Alcohol-related hepatitis induces a specific fibrosis profile through YAP activation in myofibroblasts

Background &amp; Aims: Yes-associated protein (YAP) impairs hepatocyte regeneration in alcohol-related hepatitis (AH), but its impact on fibrogenesis remains to be characterized. Our aim here was to describe fibrogenesis and investigate the impact of altered hepatocytes on fibrogenic mechanisms during AH. Methods: Fibrosis, YAP and alpha smooth muscle actin (αSMA) distribution were assessed by immunostaining. Extracellular matrix (ECM) composition in severe AH (n = 22), alcohol-related cirrhosis without AH (Cirrh, n = 24), and healthy livers (CTRL, n = 15) was assessed by PCR, and bulk and spatial proteomics. The impact of organoids generated from AH and Cirrh livers on myofibroblasts was also evaluated. Cirrh organoids were transduced with constitutively active YAP (to mimic AH organoids) and co-cultured with myofibroblasts in a 3D co-culture model. In a 2D model, myofibroblasts were treated with growth differentiation factor 15 (GDF15) or cultured with medium from transduced (YAP medium) and non-transduced hepatocytes. Results: AH livers presented with both perilobular (as in Cirrh livers) and specifically intralobular fibrosis. Bulk proteomics and PCR showed a specific ECM protein signature (e.g. laminin A2 was increased (p <0.05) whereas vitronectin was decreased (p <0.001)) in AH. Spatial proteomics showed differences in ECM composition between intralobular and perilobular areas in AH and Cirrh livers. AH organoids also overexpressed YAP (p <0.01). In the 3D model, AH organoids induced greater activation (e.g. αSMA mRNA level increased by threefold; p <0.001) of co-cultured myofibroblasts. YAP-transduced Cirrh organoids also induced greater activation of co-cultured myofibroblasts: for example, the αSMA mRNA level increased by 1.5-fold (p <0.05). In the 2D model, YAP medium also induced increased myofibroblast activation (αSMA mRNA level increased by 1.5-fold; p <0.01) and proliferation (23% increase; p <0.05), in part, through GDF15. Conclusion: AH displays a specific fibrosis profile and protein signature. AH organoids and YAP-transduced Cirrh organoids induce myofibroblast activation. Thus, hepatocyte YAP activation has an important role in the fibrogenesis observed in AH. Impact and implications: Aberrant activation of YAP in hepatocytes contributes to impaired regeneration in alcohol-related hepatitis (AH) by driving their transdifferentiation toward a cholangiocyte-like phenotype. Preliminary data suggest that the extracellular matrix in AH differs from cirrhosis, with an increased laminin-to-fibronectin ratio. In a 3D co-culture model of liver organoids and myofibroblasts, AH organoids increased activation and proliferation of myofibroblasts compared with cirrhosis organoids. Aberrant YAP activation in altered hepatocytes is involved in the activation and proliferation of myofibroblasts in AH livers. Thus, inhibiting YAP in hepatocytes could be an attractive approach for the development of AH treatments to improve hepatic regeneration and decrease fibrogenesis.