Cdc42-mediated inhibition of GSK-3β improves angio-architecture and lumen formation during VEGF-driven pathological angiogenesis.

Vascular endothelial growth factor-A (VEGF) typically induces abnormal angiogenesis in the adult, thereby aggravating disease pathology and limiting utility of VEGF for therapeutic angiogenesis. To identify strategies for rectifying defects in pathological VEGF neovessels, we investigated consequences of modulating the Rho GTPase Cdc42. In a mouse skin model of VEGF-driven pathological angiogenesis, transduction with active Cdc42 (L28Cdc42) markedly improved VEGF neovessels, as measured by increased lumen formation, enlarged vessel diameter, and enhanced perfusion of macromolecular tracers. Conversely, transduction with dominant negative Cdc42 (N17Cdc42) impaired endothelial cell (EC) assembly into lumenized blood vessels and reduced neovessel diameter and tracer perfusion. In vitro, active Cdc42 improved coordination between actin filaments and microtubules and enhanced formation of vascular cords, suggesting that active Cdc42 rectifies defects in angiogenesis by improving cytoskeletal dynamics and capillary morphogenesis. Analyses of Cdc42 signaling in microvascular ECs indicated that active Cdc42 also inhibits glycogen synthase kinase-3β (GSK-3β), a multi-functional serine/threonine protein kinase. Pharmacological inhibition of GSK-3β improved vascular cord formation in vitro and promoted proper neovessel formation in vivo comparably to active Cdc42, thus linking GSK-3β inhibition to the mechanism by which active Cdc42 rectifies pathological neovascularization. These studies identify activation of Cdc42 and inhibition of GSK-3β as novel strategies for correcting abnormalities associated with VEGF-driven angiogenesis, and they suggest new approaches for achieving improved therapeutic neovascularization with VEGF.