Rnd3功能缺陷对糖尿病内皮祖细胞血管新生功能的影响及其机制

To date, it is still unclear about the mechanisms regarding the decrease in number and dysfunction of the endothelial progenitor cell (EPC) in diabetic patients, which limited the prospects in application of activated endogenous EPC for treatment of diabetic vascular complications. Hypoxia inducible factor-1α (HIF-1α) is a key transcriptional factor that involves in activating the EPC, and stabilization of HIF-1α is essential for EPC activity. However, the expression and activity of HIF-1α were decreased in diabetes mellitus. Our previous studies have showed that downregulation of Rnd3 would result in an increase in HIF-1α hydroxylation and a subsequent HIF-1α degradation through ubiquitin proteasomal system (UPS), in turn following an impaired response to angiogenesis post tissue injured. Interestingly, we found that the decreased Rnd3 was occurred in diabetic EPC; besides, bioinformatics analysis further showed that there existed the binding sites of HIF-1α in the promoter of Rnd3. In this proposal, we hypothesize that the deficiency of Rnd3 may compromise the HIF-1α/Rnd3 signal feedback loop in diabetic EPC, which may be a new mechanism involved in the diabetic EPC dysfunction. In this project, we aim to explore the effects of dysregulation of Rnd3 on the biofunction of diabetic EPC as well as the molecular mechanism related to the diabetic vascular injury repair combining in vitro experiments and diabetic hindlimb ischemia mice models, and to demonstrate the effect of targeting HIF-1α/Rnd3 signal feedback loop on the biological function of EPC. The findings of this project would provide a potentially new target and strategy for treatment of diabetic wound healing based on EPC infusion.

糖尿病个体内皮祖细胞（EPC）数量减少且功能下降但机制不明，限制了活化内源性EPC治疗糖尿病血管并发症的前景。低氧诱导因子-1α（HIF-1α）是体内活化EPC的关键转录因子，稳定HIF-1α对EPC活性至关重要，但糖尿病时HIF-1α表达下降。课题组发现Rnd3表达下降可导致HIF-1α泛素化降解增多而影响损伤组织的血管新生，而糖尿病EPC中Rnd3表达下降；进一步生物学信息分析显示Rnd3启动子上具有HIF-1α的结合位点。由此，本项目假设糖尿病情况下EPC中Rnd3表达不足将引起HIF-1α/Rnd3信号反馈环路功能缺陷，这可能是导致糖尿病EPC生物学功能障碍的新机制。本项目拟从体内、体外两个层面研究Rnd3影响EPC功能及其在修复糖尿病血管损伤中的作用并探讨其分子机制，明确靶向干预HIF-1α/Rnd3信号反馈环路对EPC生物学功能的影响，为糖尿病伤口愈合治疗提供新靶点与新策略。

血管内皮细胞功能紊乱是糖尿病血管并发症的根本原因。糖尿病伤口局部血管新生及伤口愈合与EPC的数量及功能密切相关。已经证实，无论是I型糖尿病还是II型糖尿病患者，其外周血EPC的数量和血管生成功能均显著下降，但相关的机制仍不明确。本项目在前期研究的基础上，假设内源性ROCK1信号抑制剂Rnd3的表达异常可能在糖尿病EPC的数量和功能上发挥作用。本项目研究发现:（1）糖尿病状态下调了EPC中Rnd3蛋白的表达。（2）项目基于CRISPR/Cas9基因编辑技术，获得Rnd3基因敲除后的杂合子SD大鼠，复制了糖尿病模型，分析了Rnd3基因敲除后EPC一般生物学特性的改变。（3）研究发现糖尿病状态明显减弱了野生型SD大鼠骨髓源性EPC的活力、迁移和旁分泌VEGF能力，促进细胞凋亡蛋白表达增加，并削弱其成血管能力，而Rnd3基因缺陷大鼠Rnd3+/−制备糖尿病模型后，其 EPC的活力、迁移、旁分泌和血管生成能力进一步下降。（4）项目原先假设HIF-1α可能与EPC中Rnd3的表达存在调控关系，但实验证明，HIF-1α不能直接上调Rnd3的表达，也未发现HIF-1α蛋白与Rnd3蛋白相互作用的趋势。（5）项目延伸研究发现血管内皮细胞中Brg1异常可能与内皮功能密切相关。总之，该项目主要基于Rnd3基因编辑模型鼠成功复制了糖尿病模型，首次发现Rnd3基因缺陷促进了糖尿病对骨髓源性EPC活力、迁移、血管生成能力的抑制作用。因此，有望通过干预Rnd3来改善糖尿病患者EPC的增殖、迁移等功能，并提高其分泌促血管生成因子和成血管的能力，为糖尿病血管并发症的防治提供新的靶点和思路。