门脉高压中Notch信号对血管内皮细胞稳态和重塑的调控、机制和转化研究

Portal hypertension resulted from liver cirrhosis can be attributed not only to the accumulation of fibrogenic tissues which leads to intrahepatic resistance, but also to the microvessel dysfunction and structural abnormalities due to endothelial instabilities and remodeling. However the mechanism for the endothelial lesion has not been fully elucidated. Notch signaling pathway regulates vascular development. In our previous studies, we have demonstrated that Notch pathway is critical for the maintenance of general vascular homeostasis in conditional knockout mice of RBP-J, the key transcription factor mediating Notch signaling. Furthermore, we found that endothelial activation of Notch signaling resulted in widening of liver sinusoid and increased fenestration. These results suggest Notch signaling might regulate endothelial homeostasis and remodeling of hepatic microvessels. However, in portal hypertension, what could be the roles of Notch signaling and underlying mechanism in endothelial homeostasis and remodeling? To address these questions, in this project, by using gene-modified mouse models and portal hypertension models in combination with latest molecular and cellular approaches, we will elucidate the function of Notch signaling pathway in endothelial dysfunction and remodeling during portal hypertension. We will also clarify the mechanisms underlying Notch-mediated endothelial homeostasis and remodeling. Moreover, we will testify the potential of Notch signaling as an intervening target for endothelial dysfunction and remodeling in portal hypertension. These studies will provide new insights into the mechanism of endothelial dysfunction and remodeling, and provide new drug targets for portal hypertension in hepatic cirrhosis..

肝硬化门脉高压的形成不仅由于纤维堆积造成的循环阻力增加，还由于血管内皮失稳态和重塑造成的血管功能和结构异常，但分子机制不清。Notch途径在调控血管发育中具有关键作用。前期研究中我们利用Notch下游关键转录因子RBP-J剔除小鼠证实Notch信号调控全身血管稳态，同时发现诱导性阻断Notch信号引起小鼠肝血窦淤塞综合征；进而预实验表明，激活内皮Notch信号可使肝血窦变宽和窗孔增加，提示Notch信号调控肝内血管内皮稳态和重塑。但门脉高压中Notch信号对血管内皮稳态和重塑有何作用？分子机制如何？对此，本课题拟采用遗传修饰小鼠和门脉高压模型，结合细胞和分子生物学方法，阐明Notch信号在肝硬化门脉高压中对血管内皮细胞稳态和重塑的调控作用并揭示其机制，探讨通过Notch信号干预门脉高压发生的可能性。这些研究将进一步发现门脉高压中血管内皮功能失稳态和结构重塑的分子机制，为其干预提供新靶点。

内皮细胞的应激应答、失稳态与重塑在多种人类疾病中发挥重要的病理生理作用。肝血窦内皮细胞（LSECs）是研究内皮细胞稳态与功能的良好模型。文献报道和我们以往的工作证实，Notch信号通路调控内皮细胞增殖、分化和功能，参与血管发生和血管形成。本研究进一步发现，（1）在LSECs中激活Notch信号，引起LSECs分化特征的丧失，包括窗孔的减少和基底膜的形成，即毛细血管化。Notch激活造成的LSECs毛细血管化伴随LSECs的增殖减少、凋亡增加和老化的增加，其原因是Notch信号的激活下调了LSECs中eNOS的表达，同时eNOS上游的VEGFR2/3表达下降而Angpt2表达上升。LSECs毛细血管化还损害了LSECs的旁分泌功能，尤其是Wnt2a和HGF的分泌，引起肝细胞稳态的破坏，并且使得肝切除诱导的肝再生不能正常完成。（2）在LSECs中激活Notch信号后诱导肝纤维化，伴随毛细血管化的进一步加重，引起明显的LSECs向平滑肌细胞样细胞的转分化。这一转分化增加了肝组织的细胞外基质沉积，进一步破坏肝血窦的结构和功能，引起门脉压的进一步升高。用g-分泌酶或RBP-J敲除阻断Notch信号可减少LSECs的转分化。（3）在血管内皮细胞中鉴定了一组Notch信号的下游非编码RNA，包括一组miRNA和一组LncRNA。对Notch调控miR-342-5p在血管内皮细胞中的作用进行了深入的研究，发现Notch信号可以通过miR-342-5p协同VEGFR信号和TGF-b受体信号通路，调控内皮细胞的增殖、迁移和转分化，影响新生血管形成。（4）进一步证实自主设计的内皮靶向可溶性Notch配体mD1R可以有效调控LSECs，影响LSECs的稳态、重塑和功能。发现mD1R可以影响其他干细胞龛内皮细胞的功能，影响组织干细胞如造血干细胞的增殖、分化和应激反应。以上研究首次证实Notch信号在LSECs功能稳态和重塑中的作用，初步揭示了其作用机制；首次发现Notch信号通过LSECs影响肝纤维化中门脉高压，为肝纤维化的干预提供了新靶点。已发表研究论著7篇，综述1篇，待发表研究论著2篇。

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