Our previous studies have demonstrated that estrogen (E2) inhibits the neointimal response to balloon injury of the rat carotid artery and have given indirect evidence that adventitial activation contributes to this injury response. The current proposal will utilize an innovative approach which employs syngeneic fibroblasts derived from the adventitia of rat carotid arteries and stably transduced (retrovirally mediated) with a beta-galactosidase (lacZ) reporter gene to define adventitial responses to endoluminal vascular injury and their modulation by E2. Proposed studies will test the novel hypotheses that adventitial activation plays an important role in the response to endoluminal vascular injury and that E2 modulates this response by reducing the translocation of adventitial cells into the neointima via an indirect mechanism involving interaction with VSMCs to alter production and release of factors competent to regulate adventitial cell homeostasis. We have shown that activated VSMCs stimulate migration/adhesion of these fibroblasts in vitro by an E2-inhibitable, estrogen receptor (ER) dependent mechanism that requires gene transcription and new protein synthesis. Preliminary observations have shown E2-inhibitable expression of osteopontin (OPN) in VSMCs and expression of alphav and beta3 integrins in fibroblasts in vitro and have suggested that treatment of fibroblasts with anti-beta3 integrin antibody inhibits migration directed by VSMC conditioned media in vitro. The proposed research, based on these exciting and provocative preliminary observations, will pursue three Specific Aims.
Aim 1 will elucidate in vitro a mechanistic role for E2 in modulating VSMC-induced activation and migration/adhesion of adventitial fibroblasts via a pathway involving ERs, OPN and its alphavbeta3 integrin receptors.
Aim 2 will establish in vivo the mechanistic role for activated adventitial fibroblasts in the vascular injury response using the balloon injury model. This effort will include establishing activation and migration of transduced (lacZ) fibroblasts introduced into the adventitia of the rat carotid artery, characterization of factors that direct these processes, and assessment of the phenotypic alterations that occur in adventitial fibroblasts and medial VSMCs in response to endoluminal vascular injury.
Aim 3 will define the in vivo mechanism whereby E2 modulates the vascular injury response via ER-dependent perturbation of VSMC-induced activation and migration of stably transduced """"""""reporter"""""""" fibroblasts (lacZ) that have been introduced into the adventitia of balloon injured carotid arteries of ovariectomized rats. The proposed research will elucidate the cellular/molecular events responsible for the protective effects of E2 on injured blood vessels. These fundamental mechanistic studies will establish more rational strategies for therapeutic intervention in vascular diseases, including the basis for future gene therapy.
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