转录因子Bach1调控血管损伤后再内皮化的作用及机制研究

Endothelial repair contributes to maintain the vascular homeostasis. We have shown that the transcription factor BTB and CNC homology 1 (Bach1) inhibited the endothelial cell proliferation and migration in human umbilical vein endothelial cells (HUVECs) and suppressed angiogenesis in mice with surgically induced hind-limb ischemia (HLI). Recently, we found that Bach1 was highly expressed in neointima after femoral artery injury in mice, and Bach1 bound to SIN3A/HDAC1 complex and suppressed vascualr endothelial growth factor (VEGF) expression in HUVECs. However, the role of Bach1 in re-endothelialization after vascular injury and whether SIN3A/HDAC1 complex is involved are largely uncharacterized. In the present study, we attempt to determine whether Bach1 inhibits the transcription of VEGF by binding to the promoter of VEGF and recruiting SIN3A/HDAC1 complex to the promoter, and reducing H3K27ac in the enhancer of VEGF, and then suppressed endothelial cell proliferation and migration in HUVECs and primary mouse vascular endothelial cells. By using femoral artery injury model in tamoxifen-inducible endothelial cell-specific Bach1–/– mice, we will investigate the role and regulation mechanisms of Bach1 in the re-endothelialization after vascular injury. This study may provide the mechanisms of Bach1 in the regulation of vascular repair after vascular injury, and give the scientific basis for the treatment of vascular diseases by targeting Bach1.

血管内皮损伤后修复有助于维持血管的稳态。我们前期研究表明，转录因子Bach1抑制人血管内皮细胞增殖、迁移和小鼠下肢缺血后血管新生。近期实验发现，Bach1在小鼠股动脉损伤后新生内膜中高表达，在人血管内皮细胞上Bach1与SIN3A/HDAC1转录复合物结合，抑制VEGF的表达。但是Bach1是否影响血管损伤后再内皮化，SIN3A/HDAC1转录复合物是否参与该过程尚不清楚。本课题拟在人脐静脉内皮细胞和小鼠原代血管内皮细胞上，研究Bach1是否通过招募SIN3A/HDAC1复合物结合在VEGF基因启动子区，减少VEGF基因增强子H3K27乙酰化，进而抑制VEGF表达和内皮细胞的增殖和迁移；在内皮特异的Bach1敲除小鼠股动脉内皮剥脱模型上，研究Bach1对血管损伤后再内皮化的影响和机制。这项研究将阐明Bach1调控血管内皮损伤修复的分子机制，为以Bach1为靶点治疗血管疾病提供理论依据。

转录因子Bach1可通过抑制Wnt/β-catenin信号传导损害缺血性损伤后的血管生成；然而，Bach1抗血管生成作用的结构域仍不清楚。本研究确定Bach1的BTB结构域在缺血性血管生成中的作用。Bach1在急性心肌梗死患者的血管内皮细胞中高表达，是缺血后的早期诱导基因。在小鼠下肢缺血模型中，用编码GFP(AdGFP)、Bach1(AdBach1)或缺乏BTB结构域(AdBach1-ΔBTB)的Bach1突变体的腺病毒对小鼠缺血下肢进行处理。Bach1过表达组的血流恢复、毛细血管密度、血管内皮生长因子(VEGF)和血红素加氧酶-1 (HO-1)的表达显着降低，活性氧水平升高，但BTB结构域缺失后对血流恢复等结果则没有改变。此外，在人内皮细胞中用AdBach1过表达组中：1) Matrigel-plug测定中毛细血管密度和血红蛋白含量的显着下降；2) 增殖、迁移、管腔形成以及VEGF和HO-1在内皮细胞中的表达均显著减少。在机制研究中，我们发现Bach1直接与TCF4结合，两者的相互作用由Bach1 BTB结构域81-89区域的片段和TCF4的N端结构域介导。同时，Bach1与组蛋白去乙酰化酶1(HDAC1)共沉淀，而Bach1的BTB结构域缺失后则不与HDAC1结合。同样，HDAC1的蛋白结合域缺失后不能与Bach1蛋白结合共沉淀。总的来说，这些结果表明，Bach1的抗血管生成活性关键依赖于其BTB结构域介导的分子间的相互作用，并且该结构域可能是血管生成治疗的药物靶点。