Endothelial Desert Hedgehog and ectopic Sonic Hedgehog act antagonistically to regulate vessel integrity and angiogenesis

Abstract

The therapeutic potential of Hedgehog (Hh) signaling agonists for vascular diseases is of growing interest. However, the molecular mechanisms underlying Hh signaling effects on the vasculature remains unclear and conflicting results have been published. Our goal is to clarify and compare the vascular effects of endothelial-derived Desert Hedgehog (eDhh) and soluble N-terminal Sonic Hedgehog (N-Shh). We first confirmed that in accordance with the literature, recombinant N-Shh is pro-angiogenic in the mouse corneal neovascularization assay. However, by using mice lacking Dhh expression in endothelial cells (ECs), we showed that eDhh limits VEGFA-dependent angiogenesis in the same model. Similarly, while we found that eDhh is necessary for blood-brain barrier (BBB) integrity, ectopic administration of soluble N-Shh induces BBB leakage. Consistent results were obtained in vitro in cultured ECs. Indeed, both Dhh knock down and Shh treatment promoted EC migration and increased ECs permeability. This first set of data demonstrates for the first time that Hedgehog ligands may induced opposite effects in ECs. Next, we found that mice lacking Smoothened (a positive regulator of Hh pathway) in ECs display a similar phenotype to those lacking eDhh, suggesting that eDhh stimulates Hh signaling in ECs while soluble N-Shh inhibits it. At a molecular level, we found that soluble Shh prevent Dhh binding with its receptor Ptch1 both in IP assays and proximity ligations assays. Altogether these results suggest that soluble Shh acts as an inhibitor of Hh signaling in endothelial cells by competing with Dhh. In conclusion the present data highlights for the first time that eDhh and soluble Shh have antagonistic properties especially in ECs. Such demonstration implies that Hh ligands cannot be used instead of another for rescue/therapeutic purposes.