Integrin-CCN1 Interaction in Vascular Thrombogenicity

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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.