Abstract

Background: The liver has a remarkable regenerative capacity. Nevertheless, under chronic liver-damaging conditions, this capacity becomes exhausted, allowing the accumulation of fibrotic tissue and leading to end-stage liver disease. Enhancing the endogenous regenerative capacity by targeting regeneration breaks is a novel therapeutic approach. We set up an in vivo functional genetic screen to identify such regeneration breaks. Integrin Alpha FG-GAP Repeat Containing 1 (ITFG1) was one of the top hits. We proofed that hepatocyte specific knockdown of Itfg1 enhances the endogenous regenerative capacity of the liver and counteracts chronic liver disease.

Methods: We conducted an in vivo functional genetic RNAi screen in the thioacetamide (TAA) driven chronic liver disease model. Two independent shRNAs targeting Itfg1 were significantly enriched. We then tested the effect of Itfg1 knockdown on mouse and human hepatocyte proliferation in vitro. Furthermore, taking advantage of the FAH knockout mouse model, we tested for Itfg1 knockdown driven faster liver repopulation, enhanced regeneration after partial hepatectomy and fibrosis reduction in chronic liver damaging conditions, including “Western Diet” driven NAFLD. For unraveling the mechanism of Itfg1 inhibition driven accelerated liver regeneration we did in depth transcriptomic and proteomic analysis.

Results: Knockdown of Itfg1 in immortalized mouse as well as human hepatocytes accelerates their proliferation and wound healing in vitro. In vivo knockdown of Itfg1 in hepatocytes, accelerates their ability to repopulate the liver of FAH deficient mice. Furthermore, in fully repopulated mice, where every hepatocyte expresses the shRNA targeting Itfg1, liver regeneration upon partial hepatectomy is accelerated. The enhanced regenerative capacity also attenuated TAA and “Western Diet” induced chronic damage. Itfg1 knockdown not only reduced fibrosis but also reduced steatosis in the NAFLD model. Our transcriptomic and proteomic analysis showed that Itfg1 knockdown affects fatty acid metabolism, the MAPK and AKT pathways.

Conclusion: We identified and validated Itfg1 as a target for enhancing the endogenous regenerative capacity of the liver. Enhancing the endogenous regenerative capacity attenuates chronic liver disease. We are currently in the process to translate our results into RNAi therapeutics.