M. R. Fenlon1, N. Malkoff1, J. Xu1, E. Mahdi1, A. Glazier1, C. Lee1, R. Oweis1, K. Asahina2, K. S. Wang1 1Children’s Hospital of Los Angeles,Surgery,Los Angeles, CA, USA 2Keck School of Medicine,Pathology,Los Angeles, CA, USA
Introduction:
Biliary Atresia (BA) is the most common cause of end-stage liver failure in children. The expansion of intrahepatic ductular reactions (DR), comprised of reactive cholangiocytes, has been strongly linked to liver fibrosis, and correlates with progression toward cirrhosis post-Kasai portoenterostomy. We previously demonstrated the expansion of Prominin-1 (Prom1)-expressing hepatic progenitor cells (HPCs) within DR and evolving fibrosis in BA. Curiously, while null mutation of Prom1 leads to decreased DR and fibrosis in a murine model of BA induced by Rhesus rotavirus, targeted ablation of preexisting Prom1+ HPCs in adult mice undergoing bile duct ligation (BDL) does not. We therefore hypothesized that de-novo Prom1 expression following HPC activation, is responsible for fibroblastic activation in cholestatic liver injury.
Methods:
Adult C57BL6 mice (8-10 weeks) were bred to heterozygosity for Prom1-Cre/ERT2 and ROSA-lsl-Diphtheria Toxin A (DTA). BDL vs sham laparotomy (day 0) was performed to induce cholestatic liver injury. DTA-mediated targeted ablation of all cells expressing Prom1 was accomplished by tamoxifen (TAM)-dependent Cre recombination. Intraperitoneal TAM vs corn oil control (Oil) was administered either day -7 (Pre-BDL) to target preexisting Prom1-expressing HPCs or day +2 (Post-BDL) to target all cells expressing Prom1, including those with de novo expression. At day +10, whole liver RNA was collected for gene expression analysis with quantitative PCR. ANOVA with post-hoc Tukey test was used for statistical analysis (α <0.05 significance).
Results:
With all experimental conditions, BDL-injured mice developed the characteristic weight loss, extra-hepatic biliary dilation, ascites and jaundice. Neither pre- nor post-Sham Prom1-expressing cell ablation significantly altered gene expression compared to Oil Sham. Pre-BDL Prom1-expressing cell ablation increased fibroblastic αSMA and Vimentin expression compared to Pre-Sham (30.5±30.6 vs 0.3±0.2, p<0.01, 10.4±5.3 vs 1.3±0.7 [WK1] [FM2] p<0.001, mean±SD, N=8) whereas Post-BDL ablation (compared to post-Sham) did not (3.8±2.3 vs 2.8±1.3, 1.1±0.4 vs 1.0±0.7, 2.9±0.9 vs 1.5±0.5, all p>0.05, N=9). Moreover, Post-BDL expression of Ck19, αSMA, and Vimentin was significantly lower compared to Pre-BDL groups (3.8±2.3 v 29.6±27.0, p<0.01, 1.1±0.4 v 30.5±30.6, p<0.001, 2.6±0.9 v 10.4±5.3 p<0.001, N=9).
Conclusion:
Post-injury Prom1-expressing cell ablation mitigates the characteristic fibroblastic upregulation of cholestatic liver injury, whereas pre-injury ablation does not. This suggests that a de novo population of Prom1-expressing cells, but not preexisting Prom1-HPCs, are involved in cholestatic liver fibrosis. Further characterization of this cellular population may identify actionable pathways in the treatment of BA.