Cholangiocyte senescence has been linked to primary sclerosing cholangitis (PSC). Persistent secretion of growth factors by senescent cholangiocytes leads to the activation of stromal fibroblasts (ASFs), which are drivers of fibrosis. The activated phenotype of ASFs is characterized by an increased sensitivity to apoptotic stimuli. Here, we examined the mechanisms of apoptotic priming in ASFs and explored a combined targeting strategy to deplete senescent cholangiocytes and ASFs from fibrotic tissue to ameliorate liver fibrosis. Using a coculture system, we determined that senescent cholangiocytes promoted quiescent mesenchymal cell activation in a platelet‐derived growth factor (PDGF)‐dependent manner. We also identified B‐cell lymphoma‐extra large (Bcl‐xL) as a key survival factor in PDGF‐activated human and mouse fibroblasts. Bcl‐xL was also up‐regulated in senescent cholangiocytes. In vitro, inhibition of Bcl‐xL by the small molecule Bcl‐2 homology domain 3 mimetic, A‐1331852, or Bcl‐xL‐specific small interfering RNA induced apoptosis in PDGF‐activated fibroblasts, but not in quiescent fibroblasts. Likewise, inhibition of Bcl‐xL reduced the survival and increased apoptosis of senescent cholangiocytes, compared to nonsenescent cells. Treatment of multidrug resistance 2 gene knockout (Mdr2−/−) mice with A‐1331852 resulted in an 80% decrease in senescent cholangiocytes, a reduction of fibrosis‐inducing growth factors and cytokines, decrease of α‐smooth muscle actin–positive ASFs, and finally in a significant reduction of liver fibrosis. Conclusion: Bcl‐xL is a key survival factor in ASFs as well as in senescent cholangiocytes. Treatment with the Bcl‐xL‐specific inhibitor, A‐1331852, reduces liver fibrosis, possibly by a dual effect on activated fibroblasts and senescent cholangiocytes. This mechanism represents an attractive therapeutic strategy in biliary fibrosis. (Hepatology 2018;67:247‐259).
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Mutations in the ABCB4 gene lead to a wide-spectrum of rare liver diseases including progressive familial intrahepatic cholestasis type 3 (PFIC3) and low-phospholipid associated cholelithiasis (LPAC) syndrome. PFIC3 patients develop symptoms during late infancy, including severe itching, jaundice, and failure to thrive. The condition may progress to liver failure during childhood or adulthood. This is a highly unmet medical condition where liver transplantation is the only option to correct this disease. Recently, exciting data suggested that restoration of the ABCB4 function via gene replacement could rescue liver phenotypes associated with ABCB4 dysfunction in a pre-clinical PFIC3 mouse model. Here, we used mRNA LNP platform to determine expression and durability of ABCB4 in the liver of wild-type mice. In addition, we generated Abcb4-/- mice to study the efficacy of systemic delivery of ABCB4 mRNA LNP. We observed a robust and durable expression of hABCB4 up to 72 hours post systemic dosing in the liver of wild-type mice. Systemic administration of hABCB4 mRNA achieved a remarkable restoration of phosphatidylcholine levels in bile, a significant decrease in liver stiffness as measured by shear wave elastography, and amelioration of liver histopathology including fibrosis and ductular reaction. We conclude that administration of hABCB4 mRNA LNPs was sufficient to ameliorate fibrosis markers in the PFIC3 mouse model. Our data suggests that gene replacement using mRNA LNP modality could provide an excellent opportunity for patients with biliary diseases.
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