血管内皮Notch1的SUMO化修饰在低氧介导的病理性血管生成中的作用和机制研究

Pathological angiogenesis contributes fundamentally to numerous malignant, inflammatory, ischaemic, infectious and immune disorders. Typically, it is modulated by local hypoxia mediated Notch and VEGF signals coordinately in vascular endothelium. Although a series of drugs targeting VEGF pathway are the most common antiangiogeneic approaches employed in clinic, it is far from the full promise of antiangiogenic therapy because of the large number of nonresponsive cases. Therefore, Notch signaling adjustment, on the other hand, can be the alternative angiogenic treatment strategy for pathological angiogenic diseases. However, current knowledge of vascular notch signaling is limited to classical molecular signaling while systematic mechanisms of pathogenesis is little known. SUMOylation is a newly discovered post-translational modification pattern, which is involved in various cellular processes. Our previous work has demonstrated the indispensable pathological role of SUMO modification in hematopoiesis and vascular networking. And in angiogenesis system, our preliminary data show a specific SUMO conjugation on Notch1 in vascular endothelial cells, which is mediated by SENP1 (major member of deSUMOylation enzyme family), and significant interference of such modification on tumor angiogenesis and hindlimb ischemia-induced angiogenesis. Thereby, we will systematically investigate the pathological function of Notch1 SUMOylation in hypoxia-mediated pathological angiogenesis by employing animal disease models and clinical samples. On the other hand, further investigation in vitro will determine the regulatory role of SUMO modification on Notch1 protein and related signalings, as well as corresponding angiogenic function of vascular endothelial cells. The accomplishment of the current project will help us to better understand the pathogenesis of angiogenesis disorder from a new angle and provide new therapeutic target and theory for medical cure of clinical vascular diseases.

病理性血管生成是导致血管异常生成疾病的主因，由组织低氧介导的血管内皮Notch与VEGF通路制衡调控。针对VEGF及受体的血管生成抑制剂在临床治疗中存在较大局限性。因此，针对内皮Notch信号的调控是完善病理性血管生成治疗的潜在策略。然而Notch在其中的分子病理机制还很缺乏。我们的前期工作发现蛋白翻译后SUMO化修饰在血液和血管生成中的重要病理功能。我们的预实验发现了血管内皮Notch1的特异性SUMO化修饰，以及去SUMO化酶SENP1介导的该修饰过程在肿瘤血管生成和后肢缺血适应性血管生成中的病理性影响。因此，我们拟在体外系统中明确血管内皮Notch1的SUMO化在低氧介导的病理性血管生成中的作用；阐明SUMO化对Notch1及其信号和血管内皮细胞血管生成功能的分子病理机制。该研究有助于我们从新的角度阐明血管异常生成疾病的发病机制，并为血管疾病的治疗提供新的靶点和理论基础。

血管是器官形成及功能维持的基础。血管生成是指从已有血管系统发育形成新生血管的过程，在此过程中血管内皮NOTCH 与VEGF 通路起到主导作用且相互制衡。我们的前期工作发现蛋白翻译后SUMO 化修饰在红细胞生成中发挥重要的功能，同时发现去SUMO 化酶--SENP1的敲除明显抑制血管发育。因此我们假设SUMO化修饰可能参与调控血管生成。研究中，我们构建SENP1血管内皮细胞（EC）组织特异性敲除小鼠（SENP1 ecKO），发现NOTCH1蛋白的SUMO化修饰在EC中明显升高，并发现新生小鼠视网膜的血管生成明显减少,明确关于SUMO化修饰可能参与调控血管生成的假设是正确的，即SUMO化修饰能够负向调控视网膜血管生成。已知NOTCH信号负向调控血管生成过程，为此我们检测了敲除或敲低SENP1蛋白对EC NOTCH信号的影响。结果证实，SENP1缺失能够增强EC中NOTCH1信号的活性，暗示SUMO化修饰调控血管生成可能是通过调控NOTCH信号实现的。为此，我们借助生物信息学工具，分析并预测NOTCH1蛋白可能会被SUMO化修饰。生化实验证实NOTCH1蛋白与成熟体NOTCH1蛋白均能够被SUMO1蛋白修饰，并且我们成功鉴定出三个主要的SUMO化修饰位点，证实血管内皮NOTCH1蛋白的SUMO 化修饰是受到SENP1介导的。重要的是，我们还发现SUMO 化修饰能够促进NOTCH1蛋白的成熟剪切，并促进成熟NOTCH1蛋白的稳定性；并且SUMO化修饰的NOTCH1蛋白与转录因子RBPJ形成更多的转录复合体，增强NOTCH1信号下游基因的表达，抑制VEGFR2蛋白的表达并抑制其信号活力，从而抑制血管生成。最后，我们构建了EC特异性诱导表达的SUMO化N1ICD小鼠,证实SUMO1-N1ICD内皮细胞特异性敲入小鼠中血管生成明显受到抑制，与SENP1 ecKO中所观察现象一致。该研究有助于我们从新的角度阐明血管异常生成疾病的发病机制，并为血管疾病的治疗提供新的靶点和理论基础。.部分研究成果发表在 《International Journal of Molecular Medicine》、《中国细胞生物学学报》，已投稿1篇正在《Developmental Cell》审稿。培养博士后1名，硕士生2名。