This is a competitive renewal application that focuses on the specific roles of estrogen receptors in vascular biology, with substantial significance for the surgical ischemic cardiovascular diseases. Using molecular approaches and transgenic mouse models, the present application focuses on the exciting new findings that both male and female estrogen receptor beta (ERbeta) knockout mice (ERbetaKO), as compared to their WT littermates, have vascular contractile abnormalities, marked reduction in vascular smooth muscle cell (VSMC) potassium currents, and progressive hypertension as they age. Substantial preliminary data are presented to support that this is related to ERbeta regulation of both the VSMC gene for inducible nitric oxide synthase (iNOS) and VSMC potassium channel genes. This application therefore tests the hypothesis that ERbeta is a critical determinant of normal vascular function and blood pressure through regulated expression of the iNOS gene and VSMC ion channel genes involved in VSMC contractile function.
Three specific Aims are proposed: SA A studies the Mechanism of ERbeta-iNOS regulation of vascular tone in intact vessels using pharmacological inhibitors of ERs and iNOS in vascular rings of carotid arteries and intact mesenteric arteries from WT, ERalphaKO and ERbetaKO mice. The role of transcription and post-transcriptional regulation in the estrogen effect on contraction are tested, as is whether postnatal inhibition of ERbeta prevents the development of hypertension and/or postnatal removal of ERalpha actions prevents or modifies the development of hypertension. SA B uses cellular and in vitro approaches to identify the transcriptional mechanism of iNOS activation by ERbeta with transient transfection assays and electrophoretic mobility shift assays (EMSA), and yeast one-hybrid screening to identify VSMC-specific DNA binding proteins and transcriptional coactivators important to ERbeta-mediated iNOS induction. SA C examines which potassium channel genes are dysfunctional in the hypertensive ERbetaKO mice using specific pharmacological inhibitors in whole-cell patch clamping studies, and protein and mRNA expression studies in primary VSMC from ERbetaKO mice and their WT littermates. These studies are expected to add substantially to our understanding of vascular regulation by estrogen and estrogen receptors and to enhance further the academic vascular surgery-training environment at our institution. Furthermore, understanding the functional importance of estrogen receptors in the vasculature for blood pressure regulation may help in the design of specific therapies for the prevention and treatment of a variety of cardiovascular diseases.
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