TSP-4 genetic variants in atherogenesis and angiogenesis

描述(由申请人提供)：动脉粥样硬化病变发展的关键是血液和血管细胞与细胞外基质(ECM)成分之间的相互作用。该提案侧重于ECM蛋白--凝血酶敏感蛋白-4(TSP-4)的作用。我们发表的研究和初步数据强烈表明TSP-4参与调节血管壁炎症，因此，TSP-4 KO小鼠的动脉粥样硬化明显受到抑制。在TSP-4KO小鼠模型中，内皮细胞(EC)机械地减少了多种白细胞黏附分子和单核细胞趋化蛋白(MCP-1)的表达，导致巨噬细胞(M？)聚集到正在发展的病变中，从而抑制了动脉粥样硬化形成中的一个关键事件。此外，在动脉粥样硬化形成中另一个重要的EC反应，血管生成，在TSP-4 KO小鼠中被抑制，这是TSP-4是促血管生成的第一个证据。叠加在这些新的观察结果上的是我们的发现(现在在许多独立研究中重复)，与A387相比，高频遗传变异P387TSP-4是动脉粥样硬化血栓形成的危险因素。需要检验的主要假设是，TSP-4激活了血管细胞中调节细胞基质动力学和血管炎症的特定分子机制和途径，并且P387变异体加强了这些促动脉粥样硬化反应，包括血管生成。一种新的表达P387 TSP-4变体的敲入小鼠将允许在体内验证这一假说。我们提出了三个具体目标：1)利用TSP-4 KO和P387 TSP-4 K-来确定TSP-4变异体在小鼠动脉粥样硬化中的作用，并结合原位杂交进行骨髓移植，以确定动脉粥样硬化的差异是否依赖于血液和/或血管细胞。2)用TSP-4 KO和P387 TSP-4 K-在小鼠体内研究TSP-4对TSP-4不同变异体的差异反应的分子机制，以及TSP-4的促血管生成活性。3)。进行翻译研究，以确定TSP-4与人类冠状动脉病变和非病变区域的动脉粥样硬化和血管生成之间的关系。我们发现了一种新的血浆生物标志物TSP-4RA，它在一小部分急性心肌梗死患者中显著升高。这一线索将被用来确定TSP-4RA在急性心肌梗死患者中是否选择性升高，是否对第二次急性心肌梗死有预后意义，以及在患者达到的水平是否影响细胞反应。我们的总体目标是确定TSP-4在血管细胞生物学中的作用， 确定其促动脉粥样硬化和促血管生成功能的分子机制，并在小鼠和人类研究中确定P387TSP-4变体是否增强了这些功能。

DESCRIPTION (provided by applicant): Critical to the development of atherosclerotic lesions is the interaction between blood and vascular cells with components of the extracellular matrix (ECM). This proposal focuses on the role of an ECM protein, thrombospondin-4 (TSP-4). Our published studies and preliminary data strongly implicate TSP-4 in regulation of inflammation in the vessel wall, and, as a consequence, atherosclerosis is markedly suppressed in the TSP- 4 KO mouse. Mechanistically reduced expression of multiple leukocyte adhesion molecules and monocyte chemotactic protein (MCP-1) by endothelial cells (EC) in TSP-4 KO mice leads to few macrophages (M¿) accumulating into developing lesions, thereby suppressing a key event in atherogenesis. Furthermore, another EC response important in atherogenesis, angiogenesis, is suppressed in TSP-4 KO mice, the first evidence that TSP-4 is a pro-angiogeneic. Superimposed on these novel observations is our finding (now replicated in numerous independent studies) that a high frequency genetic variant, P387 TSP-4 as contrasted to A387, is an atherothrombotic risk factor. The primary hypothesis to be tested is that TSP-4 activates specific molecular mechanisms and pathways in vascular cells that regulate cell-matrix dynamics and vascular inflammation and that the P387 variant accentuates these pro-atherogenetic responses, including angiogenesis. A new knock-in mouse expressing P387 TSP-4 variant will permit testing this hypothesis in vivo. Three specific aims are proposed: 1) To define the role of the TSP-4 variants in atherosclerosis using TSP-4 KO and P387 TSP-4 K-In mice and to perform bone marrow transplantation in combination with in situ hybridization to determine if differences in atherosclerosis are dependent on blood and/or vascular cells. 2) To identify the molecular mechanisms underlying the differential responses of EC to the TSP-4 variants and characterize the proangiogenic activity of TSP-4 in vivo using TSP-4 KO and P387 TSP-4 K-In mice. 3). To perform translational studies to determine relationships between TSP-4, atherogenesis and angiogenesis in lesioned and non-lesioned areas of human coronary arteries. We found a new plasma biomarker, TSP-4RA, that was markedly elevated in a small panel of AMI patients. This lead will be followed to determine if TSP-4RA is selectively elevated in AMI patients, is prognostic for second AMI, and, at levels attained in patients, influences cellular responses. Our overall goals are to establish the roles of TSP-4 in vascular cell biology, to identify the molecular mechanisms underlying its proatherogenic and proangiogenic functions, and to determine whether these functions are enhanced by the P387 TSP-4 variant in mouse and human studies.