接头蛋白GAB1通过SAPKs信号通路调节血管平滑肌细胞自噬参与动脉粥样硬化的机制研究

Atherosclerosis is the major cause of myocardial infarction, stroke and peripheral vascular disease. The balance disorder between cell proliferation and death caused by excessive activation of autophagy in vascular smooth muscle cells (VSMCs) plays an important role in the generation of atherosclerotic plaque. However, the detailed mechanisms remain unknown. Our preliminary data have revealed that the GAB1, a docking protein functioning as a downstream molecule of receptor tyrosine kinase was significantly downregulated in VSMCs obtained from arteriosclerosis obliterans patients. Moreover, we found the expression level of GAB1 within VSMCs is negatively associated with the extent of host autophagy. Signaling transduction study found that activation of p38 and JNK, two major downstream of GAB1, were involved in the regulation of autophagic death of VSMCs. Therefore, we hypothesize that dysregulation of signaling transduction in VSMC caused by loss of GAB1 is the major cause of the enhancement of VSMC autophagic death, and excessive VSMC death contributes to the onset of atherosclerosis. In order to unravel this hypothesis, we propose to use VSMC (shGAB1) stable cell line, VSMC-specific GAB1 knockout mice and patient samples to study the mechanism by which GAB1 regulates autophagy machinery of VSMCs via a series of cellular biology and molecular biology methods. Furthermore, the project will evaluate the functional role of VSMC autophagy in atherosclerosis, which provides evidence for developing potential drug targets.

动脉粥样硬化是导致心肌梗死、脑卒中和周围血管疾病的主要原因。血管平滑肌细胞 （Vascular smooth muscle cell, VSMC）自噬过度激活导致的增殖-死亡失衡在动脉硬化形成中发挥了重要作用，但具体机制目前尚未明确。我们前期研究发现下肢动脉硬化闭塞症患者VSMC中，接头蛋白GAB1 (酪氨酸激酶下游信号分子) 表达缺失与其自噬过度激活密切相关，GAB1介导的下游信号分子p38和JNK参与了该细胞自噬性死亡的调控。由此我们推论GAB1缺失导致的下游信号通路失调是VSMC 自噬性死亡增加的主要病因，并进一步促进了动脉粥样硬化的发生发展。为了验证该假设，本项目拟通过建立VSMC-GAB1特异敲除细胞系和动脉GAB1特异性敲除小鼠模型，在细胞、动物和临床上，通过细胞生物学和分子生物学手段，阐明GAB1调节VSMC自噬的机制，并验证VSMC自噬在动脉粥样硬化中所发挥的重要作用，为新型靶向药物的开发提供理论依据。

下肢动脉硬化闭塞症是一种常见的因动脉粥样硬化导致的慢性动脉闭塞性疾病。在病理生理上，动脉粥样硬化是一种慢性的血管壁炎症性疾病，由血管内皮细胞、巨噬细胞和血管平滑肌细胞先后发生增殖与死亡失衡而导致动脉粥样硬化的发生。随着疾病的进展，上述细胞在疾病的不同阶段可发生不同程度的自噬、凋亡或坏死，导致斑块形成、血管狭甚至闭塞。自噬作为一种新型的细胞死亡方式，也被发现在动脉粥样硬化中扮演着重要角色。因此，自噬调节缺陷的机制研究也成为了动脉病领域的研究热点，被广泛关注。在本研究中，我们发现作为细胞内信号通路调节蛋白的接头蛋白1（GAB1）的蛋白表达量在动脉粥样硬化病变血管中发生了显著的下调。GAB1缺失直接导致了细胞内信号通路的调节紊乱，表现为Akt通路的失活与ERK信号通路、JNK信号通路和p38信号通路的异常激活。四条信号通路共同参与了宿主血管内皮细胞自噬的调控。GAB1的缺失直接导致了自噬接头蛋白p62的表达下降，和自噬标志物LC3II的表达上升。在功能学研究中，我们也发现GAB1缺失所致的血管内皮细胞自噬性死亡抑制了细胞的增殖与迁移。综上所述：我们的研究首先揭示了GAB1在动脉粥样硬化病变中通过调节血管内皮细胞自噬参与疾病发生发展与转归的作用机制。基于GAB1的靶向药物开发有望成为调节血管内皮细胞功能的一个新方向。