PARP1通过影响E2F1调控血管内皮细胞氧化应激损伤的分子机制研究

eNOS uncoupling is critically implicated in oxidative stress (OS) in ECs, and plays a causal role in the hypertension development. We previously found that DHFR was transcriptional downregulated by E2F1, which consequently mediated eNOS uncoupling and endothelial dysfunction. PARP1 is sensitive to OS in various cell types, and is involved in hypertension and endothelial dysfunction, but the mechanism is yet to be fully elucidated. Our previous results showed that 3-AB, a specific PARP1 inhibitor, was sufficient to inhibit endothelial cell injury induced by hydrogen peroxide (H2O2), as indicated by improved cytoskeleton, decreased superoxide production and elevated nitric oxide (NO) production. Interestingly, the transcription function of E2F1, which was downregulated by H2O2 and leaded to eNOS uncoupling, was significantly restored by 3-AB, suggesting that PARP1 might play a critical role in endothelial function by regulating E2F1. In this study, we will determine the potential effects of PARP1 on E2F1 and reveal the molecular mechanism associated with eNOS uncoupling. By using the spontaneously hypertensive rats (SHR) and the mice hypertension model that induced by Angiotensin II (Ang II), the protective role of PARP1 inhibitor on vascular injury as well as the mechanism based on E2F1 will be further investigated. Our findings will explain the molecular basis of eNOS uncoupling leaded by PARP1-E2F1 interaction under oxidative stress, and raise the possibility that the pathway may be utilized as a target to prevent endothelial dysfunction in cardiovasular disease.

氧化应激引起的内皮损伤与高血压的发生发展互为因果，而eNOS解偶联是内皮氧化应激的重要因素。我们前期研究发现E2F1可从转录水平调控DHFR的表达进而介导eNOS解偶联，参与了高血压的发生发展及内皮损伤。PARP1能被氧化应激激活，并与内皮氧化损伤密切相关，但其机制尚未完全明了。我们前期实验结果表明PARP1抑制剂3-AB可逆转过氧化氢诱导的内皮损伤，表现为抑制细胞骨架改变、抑制活性氧的生成、促进NO的合成等，并改善E2F1的转录活性及靶基因DHFR表达，提示PARP1可能通过调节E2F1转录活性影响eNOS解偶联进而介导内皮损伤，但其具体机制有待进一步研究。本项目我们将首先运用内皮细胞氧化损伤模型进一步研究PARP1对E2F1的影响及其调控机制，探讨其对eNOS解偶联及内皮损伤的作用，并通过自发性高血压大鼠和AngII诱导的小鼠高血压模型进行相关探讨与验证。

氧化应激引起的内皮损伤与高血压的发生发展互为因果，而eNOS解偶联是内皮氧化应激的重要因素。我们前期研究发现E2F1可从转录水平调控DHFR的表达进而介导eNOS解偶联，参与了高血压的发生发展及内皮损伤。PARP1是PARP家族的主要成员，可通过催化底物的聚ADP核糖基化参与心血管疾病的发病进程。在本项目中，通过内皮细胞氧化应激模型及高血压动物模型，我们证实了PARP1能被氧化应激激活，其与内皮氧化损伤密切相关。通过RNA干扰技术敲低PARP1或使用PARP1特异性抑制剂，可显著改善内皮损伤。进一步的研究发现，PARP1介导内皮损伤的作用与其使E2F1 聚ADP核糖基化影响E2F1转录活性进而影响eNOS解偶联有关，同时我们也探讨了E2F1发生PAR化的潜在位点。本项目重点探讨了PARP1与E2F1的互作关系及E2F1的聚ADP核糖基化修饰在内皮细胞中的作用，为防治内皮损伤相关的心血管疾病提供新的理论依据。