Elvira Haas

Regulatory Role of G Protein–Coupled Estrogen Receptor for Vascular Function and Obesity

We found that the selective stimulation of the intracellular, transmembrane G protein-coupled estrogen receptor (GPER), also known as GPR30, acutely lowers blood pressure after infusion in normotensive rats and dilates both rodent and human arterial blood vessels. Stimulation of GPER blocks vasoconstrictor-induced changes in intracellular calcium concentrations and vascular tone, as well as serum-stimulated cell proliferation of human vascular smooth muscle cells. Deletion of the GPER gene in mice abrogates vascular effects of GPER activation and is associated with visceral obesity. These findings suggest novel roles for GPER in protecting from cardiovascular disease and obesity.

Differential Effects of 17β-Estradiol on Function and Expression of Estrogen Receptor α, Estrogen Receptor β, and GPR30 in Arteries and Veins of Patients With Atherosclerosis

Venous complications have been implicated in the adverse effects of hormone replacement therapy. This study investigated acute effects of the natural estrogen, 17&bgr;-estradiol, on function, estrogen receptors/GPR30 expression, and kinase activation in vascular rings and cultured smooth muscle cells from arteries and veins of patients with coronary artery disease. Changes in vascular tone of internal mammary arteries and saphenous veins exposed to the steroid were recorded. 17&bgr;-Estradiol caused concentration-dependent, endothelium-independent relaxation in arteries (P<0.05 versus solvent control) but not in veins (P not significant). 17&bgr;-Estradiol enhanced contractions to endothelin-1 in veins but not in arteries. The novel membrane estrogen receptor GPR30 was detected in both vessels. Moreover, gene expression of estrogen receptor &bgr; was 10-fold higher than that of estrogen receptor &agr; or GPR30 (P<0.05). Expression of all 3 of the receptors was reduced after exposure to 17&bgr;-estradiol in arteries but not in veins (P<0.05). Basal phosphorylation levels of extracellular signal-regulated kinase were higher in venous than in arterial smooth muscle cells and were increased by 17&bgr;-estradiol in arterial cells only. In summary, this is the first study to report that, in human arteries but not in veins, 17&bgr;-estradiol acutely affects vascular tone, estrogen receptor expression, including GPR30, and extracellular signal-regulated kinase phosphorylation. These data indicate that effects of natural estrogens in humans differ between arterial and venous vascular beds, which may contribute to the vascular risks associated with menopause or hormone therapy.

Non-genomic regulation of vascular cell function and growth by estrogen

Estrogens exert rapid, non-genomic effects, which are mediated by plasma membrane-associated estrogen receptors (mER) mERalpha and mERbeta, and the intracellular transmembrane G protein-coupled estrogen receptor (GPER). Membrane-initiated responses contribute to transcriptional activation, resulting in a complex interplay of nuclear and extra-nuclear mechanisms that mediate the acute physiological responses to estrogens. Non-genomic estrogen signaling also activates a variety of intracellular estrogen signaling pathways that regulate vascular function and cell growth involving rapid but also long-term effects. This review discusses recent advances in understanding of the mechanisms of non-genomic estrogen receptor signaling in the vascular wall.

Distinct Roles of Estrogen Receptors α and β Mediating Acute Vasodilation of Epicardial Coronary Arteries

This study investigated the contribution of estrogen receptors (ERs) &agr; and &bgr; for epicardial coronary artery function, vascular NO bioactivity, and superoxide (O2−) formation. Porcine coronary rings were suspended in organ chambers and precontracted with prostaglandin F2&agr; to determine direct effects of the selective ER agonists 4,4′,4″-(4-propyl-[1H]pyrazole-1,3,5-triyl)tris-phenol (PPT) or 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN) or the nonselective ER agonist 17&bgr;-estradiol. Indirect effects on contractility to U46619 and relaxation to bradykinin were assessed and effects on NO, nitrite, and O2− formation were measured in cultured cells. Within 5 minutes, selective ER&agr; activation by PPT, but not 17&bgr;-estradiol or the ER&bgr; agonist DPN, caused rapid, NO-dependent, and endothelium-dependent relaxation (49±5%; P<0.001 versus ethanol). PPT also caused sustained endothelium- and NO-independent vasodilation similar to 17&bgr;-estradiol after 60 minutes (72±3%; P<0.001 versus ethanol). DPN induced endothelium-dependent NO-independent relaxation via endothelium-dependent hyperpolarization (40±4%; P<0.01 versus ethanol). 17&bgr;-Estradiol and PPT, but not DPN, attenuated the responses to U46619 and bradykinin. All of the ER agonists increased NO and nitrite formation in vascular endothelial but not smooth muscle cells and attenuated vascular smooth muscle cell O2− formation (P<0.001). ER&agr; activation had the most potent effects on both nitrite formation and inhibiting O2− (P<0.05). These data demonstrate novel and differential mechanisms by which ER&agr; and ER&bgr; activation control coronary artery vasoreactivity in males and females and regulate vascular NO and O2− formation. The findings indicate that coronary vascular effects of sex hormones differ with regard to affinity to ER&agr; and ER&bgr;, which will contribute to beneficial and adverse effects of hormone replacement therapy.

Activation of Pro-Inflammatory and Anti-Inflammatory Cytokines in Host Organs During Chronic Allograft Rejection: Role of Endothelin Receptor Signaling

This study investigated whether allograft rejection is associated with local inflammatory activation in host organs and whether endothelin ETA receptor signaling is involved. Expression of IL‐1β, IL‐1ra, IL‐6, IL‐10 and TNF‐α was investigated in host liver, lung and native heart in a rat model of chronic rejection 8 weeks after heterotopic cardiac transplantation in the absence of immunosuppression. In the presence of rejection, circulating levels of cytokines increased, while tissue level activation was dependent on the organ involved. Similarly, tissue‐specific regulatory patterns were observed regarding transcriptional activation. Although chronic ETA receptor blockade did not reduce transplant vasculopathy or tissue protein expression, treatment had pronounced effects on plasma levels and transcriptional regulation of chemokines. These data provide evidence for distinct pro‐inflammatory local activation in host organs during chronic rejection and suggest a role for ETA receptors contributing to regulation of cytokine plasma levels and transcriptional activity.

Hormone Replacement Therapy and Atherosclerosis in Postmenopausal Women: Does Aging Limit Therapeutic Benefits?

Despite the clear-cut epidemiological evidence of protective effects of endogenous estrogens in premenopausal women,1,2 the results of randomized clinical trials using conjugated equine estrogens and medroxyprogesterone acetate instead of natural hormones have led to a paradigm shift in the usefulness of hormone treatment in postmenopausal women.3,4 One of the main criticisms in addition to the types of drugs chosen for treatment was the age of the patients. Indeed, in both WHI trial and HERS study, treatment of patients was initiated in women many years beyond menopause.5 In fact, the number of years since menopause was an independent indicator for nonfatal myocardial infarction or coronary related death.5 In this context, it appears of interest that heart disease may contribute to menopausal age and that menopausal age actually may be an indicator of cardiovascular risk.6,7 See page 1782 It was noted as early as 1952 that the natural endogenous estrogen 17β-estradiol inhibits experimental atherosclerosis,8 and oral estrogens were even unsuccessfully evaluated to treat coronary artery disease in male patients.9–11 However, at the time little was known about the mechanisms by which sex steroid hormones affect vascular homeostasis and thrombogenesis. During the past 2 decades, considerable advances were made in the understanding of how natural estrogens act on the vasculature. 17β-estradiol causes rapid and endothelium-independent dilation of coronary arteries of men and women,12 and chronic treatment with 17β-estradiol inhibits experimental atherosclerosis in males and females.13,14 On the other hand, treatment with conjugated equine estrogens, which contain more than 30 different steroid compounds including testosterone and substances of still undefined vascular activity (Table), have shown less favorable effects,2–4 as has been reported for the synthetic progestin medroxyprogesterone acetate.15 With the identification of molecular targets of 17β-estradiol and other estrogens it now appears that …

Need for research on estrogen receptor function: importance for postmenopausal hormone therapy and atherosclerosis.

BACKGROUND Cardiovascular disease is the leading cause of morbidity and mortality in men and women worldwide. Although rare in premenopausal women, its incidence rises sharply after menopause, indicating atheroprotective effects of endogenous estrogens. OBJECTIVE This review discusses the differential effects of estrogen receptor function on atherosclerosis progression in pre- and postmenopausal women, including aspects of gender differences in vascular physiology of estrogens and androgens. METHODS Recent advances in the understanding of the pathogenesis of atherosclerosis, estrogen receptor function, and hormone therapy are reviewed, with particular emphasis on clinical and molecular issues. RESULTS Whether hormone therapy can improve cardiovascular health in postmenopausal women remains controversial. Current evidence suggests that the vascular effects of estrogen are affected by the stage of reproductive life, the time since menopause, and the extent of subclinical atherosclerosis. The mechanisms of vascular responsiveness to sex steroids during different stages of atherosclerosis development remain poorly understood in women and men. CONCLUSION In view of the expected increase in the prevalence of atherosclerotic vascular disease worldwide due to population aging, research is needed to determine the vascular mechanism of endogenous and exogenous sex steroids in patients with atherosclerosis. Such research may help to define new strategies to improve cardiovascular health in women and possibly also in men.

A WD-FYVE protein binds to the kinases Akt and PKCζ/λ

WD (tryptophan-aspartic acid dipeptide)-repeat proteins play a central role in signal transduction cascades by co-ordinating the interaction of key signalling molecules. We identified a novel propeller-FYVE [domain identified in Fab1p, YOTB, Vac1p and EEA1 (early endosome antigen 1)] protein, ProF, which is expressed in various cell lines and tissues and consists of seven WD-repeats and a FYVE domain. WD-repeat proteins offer a platform for protein-protein interactions by folding into a seven-bladed propeller-like structure, while the FYVE domain binds to phosphatidylinositol 3-phosphate present mainly on intracellular membranes. The ProF protein partially co-localizes with EEA1 on vesicular structures and binds to the protein kinases Akt and PKCzeta/lambda (protein kinase Czeta/lambda) via its WD-repeat propeller. ProF interacts more strongly with the kinases after hormonal stimulation. Endogenously expressed ProF and the two kinases interact in brain and in the preadipocyte cell line 3T3-L1, suggesting a role in secretory vesicular processes. In summary, we describe a new binding partner for kinases, located on vesicular structures in specialized cells, which may play a role for the spatial organization of signalling cascades.

Rictor in Perivascular Adipose Tissue Controls Vascular Function by Regulating Inflammatory Molecule Expression

Objective—Perivascular adipose tissue (PVAT) wraps blood vessels and modulates vasoreactivity by secretion of vasoactive molecules. Mammalian target of rapamycin complex 2 (mTORC2) has been shown to control inflammation and is expressed in adipose tissue. In this study, we investigated whether adipose-specific deletion of rictor and thereby inactivation of mTORC2 in PVAT may modulate vascular function by increasing inflammation in PVAT. Approach and Results—Rictor, an essential mTORC2 component, was deleted specifically in mouse adipose tissue (rictorad−/−). Phosphorylation of mTORC2 downstream target Akt at Serine 473 was reduced in PVAT from rictorad−/− mice but unaffected in aortic tissue. Ex vivo functional analysis of thoracic aortae revealed increased contractions and impaired dilation in rings with PVAT from rictorad−/− mice. Adipose rictor knockout increased gene expression and protein release of interleukin-6, macrophage inflammatory protein-1&agr;, and tumor necrosis factor-&agr; in PVAT as shown by quantitative real-time polymerase chain reaction and Bioplex analysis for the cytokines in the conditioned media, respectively. Moreover, gene and protein expression of inducible nitric oxide synthase was upregulated without affecting macrophage infiltration in PVAT from rictorad−/− mice. Inhibition of inducible nitric oxide synthase normalized vascular reactivity in aortic rings from rictorad−/− mice with no effect in rictorfl/fl mice. Interestingly, in perivascular and epididymal adipose depots, high-fat diet feeding induced downregulation of rictor gene expression. Conclusions—Here, we identify mTORC2 as a critical regulator of PVAT-directed protection of normal vascular tone. Modulation of mTORC2 activity in adipose tissue may be a potential therapeutic approach for inflammation-related vascular damage.

Inflammation and Atherosclerosis

See related article, pages 792–801 The deleterious effects of high low-density lipoprotein (LDL) cholesterol levels on atherosclerosis has been known for almost a century,1 yet plasma cholesterol continues to be a challenge for clinicians in the treatment and prevention of cardiovascular disease.2,3 Atherogenesis involves uptake of cholesterol in the vascular wall, followed by inflammatory activation and growth of vascular smooth muscle cells.4,5 Indeed, proinflammatory mediators such as interleukins and cytokines stimulate vascular cell growth and atherogenesis (reviewed in4), whereas inhibition of inflammatory pathways attenuates cell growth and atherosclerosis.6 Therefore, we now view atherosclerosis as a vascular inflammatory process7 as was already proposed by Virchow8 and later by Anitschkow who noticed an “infiltrative character” of atherosclerotic lesions of cholesterol-fed animals.9 Differentiation and growth of vascular smooth muscle cells, a prerequisite of atherosclerosis progression, depends on a fine-tuned balance between activators and inhibitors of cell growth.10 In the 1980s, Libby and colleagues reported that LDL cholesterol enhances growth factor–stimulated proliferation of …