Reduced Uterine Perfusion Pressure Research Articles

Abstract 029: Hypertensive Disorder Of Pregnancy Results In Loss Of Pancreatic Beta Cell Area In Female Rat Offspring Without Development Of Glucose Intolerance

Hypertensive disorders of pregnancy can result in increased risk for type 2 diabetes (T2D) in offspring. Reduced pancreatic beta cell area can also influence development of glucose intolerance or T2D. Using a rat model of reduced uterine perfusion pressure (RUPP) which leads to hypertension during pregnancy and intrauterine growth restriction, we previously demonstrated reduced beta cell area in female RUPP offspring vs Sham at embryonic day 19 persisting to postnatal day (PD)13. To determine if this alteration in beta cell area is associated with phenotypic changes in glucose handling as offspring mature, we assessed IPGTT (intraperitoneal glucose tolerance test), OGTT (oral glucose tolerance test) and ITT (insulin tolerance test) in PD14, PD28, 8 week and 6 month female offspring (n=5-11/group). Our results demonstrate no change in fasting blood glucose and no alteration in glucose handling with IP glucose administration at any age. However, peak glucose following oral glucose administration was significantly less in RUPP offspring at PD14 (196.3+/-8.6 mg/dl, RUPP; 218.3+/-9.5 mg/dl, Sham; p=0.05), PD28 (220.0+/-4.1 mg/dl, RUPP; 280.0+/-12.8 mg/dl, Sham; p<0.05) and 6 months (139.6+/-3.8 mg/dl, RUPP; 150.9+/-4.5 mg/dl, Sham; p<0.05) compared to Sham, suggesting the incretin effect on pancreas and non-pancreas tissues is more effective in controlling blood glucose in RUPP offspring vs Sham. In addition, the decrease in glucose level in response to insulin injection in ITT was significantly greater in RUPP offspring vs Sham at PD28 (58.9+/-5.0 mg/dl, RUPP; 78.3+/-8.9 mg/dl, Sham; p<0.05) suggesting increased insulin sensitivity in non-pancreatic tissues in RUPP compared to Sham offspring. Our studies demonstrate female offspring of hypertensive pregnancies are not glucose intolerant by 6 months of age. Despite reduced pancreatic beta cell area in female RUPP offspring early in development, glucose handling was maintained and even improved after oral glucose challenge in older RUPP offspring. Overall, these data suggest that increased peripheral insulin sensitivity may reduce the demand for beta cell function, and our studies reinforce the idea that adverse pregnancy can have a lasting effect on development and function of the pancreas.

Abstract P426: Novel Peptide B7-33 Demonstrates Efficacy In Rat Models Of Preeclampsia

Background: Preeclampsia (PreE) is a hypertensive pregnancy disorder, which occurs in approximately 10% of all gestations. The literature suggests potential therapeutic role of H2 relaxin in PreE. Due to the complex insulin-like structure of relaxin (A- and B- chains, 53 amino acids, 3 disulfide bonds), a novel H2 relaxin B-chain-only peptide variant B7-33 (27 amino acids without any disulfide bonds) has recently been developed. Objective: This single-chain peptide displayed equivalent efficacy to the natural H2 relaxin in several functional assays both in vitro and in vivo . The Aim of this study is to evaluate whether B7-33 attenuates preE syndrome in rat models of PreE. Methods: Rat Model 1. The efficacy of B7-33 was evaluated in the Reduction of Uterine Perfusion Pressure (RUPP) model as described previously. RUPP rats are randomly assigned to 4 groups (N=8/group): 1) vehicle, 2) B733; 3) B733-Fc; and 4) B733-HSA. Rats are dosed twice weekly (i.v.) from GD10 to GD 20. Rat Model 2. Timed-pregnant rats are used, and the model is conducted as previously described. DOCA and saline administration begin at approximately GD2. Three groups of animals are studied (n=8 per group). 1) normal pregnant rats; 2) pregnant animals injected (i.p.) initially with 12.5 mg of DOCA, followed by a weekly injection of 6.5 mg, and whose drinking water was replaced with 0.9% saline; 3) rats administered DOCA and saline as for Group 2, and given B733 35 ug i.p. biweekly from GD10-20. BP, proteinuria and inflammatory markers were evaluated. Statistical comparisons were performed using analysis of variance with Duncan’s post hoc test. Results: RUPP rats have increased MAP, plasma TNF-α, and plasma sFlt-1 along with decreased NO index compared to normal pregnancy. Treatment with B733 and B7-33 fusion proteins lowers MAP, TNF-α, and sFlt-1 back to that of normal pregnancy. B7-33 data are consistent with earlier data with serelaxin in the RUPP model. All fusions tested ameliorate hypertension in the RUPP animals. B7-33 normalizes BP and proteinuria in the DOCA rat model of preE. Conclusion: Both B7-33 and B7-33 fusion proteins attenuate preE syndrome in RUPP and DOCA rat models of PreE. We conclude that B7-33 is an ideal candidate for development as a novel therapeutic in preE.

Abstract P205: G Protein-Coupled Estrogen Receptor Stimulation Attenuates Hypertension And Cardiac Dysfunction In The Reduced Uterine Perfusion Pressure Model Of Preeclampsia

Preeclampsia (PE) is a disorder of pregnancy associated with maternal hypertension and cardiac dysfunction. Women with a history of PE have increased risk of cardiovascular disease and mortality that persist well beyond the post-partum period. Evidence suggests that the new estrogen receptor (GPER) is a beneficial regulator of placental development. Other studies have shown the role of GPER in promoting vasodilation and cardioprotection. However, the cardiovascular effects of GPER in an animal model of PE have not yet been determined. Thus, we hypothesize that the activation of GPER by its selective agonist G-1 is associated with the regulation of hypertension and cardiac dysfunction in rats following placental insufficiency. The reduced uterine perfusion pressure (RUPP) model was used to induce placental insufficiency in the dam by placing silver clips on abdominal aorta and uterine arteries on gestational day (GD) 14 of a 19-day gestation. RUPP rats received G-1 (100 μg/kg/day) or vehicle subcutaneously by osmotic minipump. Echocardiography was performed on GD18 to assess the effectiveness of G-1 on heart function in PE dams. On GD19, rats had carotid catheters placed, and hemodynamic measurements were made. RUPP rats exhibited hypertension and cardiac alterations, including global myocardial motion dyssynchrony, and reduced left ventricular systolic and diastolic function when compared with SHAM animals. G-1 treatment significantly attenuated hypertension and cardiac functional aberrations caused by PE in RUPP rats, a finding that may have important implications for the ongoing clinical evaluations of new drugs for the treatment of PE and its comorbidities.

OP07_4. Aspirin treatment improves reduced uterine perfusion pressure (RUPP)-induced cardiac dysfunction in pregnant rats

OP07_4. Aspirin treatment improves reduced uterine perfusion pressure (RUPP)-induced cardiac dysfunction in pregnant rats

Abstract P420: Changes In Cardiac Oxidative Stress Is Protected In Intrauterine Growth Restricted Females Vs Males Before The Development Of Hypertension And Cardiovascular Diseases

Preeclampsia (PE) is a pregnancy complication characterized by hypertension (HTN) typically during the third trimester. PE is the leading cause for intrauterine growth restricted (IUGR) offspring (OFS). Women with PE and their OFS (IUGR) are at a higher risk for developing cardiovascular diseases (CVD) later in life. Two major factors that contribute to the development of CVDs are HTN and oxidative stress (OS). Studies show that human and animals adult IUGR OFS have a higher risk of developing HTN and CVDs compared to females later in life. The rationale for these sex differences is unknown; and we hypothesize that male IUGR rats will have elevated cardiac oxidative stress, while female IUGR will have lower cardiac OS .To test, we will examine sex differences between 12 week IUGR and control (CON) OFS that are not hypertensive, to determine if there are any change in cardiac OS before the development of HTN and CVD. Methods: Sprague Dawley rats were divided into 4 groups: IUGR male, IUGR female, and control (CON) male and female. IUGR rats were derived from the OFS reduced uterine perfusion pressure (RUPP) PE rat model. CON OFS were from normal pregnant rats. At 12wk, BP was measured via carotid catheterization. Heart tissue was collected to assess oxidative stress, via examining reactive oxygen species (ROS) Heat shock protein 1 (HSP-1) and hydrogen peroxide (H2O2). Antioxidants were measured via manganese superoxide dismutase (MnSOD), copper zinc superoxide dismutase (CuZnSOD) and antioxidant capacity (AC). There were no significant changes in HSP-1, CuZnSOD, and H2O2 between male and female OFS. However, female IUGR OFS had a 20% increase in antioxidant MnSOD (119.9 ± 6.133 vs 93.98 ± 5.197 %MnSOD U/protein/CON, p <0.05) compared to CON in the heart. Male OFS had no significant changes for MnSOD. IUGR female AC had a 2-fold increase in activity (22.13 ± 1.019 vs 14.15 ± 1.852 mM Trolox/mg protein, p <0.05) compared to its CON. Furthermore, IUGR male OFS had a slight decrease in AC compared to CON male (11.00 ± 1.420 vs 13.24 ± 0.4676 mM Trolox/mg protein, ns). The significant increase of AC and MnSOD in IUGR female OFS suggests that females may be more protected from OS, via mitochondria-specific antioxidants compared to males.

Abstract P202: Changes In Cerebral Mitochondrial Function In Intrauterine Growth-Restricted Offspring Before The Development Of Hypertension And Cerebrovascular Dysfunction

Background: Preeclampsia (PE), new-onset gestational hypertension (HTN) affects 3-8% of all births in the USA. PE is the leading cause for intrauterine growth restriction (IUGR). IUGR children have increased risks of HTN and cerebrovascular disease (CVD) later in life. Prior studies in our lab show adult male IUGR rat offspring (OS) to develop HTN, CVD, and cerebral mitochondrial dysfunction (mtDys) at 17 weeks while female IUGR OS exhibit CVD. The role of mtDys in HTN is unknown and is the focus of our study. We hypothesize that IUGR OS will have mtDys prior to HTN. Methods: Our surgical PE animal model utilized the reduced-uterine-perfusion pressure (RUPP) surgery at GD 14. OS from NP served as controls (CON) while OS from RUPP mothers were IUGR. At 12 weeks, OS BP was measured alongside cerebral mtDys through mitochondrial fusion and fission, electron-transport chain (ETC) proteins, and respiration. Results: BP did not change between sexes or among IUGR and CON offspring. Male IUGR OS showed elevated mtRes (State 2: 199 ± 1.08 v. 76 ± 1.13 pmol/mg, p < .05; State 3: 555 ± 3.02 v. 245 ± 1.77 pmol/mg, p < .05; State 4: 346 ± 1.66 v. 85 ± 1.12 pmol/mg, p < .05). Female IUGR and CON OS had no changes in BP or mtRes. Male IUGR OS had increased mito-fusion proteins MFN-1 (176 ± 1.68 v. 100 ± .57 IU/protein/CON %, p < .05), MFN-2 (155 ± 1.36 v. 100 ± .74 IU/protein/CON %, p < .05) and OPA-1 (166 ± 1.99 v. 100 ± .73 IU/protein/CON %, p < .05). DRP-1 had no changes in males; females had no changes in fusion or fission. Male IUGR OS had changes in ETC proteins (CI: 8 ± 9.34 v. 100 ± .82 IU/protein/CON %, p < .05; CII: 23 ± .53 v. 100 ± .52 IU/protein/CON %, p < .05; CIII: 26 ± .23 v. 100 ± .41 IU/protein/CON %, p < .05), as did female IUGR OS (CI: 12 ± .24 v. 100 ± .86 IU/protein/CON %, p < .05; CII: 29 ± .39 v. 100 ± .61 IU/protein/CON %, p < .05; CIII: 39 ± .56 v. 100 ± .62 IU/protein/CON %, p < .05; CIV: 76 ± .74 v. 100 ± .73 IU/protein/CON %, p < .05) vs. CON OS. Summary: Male IUGR OS have elevated mtRes, changed ETC proteins, and fusion proteins that may lead to mtDys and HTN development. This study is clinically relevant because specific cerebral therapy for mtDys for IUGR OS may prevent HTN and CVD development. Future studies will examine changes in mtDys causing male HTN and protection from HTN in females.

RUPP Th17s cause hypertension and mitochondrial dysfunction in the kidney and placenta during pregnancy.

Preeclampsia (PE), new-onset hypertension (HTN), and organ dysfunction during the second half of pregnancy, is associated with an increase in inflammatory immune cells, including T helper 17 (Th17) cells. Studies have demonstrated that mitochondrial (mt) dysfunction is important in the pathogenesis of PE though causative factors have yet to be fully identified. Although Th17 cells, natural killer (NK) cells, and mt dysfunction contribute to HTN in the reduced uterine perfusion pressure (RUPP) rat model, the role of Th17 cells or IL-17 in mt dysfunction is unknown. Therefore, we hypothesize that RUPP stimulated Th17 cells cause HTN and mt dysfunction, which is alleviated with the blockade of IL-17. On gestational day 12 (GD12), RUPP Th17 cells were transferred into normal pregnant (NP) Sprague Dawley rats. A subset of NP+RUPPTh17 rats received IL-17RC (100pg/day) on GD14-19. Blood pressure (MAP), NK cells, and mt function were measured on GD19 in all groups. MAP increased in response to NP+RUPP Th17 compared to NP rats and was lowered with IL-17RC. Circulating and placental NK cells increased with NP+RUPP Th17 compared to NP and were lowered with IL-17RC. Renal mtROS increased in NP+RUPP Th17 compared to NP and was normalized with IL-17RC. Similar to PE women, placental mtROS decreased in NP+RUPP Th17 and was normalized with IL-17RC. Our results indicate that IL-17RC inhibition normalizes HTN, NK cell activation, and multi-organ mt dysfunction caused by Th17 cells stimulated in response to placental ischemia.

Endothelin-1 production via placental (pro)renin receptor in a mouse model of preeclampsia

IntroductionPreeclampsia (PE) pathogenesis is explained by the two-stage disorder theory. However, mechanisms underlying hypertension and proteinuria in PE remain unclear. The role of (pro)renin receptor (PRR) in PE pathology has received special attention. We examined endothelin-1 (ET-1) production via placental PRR in a PE mouse model. MethodsAt 14.5 day-post-coitum (DPC), we performed a reduced uterine perfusion pressure (RUPP) operation, ligating the uterine arteriovenous vessels in female mice. We also infused these mice with a PRR inhibitor, decoy peptide in the handle region of prorenin (HRP) for mice (NH2-RIPLKKMPSV-COOH). At 18.5 DPC, blood, urine, and placenta were collected; fetus and placenta were weighed. We evaluated placental hypoxia using quantitative polymerase chain reaction (PCR), with hypoxia-inducible factor-1α (HIF-1α) as index. We also evaluated PRR, transforming growth factor-β1 (TGF-β1), and ET-1 expression in the placenta using quantitative PCR and western blotting. ET-1 concentration in blood plasma was assessed using enzyme-linked immunosorbent assay. ResultsBlood pressure and proteinuria significantly increased, and fetal and placental weights decreased in RUPP mice. HIF-1α, PRR, TGF-β1, and ET-1 expressions considerably increased in RUPP mice placentas. ET-1 concentration in RUPP mice blood plasma was markedly increased. PRR inhibitor suppressed these changes. DiscussionIn PE model mice that underwent RUPP treatment, placental hypoxia increased PRR and ET-1 expression suggesting a causative relationship between ET-1 and intracellular PRR signaling. RUPP treatment, when combined with HRP, reversed the effect of elevated ET-1 levels in the model. This study may help to elucidate the pathogenesis of PE considering PRR and ET-1.

Inhibition of caspase 1 reduces blood pressure, cytotoxic NK cells, and inflammatory TH17 cells in placental ischemic rats

Preeclampsia (PE) is characterized by maternal hypertension, fetal growth restriction (FGR), and increased inflammation and immune activation, including cytotoxic NK cells (cNKs) and inflammatory T-Helper17 cells (T H 17s). Caspase 1 activity directly leads to release of the inflammatory cytokines IL1β and IL18. Using the reduced uterine perfusion pressure (RUPP) rat model of placental ischemia, we tested the hypothesis that inhibition of caspase 1 with Belnacasan in RUPP rats will attenuate PE pathophysiology. On gestation day (GD) 14, timed pregnant 10–12-week-old Sprague Dawley rats underwent the RUPP or Sham procedure and received either vehicle or Belnacasan (50mg/kg) daily i.p. from GD14 through GD19: RUPP + vehicle, RUPP + Belnacasan, Sham + vehicle, Sham + Belnacasan. Carotid catheters were placed on GD18. On GD19, MAP was measured, animals were euthanized, and blood and tissues were collected for analysis. Bioplex and flow cytometry analysis were performed on placental tissues. Placental IL1β was markedly increased in RUPP rats vs. Sham rats (44.2 vs. 21.67 pg/mg, p=0.0009). Belnacasan treatment significantly reduced IL1β in RUPP rats (21.90 pg/mg; p=0.0006 vs RUPP). Likewise, placental IL18 was increased in RUPP rats vs Sham (27.1 vs 6.7 pg/mg, p=0.007), and inhibition of caspase 1 reduced RUPP IL18 (6.2, p=0.01 vs RUPP) to Sham levels. No significant differences in IL1β or IL18 were observed in treated Sham rats. These data show the administered Belnacasan dose is effective against Caspase 1 activity. Caspase 1 inhibition reduced placental cNKs in treated-RUPP vs control RUPP (0.155% vs. 0.362 % gated; p=0.043). Belnacasan treatment also reduced placental T H 17s in RUPP rats vs. control RUPPs (1.053% vs 7.25% gated; p=0.003). Increased MAP was observed in RUPP rats vs Sham (131.5 mmHg vs 104.8 mmHg; p<0.0001) and was reduced in RUPP + Belnacasan (117.5 mmHg; p=0.0016 vs RUPP). No changes were observed in MAP of Sham rats treated with Belnacasan. Placental reactive oxygen species (ROS) were elevated in RUPP vs Sham (456.4 vs 198.8 RLU/min/mg, p=0.029). Caspase 1 inhibition significantly reduced ROS in treated RUPP rats (210.3, p=0.031) vs RUPP), and had no effect on ROS in treated Sham rats (156.3). Caspase 1 inhibition had no effect on fetal weight or placental efficiency in any groups. Additional analyses will include assessment of other cytokines in blood and tissue, as well as vascular function in all groups. In conclusion, caspase 1 inhibition reduces placental T H 17 and cNK populations and reduces MAP in RUPP rats. These data suggest that caspase 1 is a key contributor to PE pathophysiology and may represent a therapeutic target to improve maternal outcomes in PE. This work was supported by National Institutes of Health Grants T32HL105324 to C. A. Shields, F31HL165852 to Xi Wang, and R01HL151407 to D. C. Cornelius. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Reduced uterine perfusion increases hippocampal cannabinoid receptor 1 expression and reduces pentylenetetrazol-induced seizures after rimonabant pre-treatment

Eclampsia, new-onset seizures in pregnant women, can affect women with preeclampsia, a hypertensive pregnancy disorder. Eclampsia is associated with high maternal and fetal morbidity and mortality. The mechanisms contributing to increased risk of seizures in preeclampsia patients are not fully known. One potential mechanism could be abnormal endocannabinoid system activity, and abnormal neuro-modulation. Indeed, increased placental cannabinoid receptor 1 (CB1R) expression and reduced serum concentration of anandamide, a ligand that activates the CB1R, have been reported in preeclampsia patients. We hypothesized that reduced uterine perfusion pressure (RUPP) in mice leads to differences in hippocampal CB1R expression and that inhibiting CB1R activity will increase seizure sensitivity in RUPP mice. Pregnant mice underwent sham (n=5) or RUPP (n=5) surgery on gestational day (GD) 13.5. On GD 18.5, the hippocampus was harvested and processed for Western blot to analyze the expression of CB1R in homogenates, synaptosomal fraction, and cytosolic fraction of the hippocampus. To determine if blocking CB1R increases sensitivity to pentylenetetrazol (PTZ)-induced seizures, additional sham (n=7) and RUPP (n=7) mice were pretreated with 10 mg/kg of rimonabant, video monitored for 15 minutes, injected with 40 mg/kg of PTZ and video monitored for an additional 30 minutes before being sacrificed. Seizures were scored using a modified 7-point Racine scale. In hippocampal homogenates, CB1R expression increased in RUPP mice (p=0.056). There was no difference in CB1R expression in the synaptosomal fraction (p=0.095); however, there was an increase in CB1R expression in the cytosolic fraction of RUPP mice (p=0.016). Immunofluorescence analysis showed increased CB1R colocalization on both glutamate- and GABA- releasing neurons within hippocampal CA1 (p=0.029; p=0.057, respectively). CB1R blockade induced a maximum seizure score of 4 in both groups but led to higher seizure scores over time in RUPP mice (p=0.025). PTZ injection after rimonabant pretreatment increased seizure scores in sham (p<0.05), non-significantly increased scores in RUPP (p=0.075), increased duration of seizures in sham but not RUPP mice (p=0.021; p=0.245, respectively), and reduced latency to seizures in sham mice (p=0.019) while there was no further decrease in seizure latency in RUPP (p=0.374) mice. Furthermore, during the first 15 minutes of PTZ exposure, RUPP mice had lower seizure scores over time than sham [p(TimexRUPP)=0.002] following CB1R blockade. These data suggest that RUPP induces abnormal CB1R expression, resulting in increased sensitivity to seizures after CB1R blockade, while protecting against worse seizures when challenged with the pro-convulsant, PTZ. NIH R00HL129192, R00HL129192-S1, COBRE Pilot Grant, T32HL105324, William Townsend Porter Pre-Doctoral Fellowship from the American Physiological Society, 1F99NS129125-01. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Placental ischemia disrupts DNA methylation patterns in distal regulatory regions in rats

Preeclampsia (PE) is a leading cause of maternal and fetal morbidity and mortality worldwide. However, the impact of PE on the organization of the functional architecture of the placental methylome remains largely unknown. We performed whole-genome bisulfite sequencing of placental DNA and applied a Hidden Markov Model to investigate epigenome-wide alterations in functional structures, including partially methylated domains (PMDs), low-methylated regions (LMRs), and unmethylated regions (UMRs), in a reduced uterine perfusion pressure (RUPP) rat model of PE. The remarkable similarity we observed between the rat and human placental DNA methylomes suggests that the RUPP rat model is appropriate to elucidate the epigenetic mechanisms underlying human PE. The notable changes in PMDs indicate RUPP-induced perturbation of the stressed placental methylome. This was probably regulated via modulation of the epigenetic modifier expression, including significant downregulation of Dnmt1 and Dnmt3a and upregulation of Tet2. More importantly, changes in RUPP-induced DNA methylation occurred predominately in LMRs (80 %), which represent active enhancers, rather than in canonical UMRs (3 %), which represent promoters, suggesting that placental ischemia disrupts enhancer DNA methylation. Our findings emphasize the role of enhancer methylation in response to PE, corroborating discoveries in human PE studies. We suggest paying more attention to enhancer regions in future studies on PE.

Inhibition of angiotensin II type 1 receptor agonistic autoantibodies by direct binding does not impact reduced uterine perfusion pressure offspring birthweight and blood pressure at adulthood

Preeclampsia, a new-onset hypertension with end-organ damage in pregnancy, is associated with maternal death and morbidity, low birthweight, and B cells producing agonistic autoantibodies to the angiotensin II type 1 receptor. Angiotensin II type 1 receptor agonistic autoantibodies are produced during pregnancy and after delivery and are in the fetal circulation of women with preeclampsia. Angiotensin II type 1 receptor agonistic autoantibodies are shown to contribute to endothelial dysfunction, renal dysfunction, hypertension, fetal growth restriction, and chronic inflammation in women with preeclampsia. The reduced uterine perfusion pressure rat model of preeclampsia exhibits these features. In addition, we have shown that the administration of a 'n7AAc', which blocks the actions of the angiotensin II type 1 receptor autoantibodies, improves preeclamptic features in the rat with reduced uterine perfusion pressure. However, the effect of a 'n7AAc' on the long-term health of the offspring of rats with reduced uterine perfusion pressure is unknown. This study aimed to test the hypothesis that inhibition of angiotensin II type 1 receptor autoantibodies during pregnancy will improve offspring birthweight and prevent increased cardiovascular risk in offspring in adulthood. To test our hypothesis, a 'n7AAc' (24 µg/d) or vehicle (saline) was given on gestation day 14 via miniosmotic pumps to sham-operated (sham) and Sprague-Dawley rat dams with reduced uterine perfusion pressure. Dams were allowed to deliver naturally, and pup weights were recorded within 12 hours after birth. Pups were aged to 16 weeks, at which time mean arterial pressure was measured and whole blood was collected to measure immune cells by flow cytometry, cytokines by enzyme-linked immunosorbent assay, and angiotensin II type 1 receptor autoantibodies by bioassay. A 2-way analysis of variance with the Bonferroni multiple comparison posthoc test was used for statistical analysis. There was no significant change in offspring birthweight of 'n7AAc'-treated male (5.63±0.09 g) or female (5.66±0.14 g) offspring from reduced uterine perfusion pressure dams compared with vehicle male (5.51±0.17 g) or female (5.74±0.13 g) offspring from reduced uterine perfusion pressure dams. In addition, 'n7AAc' treatment did not affect the birthweight of sham male (5.83±0.11 g) or female (5.64±0.12) offspring compared with vehicle sham male (5.811±0.15 g) or female (5.40±0.24 g) offspring. At adulthood, mean arterial pressure was unchanged in 'n7AAc' treated-male (133±2 mm Hg) and female (127±3 mm Hg) offspring from reduced uterine perfusion pressure dams compared with vehicle male (142±3 mm Hg) and female (133±5 mm Hg) offspring from reduced uterine perfusion pressure dams, the 'n7AAc'-treated sham male (133±3 mm Hg) and female (135±3 mm Hg) offspring, and vehicle sham male (138±4 mm Hg) and female (130±5 mm Hg) offspring. The circulating angiotensin II type 1 receptor autoantibodies were increased in vehicle male (10±2 ΔBPM) and female (14±2 ΔBPM) offspring from reduced uterine perfusion pressure dams and 'n7AAc'-treated male (11±2 ΔBPM) and female (11±2 ΔBPM) offspring from reduced uterine perfusion pressure dams compared with vehicle sham male (1±1 ΔBPM) and female (-1±1 ΔBPM) offspring and 'n7AAc'-treated sham male (-2±2 ΔBPM) and female (-2±2 ΔBPM) offspring. Our findings indicated that perinatal 7-amino acid sequence peptide treatment does not negatively impact offspring survival or weight at birth. Perinatal 'n7AAc' treatment did not prevent increased cardiovascular risk in offspring, but it also did not cause an increased cardiovascular risk in offspring with reduced uterine perfusion pressure compared with controls. Furthermore, perinatal 'n7AAc' treatment did not affect endogenous immunologic programming as observed by no change in circulating angiotensin II type 1 receptor autoantibodies in either sex of adult offspring from reduced uterine perfusion pressure dams.

Specific HIF-2α (Hypoxia-Inducible Factor-2) Inhibitor PT2385 Mitigates Placental Dysfunction In Vitro and in a Rat Model of Preeclampsia (RUPP).

Preeclampsia is one of the leading causes of maternal mortality worldwide and is strongly associated with long-term morbidity in mothers and newborns. Referred to as one of the deep placentation disorders, insufficient remodeling of the spiral arteries during the first trimester remains a major cause of placental dysfunction. Persisting pulsatile uterine blood flow causes abnormal ischemia/reoxygenation phenomenon in the placenta and stabilizes the HIF-2α (hypoxia-inducible factor-2α) in the cytotrophoblasts. HIF-2α signaling impairs trophoblast differentiation and increases sFLT-1 (soluble fms-like tyrosine kinase-1) secretion, which reduces fetal growth and causes maternal symptoms. This study aims to evaluate the benefits of using PT2385-an oral specific HIF-2α inhibitor-to treat severe placental dysfunction. To evaluate its therapeutic potential, PT2385 was first studied in primary human cytotrophoblasts isolated from term placenta and exposed to 2.5% O2 to stabilize HIF-2α. Viability and luciferase assays, RNA sequencing, and immunostaining were used to analyze differentiation and angiogenic factor balance. The ability of PT2385 to mitigate maternal manifestations of preeclampsia was studied in the selective reduced uterine perfusion pressure model performed in Sprague-Dawley rats. In vitro, RNA sequencing analysis and conventional techniques showed that treated cytotrophoblast displayed an enhanced differentiation into syncytiotrophoblasts and normalized angiogenic factor secretion compared with vehicle-treated cells. In the selective reduced uterine perfusion pressure model, PT2385 efficiently decreased sFLT-1 production, thus preventing the onset of hypertension and proteinuria in pregnant dams. These results highlight HIF-2α as a new player in our understanding of placental dysfunction and support the use of PT2385 to treat severe preeclampsia in humans.

Inhibiting B cell activating factor attenuates preeclamptic symptoms in placental ischemic rats.

Preeclampsia (PE), new-onset hypertension during pregnancy, is associated with a pro-inflammatory state with activated T cells, cytolytic natural killer (NK) cells, dysregulated complement proteins, and B cells secreting agonistic autoantibodies to the angiotensin II type-1 receptor (AT1-AA). The reduced uterine perfusion pressure (RUPP) model of placental ischemia recapitulates these features of PE. Blocking CD40L-CD40 communication between T and B cells or B cell depletion with Rituximab prevents hypertension and AT1-AA production in RUPP rats. This suggests that T cell-dependent B cell activation contributes to the hypertension and AT1-AA associated with PE. B2 cells maturing into antibody producing plasma cells are the product of T cell-dependent B cell-interactions and B cell Activating Factor (BAFF) is an integral cytokine in the development of B2 cells specifically. Thus, we hypothesize that BAFF blockade will selectively deplete B2 cells, therefore reducing blood pressure, AT1-AA, activated NK Cells, and complement in the RUPP rat model of PE. Gestational Day (GD) 14 pregnant rats underwent the RUPP procedure, and a subset were treated with 1mg/kg Anti-BAFF antibodies via jugular catheters. On GD19, blood pressure was measured, B cells and NK cells were measured by flow cytometry, AT1-AA was measured by cardiomyocyte bioassay, and complement activation was measured by ELISA. Anti-BAFF therapy attenuated hypertension, AT1-AA, NK cell activation, and APRIL levels in RUPP rats without negatively impacting fetal outcomes. This study demonstrates that B2 cells contribute to hypertension, AT1-AA, and NK cell activation in response to placental ischemia during pregnancy.

NLRP3 inhibition improves maternal hypertension, inflammation, and vascular dysfunction in response to placental ischemia.

Preeclampsia (PE) is a pregnancy-specific hypertensive disorder with end-organ damage that presents after 20 wk of gestation. PE pathophysiology often includes vascular dysfunction and increased inflammation that continues to damage patient health even after PE resolves. Currently, there is no cure for PE beyond delivery of the fetal-placental unit. Previous clinical studies have identified elevated placental NLRP3 expression in patients with PE and suggest NLRP3 as a potential therapeutic target. In this study, we examined the effect of NLRP3 inhibition on PE pathophysiology in the reduced uterine perfusion pressure (RUPP) model rat using MCC950 (20 mg/kg/day) or esomeprazole (3.5 mg/kg/day). We hypothesized that increased NLRP3 in response to placental ischemia impairs anti-inflammatory IL-33 signaling to induce T-helper 17 cell (TH17) and cytolytic NK cell (cNK) activation, which is known to mediate oxidative stress and vascular dysfunction leading to maternal HTN and intrauterine growth restriction. RUPP rats had significantly higher placental NLRP3 expression, maternal blood pressure, fetal reabsorption rate, vascular resistance, oxidative stress, cNKs and TH17s, and decreased IL-33 compared with normal pregnant (NP) rats. NLRP3 inhibition, with either treatment, significantly reduced placental NLRP3 expression, maternal blood pressure, fetal reabsorption rates, vascular resistance, oxidative stress, cNK, and TH17 populations in RUPP rats. Based on our findings, NLRP3 inhibition reduces PE pathophysiology and esomeprazole may be a potential therapeutic for PE treatment.

Human umbilical cord mesenchymal stem cell-derived extracellular vesicles loaded with TFCP2 activate Wnt/β-catenin signaling to alleviate preeclampsia.

Failures in invasive extravillous trophoblasts (EVTs) migration into the maternal uterus have been noticed in preeclampsia (PE). Human umbilical cord mesenchymal stem cell (hUCMSC)-derived extracellular vesicles (EVs) have been highlighted for the role as a potential therapeutic method in PE. This study intends to investigate the mechanistic basis of hUCMSCs-derived EVs loaded with bioinformatically identified TFCP2 in the activities of EVTs of PE. Primary human EVTs were exposed to hypoxic/reoxygenation (H/R) to mimic the environment encountered in PE. The in vivo PE-like phenotypes were induced in mice by reduced uterine perfusion pressure (RUPP) surgery. CCK-8, Transwell and flow cytometry assays were performed to detect proliferation, migration, invasion and apoptosis of H/R-exposed EVTs. More importantly, EVs were extracted from hUCMSCs and transduced with ectopically expressed TFCP2, followed by co-culture with EVTs. TFCP2 was found to be down-regulated in the preeclamptic placental tissues and in H/R-exposed EVTs. hUCMSCs-EVs loaded with TFCP2 activated the Wnt/β-catenin pathway, thereby promoting the proliferative, migratory, and invasive potential of EVTs. Furthermore, overexpression of TFCP2 alleviated PE-like phenotypes in mice, which was associated with activated Wnt/β-catenin pathway. From our data we conclude that hUCMSCs-EVs overexpressing TFCP2 may be instrumental for the therapeutic targeting and clinical management of PE.

Elevated PGT promotes proliferation and inhibits cell apoptosis in preeclampsia by Erk signaling pathway

Prostaglandins participate in maternal recognition of pregnancy, implantation and maintenance of gestation. Prostaglandin transporter (PGT), as a candidate molecule of prostaglandin carriers, might be involved in the pathogenesis of preeclampsia. In preeclampsia (PE) patients’ placental tissue, we identified PGT by RNA sequencing, measured its expression pattern by quantitative real-time PCR and Western blot. PGT was found to be upregulated in preeclamptic placental tissue. The expression pattern of PGT in PE was double confirmed by eight Gene Expression Omnibus (GEO) databases. In abortion tissues at 6–8 weeks, we then observed the cellular location of PGT by Immunofluorescence technique (IF) and found PGT located in trophoblast cell of the placenta of early pregnancy. In vitro studies revealed that forced expression of PGT in HTR8/Sveno cell inhibited its apoptosis, but promoted its proliferation by activating Erk signaling. In vivo study, we used reduced uterine perfusion pressure (RUPP) rat model and L-NAME-induced preeclampsia-like rats to study the possible role of PGT in preeclampsia. And PGT was found to be upregulated in both preeclampsia rat models by Immunohistochemical (IHC) staining. Newly identified PGT plays an important role in trophoblast proliferation via Erk signaling, providing new insights for understanding the pathogenesis of PE.

Comparison of different modified operations in the reduced uteroplacental perfusion pressure rat model of preeclampsia

IntroductionAnimal models are indispensable tools in studying the mechanisms underlying the diseases. Rat models with reduced uterine perfusion pressure (RUPP) were able to mimic the pathophysiological traits of placental ischemia and hypoxia in preeclampsia (PE). However, ischemic injury can lead to a cascade of damage to lower limb ischemia in RUPP. Therefore, the aim of our study was to compare three modified surgical procedures of reducing uteroplacental perfusion pressure, and to provide a reference for the recognition of different PE phenotypes in the future. Material and methodsTo establish a specific uteroplacental malperfusion model of PE in rats, we bilaterally ligated uterine vessels (UU), ovarian vessels distal to ovarian branches (OO), or both (sRUPP) at 13.5 days post coitum. 21 Sprague-Dawley rats in total were used and were divided into four groups: Sham (n = 4), UU (n = 6), OO (n = 5) and sRUPP (n = 8). ResultsThe results showed that the OO and sRUPP groups could successfully mimic the phenotypes of PE while UU group not. Then, autophagy, apoptosis, and synthesis of unsaturated fatty acids were increased in both the OO and sRUPP groups compared with the Sham group, while inflammation were not statistically different. ConclusionsThe OO and sRUPP groups could successfully establish the rat model of PE while the UU group not. Notably, between the OO and sRUPP groups, the OO group has a higher fetal survival rate and might be more suitable for studying fetal-related questions, while the sRUPP group has a heavier phenotypic profile and is more suitable for studying maternal phenotypes related to PE.

PS-B12-4: SOLUBLE (PRO)RENIN RECEPTOR LEVEL INCREASED IN A RAT MODEL OF REDUCED UTERINE PERFUSION PRESSURE

Objective: The (pro)renin receptor [(P)RR)] is a multifunctional protein with roles in angiotensin II-dependent and -independent intracellular cell signaling and as an adaptor protein between the Wnt receptor complex and vacuolar proton-translocating adenosine triphosphatase. The (P)RR is cleaved from the full length (P)RR to generate soluble (P)RR [s(P)RR], reflecting the status of the tissue activity of the (P)RR. We have shown that maternal blood s(P)RR concentrations in early pregnancy are associated with increased blood pressure in late pregnancy, while higher levels of s(P)RR in cord blood in babies are at lower risk of low birth weight. We have also reported that intracellular s(P)RR is increased by hypoxia and interacts with pyruvate dehydrogenase β; subunit (PDHB) to stabilize PDHB protein and pyruvate dehydrogenase (PDH) activity in trophoblast cells, leading to our hypothesis that (P)RR may help maintain oxidative metabolism and efficient energy production during placental ischemia. In this in vivo study, we investigated effects of reduced uterine perfusion pressure (RUPP) on PDH activity and s(P)RR levels in rats. Design and method: RUPP was induced by placing sliver clips around the aorta above the iliac bifurcation and the bilateral uterine arcades in pregnant Sprague Dawley rats on gestational day 14 (RUPP rats). Weights of placentas and infants were measured on gestational day 21. mRNA expressions of (P)RR, hypoxia-inducible factor 1-alpha (HIF1a), and PDHB, and protein levels of HIF1a, (P)RR, s(P)RR, PDHB, lactate, and pyruvate, and activity of PDH in placentas were measured on gestational day 21. Result: Weight of placentas and infants were significantly decreased in RUPP rats when compared with those of sham rats. mRNA and protein levels of HIF1a were significantly higher in RUPP rats than in control sham rats, indicating that ischemia was actually induced by RUPP. mRNA levels of (P)RR and PDHB were not significantly different between the groups. The protein level of (P)RR was not significantly different between the groups while s(P)RR protein level was significantly higher in RUPP rats than in controls. Concentrations of lactate or pyruvate and PDH activity were not significantly different between the groups. Conclusion: PDH activity was not decreased even under the condition of placental ischemia. s(P)RR but not full length (P)RR was increased by placental ischemia. These data suggested that s(P)RR might be increased by placental ischemia to maintain mitochondrial oxidative metabolism in rats.

Decreased expression of caveolin-1 have relevance to promoted senescence in preeclamptic placenta.

To assess the association between altered expression of caveolin-1 and p53/p21, as indicatives of cellular senescence, in preeclamptic placenta. Placental tissues and serum were collected from rats (Sham and reduced uterine perfusion pressure group) at 18.5days post coitum and humans (normotensive pregnant and preeclampsia groups). The concentration and expression of caveolin-1 were measured in the collected tissues, and the correlation between p53 and p21 expression was evaluation. Placental mRNA expression and serum concentration of caveolin-1 were measured using qRT-PCR and ELISA, respectively. Altered expressions of caveolin-1 and p53/p21 were revealed and quantified by immunohistochemistry. The association between these changes was investigated using correlation analysis. Placental mRNA expressions and serum concentrations of caveolin-1 were significantly decreased in reduced uterine perfusion pressure and preeclampsia groups. The expressions of caveolin-1 and p53/ p21 were significantly altered in placenta complicated with preeclampsia. Correlation analysis revealed a significant inverse association between changes in caveolin-1 and p53/p21. Subsequently, these results were obtained by investigating the preeclampsia onset time. These results revealed that the expression of caveolin-1 profoundly decreases in the placenta and serum of preeclampsia. These factors contribute to the mechanism of accelerated cellular senescence in placenta, which is one of the various etiologies of preeclampsia.