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Angiogenesis, the growth of new blood vessels from pre-existing vessels, is an essential step in solid tumor progression and the formation of metastases. The development of a new vasculature requires an integration of multiple signals from secreted factors such as Vascular Endothelial Growth Factor (VEGF) and angiopoietins as well as cell-matrix adhesions. Recent evidence suggests that the Hedgehog family of secreted growth factors may function in the regulation of vasculogenesis and angiogenesis during embryonic development. Hedgehog signaling has also been shown to be aberrantly reactivated leading to enhanced tumor growth in several cancers including basal cell carcinoma, medulloblastoma, and cancers of the prostate, colon, and pancreas. Therefore, we chose to investigate how Hedgehog signaling may be involved in regulating angiogenesis. Analysis of human and murine endothelial cells in vitro and in vivo by RT-PCR and in situ hybridization demonstrated the expression of Patched-1, the Hedgehog transmembrane receptor, as well as the serpentine receptor Smoothened. Investigation of two murine endothelial cell lines, bEND3 and EOMA, indicated that bEND3 cells predominantly express Gli3 while EOMA cells predominantly express Gli1. Addition of Shh-N to monolayers of bEND3 cells induced the cells to assemble into capillary-like structures, suggesting that Hedgehog signaling may mediate this morphogenic process. Using a three-dimensional collagen gel matrix, we found that inhibition of the Hedgehog signaling pathway with cyclopamine, a specific inhibitor of Hedgehog signaling, prevented endothelial cell capillary formation in a dose-dependent manner between 1 and 10 μM. Additionally, inhibition of Hedgehog signaling by 10 μM cyclopamine treatment blocked HUVEC formation of capillary structures on Matrigel. Treatment of endothelial cell monolayers with cyclopamine resulted in an increase in actin stress fiber formation, suggesting that Hedgehog signaling may play a role in the organization of the actin cytoskeleton. The Rho family of small GTPases are the primary regulators of the actin cytoskeleton, and levels of active RhoA and Rac1 are significantly increased following cyclopamine treatment. Furthermore, GLI3 nuclear localization is increased in cyclopamine treated endothelial cells. Our results suggest that by inhibiting the Hedgehog signaling pathway, endothelial cells undergo a reorganization of the actin cytoskeleton, thereby preventing angiogenesis from occurring. In conclusion, the evidence we present suggests that the Hedgehog signaling pathway plays a critical role in angiogenesis and may be a potential therapeutic target in the prevention of tumor angiogenesis.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]