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Introduction Myofibroblasts in SSc fibrotic tissues arise from differentiation of a variety of cell types in response to different stimuli, including TGFβ, inflammatory cytokines and hypoxia. In this study, we explored the mechanisms by which TGFβ promotes transdifferentiation of endothelial cells (ECs) by the process of endothelial-to-mesenchymal transition (endoMT), suggested by the presence of EC markers in some fibroblast subpopulations enriched in SSc skin.Material and Methods Human umbilical vein ECs and human dermal microvascular ECs from different donors were stimulated with 10-50 ng/ml TGFβ2 or 1 ng/ml Shh for 5 days and lysed for protein and RNA extraction at different time points or fixed for immunofluorescence. qPCR of selected transcripts and RNA-seq and bioinformatic analysis were performed with at least 3 biological replicates. The GLI1/GLI2 inhibitor Glabrescione B was used at 1 μM in some experiments.Results Treatment of HUVEC and HDMEC cells with TGFβ resulted in gradual increase of endoMT markers, including COL1A1, ACTA2, and ZEB1 mRNA, increased α-SMA staining and reduced VE-cadherin in cell-cell contacts. Moreover, TGFβ caused upregulation of the Hedgehog pathway transcription factors GLI1 and GLI2 in ECs. Remarkably, pharmacological inhibition of GLI1/2 with Glabrescione B prevented TGFβ-induced endoMT and treatment of the cells with Sonic Hedgehog (Shh) was sufficient to induce endoMT. These results indicate that GLI1 and GLI2 are common effectors of TGFβ and Shh in the process of endoMT. RNA-seq analysis of HUVEC and HMDECs treated with Shh, TGFβ or TGFβ plus Glabrescione B for 5 days showed 408 DEGs shared by TGFβ and Shh. Reactome enrichment analysis revealed that GLI-dependent genes were related to extracellular matrix formation/organisation and collagen synthesis, formation and degradation. Two SSc hub genes identified by bioinformatic analysis in multiple gene expression profiles (SULF1 and COL5A2) were upregulated in a GLI-dependent manner. Interestingly, genes upregulated by TGFβ that were independent of GLI were related to interferon signalling and not to extracellular matrix biology.Conclusions This study demonstrates that TGFβ induces endoMT and ECM remodelling through upregulation of the GLI transcription factors. Moreover, activation of GLI proteins by Shh is sufficient to induce endoMT, a novel function of this morphogen pathway. These findings suggest that known augmented Shh and TGFβ signalling in the skin of SSc patients might contribute to microvasculature loss and increase myofibroblast numbers through endoMT.