c-Fos通过调控ABCA1表达介导肺炎衣原体感染诱导的肌源性泡沫细胞形成

Foam cells are characteristic of the atherosclerotic lesion. However, the mechanisms of VSMC-derived foam cell formation have not yet been elucidated. Few studies have reported a role of c-Fos, one of the significant transcription factors of lipid metabolism, in VSMC-derived foam cell formation, and the related mechanism of c-Fos in this process. Based on our preliminary results that c-Fos was closely associated with Chlamydia pneumoniae (C.pn) infection-induced formation of VSMC-derived foam cells, in this study, we aim to further explore the molecular mechanisms underlying VSMC-derived foam cell formation by using the human atherosclerotic plaque specimens, animal models and cell models: Firstly, the expressions and activation of c-Fos and ABCA1 in VSMC-derived foam cells induced by C.pn infection will be determined by multiple approaches including pathological techniques and molecular biology technologies; Then, the roles of c-Fos in the formation of VSMC-derived foam cells and atherosclerotic plaques will be clarified by means of gene engineering and drugs which can change the expressions of c-Fos; Furthermore, the molecular mechanisms by which c-Fos can regulate the expression of ABCA1 will be demonstrated using the methods of reporter gene, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation assay (ChIP). Thus, it will be demonstrated that c-Fos mediates C.pn infection-induced VSMC-derived foam cell formation through regulating the expression of ABCA1, which will elucidate the new mechanism by which C.pn infection promotes the initiation and progression of atherosclerosis by inducing VSMC-derived foam cell formation.

泡沫细胞是动脉粥样硬化（AS）病变的特征性病理细胞，但肌源性泡沫细胞的形成机制尚未明确。c-Fos作为调节脂代谢的重要转录因子，在肌源性泡沫细胞形成中的作用及机制鲜有报道。我们在前期研究发现c-Fos与肺炎衣原体（C.pn）感染诱导的肌源性泡沫细胞形成密切相关的基础上，拟进一步从临床标本、动物模型及细胞模型三个层面探讨肌源性泡沫细胞的形成机制：运用病理学、分子生物学等技术检测C.pn感染诱导的肌源性泡沫细胞中c-Fos和ABCA1的表达及活化；利用基因工程手段和药物改变c-Fos表达，明确c-Fos在C.pn感染诱导的肌源性泡沫细胞及AS斑块形成中的作用；使用报告基因、EMSA及ChIP实验阐明c-Fos调控ABCA1表达的分子机制。由此证实c-Fos通过调控ABCA1表达介导C.pn感染诱导的肌源性泡沫细胞形成，以期揭示C.pn感染通过诱导肌源性泡沫细胞形成促进AS发生发展的新机制。

动脉粥样硬化（AS）引发的心脑血管疾病严重威胁着人类健康。泡沫细胞是AS形成的重要病理标志，肌源性泡沫细胞是AS病变中泡沫细胞的重要来源，而其形成机制目前尚不完全清楚。新近研究显示，转录因子c-Fos可以参与调节脂质代谢，但鲜有报道其参与肌源性泡沫细胞形成。为此，我们构建高脂饮食诱导的小鼠AS模型，证实c-Fos的表达在AS病变中明显增多，而血管平滑肌细胞（VSMC）特异性c-Fos缺失后，小鼠AS病变明显减轻。基于此，我们进一步探讨c-Fos促进AS病变形成的机制：oxLDL刺激后，Western blot实验检测c-Fos蛋白表达及磷酸化水平，结果显示，oxLDL刺激可使c-Fos蛋白表达及磷酸化水平均升高；利用RNA干扰技术沉默c-Fos、使用磷酸化位点突变技术抑制c-Fos蛋白Ser32和Thr232位点磷酸化并行oxLDL刺激，胆固醇定量分析实验、Filipin荧光染色和油红O染色检测结果均显示，VSMC中脂质聚积以及泡沫细胞形成明显减少。上述结果表明，c-Fos参与oxLDL诱导的肌源性泡沫细胞的形成。随后，RT-PCR和Western blot实验检测oxLDL诱导的肌源性泡沫细胞中CD36、LOX1、SR-A的mRNA和蛋白表达，结果发现，LOX1的表达明显增多；RNAi技术沉默LOX1并行oxLDL刺激，检测VSMC中脂质聚积以及泡沫细胞形成情况，结果提示，LOX1可介导oxLDL诱导的肌源性泡沫细胞的形成，并在动物实验中得到证实；沉默c-Fos并行oxLDL刺激，RT-PCR和Western blot实验结果均显示，c-Fos表达缺失后肌源性泡沫细胞中LOX1表达明显减少。为探讨c-Fos对LOX1的调控机制，免疫荧光染色和Western blot实验检测VSMC中c-Fos蛋白的分布，结果显示，oxLDL促进VSMC中c-Fos蛋白发生明显核转位；染色质免疫共沉淀实验结果表明，oxLDL刺激后，c-Fos与LOX1启动子结合显著增强；双荧光素酶报告基因实验证实，oxLDL刺激后c-Fos对LOX1的转录调控作用也明显增强。综上，本项目证实c-Fos通过调控LOX1的转录促使oxLDL诱导肌源性泡沫细胞形成，从而促进AS病变形成，揭示了肌源性泡沫细胞形成的新机制，为开发新的AS干预和治疗药物提供重要靶点。