ADVENTITIAL RESPONSE TO VASCULAR INJURY: ESTROGEN

我们以前的研究表明，雌激素(E2)抑制了大鼠颈动脉球囊损伤后的新生内膜反应，并间接证明外膜激活参与了这种损伤反应。目前的建议将利用一种创新的方法，使用来自大鼠颈动脉外膜的同种成纤维细胞，并稳定地转导(逆转录病毒介导)β-半乳糖苷酶(LacZ)报告基因，以确定外膜对血管内损伤的反应及其受E2的调节。建议的研究将检验新的假设，即外膜激活在血管内损伤的反应中起重要作用，而E2通过与VSMCs相互作用的间接机制减少外膜细胞向新生内膜的移位来调节这种反应，从而改变有能力调节外膜细胞动态平衡的因子的产生和释放。我们已经证明，激活的VSMCs在体外通过E2可抑制的雌激素受体(ER)依赖的机制刺激这些成纤维细胞的迁移/黏附，该机制需要基因转录和新的蛋白质合成。初步观察表明，骨桥蛋白(OPN)在VSMC中的表达可被E2抑制，在体外成纤维细胞中可表达αv和β3整合素，提示抗β3整合素抗体处理成纤维细胞可抑制VSMC条件培养液在体外诱导的迁移。这项拟议的研究基于这些令人兴奋和具有挑衅性的初步观察，将追求三个具体目标。目的1通过ER、OPN及其αvbeta3整合素受体途径，在体外阐明E2对VSMC诱导的外膜成纤维细胞活化和迁移/黏附的调节机制。目的2利用球囊损伤模型，在体内建立血管外膜成纤维细胞活化在血管损伤反应中的作用机制。这项工作将包括建立导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VSMCs在腔内血管损伤时发生的表型变化。目的3将明确体内机制，即雌二醇通过内质网干扰VSMC诱导的稳定转导的“报告”成纤维细胞(LacZ)的激活和迁移来调控血管损伤反应，LacZ已被引入去卵巢大鼠颈动脉球囊损伤的外膜。这项拟议的研究将阐明E2对受损血管的保护作用所涉及的细胞/分子事件。这些基础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.