中国汉族人群冠心病新易感基因NPR-C调控动脉粥样硬化发生和易损斑块作用及机制研究

We have performed a genome-wide association study in 293 patients with coronary artery disease (CAD) and 293 sex/age matched control persons from Shanghai, China. Meantimes, a ase-controls (2046/2144) replication study from 4 different cohorts (including Shandong, Hubei, Xian and Sichuan province) and a copy number variants (CNVs) study through a two-cohort analysis (Cohort 1: 293/293; Cohort 2: 1022/714 in Shanghai) have also been performed. Finally, 6 single nucleotide polymorphisms (SNPs) loci rs700926, rs1833529, rs2270915, rs17541471, rs3792758 and rs696831 denoted as natriuretic peptide receptor C (NPR-C) were identified associated with CAD. These associations still remained significant after adjusting for significant risk factors(age, gender, smoking, hypertension and diabetes) for CAD (rs700926: Padj = 1.2×10-6; rs1833529: Padj =1.0×10-5; rs2270915: Padj =0.9×10-5; rs17541471: Padj =2.0×10-5; rs3792758: Padj =0.026 and rs696831: Padj =0.041). Here, we report for the first time new susceptibility loci of NPR-C for CAD in Chinese Han Population. We also identified cell immunolocalization of NPR-C and its differential expression in human coronary arteries correlates with atherosclerotic lesions and normal control of autopsy. NPR-C, at the time of its discovery, as a clearance receptor of natriuretic peptide family. Recently, a wealth of evidence has modified the initial view and suggests that NPR-C appears to be involved in the pathophysiology of some cardiovascular diseases. On account of them, it is hypothesized that NPR-C was involved in atherosclerosis. To validate this hypothesis, we plan to crossbreed 20 mice that lack NPR-C (NPR-C-/-) with apolipoprotein E knockout (ApoE-/-) mice, then compare atherosclerotic lesions in NPR-C-/-/ApoE-/- mice with that in sex/age matched NPR-C+/+/ApoE-/- mice (n=20). To investigate the relationship between NPR-C gene overexpression or silencing and athersclerotic plaque vulnerability and declare NPR-C signaling in vulnerable plaque, carotid atherogenesis will be initiated in 12-week-old male ApoE-/- mice by placement of a perivascular silastic collar. The resulting plaques will be incubated transluminally with recombinant adenovirus carrying full-length human p53 to induce vulnerable plaques. Simultaneously, the recombinant adenovirus carrying mouse NPR-C (Ad-NPR-C) and SiNPR-C (Ad-SiNPR-C) will be constructed separately. In vivo, Ad-NPR-C and Ad-SiNPR-C will be respectively injected into local carotid vulnerable plaque of the each mice (n=30 each group). The other mice injected separately with Ad-GFP and saline will be served as vehicle controls. In vitro, the adenovirus will also be transfected into CNP pre-incubated vascular smooth muscle cells and endothelial cells derived from ApoE-/- mice. The radiological and histologic characters of the above experimental vulnerable plaques and its molecular mechanism will be investigated by histological and electrophysiology and molecular biology technology.

我们前期应用全基因组关联研究、case-control验证及拷贝数变异检测(Case/Control：3151/3361)发现钠尿肽C型受体（NPRC）是目前未报道的中国汉族人群冠心病新易感基因；确立了正常人和有动脉粥样硬化（AS）斑块冠状动脉NPRC的细胞定位和差异表达。拟揭示NPRC基因在AS疾病中的作用和分子机制：杂交筛选NPRC-/-/ApoE-/-双基因敲除小鼠，与同周龄/性别NPRC野生型ApoE-/-小鼠AS病变比较以明确NPRC与AS进程关系；为探讨NPRC与斑块易损性关系，将P53基因局部转染已置入硅胶环的12周龄雄性ApoE-/-小鼠颈动脉以建立易损斑块模型，构建NPRC过表达和干扰病毒载体，在体定位转染小鼠颈动脉套管局部；体外转染CNP预刺激的ApoE-/-小鼠血管平滑肌和内皮细胞，设立对照，应用病理学、电生理学和分子生物学等技术对斑块影像学特征及其分子机制进行研究。

动脉粥样硬化性心血管病是常见复杂疾病。我们前期的全基因组关联研究、case-control验证及拷贝数变异检测(Case/Control：3151/3361)发现钠尿肽C型受体（NPR-C）是目前未报道的中国汉族人群冠心病新易感基因。然而NPR-C基因与AS关系尚不明确。为验证 “NPR-C基因有抗AS、减缓AS病变进程和稳定AS易损斑块作用”假说，本课题开展了如下研究，进一步证实NPR-C的8个SNP位点与中国汉族人群冠心病易感性密切关联，同时证实中国汉族人群基因组的多样性，发现中国中部汉族人群与南、北方人群有完全不同的NPR-C关联SNP位点，其中在中国南、北方汉族人群存在关联的NPR-C阳性SNP位点在以上海为代表的中国中部人群未得到一致结论；反之，NPR-C的另2个SNP位点也仅在中国中部人群中具有阳性价值。杂交筛选出NPR-C-/-/ApoE-/-双基因敲除小鼠，与同周龄/性别NPR-C野生型ApoE-/-小鼠AS病变比较，前者AS病变加重，AS病程进展迅速，表明NPR-C基因能抑制ApoE-/-小鼠AS进程；分别构建NPR-C过表达和干扰病毒载体，将NPR-C过表达病毒经ApoE-/-小鼠尾静脉转染，结果显示NPR-C过表达能减少ApoE-/-小鼠主动脉根部AS病灶面积，减少血管平滑肌细胞和胶原纤维增生，抑制AS进程；为进一步探讨NPR-C与斑块易损性关系，12周龄雄性ApoE-/-小鼠颈动脉置入硅胶环以建立易损斑块模型，在体定位转染NPR-C过表达和干扰病毒于小鼠颈动脉套管局部，结果显示局部转染NPR-C改善斑块成分，降低斑块易损指数，抑制炎性因子表达，增加AS斑块稳定性。由于本项目申请较早，在项目批准和实施过程中陆续有CNP干预平滑肌细胞和内皮细胞的报道，并揭示了NPR-C经MAPK和 PI3-kinase信号通路抑制平滑肌细胞增殖的分子机制；同样，我们证实NPR-C活化抑制血管平滑肌细胞的增殖和移行；但为避免重复研究，我们主要探讨NPR-C活化在巨噬细胞中的作用，结果显示C-ANP4-23特异激活NPR-C能抑制巨噬细胞炎性活化和凋亡，促进巨噬细胞向M2表型转化，并依赖于Gi蛋白-AC-cAMP活化通路。总之，本课题证实了NPR-C抗AS作用及其可能的分子机制，为AS疾病治疗提供可靠靶点。该项目资助3名硕士生毕业，发表SCI论文2篇。