Endogenous hydrogen sulfide (H2S) synthesized via metabolizing L-cysteine by cystathionine-beta-synthase (CBS) and cystathionine-gamma-lyase (CSE) is a potent vasodilator and angiogenic factor. The objectives of this study were to determine if human uterine artery (UA) H2S production increases with augmented expression and/or activity of CBS and/or CSE during the menstrual cycle and pregnancy and whether exogenous H2S dilates UA. Uterine arteries from nonpregnant (NP) premenopausal proliferative (pPRM) and secretory (sPRM) phases of the menstrual cycle and pregnant (P) women were studied. H2S production was measured by the methylene blue assay. CBS and CSE mRNAs were assessed by quantitative real-time PCR, and proteins were assessed by immunoblotting and semiquantitative immunofluorescence microscopy. Effects of H2S on rat UA relaxation were determined by wire myography ex vivo. H2S production was greater in NP pPRM and P than NP sPRM UAs and inhibited by the specific CBS but not CSE inhibitor. CBS but not CSE mRNA and protein were greater in NP pPRM and P than NP sPRM UAs. CBS protein was localized to endothelium and smooth muscle and its levels were in a quantitative order of P >NP UAs of pPRM>sPRM. CSE protein was localized in UA endothelium and smooth muscle with no difference among groups. A H2S donor relaxed P > NP UAs but not mesentery artery. Thus, human UA H2S production is augmented with endothelium and smooth muscle CBS upregulation, contributing to UA vasodilation in the estrogen-dominant physiological states in the proliferative phase of the menstrual cycle and pregnancy.

Adequate maternal vascular adaptations and blood supply to the uterus and placenta are crucial for optimal oxygen and nutrient transport to growing fetuses of eutherian mammals, including humans. Multiple factors contribute to hemodynamics and structuring of placental vasculature essential for term pregnancy with minimal complications. In women, failure to achieve or sustain favorable pregnancy progression is, not surprisingly, associated with high incidence of antenatal complications, including preeclampsia, a hypertensive disorder of pregnancy. While the pathogenesis of preeclampsia in women remains unknown, a role for androgens is emerging. The relationship between androgens and maternal cardiovascular and placental function deserves particular consideration because testosterone levels in the circulation of preeclamptic women are elevated approximately two- to three-fold and are positively correlated with vascular dysfunction. Preeclampsia is also associated with elevated placental androgen receptor (AR) gene expression. Studies in animal models mimicking the pattern and level of increase of adult female testosterone levels to those found in preeclamptic pregnancies, replicate key features of preeclampsia, including gestational hypertension, endothelial dysfunction, exaggerated vasoconstriction to angiotensin II, reduced spiral artery remodeling, placental hypoxia, decreased nutrient transport and fetal growth restriction. Taken together, these data strongly implicate AR-mediated testosterone action as an important pathway contributing to clinical manifestation of preeclampsia. This review critically addresses this hypothesis, taking into consideration both clinical and preclinical data.

Introduction Blood pressure is lower in females than males. Angiotensin II type-2 receptor (AT2R) induces vasodilation. This study determined whether sex differences in vascular AT2R expression occur and if androgens exert control on AT2R expression in the vasculature. Methods AT2Rs in the aorta of male and female Sprague-Dawley rats were examined following alteration in androgen levels by gonadectomy or hormone supplementation. Results AT2R mRNA and protein expression levels were lower in aorta of males than females. In males, testosterone withdrawal by castration significantly elevated AT2R mRNA and protein levels and testosterone replacement restored them. In females, increasing androgen levels decreased AT2R mRNA and protein expression and this was attenuated by androgen receptor blocker flutamide. Ex vivo, dihydrotestosterone downregulated AT2R in endothelium-intact but not -denuded aorta. Dihydrotestosterone-induced AT2R downregulation in isolated aorta was blocked by androgen receptor antagonist. Furthermore, blockade of ERK1/2 but not p38 MAP kinase or TGFβ signaling with specific inhibitors abolished dihydrotestosterone-induced AT2R downregulation. Conclusion Androgens downregulates AT2R expression levels in aorta, in vivo and ex vivo. The androgen receptor-mediated ERK1/2 MAP kinase-signaling pathway may be a key mechanism by which testosterone downregulates AT2R expression, implicating androgens’ contributing role to gender differences in vascular AT2R expression.

Hyperandrogenism is associated with hyperinsulinemia and insulin resistance in adult females. We tested whether androgens dysregulate pancreatic beta cell function to induce hyperinsulinemia through transcriptional regulation of insulin gene (Ins) in the islets. Adult female Wistar rats implanted with dihydrotestosterone (DHT; 7.5-mg, 90-d release) or placebo pellets were examined after 10 weeks. DHT exposure increased plasma DHT levels by 2-fold similar to that in polycystic ovary syndrome in women. DHT exposure induced hyperinsulinemia with increased HOMA-IR index in fasting state and glucose intolerance and exaggerated insulin responses following glucose tolerance test. DHT females had no change in islet number, size and beta cell proliferation/apoptosis but exhibited significant mitochondrial dysfunction (higher ADP/ATP ratio, decreased mtDNA copy number, increased reactive oxygen production and downregulation of mitochondrial biogenesis) and enhanced glucose-stimulated insulin secretion. Ins expression was increased in DHT islets. In vitro incubation of control islets with DHT dose dependently stimulated Ins transcription. Analysis of Ins1 gene revealed a putative androgen responsive element in the promoter. Chromatinimmunoprecipitation assays showed that androgen receptors bind to this element in response to DHT stimulation. Furthermore, reporter assays showed that the promoter element is highly responsive to androgens. Insulin-stimulated glucose uptake in skeletal muscle was decreased with associated decrease in IRβ expression in DHT females. Our studies identified a novel androgenmediated mechanism for the control of Ins expression via transcriptional regulation providing a molecular mechanism linking elevated androgens and hyperinsulemia. Decreased IRβ expression in the skeletal muscles may contribute, in part, to glucose intolerance in this model. -cell hyperinsulinemia, 2018, Vol. 98, No. 4 Summary SentenceElevated androgen levels lead to hyperinsulinemia despite mitochondrial dysfunction in pancreatic islets; hyperandrogenism directly controls the expression of Ins1 in pancreatic islets by positively regulating Ins1 transcription which might play an underlying role in hyperinsulinemia.

Normal pregnancy is associated with decreased uterine vascular contraction and increased blood flow even though angiotensin II (AngII) levels are increased. AngII not only activates the angiotensin type 1 receptor (AT1R) to mediate vasoconstriction but also angiotensin type 2 receptor (AT2R) to cause vasodilation. We hypothesized that upregulation of AT2R expression and function accounts for increased uterine artery blood flow during pregnancy. Virgin, pregnant rats at different days of gestation and post-partum Sprague-Dawley rats were used to determine uterine artery hemodynamics using micro ultrasound and plasma angiotensin II levels by ELISA. Isolated uterine arteries were examined for AT1R and AT2R expression and isometric contraction/relaxation. Plasma AngII levels were steady up to mid-pregnancy, increased as pregnancy advanced, reaching a peak in late pregnancy, and then restored to pre-pregnant levels after delivery. The pattern of increase in AngII levels mirrored a parallel increase in uterine blood flow. AT1R expression did not change but AT2R expression increased during pregnancy correlating with uterine blood flow increase. Treatment with the AT2R antagonist PD123319 reduced uterine arterial blood flow. Vasoconstriction to angiotensin II was blunted in pregnant rats. Treatment with PD123319 caused greater enhancement of AngII contraction in pregnant than virgin rats. Ex vivo exposure of estradiol to uterine arterial rings dose dependently upregulated AT2R expression, that was inhibited by estrogen receptor antagonist. These results demonstrate that elevated AngII levels during gestation induces increase in uterine blood flow via heightened AT2R-mediated signaling. Estrogens appear to directly upregulate uterine vascular AT2R independent of any endogenous factors.