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

摘要 血管平滑肌细胞（SMC）迁移是血管新生内膜形成的关键事件， 溶血磷脂酸（LPA）是动脉粥样硬化和再狭窄的重要脂质介质， 导致动脉粥样硬化的细胞事件。在上一次资助期间，使用LPA受体敲除 在小鼠模型中，我们鉴定出LPA受体1是介导LPA诱导的SMC的主要细胞膜受体 迁移和新生内膜形成。我们还发现了基质细胞蛋白Cyr 61作为关键分子， 介导LPA信号传导，导致SMC迁移和新生内膜形成。这些发现建立了一个新的 Cyr 61桥接LPA和整合素信号通路，导致粘着斑激酶（FAK）的概念 活化/SMC迁移和新生内膜形成。在此资助期内，我们建议延长我们的调查 确定LPA-Cyr 61诱导SMC迁移的分子机制和LPA-Cyr 61通路 对动脉粥样硬化的贡献。最近的谱系追踪研究表明，大部分巨噬细胞 小鼠和人动脉粥样硬化病变中的标记物阳性细胞是血管SMC来源的细胞，这表明 SMC的迁移/增殖和分化在动脉粥样硬化的形成中起重要作用。了解SMC 迁移机制将有助于理解动脉粥样硬化的形成。迄今为止，人们对 在极化前沿驱动激酶的迁移的性质和时空调节 的SMC。有趣的是，我们最近的研究揭示了几个令人兴奋的新发现：1）新的伪足- 在LPA时，富集的非典型激酶1（PEAK 1）激活定位于SMC的极化前缘 处理; 2）Cyr 61诱导PEAK 1活化和3）PEAK 1的敲低阻断SMC迁移， 提示PEAK 1是Cyr 61下游的关键介质，调节SMC迁移。此外，委员会认为， 我们发现PEAK 1与FAK相互作用，FAK是细胞迁移的关键调节因子。我们还观察到 磷酸化PEAK 1水平在ApoE-/-动脉粥样硬化病变中高度增加。小说的功能 激酶PEAK 1在血管疾病中作用尚不清楚。我们的数据表明，PEAK 1是FAK的新介质 激活和SMC迁移。基于这些新的观察结果，我们假设LPA受体- Cyr 61-integrin-PEAK 1-FAK轴介导平滑肌细胞骨架重组/迁移和损伤形成 动脉粥样硬化这些假设将在以下具体目标中得到检验。目标1：确定新角色 PEAK 1/FAK相互作用对Src/PEAK 1/FAK/Paxillin/Rac通路的激活和SMC迁移的影响。 目的2：剖析Cyr 61与整合素-粘附复合物信号转导相互作用的新机制，导致 使用来自新Cyr 61 dm/dm敲入的独特Cyr 61突变SMC的膜突出和细胞迁移 小鼠目的3：利用我们的研究建立LPA受体和Cyr 61在动脉粥样硬化发展中的作用。 创新的LPA受体/ApoE敲除、Cyr 61 dm/dm/ApoE-/-和SMC谱系追踪小鼠模型。的 预期提出的研究将确定预防和治疗动脉粥样硬化的新靶点

ABSTRACT Vascular smooth muscle cell (SMC) migration is a critical event in neointimal formation in the development of atherosclerosis and restenosis, Lysophosphatidic acid (LPA), is emerging as an important lipid mediator of the cellular events contributing to atherosclerosis. During the last grant period, using an LPA receptor knockout mouse model, we identified LPA receptor 1 as the major cell membrane receptor mediating LPA-induced SMC migration and neointimal formation. We also discovered the matricellular protein Cyr61 as the key molecule mediating LPA signaling, leading to SMC migration and neointimal formation. These findings established a new concept that Cyr61 bridges the LPA and integrin signaling pathways, leading to focal adhesion kinase (FAK) activation/SMC migration and neointimal formation. In this grant period, we propose to extend our investigation to identify the molecular mechanism by which LPA-Cyr61 induces SMC migration and the LPA-Cyr61 pathway contribution to atherogenesis. Very recently lineage-tracing studies reveal that a large portion of macrophage marker-positive cells in mouse and human atherosclerotic lesions are vascular SMC-derived cells, suggesting SMC migration/proliferation and differentiation play important roles in atherogenesis. Understanding of SMC migration mechanism will significantly contribute to understanding atherogenesis. To date, little is known about the nature and spatial-temporal regulation of the migration which drives kinases in the polarized leading edge of SMCs. Interestingly, our recent study revealed several exciting novel findings: 1) the novel pseudopodium- enriched atypical kinase 1 (PEAK1) activation is localized in the polarized leading edge of SMCs upon LPA treatment; 2) Cyr61 induces PEAK1 activation and 3) knockdown of PEAK1 blocked SMC migration, suggesting PEAK1 functions as a downstream key mediator of Cyr61, regulating SMC migration. Furthermore, we discovered that PEAK1 interacts with FAK, a key regulator of cell migration. We also observed that phosphorylated PEAK1 levels are highly increased in ApoE-/- atherosclerotic lesions. The function of the novel kinase PEAK1 in vascular disease is unknown. Our data suggest that PEAK1 is the novel mediator for FAK activation and SMC migration. Based on these new observations, we hypothesize that the LPA receptor- Cyr61-integrin-PEAK1-FAK axis mediates SMC cytoskeleton reorganization/migration and lesion formation in atherosclerosis. These hypotheses will be tested in the following specific aims. Aim 1: Determine the novel role of PEAK1/FAK interaction on the activation of the Src/PEAK1/FAK/Paxillin/Rac pathway and SMC migration. Aim 2: Dissect the novel mechanism by which Cyr61 interacts integrin-adhesion complex signaling, leading to membrane protrusion and cell migration using unique Cyr61 mutant SMCs from novel Cyr61dm/dm knock-in mice. Aim 3: Establish the roles of LPA receptors and Cyr61 in the development of atherosclerosis using our innovative LPA receptor/ApoE knockout, Cyr61dm/dm/ApoE-/- and SMC lineage tracing mouse models. The proposed studies are expected to identify novel targets for prevention and treatment of atherogenesis