The role of metalloprotease-PAR1 signaling in vascular injury and repair

描述(由申请人提供)：心血管疾病(CVD)是美国最主要的死亡原因。导致心血管疾病的主要潜在病理因素是动脉粥样硬化。动脉粥样硬化的动态本质涉及血管壁的重塑，以适应血管顺应性的变化。最初，这个过程是自适应的，后来它 就会变得有害。血管重塑的一个重要组成部分包括动脉粥样硬化斑块内及其周围的平滑肌细胞(SMC)的表型变化。血管损伤后，SMC发生巨大变化；细胞增殖、侵袭，并改变其代谢特性。SMC行为背后的分子机制还知之甚少。病理性SMC表型的一个重要特征是产生允许SMC侵袭和血管重塑的蛋白酶。基质金属蛋白酶(MMPs)是一个内肽酶家族，主要负责细胞外基质的降解，并在血管重塑和SMC行为调节中发挥重要作用。MMP1是血管修复过程中的关键分子，在动脉粥样硬化斑块中经常过度表达。已发表的数据表明，MMP1是一种重要的蛋白水解酶激活受体1(PAR1)激活剂。SMC高水平表达PAR1，在动脉粥样硬化斑块中表达增加。PAR1信号在SMC中的作用代表了血管生理学中一个有趣的悖论：在损伤期间，细胞只与天然激活剂凝血酶接触，这引发了许多关于PAR1在SMC中的作用的问题。此外，MMP1和凝血酶在不同的位置裂解和激活PAR1，导致假设凝血酶和MMP1不同地激活PAR1来调节血管损伤后的SMC活性。该项目的长期目标是更好地了解 适应不良血管重塑的病理生理学。近期的目标是研究凝血酶-MMP1-PAR1信号轴在血管修复中的作用机制。为了实现这些目标，已经确定了两个具体目标。第一，研究凝血酶与MMP1激活的PAR1信号在血管修复中的作用；第二，探讨PAR1信号在小鼠血管损伤模型中的作用。为了实现目标1，将使用PAR1和MMP1激动剂/拮抗剂对SMC进行增殖、迁移和侵袭的体外检测。此外，我们还将探讨凝血酶与MMP1激活的PAR1在SMC中的作用机制。此外，还将探讨凝血酶-MMP1-PAR1自分泌轴的作用。血管修复的小鼠模型将用于研究目标2。将在PAR1和MMP1基因敲除小鼠上进行颈动脉钢丝损伤，并将分析血管新内膜病变的形成。最后，将测试以PAR1为靶点的多肽作为治疗和预防血管新生内膜形成的疗法的有效性。 公共卫生相关性：心血管疾病(CVD)，包括心脏病发作、中风和动脉瘤，在美国仍然是主要的死亡原因。这项工作的重点是了解心血管疾病中潜在的血管损伤机制。长期目标是设计治疗心血管疾病的靶向疗法。

DESCRIPTION (provided by applicant): Cardiovascular disease (CVD) is the single leading cause of death in America. The major underlying pathology contributing to CVD is atherosclerosis. The dynamic nature of atherosclerosis involves remodeling of the blood vessel wall to accommodate for changes in vessel compliance. Initially, this process is adaptive, later it becomes injurious. An important component of vascular remodeling includes the phenotypic modulation of smooth muscle cells (SMCs) in and around the atherosclerotic plaque. SMCs undergo dramatic changes following blood vessel injury; cells proliferate, become invasive, and modify their metabolic properties. The molecular mechanisms underlying SMC behavior are poorly understood. An important characteristic of the pathologic SMC phenotype is the production of proteases that allow for SMC invasion and vessel remodeling. Matrix metalloproteases (MMPs) are a family of endopeptidases that are largely responsible for the degradation of extracellular matrix, and play important roles in vascular remodeling and modulation of SMC behavior. MMP1 is a key player in the process of vascular repair, and is often over-expressed in atherosclerotic plaques. Published data show that MMP1 is an important activator of protease-activated receptor 1 (PAR1). SMCs express high levels of PAR1, and expression is increased in atherosclerotic plaques. The role of PAR1 signaling in SMCs represents an interesting paradox in vascular physiology; the cells only contact the natural activator, thrombin, during times of injury, which raises numerous questions about the role of PAR1 on SMCs. Furthermore, MMP1 and thrombin cleave and activate PAR1 at differing sites, leading to the hypothesis that thrombin and MMP1 differentially activate PAR1 to modulate SMC activity following vascular damage. The long-term goal of this project is to better understand the pathophysiology of maladaptive vascular remodeling. The immediate objective is to investigate the mechanism of a thrombin-MMP1-PAR1 signaling axis in vascular repair. To accomplish these goals, two specific aims have been identified. First, to characterize the role of thrombin- vs MMP1-activated PAR1 signaling in vascular repair, and second, explore the contribution of PAR1 signaling in mouse models of vascular injury. To accomplish aim 1, in vitro assays for proliferation, migration, and invasion will be performed on SMCs utilizing PAR1 and MMP1 agonists/antagonists. Furthermore, we will interrogate the mechanism of thrombin- vs MMP1-activated PAR1 in SMCs. Additionally, the role of a thrombin-MMP1-PAR1 autocrine axis will also be explored. Mouse models of vascular repair will be used to investigate aim 2. Carotid artery wire injury will be performed on PAR1 and MMP1 knock-out mice, and vessels will be analyzed for neointimal lesion formation. Finally, the efficacy of PAR1 targeted pepducins will be tested as therapies for the treatment and prevention of vascular neointimal formation. PUBLIC HEALTH RELEVANCE: Cardiovascular disease (CVD), including heart attacks, strokes, and aneurysms, remains the leading cause of death in America. This work focuses on understanding the mechanisms underlying blood vessel damage in CVD. The long-term goal is to design targeted therapies for the treatment of CVD.