Nuclear FAK-mediated VSMC differentiation via epigenetic reprograming invascular diseases

项目总结

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.