Gestational intermittent hypoxia induces endothelial dysfunction and hypertension in pregnant rats: role of endothelin type B receptor†.

Obstructive sleep apnea is a recognized risk factor for gestational hypertension, yet the exact mechanism behind this association remains unclear. Here, we tested the hypothesis that intermittent hypoxia, a hallmark of obstructive sleep apnea, induces gestational hypertension through perturbed endothelin-1 signaling. Pregnant Sprague-Dawley rats were subjected to normoxia (control), mild intermittent hypoxia (10.5% O2), or severe intermittent hypoxia (6.5% O2) from gestational days 10-21. Blood pressure was monitored. Plasma was collected and mesenteric arteries were isolated for myograph and protein analyses. The mild and severe intermittent hypoxia groups demonstrated elevated blood pressure, reduced plasma nitrate/nitrite, and unchanged endothelin-1 levels compared to the control group. Western blot analysis revealed decreased expression of endothelin type B receptor and phosphorylated endothelial nitric oxide synthase, while the levels of endothelin type A receptor and total endothelial nitric oxide synthase remained unchanged following intermittent hypoxia exposure. The contractile responses to potassium chloride, phenylephrine, and endothelin-1 were unaffected in endothelium-denuded arteries from mild and severe intermittent hypoxia rats. However, mild and severe intermittent hypoxia rats exhibited impaired endothelium-dependent vasorelaxation responses to endothelin type B receptor agonist IRL-1620 and acetylcholine compared to controls. Endothelium denudation abolished IRL-1620-induced vasorelaxation, supporting the involvement of endothelium in endothelin type B receptor-mediated relaxation. Treatment with IRL-1620 during intermittent hypoxia exposure significantly attenuated intermittent hypoxia-induced hypertension in pregnant rats. This was associated with elevated circulating nitrate/nitrite levels, enhanced endothelin type B receptor expression, increased endothelial nitric oxide synthase activation, and improved vasodilation responses. Our data suggested that intermittent hypoxia exposure during gestation increases blood pressure in pregnant rats by suppressing endothelin type B receptor-mediated signaling, providing a molecular mechanism linking intermittent hypoxia and gestational hypertension.

Elevated gestational testosterone impacts vascular and uteroplacental function.

Maternal vascular adaptations to establish an adequate blood supply to the uterus and placenta are essential for optimal nutrient and oxygen delivery to the developing fetus in eutherian mammals, including humans. Numerous factors contribute to maintaining appropriate hemodynamics and placental vascular development throughout pregnancy. Failure to achieve or sustain these pregnancy-associated changes in women is strongly associated with an increased risk of antenatal complications, such as preeclampsia, a hypertensive disorder of pregnancy. The precise etiology of preeclampsia is unknown, but emerging evidence points to a potential role for androgens. The association between androgens and maternal cardiovascular and placental function merits particular attention due to the notable 2- to 3-fold elevated plasma testosterone (T) levels observed in preeclampsia. T levels in preeclamptic women positively correlate with vascular dysfunction, and preeclampsia is associated with increased androgen receptor (AR) levels in placental tissues. Moreover, animal studies replicating the pattern and magnitude of T increase observed in preeclamptic pregnancies have reproduced key features of preeclampsia, including gestational hypertension, endothelial dysfunction, heightened vasoconstriction to angiotensin II, impaired spiral artery remodeling, placental hypoxia, reduced nutrient transport, and fetal growth restriction. Collectively, these findings suggest that AR-mediated activity plays a significant role in the clinical presentation of preeclampsia. This review critically evaluates this hypothesis, considering both clinical and preclinical evidence.

PFOS Impairs Mitochondrial Biogenesis and Dynamics and Reduces Oxygen Consumption in Human Trophoblasts.

Perfluorooctane sulfonate (PFOS), a synthetic chemical used in various commercial applications and industrial settings, has led to contamination of drinking water and has been detected in the bloodstream of pregnant women with gestational complications. Recent investigations have indicated that PFOS disrupts placental function; however, the mechanism remains elusive. Given the significant abundance of mitochondria in the placenta, which play a pivotal role in fulfilling the heightened energy requirements of pregnancy, our research aimed to examine the repercussions of PFOS exposure on mitochondrial dynamics within placental trophoblasts. Specifically, human trophoblasts (HTR-8/SVneo) were exposed to environmentally relevant concentrations of PFOS ranging from 0.1 to 50 µM for 48 hours. Findings revealed that PFOS exposure elicited a concentration-dependent decrease in basal, maximal, and ATP-linked respiration. PFOS inhibited the activity of electron transport complexes I, II, and III, resulting in diminished ATP production. Furthermore, PFOS reduced mitochondrial DNA copy number, indicating less mitochondrial content. Concurrently, there was a downregulation in the expression of mitochondrial biogenesis-related genes, including PGC-1α, NRF1, and NRF2. Notably, PFOS perturbed mitochondrial dynamics by suppressing the expression of fission-related genes (FIS1 and DRP1) and fusion-related genes (MFN1 and MFN2). In summary, our findings suggest that PFOS exposure leads to a decline in mitochondrial content and compromises the bioenergetic capacity of trophoblasts by impairing cellular respiration. This reduction in mitochondrial biogenesis and alterations in fission/fusion dynamics induced by PFOS may contribute to mitochondrial dysfunction in trophoblasts. Consequently, strategies that preserve mitochondrial function in trophoblasts may mitigate PFOS-induced impairment of placental energy metabolism.

Airway surveillance and lung viral control by memory T cells induced by COVID-19 mRNA vaccine.

Although SARS-CoV-2 evolution seeds a continuous stream of antibody-evasive viral variants, COVID-19 mRNA vaccines provide robust protection against severe disease and hospitalization. Here, we asked whether mRNA vaccine-induced memory T cells limit lung SARS-CoV-2 replication and severe disease. We show that mice and humans receiving booster BioNTech mRNA vaccine developed potent CD8 T cell responses and showed similar kinetics of expansion and contraction of granzyme B/perforin-expressing effector CD8 T cells. Both monovalent and bivalent mRNA vaccines elicited strong expansion of a heterogeneous pool of terminal effectors and memory precursor effector CD8 T cells in spleen, inguinal and mediastinal lymph nodes, pulmonary vasculature, and most surprisingly in the airways, suggestive of systemic and regional surveillance. Furthermore, we document that: (a) CD8 T cell memory persists in multiple tissues for > 200 days; (b) following challenge with pathogenic SARS-CoV-2, circulating memory CD8 T cells rapidly extravasate to the lungs and promote expeditious viral clearance, by mechanisms that require CD4 T cell help; and (c) adoptively transferred splenic memory CD8 T cells traffic to the airways and promote lung SARS-CoV-2 clearance. These findings provide insights into the critical role of memory T cells in preventing severe lung disease following breakthrough infections with antibody-evasive SARS-CoV-2 variants.

Restoring Angiotensin Type 2 Receptor Function Reverses PFOS-Induced Vascular Hyper-Reactivity and Hypertension in Pregnancy.

Perfluorooctane sulfonic acid (PFOS) exposure during pregnancy induces hypertension with decreased vasodilatory angiotensin type-2 receptor (AT2R) expression and impaired vascular reactivity and fetal weights. We hypothesized that AT2R activation restores the AT1R/AT2R balance and reverses gestational hypertension by improving vascular mechanisms. Pregnant Sprague-Dawley rats were exposed to PFOS through drinking water (50 µg/mL) from gestation day (GD) 4-20. Controls received drinking water with no detectable PFOS. Control and PFOS-exposed rats were treated with AT2R agonist Compound 21 (C21; 0.3 mg/kg/day, SC) from GD 15-20. In PFOS dams, blood pressure was higher, blood flow in the uterine artery was reduced, and C21 reversed these to control levels. C21 mitigated the heightened contraction response to Ang II and enhanced endothelium-dependent vasorelaxation in uterine arteries of PFOS dams. The observed vascular effects of C21 were correlated with reduced AT1R levels and increased AT2R and eNOS protein levels. C21 also increased plasma bradykinin production in PFOS dams and attenuated the fetoplacental growth restriction. These data suggest that C21 improves the PFOS-induced maternal vascular dysfunction and blood flow to the fetoplacental unit, providing preclinical evidence to support that AT2R activation may be an important target for preventing or treating PFOS-induced adverse maternal and fetal outcomes.

Elevated Maternal Testosterone Levels Alter PFOA Elimination and Tissue Distribution in Pregnant Rats.

Perfluorooctanoic acid (PFOA) is an enduring synthetic chemical that harms human health. Recent studies indicate heightened bioaccumulation of PFOA, particularly in pregnant women experiencing preeclampsia. Since plasma testosterone levels are elevated in pregnant women with preeclampsia, we hypothesized that hyperandrogenic conditions during pregnancy may hinder PFOA elimination and contribute to their higher body burden. Pregnant Sprague-Dawley rats were s/c injected with vehicle or testosterone propionate from gestational day (GD) 15 to 20 to increase plasma testosterone levels by 2-fold, similar to levels in preeclampsia. On GD 16, [14C]-PFOA (9.4 pmol/kg) was given intravenously, and subsequently, 14C radioactivity was measured in maternal blood, urine, feces, and tissues. PFOA was primarily eliminated through urine; however, less PFOA was excreted in urine of pregnant rats with elevated testosterone levels than controls. Fecal excretion of PFOA was minimal and did not significantly differ between groups. The total elimination of PFOA (urine plus feces) was significantly reduced by 12% in pregnant rats with elevated testosterone levels. In controls, PFOA distribution was highest in placenta, followed by the kidneys, liver, brain, heart, lungs, and spleen. Pregnant rats with elevated testosterone levels displayed 12% higher concentrations of PFOA in these tissues than controls. Furthermore, the renal expression of Oat2 and Oat3 was significantly decreased, while Oatp1 and Oat-k expression was significantly increased in pregnant rats with elevated testosterone levels than controls. In conclusion, elevated maternal testosterone levels decrease urinary elimination of PFOA, possibly through altered expression of renal transporters leading to increased tissue concentrations of PFOA in pregnant rats.

Whole-Genome Transcriptome Profiling in PFOS-treated Uterine Artery Endothelial Cells Isolated from Pregnant Women.

Empirical evidence from human studies has demonstrated a correlative relationship between perfluorooctane sulfonate (PFOS) exposure and increased risks of preeclampsia and fetal developmental complications. Although experimental and circumstantial data suggest that PFOS induces endothelial dysfunction, leading to decreased uterine arterial blood flow and gestational hypertension, the precise regulatory mechanisms responsible for this effect remain unknown. To address this issue, we treated human uterine artery endothelial cells (hUAECs) isolated from pregnant women with 10 µmol/L PFOS or vehicle and conducted comparative transcriptomic analyses. We identified a total of 19 differentially expressed genes, 9 of which were upregulated and 10 were down-regulated in PFOS-treated pregnant hUAECs. Pre-ranked gene set enrichment analysis unveiled a distinct set of activated genes involved in osmotic stress, cellular stress response, translation regulation, metabolic regulation, and oxidation-reduction processes in PFOS-treated pregnant hUAECs. Furthermore, PFOS treatment resulted in the downregulation of genes implicated in cardiac muscle cell proliferation, embryonic morphogenesis, and muscle cell proliferation. In addition, we observed differential splicing events in 2678 genes in hUAECs exposed to PFOS, with cross-comparison analysis revealing 4 genes that were both differentially expressed and alternatively spliced and were implicated in oxidative stress and cardiac development. In conclusion, this study provides a comprehensive understanding of the molecular mechanisms underlying PFOS-induced gestational uterine artery endothelial dysfunction during pregnancy, offering a valuable resource for future research in this field.

Placental Fatty Acid Metabolism and Transport in a Rat Model of Gestational Diabetes Mellitus.

Gestational diabetes mellitus (GDM) is a form of heightened insulin resistance triggered during gestation. This study examines how insulin resistance alters placental long-chain polyunsaturated fatty acid (LCPUFA) transport and metabolism in a rat model of lean GDM. Pregnant Sprague Dawley rats were administered with S961, an insulin receptor antagonist (30 nmol/kg s.c. daily), or vehicle from gestational day (GD) 7 to 20. Daily maternal body weight, food, and water intake were measured. Blood pressure assessment and glucose tolerance test were done on GD20. Fetal plasma and placenta were collected on GD20 and processed for fatty acid measurement using LC-mass spectrometry. The expression of fatty acid metabolism-related genes in the placenta was assessed using RT2 Profiler PCR arrays. The results were validated by qRT-PCR. Blockade of insulin receptors with S961 in pregnant rats resulted in glucose intolerance with increased fasting glucose and insulin levels. Maternal body weight gain and food and water intake were not affected; however, S961 significantly increased maternal blood pressure and heart rate. The placenta n3 and n6 LCPUFA concentrations were significantly decreased by 8% and 11%, respectively, but their levels in the fetal plasma were increased by 15% and 4%. RT2 profiler arrays revealed that placental expressions of 10 genes related to fatty acid ß-oxidation (Acaa1a, Acadm, Acot2, Acox2, Acsbg1, Acsl4, Acsm5, Cpt1b, Eci2, Ehhadh) and 3 genes related to fatty acid transport pathway (Fabp2, Fabp3, Slc27a3) were significantly upregulated. In summary, lack of insulin action increased the expression of genes related to placental fatty acid ß-oxidation and transport with an increased transfer of LCPUFA to the fetus. The increased lipid levels routed toward the fetus may lead to fat adiposity and later-life metabolic dysfunction.

Perfluorooctane sulfonic acid modulates expression of placental steroidogenesis-associated genes and hormone levels in pregnant rats.

Perfluorooctane sulfonate (PFOS) is a widespread and persistent chemical in the environment. Reports show that PFOS is a potential endocrine disruptor; however, the possible effects of PFOS on placental endocrine function are unclear. This study aimed to investigate the endocrine-disrupting effects of PFOS on the placenta in pregnant rats and its potential mechanism. Pregnant rats from gestational days 4-20 were exposed to 0, 10, and 50 µg/mL PFOS through drinking water followed by analysis of various biochemical parameters. PFOS dose-dependently decreased fetal and placental weight in both sexes, with a specific decrease in weight of labyrinth but not junctional layer. Plasma progesterone (↑166%), aldosterone (↑201%), corticosterone (↑205%), testosterone (↑45%), luteinizing hormone (↑49%) levels were significantly increased, while estradiol (↓27%), prolactin (↓28%) and hCG (↓62%) levels were reduced in groups exposed to higher doses of PFOS. Real-time quantitative reverse transcriptase-polymerase chain reaction analysis revealed a significant increase in mRNA levels of placental steroid biosynthesis enzymes, including Cyp11A1 and 3ß-HSD1 in male placenta and StAR, Cyp11A1, 17ß-HSD1 and 17ß-HSD3 in female placenta of PFOS dams. Cyp19A1 expression in ovaries was significantly decreased in PFOS dams. mRNA levels for placental steroid metabolism enzyme UGT1A1 increased in male but not in female placenta of PFOS dams. These results suggest that the placenta is a target tissue of PFOS and PFOS-induced dysregulation in steroid hormone production might be related to the altered expression of hormone biosynthesis and metabolism enzyme genes in the placenta. This hormone disruption might affect maternal health and fetal growth.

An Endogenous Aryl Hydrocarbon Receptor Ligand Induces Preeclampsia-like Phenotypes: Transcriptome, Phosphoproteome, and Cell Functions.

Background: Preeclampsia (PE) is one hypertensive disorder and a leading cause of maternal and fetal mortality and morbidity during human pregnancy. Aryl hydrocarbon receptor (AhR) is a transcription factor, which regulates vascular functions. Exogenous and endogenous AhR ligands can induce hypertension in animals. However, if dysregulation of endogenous AhR ligands contributes to the pathophysiology of PE remains elusive. Methods: We measured AhR activities in human maternal and umbilical vein sera. We also applied physiological, cellular, and molecular approaches to dissect the role of endogenous AhR ligands in vascular functions during pregnancy using pregnant rats and primary human umbilical vein endothelial cells (HUVECs) as models. Results: PE elevated AhR activities in human umbilical vein sera. Exposure of pregnant rats to an endogenous AhR ligand, 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) increased blood pressure and proteinuria, while decreased uteroplacental blood flow and reduced fetal and placental weights, all of which are hallmarks of PE. ITE dampened vascular growth and fetal sex-specifically altered immune cell infiltration in rat placentas. ITE also decreased cell proliferation and cell monolayer integrity in HUVECs in vitro . RNA sequencing analysis revealed that ITE dysregulated transcriptome in rat placentas and HUVECs in a fetal sex-specific manner. Bottom-up phosphoproteomics showed that ITE disrupted phosphoproteome in HUVECs. These ITE-dysregulated genes and phosphoproteins were enriched in biological functions and pathways which are highly relevant to diseases of heart, liver, and kidney, vascular functions, inflammation responses, cell death, and kinase inhibition. Conclusions: Dysregulation of endogenous AhR ligands during pregnancy may lead to the development of PE with underlying impaired vascular functions, fetal sex-specific immune cell infiltration and transcriptome, and phosphoproteome. Thus, this study has provided a novel mechanism for the development of PE and potentially other forms of hypertensive pregnancies. These AhR ligand-activated genes and phosphoproteins might represent promising therapeutic and fetal sex-specific targets for PE-impaired vascular functions.

Gestational Intermittent Hypoxia Programs Hypertensive Response in Female Rat Offspring: Impact of Ovaries.

Obstructive sleep apnea (OSA) is a chronic condition frequently observed in pregnant women. We have shown that gestational intermittent hypoxia (GIH), a hallmark of OSA, leads to sex-specific impairment in the endothelium-dependent relaxation response and an increase in blood pressure in adult male but not female rat offspring. The present study tested the hypothesis that functional ovaries normalize GIH-induced hypertensive response in female offspring. Experiments were done in female offspring of pregnant rats exposed to normoxia or GIH (FIO2 21-10.5% from gestational days 10 to 21). Ovariectomy and sham surgery were performed at 5 weeks of age. Pups born to GIH dams were significantly smaller than the controls, but they exhibited catch-up growth and were similar to controls by 5 weeks of age. Ovariectomy significantly exacerbated bodyweight gain to a similar extent in both control and GIH offspring. Marked increases in blood pressure were observed in pre-pubertal GIH offspring compared to controls; however, after puberty, blood pressure in GIH offspring progressively decreased and became normotensive at adulthood. Ovariectomy led to the maintenance of higher blood pressure in post-pubertal GIH offspring with no significant effect in controls. Vascular contractile and relaxation responses were not affected in the GIH and control offspring; however, ovariectomy selectively decreased endothelium-dependent relaxation response along with a decrease in endothelial nitric oxide synthase expression in the GIH offspring. These findings suggest that functional ovaries are crucial in protecting females against GIH-mediated endothelial dysfunction and hypertension in adulthood.

Maternal perfluorooctane sulfonic acid exposure during rat pregnancy causes hypersensitivity to angiotensin II and attenuation of endothelium-dependent vasodilation in the uterine arteries †.

Epidemiological studies show a strong association between environmental exposure to perfluorooctane sulfonic acid (PFOS) and preeclampsia and fetal growth restriction; however, the underlying mechanisms are unclear. We tested the hypothesis that gestational PFOS exposure leads to pregnancy complications via alterations in uterine vascular endothelium-independent angiotensin II-related mechanisms and endothelium-derived factors such as nitric oxide. Pregnant Sprague-Dawley rats were exposed to PFOS 0.005, 0.05, 0.5, 5, 10, and 50 µg/mL through drinking water from gestational day 4 to 20, and dams with PFOS 50 µg/mL were used to assess mechanisms. PFOS exposure dose dependently increased maternal blood pressure but decreased fetal weights. Uterine artery blood flow was lower and resistance index was higher in the PFOS dams. In PFOS dams, uterine artery contractile responses to angiotensin II were significantly greater, whereas contractile responses to K+ depolarization and phenylephrine were unaffected. Plasma angiotensin II levels were not significantly different between control and PFOS dams; however, PFOS exposure significantly increased Angiotensin II type 1 receptor (AGTR1) and decreased AGTR2 protein levels in uterine arteries. Endothelium-dependent relaxation response to acetylcholine was significantly reduced with decreased endothelial nitric oxide synthase expression in the uterine arteries of PFOS dams. Left ventricular hypertrophy and fibrosis were observed, along with increased ejection fraction and fractional shortening in PFOS dams. These results suggest that elevated maternal PFOS levels decrease uterine blood flow and increase vascular resistance via heightened angiotensin II-mediated vasoconstriction and impaired endothelium-dependent vasodilation, which provides a molecular mechanism linking elevated maternal PFOS levels with gestational hypertension and fetal growth restriction.

AT2R activation increases in vitro angiogenesis in pregnant human uterine artery endothelial cells.

Angiogenesis is vital during pregnancy for remodeling and enhancing vasodilation of maternal uterine arteries, and increasing uterine blood flow. Abnormal angiogenesis is associated with decreased uteroplacental blood flow and development of pregnancy disorders such as gestational hypertension, preeclampsia, fetal growth restriction, preterm delivery, stillbirth, and miscarriage. The mechanisms that contribute to normal angiogenesis remain obscure. Our previous studies demonstrated that expression of the angiotensin type 2 receptor (AT2R) is increased while the angiotensin type 1 receptor (AT1R) is unchanged in the endothelium of uterine arteries, and that AT2R-mediated pregnancy adaptation facilitates enhanced vasodilation and uterine arterial blood flow. However, the role of AT2R in regulating angiogenesis during pregnancy has never been studied. This study examines whether or not AT2R activation induces angiogenesis and, if so, what mechanisms are involved. To this end, we used primary human uterine artery endothelial cells (hUAECs) isolated from pregnant and nonpregnant women undergoing hysterectomy. The present study shows that Compound 21, a selective AT2R agonist, induced proliferation of pregnant-hUAECs, but not nonpregnant-hUAECs, in a concentration-dependent manner, and that this C21-induced mitogenic effect was blocked by PD123319, a selective AT2R antagonist. The mitogenic effects induced by C21 were inhibited by blocking JNK-but not ERK, PI3K, and p38-signaling pathways. In addition, C21 concentration dependently increased cell migration and capillary-like tube formation in pregnant-hUAECs. The membrane-based antibody array showed that C21 increased expression of multiple angiogenic proteins, including EGF, bFGF, leptin, PLGF, IGF-1, and angiopoietins. Our qPCR analysis demonstrates that C21-induced increase in expression of these angiogenic proteins correlates with a proportional increase in mRNA expression, indicating that AT2R activates angiogenic proteins at the transcriptional level. In summary, the present study shows that AT2R activation induces angiogenesis of hUAECs in a pregnancy-specific manner through JNK-mediated pathways with associated transcriptional upregulation of multiple proangiogenic proteins.

Hyperandrogenism diminishes maternal-fetal fatty acid transport by increasing FABP4-mediated placental lipid accumulation†.

Long-chain polyunsaturated fatty acids (LCPUFAs) are critical for fetal brain development. Infants born to preeclamptic mothers or those born growth restricted due to placental insufficiency have reduced LCPUFA and are at higher risk for developing neurodevelopmental disorders. Since plasma levels of testosterone (T) and fatty acid-binding protein 4 (FABP4) are elevated in preeclampsia, we hypothesized that elevated T induces the expression of FABP4 in the placenta leading to compromised transplacental transport of LCPUFAs. Increased maternal T in pregnant rats significantly decreased n-3 and n-6 LCPUFA levels in maternal and fetal circulation, but increased their placental accumulation. Dietary LCPUFAs supplementation in T dams increased LCPUFA levels in the maternal circulation and further augmented placental storage, while failing to increase fetal levels. The placenta in T dams exhibited increased FABP4 mRNA and protein levels. In vitro, T dose-dependently upregulated FABP4 transcription in trophoblasts. Testosterone stimulated androgen receptor (AR) recruitment to the androgen response element and trans-activated FABP4 promoter activity, both of which were abolished by AR antagonist. Testosterone in pregnant rats and cultured trophoblasts significantly reduced transplacental transport of C14-docosahexaenoic acid (DHA) and increased C14-DHA accumulation in the placenta. Importantly, FABP4 overexpression by itself in pregnant rats and trophoblasts increased transplacental transport of C14-DHA with no significant placental accumulation. Testosterone exposure, in contrast, inhibited this FABP4-mediated effect by promoting C14-DHA placental accumulation.

Gestational Intermittent Hypoxia Induces Sex-Specific Impairment in Endothelial Mechanisms and Sex Steroid Hormone Levels in Male Rat Offspring.

Obstructive sleep apnea (OSA) is highly prevalent during gestation and is linked with adverse fetal outcomes. We examined whether gestational intermittent hypoxia (GIH), the main feature of OSA, leads to sex-specific alterations in cardiovascular function and vascular mechanisms in the offspring. Pregnant rats exposed to intermittent hypoxia or ambient air from gestation days 10 to 21 and their offspring were used for the study. GIH exposure did not affect water and food intake in dams. Compared to controls, the male and female offspring born to GIH dams were smaller in weight by 14% and 12%, respectively, and exhibited catch-up growth. Cardiac function was not affected in either GIH males or females. At 12 weeks of age, blood pressure was increased in GIH males, but not GIH females, compared to their control counterparts. While mesenteric arterial contractile responses to phenylephrine and endothelin were unaffected in GIH males and females, relaxation response to acetylcholine was reduced in GIH males but not GIH females. Relaxation to sodium nitroprusside was unaffected in both GIH males and females. Total eNOS expression was not affected, but phospho(Ser1177)-eNOS levels were decreased in GIH males. eNOS expression and its phosphorylation status were unaffected in GIH females. Serum testosterone and estradiol levels were higher in GIH males but were unaltered in GIH females. Together, these findings suggest that GIH leads to a sex-specific increase in blood pressure in adult male offspring with blunted endothelium-mediated relaxation, decreased eNOS activity, and elevated sex steroid hormone levels.

Activation of angiotensin type 2 receptor attenuates testosterone-induced hypertension and uterine vascular resistance in pregnant rats†.

Preeclampsia is a pregnancy-related hypertensive disorder with unclear mechanisms. While hypersensitivity to angiotensin II via vasoconstrictive angiotensin type-1 receptor (AT1R) is observed in preeclampsia, the importance of vasodilatory angiotensin type-2 receptor (AT2R) in the control of vascular dysfunction is less clear. We assessed whether AT1R, AT2R, and endothelial nitric oxide synthase (eNOS) expression are altered in placental vessels of preeclamptic women and tested if ex vivo incubation with AT2R agonist Compound 21 (C21; 1 µM) could restore AT1R, AT2R, and eNOS balance. Further, using a rat model of gestational hypertension induced by elevated testosterone, we examined whether C21 (1 µg/kg/day, oral) could preserve AT1R and AT2R balance and improve blood pressure, uterine artery blood flow, and vascular function. Western blots revealed that AT1R protein level was higher while AT2R and eNOS protein were reduced in preeclamptic placental vessels, and AT2R agonist C21 decreased AT1R and increased AT2R and eNOS protein levels in preeclamptic vessels. In testosterone dams, blood pressure was higher, and uterine artery blood flow was reduced, and C21 treatment reversed these levels similar to those in controls dams. C21 attenuated the exaggerated Ang II contraction and improved endothelium-dependent vasorelaxation in uterine arteries of testosterone dams. These C21-mediated vascular effects were associated with decreased AT1R and increased AT2R and eNOS protein levels. C21 also increased serum nitrate/nitrite and bradykinin production in testosterone dams and attenuated the fetoplacental growth restriction. Thus, AT1R upregulation and AT2R downregulation are observed in preeclampsia and testosterone model, and increasing AT2R activity could help restore AT1R and AT2R balance and improve gestational vascular function.

Perfluorooctane sulfonic acid (PFOS) exposure during pregnancy increases blood pressure and impairs vascular relaxation mechanisms in the adult offspring.

Perfluorooctanesulfonate (PFOS) is a persistent environmental agent. We examined whether PFOS exposure during pregnancy alters blood pressure in male and female offspring, and if this is related to sex-specific changes in vascular mechanisms. PFOS was administered through drinking water (50 µg/mL) to pregnant Sprague-Dawley rats from gestational day 4 until delivery. PFOS-exposure decreased maternal weight gain but did not significantly alter feed and water intake in dams. The male and female pups born to PFOS mothers were smaller in weight by 29 % and 27 %, respectively. The male PFOS offspring remained smaller through adulthood, but the female PFOS offspring exhibited catch-up growth. The blood pressure at 12 and 16 weeks of age was elevated at similar magnitude in PFOS males and females than controls. Mesenteric arterial relaxation to acetylcholine was reduced in both PFOS males and females, but the extent of decrease was greater in females. Relaxation to sodium-nitroprusside was reduced in PFOS females but unaffected in PFOS males. Vascular eNOS expression was not changed, but phospho(Ser1177)-eNOS was decreased in PFOS males. In PFOS females, both total eNOS and phospho(Ser1177)-eNOS expression were reduced. In conclusion, PFOS exposure during prenatal life (1) caused low birth weight followed by catch-up growth only in females (2) lead to hypertension of similar magnitude in both males and females; (2) decreased endothelium-dependent vascular relaxation in males but suppressed both endothelium-dependent and -independent relaxation in females. The endothelial dysfunction is associated with reduced activity of eNOS in males and decreased expression and activity of eNOS in females.

Testosterone Decreases Placental Mitochondrial Content and Cellular Bioenergetics.

Placental mitochondrial dysfunction plays a central role in the pathogenesis of preeclampsia. Since preeclampsia is a hyperandrogenic state, we hypothesized that elevated maternal testosterone levels induce damage to placental mitochondria and decrease bioenergetic profiles. To test this hypothesis, pregnant Sprague-Dawley rats were injected with vehicle or testosterone propionate (0.5 mg/kg/day) from gestation day (GD) 15 to 19. On GD20, the placentas were isolated to assess mitochondrial structure, copy number, ATP/ADP ratio, and biogenesis (Pgc-1α and Nrf1). In addition, in vitro cultures of human trophoblasts (HTR-8/SVneo) were treated with dihydrotestosterone (0.3, 1.0, and 3.0 nM), and bioenergetic profiles using seahorse analyzer were assessed. Testosterone exposure in pregnant rats led to a 2-fold increase in plasma testosterone levels with an associated decrease in placental and fetal weights compared with controls. Elevated maternal testosterone levels induced structural damage to the placental mitochondria and decreased mitochondrial copy number. The ATP/ADP ratio was reduced with a parallel decrease in the mRNA and protein expression of Pgc-1α and Nrf1 in the placenta of testosterone-treated rats compared with controls. In cultured trophoblasts, dihydrotestosterone decreased the mitochondrial copy number and reduced PGC-1α, NRF1 mRNA, and protein levels without altering the expression of mitochondrial fission/fusion genes. Dihydrotestosterone exposure induced significant mitochondrial energy deficits with a dose-dependent decrease in basal respiration, ATP-linked respiration, maximal respiration, and spare respiratory capacity. In summary, our study suggests that the placental mitochondrial dysfunction induced by elevated maternal testosterone might be a potential mechanism linking preeclampsia to feto-placental growth restriction.

Elevated Glucose and Insulin Levels Decrease DHA Transfer across Human Trophoblasts via SIRT1-Dependent Mechanism.

Gestational diabetes mellitus (GDM) results in reduced docosahexaenoic acid (DHA) transfer to the fetus, likely due to placental dysfunction. Sirtuin-1 (SIRT1) is a nutrient sensor and regulator of lipid metabolism. This study investigated whether the high glucose and insulin condition of GDM regulates DHA transfer and expression of fatty acid transporters and if this effect is related to SIRT1 expression and function. Syncytialized primary human trophoblasts were treated with and without glucose (25 mmol/L) and insulin (10-7 mol/L) for 72 h to mimic the insulin-resistance conditions of GDM pregnancies. In control conditions, DHA transfer across trophoblasts increased in a time- and dose-dependent manner. Exposure to GDM conditions significantly decreased DHA transfer, but increased triglyceride accumulation and fatty acid transporter expression (CD36, FABP3, and FABP4). GDM conditions significantly suppressed SIRT1 mRNA and protein expression. The SIRT1 inhibitor decreased DHA transfer across control trophoblasts, and recombinant SIRT1 and SIRT1 activators restored the decreased DHA transport induced by GDM conditions. The results demonstrate a novel role of SIRT1 in the regulation of DHA transfer across trophoblasts. The suppressed SIRT1 expression and the resultant decrease in placental DHA transfer caused by high glucose and insulin levels suggest new insights of molecular mechanisms linking GDM to fetal DHA deficiency.