内皮间质转化参与泡沫细胞旁分泌因子CCL20促动脉粥样硬化的机制研究

Endothelial barrier disruption and monocytes infiltrating into subendothelial are the key factors for atherogenesis and its development. Atherosclerosis (AS) as the vascular imflammatory disease, foam cells play a pivotal role in its development and the maintenance of local vessel nonresolving inflammation. Endothelial cell will lose the endothelial phenotypes and be replaced by interstitial cells’ phenotypes, which is called as endothelial-mesenchymal transition (EndMT) when stimulated by inflammation or other stimulation. EndMT can result in endothelial barrier disruption, and be beneficial for monocytes infiltrating into subendothelium. We have found that the supernatant from cultured foam cells could induce EndMT in endothelial cells, and EndMT could also found in the surface of atherosclerotic plaque in high fat-fed Ldlr-/- mice. In addition, the EndMT was related to the development of AS. We further performed the gene chip analysis of inflammatory factors in foam cells and atherosclerotic plaque. It was found that many inflammatory factors including chemokine CCL20 had very high expression, and inhibiting CCL20 could attenuate the EndMT induced by foam cell supernatant. So we speculated that foam cell could promote EndMT and subsequent endothelial barrier dysfunction, and promote AS development by producing paracrine factor CCL20. Accordingly, the present project aimed to determine the relation among EndMT, CCL20, and AS and the possible mechanisms in Ldlr-/- AS mice induced by high-fat diet, which would help to further explore the new mechanism of AS and search new therapeutic target point for the prevention and treatment of AS.

内皮屏障破坏及单核细胞内皮下浸润是动脉粥样硬化（AS）发生发展的关键。AS是血管炎症性疾病，泡沫细胞是维持血管局部非可控炎症并促AS进展的关键细胞。内皮细胞在炎症等因素作用下内皮表型消失，代以间质细胞表型，发生内皮间质转化（EndMT）而使内皮屏障受损。我们发现泡沫细胞上清可诱导内皮细胞发生间质转化，高脂喂养的Ldlr-/-小鼠AS斑块表面内皮亦存在EndMT，并与AS进程相关。通过泡沫细胞和AS斑块的炎症因子基因芯片共分析发现包括趋化因子CCL20在内的多种炎症相关因子高表达，抑制CCL20可减轻泡沫细胞上清诱导的EndMT。故推测泡沫细胞通过旁分泌CCL20作用于内皮趋化因子受体CCR6使其发生EndMT而致内皮屏障受损，促AS进展。本项目拟以Ldlr-/-小鼠高脂饮食致AS模型观察EndMT、CCL20与AS的关系，并探讨其机制，为深入认识AS进展提供新思路，并为AS防治提供新靶点。

近期，内皮间质转化（EndMT）被证实在动脉粥样硬化的发生发展中起重要作用，但其分子机制尚不清楚。在本研究中，通过扫描电镜直接观察到人主动脉粥样硬化斑块表面内皮的正常结构被破坏，可见局部缺损，有血小板、白细胞等粘附，暴露出条束状、排列紊乱的纤维胶原组织，免疫组织荧光提示该处内皮发生EndMT。在进一步研究中，我们发现M1型巨噬细胞来源的泡沫细胞（M1-FC）上清可以诱导HAECs发生EndMT，而M2型巨噬细胞来源的泡沫细胞（M2-FC）上清无明显诱导作用。接着，我们采用蛋白芯片和ELISA鉴定出M1-FC上清中多种细胞因子水平明显升高，其中C-C基序趋化因子配体4（CCL-4）可诱导EndMT发生，并增加内皮通透性和单核细胞粘附作用。此外，CCL-4中和抗体完全消除M1-FC上清诱导EndMT的作用。同时，CCL-4可激活其受体C-C基序趋化因子受体5（CCR-5），并且上调转化生长因子-β（TGF-β）的表达。进一步的研究发现，CCR-5拮抗剂和shRNA介导的TGF-β敲低可以逆转CCL-4诱导的EndMT。综上，本研究结果发现，M1-FCs通过上调CCL-4表达诱导EndMT，并增加内皮通透性和单核细胞粘附作用，这些结果对于阐明EndMT在动脉粥样硬化发生发展中的重要作用具有指导意义。