Endothelial RhoA is essential for embryonic and postnatal vascular development by regulating KLF2-Notch1 signaling

The small GTPase RhoA has diverse cellular functions, including in actin fiber reorganization. However, its role in angiogenesis remains elusive. To reveal this, we generated mice with endothelium-specific constitutive or tamoxifen-inducible conditional knockout (cKO) of RhoA. Both constitutive and inducible RhoA cKO mice all died by embryonic day 11.5. They displayed severe abnormalities in cephalic plexus vasculature and intersomitic vessels. Inducible RhoA cKO mice also had abnormal lymphatic vessels at mid-stage embryogenesis and showed significant reductions in radial outgrowth and density of vascular plexus in the retina on postnatal day 10. Blood flow recovery and vascular repair in ischemic hindlimbs were also impaired in adult inducible RhoA cKO mice. The expression of a stalk cell marker Notch1 was reduced, but that of a tip cell marker Dll4 was unchanged in the branched vessels of RhoA cKO embryos. By luciferase analysis and a database search, KLF2 was identified as a negative regulator of Notch1 expression by binding to the NOTCH1 gene promoter. KLF2 was phosphorylated by the RhoA-ROCK pathway, which inhibited KLF2 nuclear translocation, resulting in increases in Notch1 transcription and expression. Endothelial RhoA supports endothelial differentiation through KLF2-Notch1 signaling and promotes embryonic and postnatal vascular development.