Scar-associated endothelial-stellate cellular crosstalk drives fibrosis resolution in MASH [snRNA-Seq]

Fibrosis, or scarring, can affect many organs including liver, lung, heart, kidney, intestines etc. and contributes to ~40% of mortality in the industrialized world. Unlike other organs, fibrosis in the liver typically resolves when the source of injury is extinguished. Elucidating the molecular mechanisms that underlie spontaneous fibrosis resolution may lead to novel antifibrotic strategies. In this study we established a robust mouse model of fibrosis regression in MASH (Metabolic dysfunction-Associated Steatohepatitis), a highly prevalent chronic liver disease worldwide, and performed single cell and in situ molecular profiling to define the molecular drivers of fibrosis regression. As fibrosis regressed, inflammatory cell numbers decreased and endothelial cell (EC) numbers increased. Prediction of cell-cell communication using the Calligraphy pipeline identified a Wnt9b-Sfrp2 crosstalk that emerged as fibrosis resolved. To investigate the Wnt9b-Sfrp2 crosstalk as a driver of fibrosis resolution we treated mice with recombinant SFRP2, a WNT inhibitor, which attenuated spontaneous fibrosis regression compared to vehicle-treated mice. Using single nucleus RNA sequencing, we identified a subset of liver ECs termed ‘Endo4’, as the source of Wnt9b. Immunostaining of the Endo4 marker VWF using tissue clearing and 3D imaging identified VWF+ vasculature surrounded by activated hepatic stellate cells (HSCs) that penetrated deep into the fibrotic septa, establishing them as de facto scar-associated ECs. Functionally, using a novel in situ protease activity-based probes, prominent serine protease activity was found to be co-localized with both scar-associated Endo4 ECs and HSCs. In summary, WNT-dependent endo-stellate crosstalk within the fibrotic niche represents a novel regulatory node in murine MASH fibrosis regression and a promising therapeutic target.