EZH2/Sox9在慢性肾脏病血管钙化中的作用和机制研究

Vascular calcification is an independent risk factor for cardiovascular events in the late stage of chronic kidney disease (CKD). Currently, the study on the mechanism of vascular calcification is not completely known, and there is no effective intervention for this disease. Histone methylation is a hot topic in the field of epigenetics, and its role in vascular calcification has not been reported. In our preliminary experiments, we found that EZH2 was highly expressed in calcified vascular tissues of CKD rats and in vitro calcification models, and EZH2 inhibitor GSK126 inhibited calcification of vascular smooth muscle cells (VSMC). We found that EZH2 regulates the expression of transcription factor Sox9 in calcified VSMC. We hypothesize that EZH2 promotes the vascular calcification through the transcription factor Sox9. Through molecular inhibitors, gene knockdown/knockout, and gene overexpression, we will explore the role of EZH2 in CKD associated vascular calcification in the in vitro cell and vascular rings culture model and in vivo 5/6 nephrectomized (ablation/infarction) rat calcification model. Moreover, with epigenetic methods such as Chip-seq and ChIP, we will study how EZH2 promotes the vascular calcification through Sox9. Therefore, we can understand the pathogenesis of CKD associated vascular calcification and provide theoretical and experimental basis for pharmaceutical intervention.

血管钙化是慢性肾脏病(CKD)患者晚期心血管事件死亡的独立危险因素，其机制尚不清楚并缺有效干预手段。甲基化转移酶EZH2介导的组蛋白甲基化是表观遗传学研究领域的热点，其在血管钙化中的作用尚未有报道。我们前期发现EZH2在CKD大鼠血管钙化组织及体外细胞钙化模型中高表达，靶向EZH2抑制剂GSK126能够抑制血管平滑肌细胞(VSMC)钙化，同时发现EZH2调控钙化VSMC中转录因子Sox9表达。为此本课题提出“EZH2通过转录因子Sox9促进血管钙化的”机制假说。拟利用VSMC、血管环体外培养及5/6肾切除大鼠血管钙化模型，通过小分子抑制剂、基因敲除/敲低、基因过表达等实验技术，探讨EZH2在CKD血管钙化中的作用，并利用ChIP-seq及ChIP等表观遗传研究方法，研究EZH2表观调控钙化转录因子Sox9的作用机制。为揭示CKD血管钙化发病机制，寻找新的药物干预靶点提供理论基础和实验依据。

血管钙化作为慢性肾脏病常见的并发症，导致心血管病变高发及死亡率增加。EZH2是参与多种肾脏疾病的关键表观遗传调控因子，在多种肾脏病动物模型中表达升高，目前关于EZH2是否参与CKD血管钙化调控尚未报道。本研究探讨EZH2在慢性肾衰血管钙化中的作用及机制。首先，我们通过构建高磷合并腺嘌呤饮食CKD血管钙化动物模型，高磷诱导大鼠胸主动脉原代平滑肌细胞及体外大鼠主动脉血管环培养等模拟慢性肾脏病血管钙化状态，采用钙含量测定、茜素红染色、免疫组化、Western blot、实时定量PCR等方法建立血管钙化检测平台，观察血管钙化中EZH2表达量的变化。结果显示：EZH2在慢性肾衰血管钙化患者桡动脉组织，原代RVSMCs，血管环钙化及慢性肾衰小鼠血管钙化过程中表达上调。其次，我们研究了EZH2对慢性肾衰血管钙化的影响。体内及体外实验结果表明，抑制EZH2能够减轻RVSMCs细胞钙化，缓解慢性肾衰小鼠主动脉钙化。同时，我们探索了EZH2促进肾纤维化的分子机制，通过RNA-Seq筛选发现糖异生限速酶PCK1为EZH2加速CKD肾纤维化的下游靶基因，激活PCK1提高肾脏糖异生水平能够缓解EZH2诱导的CKD肾纤维化。本项目为慢性肾脏病血管并发症的机制研究提供了新的思路，提示EZH2可能为治疗慢性肾衰血管钙化的潜在靶点。