Integrin-CCN1 Interaction in Vascular Thrombogenicity

描述（由申请人提供）：CCN1 (CYR61)， CCN家族基质细胞调节蛋白的新型整合素配体，支持细胞粘附，促进细胞迁移并增强生长因子诱导的细胞增殖。尽管CCN1在健康成人血管中的表达水平较低，但在动脉粥样硬化和增殖性再狭窄等血管疾病中，其表达水平上调。移行单核细胞和血管平滑肌细胞（VSMCs）在血管疾病的发生发展中起重要作用。最近，我们发现单核细胞和VSMCs分别通过整合素α - m - β -2和α -6- β - 1粘附于CCN1。此外，CCN1诱导单核细胞基因表达，导致白细胞介素1- β (il -1- β)、单核细胞趋化蛋白1 （MCP-1）和组织因子上调。基于这些观察，我们假设：CCN1在血管壁的表达调节单核细胞和VSMC功能，导致动脉粥样硬化和血栓形成的风险增加。在这项提议中，我们的目标是：1)表征CCN1与整合素α - m - β -2和α -6- β -1的相互作用。我们已经鉴定出CCN1中的H2和T1序列分别是α - m - β -2和α -6- β -1的新结合位点。通过诱变，我们将确定这些序列中介导整合素相互作用的关键残基，并创建α - m - β -2或α -6- β -1结合缺陷的CCN1突变体。2)检测CCN1诱导单核细胞迁移的能力。我们还将表征ccn1诱导的单核细胞中il -1- β、MCP-1和组织因子的基因表达。在这个过程中所涉及的信号通路将被研究。3)评价CCN1刺激VSMC表型改变和增殖的能力。ccn1诱导的组织因子及其抑制剂在VSMCs中的表达也将被检测。4)确定体内CCN1表达对血管疾病发展的影响。我们将产生转基因小鼠，其中强力霉素可调节动脉SMCs中CCN1的诱导表达。我们将研究CCN1在血管壁上的表达是否会诱导单核细胞迁移、VSMC增殖和组织因子表达。利用这种转基因小鼠模型，我们将在光化学诱导的血管损伤模型中检测CCN1在血管壁上的表达是否会加速饮食诱导的动脉粥样硬化和闭塞血栓的形成。这些研究将为血管疾病发展的机制基础提供新的见解。

DESCRIPTION (provided by applicant): CCN1 (CYR61), a novel integrin ligand of the CCN family of matricellular regulatory proteins, supports cell adhesion, promotes cell migration and enhances growth factor-induced cell proliferation. Whereas CCN1 is present at low levels in healthy adult blood vessels, its expression is upregulated in vascular diseases such as atherosclerosis and proliferative restenosis. Transmigrated monocytes and vascular smooth muscle cells (VSMCs) play important roles in vascular disease development. Recently, we showed that monocytes and VSMCs adhere to CCN1 through integrin alpha-M-beta-2 and alpha-6-beta1, respectively. Moreover, CCN1 induces gene expression in monocytes, resulting in upregulation of interleukin 1-beta (IL-1-beta), monocyte chemotactic protein-1 (MCP-1), and tissue factor. Based on these observations, we hypothesize that: CCN1 expression in the vessel wall modulates monocyte and VSMC function, leading to an increased risk of atherosclerosis and thrombosis. In this proposal, we aim: 1) To characterize CCN1 interaction with integrins alpha-M-beta-2 and alpha-6-beta-1. We have identified the H2 and T1 sequences in CCN1 as novel binding sites for alpha-M-beta-2 and alpha-6-beta-1 respectively. By mutagenesis, we will define critical residues in these sequences mediating integrin interaction and create CCN1 mutants defective in alpha-M-beta-2 or alpha-6-beta-1 binding. 2) To examine the ability of CCN1 to induce monocyte transmigration. We will also characterize CCN1-induced gene expression in monocytes focusing on IL-1-beta, MCP-1 and tissue factor. The signaling pathways involved in this process will be investigated. 3) To evaluate the ability of CCN1 to stimulate VSMC phenotype change and proliferation. CCN1-induced expression of tissue factor and its inhibitor in VSMCs will also be examined. 4) To determine the in vivo effect of CCN1 expression on vascular disease development. We will generate transgenic mice in which doxycycline regulates inducible expression of CCN1 in arterial SMCs. We will examine whether CCN1 expression in vessel walls induces monocyte transmigration, VSMC proliferation, and tissue factor expression. Using this transgenic mouse model, will examine whether CCN1 expression in vessel walls accelerates diet-induced atherosclerosis and the formation of occluding thrombi in a photochemically induced vascular injury model. These studies will provide new insights into the mechanistic basis of the development of vascular diseases.