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

描述（由申请人提供）：溶血磷脂酸（LPA）是氧化低密度脂蛋白（LDL）的重要生物活性脂质成分，由活化的血小板产生。LPA在人动脉粥样硬化病变中高水平积累，诱导血管平滑肌细胞（SMC）增殖、迁移和血管内膜形成。因此，LPA被认为是动脉粥样硬化的危险因素。然而，LPA促进动脉粥样硬化的分子机制尚不清楚。为了鉴定LPA的新型致病介质，我们的基因芯片分析显示，LPA诱导了一组蛋白的表达，包括IL-6、IL- 8、Tuftelin和Cyr61，这些蛋白与SMC的增殖和迁移有关。我们的数据进一步表明，cyr61特异性抗体，而不是其他抗体，几乎完全阻断了lpa诱导的SMC迁移。重要的是，我们观察到LPA刺激Cyr61在血管内膜病变中的表达。这些数据有力地支持Cyr61在lpa诱导的血管内膜形成中的新作用。为此，我们的研究揭示了1)针对Cyr61和特定整合素的抗体阻断了lpa诱导的SMC迁移；2) LPA诱导一系列蛋白激酶的激活，包括PKD、MAPK和新型酪氨酸激酶JAK2、Lyn和Txk；4)敲除LPA受体1阻断这些激酶的LPA活化；5) PPAR3拮抗剂不抑制lpa诱导的Cyr61表达和SMC的增殖和迁移。基于这些新发现，我们假设LPA通过其特定的细胞表面受体激活特定的细胞内信号通路，导致Cyr61的重新表达，而Cyr61反过来激活整合素通路，导致SMC增殖/迁移和血管内膜形成。目的1：确定LPA触发的导致细胞Cyr61表达的细胞内新途径。目的2：利用新型LPA受体敲除细胞和siRNA方法确定特异性LPA受体以及Cyr61和整合素在LPA诱导的SMC细胞反应中的作用。目的3：利用新型LPA受体敲除小鼠模型和独特的siRNA方法，确定LPA受体的基因缺失是否会减少LPA诱导的Cyr61的产生和新内膜的形成，并确定Cyr61和特异性整合素在LPA诱导的血管壁重塑中的新作用。迄今为止，介导lpa诱导Cyr61表达的特定细胞表面受体尚不清楚。在血管壁细胞中介导lpa诱导Cyr61表达的分子级联从未被研究过。更重要的是，Cyr61是否以及如何介导LPA诱导的SMC增殖/迁移和血管重构是完全未知的。因此，本研究将确定一种新的趋同机制，通过Cyr61将LPA/GPCR通路和整合素通路相互连接，介导LPA诱导的血管壁重构。从这些研究中获得的信息将为理解生物活性脂质LPA和氧化LDL促进血管新生内膜形成和动脉粥样硬化的机制带来新的突破。

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.