Matrix Metalloprotease-PAR1 Regulation of Atherosclerosis

项目摘要 新出现的证据表明，不适当的基质金属蛋白酶(MMP)活性可能是导致 动脉粥样硬化和血管炎症的发病机制。尽管MMP贡献了数据 动脉粥样硬化病变重塑和不良预后，基本上对基质金属蛋白酶的作用一无所知。 作为主动信号分子在动脉粥样硬化背景下控制血管细胞的行为 疾病。我们最近有了一个意想不到的发现，即基质金属蛋白酶-1裂解并激活了由酶激活的 受体-1(PAR1)在血管中的信号转导。这具有重大意义，因为这是第一次直接报道 血管中主要胶原酶的信号功能。值得注意的是，我们发现基质金属蛋白酶-1激活了PAR1 通过在与标准凝血酶裂解位点不同的位置切割受体，从而产生更长的 系留配体偏向于不同光谱的G蛋白信号通路。这方面的研究 提案将侧重于MMP1-PAR1在动脉粥样硬化发展中完全未被探索的作用 斑块。此外，PAR1在动脉粥样硬化中的参与(与蛋白水解酶激动剂无关)作为一种 慢性进行性炎症性疾病，本质上是未知的。目标1中要检验的中心假设是 内皮细胞基质金属蛋白酶-1首先通过PAR1作为活性信号分子触发内皮炎症 单核细胞进入早期斑块。在晚期病变中，巨噬细胞的MMP1自动刺激PAR1至 通过分泌单核细胞趋化蛋白-1和其他介质来维持慢性炎症和有丝分裂状态。我们将使用 基于细胞的实验，以及Mmp1a或Par1基因缺陷的高脂血症小鼠模型。我们 将确定MMP1a和PAR1在小鼠内皮细胞中对单核细胞黏附和 切变流条件下分离的小鼠心脏内皮细胞体外迁移 Mmp1a-/-和Par1-/-小鼠，以及高脂饮食后ApoE-/-小鼠的单核细胞。目标2将考察 冠心病和急性冠脉病变患者循环单核细胞表面MMP1-PAR1系统的研究 接受经皮冠状动脉介入治疗(PCI)的综合征。基线前患者的单核细胞 对于那些正在接受PAR1肽治疗的患者，PZ-128将用于平行钢板 (动脉切变)流动室，以确定通过内皮单分子层的转运。我们将决定 具有超活性MMP1启动子多态性的受试者是否表现出更高的MMP1表达 在它们的单核细胞上形成了‘MMP1-PAR1’表型与‘Tf-PAR1’表型。AIM 3将使用领养 从Par1-/-ApoE-/-和Mmp1a-/-ApoE-/-小鼠移植骨髓来源的细胞以帮助确定细胞- MMP1-PAR1系统在动脉粥样硬化形成中的类型特异性病理生物学(例如内皮细胞与白细胞)。我们的 内皮和单核细胞MMP1-PAR1信号的病理生理相关性 巨噬细胞，以及将这些事件与动脉粥样硬化斑块发展联系起来的机制将提供 框架，以推进未来的治疗，可以阻止或逆转动脉粥样硬化的进展。

Project Summary Emerging evidence suggests that inappropriate matrix metalloprotease (MMP) activity may underlie the pathogenesis of atherosclerosis and vascular inflammation. Despite data that MMPs contribute to atherosclerotic lesion remodeling and poor outcomes, essentially nothing is known regarding the role of MMPs as active signaling molecules in controlling the behavior of vascular cells in the context of atherosclerotic disease. We recently made the unanticipated discovery that MMP-1 cleaves and activates protease-activated receptor-1 (PAR1) signaling in blood vessels. This is of major import as it was the first report of a direct signaling function of the principal collagenase in blood vessels. Notably, we found that MMP-1 activates PAR1 by cleaving the receptor at a distinct site from the canonical thrombin cleavage site which generates a longer tethered ligand that is biased towards a different spectrum of G protein signaling pathways. The studies in this proposal will focus on the completely unexplored role of MMP1-PAR1 in the development of atherosclerotic plaques. Furthermore, the involvement of PAR1 in atherosclerosis (regardless of the protease agonist) as a chronic evolving inflammatory disease, is essentially unknown. The central hypothesis to be tested in aim 1 is that endothelial MMP-1 first acts as an active signaling molecule via PAR1 to trigger endothelial inflammation and monocyte entry into early plaques. In advanced lesions, MMP1 from macrophages autostimulates PAR1 to perpetuate a chronic inflammatory and mitogenic state by secretion of MCP-1 and other mediators. We will use both cell-based experiments, and hyperlipidemic mouse models with genetic deficiency of Mmp1a or Par1. We will determine the requirement of MMP1a and PAR1 in mouse endothelial cells for monocyte adhesion and transmigration under shear flow conditions in vitro using mouse heart endothelial cells isolated from of Mmp1a-/- and Par1-/- mice, and monocytes from ApoE-/- mice after high fat diet. Aim 2 will examine the role of the MMP1-PAR1 system on circulating monocytes in subjects with coronary artery disease and acute coronary syndromes undergoing percutaneous coronary interventions (PCI). Monocytes from patients at baseline prior to PCI and from those being treated with a novel PAR1 pepducin, PZ-128, will be used in Parallel Plate (arterial-shear) flow chambers to determine transmigration through endothelial monolayers. We will determine whether subjects with a super-active Mmp1 promoter polymorphism exhibit higher expression of MMP1 that contributes to a `MMP1-PAR1' phenotype vs `TF-PAR1' phenotype on their monocytes. Aim 3 will use adoptive transfer of bone marrow-derived cells from Par1-/-ApoE-/- and Mmp1a-/-ApoE-/- mice to help define the cell- type specific pathobiology (e.g. endothelium vs leukocytes) of the MMP1-PAR1 system in atherogenesis. Our understanding of the pathophysiologic relevance of MMP1-PAR1 signaling on endothelium and monocytes/ macrophages, and the mechanism linking these events to atherosclerotic plaque development will provide a framework to advance future therapies that could halt or reverse the progression of atherosclerosis.