Autophagy and VSMC migration

 描述（由申请人提供）：作为对动脉粥样硬化前因子（如氧化脂质）或治疗干预（如血管成形术、支架或旁路手术）的反应，血管平滑肌细胞（VSMC）迁移至内膜，导致内膜增生、再狭窄、移植物失效或动脉粥样硬化。了解VSMC迁移的机制一直是生物医学研究的主要焦点。我们令人兴奋的初步数据表明，在响应或线介导的颈动脉损伤，自噬相关（Atg）蛋白的表达增加与增加VSMC迁移和内膜增生。药理学和遗传学抑制自噬抑制VSMC迁移。从机制上讲，血管自噬的抑制导致氨基酸合成蛋白5（GCN 5）的积累，GCN 5是一种普遍存在的促进转录激活的组蛋白乙酰转移酶（HAT）。此外，我们发现GCN 5的积累与α-微管蛋白乙酰化增加、微管稳定和VSMC迁移抑制有关。核心假设是GCN 5的自噬依赖性降解通过抑制α-微管蛋白乙酰化促进VSMC迁移。将在动物模型和培养的细胞中使用功能获得/丧失策略来测试该假设。目的1将通过使用smMHC/eGFP转基因小鼠表征血管自噬、VSMC迁移和新生内膜增生的空间和时间动力学，确定自噬缺陷是否抑制VSMC迁移和新生内膜形成以响应于颈动脉结扎和/或血管损伤，来确定自噬在调节VSMC迁移中的作用（beclin 1 +/-），并检查增强的VSMC自噬是否促进VSMC迁移，并在Atg 7 SMC特异性转基因小鼠（Atg-TG）中响应血管损伤而加剧新生内膜增生和动脉粥样硬化。此外，自噬在调节VSMC迁移中的作用将在培养的主动脉环和VSMC中使用功能获得和丧失方法来确定。目的2是阐明自噬促进VSMC迁移的机制。通过验证自噬通过促进GCN 5降解和减少α-微管蛋白乙酰化促进VSMC迁移的假说。这项拟议的研究将表征自噬降解VSMC中GCN 5的分子机制，检查GCN 5是否乙酰化α-微管蛋白并稳定微管，并确定自噬抑制是否通过增加beclin+/-小鼠中GCN 5介导的α-微管蛋白乙酰化来抑制VSMC迁移，这些小鼠具有导丝介导的颈动脉损伤，颈动脉结扎和动脉粥样硬化，和Apoe-/-/beclin 1 +/-小鼠，用高FT饮食喂养。

 DESCRIPTION (provided by applicant): In response to pro-atherosclerotic factors such as oxidized lipids, or to therapeutic interventions such as angioplasty, stents, or bypass surgery, vascular smooth muscle cells (VSMC) migrate to the intima, resulting in intimal hyperplasia, restenosis, graft failure, or atherosclerosis. Understanding the mechanisms involved in VSMC migration has been a major focus of biomedical research. Our exciting preliminary data show that in response to or wire-mediated carotid artery injury, increased expression of autophagy-related (Atg) proteins is associated with increased VSMC migration and intimal hyperplasia. Pharmacological and genetic suppression of autophagy inhibits VSMC migration. Mechanistically, suppression of vascular autophagy leads to the accumulation of amino acid synthesis protein 5 (GCN5), a ubiquitous histone acetyltransferase (HAT) that promotes transcriptional activation. Furthermore, we found that GCN5 accumulation is associated with increased α-tubulin acetylation, microtubule stabilization, and inhibition of VSMC migration. The central hypothesis is that autophagy-dependent degradation of GCN5 promotes VSMC migration via inhibition of α-tubulin acetylation. This hypothesis will be tested using gain-/lossof-function strategies in both animal models and cultured cells. Aim 1 will determine the role of autophagy in regulating VSMC migration in response to vascular injury in vivo by characterizing the spatial and temporal dynamics of vascular autophagy, VSMC migration, and neointimal hyperplasia using smMHC/eGFP transgenic mice, determining whether autophagy deficiency inhibits VSMC migration and neointimal formation in response to carotid ligation and/or vascular injury using Beclin1 heterozygous mice (beclin1+/-), and examining whether enhanced VSMC autophagy promotes VSMC migration and exacerbates neointimal hyperplasia and atherosclerosis in response to vascular injury in Atg7 SMC-specific transgenic mice (Atg-TG). In addition, the role of autophagy in regulating VSMC migration will be determined using gain- and loss-of-function approaches in cultured aortic rings and VSMCs. Aim 2 is to delineate the mechanism by which autophagy promotes VSMC migration. By test the hypothesis that autophagy promotes VSMC migration by enhancing GCN5 degradation and reducing α-tubulin acetylation. The proposed study will characterize the molecular mechanism by which autophagy degrades GCN5 in VSMCs, examine whether GCN5 acetylates α-tubulin and stabilizes microtubules, and determine whether autophagy suppression inhibits VSMC migration by increasing GCN5-mediated acetylation of α-tubulin in beclin+/- mice of wire-mediated carotid artery injury, carotid ligation, and atherosclerosis, and Apoe-/-/beclin1+/- mice fed with a high-ft diet.