MiRNA-223调控血管内皮活化在动脉粥样硬化发生中的作用及机制研究

The vascular endothelial cell phenotype transition from quiescence to activation is closely associated with angiogenesis and inflammation reaction. Identify and study new genes regulating the endothelial phenotype switch are of importance in discovering novel therapeutic targets for cardiovascular diseases. Applicant reported miR-223 play a crucial role in maintaining endothelial quiescent property and regulating growth factor induced angiogenesis (Circ Res 2013 cover story). Our preliminary data demonstrated endothelial miR-223 was reduced by Disturbed Shear.Stress (DDS)and deletion of miR-223 would further accelerate ApoE knockout induced arthrosclerosis plug formation in a carotid artery partial ligation model. However, the mechanism of miR-223 medicated pro-quiescence, anti-inflammation as well as clinical significance remains largely unknown. Current project is planning to analyze miR-223 expression changes in response to DDS and inflammation stimulation in levels of molecule, cell cultures and animal models, and the corresponding mechanism will be disclosed by a miR-223 promoter reporter assay. By setting up carotid artery partial ligation model and high fat diet feeding model, ApoE-/-miR-223-/y mice were used to verify the importance of miR-223 during arthrosclerosis development. We will verified miR-223 targets with aid of bio-informatics approach and functional assays, and disclose the importance of miR-223 and its targets during arthrosclerosis development as well as the corresponding molecular mechanism. With samples analysis from arthrosclerosis patients, we will demonstrate the clinical importance of miR-223. This project will provide theoretical proofs and new thought supporting miR-223 as a novel target in atherosclerosis therapy.

血管内皮细胞由静息到活化的表型转变与血管新生和炎症反应密切相关。前期研究证实miR-223明显维持内皮静息表型并抑制生长因子诱导血管新生(Circ Res，2013封面)。预实验发现扰动剪切力(DSS)下调内皮miR-223表达且miR-223缺失加速ApoE敲除小鼠颈动脉粥样硬化斑块形成，但其促内皮静息、抗炎机制和临床相关性目前尚不清楚。本课题拟在分子、细胞和动物模型水平，分析miR-223在DSS和炎症刺激诱发的内皮活化中的表达变化并利用miR-223启动子报告基因揭示调控机制；建立小鼠颈动脉部分结扎和高脂诱导模型，利用ApoE-/- miR-223-/y敲除小鼠，明确其在动脉粥样硬化发生中的重要作用；利用生物信息分析和验证实验，明确靶点基因，阐明miR-223调控动脉粥样硬化发生发的分子机制，并通过临床样本分析明确临床意义。本项目将为动脉粥样硬化靶向治疗新靶点筛选提理论依据和新思路。

MiRNA-223的表达异常参与动脉粥样硬化的病理发生，然而其起效细胞类型及作用机制尚未阐明。本项目通过使用系列实验动物模型获得miRNA-223细胞特异性的调控动脉粥样硬化、内皮血管新生,胆固醇稳态调节等方面的直接体内证据；在分子水平挖掘miRNA223新颖靶点基因及其对内皮细胞生物学活性作用和机制探究。在研过程中开拓了以器官互作为切入点的动脉粥样硬化研究思路并加以实验证实，为解决近年来针对miRNA-223通过调节胆固醇代谢影响机体固醇稳态调节及血管病变相关性的科学假设提供直接实验证据。. 通过miRNA-223条件敲除小鼠品系，进一步建立细胞特异性敲除小鼠品系，包括：全身和内皮，肝脏、血小板、髓系等细胞类型的特异性敲除小鼠品系以及ApoE-/- miRNA223 KO; ApoE-/-, 223CKO双敲小鼠品系；发现APOE-/-miRNA-223ko较APOE-/-小鼠出现更为严重的动脉粥样硬化表型及胆囊固醇型结石；肝脏细胞特异性敲除和敲低miRNA-223表达则会加重胆结石表型，但对血浆中胆固醇无显著影响，该表型也已归就为miRNA-223对SR-BI,ABCG5,ABCG8的靶向调控作用导致过度的胆固醇由肝细胞向胆汁分泌。反之，肝细胞特异性过表达miRNA-223则可有效抑制胆固醇在胆囊中的积累减轻结石进展。与此同时，AAVcre注射APOE-/- miRNA-223cko小鼠实现的肝脏特异性敲除miRNA-223,或AAV-U6-miRNA-223介导的APOE-/-小鼠肝细胞特异性过表达miRNA-223,均对小鼠血浆脂质成份和动脉粥样硬化表型均没有影响，内皮特异性敲除miRNA-223后表现出加速缺血性股动脉新生和重构，并鉴定出Septin2，MEG3,MALAT1为mirna-223新颖靶点,并对上述基因对内皮生物学功能、机制进行初步挖掘。髓系敲除miRNA-223，显著改变骨髓前体细胞和外周血白细胞的比例，加速饮食诱发的肝脏纤维化，加重血管紧张素II诱导的心肌肥大；血小板特异性敲除miRNA-223后，则可促进颈总损伤造成的血栓发生并伴有显著减弱的血小板、白细胞互作事件。.以该项目为依托，建立以miRNA223为研究对象的细胞特异性为切入点的多角度的针对动脉粥硬化等心血管相关疾病的研究体系，为后续深入探究和临床转化打下坚实基础。