ADVENTITIAL RESPONSE TO VASCULAR INJURY: ESTROGEN

我们之前的研究表明，雌激素（E2）抑制大鼠颈动脉球囊损伤的新内膜反应，并间接证明了外膜激活有助于这种损伤反应。目前的提议将利用一种创新的方法，利用来自大鼠颈动脉外膜的同基因成纤维细胞，通过β -半乳糖苷酶（lacZ）报告基因稳定地转导（逆转录病毒介导），来定义腔内血管损伤的外膜反应及其E2的调节。拟议的研究将测试新的假设，即外膜活化在腔内血管损伤的反应中起重要作用，E2通过与VSMCs相互作用的间接机制减少外膜细胞向新内膜的易位来调节这种反应，从而改变调节外膜细胞稳态的因子的产生和释放。我们已经证明，激活的VSMCs通过e2抑制、雌激素受体（ER）依赖的机制在体外刺激这些成纤维细胞的迁移/粘附，这需要基因转录和新的蛋白质合成。初步观察显示e2可抑制VSMC中骨桥蛋白（OPN）的表达和体外成纤维细胞中α和β 3整合素的表达，并提示抗β 3整合素抗体处理成纤维细胞可抑制体外VSMC条件培养基指导下的迁移。拟议的研究，基于这些令人兴奋和挑衅性的初步观察，将追求三个具体目标。目的1将阐明E2在体外通过涉及内质网、OPN及其α β 3整合素受体的途径调节vsmc诱导的成纤维细胞的激活和迁移/粘附中的机制作用。目的2将利用球囊损伤模型在体内建立活化的外层成纤维细胞在血管损伤反应中的机制作用。这项工作将包括建立引入大鼠颈动脉外膜的转导（lacZ）成纤维细胞的激活和迁移，表征指导这些过程的因素，以及评估外膜成纤维细胞和内侧vsmc在响应腔内血管损伤时发生的表型改变。目的3将定义E2调节血管损伤反应的体内机制，通过er依赖性扰动vsmc诱导的稳定转导的“报告”成纤维细胞（lacZ）的激活和迁移，这些成纤维细胞已被引入到去卵巢大鼠的颈动脉外膜损伤中。该研究将阐明E2对损伤血管的保护作用的细胞/分子事件。这些基本的机制研究将为血管疾病的治疗干预建立更合理的策略，包括为未来的基因治疗奠定基础。

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.