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。

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.