转录因子ER71介导内皮祖细胞生成在脑缺血损伤修复中的作用

Stroke is one of the three major leading causes of death. Developing alternative therapies to restore neurological function and promote angiogenesis therefore, will benefit stroke patients who do not fall under the guidelines for rt-PA therapy. Endothelial progenitor cells (EPCs) are implicated in embryonic/postnatal vasculogenesis, angiogenesis, as well as a range of pathological conditions, suggesting a natural therapeutic role for EPCs in the cerebrovascular diseases (CVD), particularly in cerebral ischemia. Flk-1 (VEGFR2) is supposed to be an early marker for EPCs, correlating with the specification and early differentiation of the endothelial cell lineage. Flk-1 positive cells derived from mouse/human embryonic stem cells (ESCs) and induced pluripotent stem cells serve as vascular progenitors. However, the pluripotency of ESCs capable of generating various cell types and the overwhelming complexity of the multiple signaling pathways involved in the process of differentiation make it extremely challenging to control EPCs differentiation in a precise and efficient manner. Flk-1 positive cells are indeed mesodermal precursor cells, which generate endothelial, blood, vascular smooth muscle, skeleton muscle cells and cardiomyocytes. Therefore, deciphering the mechanisms how the ESCs specify to EPCs, and finally commit to vascular cells will contribute to obtain a celluar source for transplantation. .Our preliminary data suggest a novel Ets transcription factor ER71 is crucial for vasculargenesis and endothelial cell generation in early embryonic stage. ER71-/- mice died early in gestation due to vessel defects. ER71 is cell autonomously required for endothelial cell generation suggested by chimeric mice study. Enforced expression of ER71 in iER71 ESCs resulted in a robust induction of Flk-1+ mesoderm. Based on these data, we propose a hypothesis that ER71 can directly specify ESCs to Flk-1+EPCs, and the robust generation of Flk-1+EPCs from iER71 ESCs can be used to rescue the injury in cerebral ischemia..The transcriptional control is critical for the endothelial cell development. Global gene expression profiles showed that endothelial genes were up-regulated by enforced ER71 expression, but down-regulated by ER71 inactivation. Intriguingly, the same gene sets were shown as potential direct binding targets of ER71 using chromatin immunoprecipitation (ChIP) -Seq analysis. Importantly, motif searches suggested other cis-regulatory elements are significantly enriched close to ER71 binding regions, such as E-box and GATA motif. In this proposal, we will further test if ER71 recruits co-activators, and regulates EPCs commitment through a combinatorial mechanisms by transcription factor interactions.

①胚胎干细胞（ESCs）来源的 Flk-1+细胞具有血管内皮祖细胞 (EPCs)的特性，有可能成为治疗缺血性脑血管病的种子细胞。但由于ESCs的多向分化潜能以及分化过程中信号调控通路的复杂性，精确诱导ESCs向EPCs定向分化极为困难。②我们前期研究发现，转录因子ER71对发育早期的血管生成具有重要作用；ER71过表达的ESCs（iEr71-ESCs）大量生成Flk-1+细胞，促进内皮细胞的生成而抑制其他细胞系的分化,表明ER71能诱导ESCs向Flk-1+EPCs定向分化。③本课题将进一步研究ER71对内皮基因表达的调控机制；拟用荧光标记iEr71-ESCs生成的Flk-1+细胞，研究其在体内外特异性生成血管组织的能力及在大鼠大脑中动脉栓塞模型所致的脑缺血损伤的修复作用，发现有效指导ESCs定向生成EPCs的关键转录因子，以期为缺血性血管病的治疗提供新策略和理论基础。

胚胎干细胞（ESCs）来源的 Flk-1＋细胞具有血管内皮祖细胞 (EPCs)的特性，有可能成为治疗缺血性脑血管病的种子细胞。但由于ESCs的多向分化潜能以及分化过程中信号调控通路的复杂性，精确诱导ESCs向EPCs定向分化极为困难。转录因子ER71对发育早期的血管生成具有重要作用； ER71可诱导ESCs向Flk-1＋EPCs定向分化。项目用荧光标记iEr71-ESCs分化生成的Flk-1＋细胞系，证实其在体外具有特异性生成血管组织的能力；应用大鼠大脑中动脉栓塞模型，探讨其对大鼠脑缺血损伤的修复作用，结果证实iEr71-Flk-1＋EPC可以增加缺血28天梗塞侧脑血流，有效减少缺血28天的脑梗死体积，显著改善运动神经功能及记忆能力。注射iEr71-Flk-1＋EPC可以减轻胼胝体萎缩，减少缺血再灌注引起的白质损伤。CD31、VE-cadherin在注射iEr71-Flk-1＋EPC组的皮层和基底节中表达明显增加，并与GFP阳性细胞有良好共定位。iEr71-ESCs来源的Flk-1+细胞具有特异性生成脑血管组织，通过减轻白质损伤改善缺血性脑损伤的能力。本研究可以为缺血性脑损伤的治疗提供新方法、新途径。