Preeclampsia (PE), the leading cause of maternal and fetal morbidity and mortality, is associated with poor fetal growth, intrauterine growth restriction (IUGR) and low birth weight (LBW). Offspring of women who had PE are at increased risk for cardiovascular (CV) disease later in life. However, the exact etiology of PE is unknown. Moreover, there are no effective interventions to treat PE or alleviate IUGR and the developmental origins of chronic disease in the offspring. The placenta is critical to fetal growth and development. Epigenetic regulatory processes such as histone modifications, microRNAs and DNA methylation play an important role in placental development including contributions to the regulation of trophoblast invasion and remodeling of the spiral arteries. Epigenetic processes that lead to changes in placental gene expression in PE mediate downstream effects that contribute to the development of placenta dysfunction, a critical mediator in the onset of PE, impaired fetal growth and IUGR. Therefore, this review will focus on epigenetic processes that contribute to the pathogenesis of PE and IUGR. Understanding the epigenetic mechanisms that contribute to normal placental development and the initiating events in PE may lead to novel therapeutic targets in PE that improve fetal growth and mitigate increased CV risk in the offspring.

Placental ischemia in preeclampsia (PE) results in hypertension and intrauterine growth restriction (IUGR). Stimulation of soluble guanylate cyclase (sGC) reduces blood pressure in the clinically relevant reduced uterine perfusion pressure (RUPP) rat model of PE implicating involvement in RUPP-induced hypertension. However, the contribution of sGC in the development of IUGR in PE is not known. Thus, this study demonstrated the efficacy of Riociguat, a sGC stimulator, in IUGR reversion in the RUPP rat model of PE, and tested the hypothesis that improvement in fetal weight occurs in association with improvement in placental perfusion, placental morphology, and placental nutrient transport protein expression. Sham or RUPP surgery was performed at gestational day 14 (G14) with administration of vehicle (Sham or RUPP) or the sGC stimulator (Riociguat, 10mg/kg/day, s.c.) (sGC-treated) until G20. Fetal weight was reduced (p=0.004) at G20 in RUPP but not sGC-treated RUPP compared to Sham, the control group. At G20, uterine artery resistance index (UARI) was increased (p=0.010) in RUPP indicating poor placental perfusion; proportional junctional zone surface area was elevated (p=0.035) indicating impaired placental development. These effects were ameliorated in sGC-treated RUPP. Placental protein expression of nutrient transporter heart fatty acid binding protein (hFABP) was increased (p=0.008) in RUPP but not sGC-treated RUPP suggesting a compensatory mechanism to maintain normal neurodevelopment. Yet, UARI (p<0.001), proportional junctional zone surface area (p=0.013), and placental hFABP protein expression (p=0.008) were increased in sGC-treated Sham suggesting a potential adverse effect of Riociguat. Collectively, these results suggest sGC contributes to IUGR in PE.

Stimulation of soluble guanylate cyclase (sGC) improves fetal growth at gestational day 20 in the reduced uterine perfusion pressure (RUPP) rat model of placental ischemia suggesting a role for sGC in the etiology of intrauterine growth restriction (IUGR). This study tested the hypothesis that stimulation of sGC until birth attenuates asymmetric IUGR mitigating increased cardiovascular risk in offspring. Sham or RUPP surgery was performed at gestational day 14 (G14); vehicle or sGC stimulator, Riociguat (10mg/kg/day, s.c.) were administered G14 until birth. Birth weight was reduced in offspring from RUPP (intrauterine growth restricted or IUGR), sGC RUPP (sGC IUGR), and sGC Sham (sGC Control) compared to Sham (Control). Crown circumference was maintained but abdominal circumference was reduced in IUGR and sGC IUGR compared to Control indicative of asymmetrical growth. Gestational length was prolonged in sGC RUPP, and survival at birth was reduced in sGC IUGR. Probability of survival to postnatal day 2 was also significantly reduced in IUGR and sGC IUGR versus Control, and in sGC IUGR versus IUGR. At 4 months of age blood pressure was increased in male IUGR and sGC IUGR, but not male sGC Control born with symmetrical IUGR. Global longitudinal strain was increased and stroke volume was decreased in male IUGR and sGC IUGR compared to Control. Thus, late gestational stimulation of sGC does not mitigate asymmetric IUGR or increased cardiovascular risk in male sGC IUGR. Furthermore, late gestational stimulation of sGC is associated with symmetrical growth restriction in sGC Control implicating contraindications in normal pregnancy.

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Placenta ischemia, the initiating factor in preeclampsia (PE), is associated with intrauterine growth restriction (IUGR) and increased blood pressure (BP) in offspring. Yet, the only treatment for PE is delivery of the baby and placenta. The Reduced Uterine Perfusion Pressure (RUPP) rat model induced by placental ischemia at gestational day 14 (G14) mimics many facets of human PE including pregnancy-specific hypertension, an increase in the agonistic ANG II Type 1 receptor autoantibody (AT1-AA), IUGR and increased BP in the offspring. Inhibition of AT1-AA using an epitope-binding inhibitory peptide ('n7AAc') attenuates increased BP at gestational day 19 in the RUPP. Yet, whether use of ‘n7aac’ improves fetal growth and mitigates increased BP in the offspring is unknown. Thus, we tested the hypothesis that maternal administration of ‘n7aac’ improves fetal growth by attenuating reduced uterine blood flow and impaired placental remodeling. Sham or RUPP surgery was performed at G14 with administration of vehicle or ‘n7aac’ (144μg/day) via mini osmotic pump until gestational day 20 (G20). At G20 uterine artery resistance index was significantly elevated in vehicle RUPP (0.69±0.02 mm/s n=10) compared to vehicle Sham (0.48±0.02 mm/s n=8) (P<0.0001) and not increased in treated RUPP (0.49±0.02 mm/s n=10) or treated Sham (0.48±0.02 mm/s n=9). Fetal weight was significantly reduced in vehicle RUPP (3.24±0.2 g) compared to vehicle Sham (3.92±0.05 g) (P=0.013) and not decreased in treated RUPP (3.70±0.04 g) or Sham (3.98±0.10 g). Litter size of viable pups at G20 was only reduced in treated RUPP (5.3±1.4) compared to vehicle Sham (11.56±0.7) (P=0.003). Importantly, using in vivo imaging, little to no auto fluorescence of rhodamine-labeled peptide (480 μg/kg/day, n=4) was detectable in the pups at G20. Thus, our results demonstrate that maternal treatment with ‘n7aac’ in the RUPP rat model of PE improve UARI, which is associated with improved fetal weight at G20 in response to placental ischemia. Whether this benefit continues to birth and mitigates increased BP in IUGR offspring is unknown but is the focus of future studies. In conclusion, inhibition of the AT1AA during PE may not only provide benefit to the mother, but may also be associated with benefit in the offspring.

Preeclampsia ( PE ) is not only detrimental to the mother and her fetus during pregnancy, but it is also associated with increased cardiovascular ( CV ) risk in the mother and her intrauterine growth restricted ( IUGR ) offspring in later life. Currently there are no known treatment options for PE beyond early delivery. Thus, there is a critical need to identify therapeutic treatments for PE. The inflammatory cytokine TNF-α is increased in women with PE. In the clinically relevant Reduced Uterine Perfusion Pressure ( RUPP ) model of PE in the rat, TNF-α is also increased and importantly, etanercept abolishes maternal hypertension. Etanercept is a soluble receptor that binds TNF-alpha to inhibit the inflammatory response. Although not yet prescribed for PE, etanercept is non-contraindicated during pregnancy. Therefore, this study tested the hypothesis that maternal administration of etanercept will mitigate IUGR in the RUPP model of PE and that improvement in fetal growth occurs in association with improved uteroplacental perfusion, placental morphology, and placental nutrient transporter protein expression. Sham or RUPP was performed at gestational day 14 ( G14 ). Dams were administered vehicle or the TNF-α inhibitor (etanercept, 0.4 mg/kg, s.c.) at G18. At G20 uterine artery resistance index ( UARI ) was significantly increased in vehicle RUPP (0.69±0.02 mm/s) vs. vehicle Sham (0.60±0.02mm/s)( p <0.05), indicative of reduced uteroplacental perfusion, but UARI was no longer increased in treated RUPP (0.65±0.03 mm/s)( p <0.05). At G20, fetal weight was significantly reduced in vehicle RUPP (3.52g±0.10) vs. vehicle Sham (3.82 g±0.09)( p <0.05). Yet, fetal weight was significantly increased in treated RUPP (3.82g±0.12) vs. vehicle RUPP ( p <0.05). Fetal brain weight did not differ in Sham or RUPP, regardless of maternal treatment. However, fetal liver weight was significantly increased in treated RUPP (0.29g ±0.02) vs. vehicle RUPP (0.24g±0.01)( p <0.05) suggesting attenuation of asymmetric growth restriction. These findings indicate that maternal administration of a TNF-α inhibitor during PE improves fetal growth in association with improved uteroplacental blood flow. Future studies are warranted to investigate the long-term benefit in IUGR offspring.

Transgender females are biological males at birth but identify as female by gender. The prevalence of the transgender population is increasing, yet the burden of increased cardiovascular (CV) risk is unclear. Sex is an independent predictor of CV risk but the role of sex hormones in mediating CV disease in the transgender population is unknown. Gender affirming therapy (GAT) in transfemales (male to female) involves administration of estradiol with a goal to achieve the female physiological range of estrogen. The aim of this study was to optimize estradiol levels in a rat model of GAT. Male Sprague Dawley (SD) rats were implanted with silastic capsules to deliver 17‐beta estradiol benzoate (E2) at doses of 2.5 (Low), 5 (Medium) and 7.5 (High) mg/day versus empty pellet (EP) starting at 13 weeks of age (n=4/group). At 16 weeks of age, E2 was determined by ELISA, lean and fat mass were measured via EchoMRI, and creatinine, BUN, lipids and liver enzymes were quantitated by Chemistry Analyzer. The different doses of E2 were compared to EP using unpaired t test (GraphPad) with p<0.05 considered significant. All doses of E2 caused significant increases in circulating estradiol versus EP (Table, p<0.001) that were equivalent to estradiol levels in age‐matched female SD rats as previously reported by our laboratory (PMID: 17724277). Food intake, body weight, and lean mass, were significantly reduced after 3 weeks of E2 compared to EP (Table, p<0.001). Administration of E2 was also associated with a significant reduction in testicular weight versus EP (Table, p<0.001). To conclude, optimization of E2 levels in the male rat is critical to the development of a rodent model that mimics GAT in male to female transgender. The use of a transfemale rodent model will allow study of the pathophysiology of increased CV risk associated with GAT across the lifespan.

Placenta ischemia, the initiating event in preeclampsia (PE), is associated with fetal growth restriction. Inhibition of the agonistic autoantibody against the angiotensin type 1 receptor, AT1-AA, using an epitope-binding inhibitory peptide ('n7AAc') attenuates increased blood pressure at gestational day (G)19 in the clinically relevant reduced uterine perfusion pressure (RUPP) model of PE. Thus, we tested the hypothesis that maternal administration of 'n7aac' does not transfer to the fetus, improves uterine blood flow and fetal growth, and attenuates elevated placental expression of miRNAs implicated in PE and FGR. Sham or RUPP surgery was performed at G14 with vehicle or 'n7aac' (144µg/day) administered via osmotic pump from G14 to G20. Maternal plasma levels of the peptide on G20 were 16.28 ± 4.4 nM, and fetal plasma levels were significantly lower at 1.15 ± 1.7 nM (p=0.0007). Uterine artery resistance index was significantly elevated in RUPP (P<0.0001), but was not increased in 'n7aac'-RUPP or 'n7aac'-Sham versus Sham. The significant reduction in fetal weight at G20 in RUPP (P=0.003) was not observed in 'n7aac'-RUPP. Yet, percent survival was reduced in RUPP (P=0.0007) and 'n7aac-RUPP (P <0.0002). Correlation analysis indicated the reduction in percent survival during gestation was specific to the RUPP (r=0.5342, p=0.043) and independent of 'n7aac'. Placental miR-155 (P=0.0091) and miR-181a (P=0.0384) expression were upregulated in RUPP at G20 but were not elevated in 'n7aac'-RUPP. Collectively, our results suggest that maternal administration of 'n7aac' does not alter fetal growth in the RUPP implicating its potential as a therapeutic for the treatment of PE.