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

Abstract

Sonic Hedgehog (Shh), when administered as recombinant protein or via gene therapy was shown to be proangiogenic. However, studies performed in mice deficient for Hedgehog (Hh) signaling pathway elements have revealed that, on the contrary, endogenous Hh signaling is necessary for endothelium integrity and limits angiogenesis, suggesting that Hh ligands may have antagonistic effects. Notably, we recently demonstrated that endothelium integrity depends on endothelial cell (EC)-derived Desert Hedgehog (Dhh). We wish to clarify mechanisms underlying regulation of angiogenesis and vascular barrier integrity by Hh ligands. First we confirmed that Shh recombinant protein induces angiogenesis in the mouse corneal angiogenesis model. Then, we investigated the role of EC-derived Dhh in the same model, and found that, unexpectedly, VEGFA-induced angiogenesis was significantly increased in the absence of EC-derived Dhh suggesting that endogenous Dhh is anti-angiogenic. Besides, we administered Shh-encoding lentiviruses in the brain of WT mice and found that ectopic Shh, on the contrary to EC-derived Dhh disrupts blood brain barrier integrity, demonstrating, once again, that EC-derived Dhh and ectopic Shh have antagonistic effects. We then performed in vitro experiments in cultured ECs and confirmed that both treatment of ECs by Shh recombinant protein and Dhh KD in ECs disrupt intercellular junction integrity and promote EC migration. Notably, Smoothened inhibition recapitulates the phenotype of Dhh deficiency both in vivo and in vitro suggesting that Dhh autocrinally activates Hh signaling in ECs while ectopic Shh inhibits it. Finally, we performed immunoprecipitation assays and found that Dhh binding to Patched-1 is decreased in the presence of Shh. Altogether these results suggest for the first time that Shh, when administered ectopically may counteract Dhh-induced blood vessel integrity and quiescence.