PTRF在动脉粥样硬化易损斑块中的作用及机制研究

Vulnerable plaque formation leads to atherosclerotic plaque rupture which is the major cause of sudden death. Therefore, how to stabilize or reverse the vulnerable plaque are the key issues that need to be solved in clinic. The vulnerable plaque is characterized by a large lipid core, thin fibrous cap and a small number of vascular smooth muscle cell (VSMC). VSMC is the sole type of cell in atherosclerotic plaques which can synthesize collagen. During the formation of inner lipid core of vulnerable plaque, VSMC has higher lipid toxicity tolerance than macrophage which leads to the long existing of lipid-laden VSMC in atherosclerotic plaques. But the role of the long existence of lipid-laden VSMC in vulnerable plaque is still unknown. .Polymerase I and transcript release factor (PTRF, also called cavin1) is a newly discovered key structural protein of caveolae. Our previous results indicated that cultured VSMC from PTRF knockout mice can resist lipid deposition and the phenotype transformation induced by high sugar plus fat, which indicated that PTRF- mediated lipid deposition had an effect on phenotype transformation of VSMC. Further study found that PTRF protein is degradated through the ubiquitin-proteasome pathway and its expression is increased in atherosclerotic plaque. In addition, PTRF is known to participate in collagen regulation, therefore we hypothesize that PTRF- mediated lipid deposition of VSMC has an impact on the vulnerable plaque. .In this project, we will first investigate the effects of PTRF mediated degradation pathway on other associated protein degradation and positioning in order to clarify the molecular mechanisms of PTRF-mediated lipid deposition of VSMC. Since VSMC phenotype transformation plays a very important role in atherosclerotic vulnerable plaque, we will explor whether PTRF has a direct or indirect effect (induced by lipid deposition) on VSMC phenotype transformation, and focus on the role of c-Src, PI3K, RhoA/ROCK-2 signaling pathways involved. Because lipid deposition leads to change of VSMC biological activity, in particular, of collagen metabolism, apoptosis and macrophage infiltration which play a very important role in the formation and rupture of vulnerable plaque. We will study the consequence and mechanism of lipid deposition of VSMC for atherosclerotic vulnerable plaque. And then we will demonstrate the role of PTRF in the formation and stability of atherosclerotic vulnerable plaque in atherosclerosis animal model from PTRF knockout mice. Finally, we will confirm the correlation of PTRF and atherosclerotic vulnerable plaque in the clinical specimens..This study can not only deepen the understanding of formation of atherosclerotic vulnerable plaque, but also provide new molecular targets for clinician to reverse or stabilize vulnerable plaque.

易损斑块破裂是导致临床急性心血管事件的关键。VSMC脂质沉积对易损斑块内脂质核形成具有重要作用，其对易损斑块的影响及机制还不清楚。我们预实验表明，聚合酶Ⅰ转录释放因子(PTRF)基因敲除的VSMC，能对抗高糖高脂诱导的脂质沉积和表型转化，提示PTRF介导脂质沉积对VSMC表型转化有影响。进一步发现，PTRF蛋白通过泛素途径降解，在AS斑块内表达增加。已知PTRF参与胶原调控，因此推测PTRF对易损斑块有影响。本项目拟：从降解途径影响相关蛋白降解与定位来阐明PTRF介导脂质沉积的分子机制；明确c-Src、PI3K、RhoA/ROCK-2信号通路在PTRF影响表型转化中的作用。从脂质沉积导致生物学功能改变入手，研究其影响AS易损斑块的分子机制；在AS模型和临床标本上证实PTRF对AS易损斑块形成及稳定具有影响。本研究可加深对AS易损斑块形成的了解，也可为临床逆转或稳定易损斑块提供新的分子靶点。

动脉粥样硬化（AS）是心血管疾病的病理基础。血管平滑肌细胞（VSMC）是动脉粥样硬化AS）斑块内唯一能够合成胶原的细胞，其功能改变在 AS 斑块演变过程中起着重要作用。聚合酶Ⅰ转录释放因子（PTRF/Cavin-1）是一种新发现的细胞质膜小凹结构蛋白，具有多种生物学功能。.我们首先在血管内膜增殖模型上证实了Cavin-1表达下降，同时伴随着蛋白酶降解途径增强。进一步使用shRNA抑制Cavin-1的表达可以促进血管内膜增生。Cavin-1对VSMC的作用与CAV-1有关，通过分别影响ERK的磷酸化和MMP-9的活性来促进VSMC的增殖和迁移，其中Cavin-1对VSMC迁移的作用强于促增殖作用，下调Cavin-1的表达，可以通过促进溶酶体降解途径来促进CAV-1的降解。这些结果首次证明Cavin-1是一个有效的防治AS形成的靶点。.接着我们发现，棕榈酸处理可以增加VSMC细胞内脂质沉积。下调Cavin-1的表达，可以抑制棕榈酸对脂质沉积的影响。进一步发现，下调VSMC上Cavin-1的表达，清道夫受体CD36和胆固醇调节元件结合蛋白(SREBP-2)的表达并无明显变化，而腺苷三磷酸结合转运蛋白G -1(ABCG-1)表达下降。说明Cavin-1对脂肪的摄入和合成影响不大，但可通过促进脂肪的转出，从而调节VSMC细胞内的脂质沉积，这对易损斑块的演变具有重要作用。.什么是Cavin-1介导导致易损斑块形成的直接原因？我们认为与胶原的代谢有关。在培养的VSMC上，下降Cavin-1的表达，能进一步促进胶原合成，说明Cavin-1能够影响胶原的合成。同时通过明胶酶谱实验，发现下降VSMC Cavin-1的表达，能够明显增加MMP-9的活性。通过以上实验证实Cavin-1可以通过影响胶原代谢对易损斑块的形成具有重要作用。进一步，我们在ApoE缺陷小鼠上，通过高脂加上血管损伤诱导易损斑块模型，发现随着易损斑块的形成，血管壁上Cavin-1 的表达下降。这个在动物模型上证实了，VSMC上Cavin-1的表达改变是导致易损伤斑块形成的一个关键因素。.综上所述，我们证实了Cavin-1在易损斑块中的作用。但是什么因素参与调节Cavin-1表达变化还不清楚。我们进一步研究发现，miRNA29a可以调控Cavin-1的表达。本研究可为临床逆转或稳定易损斑块提供新的分子靶点。

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