Scar-associated endothelial-stellate cellular crosstalk drives fibrosis resolution in MASH

Fibrosis, or scarring, can affect many organs including liver, lung, heart, kidney, intestines etc. and is responsible for ∼40% of mortality in the industrialized world. Compared to other organs, fibrosis in the liver typically resolves when the source of injury is extinguished. Elucidating the molecular mechanisms that underlie spontaneous fibrosis resolution in the liver may lead to novel antifibrotic strategies for all organs. In this study we established a robust mouse model of fibrosis regression in MASH (Metabolic dysfunction-Associated Steatohepatitis), a highly prevalent chronic liver diseases worldwide, and performed single cell and in situ molecular profiling of the liver to define novel drivers of fibrosis regression. As fibrosis regressed, we detected a reduction of inflammatory cells and an expansion of endothelial cells. Prediction of cell-cell communication using the Calligraphy pipeline identified a Wnt9b-Sfrp2 crosstalk that emerges as fibrosis resolved in our model. To establish the Wnt9b-Sfrp2 crosstalk as a driver of fibrosis resolution we treated mice with recombinant Sfrp2, which slowed spontaneous fibrosis regression compared to vehicle treated mice. From our single cell datasets we identified a subset of endothelial cells, termed “Endo4”, as the source of Wnt9b. Immunostaining of the Endo4 marker VWF using tissue clearing and 3D imaging revealed VWF+ vasculature enveloped by activated hepatic stellate cells (HSCs) that penetrated deep into the fibrotic septa, establishing Endo4 as de facto scar-associated endothelial cells and providing a structural basis of their cellular crosstalk with HSCs. Finally, using a recently developed in situ protease activity screen, prominent serine protease activity co-localized with both scar-associated Endo4 cells and HSCs. In summary, we uncovered an WNT-dependent endo-stellate crosstalk within the fibrotic niche as a novel regulatory node underlying murine MASH fibrosis regression, and a promising therapeutic target.