Notch通路在成年心肌微血管内皮向间质细胞转化的作用及其机理研究

Endothelial-to-mesenchymal cell transition (EndMT) is a process in which endothelial cells lose their cell-type specific characteristics and gain a mesenchymal cell phenotype. EndMT involves in a variety of pathological conditions including adult cardiac fibrosis. The Notch signaling pathway is crucial in the regulation of EndMT during embryonic cardiac development. However, its roles in adult vascular system have not been fully studied due to the embryonic lethal phenotype of knockout or constitutively activation of Notch1 gene. Previously, we combined the tetracycline-off and Cre/loxP systems to gain temporal and spatial control of gene expression. In our system, Notch1 can be specifically deleted or constitutively activated in endothelial cells postnatally in the mice. In addition, we found Notch1 downregulates ERG transcription factor in endothelial cells. Thus, we hypothesize that Notch signaling mediates adult cardiac EndMT through regulation of Snail, Slug and ERG transcriptional factors. The proposed project will use laser-capture microdissection and small-cell-number ChIP to collect and analyze the endothelial cells from human cardiac tissue and adult hearts from Notch1 deletion or constitutively activation mice. The cardiac endothelial cells from these adult mice will be separated for in vitro EndMT analyses. These Notch1 genetic modified mice will also be examined by cardiac fibrosis models. This study should establish the role of Notch signaling in EndMT of adult cardiac microvasculature, and provide essential information for developing new therapeutic approaches for EndMT-related pathological conditions such as cardiac fibrosis.

血管内皮细胞向间质细胞化(endothelial-to-mesenchymal transition, EndMT)是成年人心血管纤维化的一个重要机制。Notch通路可以介导胚胎期的EndMT，但由于动物模型的胚胎致死性，在成年心血管EndMT中的作用尚不明确。申请人前期结合Tet-off和Cre/loxP系统，成功的在成年小鼠内皮细胞中实现了Notch1基因敲除和持续激活，并发现Notch1可调控ERG转录因子。本课题结合既往的研究基础，假设：Notch通路通过调节Snail，Slug, ERG等转录因子的水平,调控成年心肌微血管EndMT和心肌纤维化。我们拟通过激光纤维切割等手段，在成年人心肌组织，Notch1敲除和持续激活小鼠及体外模型中探查Notch1对成年心肌微血管EndMT的作用及机制。本研究将揭示成年心肌微血管EndMT的分子机制，为心肌纤维化等疾病的防治提供新思路和新靶点。

血管内皮细胞向间质细胞转化（EndMT）是成年心血管纤维化演变的一个重要病理过程，Notch信号通路在心血管系统发育过程中调控EndMT，但其成年血管内皮细胞中的作用尚不明确。本项目研究通过人类心肌纤维化样本检测，体外细胞实验和构建分析在成年小鼠体内血管内皮细胞中激活Notch通路或敲除Notch1基因的动物模型，发现Notch信号通路激活显著降低内皮细胞标志物表达，而上调间质细胞标志物表达；Notch1基因敲除在成年动物血管内皮细胞中对EndMT作用不明显。此外，我们发现Notch信号通路在成年小鼠血管内皮细胞内调控ERG转录因子的表达，而ERG正向调控成年血管内皮细胞中vWF和VE-cadherin的表达。我们还发现Notch信号通路转录调控Netrin4基因的表达，并通过调节转录因子GATA3调控Tie2基因的表达。本研究部分揭示了成年心肌微血管EndMT的分子机制，并为研究Notch信号通路调节的血管稳态提供了新的思路。