Renault, M.-A., Roncalli, J., Tongers, J., Thorne, T., Misener, S., Volpert, O.-L., Metha, S., Burg, A., Luedemann, C., Qin, G., Kishore, R., and Losordo, D.-W.
Rationale and Objectives: The morphogen Sonic Hedgehog (Shh) is known to promote neovascularization in adults via indirect induction of pro-angiogenic cytokine expression by fibroblasts. Direct effects of Shh on endothelial cell (EC) function and angiogenesis, however, have not been characterized. Accordingly, we performed a series of in vitro and in vivo studies to evaluate the direct effects of Shh on EC and to investigate certain mechanisms by which Shh modulates angiogenesis. Methods and Results: Our data disclose that Shh promotes capillary morphogenesis (tube length on Matrigel™ increased to 271±50 % of the length in untreated cells), induces EC migration (191±35 %) and increases EC expression of matrix metalloproteinase 9 (MMP-9) and osteopontin (OPN), which are shown to be essential for Shh-induced angiogenesis both in vitro and in vivo. Shh effects in ECs, however, occur while Gli-dependent transcription is not modulated. Furthemore, our studies show that changes in gene expression and EC migration are mediated by Shh induction of Rho. The Rho dependence of Shh-induced EC angiogenic activity is documented in vitro using dominant negative constructs for RhoA and Rho kinase (ROCK), showing that RhoA and ROCK blockade attenuates Shh induced migration and tube formation, as well as the Shh induced expression of MMP-9 and OPN. In vivo in the mouse corneal angiogenesis model pharmacologic inhibition of ROCK blocks Shh induced angiogenesis, confirming the ROCK dependence of this process. Furthermore, in MMP-9 and OPN null mice Shh induced angiogenesis also blocked, indicating that Shh induced angiogenesis is also dependent on MMP-9 and OPN expression Conclusion: These data elucidate an entirely novel, “nonclassical” pathway by which Shh directly modulates EC phenotype and angiogenic activity. [Copyright &y& Elsevier]