Matrix signaling in endothelial cell dysfunction

动脉粥样硬化易发部位的动脉微环境为内皮细胞的激活做好了准备。 系统性动脉粥样硬化因子的作用，部分是通过增加血管内皮细胞受干扰部位的NF-B的表达 流。此外，紊乱的血流模式允许内皮下基质重塑，并从 我的团队和其他人已经证明，纤维连接蛋白的沉积增强了内皮细胞的促炎作用 对扰动流动和氧化低密度脂蛋白均有反应。纤维连接蛋白作用机制的研究现状 动脉粥样硬化部位的沉积集中于纤维连接蛋白表达的改变。然而，我们的初步数据显示 氧化的低密度脂蛋白可诱导纤维连接蛋白沉积，这种沉积是由Talin1依赖的整合素动态激活驱动的 没有改变纤维连接蛋白的表达，提示了一种新的动脉粥样硬化基质重塑模型。 在之前的授权期中，我们证明了整合素特异的信号转导通路以不同的方式调节流和 氧化低密度脂蛋白诱导的NF-B信号和促炎基因的表达 初步数据显示，抑制或删除内皮细胞纤维连接蛋白结合整合素会早期减少 体内致动脉粥样硬化性炎症。尽管整合素介导的NF-B激活在多发性骨髓瘤中的重要性 系统中，将整合素与NF-B激活联系起来的信号通路仍然相对未知。The IB IKK激活典型的NF-B信号，Flow和oxLDL均激活IKK依赖的NF-B 激活。非蛋白水解性泛素化(K63连接和MET1连接的泛素链)驱动 经典地将IKK与其上游激活子偶联的信号微域通过招募 泛素结合蛋白，如必需的IKK结合伙伴IKK。我们的初步数据显示 与IKK靶向和钝化K63相关的整合素黏附复合体中K63的强泛素化 泛素化可阻止ox低密度脂蛋白诱导的NF-B激活。此外，我们发现内皮细胞缺乏 纤维连接蛋白结合的整合素对促炎刺激的反应性降低，这表明 整合素信号参与了与动脉粥样硬化表型相关的内皮启动。这项研究 这项提案中概述的将测试内皮细胞中动态整合素激活驱动的假设 纤维连接蛋白沉积和整合素特异性信号促进内皮细胞激活。我们会做到这一点的 通过研究体外调节Talin1依赖的整合素激活和纤维连接蛋白沉积的机制 并在体内使用整合素激活缺陷的内皮talin1L325R转基因小鼠(目标1)。我们会 IKK/NF-B活化中整合素依赖的非蛋白水解性泛素化调控机制的研究 并描述了纤维连接蛋白结合整合素如何激活内皮细胞(目标2)。最后，我们 将利用可诱导的内皮细胞特异性缺失模型来确定内皮纤维连接蛋白结合 整合素在体内促进内皮细胞启动，促进早期基质后动脉粥样硬化斑块的进展 重塑和血流诱导的动脉性炎症(目标3)。

The arterial microenvironment at atherosclerosis-prone sites primes the endothelium for activation by a variety of systemic atherogenic factors, in part through enhanced endothelial NF-B expression at sites of disturbed flow. Additionally, disturbed flow patterns are permissive for subendothelial matrix remodeling, and work from my group and others have shown that fibronectin deposition enhances the endothelial proinflammatory response to both disturbed flow and oxidized LDL. Current research into the mechanisms of fibronectin deposition at atheroprone sites focus on altered fibronectin expression. However, our preliminary data show that oxidized LDL elicits robust fibronectin deposition driven by dynamic talin1-dependent integrin activation without altered fibronectin expression, suggesting a novel model for atherogenic matrix remodeling. In the previous grant period, we demonstrated that integrin-specific signaling differentially mediates flow and oxidized LDL induced NF-B signaling and proinflammatory gene expression, and our published and preliminary data show that inhibiting or deleting endothelial fibronectin-binding integrins reduces early atherogenic inflammation in vivo. Despite the importance of integrin-mediated NF-B activation in multiple systems, the signaling pathways linking integrins to NF-B activation remain relatively unknown. The IB kinase IKK activates canonical NF-B signaling, and both flow and oxLDL stimulate IKK-dependent NF-B activation. Nonproteolytic ubiquitination (K63-linked and Met1-linked ubiquitin chains) drives the formation of signaling microdomains that classically couple IKK to its upstream activators through the recruitment of ubiquitin-binding proteins, such as obligatory IKK-binding partner IKK. Our preliminary data demonstrate robust K63 ubiquitination in integrin adhesion complexes associated with IKK targeting, and blunting K63 ubiquitination prevents oxLDL-induced NF-B activation. In addition, we show that endothelial cells lacking fibronectin-binding integrins display reduced responsiveness to proinflammatory stimuli, suggesting that integrin signaling contributes to endothelial priming associated with the atheroprone phenotype. The research outlined in this proposal will test the hypothesis that dynamic integrin activation in endothelial cells drives fibronectin deposition and integrin-specific signaling to promote endothelial activation. We will accomplish this by examining the mechanisms regulating talin1-dependent integrin activation and fibronectin deposition in vitro and in vivo using the endothelial talin1 L325R transgenic mice deficient for integrin activation (Aim 1). We will delineate the mechanisms regulating integrin-dependent nonproteolytic ubiquitination in IKK/NF-B activation and characterize how fibronectin-binding integrins prime the endothelial cells for activation (Aim 2). Lastly, we will utilize inducible endothelial-specific deletion models to determine whether endothelial fibronectin-binding integrins contribute to endothelial priming in vivo, to atherosclerotic plaque progression following early matrix remodeling, and to flow-induced arteriogenic inflammation (Aim 3).