Placental Ischemia-induced Hypertension Research Articles

Placental ischemia-upregulated angiotensin II type 1 receptor in hypothalamic paraventricular nucleus contributes to hypertension in rat.

Preeclampsia (PE) is associated with increased angiotensin II sensitivity and poor neurological outcomes marked by temporal loss of neural control of blood pressure. Yet the role of centrally expressed angiotensin II type 1 receptor (AT1R) within the paraventricular nucleus of the hypothalamus (PVN) in the PE model is not understood. In a PE rat model with reduced placental perfusion pressure (RUPP) induced on gestational day 14 (GD14), the PVN expression and cellular localization of AT1R were assessed using immunofluorescence and western blotting. The sensitivity of RUPP to acute angiotensin II infusion was assessed. AT1R was antagonized by losartan (100µg/kg/day) for 5days intracerebroventricularly (ICV). Hemodynamic data and samples were collected on GD19 for further analysis. RUPP upregulated (p < 0.05) mRNA and protein of AT1R within the PVN and lowered (p < 0.05) circulating angiotensin II in rats. RUPP increased neural and microglial activation. Cellular localization assessment revealed that AT1R was primarily expressed in neurons and slightly in microglia and astrocytes. Infusion of 100ng/kg as bolus increased the mean arterial pressure (MAP in mmHg) in both RUPP and Sham. ICV losartan infusion attenuated RUPP-increased MAP (113.6 ± 6.22 in RUPP vs. 92.16 ± 5.30 in RUPP + Los, p = 0.021) and the expression of nuclear transcription factor NF-κB, tyrosine hydroxylase (TH), NADPH oxidase 4 (NOX4) and reactive oxygen species (ROS) in the PVN. Our data suggest that centrally expressed AT1R, within the PVN, contributes to placental ischemia-induced hypertension in RUPP rats highlighting its therapeutic potential in PE.

OP19_2. Luteolin, a natural flavonoid, attenuates the development of placental ischemia-induced hypertension in pregnant rats

OP19_2. Luteolin, a natural flavonoid, attenuates the development of placental ischemia-induced hypertension in pregnant rats

Treatment with the flavonoid, luteolin, attenuates the development of placental ischemia-induced hypertension in pregnant rats

Preeclampsia (PE) is a disease of hypertension and systemic vasoconstriction during pregnancy. Placental ischemia is implicated in PE by release of pro-hypertensive factors, like soluble Fms-like tyrosine kinase-1 (sFlt-1). Recently, we identified the bioflavonoid, luteolin, as a potent inhibitor of sFlt-1 and hypoxia-inducible factor 1α (HIF-1α, a major positive regulator of sFlt-1) in human placental cell lines and explants. However, the impact of luteolin in models of PE is unknown. We tested the hypothesis that luteolin attenuates placental ischemia-induced hypertension and lowers circulating sFlt-1 levels. Sprague-Dawley rats were started on luteolin treatment at gestational day 14 (20 mg/kg per day) or vehicle (0.5 g peanut butter) as well as placed in Reduced Uterine Perfusion Pressure (RUPP) placental ischemic or Sham surgery groups. The ensuing 4 experimental groups were: Sham+vehicle (N=14), Sham+luteolin (N=15), RUPP+vehicle (N=14), and RUPP+luteolin (N=15). On day 18, rats were implanted with carotid catheters for measurement of conscious mean arterial blood pressure (MAP) on gestational day 19. Luteolin treatment significantly attenuated RUPP-induced rises in maternal MAP (RUPP+vehicle: 118±2 vs. RUPP+luteolin: 108±1 mmHg, P&lt;0.05) with no effect in Sham rats (Sham+vehicle: 105±1 mmHg; and Sham+luteolin: 104±2 mmHg, P&gt;0.05). Circulating levels of sFlt-1 were elevated in RUPP+vehicle: 1.66±0.10 pg/mL vs. Sham+vehicle: 1.01±0.08 pg/mL (P&lt;0.05) that were significantly reduced with luteolin treatment only in RUPP+luteolin: 1.09±0.11 pg/mL and not Sham+luteolin: 1.26±0.11 pg/mL (P&lt;0.05). Total fetal weight was lower in RUPP+vehicle: 14±2 g vs. Sham+vehicle: 26±2 g (P&lt;0.05) but was not impacted by luteolin treatment in either group (RUPP+luteolin: 12±1 g or Sham+luteolin: 26±2 g, P&gt;0.05). Total placental weight was less in RUPP+vehicle: 3.3±0.6 g vs. Sham+vehicle: 6.4±0.4 g (P&lt;0.05), which was not altered by luteolin treatment in either group (RUPP+luteolin: 3.1±0.4 g or Sham+luteolin: 6.1± 0.5 g, P&gt;0.05). Overall, luteolin significantly attenuated placental ischemia-induced hypertension and reduced circulating sFlt-1 levels suggesting that naturally occurring flavonoids can be developed as therapeutics for PE. This work was supported by NIH 1R56HL157579-01. 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.

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

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

Abstract P349: Decreased Complement Component C3 In Islets Is Associated With A Reduction In Pancreatic Beta Cell Area In Rat Offspring Following Chronic Placental Ischemia-induced Hypertension

Gestational hypertension with or without low birth weight is associated with increased risk for Type 2 diabetes in offspring. Pancreatic beta cell mass is set very early in life and can influence whether an individual develops Type 2 diabetes. We previously demonstrated that postnatal day (PD)13 female offspring of rats subjected to chronic placental ischemia-induced hypertension had significantly reduced beta cell area. Previous studies in humans have noted increased circulating C3 in Type 2 diabetics, as well as an important role for intracellular complement C3 in maintaining integrity of the beta cell in the islet. Thus, we hypothesized that decreased C3 in islets is associated with reductions in beta cell area in rat offspring following placental insufficiency. The rat Reduced Uterine Perfusion Pressure (RUPP) model was used to mechanically induce placental insufficiency in the dam by placing silver clips on abdominal aorta and uterine arteries on gestation day 14 of a 21-day gestation, resulting in hypertension in the dam. Pancreatic islets and acinar were isolated by collagenase perfusion and hand picking from PD13 female offspring of RUPP and Sham dams. The concentration of C3 in serum, isolated islets and acinar was determined by ELISA. C3 was significantly increased in serum of female RUPP compared to female Sham offspring. In contrast, C3 in islets was significantly decreased in RUPP female offspring compared to Sham with no significant differences detected in acinar C3 concentrations. Thus, these data suggest that decreased C3 in islets of PD13 offspring may contribute to reduction of beta cell area and long-term susceptibility for Type 2 diabetes.

NLRP3 inflammasome inhibition ameliorated placental ischemia‐induced hypertension

Preeclampsia (PE), a pregnancy-specific syndrome characterized by de novo hypertension and vascular dysfunction, is widely accepted to be linked to placental ischemia-induced release of inflammatory cytokines and damage-associated molecular patterns. While placental damage and dysfunction caused by excessive inflammation can promote the occurrence of PE, the mechanisms whereby inflammation impacts placental ischemia-induced hypertension are not fully understood. Here we found that inflammation mediated by NLRP3 inflammasome occurs in the placenta predominantly from early onset PE, as evidenced by elevated levels of active caspase-1 and IL-1β(p<0.05). We further tested the hypothesis that inhibiting the activation of NLRP3 inflammasome can rescue the pregnancy outcome of PE. During pregnancy in rat, placental ischemia induced hypertension, proteinuria and excessive inflammatory cytokines production, whereas NLRP3 inflammasome inhibitor (MCC950, 10mg/kg/d) ameliorated the hypentension in RUPP model (117±2 vs. 101±1 mmHg, P<0.05), but exerts no beneficial effect on fetus (2.21±0.36 vs.2.09±0.49g) and placenta weights (0.40±0.06 vs.0.37±0.05g). Plasma levels of NLRP3 inflammasome associated cytokines, including IL-1β, were significantly elevated in RUPP rats compared to Sham rats, while inhibition of NLRP3 inflammasome activation attenuated the aberrant cytokines levels. Western blot also showed that MCC950 can inhibit the placental ischemia-induced expression levels of cleaved-CASP1 and mature IL-1β in the placenta. These findings suggest a promising role of NLRP3 in the development of PE and inhibiting NLRP3 inflammasome may provide a new target for the treatment of preeclampsia.

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

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

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.

Abstract 2: Increased Complement Regulator CD55 Is Associated With A Reduction In Pancreatic Beta Cell Area In Islets Of Rat Offspring Following Chronic Placental Ischemia-induced Hypertension

Placental insufficiency can result in gestational hypertension and/or intrauterine growth restriction (IUGR) in the offspring. Gestational hypertension with or without low birth weight is associated with increased risk for Type 2 diabetes in the offspring. Pancreatic β cell mass, set very early in life, can influence whether an individual develops Type 2 diabetes. Our previous studies demonstrated a significant reduction in β cell area in embryonic day 19 and postnatal day (PD)13 female offspring of rats subjected to chronic placental ischemia-induced hypertension. Previous studies in human islets demonstrated important roles for complement C3 and activation product C3a in determining β cell area, with increased C3a generation associated with prevention of islet cell atrophy. Thus, we hypothesized that increases in self-proteins that limit complement system activation, i.e. complement regulators, are associated with reductions in β cell area in the offspring following placental insufficiency. The Reduced Uterine Perfusion Pressure (RUPP) model was used to induce placental insufficiency in the dam by placing silver clips on the abdominal aorta and uterine arteries on gestation day 14 of a 21 day gestation to mechanically reduce placental perfusion. This results in hypertension in the dam and IUGR in the offspring. Islets from the pancreas of (PD)13 female offspring of RUPP and Sham dams were isolated by collagenase perfusion and hand picking. The transcript message for complement regulators CD55 and Crry (regulators of C3 activation) and CD59 (terminal pathway regulator) was determined using qRT-PCR. Complement regulator CD55 was significantly increased in female RUPP offspring (2.01 + 0.29 fold) compared to female Sham offspring (1.00 + 0.13 fold change; p &lt; 0.05; 6-13 pups from 4-7 dams; mixed model ANOVA including litter effect), whereas the Crry increase of 1.38 + 0.20 fold in female RUPP vs Sham offspring was not significant (p = 0.22). No significant change was observed in message for CD59 or C3. Thus, these data suggest increased islet complement regulator CD55 may limit complement activation in islets of PD13 offspring following placental ischemia and thereby contribute to reduction of β cell area and long term susceptibility for Type 2 diabetes.

Abstract P215: Hyperglycemic Rats Have Increased Fetal Demise But Not Hypertension During Pregnancy

While obesity is a major cause for pregnancy complications including preeclampsia and fetal demise, it is not exactly clear the precise obesity-related metabolic factors that promote these adverse outcomes. Epidemiological studies have pointed toward hyperglycemia as one such factor. Therefore, we tested the hypothesis that hyperglycemic rats have hypertension and fetal demise during pregnancy. For this purpose, we utilized the type II diabetic model, the Goto-Kakizaki (GK) rat (N=16), compared to normoglycemic Wistar Hannover (WH) rats (N=9), which were maintained on Envigo 8640 standard chow. The GK rat allows for assessment of hyperglycemia on pregnancy without confounding obesity. Maternal fasting glucose levels were significantly greater (P&lt;0.05) in GK (97±8 mg/dL) vs. WH (72±9 mg/dL) rats by gestational day 19. Body weight was lower (P&lt;0.05) in GK (248±4 g) versus WH (289±4 g) pregnant rats, whereas perirenal fat (1.56±0.07 g vs. 1.38±0.07 g, P&gt;0.05) and circulating levels of the adipokine, leptin (1.6±0.2 ng/mL vs. 2.2±0.3 ng/mL, P&gt;0.05) were similar between GK and WH pregnant groups, respectively. Endothelial-dependent relaxation to acetylcholine (sensitivity as logEC50: -5.2±0.3 M vs -5.2±0.4 M) and endothelial-independent relaxation to the nitric oxide-donor sodium nitroprusside (logEC50: -7.2±0.2 M vs. -7.5±0.1 M) were similar (P&gt;0.05) in uterine arteries isolated from GK and WH rats, respectively. It was then determined if reduced uterine perfusion pressure (RUPP)-induced placental ischemia, a significant contributor to the development of preeclampsia, promoted greater maternal hypertension in GK rats. RUPP was conducted on gestational day 14 and blood pressure assessed on day 19. RUPP produced hypertension to a similar extent (P&gt;0.05) in GK (116±5 mmHg vs. Sham 102±5 mmHg) and WH (124±4 mmHg vs. Sham 100±2 mmHg) groups. Blood pressure was similar under Sham conditions. Fetal demise was already greater in Sham GK vs. Sham WH pregnant rats (% absorptions: 13±2 vs. 2±2, P&lt;0.05) but increased similarly following RUPP in GK (61±11 %) and WH (65±5 %) pregnant rats. In conclusion, these data suggest that high glucose levels promote fetal demise during pregnancy but do not exaggerate the outcomes of placental ischemia-induced hypertension.

Impact of chronic hyperleptinemia on placental ischemia‐induced hypertension in pregnant rats

Obesity is a risk factor for preeclampsia, which is new‐onset hypertension during pregnancy. The mechanisms linking obesity and preeclampsia are poorly understood. The adipokine, leptin, is elevated in the circulation in proportion to fat mass and elicits hypertension when infused into non‐pregnant animals. However, less is studied about how hyperleptinemia impacts blood pressure regulation during pregnancy. A contributing factor to hypertension during preeclampsia is placental ischemia. Therefore, we tested the hypothesis that chronic hyperleptinemia exaggerates placental ischemia‐induced hypertension. On gestational day 14, timed‐pregnant Sprague‐Dawley rats were implanted with osmotic pumps to intravenously deliver recombinant rat leptin at 1.0 ug/kg per min or vehicle and placental ischemia was produced by the reduced uterine perfusion pressure (RUPP) procedure or Sham surgery. By day 19, leptin levels were elevated (P&lt;0.05) in Sham (leptin‐infused, N=9: 41.0±2.8 ng/mL vs vehicle, N=23: 0.8±0.1 ng/mL) and RUPP (leptin‐infused, N=9: 29.7±7.2 ng/mL vs vehicle, N=17: 1.0±0.1 ng/mL). RUPP elicited hypertension (Sham+vehicle: 100±1 mmHg vs RUPP+vehicle: 122±3 mmHg mean arterial blood pressure, P&lt;0.05). In contrast to our hypothesis, hyperleptinemia attenuated RUPP hypertension (RUPP+leptin: 109±4 mmHg, P&lt;0.05 vs RUPP+vehicle) and did not alter blood pressure in Sham rats (Sham+leptin: 100±6 mmHg vs Sham+vehicle). Therefore, we examined whether hyperleptinemia was vasoprotective by promoting vasorelaxation during pregnancy in isolated third‐order mesenteric arteries. Although endothelial‐dependent vasorelaxation to acetylcholine (measured as logEC50 sensitivity) was similar within Sham (vehicle: −6.8±0.1 M vs leptin: −6.9±0.2 M) and RUPP (vehicle: −7.0±0.2 M vs leptin: −7.0±0.3 M) groups, endothelial‐independent vasorelaxation to the nitric oxide (NO)‐donor, sodium nitroprusside, was increased (P&lt;0.05) in both Sham (vehicle: −6.0±0.2 M vs leptin: −6.8±0.3 M) and RUPP (vehicle: −6.9±0.2 M vs leptin: −7.6±0.01 M) groups. This suggested that NO‐cyclic guanosine monophosphate (cGMP) signaling was increased. Plasma levels of cGMP were elevated (P&lt;0.05) within Sham (vehicle: 1.6±0.3 pmol/mL vs leptin: 3.9±0.6 pmol/mL) and RUPP (vehicle: 3.6±0.8 pmol/mL vs leptin: 5.8±1.1 pmol/mL) groups. In contrast to our original hypothesis, chronic hyperleptinemia does not produce hypertension during pregnancy likely via promoting vascular NO‐cGMP signaling.Support or Funding InformationHL130577

Biopolymer-Delivered, Maternally Sequestered NF-κB (Nuclear Factor-κB) Inhibitory Peptide for Treatment of Preeclampsia.

Preeclampsia is a hypertensive disorder of pregnancy that causes significant acute and long-term risk to the mother and the baby. The multifaceted maternal syndrome is driven by overproduction of circulating anti-angiogenic factors, widespread inflammation, and endothelial dysfunction. Nuclear factor-κB (NF-κB) is a transcription factor that plays a central role in the inflammatory response. Its activity is increased in the preeclamptic placenta, and it promotes the systemic endothelial dysfunction present in preeclampsia. There is an acute need for new therapeutics targeted to the causative pathways of preeclampsia. Our group has developed a drug delivery system based on the bioengineered protein ELP (elastin-like polypeptide) that is capable of stabilizing therapeutics in the maternal circulation and preventing their placental transfer. Here we used the ELP carrier system to deliver a peptide known to inhibit the NF-κB pathway. This polypeptide, containing a cell-penetrating peptide and an NF-κB inhibitory peptide derived from the p50 nuclear localization sequence (abbreviated SynB1-ELP-p50i), blocked NF-κB activation and prevented TNF-α (tumor necrosis factor alpha)-induced endothelin production in vitro. Fusion of the p50i peptide to the SynB1-ELP carrier slowed its plasma clearance and prevented its placental transfer in pregnant rats, resulting in increased deposition in the maternal kidney, liver, and placenta relative to the free peptide. When administered in a rat model of placental ischemia, SynB1-ELP-p50i partially ameliorated placental ischemia-induced hypertension and reduced placental TNF-α levels with no signs of toxicity. These data support the continued development of ELP-delivered NF-κB inhibitors as maternally sequestered anti-inflammatory agents for preeclampsia therapy.

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

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

Hypertension Editors' Picks Preeclampsia.

Hypertension Editors' Picks Preeclampsia.

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.

282. A novel NF-kB inhibitory peptide-based therapeutic for the management of preeclampsia

Preeclampsia (PE) is a multifactorial disease in which little progress has been made to develop adequate therapy. One hallmark of this disease is a rise in systemic maternal inflammatory factors, many of which signal through the NF-kB pathway. We hypothesize that administration of a novel NF-κB antagonist, ELP-p50i, will attenuate the hypertension and inflammation in the preclinical RUPP model of placental insufficiency. Animals were received on GD11 and received the sham or RUPP surgery on GD14 along with drug or vehicle administration. On GD18, carotid catheters were placed for blood pressure measurement on GD19. Aortic ET-1 was measured by qRT-PCR using Taq primers specific for rat ET-1 and normalized to rat β-actin (Thermofisher). Placental TNF-α was measured using a commercial ELISA specific for rat TNF-α) (R&D). RUPP animals had significantly increased blood pressure (122.2 ± 2.56 mmHg vs 99.3 ± 3.83 mmHg, p < 0.01). ELP-p50i administration significantly reduced the blood pressure in RUPP animals (122.2 ± 2.56 mmHg vs 110.9 ± 1.61 mmHg, p < 0.05). Additionally, there is a trend to increase aortic endothelin-1 expression in RUPP compared to controls (1.4 ± 0.2 vs 1.16 ± 0.17-fold change, p = 0.47, n = 4), and ELP-p50i treated rats had significantly reduced endothelin-1 expression (1.4 ± 0.2 vs 0.66 ± 0.11-fold change, p < 0.05). Additionally, placental TNF-α trended upward in RUPP animals compared to normal pregnant (25.3 ± 1.7 pg/mg vs 21.8 ± 1.5 pg/mg, p = 0.14) and this increase in TNF-α was completely abolished by ELP-p50i treatment (25.3 ± 1.7 pg/mg vs 19.5 ± 0.8 pg/mg, p < 0.5). Our data suggest that ELP-p50i is able to attenuate placental ischemia-induced hypertension and correct symptoms such as endothelial dysfunction and increased inflammation, potentially creating a therapeutic option for preeclampsia.

Abstract P179: Placental Ischemia-Induced Hypertension is Attenuated in Melanocortin-4 Receptor (MC4R)-Deficient Pregnant Rats

The incidence of preeclampsia (PE), a pregnancy-specific disorder of new-onset hypertension that is linked to placental ischemia, has increased over the last several decades. While obesity is a major risk factor for PE, the mechanisms whereby obesity impacts placental ischemia-induced hypertension are not fully understood. We recently reported that placental ischemia-induced hypertension is partially dependent upon an intact sympathetic nervous system (SNS). In various hypertensive models including obesity, blockade of MC4R signaling leads to increased food intake and body weight but protects against the development of hypertension via suppression of the SNS. 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. Wild-type (WT) and MC4R heterozygous-deficient rats were mated with genotype-matched males at ~12 wks old. All rats were maintained on NIH31 chow diet. On gestational day 14, rats were subjected to chronic placental ischemia via the reduced uterine perfusion pressure (RUPP) procedure or Sham surgery. By day 19, conscious mean arterial blood pressure (MAP, carotid catheter) was elevated in RUPP WT (N=21) over Sham WT rats (N=14) (117±2 vs. 101±1 mmHg, P&lt;0.05) with reduced average fetus (1.71±0.04 vs. 1.89±0.05 g, P&lt;0.05) but no change in average placenta weights (0.46±0.02 vs. 0.47±0.01 g), respectively. In Sham MC4R-def vs. Sham WT, respectively, there was increased (P&lt;0.05) body weight (366±8 vs. 337±6 g), total body fat mass (72±4 vs. 44±2 g), and circulating leptin adipokine levels (6.9±0.7 vs. 3.3±0.3 ng/mL) but with similar average fetus (1.92±0.02 vs. 1.89±0.05 g) and placenta weights (0.49±0.01 vs. 0.47±0.01 g). Even though blood pressure was elevated in Sham MC4R-def (109±2 mmHg, P&lt;0.05) over Sham WT, the hypertensive response to RUPP was attenuated in MC4R-def rats (114±2 mmHg). RUPP did not alter leptin levels in MC4R-def rats (6.4±1 ng/mL). In conclusion, these data suggest that MC4R heterozygous-def pregnant rats are obese and have higher blood pressure during pregnancy. However, placental ischemia may require a full complement of MC4R expression to elicit a hypertensive response.

Depletion of B1 and B2 lymphocytes in placental ischemia‐induced hypertension in the rat

Preeclampsia is a pregnancy specific disease characterized by new‐onset maternal hypertension and increased systemic inflammation, including increased complement activation. Although the exact pathophysiology is unknown, abnormal arterial remodeling and placental insufficiency have been implicated as initiating events. Studies of others have provided evidence for the importance of the B cell in placental ischemia‐induced hypertension, but contribution of the B1 lineage of innate B lymphocytes vs. the traditional B2 cell is unknown. Our previous studies showed increased peritoneal B1 cells following placental ischemia. Thus, we hypothesized that IgM producing B1 cells are the important B cell responsible for complement activation and hypertension following chronic placental ischemia in the reduced uterine placental perfusion (RUPP) rat model. The effect of anti‐CD20 antibody treatment on placental ischemia‐induced hypertension and B cell subpopulations in peritoneal cavity, spleen and blood was determined. Anti‐murine CD20 mAb (5 mg/kg, Clone 5D2, Genentech) or corresponding mu IgG2a isotype control were administered IP to timed pregnant Sprague Dawley rats (Charles River) on gestation day (GD)10 and 13. On GD14, rats underwent either Sham surgery or clip placement on ovarian arteries and abdominal aorta to reduce uterine perfusion pressure (RUPP) resulting in increased maternal blood pressure on GD19 (n=4–14 animals/group). Mean arterial pressure was measured from a carotid catheter in unanesthetized, restrained animals and increased from 91 ± 4 to 104 ± 4 mm Hg with RUPP in isotype treated animals and was not significantly attenuated in RUPP anti‐CD20 treated animals (100 ± 3 mm Hg). On GD19, peritoneal cavity was lavaged with PBS, spleen removed and mechanically disrupted, blood obtained from abdominal aorta, and B1 (IgM+ CD11b+) and B2 (IgM+ CD11b‐) lymphocytes enumerated by flow cytometry. Data is expressed as % of total lymphocytes. Both peritoneal B1 and B2 cells increased following placental ischemia (**) with no change in blood or spleen. When evaluated at GD19, anti‐CD20 treatment significantly depleted B2 cells in all compartments and B1 cells in peritoneum and blood (*) with no effect on RUPP‐induced hypertension. A separate group of rats was used to determine if anti‐CD20 treatment effectively depleted B cells at time of RUPP surgery (GD14). On GD14, anti‐CD20 treatment reduced B1 cells in peritoneal cavity and blood and B2 cells in all compartments compared to isotype treated GD14 animals (*). However, comparing anti‐CD20 treatment at GD14 to GD19, B1 and B2 cells in peritoneal cavity were significantly greater at GD14 (#), indicating incomplete peritoneal B cell depletion at time of RUPP surgery. These data suggest that sufficient B1 and B2 cells may be present in the peritoneal cavity at time of induction of placental ischemia to contribute to hypertension, and that placental ischemia induced hypertension occurs despite significant depletion of splenic B2 cells and peripheral B1/B2 cells.Support or Funding InformationNIH HL109843 and American Heart Association Grant in Aid %B1 %B2 Peritoneal Spleen Blood Peritoneal Spleen Blood GD19 Sham Isotype 3.0 ± 0.6 19.9 ± 1.1 4.6 ± 0.8 1.4 ± 0.4 35.7 ± 2.6 21.4 ± 7.0 GD19 Sham AntiCD20 1.6 ± 0.7* 17.2 ± 2.0 0.7 ± 0.3* 0.2 ± 0.04* 7.5 ± 1.7* 1.5 ± 0.5* GD19 RUPP Isotype 4.9 ± 1.2** 20.8 ± 1.9 7.8 ± 1.5 2.4 ± 0.6** 37.2 ± 2.1 21.9 ± 1.6 GD19 RUPP AntiCD20 1.4 ± 0.6* 17.8 ± 1.2 1.1 ± 0.3* 0.3 ± 0.1* 7.0 ± 0.9* 1.5 ± 0.2* GD14 Isotype 18.1 ± 3.1 14.7 ± 2.6 6.4 ± 2.2 7.7 ± 1.6 47.9 ± 5.6 33.4 ± 2.2 GD14 AntiCD20 5.9 ± 2.6*# 11.0 ± 4.0 2.0 ± 0.6* 3.3 ± 0.9*# 6.6 ± 1.1* 2.7 ± 0.8* p&lt;0.05: vs Isotype; RUPP &gt; Sham Isotype; GD14 AntiCD20 &gt; GD19 AntiCD20 This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Placental Ischemia‐Induced Hypertension Is Abolished by Adrenergic Receptor Blockade

Hypertensive disorders of pregnancy are the number one cause of pregnancy‐related deaths in the United States. Preeclampsia is a disorder of maternal hypertension and vascular dysfunction presenting in the second half of pregnancy along with fetal growth restriction. Currently, there are no steadfast therapies besides early delivery of the fetus and ischemic placenta, which releases pro‐hypertensive factors into the maternal circulation. The sympathetic nervous system might be a novel target in treating preeclampsia, as sympathetic nervous activity is found to be increased in preeclamptic vs. normal pregnant women. However, it is unknown if the sympathetic nervous system mediates the development of placental ischemia‐induced hypertension. In this study, the hypothesis was tested that adrenergic receptor blockade prevents placental ischemia‐induced hypertension. Wistar Hannover rats were randomized to receive reduced uterine perfusion pressure (RUPP, N=15) or Sham RUPP (N=20) surgeries on gestational day 14 and examined at day 19. In RUPP vs. Sham rats, respectively, mean arterial blood pressure (MAP) was increased (121 ± 3 vs. 102 ± 1 mmHg, P&lt;0.05) without a change in heart rate (421 ± 7 vs. 423 ± 4 bpm). Average fetal weights (1.87 ± 0.04 vs. 2.11 ± 0.03 g) and placental weights (0.50 ± 0.02 vs. 0.57 ± 0.01 g) were lower (P&lt;0.05) in RUPP dams. In RUPP over Sham rats, vasoconstriction induced by the alpha‐adrenergic receptor agonist, phenylephrine, was greater in isolated renal interlobar arteries (Emax: 322 ± 19 vs. 251 ± 12 % of KCl response). The Emax response to KCl was not different between RUPP or Sham rats, in sequence: 100 ± 24 vs. 87 ± 14 % increase in force. A subset of RUPP (N=9) and Sham (N=9) rats received combined terazosin and propranolol (3 mg/kg per day of each, subcutaneous osmotic minipump) to block alpha‐ and beta‐adrenergic receptors, respectively, beginning the day of surgery on gestational day 14. By day 19, adrenergic blockade abolished the development of hypertension in RUPP rats (105 ± 2 mmHg, P&lt;0.05) and reduced their heart rate (343 ± 21 bpm, P&lt;0.05) with a lesser reduction detected in treated Sham rats for MAP (97 ± 2 mmHg, P&lt;0.05) and heart rate (387 ± 9 bpm, P&lt;0.05). Treatment did not alter average fetal weights (1.89 ± 0.05 and 2.11 ± 0.03 g) or placental weights (0.47 ± 0.02 and 0.50 ± 0.03 g) in RUPP and Sham rats, in turn, compared to their untreated counterparts. In conclusion, placental ischemia‐induced hypertension depends on activation of the sympathetic nervous system. The mechanisms responsible for this enhanced sympathetic nerve activity and adrenergic receptor signaling remain unknown but are likely to involve factors released from the ischemic placenta.Support or Funding Information4R00HL130577‐02This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Heme oxygenase-1 is a potent inhibitor of placental ischemia-mediated endothelin-1 production in cultured human glomerular endothelial cells.

Preeclampsia is a pregnancy-specific disorder of maternal hypertension and reduced renal hemodynamics linked to reduced endothelial function. Placental ischemia is thought to be the culprit of this disease, as it causes the release of factors like tumor necrosis factor (TNF)-α that induce vascular endothelin-1 (ET-1) production. Interestingly, placental ischemia-induced hypertension in rats [reduced uterine perfusion pressure (RUPP) model] is abolished by ETA receptor blockade, suggesting a critical role for ET-1. Although it has been found that systemic induction of heme oxygenase (HO)-1 is associated with reduced ET-1 production and attenuated hypertension, it is unclear whether HO-1 directly modulates the increased ET-1 response to placental factors. We tested the hypothesis that HO-1 or its metabolites inhibit ET-1 production in human glomerular endothelial cells induced by serum of RUPP rats or TNF-α. Serum (5%) from RUPP hypertensive (mean arterial blood pressure 119 ± 9 mmHg) vs. normotensive pregnant (NP, 101 ± 6 mmHg, P < 0.001) rats increased ET-1 production (RUPP 168.8 ± 18.1 pg/ml, NP 80.3 ± 22.7 pg/ml, P < 0.001, n = 12/group). HO-1 induction [25 µM cobalt photoporphyrin (CoPP)] abolished RUPP serum-induced ET-1 production (1.6 ± 0.8 pg/ml, P < 0.001), whereas bilirubin (10 µM) significantly attenuated ET-1 release (125.3 ± 5.2 pg/ml, P = 0.005). Furthermore, TNF-α-induced ET-1 production (TNF-α 31.0 ± 8.4 vs. untreated 7.5 ± 0.4 pg/ml, P < 0.001) was reduced by CoPP (1.5 ± 0.8 pg/ml, P < 0.001) and bilirubin (10.5 ± 4.3 pg/ml, P < 0.001). These results suggest that circulating factors released during placental ischemia target the maternal glomerular endothelium to increase ET-1, and that pharmacological induction of HO-1 or bilirubin could be a treatment strategy to block this prohypertensive pathway in preeclampsia.