Nuclear FAK-mediated VSMC differentiation via epigenetic reprograming invascular diseases

通过表观遗传重编程血管疾病中核 FAK 介导的 VSMC 分化

• 批准号: 10618482
• 负责人: Steve Lim
• 金额: $ 47.54万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-16 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10618482
• 关键词: Apolipoprotein E; Area; Arterial Fatty Streak; Arteries; Atherosclerosis; Biochemical; CD47 gene; Cardiovascular system; Cell Cycle; Cell Differentiation process; Cell Nucleus; Cell Proliferation; Cells; Complex; Cyclin D1; DNA Methylation; DNA Modification Methylases; Data; Deacetylase; Disease; Epigenetic Process; Failure; Focal Adhesion Kinase 1; Gene Expression; Genes; Genetic; Genetic Transcription; Growth Factor; Histones; Hyperlipidemia; Inflammatory; Injury; Integrins; Intervention; Intervention Studies; Lead; Lesion; Mediating; Modeling; Molecular; Molecular Biology; Mus; Nuclear; Nucleosomes; Pharmacology; Phenotype; Play; Protein Tyrosine Kinase; Proteomics; Repression; Role; Signal Transduction; Smooth Muscle Actin Staining Method; Smooth Muscle Myocytes; Stimulus; Testing; Therapeutic; Thick; Ubiquitination; Vascular Diseases; Vascular Smooth Muscle; cell dedifferentiation; cell motility; cell type; chromatin remodeling; epigenetic regulation; histone modification; insight; kinase inhibitor; macrophage; mouse model; novel; oxidized lipid; prevent; promoter; response; tool; transdifferentiation; vascular injury; vascular smooth muscle cell proliferation; western diet

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.

项目摘要 血管平滑肌细胞（VSMC）可以去分化为高度增殖状态， 血管损伤或转分化为巨噬细胞样细胞（MLCs）时的基因表达 动脉粥样硬化进展。这些类型的表型转换是由多个转录和 表观遗传变化尽管VSMC的去分化和转分化在血管内皮细胞中有恶性作用， 然而，针对恶性VSMC表型转换的直接干预研究一直缺乏。期间 VSMC去分化、基质和生长因子分泌增加改变整合素信号传导并导致 异常的粘着斑激酶（FAK）活化，其促进VSMC增殖。我们证明了 FAK是无活性的，主要位于健康动脉的VSMC的核内。然而，血管损伤 促进FAK活化和胞浆重定位，从而增加细胞周期。同时我们也观察到 FAK的激活抑制了VSMC收缩基因的表达， FAK对VSMC收缩基因的调控尚不清楚。我们的初步数据表明，抑制FAK VSMCs中的催化活性诱导FAK的核定位并增加收缩基因转录。 通过生物化学和蛋白质组学的研究，我们确定了两个独立的表观遗传抑制 DNA甲基转移酶3A（DNMT 3A）和核小体重塑和脱乙酰酶（NuRD） 复杂的，作为核FAK相互作用的伙伴。重要的是，我们发现FAK抑制降低了DNMT 3A 和NuRD组分表达，这与DNA甲基化降低和DNA甲基化增加有关。 收缩基因启动子中的活性组蛋白标记（H3 K27 ac和H4 ac）。使用遗传FAK细胞质 我们发现，FAK核定位是DNMT 3A减少所必需的， NuRD复合物。此外，FAK抑制阻断ApoE-/-小鼠中晚期动脉粥样硬化病变的形成 DNMT 3A和NuRD组分表达降低。这与较厚的平滑肌有关 肌动蛋白阳性面积，表明FAK抑制增加VSMC分化和斑块稳定性 与对照相比。我们的研究结果提示，损伤或高脂血症刺激后FAK的激活可能 有助于VSMC表型转换可能通过表观遗传调控。我们假设FAK 催化抑制迫使FAK核定位并通过减少表达促进VSMC分化 DNMT 3A和NuRD的组合。目的1：阐明核FAK介导VSMC增殖的分子机制 通过DNA甲基化和组蛋白修饰的表观遗传调节的表型转换。在目标2中，我们将 研究DNMT 3A和NuRD复合物在血管损伤后VSMC去分化中的作用。在Aim中 3、研究FAK抑制剂对VSMC转分化的阻断作用及对斑块形成的促进作用 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DNMT 3A和NuRD复合物稳定性通过FAK介导的表观遗传控制进行转换。