Autophagy and VSMC migration

 描述(申请人提供)：为了应对氧化脂质等促动脉粥样硬化因素，或血管成形术、支架或搭桥手术等治疗干预措施，血管平滑肌细胞(VSMC)迁移至内膜，导致内膜增生、再狭窄、移植物失败或动脉粥样硬化。了解VSMC迁移的机制一直是生物医学研究的重点。我们令人兴奋的初步数据表明，在对颈动脉损伤的反应中，自噬相关(ATG)蛋白的表达增加与VSMC迁移和内膜增生有关。自噬的药物和遗传抑制抑制了VSMC的迁移。从机制上讲，抑制血管自噬导致氨基酸合成蛋白5(GCN5)的积累，GCN5是一种普遍存在的组蛋白乙酰转移酶(HAT)，可促进转录激活。此外，我们发现GCN5的积累与α-微管蛋白乙酰化的增加、微管的稳定和抑制血管平滑肌细胞的迁移有关。中心假设是依赖自噬的GCN5降解通过抑制α-微管蛋白乙酰化来促进血管平滑肌细胞的迁移。这一假设将在动物模型和培养细胞中使用功能增减策略进行验证。目的1通过使用smMHC/EGFP转基因小鼠研究血管自噬、VSMC迁移和新生内膜增生的时空动态，利用Beclin1杂合子小鼠(Beclin1+/-)确定自噬缺陷是否抑制颈动脉结扎和/或血管损伤反应中VSMC的迁移和新生内膜形成，以及在ATG7特异性转基因小鼠(ATGTG)中检测增强的VSMC自噬是否促进VSMC迁移并加剧血管损伤后新生内膜增生和动脉粥样硬化，以确定自噬在血管损伤反应中调节VSMC迁移的作用。此外，在培养的主动脉环和VSMC中，自噬在调节VSMC迁移中的作用将通过获得和丧失功能的方法来确定。目的2描述自噬促进VSMC迁移的机制。通过验证自噬通过促进GCN5降解和减少α-微管蛋白乙酰化促进血管平滑肌细胞迁移的假说。这项拟议的研究将描述自噬降解VSMC中GCN5的分子机制，检测GCN5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GCN5介导的α-微管蛋白的乙酰化来抑制血管平滑肌细胞的迁移，在电线介导的颈动脉损伤、颈动脉结扎和动脉粥样硬化的BECLIN+/-小鼠和高脂饮食的APOE-/-/BELIN1+/-小鼠中。

 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.