Matrix signaling in endothelial cell dysfunction

描述（由申请人提供）：内皮细胞向功能障碍表型的转变受到多种环境因素的调节，包括系统性危险因素（如氧化LDL）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子。过渡基质蛋白（如纤维连接蛋白）在动脉粥样硬化早期在内皮下基质中积累，通过增强血流诱导的通透性和促炎基因表达，使内皮细胞功能障碍。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刺激，包括血流紊乱和氧化LDL，激活Rac/cdc42效应p21活化激酶（PAK）， PAK抑制剂在体外和体内动脉粥样硬化易发部位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激活，这表明基底膜的信号抑制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基质沉积区域。蛋白激酶A （PKA）在悬浮细胞中磷酸化并抑制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的良好候选物。初步数据显示，基底膜蛋白增强了血流诱导的PKA激活，抑制基底膜蛋白细胞中的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依赖性信号抑制PAK并限制内皮细胞功能障碍，而过渡基质沉积则使内皮细胞进展为功能障碍表型。本研究将通过确定基质信号调节PKA激活的机制（目的1）和探索PKA依赖性PAK抑制的分子机制（目的2）来验证这一假设。为了说明这种跨越多种动脉粥样硬化信号的新型信号轴的广泛范围，我们将确定通过PKA和PAK途径的基质成分和信号如何影响氧化ldl诱导的内皮细胞功能障碍（目的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型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. PUBLIC HEALTH RELEVANCE: Atherosclerosis, a chronic inflammatory disease of the vessel wall, is the leading cause of death in developed countries. Our research suggests that changes in the local extracellular matrix may serve as a form of tissue memory. Normal components of the tissue matrix limit cellular responsiveness to transient injurious stimuli, while transitional matrix deposition in response to chronic stimuli enhances cellular responsiveness to propagate tissue remodeling. Understanding the molecular mechanisms by which matrix composition affects cell physiology could provide novel therapeutic targets to limit chronic inflammatory diseases, such as atherosclerosis and arthritis.