Abstract
BackgroundHypoxia plays a key role in ischaemic and neovascular disorders of the retina. Cellular responses to oxygen are mediated by hypoxia-inducible transcription factors (HIFs) that are stabilised in hypoxia and induce the expression of a diverse range of genes. The purpose of this study was to define the cellular specificities of HIF-1alpha and HIF-2alpha in retinal ischaemia, and to determine their correlation with the pattern of retinal hypoxia and the expression profiles of induced molecular mediators.Methodology/Principal FindingsWe investigated the tissue distribution of retinal hypoxia during oxygen-induced retinopathy (OIR) in mice using the bio-reductive drug pimonidazole. We measured the levels of HIF-1alpha and HIF-2alpha proteins by Western blotting and determined their cellular distribution by immunohistochemistry during the development of OIR. We measured the temporal expression profiles of two downstream mediators, vascular endothelial growth factor (VEGF) and erythropoietin (Epo) by ELISA. Pimonidazole labelling was evident specifically in the inner retina. Labelling peaked at 2 hours after the onset of hypoxia and gradually declined thereafter. Marked binding to Müller glia was evident during the early hypoxic stages of OIR. Both HIF-1alpha and HIF-2alpha protein levels were significantly increased during retinal hypoxia but were evident in distinct cellular distributions; HIF-1alpha stabilisation was evident in neuronal cells throughout the inner retinal layers whereas HIF-2alpha was restricted to Müller glia and astrocytes. Hypoxia and HIF-alpha stabilisation in the retina were closely followed by upregulated expression of the downstream mediators VEGF and EPO.Conclusions/SignificanceBoth HIF-1alpha and HIF-2alpha are activated in close correlation with retinal hypoxia but have contrasting cell specificities, consistent with differential roles in retinal ischaemia. Our findings suggest that HIF-2alpha activation plays a key role in regulating the response of Müller glia to hypoxia.
Highlights
Ischaemia is common to the major causes of blindness including diabetes and retinopathy of prematurity
The spatial and temporal pattern of hypoxia in oxygen-induced retinopathy (OIR) To investigate the effect of hypoxia on the hypoxia-inducible transcription factors (HIFs) pathway in the retina we used a well-characterised mouse model of oxygeninduced retinopathy (OIR) [21]
We have found that HIF-2alpha is expressed at a higher level than HIF-1alpha in the retina, and during retinal ischaemia is activated in restricted populations of cells within the area of inner retinal hypoxia, consistent with Muller glia in the inner nuclear layer and astrocytes in the nerve fibre layer
Summary
Ischaemia is common to the major causes of blindness including diabetes and retinopathy of prematurity. Ischaemia induces powerful endogenous responses to protect against tissue injury, including compensatory changes in blood flow, paracrine expression of neurotrophic factors and angiogenesis. There is an unmet need for therapies that promote endogenous protective responses and prevent harmful angiogenesis The development of such strategies depends on a clear understanding of oxygen sensing mechanisms in the retina and the roles of downstream mediators. Dimerisation of the stabilised HIF-alpha subunit with the HIF-beta subunit enables the transcriptional activity of a wide range of genes including those involved in cellular metabolism, hypoxia tolerance and angiogenesis, such as vascular endothelial growth factor (VEGF) and erythropoietin (EPO) [6,7,8]. Cellular responses to oxygen are mediated by hypoxia-inducible transcription factors (HIFs) that are stabilised in hypoxia and induce the expression of a diverse range of genes. The purpose of this study was to define the cellular specificities of HIF-1alpha and HIF-2alpha in retinal ischaemia, and to determine their correlation with the pattern of retinal hypoxia and the expression profiles of induced molecular mediators
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