Nuclear FAK-mediated VSMC differentiation via epigenetic reprograming invascular diseases

项目摘要 血管平滑肌细胞(VSMCs)可以去分化为高度增殖的状态，但收缩能力较弱 血管损伤或转分化为巨噬细胞样细胞(MLCs)的基因表达 动脉粥样硬化进展。这些类型的表型转换是由多个转录和 表观遗传变化。血管内皮细胞脱分化和转分化的恶性影响 针对恶性VSMC表型转换的直接干预性研究尚不多见。在.期间 VSMC去分化、基质和生长因子分泌增加改变整合素信号并导致 粘着斑激酶(FAK)异常激活，促进VSMC增殖。我们证明了这一点 FAK是不活跃的，主要定位于健康动脉的VSMCs的细胞核内。然而，血管损伤 促进FAK活化和胞浆重定位，促进细胞周期。同时我们也观察到 FAK的激活抑制了VSMC收缩基因的表达，其基本机制是 FAK对VSMC收缩基因的调控尚不清楚。我们的初步数据表明，对FAK的抑制 VSMCs的催化活性诱导了FAK的核定位和收缩基因转录的增加。 通过生化和蛋白质组学研究，我们确定了两个独立的表观遗传抑制 机制、DNA甲基转移酶3A(DNMT3A)与核小体重构和脱乙酰酶(NuRD) 复杂的，作为核FAK互动的合作伙伴。重要的是，我们发现FAK抑制减少了DNMT3A 和NuRD组分的表达，这与DNA甲基化减少和增加有关 收缩基因启动子中的活化组蛋白标记(H3K27ac和H4ac)。利用遗传FAK细胞质 (Cyto)受限的VSMCs，我们发现FAK核定位是减少DNMT3A和 努德情结。此外，抑制FAK可阻止ApoE-/-小鼠晚期动脉粥样硬化病变的形成 DNMT3A和NuRD组分表达降低。这与较厚的平滑肌有关。 肌动蛋白阳性区，提示抑制FAK可增加VSMC分化和斑块稳定性 与对照组相比。我们的结果提示，损伤或高脂血症刺激时FAK的激活可能 可能通过表观遗传调控促进VSMC表型转换。我们的假设是FAK 催化抑制增强FAK核定位并通过减少表达促进VSMC分化 DNMT3A和NuRD。在目标1中，我们将阐明核FAK介导的VSMC的分子机制 通过DNA甲基化和组蛋白修饰的表观遗传调控进行表型转换。在目标2中，我们将 探讨DNMT3A和NuRD复合体在血管损伤VSMC去分化中的作用。在AIM 3、我们将评估抑制FAK对阻断VSMC转分化和促进斑块形成的作用 早期和晚期动脉粥样硬化的稳定性。这项研究将为VSMC表型提供新的见解 通过DNMT3A和NuRD复合体稳定的FAK介导的表观遗传控制的开关。

Project Summary Vascular smooth muscle cells (VSMCs) can dedifferentiate into a highly proliferative state with less contractile gene expression upon vessel injury or transdifferentiate into macrophage-like cells (MLCs) during atherosclerosis progression. These types of phenotypic switching are driven by multiple transcriptional and epigenetic changes. Despite vicious effects of VSMC dedifferentiation and transdifferentiation in vascular diseases, direct interventional studies that target vicious VSMC phenotype switching have been lacking. During VSMC dedifferentiation, increased secretion of matrix and growth factors alter integrin signaling and lead to aberrant focal adhesion kinase (FAK) activation, which promotes VSMC proliferation. We demonstrated that FAK is inactive and primarily localized within the nuclei of VSMCs of healthy arteries. However, vessel injury promoted FAK activation and cytoplasmic relocalization which increased cell cycling. While we also observed that FAK activation suppresses expression of VSMC contractile genes, the underlying mechanism by which FAK regulates VSMC contractile genes is not known. Our preliminary data demonstrated that inhibition of FAK catalytic activity in VSMCs induced nuclear localization of FAK and increased contractile gene transcription. Through biochemical and proteomics studies, we identified two independent epigenetic repression machineries, DNA methyltransferase 3A (DNMT3A) and the nucleosome remodeling and deacetylase (NuRD) complex, as nuclear FAK-interacting partners. Importantly, we found that FAK inhibition decreased DNMT3A and NuRD component expression, which was associated with decreased DNA methylation and increased active histone marks (H3K27ac and H4ac) in the contractile gene promoters. Using genetic FAK cytoplasmic (Cyto) restricted VSMCs, we found that FAK nuclear localization is required for reduction of DNMT3A and NuRD complex. Furthermore, FAK inhibition blocked advanced atherosclerotic lesion formation in ApoE-/- mice with decreased DNMT3A and NuRD component expression. This was associated with a thicker smooth muscle actin positive area, suggesting that FAK inhibition increased VSMC differentiation and plaque stability compared to control. Our results implicated that FAK activation upon injury or hyperlipidemia stimulation may contribute to VSMC phenotype switching potentially via epigenetic regulation. Our hypothesis is that FAK catalytic inhibition forces FAK nuclear localization and promotes VSMC differentiation via reduced expression of DNMT3A and NuRD. In Aim 1, we will elucidate the molecular mechanism of nuclear FAK-mediated VSMC phenotypic switching via epigenetic modulation of DNA methylation, and histone modification. In Aim 2, we will investigate the role of DNMT3A and the NuRD complex in VSMC dedifferentiation upon vascular injury. In Aim 3, we will evaluate the effect of FAK inhibition on blocking VSMC transdifferentiation and promoting plaque stability in early and advanced atherosclerosis. This study will provide new insights into VSMC phenotype switching via FAK-mediated epigenetic control through DNMT3A and NuRD complex stability.