This thesis sought to investigate novel methods for protecting the liver from ischaemia reperfusion injury in the context of liver transplantation. Research in the heart, brain and kidneys has suggested that hypoxia inducible factor (HIF) may play a key role in the delayed phase of ischaemic preconditioning and can protect organs for up to 3 days. However, although there is good evidence for the potential of HIF to protect organs from ischaemia, the HIF pathway still presents some what of a paradox because it targets both pro-death (e.g. BNIP3,NIX) as well as pro-survival genes (e.g. HO-I, EPO). HIF is primarily controlled by 3 oxygen dependent prolyl hydroxylases (PHD 1 , PHD2, PHD3), and inhibition of these prolyl hydroxylases leads to HIF activation. It was hypothesised that differential inhibition of PHD 1,2 or 3 may result in selective gene regulation and may confer greater or less protection against ischaemia reperfusion injury. To investigate this hypothesis mouse embryonic fibroblasts (MEFs) were isolated from PHDl, 2, and 3 knock-out (KO) embryos and compared to MEFs derived from WT littermate controls. In these MEFs, cell growth and proliferation, as well as cell survival following exposure to anoxia and inducers of apoptosis was studied. The principal findings were that PHD2 is the dominant regulator of HIF in normoxia. PHD2 knock-out MEFs exhibited glycolytic metabolism and had a lower oxygen consumption compared to wild-type MEFs. Gene array studies confirmed the dominant role of PHD2 but also demonstrated that PHD 1 upregulates a number of HIF target genes, albeit to a lesser extent than PHD2. There were no differences, however, in susceptibility to hypoxic injury in the PHDl, 2, and 3 knock-out MEFs compared to wild-type controls. A further aim of the study was to investigate whether prolyl hydroxylase inhibition using dimethyloxalyglycerine (DMOG) may protect the liver in a rodent model of ischaemia reperfusion injury. DMOG effectively upregulated HIF and IllF target genes. Serum transaminases (AST and AL T) were significantly lower in the DMOG treated animals compared to the normal saline treated controls 24 hours following ischaemia. This protection was similar to the protection conferred by surgically induced ischaemic preconditioning. This thesis provides important insights into the individual function of the prolyl hydroxylases and provides preliminary evidence that prolyl hydroxylase inhibitors may be useful in the treatment of ischaemia reperfusion injury in liver transplantation.

University of Macau / Institute of Chinese Medical Sciences