Hedgehog proteins activate pro-angiogenic responses in endothelial cells through non-canonical signaling pathways

The Hedgehog (Hh) pathway orchestrates developmental and homeostatic angiogenesis. The three Hh isoforms- Sonic Hedgehog (Shh), Indian Hedgehog (Ihh) and Desert Hedgehog (Dhh)-signal through Patched-1 (PTCH1) and Smoothened (SMO), to activate the Gli transcription factors with a characteristic rank of potency (Shh>>Ihh>Dhh). To dissect the mechanisms through which Hh proteins promote angiogenesis, we analyzed processes inherent to vessel formation in endothelial cells. We found that none of the Hh ligands were able to induce Gli-target genes in human umbilical vein (HUVEC) or human cardiac microvascular endothelial cells (HMVEC), suggesting that endothelial cells do not respond to Hh through the canonical pathway. However, our results show that the three Hh proteins promote endothelial cell tubulogenesis in 3D cultures in a SMO- and Gi protein-dependent manner. Consistent with the required cytoskeletal rearrangement for tubulogenesis, Shh, Ihh, and Dhh all stimulated the small GTPase RhoA and the formation of actin stress fibers. This effect, which was mediated by SMO, Gi proteins, and Rac1, defines a new non-canonical Hh pathway. In addition to regulating the actin cytoskeleton, the Hh ligands promoted survival through inhibition of the pro-apoptotic effect of PTCH1 in a SMO-independent manner. Altogether, our results support the existence of Gli-independent Hh responses in endothelial cells that regulate tubulogenesis and apoptosis. The identification of novel non-canonical responses elicited by Hh proteins in endothelial cells highlights the complexity of the Hh signaling pathway and reveals striking differences in ligand strength for transcriptional and non-transcriptional responses.