kallistatin 抗动脉粥样硬化的作用及其机制研究

Kallistatin (KS), a serine proteinase inhibitor, was downregulated expression by oxidative stress. However, no result has demonstrated that KS has anti-atherosclerotic effects. In our preliminary experiments, the contents of Kallistatin in coronary heart disease patients are statistically significant negative correlation the severity of coronary artery stenosis. Adenovirus-mediated overexpression of KS (Ad.KS) inhibits shear-stress induced carotid plaque formation in apoe-/- mice. In this experiment, we will generate the mice model with a deletion of KS in the whole-body (KSKO) and overexpression KS on an atherosclerotic prone background (apolipoprotein E-/-) and investigate the effects of KS deficiency or overexpression on atherosclerosis (KSKOapoE-/-, Ad.KSapoE-/-). Partial ligation of the left internal and external carotid arteries was performed in 12-week-old KSKOapoE-/-, Ad.KSapoE-/- and apoE-/- mice on a high fat diet, the carotid artery plaque volumes will be quantified by microMRI and histology for correlation analysis. Inflammatory cell subtypes are studied by immunohistochemistry. HUVECs and macrophage will be studied using adenovirus transfection, RNA interference, real time PCR, Western blotting, and signal transduction inhibitors and agonists. We try to clarify the novel mechanism of KS inhibit atherosclerotic plaque formation. KS decreases cellular levels of ROS through activating AKT/SIRT1/eNOS pathway, antioxidant-encoding genes Mn-SOD and catalase and mitochondria and peroxisome function. KS induces arginase1 expression in macrophages, enriches macrophages in markers of the M2, anti-inflammatory. Identification of the exact mechanisms of KS inhibition atherosclerosis may have important implications on the development of novel molecular therapies for the prevention of coronary heart disease.

Kallistatin(KS)是一种丝氨酸蛋白酶抑制剂，高氧化应激(ROS)抑制内皮细胞KS表达。我们首次发现冠心病血浆KS水平显著下降，与动脉粥样硬化（AS）严重程度正相关，KS腺病毒（Ad.KS）转染抑制鼠颈动脉AS形成。我们拟构建KS全身敲除小鼠与apoE-/-杂交，KS高表达apoE-/-鼠，建立颈动脉AS模型，检测斑块体积，免疫组化检测炎症细胞亚型。采用Ad.KS或RNA干扰研究KS对炎症因子诱导的内皮及巨噬细胞的作用，激活或阻断信号通路的调控单元以验证相关通路。证明KS是维持血管功能的内源蛋白，高表达KS抑制AS，KS敲除促进AS形成的假说。阐明KS通过 AKT/沉默信息调节因子1/eNOS通路, 活化抗ROS酶，促进线粒体、过氧化酶体功能，抑制ROS。KS活化精氨酸酶1启动子促其表达，促进M2型巨噬细胞极化，抑制炎症反应。KS抗AS新机制的揭示，为治疗AS提供新靶标。

本研究首先在临床研究中发现在冠心病患者血清中kallistatin的水平明显降低，与冠心病的严重程度及氧化应激呈负相关。接下来通过尾静脉注射含有人类kallistatin基因的腺病毒载体在颈动脉结扎的ApoE-/-小鼠中发现，kallistatin 基因转染后抑制斑块形成和M1炎症细胞极化。进一步通过构建serpina3c和Apoe双基因敲除小鼠然后高质饮食喂养12周建立小鼠动脉粥样硬化模型。动物实验结果表明kallistatin/serpina3c基因敲除后促进动脉粥样硬化斑块形成，促进主动脉斑块中的血管平滑肌细胞（VSMC）增值和巨噬细胞浸润；体外实验表明serpina3c抑制VSMC增值，并且利用蛋白建模和免疫共沉淀的方法证实serpina3c与凝血酶相互结合并抑制其活性。分子机制研究方面发现serpina3c通过抑制凝血酶/PAR-1系统导致细胞内介导的VSMC增殖中的ERK1/2和JNK信号通路抑制从而起到保护作用。该研究结果强调了serpina3c作为一种新型凝血酶抑制剂在动脉粥样硬化发展中通过抑制VSMC增值起到保护作用。除此以外，该研究意外发现serpina3c基因敲除后小鼠脂肪组织重量下降、脂肪细胞减小，肝脏异位脂肪沉积增加。并且脂肪组织PPARγ、CEBPβ、vaspin、leptin的mRNA表达下降。说明脂肪组织分泌的serpina3c对脂肪分化具有重要作用。本项目通过临床试验和基础实验证实了丝氨酸蛋白酶抑制剂kallistatin/serpina3c在动脉粥样硬化疾病发展中的作用，并进一步说明相关机制，对理解动脉粥样硬化的发病机制提供新思路，为防止动脉粥样硬化提供新的治疗靶标。其次，来源于脂肪组织的serpina3c对脂肪分化具有重要调控作用值得我们重点关注，为我们进一步研究脂肪来源的serpina3c在AS中的作用提供新的方向。