血管重构是许多重要血管疾病共同的病理环节，而内皮细胞功能异常是血管重构的重要原因。MiR-126是内皮细胞特异高表达的微小RNA并参与内皮细胞功能调节。我们前期研究发现miR-126位于其宿主基因EGFL7的内含子中，二者协同调控血管发育；敲除内皮细胞miR-126明显促进肺动脉高压中血管重构，因此miR-126和其宿主基因EGFL7可能参与调控血管重构。为验证该科学假设，我们建立了miR-126敲除、EGFL7突变、miR-126/EGFL7双突变等三种小鼠品系，并以此为基础，探索miR-126和EGFL7在血管重构中的作用、在内皮-平滑肌细胞相互作用中的调控方式，明确二者协同作用的细胞学和分子基础。此外，内皮细胞中miR-126存在序列多态性，能够影响靶基因翻译或自身降解，进而介导血管重构过程；该序列多态性的改变可能是血管重构的重要特征，具有潜在的临床应用价值。

Dysfunction of Endothelial cell is a major cause of vascular remodeling. MiRNA, which plays an essential role in functional regulation of endothelial cell, might be involved in vascular remodeling. We found that miR-126 was the most abundant miRNA in endothelial cells. In monocrotaline-induced pulmonary hypertension, miR-126 knockout promoted vascular remodeling. MiR-126 located in the seven introns of EGFL7. During vascular development, miR-126/EGFL7 have a synergistic role. To verify the synergies of miR-126 and EGFL7 in vascular remodeling, we constructed miR-126 knockout, EGFL7 mutation, and miR-126/EGFL7 double mutant mice. In this project, we will verify the function of miR-126 and EGFL7 in vascular remodeling, explore the cellular interaction function of miR-126/EGFL7 between endothelial cells and smooth muscle cells, and clarify the signaling pathways regulated by miR-126/EGFL7. We also found that polymorphism exists in miR-126 sequence, namely isomiR-126, which might be involved in interactions between endothelial cells and smooth muscle cell. We will investigate the biological significance and clinical applications of this sequence polymorphism, which might have potential roles in cell-cell interaction, or serve as predicting biomarker for vascular remodeling diseases in the plasma.

课题组利用miR-126单敲小鼠、EGFL7单敲小鼠和miR-126 & EGFL7双敲除小鼠，首先对三种基因型敲除小鼠胚胎发育过程进行分析，发现miR-126和EGFL7敲除小鼠胚胎头部均发生血管数量减少及局部出血，而miR-126和EGFL7双敲除小鼠胚胎头部和背部严重出血、全身严重水肿。基于miR-126 和 EGFL7在内皮细胞表达的特性，课题组对分选出的内皮细胞进行非编码RNA测序，通过生物信息学分析，发现一批相关联的circRNA-miRNA-mRNA可能在内皮发育过程中起调控作用。同时利用单细胞测序的方法对1000个内皮细胞进行测序分析，发现了多种内皮细胞亚型，可用于血管新生和重构过程中机制的探讨。建立了稳定的股动脉损伤和后肢缺血模型，以研究miR-126和EGFL7在血管稳态和重构中的作用机制。利用小鼠后肢缺血模型，发现单敲miR-126和EGFL7以及双敲小鼠均表现出血流恢复障碍，且双敲小鼠血流恢复障碍表型更明显，提示miR-126和EGFL7在血管新生过程中具有协同作用。通过小鼠股动脉损伤模型，发现单敲miR-126和EGFL7小鼠股动脉在损伤恢复时出现中膜异常增厚，检测发现这种病理性增厚是血管中膜平滑肌细胞增加所致。而在miR-126和EGFL7双敲的小鼠中，并没有发现中膜增厚的叠加现象，提示在血管损伤修复过程中miR-126和EGFL7具有不同的作用机制。课题组创新性地使用蛋白质组学的方法，对小鼠股动脉损伤后的血管进行多种蛋白质修饰的检测，发现了一批蛋白的巴豆酰化修饰在血管修复中变化显著，提示miR-126和EGFL7可能通过调控上述蛋白的巴豆酰化进而影响血管中膜平滑肌细胞的表型转化。miR-155通过oxLDL/YY1/HDACs/miR-155/HBP1信号通路调控动脉粥样硬化形成，证明了miR-155是治疗动脉粥样硬化的一个潜在分子靶点，相关内容已申请专利，且在国际心血管专业权威期刊Cardiovascular Research上发表。miR-126分别通过Flt4和Cxcl12a信号通路协同参与淋巴管的出芽和延伸，该研究结果已在国际血管生物学权威期刊Arteriosclerosis, Thrombosis, and Vascular Biology上发表。这些发现阐述了miRNA以及与其相互作用的蛋白在血管稳态与重构中的机制。