Rationale: The highly conserved NOTCH (neurogenic locus notch homolog protein) signaling pathway functions as a key cell–cell interaction mechanism controlling cell fate and tissue patterning, whereas its dysregulation is implicated in a variety of developmental disorders and cancers. The pivotal role of endothelial NOTCH in regulation of angiogenesis is widely appreciated; however, little is known about what controls its signal transduction. Our previous study indicated the potential role of post-translational SUMO (small ubiquitin-like modifier) modification (SUMOylation) in vascular disorders.

Objective: The aim of this study was to investigate the role of SUMOylation in endothelial NOTCH signaling and angiogenesis.

Methods and Results: Endothelial SENP1 (sentrin-specific protease 1) deletion, in newly generated endothelial SENP1 (the major protease of the SUMO system)–deficient mice, significantly delayed retinal vascularization by maintaining prolonged NOTCH1 signaling, as confirmed in cultured endothelial cells. An in vitro SUMOylation assay and immunoprecipitation revealed that when SENP1 associated with N1ICD (NOTCH1 intracellular domain), it functions as a deSUMOylase of N1ICD SUMOylation on conserved lysines. Immunoblot and immunoprecipitation analyses and dual-luciferase assays of natural and SUMO-conjugated/nonconjugated NOTCH1 forms demonstrated that SUMO conjugation facilitated NOTCH1 cleavage. This released N1ICD from the membrane and stabilized it for translocation to the nucleus where it functions as a cotranscriptional factor. Functionally, SENP1-mediated NOTCH1 deSUMOylation was required for NOTCH signal activation in response to DLL4 (Delta-like 4) stimulation. This in turn suppressed VEGF (vascular endothelial growth factor) receptor signaling and angiogenesis, as evidenced by immunoblotted signaling molecules and in vitro angiogenesis assays.

Conclusions: These results establish reversible NOTCH1 SUMOylation as a regulatory mechanism in coordinating endothelial angiogenic signaling; SENP1 acts as a critical intrinsic mediator of this process. These findings may apply to NOTCH-regulated biological events in nonvascular tissues and provide a novel therapeutic strategy for vascular diseases and tumors.

基本原理：高度保守的NOTCH（神经源性基因座缺口同源蛋白）信号传导途径是控制细胞命运和组织模式的关键细胞间相互作用机制，而其失调与多种发育障碍和癌症有关。内皮NOTCH在调节血管生成中的关键作用已广为人知。然而，对于控制其信号转导的知之甚少。我们先前的研究表明翻译后SUMO（小的泛素样修饰剂）修饰（SUMOylation）在血管疾病中的潜在作用。

目的：本研究的目的是研究SUMOylation在内皮NOTCH信号传导和血管生成中的作用。

方法和结果：在新产生的内皮SENP1中删除了内皮SENP1（Sentrin特异性蛋白酶1）（SUMO系统的主要蛋白酶）缺陷型小鼠，通过维持延长的NOTCH1信号传导，大大延迟了视网膜血管形成，这在培养的内皮细胞中得到了证实。体外SUMOylation分析和免疫沉淀显示，当SENP1与N1ICD（NOTCH1细胞内结构域）相关时，它充当保守赖氨酸上N1ICD SUMOylation的去SUMO化酶。天然和SUMO共轭/非共轭NOTCH1形式的免疫印迹和免疫沉淀分析以及双荧光素酶测定法表明，SUMO共轭促进了NOTCH1的裂解。这将N1ICD从膜中释放出来并使其稳定，以便易位至细胞核，在细胞核中它起共转录因子的作用。在功能上 SENP1介导的NOTCH1去SUMOylation是NOTCH信号激活响应DLL4（Delta-like 4）刺激所必需的。继而抑制了VEGF（血管内皮生长因子）受体的信号传导和血管生成，如免疫印迹信号分子和体外血管生成测定所证明的那样。

结论：这些结果建立了可逆的NOTCH1 SUMOylation作为协调内皮血管生成信号的调节机制。SENP1充当了此过程的关键内在中介。这些发现可能适用于非血管组织中NOTCH调节的生物学事件，并为血管疾病和肿瘤提供了一种新颖的治疗策略。