Mechanical activation of Hippo/TGFb pathways lead human saphenous vein progenitors toward pro-fibrotic differentiation in aorto-coronary bypass failure

Abstract

Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Ricerca Corrente Introduction We previously demonstrated that mechanical stress deriving from coronary flow/pressure patterns in the human saphenous vein (SV) conduits induce release of Thrombospondin-1 (TSP-1) by smooth muscle cells, and this activates adventitial progenitors (SVPs) pathologic activation. Purpose In this study, we show a cooperation of the TGF-β/TSP-1 signaling with the mechanically-activated Hippo transcriptional pathway in fibrotic SVPs commitment. Methods Human derived-SVPs were isolated using a MACS based protocol with a positive selection for CD34 and a negative depletion of CD31+ cells. We performed an RNA-seq analysis of SVPs subjected to 10% uniaxial deformation (n=5), followed by differential gene expression and pathway analyses. We validated results in vitro and in two animal models of vein arterialization. Results A response of SVPs to mechanical stimulation was assessed from variations in cell alignment, circularity and area. The susceptibility of SVPs to uniaxial strain was revealed by a trend of the cells to orientate in orthogonal direction to the strain field and changes in cell shape. Mechanically stimulated cells for 72 hrs showed a significant increase in their motility as verified by migration assays in the presence of medium supplemented with 10% serum. RNA-seq analysis of the total transcriptome expressed in these cells with/without mechanical stimulation was performed. The differentially expressed genes (DEGs) analysis highlighted a maximum variation of the transcriptome at 72hrs of mechanical stimulation vs. static controls with n=819 DEGs. A gene enrichment analysis revealed an involvement of the HIPPO/YAP/TEAD and of the TGF-β/SMAD transcriptional circuitries in mechanically-stimulated cells. In keeping, immunofluorescence and RT-qPCR showed an increase in YAP nuclear translocation and activity. We treated cells with a cytoskeleton inhibitor (Forskolin, FRSK) and a drug (Verteporfin, VTP) that prevents the interaction of the YAP/TAZ complex with TEADs. Both drugs inhibited expression of YAP-transcriptional targets and cellular motility in response to serum. We then treated cells with TGF-β1, TSP-1 alone or in combination. Under these conditions we observed an increased expression of YAP targets and CollA1, a higher amount of Collagen secretion in the supernatant and a higher association of YAP with pSMAD3. All these effects were blunted by VTP. YAP nuclear localization was finally validated in two models of vein arterialization in mice and pigs. Conclusions Our data suggest a convergent activation of Hippo/TGF-β pathways in the failure of the aorto-coronary bypass and highlight a future novel strategy to limit its progression in patients.