gelatinolytic activity of MMP-2 and -9 and the inflammatory response in the wall of an aneurysm, consequently decreasing the formation and development of AAAs. METHODS: Male Wistar rats were divided into the following four groups: aneurysm (A); control (C); aneurysm+doxycycline (A+D) and control+doxycycline (C+D), with 24 animals per group subdivided into n=6 animals at different time points [1, 3, 7, and 15 days postsurgery (dps)]. The (A) and (A+D) groups simultaneously received the injury and extrinsic stenosis of the aortic wall. The (C) and (C+D) groups received sham operation. The treated animals received doxycycline via gavage (30 mg/kg/day) from 48 h before surgery until the end of experiment. At 1, 3, 7, and 15 dps, the animals were euthanized, and the aortas were collected for morphological analyses, immunohistochemistry, and zymography. RESULTS: The animals from the (A) group developed AAAs. However, the animals treated with doxycycline showed a 85% decrease in AAA development, which was associated with a large reduction in gelatinolytic activity of MMP-2 and -9, and decreased inflammatory response (P<.05). CONCLUSIONS: These results suggest that pretreatment with doxycycline before surgery inhibited the activity of MMP-2 and -9, as well as the inflammatory response, and may play an important role in the prevention of the development of AAAs.

Altered matrix metalloproteinase-2 and -9 expression/activity links placental ischemia and anti-angiogenic sFlt-1 to uteroplacental and vascular remodeling and collagen deposition in hypertensive pregnancy.

Preeclampsia is a complication of pregnancy manifested as maternal hypertension and often fetal growth restriction. Placental ischemia could be an initiating event, but the linking mechanisms leading to hypertension and growth restriction are unclear. We have shown an upregulation of matrix metalloproteinases (MMPs) during normal pregnancy (Norm-Preg). To test the role of MMPs in hypertensive-pregnancy (HTN-Preg), maternal and fetal parameters, MMPs expression, activity and distribution, and collagen and elastin content were measured in uterus, placenta and aorta of Norm-Preg rats and in rat model of reduced uteroplacental perfusion pressure (RUPP). Maternal blood pressure was higher, and uterine, placental and aortic weight, and the litter size and pup weight were less in RUPP than Norm-Preg rats. Western blots and gelatin zymography revealed decreases in amount and gelatinase activity of MMP-2 and MMP-9 in uterus, placenta and aorta of RUPP compared with Norm-Preg rats. Immunohistochemistry confirmed reduced MMPs in uterus, placenta and aortic media of RUPP rats. Collagen, but not elastin, was more abundant in uterus, placenta and aorta of RUPP than Norm-Preg rats. The anti-angiogenic factor soluble fms-like tyrosine kinase-1 (sFlt-1) decreased MMPs in uterus, placenta and aorta of Norm-Preg rats, and vascular endothelial growth factor (VEGF) reversed the decreases in MMPs in tissues of RUPP rats. Thus placental ischemia and anti-angiogenic sFlt-1 decrease uterine, placental and vascular MMP-2 and MMP-9, leading to increased uteroplacental and vascular collagen, and growth-restrictive remodeling in HTN-Preg. Angiogenic factors and MMP activators may reverse the decrease in MMPs and enhance growth-permissive remodeling in preeclampsia.

Downregulation of microvascular endothelial type B endothelin receptor is a central vascular mechanism in hypertensive pregnancy.

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, [Ca(2+)]i, and endothelin receptor type-A and ETBR levels. BP, ET-1- and potassium chloride-induced vasoconstriction, and [Ca(2+)]i were greater in RUPP than in Norm-Preg rats. Endothelium removal or microvessel treatment with ETBR antagonist BQ-788 enhanced ET-1 vasoconstriction and [Ca(2+)]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 [Ca(2+)]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.

Enhanced endothelin receptor type B-mediated vasodilation and underlying [Ca²âº]i in mesenteric microvessels of pregnant rats.

BACKGROUND AND PURPOSE: Normal pregnancy is associated with decreased vascular resistance and increased release of vasodilators. Endothelin-1 (ET-1) causes vasoconstriction via endothelin receptor type A (ET(A)R), but could activate ET(B)R in the endothelium and release vasodilator substances. However, the roles of ET(B)R in the regulation of vascular function during pregnancy and the vascular mediators involved are unclear. EXPERIMENTAL APPROACH: Pressurized mesenteric microvessels from pregnant and virgin Sprague-Dawley rats were loaded with fura-2/AM for simultaneous measurement of diameter and [Ca²âº]i. KEY RESULTS: High KCl (51 mM) and phenylephrine (PHE) caused increases in vasoconstriction and [Ca²âº]i that were similar in pregnant and virgin rats. ET-1 caused vasoconstriction that was less in pregnant than virgin rats, with small increases in [Ca²âº]i. Pretreatment with the ET(B)R antagonist BQ-788 caused greater enhancement of ET-1-induced vasoconstriction in pregnant rats. ACh caused endothelium-dependent relaxation and decreased [Ca²âº]i, and was more potent in pregnant than in virgin rats. ET-1 + ET(A)R antagonist BQ-123, and the ET(B)R agonists sarafotoxin 6c (S6c) and IRL-1620 caused greater vasodilation in pregnant than in virgin rats with no changes in [Ca²âº]i, suggesting up-regulated ET(B)R-mediated relaxation pathways. ACh-, S6c- and IRL-1620-induced relaxation was reduced by the NO synthase inhibitor Nω-nitro-L-arginine methyl ester, and abolished by tetraethylammonium or endothelium removal. Western blots revealed greater amount of ET(B)R in intact microvessels of pregnant than virgin rats, but reduced levels in endothelium-denuded microvessels, supporting a role of endothelial ET(B)R. CONCLUSIONS AND IMPLICATIONS: The enhanced ET(B)R-mediated microvascular relaxation may contribute to the decreased vasoconstriction and vascular resistance during pregnancy.

Vascular endothelin receptor type B: structure, function and dysregulation in vascular disease.

Endothelin-1 (ET-1) is a major regulator of vascular function, acting via both endothelin receptor type A (ET(A)R) and type B (ET(B)R). Although the role of ET(A)R in vascular smooth muscle (VSM) contraction has been studied, little is known about ET(B)R. ET(B)R is a G-protein coupled receptor with a molecular mass of ~50 kDa and 442 amino acids arranged in seven transmembrane domains. Alternative splice variants of ET(B)R and heterodimerization and cross-talk with ET(A)R may affect the receptor function. ET(B)R has been identified in numerous blood vessels with substantial effects in the systemic, renal, pulmonary, coronary and cerebral circulation. ET(B)R in the endothelium mediates the release of relaxing factors such as nitric oxide, prostacyclin and endothelium-derived hyperpolarizing factor, and could also play a role in ET-1 clearance. ET(B)R in VSM mediates increases in [Ca(2+)](i), protein kinase C, mitogen-activated protein kinase and other pathways of VSM contraction and cell growth. ET-1/ET(A)R signaling has been associated with salt-sensitive hypertension (HTN) and pulmonary arterial hypertension (PAH), and ET(A)R antagonists have shown some benefits in these conditions. In search for other pathogenetic factors and more effective approaches, the role of alterations in endothelial ET(B)R and VSM ET(B)R in vascular dysfunction, and the potential benefits of modulators of ET(B)R in treatment of HTN and PAH are being examined. Combined ET(A)R/ET(B)R antagonists could be more efficacious in the management of conditions involving upregulation of ET(A)R and ET(B)R in VSM. Combined ET(A)R antagonist with ET(B)R agonist may need to be evaluated in conditions associated with decreased endothelial ET(B)R expression/activity.