Normal Pregnant Rats Research Articles

Antihypertensive responses of vasoactive androgens in an in vivo experimental model of preeclampsia

Dehydroepiandrosterone (DHEA), testosterone (TES) and its 5-reduced metabolites induce a nongenomic vasorelaxation in several vascular beds of mammals; similarly these hormones produce systemic hypotensive and antihypertensive responses in normotensive and hypertensive male rats. Thus, it was hypothesized that the antihypertensive response of androgens, whose levels are elevated during gestation, protect against gestational hypertension. An animal model of preeclampsia was induced in female Wistar rats using DOCA-salt-treated pregnant (PT) and normal pregnant (NP) rats. In vivo experiments in conscious rats revealed that bolus intravenous injections of DHEA, TES, 5α- or 5β-dihydrotestosterone (-DHT) log −1.0 to 2.0μmolk−1min−1, produced substantial transient reductions in arterial blood pressure (BP), without significant changes in heart rate (HR). Mean arterial blood pressure (MAP) was reduced significantly in both groups. PT rats were more sensitive to the antihypertensive responses of androgens than NP. DHEA and 5β-DHT were the most potent to reduce MAP: 66±07 and 69±2.0mmHg in PT but only 33±0.5 and 35±1.2mmHg in NP rats, respectively. In isolated aortas of PT and NP, the concentration-response curves to each androgen (0.1–100μM) indicated that KCl-induced pre-contraction is more sensitive to all androgens than phenylephrine (Phe) pre-contractions. Notably, 5β-DHT is the greatest vasorelaxant with KCl-induced contraction than with Phe contraction of both groups, suggesting a preferential blockade on L-VOCCs. TES exhibited minor vasorelaxing effect of aortas pre-contracted with KCl, compared to its precursor DHEA and its 5-reduced metabolites. These data show that these androgens exert acute vasorelaxing effects in vitro and remarkably, reduce the BP in vivo in PT and NP at term pregnancy. Moreover, a deficit in feto-placental androgen production during pregnancy may trigger the development of preeclampsia or gestational hypertension.

Oxidative Stress in the Rostral Ventrolateral Medulla Contributes To Cardiovascular Regulation in Preeclampsia.

Background: It has been demonstrated that preeclampsia, a pregnancy-specific hypertension disorder, is characterized by high blood pressure (BP) and sympathetic overactivity. Increased reactive oxygen species (ROS) in the rostral ventrolateral medulla (RVLM), a key region for controlling sympathetic tone, has been reported to contribute to high level of BP and sympathetic outflow. The aim of the present study was to determine the role of the RVLM ROS in mediating the preeclampsia-associated cardiovascular dysfunction.Methods: The animal model of preeclampsia was produced by administration of desoxycorticosterone acetate (DOCA) to pregnant rats.Results: Compared with normal pregnant rats without DOCA treatment (NP), the protein concentration and norepinephrine excretion in 24-h urine, as well as BP in pregnant rats with DOCA treatment (PDS) were significantly increased. The levels of superoxide anion and the protein expression of NADPH oxidase subtype (NOX4) in the RVLM were significantly increased in PDS than in NP groups. Furthermore, microinjection of the superoxide dismutase (SOD) mimic Tempol (5 nmol) into the RVLM significantly decreased BP, heart rate, and renal sympathetic never activity in PDS but not in NP group.Conclusion: The present data suggest that high BP and sympathetic overactivity in preeclampsia rats is associated with increased oxidative stress in the RVLM via upregulation of NOX4 expression.

SERUM VASPIN LEVELS IN NORMAL PREGNANT AND GESTATIONAL DIABETIC ALBINO RAT MODEL

Background:Gestational diabetes mellitus (GDM) is a major pregnancy complication with increased blood glucose levels and insulin resistance. Vaspin is a visceral adipokine that is synthesized and secreted by abdominal fat tissue and one of the superfamily of serine protease inhibitors. Data proposed vaspin levels in normal pregnancy and GDM are still controversial. Objective : To assess serum vaspin levels in both of late pregnancy and experimentally-induced GDM in relation to certain metabolic parameters in albino rats. Material and methods: Thirty six virgin female albino rats of a local strain weighing 100-150 g were divided randomly into three equals groups. Group I (Control); virgin rats were fed normal diet. Group II (normal pregnant); rats were fed normal diet for five weeks before induction of pregnancy. Group III (experimentally-induced GDM); rats were fed fatty-sucrose diet (FSD) for five weeks before induction of pregnancy, then injected intraperitoneally with streptozotocin (25 mg/kg) on the 7th day of gestation. In all groups, serum vaspin, body mass index (BMI), serum estradiol, progesterone, total triglycerides (TG), total cholesterol (TC), low density lipoproteins-cholesterol (LDL-c), high density lipoproteins-cholesterol (HDL-c), glucose, insulin levels and calculated homeostatic model assessment of insulin resistance index (HOMA-IR) were estimated. Results: Normal pregnant rats showed a significant increase in BMI, serum vaspin, TG, TC, LDL-c, insulin levels and HOMA-IR compared to controls. Additionally, serum vaspin levels were significantly and positively correlated with BMI, serum glucose, TG levels and HOMA-IR. GDM-induced rats had significantly increased levels of serum vaspin, TC, TG, LDL-c, glucose, BMI and HOMA-IR, while decreased insulin and HDL-c levels compared to normal pregnant and control rats. Moreover, serum vaspin levels showed significant positive correlations with BMI, HOMA-IR, glucose, TG, TC levels, and an inverse correlation with HDL-c levels in GDM-induced group. Conclusion: Serum vaspin levels increased in both of late pregnancy and experimentally-induced GDM, but more in the later possibly for compensating GDM-induced insulin resistance and dyslipidemia.

Angiogenic imbalance and diminished matrix metalloproteinase-2 and -9 underlie regional decreases in uteroplacental vascularization and feto-placental growth in hypertensive pregnancy

Preeclampsia is a form of hypertension-in-pregnancy (HTN-Preg) with unclear mechanism. Generalized reduction of uterine perfusion pressure (RUPP) could be an initiating event leading to uteroplacental ischemia, angiogenic imbalance, and HTN-Preg. Additional regional differences in uteroplacental blood flow could further affect the pregnancy outcome and increase the risk of preeclampsia in twin or multiple pregnancy, but the mechanisms involved are unclear. To test the hypothesis that regional differences in angiogenic balance and matrix metalloproteinases (MMPs) underlie regional uteroplacental vascularization and feto-placental development, we compared fetal and placental growth, and placental and myoendometrial vascularization in the proximal, middle and distal regions of the uterus (in relation to the iliac bifurcation) in normal pregnant (Preg) and RUPP rats. Maternal blood pressure and plasma anti-angiogenic soluble fms-like tyrosine kinase-1 (sFlt-1)/placenta growth factor (PIGF) ratio were higher, and average placentae number, placenta weight, litter size, and pup weight were less in RUPP than Preg rats. The placenta and pup number and weight were reduced, while the number and diameter of placental and adjacent myoendometrial arteries, and MMP-2 and MMP-9 levels/activity were increased, and sFlt-1/PlGF ratio was decreased in distal vs proximal uterus of Preg rats. In RUPP rats, the placenta and pup number and weight, the number and diameter of placental and myoendometrial arteries, and MMP-2 and -9 levels/activity were decreased, and sFlt-1/PlGF ratio was increased in distal vs proximal uterus. Treatment with sFlt-1 or RUPP placenta extract decreased MMP-2 and MMP-9 in distal segments of Preg uterus, and treatment with PIGF or Preg placenta extract restored MMP levels in distal segments of RUPP uterus. Thus, in addition to the general reduction in placental and fetal growth during uteroplacental ischemia, localized angiogenic imbalance and diminished MMP-2 and MMP-9 could cause further decrease in placental and myoendometrial vascularization and placental and fetal growth in distal vs proximal uterus of HTN-Preg rats. Regional differences in uteroplacental perfusion, angiogenic balance and MMPs could be a factor in the incidence of preeclampsia in multiple pregnancy.

Abstract P271: Interleukin-4 Supplementation Improves Chronic Inflammation and Hypertension in Response to Placental Ischemia During Pregnancy

Preeclampsia (PE), new onset hypertension in response to placental ischemia during pregnancy, is associated with chronic inflammation characterized by elevated tumor necrosis factor (TNF-α), interleukin-6 (IL-6), agonistic autoantibody to the Ang II type 1 receptor (AT1-AA). In addition, evidence for a shift towards proinflammatory CD4+TH1 vs CD4+TH2 profile exist in PE. Therefore we tested the hypothesis that IL-4, a cytokine essential for TH2 differentiation and proliferation, would improve pregnancy outcomes in response to placental ischemia (RUPP) by shifting the immune profile toward CD4+TH2. Interleukin-4, 600 ng/day, was administered via a mini-osmotic pump on day 14 of gestation into normal pregnant (NP) rats and into RUPP rats immediately following the reduced uterine perfusion pressure (RUPP) procedure and carotid catheters were inserted on gestation day 18. Blood pressure (MAP), TNF-α, IL-6, AT1-AA and circulating TH2 cells were measured on GD 19. MAP in NP rats (n=10) was 96 + 2, 101 + 2 in NP+IL-4 rats (n=14), 130 + 4 in RUPP rats (n=6), which improved to 108 + 2 mmHg in RUPP+IL-4 rats (n=15), p< 0.05. Plasma levels of TNF-α and IL-6 were 25 + 6, 29 + 3 in NP rats (n=7-8/group), 116 + 30, 224 + 62 in RUPP rats (n=9-10/group), which decreased to 23 + 5 and 135 + 48 pg/mL in RUPP+IL-4 rats (n=5-7/group), p<0.05. Importantly, plasma levels of AT1-AA were 0.3 + 0.4 in NP rats (n=8), 0.3 + 0.2 in NP+IL-4 rats (n=5), 18 + 0.3 in RUPP rats (n=9), which were blunted to 4 + 1 bpm in RUPP+IL-4 rats (n=14), p<0.05. Circulating TH2 cells were 17.3 ± 2.7 in NP rats (n=3), 9.0 ± 2.0 in RUPP rats (n=5), which improved to 13.4 ± 3.2 % gated in RUPP+IL-4 rats (n=9). This study illustrates that administration of IL-4 decreases TNF-α, IL-6, AT1-AA, possibly by increasing TH2/IL-4 in RUPP rats, which ultimately led to a reduction in hypertension in response to placental ischemia of pregnancy.

Abstract P265: Placental Ischemia Causes Renal Mitochondrial Impairment in Reduced Uterine Perfusion Pressure Rats

Introduction: Preeclampsia (PE) is characterized by new onset hypertension in pregnancy and is associated with renal dysfunction, proteinuria and is believed to be initiated by placental ischemia. Mitochondrial dysfunction is an important source of reactive oxygen species (ROS) generation. To better understand the role of mitochondrial dysfunction in hypertension during PE we examined renal mitochondrial expression in the kidney of RUPP rats. Methods: Female Sprague Dawley rats were dived into two groups; normal pregnant (NP) and RUPP rats. On gestational day (GD) 14, RUPP surgery was performed, GD18 carotid catheters were inserted, and GD19 conscious blood pressure (MAP) was measured, renal mitochondria were isolated for respiration and analysis. Respiration measurements were performed on intact isolated mitochondria under glutamate/malate as complex I substrate using Oroboros Oxygraph-2K. Oxidative phosphorylation was analyzed by western blot using total Oxphos cocktail antibody and VDAC. Results: MAP was elevated in RUPP (n=8) vs NP rats (n=10) (125±6 vs. 99±2 mmHg, p<0.05). RUPP (n=3) renal mitochondria show reduced expression of Complex I (0.5±0.02 vs 0.8±0.09, p<0.05) and Complex II (0.7±0.03 vs 1±0.08, p<0.05) compared to NP mitochondria. State 3 (624±112 vs 878±9 pmol/sec/mg, p=0.22) and maximal (496±87vs 748±75 pmol/sec/mg, p=0.14) respiration rates trended towards reduction in RUPPs (n=4) compared to NP (n=2). Conclusion: Reduced expression of Complex I and II expression with reduced respiratory rates indicate mitochondrial impairment in the RUPP kidneys. Although, renal mitochondrial mediated oxidative stress may be one mechanism of hypertension in the RUPP rat model of PE, further exploration of oxidative stress in the kidney is necessary to fully understand the contribution of mitochondrial mediated oxidative stress to pathology of PE.

Abstract P269: Circulating Factors in Response to Placental Ischemia Cause Vascular Endothelial Mitochondrial Oxidative Stress

Introduction: Preeclampsia (PE) is associated with placental ischemia, new onset hypertension, oxidative stress and endothelial dysfunction. Factors linking placental ischemia with endothelial dysfunction and hypertension are not completely understood. Mitochondrial (mt) dysfunction (dys) is a major source of reactive oxygen species (ROS) and we have shown that placental ischemia causes oxidative stress in RUPP rats. We hypothesize that circulating factors in RUPP rats cause vascular endothelial mt dys and mt ROS as a contributor to endothelial dysfunction and hypertension during pregnancy. Methods: Female Sprague Dawley rats were dived into two groups; normal pregnant (NP) and RUPP rats. On gestational day (GD) 14, RUPP surgery was performed, GD18 carotid catheters were inserted, and GD19 conscious blood pressure (MAP) was measured. GD 19 placentas were collected and mitochondria were isolated for respiration and ROS measurements. Mt ROS was measured spectrophotometrically in HUVECs incubated with 10% serum from NP or RUPP rats using MitoSox Red. Results: MAP was elevated in RUPP (n=9) compared to NP rats (n=9) (122±2 vs. 104±2 mmHg, p<0.05). State 3 (313±16 vs 423±15 pmol/sec/mg, p<0.05) and maximal (244±13 vs 300±11 pmol/sec/mg, p<0.05) respiration rates were significantly reduced in placental mitochondria from RUPP (n=7) vs NP (n=8) rats. RUPP placental mitochondria show 35-fold increase in ROS production compared NP mitochondria (p<0.05). HUVECs incubated with RUPP (n=7) serum showed significantly increased ROS vs NP (n=7) serum (9±3 vs 3±1, % gated, p=0.05). Conclusion: Reduced placental mitochondrial respiration and increased mt ROS support the hypothesis that mt dys and mt ROS occurs in response to placental ischemia. Importantly, increased ROS from endothelial cells in response to RUPP serum indicate the importance of circulating factors to cause vascular mt dyst and mt ROS.

Abstract P267: Placental Ischemia Causes Cardiac Structural and Functional Abnormalities in the Reduced Uterine Perfusion Pressure Rat Model

Preeclampsia (PE) is a disorder prevalent in 5-7% of pregnancies. It is characterized by maternal hypertension and endothelial dysfunction. While it has been shown that systolic and diastolic function is impaired in women with PE, the exact mechanisms responsible for the cardiac dysfunction in PE have yet to be fully elucidated. PE is thought to develop in response to placental ischemia and ultimately the release of anti-angiogenic and pro-inflammatory factors into the maternal circulation. However, the impact of placental ischemia on cardiac function is unclear. Therefore the aim of this study was to assess cardiac structure and function in response to placental ischemia utilizing the Reduced Uterine Perfusion Pressure (RUPP) rat model. Briefly in this model, silver clips are placed on the abdominal aorta and branches of the ovarian arteries on gestational day (GD) 14 to induce placental ischemia. For this study, the RUPP ( n =8) group underwent surgery on GD 14, and both normal pregnant (NP, n =8) and RUPP rats had carotid catheters placed on GD 18. Blood pressure and echocardiography measurements, and tissue harvest were performed on GD 19. The RUPP group had significantly increased mean arterial pressure compared to the NP group on GD 19 (123±3 vs. 97±2 mmHg, P<0.01). RUPP rats had lower mean left ventricular ejection fraction (60±2 vs. 78±2 %, P<0.01) and fractional shortening (46±3 vs. 56±1 %, P=0.05), in addition to cardiac hypertrophy (0.97±0.04 vs. 0.91±0.02 g, P=0.02). These data were accompanied by increased cardiomyocyte surface area (348±36 vs. 289±23 μm 2 , P=0.03). In conclusion, this study shows that the RUPP rat develops cardiac structural and functional abnormalities after only five days of placental ischemia. Furthermore, these data suggest that the RUPP model could be useful in investigating and understanding the mechanisms which underpin these cardiac changes in PE patients.

P 38 Proteinase activated receptor 2 (PAR2) on blood pressure and electrolyte handling in the normal pregnant rat

Objectives Healthy pregnancy is characterized by avid aldosterone-mediated renal sodium retention driving a ∼40% plasma volume expansion in the presence of decreased or normal blood pressure. Incredibly, gestational potassium retention occurs in the face of this high circulating aldosterone, decreased renal NCC abundance/ phosphorylation, and increased renal ENaC activity, all of which are potent kaliuretic factors. The mechanisms permitting these opposing adaptations are largely unknown. Research in non-pregnant rats has shown that activation of proteinase activated receptor type 2 (PAR2) can produce vasodilation, inhibit potassium secretion via the renal outer medullary potassium channel (ROMK) in principal cells, and stimulate thiazide sensitive sodium chloride reabsorption (pendrin/NDCBE-mediated) in intercalated cells of the cortical collecting duct. Here, we investigate renal PAR2 activation in virgin and normal pregnant rats. Methods We measured the mRNA expression of PAR2 in the renal cortex, outer medulla, and inner medulla of virgin (V), mid- (MP), and late pregnant (LP) rats using quantitative real-time PCR. We also measured in vivo blood pressure (BP), natriuretic, and kaliuretic responses to 0.1 mg/kg, 0.3 mg/kg, and 1 mg/kg doses of PAR2 agonist peptide (SLIGRL-NH2) in anesthetized V and LP rats. Results We found that PAR2 mRNA was increased in the inner medulla of MP and LP rats. We also found that LP rats had larger decreases in BP compared to baseline than V rats at 0.1 and 0.3 mg/kg doses of agonist peptide. LP rats also had increased net sodium retention compared to virgin rats following 0.3 mg/kg and 1 mg/kg agonist peptide, but decreased net potassium retention compared to virgins with all doses. Conclusions PAR2 mRNA (an index of activity) is increased in the pregnant rat and activation of this receptor causes larger decreases in BP and increases in sodium retention compared to virgin controls. Further studies will determine the mechanism involved as well as the physiological role of increased renal PAR2 in pregnancy.

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.

Increased retinol-free RBP4 contributes to insulin resistance in gestational diabetes mellitus.

Retinol-binding protein 4 (RBP4) is a circulating retinol transporter that is strongly associated with insulin resistance. The aim of this study was to evaluate the RBP4 and retinol level in rat model of gestational diabetes mellitus and the relationship between retinol-free RBP4 (apo-RBP4), retinol-bound RBP4 (holo-RBP4) and insulin resistance. Pregnant rats were administered streptozotocin to induce diabetes. The RBP4 and retinol levels were evaluated in GDM and normal pregnant rats. After then, normal pregnant rats were divided into two groups to receive either apo-RBP4 or vehicle injection. The metabolic parameters and insulin signaling in adipose tissue, skeletal muscle and liver were determined in apo-RBP4 and control groups. Primary human adipocytes were cultured in vitro with different proportions of apo-RBP4 and holo-RBP4 for 24h. The interaction between RBP4 and STRA6 was assessed by co-immunoprecipitation, and the expression of JAK-STAT pathway and insulin signaling were detected by Western blotting and immunofluorescence. We found increases in serum RBP4 levels and the RBP4:retinol ratio but not in the retinol levels in GDM rats. Exogenous apo-RBP4 injection attenuated insulin sensitivity in pregnant rats. In vitro, a prolonged interaction between RBP4 and STRA6 was observed when apo-RBP4 was present. In response to increased apo-RBP4 levels, cells showed elevated activation of the JAK2/STAT5 cascade and SOCS3 expression, decreased phosphorylation of IR and IRS1, and attenuated GLUT4 translocation and glucose uptake upon insulin stimulation. Apo-RBP4 is a ligand that activates the STRA6 signaling cascade, inducing insulin resistance in GDM.

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.

Dysregulation of the Fas/FasL system in an experimental animal model of HELLP syndrome.

Placental FasL is up-regulated in women with HELLP (hemolysis elevated liver enzyme and low platelet) syndrome and has been proposed to contribute to the liver damage seen in these patients. This study aimed to determine if an experimental rodent model of HELLP also had dysregulation of Fas/FasL compared to normal pregnant (NP) rats. We also set out to determine if blockade of the endothelin system regulated Fas/FasL expression in HELLP rats. On gestational day (GD) 12, sEng (7ug/kg) and sFlt-1 (4.7ug/kg) infusion began via mini-osmotic pump into NP rats. On GD19 plasma and tissue were collected and FasL and Fas were measured via enzyme linked immunosorbent assay and gene expression via real-time PCR. HELLP rats had significantly more circulating and placental FasL compared to NP rats, whereas hepatic FasL was decreased and placental Fas was increased compared to NP rats. Administration of an endothelin A receptor antagonist (ETA) beginning on GD12 significantly decreased placental expression of Fas in HELLP rats. Liver mRNA transcript of Fas was significantly increased in HELLP rats compared to NP rats. These data suggest that rats in this experimental model of HELLP syndrome have abnormal expression of the Fas/FasL system. Future studies will examine the sources of Fas/FasL dysregulation in this model and if blockade could reduce some of the inflammation and hypertension associated with HELLP syndrome.

Reduction of Uterine Perfusion Pressure Induced Redistribution of Endothelin Receptor Type-B Between the Intima and Media Contributes to the Pathogenesis of Pregnancy-Induced Hypertension

Studies have shown that a change in endothelin receptor expression in the artery is related to pregnancy-induced hypertension (PIH). However, the mechanism underlying this change remains unclear. To test whether the distribution of endothelin receptor type-A (ETAR) and type-B (ETBR) plays an important role in PIH, a reduction of uterine perfusion pressure (RUPP) rat model was used to mimic some of the features of PIH; the resulting variable endothelin receptor expression was investigated in the media and intima of the aorta. Single vascular smooth muscle cells (VSMCs) were isolated from RUPP and normal pregnant (NP) rats to study the effect of ETAR and ETBR in smooth muscle cells. Compared with NP rats, RUPP rats had a significant redistribution of ETBR expression in the intima and media, while there was no significant difference in ETAR expression between the two groups. ETBR upregulation in VSMCs enhanced cellular contraction and contributed to PIH. The TNF-α plasma levels in RUPP rats were two-fold higher than those of NP rats, which upregulated the expression of ETBR in VSMCS through the NF-κB pathways in RUPP rats. Redistribution of ETBR between the media and intima played an important role in the pathogenesis of PIH.

Vitamin D supplementation reduces some AT1-AA-induced downstream targets implicated in preeclampsia including hypertension.

Autoantibodies to the ANG II type I receptor (AT1-AA) are associated with preeclampsia (PE). We found that vitamin D supplementation reduced AT1-AA and blood pressure (MAP) in the RUPP rat model of PE. However, it was undetermined whether the decrease in AT1-AA was the mechanism whereby vitamin D lowered MAP or if it were through factors downstream of AT1-AA. Uterine artery resistance index, placental ET-1, and soluble FMS-like tyrosine kinase-1 are increased with AT1-AA-induced hypertension and are considered markers of PE in pregnant women. Therefore, we hypothesized that vitamin D would reduce PE factors during AT1-AA-induced hypertension and could lower blood pressure in a model of hypertension during pregnancy without PE features. Either ANG II (50 ng·kg-1·day) or AT1-AA (1:40) was infused from gestational day (GD) 12-19. vitamin D2 (VD2, 270 IU/day) or vitamin D3 (VD3, 15 IU/day) was administered orally from GD14-GD18. MAP (mmHg) increased in AT1-AA (121 ± 4) and ANG II (113 ± 1)-infused pregnant rats compared with normal pregnant rats (NP) (101 ± 2) but was lower in AT1-AA+VD2 (105 ± 2), AT1-AA+VD3 (109 ± 2), ANG II+VD2 (104 ± 4), and ANG II+VD3 (104 ± 3). VD2 and/or VD3 improved PE features associated with AT1-AA during pregnancy, while ANG II did not induce such features, supporting the hypothesis that AT1-AA induces PE features during pregnancy, and these are improved with vitamin D. In this study, we demonstrate that vitamin D improved many factors associated with PE and reduced blood pressure in a hypertensive model without PE features, indicating that vitamin D could be beneficial for various hypertensive disorders of pregnancy.

Vitamin D restores angiogenic balance and decreases tumor necrosis factor-α in a rat model of pre-eclampsia.

Deficiency of vitamin D is correlated with pre-eclampsia (PE), a hypertensive disorder of pregnancy, and is characterized by angiogenic imbalance and inflammation. The aim of this study was to investigate whether vitamin D supplementation can restore the angiogenic balance and ameliorate inflammation in a rat model of PE. PE was induced using l-nitroarginine methylester. Normal pregnant and PE-induced rats were supplemented with vitamin D on gestation days 14-19. Blood pressure was significantly increased in PE-induced rats compared with normal pregnant rats (P < 0.05), and vitamin D supplementation ameliorated this difference. In addition, rats from the PE group had lower vascular endothelial growth factor (VEGF; P < 0.01), and higher plasma-soluble FMS-like tyrosine kinase-1 (sFlt-1) and tumor necrosis factor-α (TNF-α; P < 0.01 for both) compared with the normal pregnant group. The vitamin D treatment group had significantly increased VEGF, and reduced sFlt-1 and TNF-α compared with the untreated PE group. Moreover, vitamin D supplementation was able to reduce the oxidative stress by lowering the plasma oxidative stress marker malondialdehyde. Vitamin D supplementation plays an important role in restoring angiogenic balance and reducing inflammation in pregnancy-induced hypertension.

Uterine focal adhesions are retained at implantation after rat ovarian hyperstimulation.

Controlled ovarian hyperstimulation is an essential component of IVF techniques to ensure proliferation and development of multiple ovarian follicles, but the effects of these hormones on the endometrium are largely unknown. During normal pregnancy in rats, there are significant changes in the basal plasma membrane of uterine epithelial cells (UECs) at the time of receptivity, including loss of focal adhesions. This enables the UECs to be removed from the implantation chamber surrounding the blastocyst, thus allowing invasion into the underlying stroma. This study investigated the influence of ovarian hyperstimulation (OH) on the basal plasma membrane of UECs during early pregnancy in the rat. Immunofluorescence results demonstrate the presence of paxillin, talin, integrin β1 and phosphorylated FAK (Y397FAK) in the basal portion of UECs at the time of implantation in OH pregnancy. TEM analysis demonstrated a flattened basal lamina and the presence of focal adhesions on the basal surface at this time in OH pregnancy. Significantly low full-length paxillin, high paxillin δ and integrin β1 were seen at the time of implantation in OH compared with those in normal pregnancy. The increase in paxillin δ suggests that these cells are less mobile, whereas the increase in integrin β1 and Y397FAK suggests the retention of a stable FA complex. Taken together with the increase in morphological focal adhesions, this represents a cell type that is stable and less easily removed for blastocyst implantation. This may be one mechanism explaining lower implantation rates after fresh embryo transfers compared with frozen cycles.

Abstract 031: Placental Ischemia Causes Mitochondrial Dysfunction in Reduced Uterine Perfusion Pressure Rats

Introduction: Placental ischemia is believed to be the initial event in the development of preeclampsia (PE). PE is characterized by new onset hypertension and is associated with reduced fetal weight and placental oxidative stress. Mitochondrial dysfunction is the major cause of reactive oxygen species (ROS) generation. We hypothesize that the placental ischemia causes mitochondrial dysfunction in the reduced uterine perfusion pressure (RUPP) rat model of PE. Thus, the purpose of this study is to examine the effect of placental ischemia on mitochondrial function. Methods: Female Sprague Dawley rats were dived into two groups; normal pregnant (NP) and RUPP rats. On gestational day (GD) 14, RUPP surgery was performed, GD18 carotid catheters were inserted, and GD19 conscious blood pressure (MAP) was measured. GD 19 placentas were collected and mitochondria were isolated for respiration measurements. Respiration measurements included: basal state (isolated mitochondria with no substrates), state 2 (glutamate and malate as complex I substrates), state 3 (ADP stimulated), leak state (oligomycin induced ATP synthase inhibition), and maximal state (FCCP stimulated uncoupled). Cytochrome C oxidase (complex IV) activity was also measured in isolated mitochondria. Results: MAP was elevated in RUPP (n=16) compared to NP rats (n=14) (119±2 vs. 100±2 mmHg, p&lt;0.05). State 3 (149±8 vs 220±12 pmol/sec/mg, p&lt;0.05) and maximal (123±8 vs 159±5 pmol/sec/mg, p&lt;0.05) respiration rates were significantly reduced in RUPP (n=5) vs NP (n=7) rats. However, basal, state 2, and state 4 respiratory rates were not different between RUPP vs NP. Respiratory control ratio (state 3/ state 4) was significantly reduced in RUPP (n=5) vs NP (n=7) (7±1 vs 10±1, p&lt;0.05). Complex IV activity was reduced in RUPP (n=5) vs. NP (n=6) rats (508±14 vs 644±17 nmol e-/min/mg, P&lt;0.05). Conclusion: Reduced mitochondrial respiration and cytochrome c oxidase activity, indicators of mitochondrial dysfunction, occur in response to placental ischemia during pregnancy. Thus, suggesting that mitochondrial dysfunction may contribute to the pathophysiology of oxidative stress and hypertension during PE.

Abstract P174: 17-hydroxyprogesterone Caproate Improves Blood Pressures and Placental Cytolytic Nk Cells in Response to Placental Ischemia During Pregnancy

Preeclampsia (PE), new onset hypertension, is characterized by decreased fetal weight, elevated cytolytic natural killer (NK) cells and placental ischemia during pregnancy. Cytolytic NK are thought to play a role in fetal demise as they have also been shown to be increased in patients suffering from miscarriage.Currently, there is no effective treatment for PE except for early delivery, making PE the leading cause for premature births worldwide. Multiple injections of 17-hydroxyprogesterone caproate (17-OHPC) is used for prevention of preterm labor, but not for management of PE. We have shown that injections of 17-OHPC to the RUPP rat model of PE improves some but not all facets of PE observed in this model. Therefore this study was designed to test the hypothesis that injections of 17-OHPC on both day 15 (GD15) and GD (18) improve outcomes of hypertension in response to placental ischemia. To do so, 17-OHPC (3.32mg/kg) was administered intraperitoneally on GD 15 and 18 to reduced uterine perfusion pressure (RUPP) rats, carotid catheters were inserted on GD 18 and blood pressure (MAP) andplacental cytolytic NK cells were measured on GD 19. MAP in normal pregnant (NP) rats (n=8) was 104±4,119± 5 in RUPP rats (n=5) and 102±5 mmHgin RUPP+17-OHPC GD15 &amp;18 (n=4), p &lt;0.05.Total number of placental NK cells was 8.5± 3 in NP, 20±2 in RUPP rats, which decreased to 4.7± 3 % in RUPP+17-OHPC GD15 &amp;18, p&lt;0.05. Activated placental NK cells was 3.4± 1.6 in NP, 10.5±2.3 in RUPP, which improved to 2.7± 2.7 % in RUPP+17-OHPCGD15 &amp; 18, p &lt;0.05.In conclusion, administration of 17-OHPC on days 15 and 18 decreased hypertension and NK cells that are associated with PE in the RUPP ratand should be considered for addition to the management of PE.

Decreased Uterine Vascularization and Uterine Arteriolar Expansive Remodeling by Matrix Metalloproteinases in Hypertensive Pregnancy

Normal pregnancy is associated with extensive remodeling of spiral arteries in order to maintain nutrients and blood supply to the growing fetus. Inadequate placentation and invasion of the uterine wall could lead to uteroplacental ischemia and hypertensive pregnancy (HTN‐Preg); however, the extent of uterine vascularization and the mechanisms involved are unclear. We hypothesized that HTN‐Preg is associated with changes in uterine vascularization and arterial remodeling by matrix metalloproteinases (MMPs). Blood pressure (BP) and fetal parameters were assessed on day‐19 in normal pregnant rats (Preg) and pregnant rats with reduced uterine perfusion pressure (RUPP), and the uteri were isolated for measurement of tissue vascularity and MMPs expression/activity. BP was greater and pups number and weight, and uterus weight were reduced in RUPP vs. Preg rats (BP 128.2±1.7 vs. 91.60±2.8mmHg; pups number 8.8±1.1 vs. 13.00±0.7; pup weight 1.48±0.059 vs. 2.02±0.02g; uterus weight 3.82±0.37 vs. 4.92±0.27g). Histological analysis of uterine tissue sections showed reduced total number and whole area of uterine arterioles in RUPP vs. Preg rats (number 5.75±0.95 vs. 11.50±0.87; area 0.05±0.01 vs. 0.12±0.02mm2). Immunohistochemistry with the endothelial cell marker CD31 showed localization in the uterine arterial intima and confirmed reduction in the number and size of uterine arterioles in RUPP vs. Preg rats. Further analysis of the uterine arterial wall showed reduction in the area of all three layers of the uterine arterial wall in RUPP vs. Preg rats (intima 0.015±0.003 vs. 0.031±0.005; media 0.012±0.002 vs. 0.038±0.005; adventitia 0.021±0.003 vs. 0.048±0.007mm2). Western blots and gelatin zymography showed a decrease in expression and activity of MMP‐2 and ‐9 in uterus and uterine arteries of RUPP vs. Preg rats. Immunohistochemistry showed decreased total amount of MMP‐2 and ‐9 in the uterus, and reduction in MMP distribution in the intima and media of uterine arteries of RUPP vs. Preg rats. Thus, in HTN‐Preg rats there is reduction in the number and size of uterine arteries and decreased expression and activity of MMP‐2 and ‐9. These results suggest that reduced uterine vascularization and uterine arteriolar expansive remodeling by MMPs could be a factor in the uteroplacental ischemia associated with HTN‐Preg and preeclampsia.Support or Funding InformationThis work was supported by grants from National Heart, Lung, and Blood Institute (HL‐65998,HL‐111775).