Reduced Uterine Perfusion Pressure Procedure Research Articles

AT1-AA Is Produced in Offspring in Response to Placental Ischemia and Is Lowered by B-Cell Depletion Without Compromising Overall Offspring Health.

Preeclampsia, new-onset hypertension during pregnancy alongside other organ dysfunction, is the leading cause of mortality for the mother and low birth weight for the baby. Low birth weight contributes to high risk of cardiovascular disorders later in life. Women with preeclampsia have activated B cells producing agonistic autoantibodies to AT1-AA (angiotensin II type I receptor). We hypothesize that rituximab, a B cell-depleting chemotherapeutic, will deplete maternal B cells in reduced uterine perfusion pressure (RUPP) rats without worsening the effect of placental ischemia on pup growth and survival. To test this hypothesis, the RUPP procedure was performed, and rituximab was continuously infused via miniosmotic pump. Maternal blood and tissues were collected. A separate group of dams were allowed to deliver, pup weights were recorded, and at 4 months of age, tissues were collected from offspring. Immune cells were measured via flow cytometry, and AT1-AA was quantified using a contraction bioassay. Blood pressure increased in RUPP rats and was normalized with rituximab treatment. RUPP offspring also had increased circulating B cells, cytolytic natural killer cells, and increased circulating AT1-AA, which were normalized with maternal rituximab treatment. This is the first study to analyze the AT1-AA in RUPP offspring, which was normalized with rituximab. Our findings indicate that perinatal rituximab lowers maternal mean arterial pressure in RUPP rats and improves birth weight, circulating AT1-AA, and circulating natural killer cells, indicating that rituximab improves adverse fetal outcomes in response to placental ischemia.

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.

Photoacoustic imaging provides an in vivo assessment of the preeclamptic placenta remodeling and function in response to therapy

IntroductionThere is a lack of effective therapeutic interventions for preeclampsia. A central factor in the etiology of the disease is the development of placental hypoxia due to abnormal vascular remodeling. However, methods to assess the impact of potential therapies on placental growth and remodeling are currently lacking. Here, we develop and validate ultrasound-guided photoacoustic imaging methods to monitor the placental response to therapeutic intervention. Establishing non-invasive tools to image placental function opens up previously unachievable understandings of placental therapeutic response. MethodsStudies were performed in the reduced uterine perfusion pressure (RUPP) rat model of preeclampsia. Preclinical research has identified tempol, a superoxide dismutase mimetic, and the phosphodiesterase inhibitor sildenafil as potential therapeutics for preeclampsia, as both improve in vivo maternal outcomes. PA images of the placental environment were acquired in RUPP rats receiving tempol (n = 8) or sildenafil (n = 8) to assess the longitudinal effects of treatment on placental oxygenation and vascular remodeling. Imaging measurements were validated with ex vivo histological analysis. ResultsSpectral photoacoustic imaging non-invasively measured placental hypoxia and impaired vascular growth two days after the RUPP procedure was implemented. Sildenafil significantly improved (p < 0.05) placental oxygenation and promoted vascular remodeling in RUPP animals, while RUPP animals treated with tempol had a diminished placental therapeutic response. DiscussionWe demonstrate that photoacoustic imaging provides in vivo measures of placental oxygenation and vascular remodeling, a previously unobtainable assessment of preeclamptic therapeutic response. These imaging tools have tremendous potential to accelerate the search for effective therapies for preeclampsia.

Maternal B Cell Depletion Reduces Blood Pressure and Improves Fetal Weights in Male Offspring of a Rat Model of Preeclampsia

Preeclampsia (PE), new onset hypertension during pregnancy, is the leading cause of death and morbidity for the mother and low birth weight in offspring. Low birth weight has been shown to cause offspring to be at high risk for cardiovascular and metabolic disorders later in life. PE has been associated with chronic immune activation including T helper cells and natural killer cells. PE women also have activated B cells producing agonistic autoantibodies to the angiotensin II type I receptor (AT1‐AA). AT1‐AA has been implicated in numerous pathways in the pathophysiology of PE. We have shown Rituximab (R), used clinically for B cell depletion, lowers mean arterial pressure (MAP), B cells, and AT1‐AA in the reduced uterine perfusion pressure (RUPP) rat model of PE. Clinical studies show no untoward effects on offspring of pregnant women maintained on R for treating lymphoma. R is not used during PE, therefore, effects of maternal B cell depletion on offspring survival and growth in response to placental ischemia is unknown. We hypothesize that R will deplete maternal B cells in RUPP rats without worsening the effect of placental ischemia on pup growth and survival. To test this hypothesis, the RUPP procedure was performed and R (250 mcg/kg) was given on gestation day (GD) 14 via mini‐osmotic pump. On GD 19, B cells were measured by flow cytometry, and MAP and pup weights were recorded. A separate group of dams were allowed to deliver, pup weights were recorded within 12 hours of birth and weekly until 16 weeks, and B cells were analyzed. A one‐way or two‐way ANOVA was used for statistical analysis. MAP increased in RUPP 123±2 (n=19, p&lt;0.05) compared to NP controls 101±1 (n=18) and was 106±3 mmHg in RUPP+R (n=8, p&lt;0.05). On GD19, maternal circulating B cells were 16±2 % (n=14) in RUPPs, 8±2 % in NP rats, (n=7, p&lt;0.05), and 5.5±1% gate in RUPP+R (n=5, p&lt;0.05). RUPP male and female offspring tended to be smaller (5.11±0.23 g, 5.19±0.14 g; n=4, n=4) at birth than NP offspring (6.09±0.15 g, 5.87±0.12 g; n=6, p&lt;0.064; n=6, p&lt;0.948) or RUPP+R offspring (5.75±0.24 g, 5.36±0.28 g; n=6, p=0.291; n=6, p&gt;0.999). At 12 weeks, male and female RUPP offspring had elevated circulating B cells (21.24±2.92, 20.04±0.72 %; n=6; n=9) compared to NP (1.38±0.23, 1.63±0.06 %; n=4, p&lt;0.05; n=3, p&lt;0.04) which was normalized in RUPP+R offspring (0.40±0.10, 0.27±0.03 % gate; n=3, p&lt;0.05; n=8, p&lt;0.05). At 16 weeks, B cells were comparable in male and female offspring from NP (0.78±0.09 %, n=10; 1.06±0.21 % gate, n=6) and RUPP+R rats (1.21±0.34% gate, n=5; 1.62±0.34% gate, n=5), but were elevated in RUPP male and female offspring (2.45±0.27% gate, n=9; 1.68±0.45% gate, n=13) compared to NP (p&lt;0.05; p&gt;0.99) and RUPP+R (p&lt;0.05; p&gt;0.99). Our findings indicate that R lowers maternal circulating B cells and MAP in RUPP rats and improves fetal weight and circulating B cells, indicating that R does not worsen adverse fetal outcomes in response to placental ischemia.

Abstract P225: Inhibiting B Cell Activating Factor Normalizes The Hypertension And Cytolytic Natural Killer Cells Associated With Placental Ischemia

Preeclampsia (PE) is new onset hypertension (HTN) during pregnancy and is associated with a pro-inflammatory state with increased T Cells, B Cells, cytolytic Natural Killer (cNK) Cells, and Agonistic Antibodies to the Angiotensin II Type 1 Receptor (AT1-AA) compared to Normal Pregnancy (NP). The Reduced Uterine Perfusion Pressure (RUPP) model of placental ischemia recapitulates these features. We have shown that blocking communication between T Cell CD40L and B Cell CD40 or B Cell depletion prevents HTN and AT1-AA in rat models of placental ischemia induced HTN. This suggests that T Cell dependent B Cell activation is involved in the HTN and AT1-AA seen in PE. B2 B cells maturing into antibody producing plasma cells are the product of T Cell Dependent B Cell Interactions. B Cell activating factor (BAFF) is an integral cytokine in the development of B2 cells specifically. Therapeutic antibodies against BAFF are used in treatment for Systemic Lupus Erythematosus with long-term treatment with Anti-BAFF antibodies reducing patient circulating B cells. Thus, we hypothesize that Anti-BAFF antibodies will selectively deplete B2 cells, therefore reducing HTN and cNK Cells in the RUPP rat model of PE. To test this hypothesis, Gestation Day (GD) 14 pregnant rats underwent the RUPP procedure and some were treated with 1 mg/kg Anti-BAFF antibodies by infusion via jugular catheters inserted 24 hours prior. On GD19, blood pressure (MAP) was measured and circulating, placental, and splenic B Cells and NK cells were measured by flow cytometry. RUPP rats had elevated MAP (119±2 mmHg, n=12) compared to NP (100±3 mmHg, n=9) which was normalized by Anti-BAFF treatment (100±3 mmHg, n=11)(p&lt;0.05). RUPP fetuses were smaller at GD19 (1.97±0.08 g; n=12) compared to NP fetuses (2.207±0.1 g)(p&lt;0.05), and this was unchanged with Anti-BAFF (1.82±0.1 g). RUPP rats had elevated cNK cells (9.1±2%, n=7) compared to NP (3.1±3%, n=3) which was attenuated with anti-BAFF therapy (3.6±1.3%, n=6). RUPP rats had elevated splenic B Cells (4±0.7%, n=8) compared to NP (2±0.7%, n=4) which was reduced by Anti-BAFF (2.8%±1.1%, n=4). In conclusion, Anti-BAFF treatment reduces B cells and cNK cells and normalizes the HTN associated with placental ischemia without worsening fetal weight in a rat model of PE.

Interferon γ neutralization reduces blood pressure, uterine artery resistance index, and placental oxidative stress in placental ischemic rats.

Preeclampsia (PE) is characterized by maternal hypertension, intrauterine growth restriction, and increased cytolytic natural killer cells (cNKs), which secrete interferon γ (IFNγ). However, the precise role of IFNγ in contributing to PE pathophysiology remains unclear. Using the reduced uterine perfusion pressure (RUPP) rat model of placental ischemia, we tested the hypothesis that neutralization of IFNγ in RUPPs will decrease placental reactive oxygen species (ROS) and improve vascular function resulting in decreased MAP and improved fetal growth. On gestation day (GD) 14, the RUPP procedure was performed and on GDs 15 and 18, a subset of normal pregnant rats (NP) and RUPP rats were injected with 10 μg/kg of an anti-rat IFNγ monoclonal antibody. On GD 18, uterine artery resistance index (UARI) was measured via Doppler ultrasound and on GD 19, mean arterial pressure (MAP) was measured, animals were euthanized, and blood and tissues were collected for analysis. Increased MAP was observed in RUPP rats compared with NP and was reduced in RUPP + anti-IFNγ. Placental ROS was also increased in RUPP rats compared with NP rats and was normalized in RUPP + anti-IFNγ. Fetal and placental weights were reduced in RUPP rats, but were not improved following anti-IFNγ treatment. However, UARI was elevated in RUPP compared with NP rats and was reduced in RUPP + anti-IFNγ. In conclusion, we observed that IFNγ neutralization reduced MAP, UARI, and placental ROS in RUPP recipients. These data suggest that IFNγ is a potential mechanism by which cNKs contribute to PE pathophysiology and may represent a therapeutic target to improve maternal outcomes in PE.

Impact of hyperleptinemia during placental ischemia-induced hypertension in pregnant rats.

The prevalence of preeclampsia and obesity have increased. Although obesity is a major risk factor for preeclampsia, the mechanisms linking these morbidities are poorly understood. Circulating leptin levels increase in proportion to fat mass. Infusion of this adipokine elicits hypertension in nonpregnant rats, but less is known about how hyperleptinemia impacts blood pressure during placental ischemia, an initiating event in the pathophysiology of hypertension in preeclampsia. We tested the hypothesis that hyperleptinemia during reduced uterine perfusion pressure (RUPP) exaggerates placental ischemia-induced hypertension. On gestational day (GD) 14, Sprague-Dawley rats were implanted with osmotic mini-pumps delivering recombinant rat leptin (1 µg/kg/min iv) or vehicle concurrently with the RUPP procedure to induce placental ischemia or Sham. On GD 19, plasma leptin was elevated in Sham + Leptin and RUPP + Leptin. Leptin infusion did not significantly impact mean arterial pressure (MAP) in Sham. MAP was increased in RUPP + Vehicle vs. Sham + Vehicle. In contrast to our hypothesis, placental ischemia-induced hypertension was attenuated by leptin infusion. To examine potential mechanisms for attenuation of RUPP-induced hypertension during hyperleptinemia, endothelial-dependent vasorelaxation to acetylcholine was similar between Sham and RUPP; however, endothelial-independent vasorelaxation to the nitric oxide (NO)-donor, sodium nitroprusside, was increased in Sham and RUPP. These findings suggest that NO/cyclic guanosine monophosphate (cGMP) signaling was increased in the presence of hyperleptinemia. Plasma cGMP was elevated in Sham and RUPP hyperleptinemic groups compared with vehicle groups but plasma and vascular NO metabolites were reduced. These data suggest that hyperleptinemia during placental ischemia attenuates hypertension by compensatory increases in NO/cGMP signaling.NEW & NOTEWORTHY Ours is the first study to examine the impact of hyperleptinemia on the development of placental ischemia-induced hypertension using an experimental animal model.

CD4+ T cells cause renal and placental mitochondrial oxidative stress as mechanisms of hypertension in response to placental ischemia.

The reduced uterine perfusion pressure (RUPP) rat model and normal pregnant (NP) rat recipients of RUPP CD4+ T cells recapitulate many characteristics of preeclampsia such as hypertension and oxidative stress. We have shown an important hypertensive role for natural killer (NK) cells to cause mitochondrial dysfunction in RUPP rats; however, the role for RUPP CD4+ T cells to stimulate NK cells is unknown. Therefore, we hypothesized that RUPP-induced CD4+ T cells activate NK cells to cause mitochondrial dysfunction/reactive oxygen species (ROS) as mechanisms of hypertension during pregnancy. We tested our hypothesis by adoptive transfer of RUPP CD4+T cells into NP rats or by inhibiting the activation of RUPP CD4+T cells with Orencia (abatacept) and examining hypertension, NK cells, and mitochondrial function. RUPP was performed on gestation day (GD) 14, and splenic CD4+ T cells were isolated on GD 19 and injected into NP rats on GD 13. In a separate group of rats, Orencia was infused and the RUPP procedure was performed. Mean arterial pressure and placental and renal mitochondrial ROS increased in RUPP (n = 7, P < 0.05) and NP+RUPP CD4+ T-cell recipients (n = 13, P < 0.05) compared with control NP (n = 7) and NP+NP CD4+ T-cell recipients (n = 5) but was reduced with Orencia (n = 13, P < 0.05). Placental and renal respiration was reduced in RUPP (n = 6, P < 0.05) and NP+RUPP CD4+ T-cell recipients (n = 6, state 3 P < 0.05) compared with NP (n = 5) and NP+NP CD4+ T-cell recipients (n = 5) but improved with Orencia (n = 9, n = 8 P < 0.05). These data indicate that CD4+ T cells, independent of NK cells, cause mitochondrial dysfunction/ROS contributing to hypertension in response to placental ischemia during pregnancy.

Abstract P208: Imaging The Longitudinal Reduction In Placental Ischemia In Response To Therapeutic Intervention For Preeclampsia

Preeclampsia is believed to be induced by abnormal vascular remodeling and the resulting placental ischemia during early development. Sildenafil, a phosphodiesterase-5 inhibitor, reduces the maternal symptoms of preeclampsia by promoting angiogenesis and enhancing NO-mediated vasodilation. However, the effect of sildenafil on in vivo placental function has not been demonstrated. We performed longitudinal spectral photoacoustic (sPA) imaging of placental therapeutic response to sildenafil in the reduced uterine perfusion pressure (RUPP) model of preeclampsia. Sprague Dawley rats were administered sildenafil (S) via drinking water beginning on gestational day (GD) 11. Imaging was performed on GD14, 16, and 18 with the RUPP procedure implemented after the first imaging session. sPA images of oxygenation show that the placental ischemia induced by the RUPP surgery (RUPP, n=8, 48%; NP, n=8, 54%), was effectively eliminated by treatment with sildenafil (RUPP+S, n=8, 53%, p&lt;0.05). In addition to improved placental oxygenation, sildenafil was also found to reduce mean arterial pressure in RUPP animals by GD18 (RUPP, 110 mmHg; RUPP+S, 98 mmHg; p&lt;0.05), consistent with prior reports. Our studies demonstrate that sPA imaging can detect changes in placental oxygenation which could be used to indicate in vivo placental therapeutic response. Figure 1: Placental hypoxia in the RUPP is improved by treatment with sildenafil on GD16. B-mode US images (a, c) of anatomy were used to manually select the placenta (p) and average placental oxygenation was calculated. A custom red-blue oxygenation colormap was then overlaid for visualization (b, d). Scale bars = 3mm.

Hypertension Editors' Picks Preeclampsia.

Hypertension Editors' Picks Preeclampsia.

LTP Nanoparticles Seeded with Plasmid for Vascular Endothelial Growth Factor Receptor 2 Improve Fetal Morphometrics in a Rat Model of Preeclampsia

Recent studies by our lab have demonstrated that the delivery of L‐Tyrosine Phosphate nanoparticles seeded with plasmid for Vascular Endothelial Growth Factor Receptor 2 (LTP VEGFR2) normalizes mean arterial pressure, uterine vessel myogenic reactivity, and fetal growth restriction in the reduced uterine perfusion pressure (RUPP) animal model. The goal of this study was to investigate fetal morphometrics in RUPP+LTP VEGFR2 dams. For the RUPP procedure, silver clips are placed on the abdominal aorta (0.2mm i.d.) below the kidneys and the utero‐ovarian arteries (0.1mm i.d.) on day 14 of pregnancy in Sprague‐Dawley rats. SHAM pregnant rats undergo the same surgery without the clip placement. On the same day as surgery, LTP nanoparticles encapsulated with plasmid DNA encoding for VEGF Receptor 2 (VEGFR2), or nonsense were injected into the uterine wall. On day 21 of gestation, the uterus was removed and fetal weights, placental weights, and head circumference were recorded on three fetuses from each dam. The fetuses were selected based on position in the uterus in relation to the site of injection of the LTP nanoparticles. Fetal position was designated as the following: Site 1 was selected as the closest to the injection site, Site 6 was distal in the same uterine horn, and Site 14 was contralateral to Site 6. Fetal weight for pups at Site 1 was significantly increased in RUPP rats treated with VEGFR2 nanoparticles compared to RUPP treated with nonsense nanoparticles (4.70g ± 0.44 vs. 3.89g ± 0.31; p&lt;0.05). Placental weight at Site 1 was significantly increased in RUPP rats treated with VEGFR2 nanoparticles compared to RUPP treated with nonsense nanoparticles (0.635g ± 0.06 vs. 0.491g ± 0.04; p&lt;0.05). Head circumference was also significantly increased in pups at site 1 in RUPP rats treated with VEGFR2 nanoparticles as compared RUPP rats treated with nonsense nanoparticles (2.37cm ± 0.32 vs. 1.45cm ± 0.07; p&lt;0.05). There were no significant differences in fetal weights, placental weights and head circumference in the SHAM group at any of the fetal positions. Furthermore, there were no significant differences when comparing between the SHAM and RUPP groups. The fetal measurements (fetal weight, placental weight and fetal head circumference) from RUPP treated with nonsense nanoparticles tended to be lower compared to the other groups (SHAM treated with VEGFR2 nanoparticles, SHAM treated with nonsense nanoparticles or RUPP treated with VEGFR2 nanoparticles; however, it failed to reach significance in this preliminary study. In conclusion, uterine injection of LTP nanoparticles with plasmid DNA encoding for VEGFR2 improved fetal morphometrics in RUPP animals.Support or Funding InformationAmerican Heart Association‐18AIREA3396032This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Melanocortin-4 Receptor Deficiency Attenuates Placental Ischemia-Induced Hypertension in Pregnant Rats.

Preeclampsia is a pregnancy-specific disorder of new-onset hypertension linked to placental ischemia. While obesity is a major risk factor for preeclampsia, not all obese pregnant women develop pregnancy-induced hypertension or preeclampsia. Previously, we reported that placental ischemia-induced hypertension is dependent upon intact signaling of the sympathetic nervous system. Moreover, in various models of obesity, blockade of MC4R (melanocortin-4 receptor) signaling protects against the development of hypertension via suppression of the sympathetic nervous system. Less is known about this pathway during obese pregnancy. Although blockade of MC4R may lead to increased body weight during pregnancy, we tested the hypothesis that placental ischemia-induced hypertension is attenuated in obese MC4R-deficient pregnant rats. On gestational day 14, MC4R wild-type or heterozygous-deficient (MC4R-def) rats were subjected to chronic placental ischemia via the reduced uterine perfusion pressure procedure or Sham surgery then examined on gestational day 19. In Sham MC4R-def versus Sham wild-type pregnant rats, there was increased body weight, fat mass, and circulating leptin levels but they had similar fetus weights. Reduced uterine perfusion pressure reduced fetus weights in both strains. Reduced uterine perfusion pressure increased blood pressure in wild-type rats but this response was significantly attenuated in MC4R-def rats, although blood pressure was elevated in Sham MC4R-def over Sham wild-type. These data indicate that while obese MC4R-def pregnant rats have higher blood pressure during pregnancy, placental ischemia-induced hypertension is attenuated in obese MC4R-def pregnant rats. Thus, obese women with abnormal MC4R signaling may be less susceptible to the development of placental ischemia-induced hypertension.

Therapeutic Effect of Long-Term Epidural Block in Rats with Pregnancy Induced Hypertension

Background Pregnancy induced hypertension (PIH) causes a variety of systemic disorders that negatively affect the maternal placenta and fetal growth. Epidural sympathetic block elicits symptoms of decreased blood pressure. This study was designed to determine the therapeutic effect of long-term epidural block in rats with PIH. Methods Forty healthy pregnant Sprague Dawley rats were randomized into four groups with each group consisting of 10 rats. On gestation day (GD) 14, rats in control group underwent a sham procedure; rats in RUPP group were operated on to obtain reduced uterine perfusion pressure (RUPP); rats in RUPP plus normal saline (NS) group were also subjected to the RUPP procedure and underwent epidural block with 25 μl normal saline twice daily until delivery; rats in RUPP plus epidural block (EB) group were treated as those in RUPP plus NS group except that an epidural block with 25 μl of 0.125% bupivacaine was administered two times per day until delivery. On GD 20, blood pressure was measured in all groups before delivery, and blood samples were collected in order to quantify the serum concentrations of vascular endothelial growth factor (VEGF) and soluble fms-like tyrosine kinase 1 (sFlt-1). Results The mean arterial pressure (MAP) of rats in RUPP group (147.6 ± 6.0 mmHg) was markedly increased when compared with control group (80.8 ± 4.6 mmHg) (p < 0.05). The MAP of rats in RUPP plus EB group (114.4 ± 7.2 mmHg) was clearly decreased in contrast with RUPP group but was still higher than in control group (p < 0.05). The variation of fetal weight in all groups followed a similar trend to that of MAP. However, there were no significant differences between control group and RUPP plus EB group with respect to placental weight (p = 0.186). Variation in MAP was positively correlated with the expression of sFlt-1 in each group but was negatively correlated with VEGF. Conclusion This study demonstrates that long-term epidural block decreases blood pressure in PIH rats and improves the serum concentrations of VEGF and sFlt-1. Taken together, long-term epidural block may have a potential role in PIH treatment.

Molecular hydrogen ameliorates several characteristics of preeclampsia in the Reduced Uterine Perfusion Pressure (RUPP) rat model

Oxidative stress plays an important role in the pathogenesis of preeclampsia. Recently, molecular hydrogen (H2) has been shown to have therapeutic potential in various oxidative stress-related diseases. The aim of this study is to investigate the effect of H2 on preeclampsia. We used the reduced utero-placental perfusion pressure (RUPP) rat model, which has been widely used as a model of preeclampsia. H2 water (HW) was administered orally ad libitum in RUPP rats from gestational day (GD) 12–19, starting 2 days before RUPP procedure. On GD19, mean arterial pressure (MAP) was measured, and samples were collected. Maternal administration of HW significantly decreased MAP, and increased fetal and placental weight in RUPP rats. The increased levels of soluble fms-like tyrosine kinase-1 (sFlt-1) and diacron reactive oxygen metabolites as a biomarker of reactive oxygen species in maternal blood were decreased by HW administration. However, vascular endothelial growth factor level in maternal blood was increased by HW administration. Proteinuria, and histological findings in kidney were improved by HW administration. In addition, the effects of H2 on placental villi were examined by using a trophoblast cell line (BeWo) and villous explants from the placental tissue of women with or without preeclampsia. H2 significantly attenuated hydrogen peroxide-induced sFlt-1 expression, but could not reduce the expression induced by hypoxia in BeWo cells. H2 significantly attenuated sFlt-1 expression in villous explants from women with preeclampsia, but not affected them from normotensive pregnancy. The prophylactic administration of H2 attenuated placental ischemia-induced hypertension, angiogenic imbalance, and oxidative stress. These results support the theory that H2 has a potential benefit in the prevention of preeclampsia.

79 Renal and cardiac disturbances 8 weeks after exposure to placental ischemia in rat dams

Introduction Women with a history of preeclampsia (PE) have an increased risk to develop cardiovascular and renal disease later in life. Pre-existing cardiovascular risk factors may play an important role, on the other hand the transient presence of placenta ischemia might induce persistent changes in the maternal cardiovascular system. Objective We aimed to assess whether exposure to placental ischemia during pregnancy enhances long-term effects on the maternal cardiovascular and renal systems in rats using the Reduced Uterine Perfusion Pressure (RUPP) model for PE. Methods At gestational day (gd) 14, Sprague Dawley rats were randomized to two groups: SHAM (n = 12) or RUPP (n = 21). The RUPP procedure involved partial clipping of the lower abdominal aorta and both ovarian arteries to reduce placental flow. Post delivery, rats were followed for 8 weeks after which we measured mean arterial pressure (MAP, carotid), GFR (transcutaneous, sinistrin), albuminuria (24-h) and cardiac function (cardiac ultrasound). All rats were sacrificed for tissue collection and mesenteric arteries were used for reactivity experiments to assess peripheral arterial function. Parametric data are presented as mean ± SD and non-parametric data as median (25–75 percentile), and were respectively analyzed with student T-test or Mann–Whitney U-test. Morphologic and transcriptional analyses are ongoing. Results The mean weight gain of the RUPP rats during pregnancy (gd13–gd19) was significantly lower compared to the SHAM operated rats [30 ± 18 g vs 63 ± 12; p Conclusions We show that exposure to experimental placenta ischemia is accompanied by subtle disturbances in maternal cardiac and renal function after recovery from PE. Our results indicate that placental ischemia itself can result in persistent changes in the cardiac and renal systems, although at this early stage no differences in vascular reactivity or blood pressure regulation were observed. Future studies should elucidate whether aging, a second hit or underlying disease aggravate target organ dysfunction after placental ischemia. Results Despite being all followed up for 27.5 yrs, the exposed women were older when they had their first pregnancy with and median age of 24.9 yrs ((5th;95th percentiles) (19.5;27.3)) vs. 24.6 yrs (19.4;27.3), p Conclusion A small extra vitamin D in fetal life from food fortification reduced risk of PE later in life. The effect was mainly seen among current smokers.

An increased population of regulatory T cells improves the pathophysiology of placental ischemia in a rat model of preeclampsia.

The reduced uterine perfusion pressure (RUPP) rat model of preeclampsia exhibits much of the pathology characterizing this disease, such as hypertension, inflammation, suppressed regulatory T cells (TRegs), reactive oxygen species (ROS), and autoantibodies to the ANG II type I receptor (AT1-AA) during pregnancy. The objective of this study was to determine whether supplementation of normal pregnant (NP) TRegs into RUPP rats would attenuate the pathophysiology associated with preeclampsia during pregnancy. CD4(+)/CD25(+) T cells were isolated from spleens of NP and RUPP rats, cultured, and injected into gestation day (GD) 12 normal pregnant rats that underwent the RUPP procedure on GD 14. On GD 1, mean arterial pressure (MAP) was recorded, and blood and tissues were collected for analysis. One-way ANOVA was used for statistical analysis. MAP increased from 99 ± 2 mmHg in NP (n = 12) to 127 ± 2 mmHg in RUPP (n = 21) but decreased to 118 ± 2 mmHg in RUPP+NP TRegs (n = 17). Circulating IL-6 and IL-10 were not significantly changed, while circulating TNF-α and IL-17 were significantly decreased after supplementation of TRegs. Placental and renal ROS were 339 ± 58.7 and 603 ± 88.1 RLU·min(-1)·mg(-1) in RUPP and significantly decreased to 178 ± 27.8 and 171 ± 55.6 RLU·min(-1)·mg(-1), respectively, in RUPP+NP TRegs; AT1-AA was 17.81 ± 1.1 beats per minute (bpm) in RUPP but was attenuated to 0.50 ± 0.3 bpm with NP TRegs. This study demonstrates that NP TRegs can significantly improve inflammatory mediators, such as IL-17, TNF-α, and AT1-AA, which have been shown to increase blood pressure during pregnancy.

Inhibition of 20‐HETE Synthesis Attenuates Elevations in Blood Pressure and Uterine Artery Resistance Index in the RUPP Rat Model of Preeclampsia

Preeclampsia (PE) is a hypertensive disorder of pregnancy afflicting 5-10% of US pregnancies. Recent data suggests the vasoactive cytochromeP450 metabolite, 20-hydroxyeicosatetraenoic (20-HETE), may play a role in hypertension in response to placental ischemia. Thus, we hypothesized that administration of HET0016, a specific 20-HETE synthesis inhibitor, would improve the pathophysiology in the reduced uterine perfusion pressure (RUPP) rat model of placental ischemia. RUPP procedure was performed on gestational day (GD) 14 and HET0016 (1mg/kg, i.p) was administered on GD14-18. Uterine Artery Resistance Index (UARI) was measured via sonography on GD18 and blood pressure (MAP) was measured on day GD19. UARI increased in RUPP compared to NP rats (0.71±0.04 vs 0.60 ±0.02), and was reduced in RUPP+HET0016 treated animals (0.56±0.07). Pup reabsorption (% of total pups at GD19) was significantly increased in RUPP versus NP rats (58±6 vs 7±6%, P<0.05). Treatment with HET0016 significantly reduced pup resorption in RUPP+HET0016 rats relative to control (21±7%, P<0.05). MAP was elevated in RUPP rats compared to NP rats (122±3 vs 104±3 mmHg, P<0.05), and was significantly decreased upon administration of HET0016 (112 ±3 mmHg, P<0.05). MAP remained unchanged in NP+HET0016 rats (106±5 mmHg). In conclusion, inhibition of 20-HETE synthesis prevented increases in UARI, fetal reabsorption, and blood pressure in the RUPP rat model of PE, thereby improving the pathology associated with hypertension in response to placental ischemia.

Abstract 292: Vitamin D Supplementation Reduces CD4+ T Cells, Blood Pressure and Autoantibodies to the AT1 Receptor (AT1-AA) in a Rat Model of Preeclampsia

Preeclampsia (PE) is characterized by the development of hypertension, endothelial dysfunction and chronic immune activation after 20 weeks of gestation. PE women exhibit elevated CD4 + T cells, proinflammatory cytokines and agonistic autoantibodies to the angiotensin II type I receptor (AT1-AA). Vitamin D deficiency is associated with increased prevalence of hypertension and chronic inflammatory diseases both in the presence and absence of pregnancy. We utilize the Reduced Uterine Perfusion Pressure (RUPP) model of PE in the pregnant rat which exhibits hypertension, endothelial dysfunction as well as increased CD4 + T cells, inflammatory cytokines and AT1-AA. Therefore, we tested the hypothesis that administration of exogenous Vitamin D2 (VD2) and Vitamin D3 (VD3) would ameliorate hypertension and or chronic inflammation associated with placental ischemia in the RUPP rat. For these studies, the RUPP procedure was performed on gestation day 14 (GD14) and normal pregnant (NP) and RUPP rats were treated daily with 50uM VD2 or VD3 via oral gavage on GD14-18. Carotid catheters were inserted on GD18 and blood pressure (MAP), blood and tissues were collected on GD19. Flow cytometry was used to evaluate CD4 + T cells in control RUPP rats and RUPP rats treated with Vitamin D. On GD19, MAP was increased in RUPP rats to 123 ± 3.5 compared to 108.8 ± 4.3 mmHg in NP rats (n=5-6, p&lt;0.05). Supplementation with either VD2 or VD3 lowered MAP to 113 ± 6.9 or 115.4 ± 2.3 mmHg, respectively (n=4-5). CD4 + T cells in RUPP compared to NP rats increased from 2.80 ± 0.95 to 8.49 ± 1.52 percent (n=8-11, p&lt;0.05). Circulating CD4 + T cells fell to 1.22 ± 0.41 percent with VD2 and to 4.59 ± 3.67 percent with VD3 (n=2-3). AT1-AA levels, measured via chronotropic response in cardiomyocytes, were 19.50 ± 0.44 beats/min in RUPP rats and decreased to 15.35 ± 0.69 with VD3, and further to 8.33 ± 0.51 with VD2 supplementation (n=5, p&lt;0.05). Taken together, these data indicate that pharmaceutical Vitamin D supplementation reduced blood pressure and markers of chronic inflammation in a rat model of PE, thereby suggesting a role for Vitamin D supplementation to improve hypertension and chronic inflammation in response to diseases associated with placental ischemia, such as PE.

Abstract 209: Hemeoxygenase-1 Induction Restores Beta ENaC Expression in Ischemic Placentas

Preeclampsia is a pregnancy-specific disease characterized by hypertension, proteinuria, and vascular abnormalities; the etiology of which is under active investigation. Utilizing the rat model of reduced uterine perfusion pressure (RUPP), our lab previously demonstrated that induction of hemeoxygenase-1 (HO-1) reduces blood pressure and oxidative stress in placental ischemic rats. While epithelial sodium channel proteins (specifically β-ENaC) are important in maintaining vascular myogenic tone and have been implicated in cytotropholast invasion, the effect of placental ischemia and induction of HO-1 on β-ENaC expression in placentas have not been established. Therefore, the purpose of this study is to test the hypothesis that placental β-ENaC expression is reduced in ischemic placentas and restored by HO-1 induction. Normal pregnant and RUPP rats were treated with the HO-1 inducer, cobalt protoporphyrin (CoPP) (n=4 per group/treatment). Blood pressure was measured and placentas were harvested on day 19 of gestation. Blood pressure increased from 101±1 to 141±4 mmHg (p&lt;0.0001) in response to the RUPP procedure but was reduced by HO-1 induction (117±2 mmHg). RUPP rats also had fewer pups compared to normal pregnant rats (14±1 vs. 4±2 pups). HO-1 induction did not prevent the reduction in pup number. Placental β-ENaC protein expression was reduced by 62% in the RUPPs compared to the normal pregnant rats (p=0.036). HO-1 induction restored placental expression of β-ENaC to the levels of the normal pregnant rats. These findings suggest that HO-1 may exert its protective effects by altering the placental environment through changes in β-ENaC expression.

The use of N-acetylcysteine for the prevention of hypertension in the reduced uterine perfusion pressure model for preeclampsia in Sprague-Dawley rats

To determine whether N-acetylcysteine attenuates abnormal changes in the reduced uterine perfusion pressure model. Twenty-four timed-pregnant Sprague-Dawley rats underwent sham surgery or the reduced uterine perfusion pressure procedure on day 15 of 22. Reduced uterine perfusion pressure animals were treated with N-acetylcysteine (100 mg/kg) or saline twice daily until delivery. On day 21 of 22, mean arterial pressure was determined and maternal tissue was collected and stored. Pups and pup brains were weighed. Statistical analysis was performed using 1-way analysis of variance and the Tukey-Kramer test with significance at P < .05. There was a significant increase in blood pressure with the reduced uterine perfusion pressure procedure (P = .016), which was alleviated by N-acetylcysteine (P = .044). There was a significant decrease in pup weight and brain weight with the reduced uterine perfusion pressure procedure (P = .043 and P = .046, respectively). N-acetylcysteine restored pup brain weight (P = .021) but had no significant effect on pup weight. N-acetylcysteine reduced blood pressure without adversely effecting fetal weight.