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调节，包括全身性风险因素（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剂。过渡性基质蛋白（例如，纤连蛋白）在动脉粥样硬化形成早期在内皮下基质中积累，并通过增强流动诱导的渗透性和促炎基因表达来引发内皮细胞功能障碍。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的刺激，包括干扰的血流和氧化的LDL，激活Rac/cdc 42效应子p21激活的激酶（PAK），并且PAK抑制剂在体外和体内动脉粥样硬化倾向部位降低内皮渗透性和促炎反应。尽管上游途径激活，但基底膜蛋白不支持PAK激活，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的区域。蛋白激酶A（PKA）磷酸化并抑制悬浮细胞中的PAK。此外，PKA降低促炎基因表达和内皮通透性，表明PKA是基质特异性PAK抑制的良好候选物。初步数据显示，基底膜蛋白增强流动诱导的PKA活化，并且抑制细胞中基底膜蛋白上的PKA足以恢复流动诱导的PAK活化和促炎反应。这些数据使我们假设，基底膜蛋白利用PKA依赖性信号来抑制PAK和限制内皮细胞功能障碍，而过渡性基质沉积引发内皮细胞进展为功能障碍表型。拟开展的工作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。为了说明这一新的信号传导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途径的信号传导如何影响氧化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的方法提供洞察内源性基底膜作为一种新的动脉粥样硬化保护剂的作用。 公共卫生相关性：动脉粥样硬化是一种血管壁慢性炎症性疾病，是发达国家的主要死亡原因。我们的研究表明，局部细胞外基质的变化可能是一种组织记忆。组织基质的正常组分限制细胞对瞬时伤害性刺激的反应，而响应于慢性刺激的过渡性基质沉积增强细胞对传播组织重塑的反应。了解基质成分影响细胞生理学的分子机制可以提供新的治疗靶点，以限制慢性炎症性疾病，如动脉粥样硬化和关节炎。

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.