NADPH氧化酶来源的活性氧在iPS细胞移植促血管新生中的作用及机制研究

Induced pluripotent stem cells (iPSCs) are a promising cell type for cell-based vascular regeneration. There is a special subject about iPSCs' research applications in 2012 published by Nature. IPSC transplantation promotes angiogenesis of ischemic tissue. However, the molecular mechanisms of iPSC transplantation linked to angiogenesis remains unknown. We found that transplanted antioxidant NAC-treated iPSCs impaired neovascularization compared with iPSC transplation in hindlimb ischemic mice. NAC treatment inhibited VEGF expression, and impaired cell adhesion and inhibited migration and proliferation of iPSCs in vitro. NADPH oxidase is major sources of reactive oxygen species (ROS) in iPSCs, we showed that NADPH oxidase subunit Nox4 and Nox2 are highly expressed in iPSCs. By scavenging of ROS with inhibition of NADPH oxidase activity, or Nox4 or Nox2 expression in iPSCs, we attempt to investigate the role of NADPH oxidase-derived ROS on the efficacy of iPSCs for angiogenesis, and their role on the paracrine effect and differentiation of iPSCs in a mouse hindlimb ischemia model. We will determine whether NADPH oxidase-derived ROS are involved in iPSC migration and proliferation by regulating the VEGF expression, and investigate the role and underling mechanisms of NADPH oxidase-derived ROS in iPSC adhesion and differentiation into vascular endothelial cells. This study will provide the scientific basis for the treatment of ischemic diseases by iPSC transplantation.

诱导性多能干细胞(iPS细胞)在血管再生方面最具有应用前景。20012年《Nature》杂志对此有专题报道，但iPS细胞移植促缺血组织血管新生的机制不清。申请人前期发现，将抑制活性氧(ROS)的iPS细胞移植入小鼠体内，小鼠缺血下肢血管新生比iPS移植组明显降低；抑制iPS细胞ROS产生抑制VEGF表达，及细胞的粘附，迁移和增殖。NADPH氧化酶是iPS细胞最主要ROS来源，其亚基Nox4和Nox2在iPS细胞中高表达。本项目拟通过抑制iPS细胞NADPH氧化酶活性或者Nox4或Nox2表达，研究：1）NADPH氧化酶来源的ROS对iPS细胞移植促小鼠缺血下肢血管新生的影响，及对体内iPS细胞旁分泌和分化的影响。2）明确其是否通过调控VEGF表达影响iPS细胞迁移和增殖。3）研究其对iPS细胞粘附和分化成血管内皮细胞的影响及分子机理。这项研究将为iPS细胞移植治疗缺血性疾病提供科学依据。

活性氧（ROS）在血管新生中起重要作用，NADPH氧化酶是诱导性多能干细胞（mouse induced pluripotent stem cells, iPSCs）最主要ROS来源，我们前期研究发现NADPH氧化酶2（Nox2）在iPSCs中高表达。但是Nox2来源的ROS是否影响iPSCs向内皮细胞分化以及影响iPSC-EC的促血管新生作用还不清楚。我们利用OKSM慢病毒感染野生型和Nox2−/−小鼠胚胎成纤维细胞使其重编程为小鼠iPSCs，建立WT miPSCs和Nox2−/−miPSCs细胞系。我们发现，Nox2−/− miPSCs来源的内皮细胞（Nox2−/−miPSC-EC）ROS产生，内皮细胞标志物，动脉内皮细胞标志物，促血管新生细胞因子和Notch信号通路分子的表达水平均明显低于野生型的miPSCs来源的内皮细胞（WT miPSC-EC）。这些基因表达水平的降低，能够被Nox2或Notch1过表达所逆转。过表达Nox2基因激活Notch信号通路，促进miPSCs向动脉内皮细胞分化，这些作用可以被ROS抑制剂或抑制Notch1表达所阻断。Nox2−/−iPSC-EC的迁移能力，增殖能力，管腔形成能力及细胞存活能力减少。在小鼠下肢缺血动物实验中，缺血下肢给予Nox2−/−miPSC-EC治疗后，小鼠血流恢复，毛细血管和小动脉密度较WT miPSC-EC治疗组明显降低。上述研究表明，Nox2产生的ROS通过激活Notch信号通路促进iPSCs向动脉内皮细胞分化，促进iPSC-EC的促血管新生作用，这为以Nox2为靶点提高iPSCs向动脉内皮细胞的分化效率提供了新策略，为心血管疾病再生治疗提供了理论基础。