Inhibition of Rho-dependent kinases ROCK I/II activates VEGF-driven retinal neovascularization and sprouting angiogenesis

1 Center for Biomedicine and Medical Technology Mannheim, Joint Research Division Vascular Biology of the Medical Faculty Mannheim, University of Heidelberg and the German Cancer Research Center, Heidelberg; 2 Medical Faculty Mannheim, Fifth Medical Department, University of Heidelberg, Mannheim; and 3 Medical Faculty Mannheim, Institute of Pharmacology and Toxicology, University of Heidelberg, Mannheim, Germany Submitted 29 September 2008 ; accepted in final form 22 January 2009 Vascular endothelial growth factor (VEGF) is an endothelial-specific growth factor that activates the small GTPase RhoA. While the role of RhoA for VEGF-driven endothelial migration and angiogenesis has been studied in detail, the function of its target proteins, the Rho-dependent kinases ROCK I and II, are controversially discussed. Using the mouse model of oxygen-induced proliferative retinopathy, ROCK I/II inhibition by H-1152 resulted in increased angiogenesis. This enhanced angiogenesis, however, was completely blocked by the VEGF-receptor antagonist PTK787/ZK222584. Loss-of-function experiments in endothelial cells revealed that inhibition of ROCK I/II using the pharmacological inhibitor H-1152 and ROCK I/II-specific small-interfering RNAs resulted in a rise of VEGF-driven sprouting angiogenesis. These functional data were biochemically substantiated by showing an enhanced VEGF-receptor kinase insert domain receptor phosphorylation and extracellular signal-regulated kinase 1/2 activation after inhibition of ROCK I/II. Thus our data identify that the inhibition of Rho-dependent kinases ROCK I/II activates angiogenesis both, in vitro and in vivo. Rho-dependent kinases ROCK I and ROCK II; vascular endothelial growth factor; retinal neovascularization; angiogenesis; endothelial signaling Address for reprint requests and other correspondence: J. Kroll, Center for Biomedicine and Medical Technology Mannheim, Joint Research Division Vascular Biology Medical Faculty Mannheim, Univ. of Heidelberg and the German Cancer Research Center, Ludolf-Krehl-Str. 13-17, 68167 Mannheim, Germany (e-mail: kroll{at}angiogenese.de )