The Role of G protein-coupled estrogen receptor in vascular function and hypertension

Regulation of arterial tone relies heavily on molecular crosstalk between endothelial and smooth muscle cells. Although it has been demonstrated that estrogen (E2) exerts protective effects against cardiovascular disease (CVD), the exact mechanisms and balance of E2 signaling in endothelial and smooth muscle cells remain unresolved. Here we characterize the G protein-coupled estrogen receptor (GPER) in the vasculature by elucidating its pathway in endothelial nitric oxide (NO) production and vascular smooth muscle maintenance of reactive oxygen species (ROS) by NADPH oxidases (NOX). NO is thought of as a vasoprotective entity, while the overproduction of ROS by NOX can lead to vascular dysfunction. These two forces counteract each other to maintain arterial tone, and understanding their mediation by GPER will provide significant insights in sex differences in vascular function. In endothelial cells, we demonstrated that GPER activation induces phosphorylation of eNOS at ser1179, and contributes to ER-dependent NO production. Inhibition of GPER through pharmacologic antagonism (G36) or genomic deletion (GPER KO) reduced NO production and vasorelaxation to E2. We also found that NO production by selective estrogen receptor modifiers and downregulators (SERMs/SERDs), known to antagonize classic ERs and agonize GPER, was only partially abrogated by GPER inhibition, suggesting that there may be E2- independent targeting effects by these compounds. In the smooth muscle we were surprised to find that the arteries of GPER KO animals exhibited a reduced contraction response to Ang II. We characterized a novel pathway in which GPER mediates genomic expression of the NOX1 subunit in smooth muscle cells. Critically, we report that GPER inhibition or deletion reduces or blocks the hypertensive response to Ang II in mice. Taken together, these data describe a counterintuitive paradigm in which GPER signaling is important for E2-mediated NO production at the endothelium through acute eNOS activation, but conversely is involved in ROS generation at the smooth muscle though expression of NOX1. In lieu of the conflicting CVD reported for E2 replacement in post-menopausal women, we propose that the signaling pathway(s) of GPER may increase ROS production through smooth muscle NOX1 expression, which may override endothelial NO production, resulting in vascular dysfunction. National Institutes of Health Vascular Physiology training grantBiomedical ScienceDoctoralUniversity of New Mexico. Biomedical Sciences Graduate ProgramProssnitz, EricHathaway, HelenResta, ThomasNikki, JerniganRobert, Orland

Regulation of Vascular Smooth Muscle Tone by Adipose-Derived Contracting Factor

<div><p>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.</p></div

Cyclooxygenase inhibition prevents perivascular adipose-dependent potentiation of contractions to serotonin in monogenic obesity.

<p>Aortic rings with perivascular adipose (PVAT) from monogenic obese GPER⁰ mice or lean WT (control) mice were pretreated with the cyclooxygenase inhibitor meclofenamate (Meclo, 1 µmol/L) prior to stimulation with increasing concentrations of serotonin. Inset: Area under the curve (AUC) of concentration-response curves is expressed as arbitrary units (AU). , control, untreated (<i>n</i> = 6); , control, meclofenamate (<i>n</i> = 7); , GPER⁰, untreated (<i>n</i> = 7); GPER⁰, meclofenamate (<i>n</i> = 6). *<i>P</i><0.001 <i>vs.</i> untreated vascular rings; †<i>p</i><0.05 <i>vs.</i> control.</p

Quantitative measurements of adipocyte-related gene expression in aortic perivascular adipose from GPER⁰ and control mice.

<p>Adipose was collected and analyzed from animals with monogenic (GPER⁰) obesity and compared to lean WT controls (<i>n</i> = 5/group). Expression levels of mRNA were calculated based on the 2^−ΔΔCT method and expressed as arbitrary units. GAPDH served as the housekeeping control.</p

Perivascular adipose and perigonadal fat mass, body weight and blood pressure in monogenic obesity (GPER⁰).

<p>A, Mass and macroscopic difference in the quantity of perivascular adipose (PVAT) surrounding the aorta (a, Aortic arch; b, Thoracic aorta; c, Abdominal aorta) of obese (GPER⁰, <i>n</i> = 5) and lean WT control mice (CTL, <i>n</i> = 8). B, Perigonadal fat weight (CTL, <i>n</i> = 8; GPER⁰, <i>n</i> = 9); C, body weight (<i>n</i> = 19/group); D, systolic and diastolic blood pressure levels in obese (GPER⁰, , <i>n</i> = 7) and lean WT mice (CTL, , <i>n</i> = 6). Fat weights are normalized to tibial length. A–C: open bars, lean WT control (CTL); solid bars, monogenic obesity (GPER⁰). *<i>p</i><0.01 <i>vs.</i> control.</p

Effect of obesity on gene expression of cyclooxygenase (COX)-1 and COX-2 isoforms in aortic perivascular adipose.

<p>Adipose was collected and analyzed from animals with monogenic (GPER⁰, <i>n</i> = 5) obesity or in diet-induced obesity (DIO, <i>n</i> = 6), as well as from lean, age-matched WT controls (<i>n</i> = 6). Expression levels of mRNA were calculated based on the 2^−ΔΔCT method and expressed as arbitrary units. GAPDH served as housekeeping control. *<i>p</i><0.01 <i>vs.</i> COX-1.</p

Effect of perivascular adipose on serotonin-induced contractions in mice with diet-induced obesity.

<p>Aortic rings with and without perivascular adipose (PVAT) from WT mice with diet-induced obesity (DIO) or lean WT mice (control) mice were exposed to increasing concentrations of serotonin. DIO animals were fed a high-fat diet for 24 weeks and compared to age-matched mice fed a standard chow (control). Inset: Area under the curve (AUC) is expressed as arbitrary units (AU). , control, without PVAT (<i>n</i> = 6); , control, with PVAT (<i>n</i> = 6); , DIO, without PVAT (<i>n</i> = 5); , DIO, with PVAT (<i>n</i> = 7). *<i>p</i><0.05 <i>vs.</i> aortic rings without perivascular adipose; †<i>p</i><0.05 <i>vs.</i> control.</p

Cyclooxygenase inhibition prevents perivascular adipose-dependent potentiation of contractions to serotonin in diet-induced obesity.

<p>Aortic rings with perivascular adipose (PVAT) from WT mice with diet-induced obesity (DIO) or lean WT mice (control) mice were exposed to increasing concentrations of serotonin in the presence or absence of the cyclooxygenase inhibitor meclofenamate (Meclo, 1 µmol/L). DIO animals were fed a high-fat diet for 24 weeks and compared to age-matched mice fed a standard chow (control). Inset: Area under the curve (AUC) is expressed as arbitrary units (AU). , control, untreated (<i>n</i> = 6); control, meclofenamate (<i>n</i> = 4); , DIO, untreated (<i>n</i> = 7); , DIO, meclofenamate (<i>n</i> = 5). *<i>p</i><0.001 <i>vs.</i> untreated vascular rings; †<i>p</i><0.05 <i>vs.</i> control.</p