The Apelin–Apelin Receptor Axis Triggers Cholangiocyte Proliferation and Liver Fibrosis During Mouse Models of Cholestasis

Background and Aims Apelin (APLN) is the endogenous ligand of its G protein–coupled receptor, apelin receptor (APJ). APLN serum levels are increased in human liver diseases. We evaluated whether the APLN–APJ axis regulates ductular reaction and liver fibrosis during cholestasis. Approach and Results We measured the expression of APLN and APJ and serum APLN levels in human primary sclerosing cholangitis (PSC) samples. Following bile duct ligation (BDL) or sham surgery, male wild‐type (WT) mice were treated with ML221 (APJ antagonist) or saline for 1 week. WT and APLN −/− mice underwent BDL or sham surgery for 1 week. Multidrug resistance gene 2 knockout (Mdr2 −/− ) mice were treated with ML221 for 1 week. APLN levels were measured in serum and cholangiocyte supernatants, and cholangiocyte proliferation/senescence and liver inflammation, fibrosis, and angiogenesis were measured in liver tissues. The regulatory mechanisms of APLN–APJ in (1) biliary damage and liver fibrosis were examined in human intrahepatic biliary epithelial cells (HIBEpiCs) treated with APLN and (2) hepatic stellate cell (HSC) activation in APLN‐treated human HSC lines (HHSteCs). APLN serum levels and biliary expression of APLN and APJ increased in PSC samples. APLN levels were higher in serum and cholangiocyte supernatants from BDL and Mdr2 −/− mice. ML221 treatment or APLN −/− reduced BDL‐induced and Mdr2 −/− ‐induced cholangiocyte proliferation/senescence, liver inflammation, fibrosis, and angiogenesis. In vitro , APLN induced HIBEpiC proliferation, increased nicotinamide adenine dinucleotide phosphate oxidase 4 (Nox4) expression, reactive oxygen species (ROS) generation, and extracellular signal–regulated kinase (ERK) phosphorylation. Pretreatment of HIBEpiCs with ML221, diphenyleneiodonium chloride (Nox4 inhibitor), N ‐acetyl‐cysteine (NAC, ROS inhibitor), or PD98059 (ERK inhibitor) reduced APLN‐induced cholangiocyte proliferation. Activation of HHSteCs was induced by APLN but reduced by NAC. Conclusions The APLN–APJ axis induces cholangiocyte proliferation through Nox4/ROS/ERK‐dependent signaling and HSC activation through intracellular ROS. Modulation of the APLN–APJ axis may be important for managing cholangiopathies.