Richards, T; (2008) The role of copper in hypoxia inducible factor-1 expression: A potential mechanism of chemical hypoxia preconditioning for ischaemia reperfusion injury. Doctoral thesis , UCL (University College London).

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Abstract

Introduction: Hypoxia Preconditioning (HPC) involves a short period of hypoxia prior to a more sustained ischaemia. This method reduces Ischaemia Reperfusion Injury (IRI) in vascular surgery. The molecular response to hypoxia and HPC is mediated by the transcription factor Hypoxia Inducible Factor 1 (HIF-1). HIF-1 activation involves cell signalling by Reactive Oxygen Species (ROS), which are normally buffered by metalloenzymes, in particular by the transition metal ion copper. We wished to assess how altering intracellular copper concentration would affect HIF-1 expression. This may provide a mechanism to artificially induce HPC and modify IRI in vivo. Methods: The effect of altering copper levels on HIF-1 was assessed in cell culture, animal models and in human liver. HIF-1 activation was studied in HEP3B cell culture HIF-1 nuclear protein by Western Blot, HIF-1 transcription activation by Northern Blot for VEGF mRNA and in a reporter construct. In vivo analysis for HIF-1 was performed in rodent models of copper excess and copper deficiency. In humans HIF-1 expression was assessed in patients with Wilson's disease and those with IRI following liver transplantation. Results: Increased copper increased HIF-1 nuclear protein. Reduced copper reduced HIF-1 nuclear protein response to hypoxia. Increased copper increased VEGF mRNA. The reporter construct confirmed these findings. Animal models produced conflicting results that may have been due to confounding factors. Copper toxic biopsies from patients with Wilson's disease showed no effect, this may reflect chronic copper exposure rather than an acute response. HIF-1 may be increased in IRI biopsies. Conclusion: In cell culture intracellular copper concentration appears to affect the HIF-1 pathway of activation and response. In vivo analysis was less conclusive. Artificial control of HIF-1 may be feasible by altering intracellular copper balance.

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