Sarah H. Lindsey

Role of estrogen in diastolic dysfunction

The prevalence of left ventricular diastolic dysfunction (LVDD) sharply increases in women after menopause and may lead to heart failure. While evidence suggests that estrogens protect the premenopausal heart from hypertension and ventricular remodeling, the specific mechanisms involved remain elusive. Moreover, whether there is a protective role of estrogens against cardiovascular disease, and specifically LVDD, continues to be controversial. Clinical and basic science have implicated activation of the renin-angiotensin-aldosterone system (RAAS), linked to the loss of ovarian estrogens, in the pathogenesis of postmenopausal diastolic dysfunction. As a consequence of increased tissue ANG II and low estrogen, a maladaptive nitric oxide synthase (NOS) system produces ROS that contribute to female sex-specific hypertensive heart disease. Recent insights from rodent models that mimic the cardiac phenotype of an estrogen-insufficient or -deficient woman (e.g., premature ovarian failure or postmenopausal), including the ovariectomized congenic mRen2.Lewis female rat, provide evidence showing that estrogen modulates the tissue RAAS and NOS system and related intracellular signaling pathways, in part via the membrane G protein-coupled receptor 30 (GPR30; also called G protein-coupled estrogen receptor 1). Complementing the cardiovascular research in this field, the echocardiographic correlates of LVDD as well as inherent limitations to its use in preclinical rodent studies will be briefly presented. Understanding the roles of estrogen and GPR30, their interactions with the local RAAS and NOS system, and the relationship of each of these to LVDD is necessary to identify new therapeutic targets and alternative treatments for diastolic heart failure that achieve the cardiovascular benefits of estrogen replacement without its side effects and contraindications.

Vasodilation by GPER in mesenteric arteries involves both endothelial nitric oxide and smooth muscle cAMP signaling

Our previous work showed that chronic activation of the membrane-bound estrogen receptor GPR30/GPER significantly lowers blood pressure in ovariectomized hypertensive mRen2.Lewis female rats which may, in part, reflect direct vasodilatory actions. The current study assessed the hypothesis that cyclic adenosine monophosphate (cAMP) signaling contributes to GPER-mediated vasorelaxation. In mesenteric resistance arteries from intact Lewis females, relaxation to 17-β-estradiol (E2; 47±3% of phenylephrine contraction vs. vehicle 89±2%, P<0.001) or G-1 (44±8%, P<0.001) was blunted to a similar extent by denuding (P<0.001) or the nitric oxide synthase inhibitor l-NAME (P<0.001). In contrast, the cyclooxygenase inhibitor indomethacin did not alter vasodilation (P>0.05). The cAMP analog Rp-cAMPS partially attenuated vasodilation (65±7%, P<0.001), while the combination of l-NAME and Rp-cAMPS exhibited additive effects to effectively abolish vasorelaxation (P>0.05 vs. vehicle). Pretreatment of endothelium-intact vessels with the adenylyl cyclase inhibitor SQ (63±6%) or the guanylyl cyclase inhibitor ODQ (62±9%) both partially inhibited the response to G-1 (P<0.01), while pretreatment with the both inhibitors completely abolished vasorelaxation (P>0.05 vs. vehicle). In denuded vessels only SQ reduced the response (88±3%, P<0.001). Moreover, G-1 significantly increased intracellular cAMP levels in cultured mesenteric smooth muscle cells (P<0.05). We conclude that GPER-dependent vasorelaxation apparently involves both endothelial release of nitric oxide which activates guanylyl cyclase and smooth muscle cell activation of adenylyl cyclase. Downstream production of cyclic nucleotides and stimulation of protein kinases may phosphorylate proteins to promote vascular smooth muscle cell relaxation. The ability of GPER to initiate these signaling pathways may contribute to the beneficial vascular effects of estrogen.

Reduced vasorelaxation to estradiol and G-1 in aged female and adult male rats is associated with GPR30 downregulation.

Previously, we reported that chronic activation of the estrogen receptor GPR30 by its selective agonist G-1 decreases blood pressure in ovariectomized hypertensive mRen2.Lewis (mRen2) rats but not intact male littermates. Furthermore, G-1 relaxes female mesenteric resistance arteries via both endothelium-dependent and -independent mechanisms. Because of the lack of a blood pressure-lowering effect by G-1 in males and the potential influence of aging on estrogen receptor expression, we hypothesized that GPR30-dependent vasodilation and receptor expression are altered in males and aged females. Thus, we assessed the response to 17β-estradiol or G-1 in mesenteric arteries obtained from 15-wk-old normotensive Lewis and hypertensive mRen2 females and males as well as 52-wk-old Lewis females. Vasodilation to 17β-estradiol (E₂) and G-1 was significantly attenuated in 15-wk-old Lewis and mRen2 males compared with age-matched females. Pretreatment of male vessels with the nitric oxide synthase inhibitor L-NAME had no significant effect on the estradiol or G-1 response. In aged females, E₂ and G-1 vasorelaxation was also significantly blunted; however, L-NAME essentially abolished the response. Associated with the reduced vascular responses, GPR30 expression in mesenteric arteries was approximately 50% lower in males and aged females compared with young females. We conclude that alterations in GPR30 expression and signaling may contribute to vascular dysfunction in aging females and a greater blood pressure in hypertensive males.

Uterine artery dysfunction in pregnant ACE2 knockout mice is associated with placental hypoxia and reduced umbilical blood flow velocity

Angiotensin-converting enzyme 2 (ACE2) knockout is associated with reduced fetal weight at late gestation; however, whether uteroplacental vascular and/or hemodynamic disturbances underlie this growth-restricted phenotype is unknown. Uterine artery reactivity and flow velocities, umbilical flow velocities, trophoblast invasion, and placental hypoxia were determined in ACE2 knockout (KO) and C57Bl/6 wild-type (WT) mice at day 14 of gestation. Although systolic blood pressure was higher in pregnant ACE2 KO vs. WT mice (102.3 ± 5.1 vs. 85.1 ± 1.9 mmHg, n = 5-6), the magnitude of difference was similar to that observed in nonpregnant ACE2 KO vs. WT mice. Maternal urinary protein excretion, serum creatinine, and kidney or heart weights were not different in ACE2 KO vs. WT. Fetal weight and pup-to-placental weight ratio were lower in ACE2 KO vs. WT mice. A higher sensitivity to Ang II [pD2 8.64 ± 0.04 vs. 8.5 ± 0.03 (-log EC50)] and greater maximal contraction to phenylephrine (169.0 ± 9.0 vs. 139.0 ± 7.0% KMAX), were associated with lower immunostaining for Ang II receptor 2 and fibrinoid content of the uterine artery in ACE2 KO mice. Uterine artery flow velocities and trophoblast invasion were similar between study groups. In contrast, umbilical artery peak systolic velocities (60.2 ± 4.5 vs. 75.1 ± 4.5 mm/s) and the resistance index measured using VEVO 2100 ultrasound were lower in the ACE2 KO vs. WT mice. Immunostaining for pimonidazole, a marker of hypoxia, and hypoxia-inducible factor-2α were higher in the trophospongium and placental labyrinth of the ACE2 KO vs. WT. In summary, placental hypoxia and uterine artery dysfunction develop before major growth of the fetus occurs and may explain the fetal growth restricted phenotype.

GPER activation ameliorates aortic remodeling induced by salt-sensitive hypertension

The mRen2 female rat is an estrogen- and salt-sensitive model of hypertension that reflects the higher pressure and salt sensitivity associated with menopause. We previously showed that the G protein-coupled estrogen receptor (GPER) mediates estrogenic effects in this model. The current study hypothesized that GPER protects against vascular injury during salt loading. Intact mRen2 female rats were fed a normal (NS; 0.5% Na(+)) or high-salt diet (HS; 4% Na(+)) for 10 wk, which significantly increased systolic blood pressure (149 ± 5 vs. 224 ± 8 mmHg;P< 0.001). Treatment with the selective GPER agonist G-1 for 2 wk did not alter salt-sensitive hypertension (216 ± 4 mmHg;P> 0.05) or ex vivo vascular responses to angiotensin II or phenylephrine (P> 0.05). However, G-1 significantly attenuated salt-induced aortic remodeling assessed by media-to-lumen ratio (NS: 0.43; HS+veh: 0.89; HS+G-1: 0.61;P< 0.05). Aortic thickening was not accompanied by changes in collagen, elastin, or medial proliferation. However, HS induced increases in medial layer glycosaminoglycans (0.07 vs. 0.42 mm(2);P< 0.001) and lipid peroxidation (0.11 vs. 0.51 mm(2);P< 0.01), both of which were reduced by G-1 (0.20 mm(2)and 0.23 mm(2); both P< 0.05). We conclude that GPERs beneficial actions in the aorta of salt-loaded mRen2 females occur independently of changes in blood pressure and vasoreactivity. GPER-induced attenuation of aortic remodeling was associated with a reduction in oxidative stress and decreased accumulation of glycosaminoglycans. Endogenous activation of GPER may protect females from salt- and pressure-induced vascular damage.

GPER–novel membrane oestrogen receptor

The recent discovery of the G protein-coupled oestrogen receptor (GPER) presents new challenges and opportunities for understanding the physiology, pathophysiology and pharmacology of many diseases. This review will focus on the expression and function of GPER in hypertension, kidney disease, atherosclerosis, vascular remodelling, heart failure, reproduction, metabolic disorders, cancer, environmental health and menopause. Furthermore, this review will highlight the potential of GPER as a therapeutic target.

Importance of Estrogen Metabolites

See related article, pp 134–140 The influence of estrogen on blood pressure is complicated and sometimes contradictory. Premenopausal women have a lower incidence of hypertension, but recent clinical trials suggest that postmenopausal hormone replacement therapy does not necessarily decrease the risk of cardiovascular disease. Sexually dimorphic hypertension is evident in multiple animal models, yet only some indicate a critical role for estrogen.1 These contradictions emphasize the importance of identifying the critical molecular signaling pathways in the cardiovascular system that are activated by estrogen and pursuing alternative pathways for their activation. One important factor that has been neglected in the study of estrogen’s cardiovascular effects is the conversion of 17-β-estradiol, the most commonly studied ovarian estrogen, to metabolites that are capable of exerting discrete physiological effects (Figure). Figure. In the study by Jennings et al,2 the ability of endogenous estrogen to counteract the blood pressure response to angiotensin II infusion is a result of the metabolism of 17-β-estradiol (17-β-E2) to 2-hydroxyestradiol (2-OHE) by cytochrome P450 1B1 (CYP1B1) and subsequent conversion by catechol-O-methyl transferase (COMT) to 2-methoxyestradiol (2-MeE2). In contrast, the production of 4-OHE and 4-methoxyestradiol (4-MeE2) exacerbates hypertension in this model. Other 17-β-E2 precursors or metabolites that may impact cardiovascular function include 27-hydroxycholesterol (27-OHC), 16α-OHE, estrone (E1), and estriol (E3). In this issue of Hypertension , Jennings et al2 …

Effect of menopausal hormone therapy on components of the metabolic syndrome

The world population is aging, and women will spend an increasing share of their lives in a postmenopausal state that predisposes to metabolic dysfunction. Thus, the prevalence of metabolic syndrome (MetS) in women is likely to increase dramatically. This article summarizes the effects of menopause in predisposing to components of MetS including visceral obesity, dyslipidemia, type 2 diabetes (T2D) and hypertension (HTN). We also summarize the effects of menopausal hormone therapy (MHT) in reversing these metabolic alterations and discuss therapeutic advances of novel menopausal treatment on metabolic function.

Analysis of Erectile Responses to Bradykinin in the Anesthetized Rat

The kallikrein-kinin system is expressed in the corpus cavernosa, and bradykinin (BK) relaxes isolated corpora cavernosal strips. However, erectile responses to BK in the rat have not been investigated in vivo. In the present study, responses to intracorporal (ic) injections of BK were investigated in the anesthetized rat. BK, in doses of 1-100 μg/kg ic, produced dose-related increases in intracavernosal pressure (ICP) and dose-related deceases in mean arterial pressure (MAP). When decreases in MAP were prevented by intravenous injections of angiotensin II (Ang II), increases in ICP, in response to BK, were enhanced. Increases in ICP, ICP/MAP ratio, and area under the curve and decreases in MAP in response to BK were inhibited by the kinin B2 receptor antagonist HOE-140 and enhanced by the angiotensin-converting enzyme (ACE) inhibitor captopril and by Ang-(1-7). Increases in ICP, in response to BK, were not attenuated by the nitric oxide (NO) synthase inhibitor (N(ω)-nitro-L-arginine methyl ester) or the soluble guanylate cyclase inhibitor (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) but were attenuated by the cyclooxygenase inhibitor, sodium meclofenamate. Decreases in MAP were not attenuated by either inhibitor. These data suggest that erectile responses are mediated by kinin B2 receptors and modulated by decreases in MAP. These data indicate that ACE is important in the inactivation of BK and that erectile and hypotensive responses are independent of NO in the penis or the systemic vascular bed. Erectile responses to cavernosal nerve stimulation are not altered by BK or HOE-140, suggesting that BK and B2 receptors do not modulate nerve-mediated erectile responses under physiologic conditions. These data suggest that erectile responses to BK are mediated, in part, by the release of cyclooxygenase products.

Transforming growth factor β1 antagonizes the transcription, expression and vascular signaling of guanylyl cyclase/natriuretic peptide receptor A - role of δEF1.

The objective of this study was to determine the role of transforming growth factor β1 (TGF‐β1) in transcriptional regulation and function of the guanylyl cyclase A/natriuretic peptide receptor A gene (Npr1) and whether cross‐talk exists between these two hormonal systems in target cells. After treatment of primary cultured rat thoracic aortic vascular smooth muscle cells and mouse mesangial cells with TGF‐β1, the Npr1 promoter construct containing a δ‐crystallin enhancer binding factor 1 (δEF1) site showed 85% reduction in luciferase activity in a time‐ and dose‐dependent manner. TGF‐β1 also significantly attenuated luciferase activity of the Npr1 promoter by 62%, and decreased atrial natriuretic peptide‐mediated relaxation of mouse denuded aortic rings ex vivo. Treatment of cells with TGF‐β1 increased the protein levels of δEF1 by 2.4–2.8‐fold, and also significantly enhanced the phosphorylation of Smad 2/3, but markedly reduced Npr1 mRNA and receptor protein levels. Over‐expression of δEF1 showed a reduction in Npr1 promoter activity by 75%, while deletion or site‐directed mutagenesis of δEF1 sites in the Npr1 promoter eliminated the TGF‐β1‐mediated repression of Npr1 transcription. TGF‐β1 significantly increased the expression of α‐smooth muscle actin and collagen type I α2 in rat thoracic aortic vascular smooth muscle cells, which was markedly attenuated by atrial natriuretic peptide in cells over‐expressing natriuretic peptide receptor A. Together, the present results suggest that an antagonistic cascade exists between the TGF‐β1/Smad/δEF1 pathways and Npr1 expression and receptor signaling that is relevant to renal and vascular remodeling, and may be critical in the regulation of blood pressure and cardiovascular homeostasis.