Inhibition of raci activity hinders vasculogenesis during early embryonic development

Whether small G protein Rac1 plays a role in regulating blood vessel formation especially endothelial cells(ECs) differentiation during early embryonic vascular development remains unclear. Murine embryonic stem cells (ESCs) lines stably expressing the constitutively active Rac1 mutant (Rac1G12V) or dominant negative Rac1 mutant (Rac1T17N) and the resulting embryoid bodies (EBs) were established as models, to observe the effects of Rac1 on the differentiation and migration of ECs. Using contrast phase microscope to observe the development and differentiation features of EBs; pull-down assay to analyze the expression of Rac1 activity; immunofluorescence staining and Western blot to inspect the differentiation markers of ECs; matrigel model to observe the assembly of endothelial network. It showed that neither increase nor suppression of Rac1 activity significantly affect the differentiation of EBs to form the typical primitive germ layers. Expression of dominant negative Rac1 did not affect ECs differentiation, but it significantly inhibited cell migration, furthermore completely blocked the assembly of vessel network. Actin stress fibers were largely absent in Rac1T17N-expressing cells. Expression of Rac1T17N inhibited FAK activity while Rac1G12V did not affect it compared with control group. These results indicated that the effect factor of Rac1 on the vasculogenesis of EBs was to inhibit cell migration which probably mediated by F-actin mechanism.