Sonic hedgehog induces angiogenesis via Rho kinase-dependent signaling in endothelial cells.
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.
Renault
Marie-Ange
MA
Feinberg Cardiovascular Research Institute, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA.
Roncalli
Jérôme
J
Tongers
Jörn
J
Thorne
Tina
T
Klyachko
Ekaterina
E
Misener
Sol
S
Volpert
Olga V
OV
Mehta
Shanu
S
Burg
Aaron
A
Luedemann
Corinne
C
Qin
Gangjian
G
Kishore
Raj
R
Losordo
Douglas W
DW
D000818:Animals; D001011:Aorta / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D017209:Apoptosis; D015153:Blotting, Western; D002417:Cattle; D002448:Cell Adhesion; D002465:Cell Movement; D049109:Cell Proliferation; D002478:Cells, Cultured; D016510:Corneal Neovascularization / Q000378:metabolism* / Q000473:pathology; D003331:Coronary Vessels / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D053823:Hedgehog Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051741:Kruppel-Like Transcription Factors / Q000502:physiology; D020780:Matrix Metalloproteinase 9 / Q000502:physiology; D051379:Mice; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D009024:Morphogenesis; D018919:Neovascularization, Physiologic / Q000502:physiology*; D009419:Nerve Tissue Proteins / Q000502:physiology; D053495:Osteopontin / Q000502:physiology; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism; D000074290:Zinc Finger Protein Gli3; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
C465571:Gli3 protein, mouse; D053823:Hedgehog Proteins; D051741:Kruppel-Like Transcription Factors; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D042461:Vascular Endothelial Growth Factor A; D000074290:Zinc Finger Protein Gli3; D053495:Osteopontin; D054460:rho-Associated Kinases; D020780:Matrix Metalloproteinase 9