Wang Shouli, Yin Zhao, Zhao Bei, Qi Yanmei, Liu Jie, Rahimi Saum A, Lee Leonard Y, Li Shaohua
Department of Cardiology, Beijing 306 Hospital, Beijing 100101, China.
Department of Cardiology, Beijing 306 Hospital, Beijing 100101, China.
Stem Cell Res. 2017 Dec;25:157-165. doi: 10.1016/j.scr.2017.11.002. Epub 2017 Nov 7.
Gravity plays an important role in normal tissue maintenance. The ability of stem cells to repair tissue loss in space through regeneration and differentiation remains largely unknown. To investigate the impact of microgravity on blood vessel formation from pluripotent stem cells, we employed the embryoid body (EB) model for vasculogenesis and simulated microgravity by clinorotation. We first differentiated mouse embryonic stem cells into cystic EBs containing two germ layers and then analyzed vessel formation under clinorotation. We observed that endothelial cell differentiation was slightly reduced under clinorotation, whereas vascular branch morphogenesis was markedly enhanced. EB-derived endothelial cells migrated faster, displayed multiple cellular processes, and had higher Cdc42 and Rac1 activity when subjected to clinorotation. Genetic analysis and rescue experiments demonstrated that Cdc42 but not Rac1 is required for microgravity-induced vascular branch morphogenesis. Furthermore, affinity pull-down assay and mass spectrometry identified Rap1GDS1 to be a Cdc42 guanine nucleotide exchange factor, which was upregulated by clinorotation. shRNA-mediated knockdown of Rap1GDS1 selectively suppressed Cdc42 activation and inhibited both baseline and microgravity-induced vasculogenesis. This was rescued by ectopic expression of constitutively active Cdc42. Taken together, these results support the notion that simulated microgravity activates Cdc42 via Rap1GDS1 to promote vascular branch morphogenesis.
重力在正常组织维持中起着重要作用。干细胞在太空中通过再生和分化修复组织损伤的能力在很大程度上仍不清楚。为了研究微重力对多能干细胞血管形成的影响，我们采用胚状体（EB）模型进行血管生成，并通过clinorotation模拟微重力。我们首先将小鼠胚胎干细胞分化为包含两个胚层的囊性EB，然后分析clinorotation条件下的血管形成。我们观察到，clinorotation条件下内皮细胞分化略有减少，而血管分支形态发生明显增强。clinorotation处理后，EB来源的内皮细胞迁移更快，表现出多个细胞突起，且Cdc42和Rac1活性更高。遗传分析和拯救实验表明，微重力诱导的血管分支形态发生需要Cdc42而不是Rac1。此外，亲和下拉分析和质谱鉴定Rap1GDS1是一种Cdc42鸟嘌呤核苷酸交换因子，clinorotation可使其上调。shRNA介导的Rap1GDS1敲低选择性抑制Cdc42激活，并抑制基线和微重力诱导的血管生成。组成型活性Cdc42的异位表达可挽救这种情况。综上所述，这些结果支持模拟微重力通过Rap1GDS1激活Cdc42以促进血管分支形态发生这一观点。
