Novel mechanism mediating LPA-induced smooth muscle cell and vascular responses

描述（由申请方提供）：溶血磷脂酸（LPA）是氧化低密度脂蛋白（LDL）的主要生物活性脂质成分，由活化血小板产生。LPA在人类动脉粥样硬化病变中以高水平积累，并诱导血管平滑肌细胞（SMC）增殖和迁移以及血管新生内膜形成。因此，LPA被认为是动脉粥样硬化的危险因素。然而，LPA促进动脉粥样硬化形成的分子机制尚不清楚。在努力确定新的致病介质LPA，我们的基因芯片分析显示，LPA诱导的一组蛋白质，包括IL-6，IL- 8，Tuftelin和Cyr 61的表达，已涉及SMC的增殖和迁移。我们的数据进一步表明，Cyr 61特异性抗体，而不是其他，几乎完全阻断LPA诱导的SMC迁移。重要的是，我们观察到LPA刺激Cyr 61在血管新生内膜病变中的表达。这些数据强烈支持Cyr 61在LPA诱导的血管新生内膜形成中的新作用。为此，我们的研究表明：1）Cyr 61和特定整合素的抗体阻断LPA诱导的SMC迁移; 2）LPA诱导的一系列蛋白激酶包括PKD、MAPK和新的酪氨酸激酶JAK 2、林恩和Txk的激活; 4）LPA受体1的敲除阻断LPA激活这些激酶; PPAR 3拮抗剂不能抑制LPA诱导的Cyr 61表达和SMC增殖、迁移。基于这些新的发现，我们假设LPA通过其特异性细胞表面受体激活特异性细胞内信号传导途径，导致Cyr 61的从头表达，这反过来激活整联蛋白途径，导致SMC增殖/迁移和血管新生内膜形成。目的1：鉴定LPA触发的导致基质细胞Cyr 61表达的新细胞内途径。目标二：使用新型LPA受体敲除细胞和siRNA方法确定特异性LPA受体以及Cyr 61和整合素在LPA诱导的SMC细胞应答中的作用。目标3：确定LPA受体的基因缺失是否减少LPA诱导的Cyr 61产生和新生内膜形成，并使用新型LPA受体敲除小鼠模型和独特的siRNA方法确定Cyr 61和特定整合素在LPA诱导的血管壁重塑中的新作用。迄今为止，介导LPA诱导的Cyr 61表达的特异性细胞表面受体尚不清楚。介导LPA诱导的Cyr 61在血管壁细胞中表达的分子级联从未被研究过。更重要的是，Cyr 61是否以及如何介导LPA诱导的SMC增殖/迁移和血管重塑是完全未知的。因此，拟议的研究将确定一种新的会聚机制，通过该机制，LPA/GPCR途径和整合素途径通过Cyr 61相互连接并介导LPA诱导的血管壁重塑。从拟议的研究中获得的信息将导致一个新的突破，了解生物活性脂质LPA和氧化低密度脂蛋白有助于血管新生内膜形成和动脉粥样硬化的机制。

DESCRIPTION (provided by applicant): Lysophosphatidic acid (LPA) is a prominent bioactive lipid component of oxidized low density lipoprotein (LDL) and is produced by activated platelets. LPA accumulates at high levels in human atherosclerotic lesions and induces vascular smooth muscle cell (SMC) proliferation and migration and vascular neointimal formation. Thus, LPA is being recognized as a risk factor for atherosclerosis. However, the molecular mechanism by which LPA contributes to atherogenesis is unclear. In an effort to identify novel pathogenic mediators of LPA, our gene microarray assays revealed that LPA induced the expression of a group of proteins, including IL-6, IL- 8, Tuftelin and Cyr61, that have been implicated in SMC proliferation and migration. Our data further demonstrated that the Cyr61-specific antibody, but not the others, almost completely blocked LPA-induced SMC migration. Importantly, we observed that LPA stimulates Cyr61 expression in vascular neointimal lesions. These data strongly support a novel role of Cyr61 in LPA-induced vascular neointimal formation. To this end, our study has revealed that 1) antibodies against Cyr61 and particular integrins blocked LPA-induced SMC migration; 2) LPA induced activation of a series of protein kinases including PKD, MAPK, and novel tyrosine kinases, JAK2, Lyn and Txk; 4) knockout of LPA receptor 1 blocked LPA activation of these kinases; and 5) LPA-induced Cyr61 expression and SMC proliferation and migration were not inhibited by PPAR3 antagonist. Based on these novel findings, we hypothesize that LPA, via its specific cell surface receptor(s), activates a specific intracellular signaling pathway, leading to de novo expression of Cyr61, which, in turn activates the integrin pathway leading to SMC proliferation/migration and vascular neointimal formation. Aim 1: Identify LPA- triggered novel intracellular pathways leading to matricellular Cyr61 expression. Aim 2: Determine the specific LPA receptors and the roles of Cyr61 and integrins in LPA-induced SMC cellular response using novel LPA receptor-knockout cells and siRNA approaches. Aim 3: Determine whether genetic depletion of LPA receptors diminishes LPA-induced Cyr61production and neointimal formation, and determine the novel roles of Cyr61 and specific integrins in LPA-induced vascular wall remodeling using novel LPA receptor knockout mouse models and the unique siRNA approach. To date, the specific cell surface receptor that mediates LPA-induced Cyr61 expression is unknown. The molecular cascades that mediate LPA-induced Cyr61 expression in the vascular wall cells have never been investigated. More importantly, whether and how Cyr61 mediates LPA- induced SMC proliferation/migration and vascular remodeling is completely unknown. Thus, the proposed study will determine a novel convergence mechanism by which the LPA/GPCR pathway and the integrin pathway are interconnected by Cyr61 and mediate LPA-induced vascular wall remodeling. The information obtained from the proposed studies will lead to a new breakthrough in understanding the mechanism by which bioactive lipid LPA and oxidized LDL contribute to vascular neointimal formation and atherogenesis.