Endothelial Cell Estrogen Receptor Alpha-mediated Mechanisms of Protection from Atherosclerosis Inflammation in Females

Tufts University Boston, Boston MA

Investigators

Abstract

PROJECT SUMMARY/ABSTRACT Enhanced understanding of sex-specific mechanisms driving cardiovascular disease (CVD) is necessary to develop precision strategies to prevent CVD, the leading cause of death for men and women. Heart attack and stroke are most often caused by the rupture of inflamed atherosclerotic (athero) plaques. Athero plaques develop from endothelial cell (EC) damage that induces expression of adhesion molecules (AMs), including intracellular AM 1 (ICAM1), E-selectin (Esel), P-selectin (Psel), and vascular AM 1 (VCAM1), resulting in leukocyte recruitment causing inflamed plaques. Women are protected from CVD relative to men, and our lab found that female athero-prone mice have reduced athero plaque inflammation compared to males, suggesting that females may be protected from CVD events by decreased plaque inflammation. Since CVD risk increases in women after menopause, estrogen (E) and estrogen receptors (ERs) have been implicated, yet the detailed mechanism remains unknown. Indeed, E treatment in postmenopausal women has been shown to reduce CVD events when began <10 years post-menopause. Prior studies suggest that ERs may regulate known drivers of EC inflammation. Specifically, (1) our lab showed that ERa inhibits mineralocorticoid receptor (MR) transcriptional activity in vitro and (2) prior studies show that ER can inhibit the pro-inflammatory transcription factor nuclear factor kappa B (NFkB), a known activator of AM expression. Whether these mechanisms contribute to protection from athero inflammation in females remains a gap in knowledge. Therefore, we propose to test the hypothesis that EC-ERa protects against athero inflammation in females by downregulating AM expression via distinct MR and NFkB-mediated mechanisms. In Aim 1, we test the hypothesis in vitro that EC-ERa inhibits AM expression by preventing EC-MR and NFkB binding to AM promoters, thus attenuating leukocyte adhesion to ECs. To test this, ER expression and activity will be modulated in primary human aortic ECs from women using ERa siRNA knock-down +/- ERa agonism with E2 and we will measure (1) ERa, MR, and NFkB enrichment on AM promoters by chromatin immunoprecipitation (ChIP)-PCR, (2) AM expression by PCR and immunoblot (IB), and (3) adhesion of fluorescent leukocytes to ECs. In Aim 2, we test the hypothesis in vivo that EC-ERa inhibits AM expression and protects from athero inflammation in females using three novel cell-specific knock-out mouse models in which ERa, MR, or both ERa and MR are specifically deleted in ECs and crossed to the athero-prone low-density lipoprotein receptor background. In female mice, we will measure (1) leukocyte trafficking by intravital microscopy, (2) plaque composition by histology, (3) AM expression by PCR and IB, and (4) ERa and NFkB enrichment on AM promoters in aortic tissue after 12 weeks of high fat diet. Successful completion of these aims will elucidate EC-ERa-mediated pathways that regulate AMs and leukocyte trafficking thereby clarifying mechanisms by which young females are protected from CVD. The detailed training plan will prepare the PI for a career as an independent physician-scientist studying mechanisms driving sex differences in human disease.