Non-alcoholic fatty liver disease (NAFLD) has become the leading cause of chronic liver disease, exposing to the risk of liver fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). Angio-genesis is a complex process leading to the development of new vessels from pre-existing vessels. Angiogenesis is triggered by hypoxia and inflammation and is driven by the action of proangiogenic cytokines, mainly vascular endothelial growth factor (VEGF). In this review, we focus on liver angiogenesis associated with NAFLD and analyze the evidence of liver angiogenesis in animal models of NAFLD and in NAFLD patients. We also report the data explaining the role of angiogenesis in the progression of NAFLD and discuss the potential of targeting angiogenesis, notably VEGF, to treat NAFLD.

ABCB4, is an adenosine triphosphate-binding cassette (ABC) transporter localized at the canalicular membrane of hepatocytes, where it mediates phosphatidylcholine secretion into bile. Gene variations of ABCB4 cause different types of liver diseases, including progressive familial intrahepatic cholestasis type 3 (PFIC3). The molecular mechanisms underlying the trafficking of ABCB4 to and from the canalicular membrane are still unknown. We identified the serine/threonine kinase Myotonic dystrophy kinase-related Cdc42-binding kinase isoform α (MRCKα) as a novel partner of ABCB4. The role of MRCKα was explored, either by expression of dominant negative mutant or by gene silencing using the specific RNAi and CRISPR-cas9 strategy in cell models. The expression of a dominant-negative mutant of MRCKα and MRCKα inhibition by chelerythrine both caused a significant increase in ABCB4 steady-state expression in primary human hepatocytes and HEK-293 cells. RNA interference and CRISPR-Cas9 knockout of MRCKα also caused a significant increase in the amount of ABCB4 protein expression. We demonstrated that the effect of MRCKα was mediated by its downstream effector, the myosin II regulatory light chain (MRLC), which was shown to also bind ABCB4. Our findings provide evidence that MRCKα and MRLC bind to ABCB4 and regulate its cell surface expression.

Gli1+ cells were previously described as myofibroblast precursors. We aimed to delineate the identity of Gli1+ cells and their contribution to wound healing in liver. Gli1+ cells were analyzed in normal mouse liver by single-cell RNA sequencing (n = 3). Genetic cell fate tracing and depletion of Gli1+ cells were achieved in male mice, undergoing cholestatic injury induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) or bile duct ligation (BDL), and metabolic dysfunction-associated steatohepatitis (MASH). Gli1 expression was examined by fluorescence in-situ hybridization in human liver (n = 16). Cell interactions were assessed using organoids. In normal liver, Gli1 demarcated a periductal subpopulation (11.8%) of portal fibroblasts, which transcriptome pertains to extracellular matrix and structure organization. In liver injury, Gli1+ fibroblasts accumulated with fibrosis as multilayers around bile ducts and discontinuously along the inner border of the ductular reaction, predominantly in biliary models and less markedly in MASH. In humans, periductal Gli1+ cells were present in normal liver and accumulated in primary sclerosing cholangitis and, to a lesser extent, in MASH, notably alongside the ductular reaction in late-stage diseases. Co-culture of cholangiocyte organoids with Gli1+ fibroblasts showed bidirectional interactions fostering proliferation of both cell types. Genetic depletion of Gli1+ cells (n ≥4/group) caused a marked reduction of fibrosis, assessed by Sirius Red area, in DDC-fed and BDL mice after 1 week (p <0.01) and of ductular reaction, assessed by cytokeratin-19+ cell count in DDC (p <0.01), although cholestatic injury persisted or even increased. Gli1 demarcates a subpopulation of fibroblasts that play a key role in the wound healing process combining portal fibrosis and ductular reaction notably in biliary diseases. The heterogeneity and the functions of hepatic fibroblasts in liver fibrosis remain poorly defined. Gli1+ fibroblasts are a transcriptionally distinct subset of hepatic fibroblasts, mainly located around bile ducts. Gli1+ fibroblasts develop bidirectional interactions with cholangiocytes, underlying portal fibrosis and ductular reaction formation, as they typically occur in biliary diseases. The therapeutic targeting of Gli1+ hepatic fibroblasts is of potential interest in biliary diseases.