ADVENTITIAL RESPONSE TO VASCULAR INJURY: ESTROGEN

我们以前的研究表明，雌激素（E2）抑制大鼠颈动脉球囊损伤的新生内膜反应，并提供了外膜激活有助于这种损伤反应的间接证据。 目前的建议将利用一种创新的方法，采用同源成纤维细胞来自外膜的大鼠颈动脉和稳定转导（逆转录病毒介导）与β-半乳糖苷酶（lacZ）报告基因，以确定外膜反应腔内血管损伤和他们的调制E2。 拟议的研究将测试新的假设，即外膜激活在腔内血管损伤的反应中起着重要作用，E2通过间接机制（涉及与VSMC相互作用，以改变能够调节外膜细胞稳态的因子的产生和释放）减少外膜细胞易位到新生内膜来调节这种反应。 我们已经表明，激活的血管平滑肌细胞刺激迁移/粘附这些成纤维细胞在体外的E2-可降解，雌激素受体（ER）依赖的机制，需要基因转录和新的蛋白质合成。 初步观察显示，体外VSMC中骨桥蛋白（OPN）的E2-可诱导表达以及成纤维细胞中α v和β 3整联蛋白的表达，并表明用抗β 3整联蛋白抗体处理成纤维细胞可抑制体外VSMC条件培养基指导的迁移。基于这些令人兴奋和挑衅性的初步观察，拟议的研究将追求三个具体目标。 目的1将阐明E2在体外通过ER、OPN及其α v β 3整合素受体调节VSMC诱导的外膜成纤维细胞活化和迁移/粘附的机制作用。 目的2将使用球囊损伤模型在体内建立活化的外膜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激活和迁移的转导（lacZ）成纤维细胞引入外膜的大鼠颈动脉，表征的因素，指导这些过程，并评估发生在外膜成纤维细胞和中膜血管平滑肌细胞的表型改变，以响应腔内血管损伤。 目的3将定义在体内的机制，E2调节血管损伤反应，通过ER依赖性扰动的VSMC诱导的激活和迁移的稳定转导的“报告”成纤维细胞（lacZ）已被引入到外膜的球囊损伤的卵巢切除大鼠颈动脉。 拟议的研究将阐明负责E2对受损血管的保护作用的细胞/分子事件。 这些基本的机制研究将建立更合理的策略，治疗干预血管疾病，包括未来的基因治疗的基础。

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.