Normal Pregnant Research Articles

Intrauterine growth-restricted pregnant rats, from placental ischemic dams, display preeclamptic-like symptoms: A new rat model of preeclampsia.

Preeclampsia (PE) is characterized by de novo hypertension (HTN) and is often associated with intrauterine growth restriction (IUGR). Hallmarks of PE are placental ischemia, decreased nitric oxide (NO) bioavailability, oxidative stress (OS), and organ damage in the kidneys and brain. This study aims to characterize a new model of PE using pregnant IUGR rats from hypertensive placental ischemic dams. It is hypothesized that pregnant IUGR rats from hypertensive placental ischemic dams will have elevated blood pressure (BP), OS, and organ damage. In this study, pregnant rats are divided into two groups: normal pregnant (NP) and hypertensive placental ischemic dams (RUPP). Offspring from NP and RUPP dams were mated at 10 weeks of age to generate pregnant IUGR (IUGR Preg) and pregnant control (CON Preg) rats. BP and other markers of PE were evaluated during late gestation. Pregnant IUGR rats had elevated BP and systemic OS. The maternal body weight of pregnant IUGR rats and their pups' weights were decreased, while the brains were enlarged with elevated OS. In summary, pregnant IUGR rats, born from hypertensive placental ischemic dams, have HTN and increased systemic and brain OS, with larger brain sizes and smaller pups. Furthermore, this study shows that pregnant IUGR rats exhibit a preeclamptic-like phenotype, suggesting a new epigenetic model of PE.

Abstract MP30: The Role of CD20+ B cells in Mediating Hypertension in Preeclampsia during Pregnancy

Pre-eclampsia (PE), new-onset hypertension after the 20 th week of pregnancy alongside other organ dysfunction, endothelial dysfunction, and immune activation. We have previously shown that CD19+ B cell adoptive transfer causes a PE phenotype in athymic nude rats. CD19 is present on all B cells including plasma cells. CD20 is absent from plasma cells but is present on mature B2 and memory B cells. The objective of this study was to determine if CD20+ B cells, excluding plasma cells, cause maternal hypertension, fetal growth restriction, and endothelial dysfunction. Three hundred thousand placental CD20+ B Cells from normal pregnant (NP) or PE patients were isolated by magnetic separation and injected i.p. into nude athymic rats on gestational day (GD) 12. On GD18, carotid catheters were inserted. On GD19, mean arterial pressure (MAP) was measured and blood and tissues were collected. Preproendothelin (PPET) expression was quantified in kidney and placental tissues using RT-PCR. A student’s t-test was used for statistical analysis. Participants whose placental B cells were used in this study had similar age, pre-pregnancy BMI, and diastolic BP on admission. PE participants had elevated systolic BP at admission (135±4 vs 120±4 mmHg) and delivery (139±6 vs 124±3 mmHg) as well as diastolic BP at delivery (79±4 vs 68±2 mmHg). PE participants also had trending decreases in fetal weight (2621±323 vs 3202±102 g) and gestational age (36±1 vs 39±0 weeks) at delivery. MAP increased from 107±4 mmHg (n=8) in the NP recipient rats to 123±2 mmHg (n=8, p<0.01) in the PE recipient rats. Pup and placental weights were unchanged following adoptive transfer. Renal PPET expression increased by 2.595±0.61 fold (ΔΔct) in PE recipient rats (n=7, p<0.05) compared to NP recipient rats (n=7). Placental PPET expression was unchanged following adoptive transfer. CD20+ B cells from women with PE contribute to hypertension and endothelial dysfunction during pregnancy. This data supports the important role of B cells in the development of PE and improves our understanding of the population of B cells responsible for contribution to the disease.

Progesterone Supplementation Improves the Maternal Syndrome of Superimposed Preeclampsia in the Dahl Salt Sensitive Rats

Introduction: Preeclampsia (PE) is characterized by new onset of hypertension after 20 weeks of gestation. It affects 5-7% of all pregnancies in the U.S., and is associated with reduced fetal weight, inflammation and hypertension (HTN). Importantly, 30% of HTN disorders in pregnancy are caused by chronic HTN that is present prior to pregnancy which increases the risk of superimposed PE (SIPE). The mechanisms responsible for the pathogenesis of PE and SIPE are unclear and currently the only treatment is early delivery of the fetus. Progesterone is important for the establishment and maintenance of pregnancy. We have previously shown that progesterone supplementation with 17-hydroxyprogesterone caproate (17-OHPC) improves inflammation, fetal weight and blood pressure in the preclinical RUPP rat model of PE, however the mechanism for this in SIPE is still unknown. Hypothesis: This study was designed to test the hypothesis that 17-OHPC reduces inflammation while improving maternal blood pressure in the preclinical pregnant Dahl Salt Sensitive (DS) rat model of SIPE. Methods: 17-OHPC (3.32mg/kg) or vehicle (Saline) was administrated intraperitoneally on gestation day (GD) 15 to normal pregnant (NP) Sprague-Dawley (SD) and pregnant Dahl Salt Sensitive (DS) rats. On GD 18, Uterine Artery Resistance Index (UARI) was measured by Vevo Doppler Ultrasound and carotid catheters were inserted. On GD 19, mean arterial blood pressure (MAP) and samples were collected. All data are expressed as mean ± standard error means (SEM). Results: MAP was 105±5 mmHg in SD+ Saline rats (n=7) and 105±4 mmHg in SD+17-OHPC rats (n=4), 137±3 mmHg in DS + Saline rats (p<0.05, n=11), which improved to 125±4 mmHg in DS+17-OHPC rats (p<0.05, n=7). Pup and Placenta weights were 2.1± 0.1 g, 0.6 ± 0.1 g in SD + saline rats and significantly reduced to 1.4±0.1 g, 0.5±0.1 g in DS+ Saline rats (p<0.05). Neither placental weight nor pup weight was affected by 17-OHPC. UARI was 0.5 ±0.1 in SD+ Saline rats (n=6) and 0.5 ±0.1 in in SD+17-OHPC (n=4), 0.7±0.1 in DS+Saline rats (n=6, p<0.05), which reduced to 0.5±0.1 in DS+17-OHPC (n=7, p<0.05). TNF-alpha levels were 3.0±1.0 pg/mL in SD+ Saline rats, 11.0±1.2 in DS + Saline rats, which reduced to 4.1±1.6 in DS+17-OHPC (n=4, p<0.05). Importantly, circulating and placental CD4+ T cells were 9.3±3.7 % Gate and 6.4± 1.6 % Gate in SD+ Saline rats (n=4), 38.9 ±2.9 % Gate and 33.1±4.3 % Gate in DS + Saline rats (n=6), which significantly reduced to 26.3 ± 0.1 % Gate and 3.4±1.2 % Gate in DS + 17-OHPC. Conclusion: Collectively, our findings demonstrate that 17-OHPC reduces inflammation and hypertension in the Dahl Salt Sensitive rat model of SIPE. Supported by NIH P20GM121334. This is the full abstract presented at the American Physiology Summit 2024 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.

IL-33 supplementation improves uterine artery resistance and maternal hypertension in response to placental ischemia.

Preeclampsia (PE), a leading cause of maternal/fetal morbidity and mortality, is a hypertensive pregnancy disorder with end-organ damage that manifests after 20 wk of gestation. PE is characterized by chronic immune activation and endothelial dysfunction. Clinical studies report reduced IL-33 signaling in PE. We use the Reduced Uterine Perfusion Pressure (RUPP) rat model, which mimics many PE characteristics including reduced IL-33, to identify mechanisms mediating PE pathophysiology. We hypothesized that IL-33 supplementation would improve blood pressure (BP), inflammation, and oxidative stress (ROS) during placental ischemia. We implanted intraperitoneal mini-osmotic pumps infusing recombinant rat IL-33 (1 µg/kg/day) into normal pregnant (NP) and RUPP rats from gestation day 14 to 19. We found that IL-33 supplementation in RUPP rats reduces maternal blood pressure and improves the uterine artery resistance index (UARI). In addition to physiological improvements, we found decreased circulating and placental cytolytic Natural Killer cells (cNKs) and decreased circulating, placental, and renal TH17s in IL-33-treated RUPP rats. cNK cell cytotoxic activity also decreased in IL-33-supplemented RUPP rats. Furthermore, renal ROS and placental preproendothelin-1 (PPET-1) decreased in RUPP rats treated with IL-33. These findings demonstrate a role for IL-33 in controlling vascular function and maternal BP during pregnancy by decreasing inflammation, renal ROS, and PPET-1 expression. These data suggest that IL-33 may have therapeutic potential in managing PE.NEW & NOTEWORTHY Though decreased IL-33 signaling has been clinically associated with PE, the mechanisms linking this signaling pathway to overall disease pathophysiology are not well understood. This study provides compelling evidence that mechanistically links reduced IL-33 with the inflammatory response and vascular dysfunction observed in response to placental ischemia, such as in PE. Data presented in this study submit the IL-33 signaling pathway as a possible therapeutic target for the treatment of PE.

Abstract 022: Il-33 Supplementation Improves Vascular Function And Maternal Hypertension In Response To Placental Ischemia

Preeclampsia (PE), a leading cause of maternal/fetal morbidity and mortality, is a hypertensive pregnancy disorder with end-organ damage that manifests after 20 weeks of gestation. PE is characterized by chronic immune activation and endothelial dysfunction. Clinical studies report reduced IL-33 signaling in PE. We use the R educed U terine P erfusion P ressure (RUPP) rat model, which mimics many PE characteristics including reduced IL-33, to identify mechanisms mediating PE pathophysiology. We hypothesized IL-33 supplementation would improve blood pressure (BP), inflammation, and oxidative stress (ROS) during placental ischemia. We implanted i.p. mini-osmotic pumps infusing recombinant rat IL-33 (1 μg/kg/day) into normal pregnant (NP) and RUPP rats from gestation day 14-19. We assessed maternal BP, cytolytic NK (cNK) and T H 17 cells, cNK cell activity, ROS, preproendothelin-1 (PPET) expression, and uterine artery resistance index (UARI). MAP (mmHg) was 104±4 in NP vs 124±7 in RUPP (p<0.05 vs NP) and lowered to 108±10 in RUPP+IL-33 (p<0.05 vs RUPP, n=8/group). Circulating cNKs (% gated) were 0.9±0.2% in NP vs 3.9±1.1% in RUPP (p<0.05 vs NP) and decreased to 1.1±0.6% in RUPP+IL-33 (p<0.05 vs RUPP, n=8/group). Circulating T H 17s (%CD4 + T cells) were 1.0±0.6% in NP vs 7.2±0.5% in RUPP (p<0.05 vs NP) and 0.8±0.4% in RUPP+IL-33 (p<0.05 vs RUPP, n=8/group). Renal T H 17s, placenta cNKs and T H 17s followed similar trends. cNK cell activity (fold change) was 3.92±0.6 fold higher in RUPP vs NP (p<0.05 vs NP) and decreased to -0.24±0.1 in RUPP+IL-33 (p<0.05 vs RUPP, n=3/group). Renal ROS (relative light units/min/mg protein) was 452±33 in NP vs 676±44 in RUPP (p<0.05) and decreased to 258±50 in RUPP+IL-33 (p<0.05 vs RUPP, n=5/group). Placental PPET (fold change) was 2.36±0.4 fold higher in RUPP vs NP (p<0.05) and decreased to 1.22±0.2 in RUPP+IL-33 (p=0.05 vs RUPP, n=6-7/group). UARI (arbitrary units) was 0.71±0.01 in RUPP vs 0.54±0.01 in NP (p<0.05) and was lowered to 0.57±0.02 in RUPP+IL-33 (p<0.05 vs RUPP, n=6/group). These findings demonstrate a role for IL-33 in controlling vascular function and maternal BP during pregnancy by decreasing inflammation, ROS, and PPET expression. These data suggest IL-33 may have therapeutic potential in managing PE.

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.

Adoptive transfer of CD4+ T cells from women with preeclampsia with or without a history of COVID during pregnancy contribute to hypertension, AT1-AA, and neurovascular dysfunction in pregnant rats

Objective: Preeclampsia (PE) is characterized by new onset hypertension during pregnancy and is associated with chronic inflammation and neurovascular dysfunction. The inflammation in PE includes activated CD4+ T cells and B cells producing agonistic antibodies against the angiotensin II type 1 receptor (AT1AA) which has also been shown to be produced in patients with COVID-19 (CV) infection. Interestingly, having had CV during pregnancy increases the incidence of a PE-like phenotype in that pregnancy. Both PE and CV have long lasting neurological implications and we have shown that AT1AA mediates neurovascular dysfunction in PE. Moreover, we have shown that CD4+ T cells from women with PE induce a PE syndrome in pregnant athymic nude recipient rats. Although CD4+T cells have been shown to contribute to neurovascular dysfunction, we do not know the role of CD4+ T cells in contributing to neurovascular dysfunction in PE or in patients with a history (Hx) CV during pregnancy. Therefore, we sought examine a role for CD4+ T cells from PE with or without a CV Hx to contribute to AT1AA in pregnancy as well as hypertension and cerebrovascular dysfunction postpartum. We utilized adoptive transfer of placental CD4+ T cells from PE and normotensive (NT) patients with or without a CV Hx during pregnancy into athymic nude recipient rats and determined the presence of a PE phenotype during pregnancy or postpartum. Methods: CD4+ T cells were isolated from placentas of normal pregnant (NP), PE, CV Hx normotensive (CV Hx NT), and Hx of CV with PE (CV Hx PE) at delivery. One million CD4+ T cells were injected i.p. into gestational day (GD) 12 nude athymic rats. At GD19 blood samples were collected to measure AT1AA by cardiomyocyte assay. At three months postpartum, MAP and CBF were measured by arterial catheter and laser Doppler flowmetry. A two-way ANOVA was used for statistical analysis. Results: In pregnant recipient rats, AT1AA was increased in PE (8±2 ΔBPM, n=4) and CV Hx PE (4±2 ΔBPM, n=4) compared to NP (-3±2 ΔBPM, n=4, p<0.05) and CV Hx NT (2±2 ΔBPM, n=2) respectively. At 3 months postpartum, MAP was increased in PE (115±2 mmHg, n=5) and CV Hx PE (111±4, n=4) compared to NP (99±3 mmHg, n=4 p<0.05) and CV Hx NT (100±4, n=5). PE and CV Hx PE had impaired autoregulation of CBF compared to CV Hx NT and NP rats. Conclusion: Our findings indicate that CD4+ T cells from PE or CV HX PE patients cause AT1-AA in pregnancy as well as hypertension and impaired CBF 3 months postpartum. Collectively, the data demonstrates that CD4+ T cells cause hypertension and inflammation in PE or CV Hx PE and may contribute to neurovascular dysfunction in patients with previous PE or CV Hx PE during pregnancy. RO1HD067541-06, P20GM121334, and AHA 19CDA34670055 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.

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.

Abstract 103: Il-33 Supplementation Improves Vascular Function And Maternal Hypertension In Response To Placental Ischemia

Preeclampsia (PE), a leading cause of maternal and fetal morbidity and mortality, is a hypertensive pregnancy disorder with end-organ damage that develops after 20 weeks of gestation. PE is characterized by chronic immune activation, endothelial dysfunction, and fetal growth restriction (FGR). Clinical studies have shown decreased IL-33 signaling in PE women. We use the Reduced Uterine Perfusion Pressure (RUPP) rat model, which mimics many characteristics of PE patients including reduced IL-33 levels, to identify the mechanisms mediating maternal hypertension and FGR in PE. We hypothesized that IL-33 supplementation would improve maternal hypertension, fetal growth, and oxidative stress (ROS) during placental ischemia in pregnant rats. We implanted intraperitoneal mini-osmotic pumps infusing recombinant rat IL-33 (1 μg/kg/day) in normal pregnant (NP) and RUPP rats from gestation day 14-19. Maternal blood pressure, fetal growth, placental and renal ROS, phosphorylated endothelial nitric-oxide synthase ( p eNOS), and uterine artery resistance index (UARI) were assessed. MAP (mmHg) increased from 104±4 in NP to 124±7 in RUPP (p<0.05) and was significantly lowered in RUPP+IL-33 to 108±10 (p<0.05, n=6-8/group). Fetal weight was significantly increased in RUPP+ IL-33 compared to RUPP (p<0.05). Placental ROS was not different among any groups. However, renal ROS (relative light units/min/mg protein) was increased from 452±33 in NP to 676±44 in RUPP (p<0.05) and was significantly decreased to 309±61 in RUPP+IL-33 (n=4-5/group, p<0.05 vs RUPP). Placental p eNOS expression was 4-fold lower in RUPP vs NP (p<0.05) and 5-fold lower in RUPP+IL-33 vs NP, n=3-5/group). UARI (arbitrary units) was 0.709±0.012 in RUPP vs NP (0.585±0.025, p<0.05) and was significantly lowered to 0.577±0.025 in RUPP+IL-33 rats (p<0.05 vs RUPP, n=5-6/group). These findings demonstrate a role for IL-33 in controlling vascular function and maternal blood pressure during pregnancy independent of eNOS activation. Further studies will quantify effector immune cells and inflammatory cytokine levels in response to IL-33 supplementation during placental ischemia. These data suggest that IL-33 may have therapeutic potential in management of PE pathophysiology during pregnancy.

Abstract 018: B Cells Induce Hypertension, AT1AA, And Complement Activation In Preeclampsia Or In Response To A Prior Covid-19 Infection During Pregnancy

Preeclampsia (PE), new onset hypertension (HTN) during pregnancy, is associated with placental ischemia and chronic inflammation that includes increased CD4+ T cells, B cells secreting agonistic autoantibodies against the angiotensin II type 1 receptor (AT1AA), and activation of the complement system. Previous studies have shown AT1-AA is produced in patients with COVID-19 infection. Interestingly, having had COVID-19 during pregnancy is associated with increased incidence of developing a PE phenotype during pregnancy. We have previously shown an important role for B cell depletion or AT1AA inhibition to attenuate HTN in rat models of PE. Collectively, this data suggests B cells contribute to PE development and that B cells may increase incidence of PE in patients with a history (Hx) of COVID-19 during pregnancy through production of the AT1AA. We hypothesize B cells from PE or CV Hx PE patients produce AT1AA resulting in HTN and complement activation in pregnancy. Placental B cells were isolated from normal pregnant (NP), PE, normotensive (NT) CV Hx, or PE CV Hx patients at delivery. B cells were transferred i.p. into pregnant athymic rats at gestation (GD) 12. On GD18, carotid catheters were inserted. On GD19, blood pressure was measured and tissues collected. PE B cell recipients had increased Mean Arterial Pressure (MAP) (115±3 mmHg n=6) compared to NP B cell recipients (97±4 mmHg n=6 p<0.05). PE B cell recipients had increased AT1AA (20±2 ΔBPM n=4) compared to NP B cell recipients (6±1 ΔBPM n=4 p<0.05). PE B cell recipients had increased markers of complement activation such as reduced plasma C4 (1302±169 μg/mL n=4) and C3 (516±45 μg/mL n=4) compared to recipients of NP B cells (2348±338 μg/mL n=4 p<0.05) and (790±66 μg/mL n=4 p<0.05) respectively. CV Hx PE B cell recipients had elevated MAP (108±3 mmHg n=4) compared to CV Hx NT B cell recipients (101±7 mmHg n=4) and increased AT1AA (24±3 ΔBPM n=3) compared to CV Hx NT B cell Recipients (4±1 ΔBPM n=4 p<0.05). Collectively, this study demonstrates an important role for B cells to cause HTN during pregnancy; and indicates that B cells contribute to a higher incidence of PE in women with a Hx of CV infection during pregnancy possibly by secreting AT1-AA.

Integrative analysis of circulating microRNAs and the placental transcriptome in recurrent pregnancy loss.

Recurrent pregnancy loss (RPL) is a major type of pathological pregnancy that still lacks reliable early diagnosis and effective treatment. The placenta is critical to fetal development and pregnancy success because it participates in critical processes such as early embryo implantation, vascular remodeling, and immunological tolerance. RPL is associated with abnormalities in the biological behavior of placental villous trophoblasts, resulting in aberrant placental function. MicroRNAs (miRNAs) are increasingly being recognized as essential regulators of placental development, as well as potential biomarkers. In this study, plasma miRNAs and placental messenger RNAs (mRNAs) from RPL patients and normal pregnant (NP) controls were sequenced and analyzed. Compared to those in NP controls, 108 circulating miRNAs and 1199 placental mRNAs were differentially expressed in RPL samples. A total of 140 overlapping genes (overlapping between plasma miRNA target genes and actual placental disorder genes) were identified, and functional enrichment analysis showed that these genes were mainly related to cell proliferation, angiogenesis, and cell migration. The regulatory network among miRNAs, overlapping genes, and downstream biological processes was analyzed by protein–protein interactions and Cytoscape. Moreover, enriched mRNAs, which were predictive targets of the differentially expressed plasma miRNAs miR-766-5p, miR-1285-3p, and miR-520a-3p, were accordingly altered in the placenta. These results suggest that circulating miRNAs may be involved in the pathogenesis of RPL and are potential noninvasive biomarkers for RPL.

The potential value of diagnostic and predictive serum biomarkers for preeclampsia.

Background: Preeclampsia (PE), one of the classes of hypertensive pregnancy disorders, is one of the three causes of maternal morbidity and mortality worldwide. The angiogenic and anti-angiogenic factors are useful markers in predicting and diagnosing PE. Aim: This study aims to detect and measure the serum level of some biomarkers [hypoxia-inducible factor-1 subunit alpha (HIF-1A), vascular endothelial growth factor (VEGF), interferon-gamma-inducible protein of 10 kDa (IP-10), matrix metalloproteinase-13 (MMP-13)] in patients with PE and their correlation with the severity of the disease, to find a good predictor for PE. Patients, Materials and Methods: This prospective study aims to monitor 48 pregnant women who address obstetric consultation and who present risk factors for PE, and a control group with characteristics similar to the study group. Patients were divided into three groups: Group I (n=15) including normal pregnant (NP) women with blood pressure <140/90 mmHg, without proteinuria, Group II (n=18) including patients with mild PE (MildPE), Group III (n=15) including patients with severe PE (SeverePE). The analysis of serum biomarkers was based on a quantitative sandwich enzyme-linked immunosorbent assay (ELISA), according to the manufacturer’s instructions. Results: In our study, we found that all biomarkers investigated have higher concentrations in the serum of patients with SeverePE and MildPE than those in the control subjects (Group I, NP), the concentrations were increasing along with the disease activity. The means concentrations of HIF-1A, VEGF, IP-10, MMP-13, better correlated with indices in SeverePE group than in MildPE group. We found that VEGF was the biomarker that best correlates with indices that assess the severity of PE. The best separation of patients with SeverePE from those with MildPE can be done with the help of MMP-13 (82% accuracy), followed by VEGF (80.40% accuracy) and the least good detection being done by dosing IP-10. Conclusions: We can say that, due to high specificity diagnostic accuracy, determination of serum concentrations of MMP-13 and VEGF, could be useful in the diagnosis and distinguishing of patients with SeverePE and may prove useful in the monitoring of the disease course.

IL17 administration in the Absence of T cells Results in Hypertension, NK cell Activation, and Reduced Pup Weight at Birth, but No Changes in Blood Pressure or Weight at Maturation of Offspring

Preeclampsia (PE) is characterized by new onset hypertension (HTN), fetal growth restriction (FGR), multi‐organ dysfunction, and increased inflammatory cytokines. Moreover, we have shown offspring of RUPP rats develop hypertension at different stages of life based on sex. More recent studies demonstrate a role for mitochondrial (mt) dysfunction/mtROS in the pathogenesis of the disease, however, causative factors for mt dysfunction are still being identified. Th17 cells stimulated by a rat model of PE have been shown to cause HTN, FGR, and mt dysfunction. One of the main cytokines produced by TH17 cells is IL‐17, which we have shown to cause HTN, mtROS, stimulate TH17 cell proliferation, and activate natural killer (NK) cells in pregnant Sprague Dawley rats. The athymic nude rat model lacks T cells, and therefore are useful in examining the differences in the roles of IL‐17 and TH17 cells in the pathophysiology of PE. We hypothesize that IL‐17 induces HTN and is associated with renal and placental mt dysfunction during pregnancy in the absence of T cells in pregnant athymic nude rats. We further hypothesize that offspring of these animals generate hypertension at maturation (16 weeks of age). IL‐17 (150 pg/day) was infused via osmotic minipumps on gestation day (GD) 14. Blood pressure (MAP) and mt function were measured on GD19 and were compared to normal pregnant (NP) rats. Pups were weaned 3 weeks after birth and weighed once a week for 16 weeks, and MAP was measured at 16 weeks. MAP increased in response to IL‐17 (NP+IL‐17, 115.3±1.5 mmHg in, n=6) compared to NP rats (95.5±3.85 mmHg, n=6, p&lt;0.001). Interestingly, mtROS in the absence of T cells is reduced in both the placenta and kidney (46±6.6%fold change in NP+IL‐17 placental mitochondria; 48.8±2.8%fold change in the kidney mitochondria, n=5) compared to NP mtROS (100±6.6%fold change in NP placental mitochondria; 100±3.7% fold change in the kidney mitochondria, (n=4). ADP production in response to IL‐17 in the absence of T cells in the placenta or the kidney increases (105.5±91 pmol of O2/sec/mg, n=3NP+IL‐17 placental mitochondria, 2016±951.2 pmol of O2/sec/mg, n=4, in NP+IL‐17 kidney mitochondria) compared to NP (15.53±1.6 pmol of O2/sec/mg, n=3 NP placenta, 1196±459.5 pmol of O2/sec/mg, n=4 NP kidney (n=4)). Pup weight at GD19 decreased in response to IL‐17 from 1.46±0.2 g in NP (n=6) to 0.98±0.07g in NP+IL‐17 (n=6) in the absence of T cells (p&lt;0.05). However, there was no change in weight after weaning and into adulthood in either male or female offspring. There was also no change in MAP in NP+IL17 (123± 2.7 mmHg, n=3) male offspring compared to NP offspring (122± 2.6 mmHg, n=4) at 16 weeks. There was also no change in female offspring MAP in response to IL‐17 (121± 12 mmHg, n=2) compared to NP (105 ± 21 mmHg, n=3) in offspring at 16 weeks. These results suggest that IL‐17 induces HTN, and FGR without the presence of T cells, and suggests that T cells induce mt dysfunction in PE. It also shows that IL‐17 may not cause hypertension of the offspring after birth.

CD4+T Cells cause increased glucose, mitochondrial dysfunction, and hypertension in a Novel Pregnant Rodent Model of Gestational Diabetes Mellitus

Hypertensive (HTN) disorders of pregnancy complicate approximately 10% of all pregnancies worldwide and contribute to maternal and fetal morbidity and mortality. HTN increases risks for greater pathophysiology in gestational diabetes mellitus (GDM) pregnant women, thereby supporting a need for controlling maternal glucose and blood pressure. GDM is a major medical complication characterized by hyperglycemia and/or β‐cell dysfunction and associated with increased inflammatory cytokines, oxidative stress, and activated CD4+ T cells. Streptozotocin (STZ) is utilized in nonpregnant rats to induce β‐cell destruction causing features of diabetes. However, STZ is not ideal for pregnancy and leads to unsuccessful pregnancy outcomes, and, therefore other ways to establish animal models of GDM must be pursued. Previously, we have shown CD4+T cells from a rat model of preeclampsia causes HTN and mitochondrial (mt) dysfunction/ROS compared to normal pregnant (NP) rats. Therefore, we hypothesize CD4+ T cells cause pancreatic β‐islet cell destruction/dysfunction via mt dysfunction/ROS leading to HTN during pregnancy. To examine our hypothesis, we adoptively transferred CD4+ T cells from STZ Dahl diabetic rats to cause features of GDM in pregnant Sprague Dawley (SD) rats. Circulating CD4+ T cells were isolated from STZ induced diabetic Dahl virgin female rats and injected into pregnant SD rats on gestational day (GD) 12. On GD18 carotid catheters were inserted. On GD19, blood pressure (MAP) and tissues were collected and glucose levels were measured after 2h fasting in STZ CD4+ T cell recipients (GDM) and NP controls. Mt respiration and mtROS was measured in isolated mitochondria using an Oxygraph 2K and by production of H2O2 using a fluorescent microplate reader, respectively. A student’s t‐test was used for statistical analysis. On GD19, MAP increased to 106±4 mmHg (n=5, p&lt;0.05) in GMD pregnant rats compared to control NP rats 90±2 mmHg (n=4). Blood glucose levels were elevated in GDM rats (138 ± 6 mg/dl, n=5, p&lt;0.05) compared to NP controls (86 ± 7 mg/dl, n=4). Placental state 3 (27±3 vs 50.3±3 pmol/sec/mg, p&lt;0.05) respiration rates, indicative of ATP production, was reduced in GDM rats (n=5) cells compared to NP controls (n=4). Placental mtROS was significantly increased in GDM rats (266 ± 34 % gated, n=5, p&lt;0.05) compared to NP rats (100 ± 48 % gated, n=4). Collectively, the data indicate adoptive transfer of STZ CD4+ T cells causes increased circulating glucose, placental mt dysfunction and mtROS and HTN during pregnancy. These data demonstrate the importance of CD4+T cells in mechanisms causing the pathophysiology of GDM, and also introduces a potential novel animal model of GDM.

NLRP3 Inhibition Improves Maternal Blood Pressure, Inflammation, and Vascular Function During Placental Ischemia

Preeclampsia (PE) is a pregnancy, hypertensive disorder with end‐organ damage, that develops after 20 weeks of gestation. PE pathophysiology often includes vascular dysfunction and increased inflammation that continues to damage patient health even after their PE resolves. Currently, there are no treatments for PE beyond delivery of the fetal‐placental unit. Previous clinical studies have identified elevated NLRP3 expression in PE placentas and suggest NLRP3 as a potential therapeutic target. In this study, we examined the effects of NLRP3 inhibition on PE pathophysiology in the reduced uterine perfusion pressure (RUPP) 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‐helper17 cell (TH17) and cytolytic NK cell (cNK) activation, which is known to mediate oxidative stress and vascular dysfunction causing 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 to 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.

Placental CD4+ T cells from preeclamptic patients cause autoantibodies to the angiotensin II type I receptor and hypertension in a pregnant rat model of preeclampsia.

Preeclampsia (PE) is a hypertensive disorder of pregnancy associated with activated CD4+ T cells and autoantibodies to angiotensin II type 1 receptor (AT1-AA). We have previously shown that CD4+ T cells isolated from women with PE cause hypertension, increased tumor necrosis factor alpha (TNF-α), endothelin-1, and soluble fms-like tyrosine kinase-1 (sFlt-1) when injected into pregnant nude-athymic rats compared to CD4+ T cells from normal pregnant (NP) women. However, the role of PE CD4+ T cells to cause AT1-AA as a mechanism of hypertension is not known. Aim: Our goal was to determine if PE CD4+ T cells stimulate AT1-AA in pregnant nude-athymic rats. CD4+ T cells were isolated from human NP and PE placentasand injected into nude-athymic rats on gestational day (GD) 12. In order to examine the role of the PE CD4+ T cells to stimulate B cell secretion of AT1-AA, a subset of the rats receiving PE CD4+ T cells were treated with rituximab on GD 14 or anti-CD40 ligand (anti-CD40L) on GD 12. On GD 19, mean arterial pressure (MAP) and tissues were obtained MAP [114 ± 1 mmHg (n = 9)] and AT1-AA [19.8 ± 0.9 beats per minute (bpm, n = 4)] were increased in NP nude + PE CD4+ T cells compared to NP nude + NP CD4+ T cells [98 ± 2 mmHg (n = 7, P < 0.05) and 1.3 ± 0.9 bpm (n = 5, P < 0.05)]. Rituximab (103 ± 2 mmHg, n = 3, P < 0.05) and anti-CD40L (102 ± 1 mmHg, n = 3, P < 0.05) lowered MAP compared to NP nude + PE CD4+ T cells. Circulating a proliferation-inducing ligand (APRIL) and placental angiotensin-converting enzyme 2 (ACE-2) activity was increased in response to PE CD4+ T cells. These results show that placental CD4+ T cells play an important role in the pathophysiology of PE, by activating B cells secreting AT1-AA to cause hypertension during pregnancy.

Angiotensin II type 1 receptor agonistic autoantibody blockade improves postpartum hypertension and cardiac mitochondrial function in rat model of preeclampsia

Women with preeclampsia (PE) have a greater risk of developing hypertension, cardiovascular disease (CVD), and renal disease later in life. Angiotensin II type I receptor agonistic autoantibodies (AT1-AAs) are elevated in women with PE during pregnancy and up to 2-year postpartum (PP), and in the reduced uterine perfusion pressure (RUPP) rat model of PE. Blockade of AT1-AA with a specific 7 amino acid peptide binding sequence (‘n7AAc’) improves pathophysiology observed in RUPP rats; however, the long-term effects of AT1-AA inhibition in PP is unknown. Pregnant Sprague Dawley rats were divided into three groups: normal pregnant (NP) (n = 16), RUPP (n = 15), and RUPP + ‘n7AAc’ (n = 16). Gestational day 14, RUPP surgery was performed and ‘n7AAc’ (144 μg/day) administered via osmotic minipump. At 10-week PP, mean arterial pressure (MAP), renal glomerular filtration rate (GFR) and cardiac functions, and cardiac mitochondria function were assessed. MAP was elevated PP in RUPP vs. NP (126 ± 4 vs. 116 ± 3 mmHg, p < 0.05), but was normalized in in RUPP + ‘n7AAc’ (109 ± 3 mmHg) vs. RUPP (p < 0.05). PP heart size was reduced by RUPP + ’n7AAc’ vs. RUPP rats (p < 0.05). Complex IV protein abundance and enzymatic activity, along with glutamate/malate-driven respiration (complexes I, III, and IV), were reduced in the heart of RUPP vs. NP rats which was prevented with ‘n7AAc’. AT1-AA inhibition during pregnancy not only improves blood pressure and pathophysiology of PE in rats during pregnancy, but also long-term changes in blood pressure, cardiac hypertrophy, and cardiac mitochondrial function PP.

Progesterone Induced Blocking Factor Reduces Hypertension and Placental Mitochondrial Dysfunction in Response to sFlt-1 during Pregnancy.

Preeclampsia (PE) is characterized by new onset hypertension in association with placental ischemia, reduced fetal weight, elevated soluble fms-like tyrosine kinase-1 (sFlt-1), and placental mitochondrial (mt) dysfunction and oxidative stress (ROS). Progesterone induced blocking factor (PIBF) is a product of progesterone signaling that blocks inflammatory processes and we have previously shown PIBF to lower mean arterial blood pressure (MAP) and sFlt-1 in a rat model of PE. Infusion of sFlt-1 causes hypertension and many characteristics of PE in pregnant rodents, however, its role in causing mt dysfunction is unknown. Therefore, we hypothesize that PIBF will improve mt function and MAP in response to elevated sFlt-1 during pregnancy. We tested our hypothesis by infusing sFlt-1 via miniosmotic pumps in normal pregnant (NP) Sprague-Dawley rats (3.7 μg·kg−1·day−1) on gestation days (GD) 13–19 in the presence or absence of PIBF (2.0 µg/mL) injected intraperitoneally on GD 15 and examined mean arterial blood pressure (MAP) and placental mt ROS on GD 19. sFlt-1 increased MAP to 112 + 2 (n = 11) compared to NP rats (98 + 2 mmHg, n = 15, p < 0.05), which was lowered in the presence of sFlt-1 (100 + 1 mmHg, n = 5, p < 0.05). Placental mtATP was reduced in sFlt-1 infused rats versus NP controls, but was improved with PIBF. Placental mtROS was elevated with sFlt-1 compared to NP controls, but was reduced with PIBF. Sera from NP + sFlt-1 increased endothelial cell mtROS, which was attenuated with PIBF. These data demonstrate sFlt-1 induced HTN during pregnancy reduces placental mt function. Importantly, PIBF improved placental mt function and HTN, indicating the efficacy of improved progesterone signaling as potential therapeutics for PE.

Omentin-1 and its relationship with inflammatory factors in maternal plasma and visceral adipose tissue of women with gestational diabetes mellitus.

To investigate the association of omentin-1 and inflammatory factors in serum and visceral adipose tissue (VAT) of women with gestational diabetes mellitus (GDM) compared to normal pregnant (NP) subjects. Furthermore, to examine their correlation with maternal clinical characteristics. We compared 116 GDM women to 115 NP women, at the time of cesarean section. Circulating omentin-1 and pro-inflammatory (IL-1β, IL-6, TNF-α), and anti-inflammatory cytokines (IL-1RA, IL-10) were examined. Moreover, their mRNA expression in VAT, along with inflammatory factors involved in the NF-κB pathway (TLR2, TLR4, NF-κB, IKκB), were examined. Circulating omentin-1 (p = 0.022) was lower and circulating IL-1-β, IL-1RA, as well as IL-10 (p = 0.005, p = 0.007, and p = 0.015, respectively), were higher in GDM compared to NP women. Omentin-1 correlated negatively with pre-pregnancy and gestational BMI, and HOMA-IR in all women, but was not associated with cytokines. TLR2, TLR4, IL-1β, IL-1RA, IL-6, IL-10 mRNA expression in VAT was lower in GDM compared with controls (p < 0.05 all). In multivariate analysis, BMI at delivery was significantly correlated to omentin-1 concentrations in all and NP subjects. In addition, omentin-1 expression was correlated to inflammatory gene expression in all, GDM and NP, women (p < 0.05 all). Serum levels and VAT gene expression of omentin-1 are not independently linked to GDM; notwithstanding, GDM women have a VAT-altered inflammatory status. In addition, no systemic association between omentin-1 and inflammatory factors was found, whereas associations between their expression in all women were observed, indicating that expression of these adipokines is linked between them regardless of GDM.

Abstract 61: Cd4+t Cells Cause Hypertension, Increased Glucose, And Mitochondrial Dysfunction In A Novel Rodent Model Of Gestational Diabetes Mellitus

Hypertensive (HTN) disorders of pregnancy increase risks for gestational diabetes mellitus (GDM) in pregnant women. GDM is characterized by hyperglycemia and β-cell dysfunction associated with increased inflammatory cytokines, oxidative stress, and activated CD4+ T cells. Streptozotocin (STZ) is used in nonpregnant rats to induce β-cell destruction causing features of diabetes. However, STZ is not ideal for pregnancy and leads to unsuccessful pregnancy outcomes, therefore other ways to establish animal models of GDM must be pursued. Previously, we showed CD4+T cells from a rat model of preeclampsia causes HTN and mitochondrial (mt) dysfunction/ROS compared to normal pregnant (NP) rats. Therefore, we hypothesize CD4+ T cells from a diabetic rat model could cause mt dysfunction/ROS and pancreatic β-islet cell destruction and lead to increased glucose and HTN during pregnancy. To examine our hypothesis, we adoptively transferred CD4+ T cells from STZ Dahl diabetic rats into pregnant Sprague Dawley (SD) rats and measured GDM features. Circulating CD4+ T cells were isolated from STZ induced diabetic Dahl virgin female rats and injected into pregnant SD rats on gestational day (GD) 12. On GD19, blood pressure (MAP) and tissues were collected and glucose levels were measured after 2h fasting in STZ CD4+ T cell recipients (GDM) and NP controls. Mt respiration and mtROS was measured in isolated mitochondria. On GD19, MAP increased to 105±0.5 mmHg (n=4, p&lt;0.05) in GMD pregnant rats compared to control NP rats 91±2.1 mmHg (n=3). Blood glucose levels were elevated in GDM rats (139 ± 7 mg/dl, n=4, p&lt;0.05) compared to NP controls (94 ± 1 mg/dl, n=3). Placental state 3 (26.4±5.9 vs 53.9±1.7 pmol/sec/mg, p&lt;0.05) respiration rates, indicative of ATP production, was reduced in GDM rats (n=4) compared to NP controls (n=3). Placental mtROS was significantly increased in GDM rats (190 ± 27.1 % gated, n=3, p&lt;0.05) compared to NP rats (100 ± 2.7 % gated, n=3). Collectively, the data indicate adoptive transfer of STZ CD4+ T cells causes increased circulating glucose, placental mt dysfunction and mtROS and HTN during pregnancy. These data demonstrate the importance of CD4+T cells in mechanisms causing the pathophysiology of GDM, and also introduces a potential novel animal model of GDM.