Sonic hedgehog induces angiogenesis via Rho kinase-dependent signaling in endothelial cells

• Published in: Journal of molecular and cellular cardiology 2010-09, Vol.49 (3), p.490-498
• Main Authors: Renault, Marie-Ange, Roncalli, Jérôme, Tongers, Jörn, Thorne, Tina, Klyachko, Ekaterina, Misener, Sol, Volpert, Olga V, Mehta, Shanu, Burg, Aaron, Luedemann, Corinne, Qin, Gangjian, Kishore, Raj, Losordo, Douglas W
• Format: Article
• Language: English
• Subjects: Angiogenesis; Animals; Aorta - cytology; Aorta - drug effects; Aorta - metabolism; Apoptosis; Blotting, Western; Cardiovascular; Cattle; Cell Adhesion; Cell Movement; Cell Proliferation; Cells, Cultured; Corneal Neovascularization - metabolism; Corneal Neovascularization - pathology; Coronary Vessels - cytology; Coronary Vessels - drug effects; Coronary Vessels - metabolism; Endothelial cells; Endothelium, Vascular - cytology; Endothelium, Vascular - drug effects; Endothelium, Vascular - metabolism; Fibroblasts - cytology; Fibroblasts - drug effects; Fibroblasts - metabolism; Gli transcription factors; Hedgehog Proteins - genetics; Hedgehog Proteins - metabolism; Humans; Ischemia; Kruppel-Like Transcription Factors - physiology; Matrix Metalloproteinase 9 - physiology; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Morphogenesis; Neovascularization, Physiologic - physiology; Nerve Tissue Proteins - physiology; Osteopontin - physiology; Reverse Transcriptase Polymerase Chain Reaction; rho-Associated Kinases - antagonists & inhibitors; rho-Associated Kinases - genetics; rho-Associated Kinases - metabolism; RNA, Messenger - genetics; RNA, Small Interfering - pharmacology; Signal Transduction; Sonic hedgehog; Vascular Endothelial Growth Factor A - genetics; Vascular Endothelial Growth Factor A - metabolism; Zinc Finger Protein Gli3
• ISSN: 0022-2828; 1095-8584
• DOI: 10.1016/j.yjmcc.2010.05.003

Abstract The morphogen Sonic hedgehog (Shh) promotes neovascularization in adults by inducing pro-angiogenic cytokine expression in fibroblasts; however, the direct effects of Shh on endothelial cell (EC) function during angiogenesis are unknown. Our findings indicate that Shh promotes capillary morphogenesis (tube length on Matrigel™ increased to 271 ± 50% of the length in untreated cells, p = 0.00003), induces EC migration (modified Boyden chamber assay, 191 ± 35% of migration in untreated cells, p = 0.00009), and increases EC expression of matrix metalloproteinase 9 (MMP-9) and osteopontin (OPN) mRNA (real-time RT-PCR), which are essential for Shh-induced angiogenesis both in vitro and in vivo . Shh activity in ECs is mediated by Rho, rather than through the “classic” Shh signaling pathway, which involves the Gli transcription factors. The Rho dependence of Shh-induced EC angiogenic activity was documented both in vitro , with dominant-negative RhoA and Rho kinase (ROCK) constructs, and in vivo , with the ROCK inhibitor Y27632 in the mouse corneal angiogenesis model. Finally, experiments performed in MMP-9- and OPN-knockout mice confirmed the roles of the ROCK downstream targets MMP-9 and OPN in Shh-induced angiogenesis. Collectively, our results identify a “nonclassical” pathway by which Shh directly modulates EC phenotype and angiogenic activity.