M. R. Fenlon1,2, J. Xu1, J. Zagory1, K. Asahina2, K. Wang1 1Children’s Hospital Los Angeles,Surgery,Los Angeles, CA, USA 2University Of Southern California,Los Angeles, CA, USA
Introduction:
In Biliary Atresia (BA), post-Kasai hepatoenterostomy survival with native liver inversely correlates with extent of liver fibrosis. We previously demonstrated an expansion of Prom1+ Hepatic Progenitor Cells (HPCs) within areas of developing ductular reactions in the Rhesus Rotavirus (RRV) model of experimental BA (Mavila, Hepatology 2014). Here, the degree of Prom1+ cell expansion correlated with the extent of fibrosis. We also previous demonstrated differential regenerative capacity of Prom1+ HPCs in neonatal mice compared to adult (Zhu, Cell, 2016). Moreover, null mutation of Prom1 leads to decreased ductular reaction and periportal fibrosis in RRV-induced BA (Zagory, unpublished data). Herein, we hypothesized that selective Prom1+ cell ablation is associated with decreases ductular reaction and liver fibrosis in an adult model of cholestasis caused by bile duct ligation (BDL).
Methods:
C57BL6 mice were bred to heterozygosity for both Prom1-Cre and inducible-Diphtheria Toxin Receptor (iDTR). 8-10 week old transgenic mice underwent BDL. Two weeks prior to BDL, tamoxifen was administered to induce cre-recombination in Prom1+ HPCs, resulting in targeted expression of iDTR. 1 week prior to BDL, Diphtheria Toxin (DT) or saline was injected to induce selective ablation of Prom1+ HPCs expressing iDTR. At 5 days post-BDL, whole liver tissue was collected and analyzed for histology and gene expression.
Results:
The extent of ductular reaction following BDL ± DT, as demonstrated by biliary epithelial marker CYTOKERATIN-19 (CK19) immunofluorescence, was comparable. Compared to sham, BDL resulted in increased Sirius red staining indicating increased deposition of excess extracellular matrix, a surrogate marker for liver injury (n = 13). Sirius red staining was greater post-BDL in DT treated mice compared to saline. Quantitative PCR analysis of hepatic fibrosis markers demonstrated trends towards increased fibrosis in BDL mice after DT ablation compared to saline group: α-Smooth Muscle Actin 4.19 vs 5.73, p=0.34; Vimentin 1.77 vs 6.31, p=0.15 (median fold expression of NS BDL and DT BDL groups).
Conclusion:
Contrary to our expected result, selective ablation of Prom1-expressing HPC leads to increased fibrosis following BDL in adult mice. This may be the consequence of differential regenerative capacity of Prom1+ HPC in adults compared to neonates. Further studies are essential to elucidate the functional role of Prom1+ HPC in infants with BA.
