EphA2 signaling in atherosclerotic fibroproliferative remodeling

项目摘要 哺乳动物基因组中最大的受体酪氨酸激酶Eph家族及其肝配蛋白配体 在多种病理条件下严重影响炎症。我们的工作首次描述了 EphA 2在动脉粥样硬化斑块中的多种细胞类型中表达，我们最近证明EphA 2 缺失减少了与斑块炎症减少相关的动脉粥样硬化斑块形成。令人惊奇的是， EphA 2缺失也降低了进展为晚期动脉粥样硬化疾病， 肌肉含量平滑肌细胞从收缩表型到合成表型的表型调节 它们在斑块发育期间积累，并且平滑肌细胞显示增强的EphA 2表达 在体外和体内的表型调节过程中。我们的初步数据显示， 促进纤连蛋白依赖性整合素信号传导，在表型分化过程中关键调节EphA 2表达 调节，而钝化EphA 2表达限制体内和体外纤连蛋白基质沉积。采取 总之，这些数据表明EphA 2和基质重塑之间的动态相互作用， 平滑肌基质沉积。 除了基质沉积，我们还证明了EphA 2在平滑肌中的表达的关键作用。 促有丝分裂信号转导（ERK 1/2，AKT）和增殖在体外和动脉粥样硬化斑块在体内。而 EphA 2与ephrinA 1的连接揭示了EphA 2致动脉粥样硬化促炎反应的多个方面，我们的研究表明， 初步数据显示EphA 2连接减少平滑肌增殖。相反，EphA 2也可以 在配体非依赖性状态下的信号传导，其促进癌症模型中的细胞增殖和迁移，并且我们 观察到EphA 2配体非依赖性信号传导（Ser 897磷酸化）在平滑肌细胞中的增加。 在体外增殖和在体内平滑肌表型调节位点。然而，机制 调节EphA 2的不同配体依赖性和配体非依赖性促有丝分裂信号传导几乎仍然是 未开发的因此，我们假设EphA 2表达和基质细胞之间的动态相互作用， 动脉粥样硬化斑块中的重塑通过以下途径驱动平滑肌侵袭和纤维增生性重塑： EphA 2配体非依赖性信号传导的激活。为了验证这一假设，我们将描述动态特征 EphA 2表达和基质组成之间的相互作用，使用细胞培养模型和平滑肌- 特异性缺失纤连蛋白和纤连蛋白结合整合素（目的1），我们将确定机制， EphA 2信号传导影响平滑肌表型（Aim 2），我们将评估EphA 2细胞类型的作用。 使用新的EphA 2条件刺激在动脉粥样硬化纤维增生性重构中的特异性表达和信号传导 EphA 2连接和激酶活性的敲除和抑制剂。成功完成这些目标将确定 一种选择性减少斑块相关炎症同时促进平滑肌的新机制- 纤维帽的依赖性稳定。

Project Summary The Eph family of receptor tyrosine kinases, the largest in the mammalian genome, and their ephrin ligands critically influence inflammation in a variety of pathological conditions. Our work provided the first description of EphA2 expression in multiple cell types in the atherosclerotic plaque, and we recently demonstrated that EphA2 deletion reduces atherosclerotic plaque formation associated with diminished plaque inflammation. Surprisingly, EphA2 deletion also reduced progression to advanced atherosclerotic disease with diminished plaque smooth muscle content. Phenotypic modulation of smooth muscle cells from a contractile to a synthetic phenotype drives their accumulation during plaque development, and smooth muscle cells show enhanced EphA2 expression during phenotypic modulation both in vitro and in vivo. Our preliminary data show that matrix remodeling that promotes fibronectin-dependent integrin signaling critically regulates EphA2 expression during phenotypic modulation, whereas blunting EphA2 expression limits fibronectin matrix deposition in vivo and in vitro. Taken together, these data suggest a dynamic interplay between EphA2 and matrix remodeling that critically regulates the smooth muscle matrix deposition. In addition to matrix deposition, we also demonstrated a critical role for EphA2 expression in smooth muscle mitogenic signaling (ERK1/2, AKT) and proliferation both in vitro and in atherosclerotic plaques in vivo. While EphA2 ligation by ephrinA1 elicits multiple aspects of EphA2's atherogenic proinflammatory responses, our preliminary data show that EphA2 ligation reduces smooth muscle proliferation. Conversely, EphA2 can also signal in a ligand-independent state, which promotes cell proliferation and migration in cancer models, and we observed an increase in EphA2 ligand-independent signaling (Ser897 phosphorylation) during smooth muscle proliferation in vitro and at sites of smooth muscle phenotypic modulation in vivo. However, the mechanisms regulating EphA2's differential ligand-dependent and ligand-independent mitogenic signaling remain virtually unexplored. Therefore, we hypothesize that dynamic interplay between EphA2 expression and matrix remodeling in the atherosclerotic plaque drives smooth muscle invasion and fibroproliferative remodeling through activation of EphA2 ligand-independent signaling. To test this hypothesis, we will characterize the dynamic interplay between EphA2 expression and matrix composition using both cell culture models and smooth muscle- specific deletion of fibronectin and fibronectin-binding integrins (Aim 1), we will determine the mechanisms by which EphA2 signaling affects smooth muscle phenotype (Aim 2), and we will assess the role of EphA2 cell-type specific expression and signaling in atherosclerotic fibroproliferative remodeling using novel EphA2 conditional knockouts and inhibitors of EphA2 ligation and kinase activity. Successful completion of these Aims will identify a novel mechanism to selectively reduce plaque-associated inflammation while promoting smooth muscle- dependent stabilization of the fibrous cap.