Mimicking Hypoxia by the Chemical Stabilization of Hif-1a in the Mouse Retina

Abstract

Purpose:: The hypoxic-preconditioned mouse retina is resistant to light-induced photoreceptor apoptosis. In the normoxic retina, iron dependent prolylhydroxylases hydroxylate the transcription factor hypoxia-inducible factor-1a (HIF-1a), which leads to its rapid degradation. Hypoxic preconditioning stabilizes HIF-1a causing the expression of downstream targets like erythropoietin (EPO). Here, we investigate whether the exogenous application of HIF-1 stabilizing compounds can be used to mimic hypoxic preconditioning with the goal to protect photoreceptors against light-induced degeneration.

Methods:: For the stabilization of HIF-1a, desferrioxamine (DFO, an iron chelator) and pyruvate (a metabolic intermediate of glycolysis), were combined and injected (s.c, and i.p, respectively) into 6-8 weeks old male mice (BALB/c and CD1) at a dose of 1g/kg. In vitro retinas were incubated in DMEM in the presence or absence of the chemicals (1mM). Real time PCR was used to investigate gene expression patterns and Western blotting was used to detect EPO in retinal extracts. Treated and untreated mice will be exposed to white fluorescent light. Light damage will be assessed biochemically through the detection of free nucleosomes using ELISA and morphologically by light microscopy.

Results:: HIF-1a is the predominant HIF-a isomer in the mouse retina. HIF-2a was expressed at about 20% and HIF-3a at about less than 5% of HIF-1a levels. Both prolylhydroxylase-1 (PHD1) and PHD2 were highly expressed. Application of DFO and pyruvate showed a synergetic effect and strongly stabilized HIF-1a in vivo. These compounds also stabilized HIF-1a in retinas cultivated in vitro. Peak expression of the downstream target Epo was around 72 hours post injection. The potential neuroprotective effect of the treatment in a light damage protocol will be presented.

Conclusions:: The predominant HIF-a isoform in the mouse retina is HIF-1a. HIF-1a can be stabilized in vivo (and in vitro) by the application of DFO and pyruvate. The treatment results in the production of HIF-1 downstream target proteins suggesting that chemical stabilization of HIF-1a mimics hypoxic preconditioning and might therefore be neuroprotective.

Keywords: neuroprotection • hypoxia • cell survival