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

说明(申请人提供)：溶血磷脂酸(LPA)是氧化型低密度脂蛋白(LDL)中一种重要的生物活性脂质成分，由活化的血小板产生。LPA在人类动脉粥样硬化病变中大量积聚，并诱导血管平滑肌细胞(SMC)增殖和迁移，以及血管新生内膜的形成。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。为了寻找新的LPA致病介质，我们的基因芯片分析发现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特异性抗体几乎完全阻断了LPA诱导的SMC迁移，而其他抗体则没有。重要的是，我们观察到LPA刺激血管新生内膜病变中Cyr61的表达。这些数据有力地支持了Cyr61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)抗Cyr61和特定整合素的抗体阻断了LPA诱导的SMC迁移；2)LPA诱导了包括PKD、MAPK和新的酪氨酸激酶JAK2、Lyn和TXK在内的一系列蛋白激酶的激活；4)LPA受体1的敲除阻止了LPA对这些激酶的激活；5)PPAR3拮抗剂不抑制LPA诱导的Cyr61的表达和SMC的增殖和迁移。基于这些新的发现，我们假设LPA通过其特定的细胞表面受体(S)激活特定的细胞内信号通路，导致Cyr61从头表达，进而激活导致SMC增殖/迁移和血管新生内膜形成的整合素途径。目的1：确定LPA触发的导致基质细胞Cyr61表达的新的细胞内途径。目的：利用新的LPA受体敲除细胞和siRNA方法，确定LPA受体的特异性以及Cyr61和整合素在LPA诱导的SMC细胞反应中的作用。目的：利用新的LPA受体敲除小鼠模型和独特的siRNA方法，确定LPA受体基因缺失是否减少了LPA诱导的Cyr61的产生和新生内膜的形成，并确定了Cyr61和特定的整合素在LPA诱导的血管壁重建中的新作用。到目前为止，介导LPA诱导的Cyr61表达的特异性细胞表面受体尚不清楚。介导LPA诱导的血管壁细胞Cyr61表达的分子级联机制从未被研究过。更重要的是，Cyr61是否以及如何介导LPA诱导的SMC增殖/迁移和血管重塑是完全未知的。因此，这项研究将确定一种新的汇聚机制，通过Cyr61将LPA/GPCR途径和整合素途径连接起来，并介导LPA诱导的血管壁重建。这些研究将为理解生物活性脂类LPA和氧化型低密度脂蛋白在血管新生内膜形成和动脉粥样硬化中的作用机制带来新的突破。 公共卫生相关性：这项提案的中心目标是揭示溶血磷脂酸(LPA)的病理作用，它参与动脉粥样硬化的发展，动脉硬化是一种类型的动脉硬化。根据美国心脏协会2009年年度报告，心血管疾病是美国头号死亡和残疾原因。了解动脉粥样硬化的基本机制为新的预防和治疗方法铺平了道路。

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. PUBLIC HEALTH RELEVANCE: The central goal of this proposal is to reveal the pathological role of lysophosphatidic acid (LPA), which is involved in the development of atherosclerosis, a type of hardening of the arteries. Cardiovascular disease is the number one cause of death and disability in the United States, according to the American Heart Association's 2009 Annual Report. Understanding the basic mechanisms of atherosclerosis paves the way for new prevention and treatment approaches.