PMO-115 ANTI-B1-integrin antibodies improve survival of isolated human hepatocytes significantly increasing both adhesion to hepatic sinusoidal endothelium under flow and engraftment in murine liver following transplantation

Introduction Hepatocyte transplantation is a potential alternative to orthotopic liver transplantation but is limited by poor survival of transplanted cells. This may be partly due to apoptosis of isolated hepatocytes following detachment from extracellular matrix with loss of β1-integrin-mediated survival signals. Anti-β1-integrin antibodies have been shown to reduce apoptosis of rat hepatocytes 1 and improve their survival in allogeneic transplantation. 2 The purpose of this study was to determine the effect of β1-integrin blocking antibodies on the survival and initial engraftment of transplanted human hepatocytes. Methods Hepatocytes were isolated from tissue obtained with ethical approval from Queen Elizabeth Hospital Birmingham. Integrin expression was confirmed using flow cytometry. Cells were incubated in suspension with anti-β1-integrin blocking antibodies or isotype matched control for 1 h. Viability and caspase three activity were assessed using flow cytometry and cleaved caspase 3 ELISA respectively. A modified flow adhesion assay was used to investigate the resistance to flow of cells adherent to sinusoidal endothelium (HSEC). An FC blocking agent was used to exclude the possibility of antibody-treated cells binding via antibody-FC receptor interactions. 1×10 6 fluorescently labelled cells were injected into C57BL/6 mice via the portal vein under general anaesthesia and the mice culled after 15 min. The livers were immediately frozen and sectioned and the number of fluorescent cells per field of view counted. Results Mean surface expression of the β1-integrin subunit on human hepatocytes was 86.8% (MFI 46.8). Hepatocytes treated with β1-integrin antibodies showed increased viability (85.4% vs 79.0% p=0.02) and reduced caspase 3 activity as demonstrated by a decrease in cleaved caspase 3 (mean 450 nm absorbance 1.37 vs 1.90, p=0.02). β1-integrin blockade significantly increased the mean percentage of cells remaining adherent to HSEC under flow compared to IgG control (30.6% vs 12.7%, p=0.03) and significantly increased the number of cells per field of view in the livers of mice following transplantation (1.6 vs 0.7, p=0.017). Conclusion β1-integrin blocking antibodies increase survival of isolated hepatocytes and improve their ability to remain adherent to HSEC under flow resulting in increased engraftment of transplanted human hepatocytes in mouse liver. The use of β1-integrin blocking antibodies may provide a means to increase the efficacy of human hepatocyte transplantation. Competing interests None declared. References 1. Pinkse , et al . J Hep 2005. 2. Kocken , et al . Lab Invest 1997.