Matrix Signaling in Endothelial Cell Dysfunction

描述(由申请人提供)：向功能障碍内皮细胞表型的转变受多种环境因素的调节，包括两个系统性风险因素(例如。氧化低密度脂蛋白)和局部血流模式。我的研究表明，局部基质成分是内皮细胞功能障碍的一种新的调节因素。过渡性基质蛋白(例如纤维连接蛋白)在动脉粥样硬化早期积聚在内皮下基质中，并通过增强血流诱导的通透性和促炎基因表达而启动内皮细胞的功能障碍。相反，基底膜蛋白限制内皮细胞功能障碍。多种致动脉粥样硬化的刺激，包括紊乱的血流和氧化的低密度脂蛋白，激活RAC/CDC42效应p21激活的激酶(PAK)，而PAK抑制剂在体外和体内动脉粥样硬化易发部位都能降低内皮通透性和促炎反应。尽管上游通路被激活，但基底膜蛋白不支持PAK的激活，这表明来自基底膜的信号抑制PAK以限制内皮细胞功能障碍。因此，PAK在体内的激活仅限于过渡性基质沉积区域。蛋白激酶A(PKA)在悬浮状态下磷酸化并抑制细胞内的PAK。此外，PKA可降低促炎基因表达和内皮通透性，提示PKA是抑制基质特异性PAK的良好候选者。初步数据表明，基底膜蛋白可促进血流诱导的PKA活化，而抑制基底膜蛋白上的细胞内PKA足以恢复血流诱导的PAK激活和促炎反应。这些数据使我们假设，基底膜蛋白利用PKA依赖的信号来抑制PAK并限制内皮细胞功能障碍，而过渡性基质沉积则启动内皮细胞进入功能障碍表型。这项拟议的工作将通过确定基质信号调节PKA激活的机制(目标1)和探索依赖PKA的PAK抑制的分子机制(目标2)来验证这一假说。为了阐明这个新的信号轴跨越多种致动脉粥样硬化信号的广泛范围，我们将确定基质组成和通过PKA和PAK通路的信号如何影响氧化低密度脂蛋白诱导的内皮细胞功能障碍(目标3)。这项工作将利用多因素方法来深入了解内源性基底膜作为一种新的动脉粥样硬化保护剂的作用。

DESCRIPTION (provided by applicant): The transition to a dysfunction endothelial cell phenotype is regulated by multiple environmental factors, including both systemic risk factors (ex. oxidized LDL) and local blood flow patterns. My research suggests that local matrix composition is a novel regulator of endothelial cell dysfunction. Transitional matrix proteins (ex. fibronectin) accumulate in the subendothelial matrix early during atherogenesis and prime endothelial cells for dysfunction by enhancing flow-induced permeability and proinflammatory gene expression. In contrast, basement membrane proteins limit endothelial cell dysfunction. Multiple atherogenic stimuli, including disturbed flow and oxidized LDL, activate the Rac/cdc42 effector p21 activated kinase (PAK), and PAK inhibitors reduce endothelial permeability and proinflammatory responses both in vitro and at atherosclerosis- prone sites in vivo. Despite activation of upstream pathways, basement membrane proteins do not support PAK activation, suggesting that signals from the basement membrane inhibit PAK to limit endothelial cell dysfunction. As such, PAK activation in vivo is restricted to regions of transitional matrix deposition. Protein kinase A (PKA) phosphorylates and inhibits PAK in cells in suspension. In addition, PKA reduces both proinflammatory gene expression and endothelial permeability, suggesting PKA is a good candidate for matrix- specific PAK suppression. Preliminary data show that basement membrane proteins enhance flow-induced PKA activation, and inhibiting PKA in cells on basement membrane proteins is sufficient to restore flow- induced PAK activation and proinflammatory responses. These data lead us to hypothesize that basement membrane proteins utilize a PKA-dependent signal to inhibit PAK and limit endothelial cell dysfunction, whereas transitional matrix deposition primes endothelial cells to progress to a dysfunctional phenotype. The proposed work will test this hypothesis by determining the mechanisms by which matrix signaling modulates PKA activation (Aim 1) and by exploring the molecular mechanisms of PKA-dependent PAK inhibition (Aim 2). To illustrate the broad scope of this novel signaling axis across multiple atherogenic signals, we will determine how matrix composition and signaling through the PKA and PAK pathways affect oxidized LDL-induced endothelial cell dysfunction (Aim 3). This work will utilize a multifactorial approach to provide insight into the role of the endogenous basement membrane as a novel atheroprotective agent.