ADMA关键代谢酶基因多态性与辛伐他汀心血管保护作用个体差异及机制研究

Statins show cardiovascular and cerebrovascular protective effects through mechanisms including lipid-lowering, anti-atherosclerosis, and vascular endothelial protection. The endogenous NO synthase inhibitor asymmetric dimethylarginine (ADMA) is involved in the pathophysiology of cardiovascular diseases through inhibiting NO production. Plasma ADMA level can affect statins responses and is also regulated by statins. Based on our previous findings that functional polymorphisms genes coding DDAH1 and AGXT2, two genes coding enzymes play critical role in ADMA metabolism, are associated with risks for and prognosis of cardiovascualr and cerebrovascular diseases, we assumed that the polymorphisms in these genes may affect the cardiovascular protective effects of simvastatin. By strictly designed clinical trial and prospective epidemiological study, we would firstly make clear whether functional polymorphisms in DDAH1 and AGXT2 affect the cardiovascular and cerebrovascular protectve effects of simvastatin as well as its effects in improving the prognosis of ischemic stroke due to atherosclerosis. And then,the mechanisms by which polymorphisms in the two genes affecting the cardiovascular protective effective of simvastatin would be investigated in primarily cultured HUVEC or HUVECs cell line. The study aims to identify new genetic markers that can be used to predict simvastatin response, and provide basis for personalized therapy for simvastatin in clinic.

他汀类药物通过调脂、抗动脉粥样硬化、血管内皮保护等多种机制发挥心脑血管保护作用，其效应受遗传因素影响。内源性NO合酶抑制物非对称二甲基精氨酸（ADMA）通过抑制NO生成，参与心血管疾病发病机制，影响药物反应性，并受他汀类药物调节。本项目在前期研究发现ADMA关键代谢酶DDAH1和AGXT2编码基因的功能性遗传多态影响心脑血管疾病易感性或预后的基础上，提出DDAH1和AGXT2基因多态性可能影响辛伐他汀的心脑血管保护效应。项目拟通过严格设计的临床试验和前瞻性流行病学调查，查明DDAH1和AGXT2功能性遗传变异对辛伐他汀心脑血管保护作用及其改善动脉粥样硬化性脑梗死预后的影响基础上，应用原代培养的人脐静脉内皮细胞（HUVEC）和内皮细胞株，研究两个基因的遗传变异影响辛伐他汀心脑血管保护作用的分子机制；旨在发现影响辛伐他汀心血管保护效应新的遗传标记，为个体化应用辛伐他汀、提高其临床疗效奠定基础。

对452例PCI术后应用阿托伐他汀/瑞舒伐他汀治疗的冠心病（CAD）患者ADMA代谢酶基因多态性与术后1年支架内再狭窄（ISR）的发生风险进行研究，发现DDAH1 rs233112和rs233113多态性可影响阿托伐他汀/瑞舒伐他汀对ISR的预防作用；基于593例CAD患者和463位健康对照样本的病例-对照研究发现，携带rs233113 T等位基因有降低CAD发病风险的趋势（P=0.072）；在1258例脑梗死（IS）患者和1103例正常对照人群中研究了AGXT2 rs37369、DDAH1 rs233113和rs233112多态性与IS易感性的关联关系，发现 rs233113 T等位和rs233112 G等位可以降低IS的发病风险，尤其是在伴有糖尿和高血压的患者，且DDAH1 rs233113 A和rs233112 A等位间存在联合作用；AGXT2 rs37369多态不影响IS的易感性，AGXT2 rs37369、DDAH1 rs233113和rs233112与IS的预后无显著关联。报告基因实验分析发现，DDAH1同时为miR-21 和miR-455-5p 的靶基因，但两个miRNA与DDAH1不同转录本的结合能力存在差异，miR-21和miR-455-5p均可靶向DDAH1 3’UTR而抑制DDAH1的表达；而DDAH1-V2/3转录本可能发挥miRNA海绵作用。颈动脉粥样硬化（AS）斑块剥离术患者AS斑块组织中DDAH1三个转录本mRNA的表达均显著降低（P分别为0.009，0.003和0.026），而miR-21和miR-455-5p的表达均显著升高（P分别为0.001和0.029）；与正常血管壁组织相比，AS斑块中miR-21和miR-455-5p与DDAH1 3个转录本的表达相关性发生改变。不同浓度的TNF-α可显著上调HUVEC-C中miR-455-5p 的表达，而阿托伐他汀可逆转 TNF-α对DDAH1 mRNA表达下调的作用；阿托伐他汀还可逆转ADMA所致的细胞迁移增加和血管新生抑制，其机制与阿托伐他汀导致HUVEC-C细胞发生细胞周期阻滞在G0/G1期、逆转ADMA所致的p21、p27表达的下调有关。基于原代培养的人脐静脉内皮细胞发现，DDAH1 rs233113T和rs233112G等位可降低DDAH1三个转录本mRNA半衰期而发挥功能。