Reduced Uteroplacental Perfusion Pressure Research Articles

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.

Sodium Nitrite Attenuates Reduced Activity of Vascular Matrix Metalloproteinase-2 and Vascular Hyper-Reactivity and Increased Systolic Blood Pressure Induced by the Placental Ischemia Model of Preeclampsia in Anesthetized Rats.

Preeclampsia is a maternal hypertension disorder associated with vascular dysfunction and fetal and placental growth restrictions. Placental ischemia is suggested as the primary trigger of preeclampsia-associated impairments of both endothelium-derived nitric oxide (NO) and the vascular activity of extracellular matrix metalloproteinase-2 (MMP-2). Reduced uteroplacental perfusion pressure (RUPP) is a placental ischemia model of preeclampsia. Reduction of sodium nitrite to NO may occur during ischemic conditions. However, sodium nitrite effects in the RUPP model of preeclampsia have not yet been investigated. Pregnant rats were divided into four groups: normotensive pregnant rats (Norm-Preg), pregnant rats treated with sodium nitrite (Preg + Nitrite), preeclamptic rats (RUPP), and preeclamptic rats treated with sodium nitrite (RUPP + Nitrite). Maternal blood pressure and fetal and placental parameters were recorded. Vascular function, circulating NO metabolites, and the gelatinolytic activity of vascular MMP-2 were also examined. Sodium nitrite attenuates increased blood pressure, prevents fetal and placental weight loss, counteracts vascular hyper-reactivity, and partially restores NO metabolites and MMP-2 activity. In conclusion, sodium nitrite reduction to NO may occur during RUPP-induced placental ischemia, thereby attenuating increased blood pressure, fetal and placental growth restriction, and vascular hyper-reactivity associated with preeclampsia and possibly restoring NO and MMP-2 activity, which underlie the blood pressure-lowering effects.

Stable adrenomedullin analog mitigates placental ischemia-induced hypertension and fetal growth restriction in rats.

Preeclampsia is a heterogeneous hypertensive disorder of pregnancy. It affects multiorgans and may lead to fetal growth restriction, organ failure, seizure, and maternal death. Unfortunately, current treatments are ineffective at delaying the progression of preeclampsia even for a few days. Clinicians are often forced to deliver preterm fetus if severe preeclampsia occurred early during pregnancy, leading to premature birth-associated complications. Preeclampsia has been associated with defects at the maternal-fetal interface and maternal vascular dysfunction. Of interest, the adrenomedullin peptide and its cognate receptors, calcitonin receptor-like receptor (CLR)/ receptor activity-modifying protein (RAMP) receptor complexes, have been shown to be important regulators of cardiovascular adaptation and feto-placental development during pregnancy. Although the exact role of adrenomedullin-CLR/RAMP signaling in different feto-maternal compartments during pregnancy and how adrenomedullin expression affects preeclampsia development remains to be clarified, we hypothesized that the sustained activation of CLR/RAMP receptors could be a promising strategy to mitigate placental ischemia-associated vascular dysfunction and fetal growth restriction under preeclampsia-like conditions. To explore this possibility, we have developed a stable adrenomedullin analog, ADE101, and investigated its effects on human lymphatic microvascular endothelial (HLME) cell proliferation, hemodynamics, and pregnancy outcomes in pregnant rats with reduced uteroplacental perfusion pressure (RUPP) induced by clipping of uterine arteries on gestation day 14. The ADE101 analog has a potent effect on CLR/RAMP2 receptor activation, and an enhanced stimulatory effect on HLME cell proliferation compared to wild-type peptides. ADE101 also exhibits a lasting effect on hemodynamics in normal and hypertensive rats. In addition, studies using the RUPP model showed that ADE101 significantly reduces placental ischemia-induced hypertension and fetal growth restriction in a dose-dependent manner. Infusion of ADE101 increased the weight of fetuses and placentas in RUPP animals to 252% and 202% of that of RUPP controls, respectively. These data suggested that long-acting adrenomedullin analog could be useful for quenching hypertension as well as the vascular ischemia-associated organ damages in preeclamptic patients.

Role of Macrophages in Developmental Programming of Pancreatic β‐Cell Area in Offspring of Gestational Hypertension

Preeclampsia is a pregnancy specific complication characterized by new‐onset maternal hypertension and a high incidence of intrauterine growth restriction (IUGR), both associated with negative outcomes for offspring. Our previous studies indicated that IUGR in conjunction with maternal hypertension in the rat Reduced Uteroplacental Perfusion Pressure (RUPP) model of chronic placental ischemia results in female offspring with reduced pancreatic β‐cell area compared to sham pregnancies at embryonic day 19, and this decrease in β‐cell area persists to postnatal day 13 (PD13). Pancreatic β‐cell area, set very early in life, can influence whether an individual develops type 2 diabetes (T2D). Consistent with reduced β‐cell area, previous studies of others have demonstrated oral glucose intolerance in 6‐12 month RUPP offspring. T2D is associated with increased macrophages in the pancreas, and we previously reported increased message for macrophage marker CD68 is associated with reduced β‐cell area in islets of female PD13 RUPP animals. Thus, we hypothesized that macrophages are crucial to the reduction in β‐cell area in RUPP offspring. To test this hypothesis, we determined if postnatal depletion of macrophages rescued the β‐cell area. Chronic placental ischemia was induced in timed‐pregnant Sprague‐Dawley rats using the RUPP model by surgical placement of clips on abdominal aorta and uterine arteries on gestation day 14 of a 21‐day gestation. Female offspring were intraperitoneally injected with control liposomes or clodronate liposomes (56 and 28 mg clodronate/kg on PD2 and PD9, respectively) to deplete macrophages. On PD13, offspring were euthanized and pancreata fixed in formalin for paraffin embedding and determination of β‐cell area. Other pancreata were perfused with collagenase and digested for islet isolation by hand‐picking. β‐cell/pancreas area was determined by immunofluorescence and image analysis. Female RUPP animals treated with control liposomes had significantly reduced β‐cell area compared to sham offspring treated with control liposomes (0.0156 vs 0.0242 β‐cell/pancreas area, n=5, p<0.05). Notably, β‐cell area of sham or RUPP offspring treated with clodronate liposomes was not different (0.0221 vs 0.0225 β‐cell/pancreas area, n= 6‐7) from sham animals treated with control liposomes (0.0242 β‐cell/pancreas area, n=5). Thus, clodronate liposome treatment rescued the defect in β‐cell area of female RUPP offspring compared to RUPP animals treated with control liposomes. Message for CD68 in islets was significantly decreased following clodronate treatment, suggesting depletion in the islet. In addition, macrophages were significantly depleted in peritoneal lavage, spleen and islets as determined by flow cytometry. Total macrophages in lavage decreased from 7.1 x 10^5 to 0.05 x 10^5 cells following clodronate treatment. Thus, our studies suggest macrophages are critical for the reduction in β‐cell area in RUPP offspring following chronic placental ischemia. Future studies will explore the influence of apoptosis, proliferation, β‐cell size and number on reduction in β‐cell area.

TNFα blockade reverses vascular and uteroplacental matrix metalloproteinases imbalance and collagen accumulation in hypertensive pregnant rats

Preeclampsia is a pregnancy-related disorder of maternal hypertension-in-pregnancy (HTN-Preg) and often fetal growth restriction (FGR). Placental ischemia could be an initiating event leading to inadequate vascular and uteroplacental remodeling and HTN-Preg; however, the molecular targets are unclear. To test the hypothesis that placental ischemia-induced release of proinflammatory cytokines target vascular and uteroplacental matrix metalloproteinases (MMPs), we tested if infusing TNFα (200 ng/kg/day) in day-14 pregnant (Preg) rats causes MMP imbalance and collagen accumulation, and if infusing TNFα decoy receptor Etanercept (0.4 mg/kg/day) in HTN-Preg rats with reduced uteroplacental perfusion pressure (RUPP) reverses MMP imbalance and collagen accumulation. On gestational day-19, blood pressure (BP) was higher in Preg + TNFα and RUPP vs Preg rats, and restored in RUPP + Etanercept rats. Gelatin zymography and Western blots revealed decreases in MMP-2 and MMP-9 and increases in MMP-1 and MMP-7 in aorta, uterus and placenta of Preg + TNFα and RUPP, that were reversed in RUPP + Etanercept rats. Collagen-I and IV were abundant in Preg + TNFα and RUPP, and were decreased in RUPP + Etanercept rats. The litter size, uterine, placenta, and pup weight were markedly reduced in RUPP, insignificantly reduced in Preg + TNFα, and slightly improved in RUPP + Etanercept rats. Thus TNFα blockade reverses the decreases in vascular and uteroplacental MMP-2 and MMP-9, and the increases in MMP-1, MMP-7 and accumulation of collagen-I and IV induced by placental ischemia and TNFα in HTN-Preg rats. Targeting TNFα using cytokine antagonists, or MMPs using MMP modulators could rectify MMP imbalance and collagen accumulation, restore vascular and uteroplacental remodeling, and improve BP in HTN-Preg and preeclampsia.

Decreased Systemic Complement Activation Product C3a is Associated with a Reduction in Pancreatic β Cell Area in Islets of Female Rat Offspring following Chronic Placental Ischemia‐induced Hypertension

Preeclampsia is a pregnancy specific condition characterized by new-onset maternal hypertension and increased systemic inflammation, including increased complement activation. Our previous studies demonstrated that inhibition of complement activation attenuated placental ischemia-induced hypertension in the Reduced Uteroplacental Perfusion Pressure (RUPP) model of gestational hypertension in rat. Hypertensive pregnancy disorders can also result in increased risk of later-life cardiovascular and metabolic disease for the offspring. In fact, recent studies have demonstrated that gestational hypertension is associated with increased risk for Type 2 diabetes in offspring. Clearly, pancreatic β cell mass, established early in life, can influence whether an individual develops Type 2 diabetes. β cell area has also been linked to complement component C3 and activation product C3a, with increased C3a generation associated with prevention of human islet cell atrophy. Our previous studies demonstrated a significant reduction in β cell area in postnatal day (PD)13 female, but not male, offspring of rats subjected to chronic placental ischemia-induced hypertension in the RUPP model. Thus, we hypothesized that decreased C3a is associated with reductions in β cell area in the female offspring following placental insufficiency. The RUPP model was used to induce placental insufficiency by placing silver clips on the abdominal aorta and uterine arteries on gestation day 14 of a 21 day gestation. Serum was collected via the abdominal aorta from PD13 pups born from RUPP and Sham pregnancies. Pancreatic islets of PD13 male and female offspring of RUPP and Sham dams were isolated by collagenase perfusion and handpicking. We previously demonstrated that the transcript message for membrane-bound complement regulator CD55 (negative regulator of C3 activation) was significantly increased in islets isolated from female, but not male, RUPP vs Sham offspring, with no change in acinar tissue. In addition, no significant change was observed in message for the parent molecule C3. The relative amount of circulating C3a in serum of PD13 offspring was determined by Western blot and expressed as C3a units/ul based on signal intensity of 1 ul of a standard pool of adult rat serum activated by yeast. Circulating C3a in female RUPP offspring (1.35 ± 0.37 units/ul) was significantly lower (p<0.05) than C3a in female Sham offspring (2.54 ± 0.44 units/ul). No difference was noted comparing C3a in serum of male offspring, RUPP vs Sham, where no change in β cell area has been observed. Thus, these data suggest an increase in CD55 may limit complement activation and C3a production in female offspring. This dysregulation of complement activation may contribute to reduction of β cell area and long-term susceptibility for Type 2 diabetes. Future studies are needed to determine if local or circulating C3a is instrumental in controlling pancreatic β cell area in offspring of hypertensive pregnancies.

Reduction in Pancreatic β Cell Area is Associated with Increased Islet Macrophage Message in Female Rat Offspring following Chronic Placental Ischemia

Placental insufficiency in a well‐nourished population can result in intrauterine growth restriction (IUGR) contributing to pathophysiology of diseases with origins early in life, including Type 2 diabetes (T2D). Certainly the risk for T2D is increased in low birth weight offspring and/or offspring of pregnancies complicated by gestational hypertension and low birth weight. Using the reduced uteroplacental perfusion pressure (RUPP) model of chronic placental ischemia in the rat, our previous studies have demonstrated β cell area is reduced and apoptosis increased in pancreatic islets of growth restricted fetus at gestation day (GD)19. Also, adult RUPP offspring have been shown to display glucose intolerance. Macrophages in pancreatic islets have been implicated as important for islet development, but whether macrophages are instrumental in T2D islet pathology and decreasing β cell area is unknown. The goal of the present study was to determine if reduction in β cell area in offspring of RUPP pregnancies persisted to postnatal day (PD)13 and was accompanied by changes in islet macrophages. On GD14, timed pregnant Sprague Dawley rats underwent either Sham surgery or RUPP surgery with clip placement on ovarian arteries and abdominal aorta to reduce uterine perfusion pressure, resulting in hypertension in the dam and IUGR. Rats were allowed to deliver pups spontaneously, and litters were culled to 8 offspring. On PD13, offspring were euthanized, and two pancreata of each sex were isolated, weighed, and fixed in neutral buffered formalin for determination of β cell area. For isolation of islets, two pancreata of each sex were inflated with collagenase via the hepatic duct. After dissection, the pancreata were further digested with collagenase at 37 C, and intact islets hand‐picked for culture overnight in media. Isolated islets were then added to RNA later for isolation of RNA and qRT PCR for the macrophage marker CD68. β cell area was determined using immunofluorescence by measuring the insulin positive area/total pancreas area in 3 sections/rat pancreas at least 100 microns apart. At PD13, β cell area was reduced in female RUPP offspring (0.012±0.001 ratio) compared to Sham (0.016±0.001 ratio) with N of 3–4 pups from at least 2 litters. Message for CD68 as an indicator of macrophages was significantly increased 2.6 ± 0.8 fold in female offspring of RUPP pregnancies compared to Sham using actin to normalize, with no significant change in CD68 in male offspring of RUPP vs Sham pregnancies. Evaluation of CD68 message in surrounding acinar tissue showed no significant change in RUPP vs Sham in either male or female offspring. Thus, our data suggest that the reduction in β cell area following chronic placental ischemia persists in female but not male offspring and is associated with increased islet macrophages. Continued studies will evaluate the potential role of macrophages in reducing β cell mass and contributing to islet cell pathology in development of T2D.Support or Funding InformationFunded in part by NIH NIDDK R03 DK114465 (EA) and American Heart Association Grant 17GRNT33650049 (JR)

Role of B1 and B2 lymphocytes in placental ischemia-induced hypertension.

Preeclampsia is a prevalent pregnancy complication characterized by new-onset maternal hypertension and inflammation, with placental ischemia as the initiating event. Studies of others have provided evidence for the importance of lymphocytes in placental ischemia-induced hypertension; however, the contributions of B1 versus B2 lymphocytes are unknown. We hypothesized that peritoneal B1 lymphocytes are important for placental ischemia-induced hypertension. As an initial test of this hypothesis, the effect of anti-CD20 depletion on both B-cell populations was determined in a reduced utero-placental perfusion pressure (RUPP) model of preeclampsia. Anti-murine CD20 monoclonal antibody (5 mg/kg, Clone 5D2) or corresponding mu IgG2a isotype control was administered intraperitoneally to timed pregnant Sprague-Dawley rats on gestation day (GD)10 and 13. RUPP or sham control surgeries were performed on GD14, and mean arterial pressure (MAP) was measured on GD19 from a carotid catheter. As anticipated, RUPP surgery increased MAP and heart rate and decreased mean fetal and placental weight. However, anti-CD20 treatment did not affect these responses. On GD19, B-cell populations were enumerated in the blood, peritoneal cavity, spleen, and placenta with flow cytometry. B1 and B2 cells were not significantly increased following RUPP. Anti-CD20 depleted B1 and B2 cells in peritoneum and circulation but depleted only B2 lymphocytes in spleen and placenta, with no effect on circulating or peritoneal IgM. Overall, these data do not exclude a role for antibodies produced by B cells before depletion but indicate the presence of B lymphocytes in the last trimester of pregnancy is not critical for placental ischemia-induced hypertension.NEW & NOTEWORTHY The adaptive and innate immune systems are implicated in hypertension, including the pregnancy-specific hypertensive condition preeclampsia. However, the mechanism of immune system dysfunction leading to pregnancy-induced hypertension is unresolved. In contrast to previous reports, this study reveals that the presence of classic B2 lymphocytes and peritoneal and circulating B1 lymphocytes is not required for development of hypertension following third trimester placental ischemia in a rat model of pregnancy-induced hypertension.

Reduced Uteroplacental Perfusion Pressure (RUPP) causes altered trophoblast differentiation and pericyte reduction in the mouse placenta labyrinth

This study characterized the effect of the reduced utero-placental perfusion pressure (RUPP) model of placental insufficiency on placental morphology and trophoblast differentiation at mid-late gestation (E14.5). Altered trophoblast proliferation, reduced syncytiotrophoblast gene expression, increased numbers of sinusoidal trophoblast giant cells, decreased Vegfa and decreased pericyte presence in the labyrinth were observed in addition to changes in maternal blood spaces, the fetal capillary network and reduced fetal weight. Further, the junctional zone was characterized by reduced spongiotrophoblast and glycogen trophoblast with increased trophoblast giant cells. Increased Hif-1α and TGF-β-3 in vivo with supporting hypoxia studies in trophoblast stem (TS) cells in vitro, support hypoxia as a contributing factor to the RUPP placenta phenotype. Together, this study identifies altered cell populations within the placenta that may contribute to the phenotype, and thus support the use of RUPP in the mouse as a model of placenta insufficiency. As such, this model in the mouse provides a valuable tool for understanding the phenotypes resulting from genetic manipulation of isolated cell populations to further understand the etiology of placenta insufficiency and fetal growth restriction. Further this study identifies a novel relationship between placental insufficiency and pericyte depletion in the labyrinth layer.

Decreased homodimerization and increased TIMP-1 complexation of uteroplacental and uterine arterial matrix metalloproteinase-9 during hypertension-in-pregnancy

Preeclampsia is a complication of pregnancy manifested as hypertension-in-pregnancy (HTN-Preg) and often intrauterine growth restriction (IUGR). Placental ischemia could be an initiating event, but the molecular mechanisms are unclear. To test the hypothesis that dimerization of matrix metalloproteinases (MMPs) plays a role in HTN-Preg and IUGR, the levels/activity of MMP-9, tissue inhibitor of metalloproteinase (TIMP-1), and their dimerization forms were measured in the placenta, uterus, and uterine artery of normal pregnant (Preg) rats and a rat model of reduced uteroplacental perfusion pressure (RUPP). Consistent with our previous report, blood pressure (BP) was higher, pup weight was lower, and gelatin zymography showed different gelatinolytic activity for pro-MMP-9, MMP-9, pro-MMP-2 and MMP-2 in RUPP vs Preg rats. Careful examination of the zymograms showed additional bands at 200 and 135kDa. Western blots with MMP-9 antibody suggested that the 200kDa band was a MMP-9 homodimer. Western blots with TIMP-1 antibody as well as reverse zymography suggested that the 135kDa band was a MMP-9/TIMP-1 complex. The protein levels and gelatinase activity of MMP-9 homodimer were decreased while MMP-9/TIMP-1 complex was increased in placenta, uterus and uterine artery of RUPP vs Preg rats. The epidermal growth factor (EGF) receptor blocker erlotinib and protein kinase C (PKC) inhibitor bisindolylmaleimide decreased MMP-9 homodimer and increased MMP-9/TIMP-1 complex in placenta, uterus and uterine artery of Preg rats. EGF and the PKC activator phorbol-12,13-dibutyrate (PDBu) reversed the decreases in MMP-9 homodimer and the increases in MMP-9/TIMP-1 complex in tissues of RUPP rats. Thus, the increased BP and decreased pup weight in placental ischemia model of HTN-Preg are associated with a decrease in MMP-9 homodimer and an increase in MMP-9/TIMP-1 complex in placenta, uterus, and uterine artery, which together would cause a net decrease in MMP-9 activity and reduce uteroplacental and vascular remodeling in the setting of HTN-Preg and IUGR. Enhancing EGFR/PKC signaling may reverse the MMP-9 unfavorable dimerization patterns and thereby promote uteroplacental and vascular remodeling in preeclampsia.

Effect of nicotine on placental ischemia-induced complement activation and hypertension in the rat

Preeclampsia is a pregnancy-specific condition manifested by new-onset maternal hypertension with systemic inflammation, including increased innate immune system complement activation. While exact pathophysiology is unknown, evidence suggests that inadequate spiral artery invasion and resulting utero-placental insufficiency is the initiating event. Cigarette smoking during pregnancy decreases the risk of preeclampsia. Nicotine, a major component of cigarettes, stimulates the efferent cholinergic anti-inflammatory pathway through peripherally expressed nicotinic acetylcholine receptors (nAChR) and is known to attenuate ischemia–reperfusion injury in kidney and liver. Prior studies indicated that complement activation was critical for placental ischemia-induced hypertension in a rat model. Thus, it was hypothesized here that nicotine was responsible for the protective effect of cigarette smoking in preeclampsia and would attenuate placental ischemia-induced systemic complement activation and hypertension. The Reduced Utero-placental Perfusion Pressure (RUPP) model in the pregnant rat was employed to induce placental ischemia, resulting in complement activation, fetal resorptions, and hypertension. On gestation day (GD)14, nicotine (1 mg/kg) or saline was administered via subcutaneous injection prior to RUPP surgery and daily through GD18. On GD19, placental ischemia significantly increased mean arterial pressure (MAP) in saline injected animals. However, the placental ischemia-induced increase in blood pressure was not evident in nicotine-treated animals and nicotine treatment significantly increased MAP variability. Circulating C3a was measured as an indicator of complement activation and increased C3a in RUPP compared to Sham persisted with nicotine treatment, as did fetal resorptions. These data suggested to us that nicotine may contribute to the decreased risk of preeclampsia with cigarette smoking, but this protective effect was confounded by additional effects of nicotine on the cardiovascular system.

Role of IgM and angiotensin II Type I receptor autoantibodies in local complement activation in placental ischemia-induced hypertension in the rat.

Preeclampsia is characterized by development of hypertension during pregnancy and reduced placental perfusion. Previous studies in a rat model of placental ischemia-induced hypertension demonstrated that inhibiting complement activation attenuated increased maternal blood pressure with C3a and C5a identified as the important products of complement activation. Given that in other forms of ischemia both natural IgM and antigen antibody complexes initiate complement activation, we hypothesized that placental ischemia exposes neoepitopes recognized by IgM to cause local complement activation and hypertension. Alternatively, we postulated that autoantibody to angiotensin II Type 1 receptor (AT1-AA) interacts with AT1 receptors to cause complement activation. Since complement activation occurs in kidney and placenta in preeclampsia, we used immunohistochemistry to determine IgM deposition and local complement activation in each organ (C3 deposition), and quantitative real-time polymerase chain reaction (qRT-PCR) to quantitate mRNA for endogenous regulators of complement activation CD55, CD59 and Complement receptor 1-related gene/protein y (Crry). On gestation day (GD)14.5, timed pregnant Sprague Dawley rats underwent Sham surgery or placement of clips on inferior abdominal aorta and ovarian arteries to create placental ischemia using the reduced utero-placental perfusion pressure (RUPP) model. As previously reported, RUPP surgery increased mean arterial pressure and circulating C3a on GD19.5. In placenta, IgM and C3 deposition increased, whereas mRNA for complement regulators Crry and CD59 decreased along with Crry protein in RUPP compared to Sham treated animals. In kidney, IgM deposition increased in animals subjected to RUPP vs Sham surgery without a significant change in C3 deposition and coincident with an increase in mRNA for CD55 and CD59. The AT1 receptor antagonist losartan prevents placental ischemia-induced hypertension as well as AT1-AA interaction with AT1 receptors. However, losartan did not attenuate complement activation as measured by circulating C3a or placental C3 deposition. Importantly, our studies indicate that following placental ischemia, complement activation is not due to AT1-AA but is associated with IgM deposition. These studies suggest a role for natural antibodies interacting with placental ischemia-induced neoepitopes to activate complement and contribute to hypertension.

Neutrophil Depletion Attenuates Placental Ischemia-Induced Hypertension in the Rat.

Preeclampsia is characterized by reduced placental perfusion with placental ischemia and hypertension during pregnancy. Preeclamptic women also exhibit a heightened inflammatory state and greater number of neutrophils in the vasculature compared to normal pregnancy. Since neutrophils are associated with tissue injury and inflammation, we hypothesized that neutrophils are critical to placental ischemia-induced hypertension and fetal demise. Using the reduced uteroplacental perfusion pressure (RUPP) model of placental ischemia-induced hypertension in the rat, we determined the effect of neutrophil depletion on blood pressure and fetal resorptions. Neutrophils were depleted with repeated injections of polyclonal rabbit anti-rat polymorphonuclear leukocyte (PMN) antibody (antiPMN). Rats received either antiPMN or normal rabbit serum (Control) on 13.5, 15.5, 17.5, and 18.5 days post conception (dpc). On 14.5 dpc, rats underwent either Sham surgery or clip placement on ovarian arteries and abdominal aorta to reduce uterine perfusion pressure (RUPP). On 18.5 dpc, carotid arterial catheters were placed and mean arterial pressure (MAP) was measured on 19.5 dpc. Neutrophil-depleted rats had reduced circulating neutrophils from 14.5 to 19.5 dpc compared to Control, as well as decreased neutrophils in lung and placenta on 19.5 dpc. MAP increased in RUPP Control vs Sham Control rats, and neutrophil depletion attenuated this increase in MAP in RUPP rats without any effect on Sham rats. The RUPP-induced increase in fetal resorptions and complement activation product C3a were not affected by neutrophil depletion. Thus, these data are the first to indicate that neutrophils play an important role in RUPP hypertension and that cells of the innate immune system may significantly contribute to pregnancy-induced hypertension.

Therapeutic effect of maternal molecular hydrogen administration in a rat model of preeclampsia

s / Placenta 35 (2014) A1eA112 A78 Conclusion: A novel biomarker to specifically and reliably predict the risk for severe preeclampsia is successfully identified. Further investigations in larger cohorts including populations of other ethnic/genetic backgrounds are necessary. P2.52. THERAPEUTIC EFFECT OF MATERNAL MOLECULAR HYDROGEN ADMINISTRATION IN A RAT MODEL OF PREECLAMPSIA Takafumi Ushida, Yukio Mano, Tomomi Kotani Nagoya university graduate school of medicine, Nagoya city, Japan Objective: Preeclampsia is a pregnancy-specific condition characterized by new-onset hypertension and proteinuria. It is also occasionally related to fetal growth restriction. Some studies suggest that placental oxidative stress plays an important role in the pathogenesis of preeclampsia. Recently, molecular hydrogen (H2) is reported to prevent a variety of diseases associated with oxidative stress in model systems and in human. Here, we studied the efficacy of H2 using a rat model of preeclampsia. Methods:We used thewell-established reduced utero-placental perfusion pressure (RUPP) model of placental ischemia-induced hypertension in rat that mimics features of preeclampsia. RUPP was performed on day 14 of pregnancy, clips were placed around the aorta below the renal arteries and on both the left and right uterine arcade at the ovarian artery. The sham group underwent laparotomy on day 14 of pregnancy without RUPP procedure. Hydrogen-saturated water (HW) was orally administered ad libitum from day 12 to 19 of pregnancy. On day 19, mean arterial pressure (MAP) was measured via carotid catheters, and then fetus and placenta were collected by cesarean section. Results:MAP and urinary protein were significantly increased in the RUPP group compared with the sham group. HW treatment attenuated both MAP and urinary protein (sham 4.73, RUPP 8.14 and RUPP+HW 5.88mg/dL, respectively). RUPP fetuses and placentas were significantly smaller than those of sham group. In the RUPP+HW group, the decrease in fetal and placental weights was improved (fetal weight, sham 2.25, RUPP 1.79 and RUPP+HW 1.98g, respectively; placental weight, sham 0.417, 0.381 and 0.416g, respectively). Conclusion: The prophylactic administration of HWsignificantly attenuated features of preeclampsia. Moreover, fetal growth was also improved. These results suggest that maternal administration of HW could have a potential benefit for the prevention of preeclampsia as a novel intra-uterine therapy. P2.53. PREECLAMPSIA IN PREGNANCIES WITH AND WITHOUT DIABETES; THE ASSOCIATIONS WITH PLACENTAL WEIGHT. A POPULATION STUDY OF 655 842 PREGNANCIES Johanne Dypvik , Ellen Marie Strom-Roum , Camilla Haavaldsen , Lars Johan Vatten , Anne Eskild a,b Department of Obstetrics and Gynecology, Akerhus University Hospital, Lorenskog, Norway; b Institute of Clinical Medicine, University of Oslo, Oslo, Norway; Department of Public Health, Norwegian University of Science and Technology, Trondheim, Norway Objectives: To study whether placental weight in pregnancies with preeclampsia differs by maternal diabetes status? Methods: The study comprised all singleton deliveries in Norway from 1999 through 2010 (n 1⁄4 655 842). Data were obtained from the Medical Birth Registry of Norway. We used z-scores of placental weight to adjust for differences in gestational age at delivery between pregnancies, and we studied the distributions of placental weight z-scores in deciles (tenths) in preeclamptic pregnancies with and without diabetes and in normotensive pregnancies with and without diabetes. Results: In pregnancies with preeclampsia, the mean placental weight was much higher in diabetic pregnancies as compared to non-diabetic pregnancies. Thus, among pregnancies with preeclampsia, diabetic pregnancies were overrepresented in the highest decile of placental weight (28.8%) whereas preeclamptic pregnancies without diabetes were underrepresented in the highest decile of placental weight (9.8%). The enlargement of the placentas in preeclamptic pregnancies with diabetes was also pronounced in pregnancies with preterm delivery, and 30.1% were in the highest decile of placental weight as compared to 5.1% of the preeclamptic pregnancies without diabetes. Conclusion: In pregnancies with preeclampsia, the placenta was large when the mother had concomitant diabetes, whereas in preeclamptic pregnancies without diabetes the placenta was small and smaller than in normotensive pregnancies. This finding suggests that the placental role in preeclampsia may differ by maternal diabetes status. P2.54. ALPHA-1 MICROGLOBULIN AS A POTENTIAL THERAPEUTIC CANDIDATE FOR THE TREATMENT OF PREECLAMPSIA Lena Erlandsson , Ludivine Doridot , Aur elien Ducat , Johann Castille , Jean-Luc Vilotte , Lena Wester Rosenl€ of , Magnus Gram, Bo Akerstr€ om , Daniel Vaiman , Stefan Hansson a a Institution for Clinical Sciences in Lund, Lund University, Department of Obstetrics and Gynecology, Lund, Sweden,; b INSERM U1016, Institute Cochin, Paris, France; c INRA UMR1313, G en etique Animale et Biologie Int egrative, Jouy-en-Josas,

A pregnancy case report of alagille syndrome; expression of NOTCH signaling in the placenta

A pregnancy case report of alagille syndrome; expression of NOTCH signaling in the placenta

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<i>Hypertension</i> Editors’ Picks

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.

Antihypertensive effects of inducible nitric oxide synthase inhibition in experimental pre‐eclampsia

Upregulation of inducible nitric oxide synthase (iNOS) has been reported in both experimental and clinical hypertension. However, although pro-inflammatory cytokines that up-regulate iNOS contribute to pre-eclampsia, no previous study has tested the hypothesis that a selective iNOS inhibitor (1400 W) could exert antihypertensive effects associated with decreased iNOS expression and nitrosative stress in pre-eclampsia. This study examined the effects of 1400 W in the reduced uteroplacental perfusion pressure (RUPP) placental ischaemia animal model and in normal pregnant rats. Sham-operated and RUPP rats were treated with daily vehicle or 1 mg/kg/day N-[3-(Aminomethyl) benzyl] acetamidine (1400 W) subcutaneously for 5 days. Plasma 8-isoprostane levels, aortic reactive oxygen species (ROS) levels and nicotinamide adenine dinucleotide phosphate (NADPH)-dependent ROS production were evaluated by ELISA, dihydroethidium fluorescence microscopy and lucigenin chemiluminescence respectively. Inducible nitric oxide synthase expression was assessed by western blotting analysis and aortic nitrotyrosine was evaluated by immunohistochemistry. Mean arterial blood pressure increased by ∼30 mmHg in RUPP rats, and 1400 W attenuated this increase by ∼50% (P < 0.05). While RUPP increased plasma 8-isoprostane levels, aortic ROS levels, and NADPH-dependent ROS production (P < 0.05), treatment with 1400 W blunted these alterations (P < 0.05). Moreover, while RUPP increased iNOS expression and aortic nitrotyrosine levels (P < 0.05), treatment with 1400 W blunted these alterations (P < 0.05). These results clearly implicate iNOS in the hypertension associated with RUPP. Our findings may suggest that iNOS inhibitors could be clinically useful in the therapy of pre-eclampsia, especially in particular groups of patients genetically more prone to express higher levels of iNOS. This issue deserves further confirmation.

Complement activation is critical for placental ischemia-induced hypertension in the rat

Preeclampsia is a major obstetric problem defined by new-onset hypertension and proteinuria associated with compromised placental perfusion. Although activation of the complement system is increased in preeclampsia compared to normal pregnancy, it remains unclear whether excess complement activation is a cause or consequence of placental ischemia. Therefore, we hypothesized that complement activation is critical for placental ischemia-induced hypertension. We employed the reduced utero-placental perfusion pressure (RUPP) model of placental ischemia in the rat to induce hypertension in the third trimester and evaluated the effect of inhibiting complement activation with a soluble recombinant form of an endogenous complement regulator, human complement receptor 1 (sCR1; CDX-1135). On day 14 of a 21-day gestation, rats received either RUPP or Sham surgery and 15mg/kg/day sCR1 or saline intravenously on days 14–18. Circulating complement component 3 decreased and complement activation product C3a increased in RUPP vs. Sham (p<0.05), indicating complement activation had occurred. Mean arterial pressure (MAP) measured on day 19 increased in RUPP vs. Sham rats (109.8±2.8mmHg vs. 93.6±1.6mmHg). Treatment with sCR1 significantly reduced elevated MAP in RUPP rats (98.4±3.6mmHg, p<0.05) and reduced C3a production. Vascular endothelial growth factor (VEGF) decreased in RUPP compared to Sham rats, and the decrease in VEGF was not affected by sCR1 treatment. Thus, these studies have identified a mechanistic link between complement activation and the pregnancy complication of hypertension apart from free plasma VEGF and have identified complement inhibition as a potential treatment strategy for placental ischemia-induced hypertension in preeclampsia.

Pravastatin Attenuates Hypertension, Oxidative Stress, and Angiogenic Imbalance in Rat Model of Placental Ischemia-Induced Hypertension

Preeclampsia is a pregnancy-specific condition characterized by an imbalance of circulating angiogenic factors and new-onset hypertension. Although current treatment options are limited, recent studies suggest that pravastatin may improve angiogenic profile and reduce blood pressure in preeclampsia. We hypothesized pravastatin would restore angiogenic balance and reduce mean arterial pressure (MAP) in rats with reduced utero-placental perfusion pressure (RUPP)-induced hypertension. Pravastatin was administered intraperitoneally (1 mg/kg per day) in RUPP (RUPP+P) and normal pregnant rats (NP+P) from day 14 to 19 of pregnancy. On day 19, MAP was measured via catheter, conceptus data were recorded, and tissues collected. MAP was increased (P<0.05) in RUPP compared with NP dams, and pravastatin ameliorated this difference. Pravastatin attenuated decreased fetal weight and plasma vascular endothelial growth factor and the RUPP-induced increased soluble fms-like tyrosine kinase-1 when compared with NP dams. Pravastatin treatment did not improve angiogenic potential in RUPP serum and decreased (P<0.05) endothelial tube formation in NP rats. RUPP rats presented with indices of oxidative stress, such as increased placental catalase activity and plasma thiobarbituric acid reactive substances along with decreased plasma total antioxidant capacity compared with NP controls, and pravastatin attenuated these effects. MAP, fetal weight, plasma vascular endothelial growth factor, and plasma soluble fms-like tyrosine kinase-1 were unchanged in NP+P compared with NP controls. The present data indicate that treatment with pravastatin attenuates oxidative stress and lowers MAP in placental ischemia-induced hypertension, but may have negative effects on circulating angiogenic potential during pregnancy. Further studies are needed to determine whether there are long-term deleterious effects on maternal or fetal health after pravastatin treatment during pregnancy-induced hypertension or preeclampsia.