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Platt, Simon (2022) The role of factor inhibiting HIF-1 in the cardiovascular responses to hypoxia. PhD thesis, University of Nottingham.

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Abstract

Many diseases of the cardiovascular system involve an element of hypoxia due to impairments in oxygen delivery. Central to the cellular responses to hypoxia is the hypoxia inducible factor (HIF) transcription factor signalling system. The HIF system is somewhat unique in that the labile α subunit is constantly expressed, but signalling constrained in an oxygen dependent manner by both prolyl hydroxylase domain (PHD) enzymes and an asparaginyl hydroxylase, factor inhibiting HIF1 (FIH1). PHD enzymes regulate the degradation of HIFα, whereas FIH1 inhibits co-factor binding needed to induce target gene expression, both in an oxygen dependent manner. This thesis aimed to understand the role of FIH1 in aspects of the cardiovascular responses to hypoxia.

In chapters three and four, a mouse model with a null mutation in the HIF1AN gene, which encodes for FIH1, was studied. Cardiac function and metabolism was assessed using Langendorff isolated heart perfusion, in combination with radioisotope tracers (3H palmitate and glucose), and comparisons made to wild type (WT) littermate controls. FIH1-/- hearts were found to have equivalent rates of fatty acid oxidation to WT controls. Furthermore, there was no evidence of alterations in mitochondrial content or function in the hearts of FIH1-/- mice. Upon exposure to chronic hypoxia (11% O2, 3 weeks duration) there was no evidence of differential response in FIH1-/- animals and no change in cardiac or skeletal muscle mitochondrial content in either genotype.

Hearts from WT and FIH1-/- mice were exposed to a Langendorff ischaemiareperfusion model. FIH1 mice recovered a greater proportion of baseline function than WT controls. This enhanced recovery was associated with increased rate of glycolysis in both the pre ischaemic and reperfusion periods and an increased degree of lactate accumulation during ischaemia.

In chapter five, to study the effect of FIH1 inhibition upon angiogenic signalling, acute treatment with the selective FIH1 inhibitor DM-NOFD was applied to cell culture models. Hypoxia response element signalling assays revealed that DM-NOFD treatment approximately doubled the HIF signal induced by hypoxia (1% O2) in HeLa cells. Novel bioluminescent techniques were combined with CRISPR Cas9 gene editing to append an 11 amino acid split luciferase tag on the C-terminal of the VEGF-A gene in human umbilical vein endothelial cells. This allowed for monitoring of VEGF-A production, and was used to assay the effect of DM-NOFD upon VEGF-A production in hypoxia (1% O2), which was not enhanced by DM-NOFD treatment.

In summary, loss of FIH1 was shown to modulate carbohydrate, but not lipid metabolism in the heart. FIH1 loss is associated with a degree of protection from ischaemic injury in the isolated heart. Although the inhibition of FIH1 in HeLa cells demonstrated a doubling of hypoxia induced HIF signalling, this did not translate to an increase in VEGF-A production by vascular endothelial cells. The techniques developed for monitoring VEGF-A production (split luciferase tag by CRISPR editing), show promise for broader application.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	Cole, Mark Woolard, Jeanette
Keywords:	Hypoxia inducible factor, HIF, Cardiovascular system
Subjects:	Q Science > QM Human anatomy R Medicine > RB Pathology
Faculties/Schools:	UK Campuses > Faculty of Medicine and Health Sciences > School of Life Sciences
Item ID:	68651
Depositing User:	Platt, Simon
Date Deposited:	31 Jul 2022 04:41
Last Modified:	31 Jul 2024 04:30
URI:	https://eprints.nottingham.ac.uk/id/eprint/68651

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