Natalie C. Fredette

G Protein-coupled Estrogen Receptor Protects from Atherosclerosis

Coronary atherosclerosis and myocardial infarction in postmenopausal women have been linked to inflammation and reduced nitric oxide (NO) formation. Natural estrogen exerts protective effects on both processes, yet also displays uterotrophic activity. Here, we used genetic and pharmacologic approaches to investigate the role of the G protein-coupled estrogen receptor (GPER) in atherosclerosis. In ovary-intact mice, deletion of gper increased atherosclerosis progression, total and LDL cholesterol levels and inflammation while reducing vascular NO bioactivity, effects that were in some cases aggravated by surgical menopause. In human endothelial cells, GPER was expressed on intracellular membranes and mediated eNOS activation and NO formation, partially accounting for estrogen-mediated effects. Chronic treatment with G-1, a synthetic, highly selective small molecule agonist of GPER, reduced postmenopausal atherosclerosis and inflammation without uterotrophic effects. In summary, this study reveals an atheroprotective function of GPER and introduces selective GPER activation as a novel therapeutic approach to inhibit postmenopausal atherosclerosis and inflammation in the absence of uterotrophic activity.

Regulation of vascular smooth muscle tone by adipose-derived contracting factor.

Obesity and arterial hypertension, important risk factors for atherosclerosis and coronary artery disease, are characterized by an increase in vascular tone. While obesity is known to augment vasoconstrictor prostanoid activity in endothelial cells, less is known about factors released from fat tissue surrounding arteries (perivascular adipose). Using lean controls and mice with either monogenic or diet-induced obesity, we set out to determine whether and through which pathways perivascular adipose affects vascular tone. We unexpectedly found that in the aorta of obese mice, perivascular adipose potentiates vascular contractility to serotonin and phenylephrine, indicating activity of a factor generated by perivascular adipose, which we designated “adipose-derived contracting factor” (ADCF). Inhibition of cyclooxygenase (COX) fully prevented ADCF-mediated contractions, whereas COX-1 or COX-2-selective inhibition was only partially effective. By contrast, inhibition of superoxide anions, NO synthase, or endothelin receptors had no effect on ADCF activity. Perivascular adipose as a source of COX-derived ADCF was further confirmed by detecting increased thromboxane A2 formation from perivascular adipose-replete aortae from obese mice. Taken together, this study identifies perivascular adipose as a novel regulator of arterial vasoconstriction through the release of COX-derived ADCF. Excessive ADCF activity in perivascular fat under obese conditions likely contributes to increased vascular tone by antagonizing vasodilation. ADCF may thus propagate obesity-dependent hypertension and the associated increased risk in coronary artery disease, potentially representing a novel therapeutic target.

Obligatory role for GPER in cardiovascular aging and disease

The G protein–coupled receptor GPER promotes hypertension and aging-induced tissue damage through reactive oxygen species. Preventing the ravages of ROS Ligand-dependent activation of the G protein–coupled estrogen receptor (GPER) has been reported to confer cardiovascular benefits. However, Meyer et al. found that genetic absence of Gper conferred protection from cardiovascular pathologies associated with aging and hypertension. GPER activity was required to increase the abundance of the enzyme Nox1 in vascular smooth muscle cells, blood vessels, and myocardium, and was associated with enhanced production of tissue-damaging superoxide. Aged mice that were deficient in Gper developed much less cardiac fibrosis and hypertrophy and also retained greater cardiovascular function. In addition, a pharmacological inhibitor of GPER reduced blood pressure, superoxide production, and Nox1 abundance in hypertensive mice. Thus, inhibitors of GPER are potential therapies for cardiovascular diseases and conditions characterized by excessive superoxide generation. Pharmacological activation of the heptahelical G protein–coupled estrogen receptor (GPER) by selective ligands counteracts multiple aspects of cardiovascular disease. We thus expected that genetic deletion or pharmacological inhibition of GPER would further aggravate such disease states, particularly with age. To the contrary, we found that genetic ablation of Gper in mice prevented cardiovascular pathologies associated with aging by reducing superoxide (⋅O2−) formation by NADPH oxidase (Nox) specifically through reducing the expression of the Nox isoform Nox1. Blocking GPER activity pharmacologically with G36, a synthetic, small-molecule, GPER-selective blocker (GRB), decreased Nox1 abundance and ⋅O2− production to basal amounts in cells exposed to angiotensin II and in mice chronically infused with angiotensin II, reducing arterial hypertension. Thus, this study revealed a role for GPER activity in regulating Nox1 abundance and associated ⋅O2−-mediated structural and functional damage that contributes to disease pathology. Our results indicated that GRBs represent a new class of drugs that can reduce Nox abundance and activity and could be used for the treatment of chronic disease processes involving excessive ⋅O2− formation, including arterial hypertension and heart failure.

G protein-coupled estrogen receptor inhibits vascular prostanoid production and activity

Complications of atherosclerotic vascular disease, such as myocardial infarction and stroke, are the most common causes of death in postmenopausal women. Endogenous estrogens inhibit vascular inflammation-driven atherogenesis, a process that involves cyclooxygenase (COX)-derived vasoconstrictor prostanoids such as thromboxane A2. Here, we studied whether the G protein-coupled estrogen receptor (GPER) mediates estrogen-dependent inhibitory effects on prostanoid production and activity under pro-inflammatory conditions. Effects of estrogen on production of thromboxane A(2) were determined in human endothelial cells stimulated by the pro-inflammatory cytokine tumour necrosis factor alpha (TNF-α). Moreover, Gper-deficient (Gper(-/-)) and WT mice were fed a pro-inflammatory diet and underwent ovariectomy or sham surgery to unmask the role of endogenous estrogens. Thereafter, contractions to acetylcholine-stimulated endothelial vasoconstrictor prostanoids and the thromboxane-prostanoid receptor agonist U46619 were recorded in isolated carotid arteries. In endothelial cells, TNF-α-stimulated thromboxane A2 production was inhibited by estrogen, an effect blocked by the GPER-selective antagonist G36. In ovary-intact mice, deletion of Gper increased prostanoid-dependent contractions by twofold. Ovariectomy also augmented prostanoid-dependent contractions by twofold in WT mice but had no additional effect in Gper(-/-) mice. These contractions were blocked by the COX inhibitor meclofenamate and unaffected by the nitric oxide synthase inhibitor l-N(G)-nitroarginine methyl ester. Vasoconstrictor responses to U46619 did not differ between groups, indicating intact signaling downstream of thromboxane-prostanoid receptor activation. In summary, under pro-inflammatory conditions, estrogen inhibits vasoconstrictor prostanoid production in endothelial cells and activity in intact arteries through GPER. Selective activation of GPER may therefore be considered as a novel strategy to treat increased prostanoid-dependent vasomotor tone or vascular disease in postmenopausal women.

Endothelin-1 but not angiotensin II contributes to functional aging in murine carotid arteries

AIMS Aging is a major risk factor for carotid artery disease and stroke. Endothelin-1 (ET-1) and angiotensin II (Ang II) are important modifiers of vascular disease, partly through increased activity of NADPH oxidase and vasoconstrictor prostanoids. Since the renin-angiotensin and endothelin systems become activated with age, we hypothesized that aging affects NADPH oxidase- and prostanoid-dependent contractions to ET-1 and Ang II. MAIN METHODS Carotid artery rings of young (4 month-old) and old (24 month-old) C57BL6 mice were pretreated with the NO synthase inhibitor L-NAME to exclude differential effects of NO. Contractions to ET-1 and Ang II were determined in the presence and absence of the NADPH oxidase-selective inhibitor gp91ds-tat or the thromboxane-prostanoid receptor antagonist SQ 29,548. Gene expression of endothelin and angiotensin receptors was measured by qPCR. KEY FINDINGS Aging reduced ET-1-induced contractions and diminished ETA but increased ETB receptor gene expression levels. Gp91ds-tat inhibited contractions to ET-1 in young and to a greater extent in old animals, whereas SQ 29,548 had no effect. Ang II-induced contractions were weak compared to ET-1 and unaffected by aging, gp91ds-tat, and SQ 29,548. Aging had also no effect on AT1A and AT1B receptor gene expression levels. SIGNIFICANCE Aging in carotid arteries decreases ETA receptor gene expression and responsiveness to ET-1, which nevertheless becomes increasingly dependent upon NAPDH oxidase activity with age; responses to Ang II and gene expression of its receptors are however unaffected. These findings suggest that physiological aging differentially regulates functional responses to G protein-coupled receptor agonists and the signaling pathways associated with their activation.

GPER is required for the age-dependent upregulation of the myocardial endothelin system

AIMS Cardiac aging is associated with progressive structural changes and functional impairment, such as left ventricular hypertrophy, fibrosis and diastolic dysfunction. Aging also increases myocardial activity of endothelin-1 (ET-1), a multifunctional peptide with growth-promoting and pro-fibrotic activity. Because the G protein-coupled estrogen receptor (GPER) regulates vascular responsiveness to ET-1, we investigated whether GPER also plays a role in the regulation of the myocardial endothelin system with aging. MAIN METHODS Young (4month-old) and aged (24month-old) wild-type and Gper-deficient (Gper(-/-)) mice were studied. Gene expression levels of prepro-ET-1, endothelin converting enzymes ECE-1 and ECE-2, and endothelin ETA and ETB receptors were determined by qPCR in left ventricular myocardium. KEY FINDINGS Aging markedly increased steady-state mRNA expression levels of ECE-1, ECE-2, ETA and ETB receptors (each p<0.001 vs. young mice). Deletion of Gper inhibited the age-dependent increase in ECE-2 and ETB receptor mRNA levels (57% and 40% reduction, respectively, each p<0.01 vs. wild-type mice), whereas gene expression of prepro-ET-1, ECE-1, and the ETA receptor was unaffected in Gper(-/-) mice. SIGNIFICANCE We identified a novel regulatory mechanism through which the endogenous Gper facilitates the age-dependent increase in myocardial expression of ECE-2 and the ETB receptor, which is compatible with an activating role of GPER for the local endothelin system with aging. Targeting GPER signaling by selective antagonists may therefore be considered a new therapeutic approach to reduce age-dependent increased ET-1 activity and the associated development of left ventricular hypertrophy, fibrosis and heart failure.

Erratum: G Protein-coupled Estrogen Receptor Protects from Atherosclerosis.

Cardiovascular disease risk is associated with excess body weight and elevated plasma free fatty acid (FFA) concentrations. This study examines how an alternate-day fasting (ADF) diet high (HF) or low (LF) in fat affects plasma FFA profiles in the context of weight loss, and changes in body composition and lipid profiles. After a 2-week weight maintenance period, 29 women (BMI 30–39.9 kg/m2) 25–65 years old were randomized to an 8-week ADF-HF (45% fat) diet or an ADF-LF (25% fat) diet with 25% energy intake on fast days and ad libitum intake on feed days. Body weight, BMI and waist circumference were assessed weekly and body composition was measured using dual x-ray absorptiometry (DXA). Total and individual FFA and plasma lipid concentrations were measured before and after weight loss. Body weight, BMI, fat mass, total cholesterol, LDL-C and triglyceride concentrations decreased (P < 0.05) in both groups. Total FFA concentrations also decreased (P < 0.001). In the ADF-LF group, decreases were found in several more FFAs than in the ADF-HF group. In the ADF-HF group, FFA concentrations were positively correlated with waist circumference. Depending on the macronutrient composition of a diet, weight loss with an ADF diet decreases FFA concentrations through potentially different mechanisms.