Serine and 1-carbon metabolism are required for HIF-mediated protection against retinopathy of prematurity (original) (raw)
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Investigative Ophthalmology & Visual Science, 2013
PURPOSE. To study the effect of systemic hypoxia-inducible factor prolyl hydroxylase inhibition (HIF PHDi) in the rat 50/10 oxygen-induced retinopathy (OIR) model. METHODS. Oxygen-induced retinopathy was created with the rat 50/10 OIR model. OIR animals received intraperitoneal injections of dimethyloxalylglycine (DMOG, 200 lg/g), an antagonist of a-ketoglutarate cofactor and inhibitor for HIF PHD, on postnatal day (P)3, P5, and P7. Control animals received intraperitoneal injections of PBS. On P14 and P21, animals were humanely killed and the effect on vascular obliteration, tortuosity, and neovascularization quantified. To analyze HIF and erythropoietin, rats at P5 were injected with DMOG (200 lg/g). Western blot or ELISA measured the levels of HIF-1 and Epo protein. Epo mRNA was measured by quantitative PCR. RESULTS. Alternating hyperoxia and hypoxia in untreated rats led to peripheral vascular obliteration on day P14 and P21. Rats that were treated with systemic DMOG by intraperitoneal injections had 3 times less ischemia and greater peripheral vascularity (P ¼ 0.001) than control animals treated with PBS injections. Neovascularization similarly decreased by a factor of 3 (P ¼ 0.0002). Intraperitoneal DMOG administration increased the levels of HIF and Epo in the liver and brain. Serum Epo also increased 6-fold (P ¼ 0.0016). Systemic DMOG had no adverse effect on growth of rats treated with oxygen. CONCLUSIONS. One of the many controversies in the study of retinopathy of prematurity is whether hyperoxia or alternating hyperoxia and hypoxia creates the disease phenotype in humans. We have previously demonstrated that PHDi prevents OIR in mice exposed to 5 days of sustained 75% oxygen followed by 5 days of 21% oxygen. The 50/10 rat experiments demonstrate that PHDi is also effective in a 24-hour alternating hyperoxia-hypoxia model. The rat OIR model further validates the therapeutic value of HIF PHDi to prevent retinopathy of prematurity because it reduces oxygen-induced vascular obliteration and retinovascular growth attenuation in prolonged and/or alternating hyperoxia.
Proceedings of the National Academy of Sciences of the United States of America, 2016
Retinopathy of prematurity (ROP) causes 100,000 new cases of childhood blindness each year. ROP is initiated by oxygen supplementation necessary to prevent neonatal death. We used organ systems pharmacology to define the transcriptomes of mice that were cured of oxygen-induced retinopathy (OIR, ROP model) by hypoxia-inducible factor (HIF) stabilization via HIF prolyl hydroxylase inhibition using the isoquinolone Roxadustat or the 2-oxoglutarate analog dimethyloxalylglycine (DMOG). Although both molecules conferred a protective phenotype, gene expression analysis by RNA sequencing found that Roxadustat can prevent OIR by two pathways: direct retinal HIF stabilization and induction of aerobic glycolysis or indirect hepatic HIF-1 stabilization and increased serum angiokines. As predicted by pathway analysis, Roxadustat rescued the hepatic HIF-1 knockout mouse from retinal oxygen toxicity, whereas DMOG could not. The simplicity of systemic treatment that targets both the liver and the e...
Oxygen-dependent diseases in the retina: Role of hypoxia-inducible factors
Experimental Eye Research, 2006
The function of the retina is sensitive to oxygen tension. Any change in the perfusion pressure of the eye affects the retina although the eye is able to autoregulate its hemodynamics. Systemic hypoxemia (lung or heart disease) or a vascular disease in the retina can cause retinal hypoxia. All the hypoxia-dependent events in cells appear to share a common denominator: hypoxia-inducible factor (HIF), which is a heterodimeric transcription factor, a protein. HIF comprises a labile a subunit (1e3), which is regulated, and a stable b subunit, which is constitutively expressed. Both are helix-loop-helix factors and belong to the PAS-domain family of transcription factors. Oxygen plays the key role in stabilizing HIF-1a and its function. When the oxygen tension is normal, HIF-1a is rapidly oxidized by hydroxylase enzymes, but when cells become hypoxic, HIF-1a escapes the degradation and starts to accumulate, triggering the activation of a large number of genes, like vascular endothelial growth factor (VEGF) and erythropoietin. HIF-1a has been shown to have, either clinically or experimentally, a mediating or contributing role in several oxygen-dependent retinal diseases such as von HippeleLindau, proliferative diabetic retinopathy, retinopathy of prematurity and glaucoma. In retinitis pigmentosa and high-altitude retinopathy, however, the evidence is still indirect. There are three different strategies available for treating retinal diseases, which have all shown promising results: retinal cell transplantation or replacement, gene replacement, and pharmacological intervention. Specifically, recent results show that the HIF pathway can be used as a therapeutic target, although there is still a long way to go from bench to clinic. HIF can be stabilized by inhibiting prolyl hydroxylase or by blocking the VHL:HIF-a complex if angiogenesis is the goal, as in retinitis pigmentosa. On the other hand, the downregulation of HIF has a pivotal role if we are to inhibit neovascularization, as in proliferative diabetic retinopathy. To date, several small-molecule inhibitors of HIF have been developed and are entering clinical trials. HIF is a remarkable example of a single transcription factor that can be regarded as a ''master switch'' regulating all the oxygen-dependent retinal diseases.
Active HIF-1 in the Normal Human Retina
Journal of Histochemistry & Cytochemistry, 2009
A unique feature of the retina is the presence of photoreceptors, which require an enormous amount of oxygen for the conversion of light to an electrical signal. Hypoxia-inducible factor-1 alpha (HIF-1α) is a transcription factor that is the master regulator of cellular adaptation to low oxygen tension. Only in hypoxic conditions is HIF-1α protein stabilized and translocated to the nucleus, where it induces transcription of target genes involved in oxygen delivery and energy metabolism. We hypothesized that HIF-1α is constitutively stabilized and active in the normal human retina. We investigated the cellular distribution of HIF-1α and the expression of its downstream targets, vascular endothelial growth factor (VEGF), glucose transporter 1 (GLUT-1), and carbonic anhydrase IX (CAIX), by immunohistochemistry and immunoblotting in the retina of normal rats and human donor eyes. Both human and rat retinas displayed prominent staining of HIF-1α in nuclei of most cell types in inner and ...
3-Hydroxypyruvate Destabilizes Hypoxia Inducible Factor and Induces Angiostasis
Investigative ophthalmology & visual science, 2018
Transcriptional analysis of retina protected by hypoxia-inducible factor (HIF) stabilization demonstrates an increase in genes associated with aerobic glycolysis. We hypothesized that since protection is associated with a change in metabolism, oxygen-induced metabolites might transduce oxygen toxicity. We used global metabolic profiling to identify retinal metabolites increased in hyperoxia compared to normoxia. Untargeted gas chromatography mass spectroscopy (GC-MS) was performed on both mouse retina samples collected in hyperoxia and on primary human retinal endothelial cells, each with and without HIF stabilization. After identifying 3-hydropxypyruvate (3OH-pyruvate) as a unique hyperoxic metabolite, endothelial cells in culture and choroidal explants were challenged with 3OH-pyruvate in order to determine how this glycolytic intermediate was metabolized, and whether it had an effect on angiogenesis. 3OH-pyruvate was one of five metabolites at least 2.0-fold elevated in hyperoxia...
Rank Order of Small Molecule Induced Hypoxiamimesis to Prevent Retinopathy of Prematurity
Frontiers in Cell and Developmental Biology, 2020
Here we rank order small molecule inhibitors of hypoxia inducible factor (HIF) prolyl hydroxylases (PHDs) using severity of oxygen induced retinopathy (OIR) as an outcome measure. Dose response analyses in cell cultures of hepatoma (Hep3B), retinal Müller cells (MIO-M1) and primary retinal endothelial cells were conducted to evaluate potency by comparing dose to HIF-1,2 protein levels by western blotting. In vivo dose response was determined using the luciferase-transgene HIF reporter (luc-ODD). Each compound was placed in rank order by their ability to reduce neovascularization and capillary drop out in the OIR mouse model. An Epas1 KO confined to retinal Müller cells was used to determine whether successful protection by HIF stabilization requires HIF-2. Two candidate small molecules can prevent OIR by stabilizing HIF-1 to prevent oxygen induced growth attenuation and vascular obliteration. Müller cell HIF-2, the mediator of pathologic retinal angiogenesis, is not required for protection. The lack of dependence on Müller cell HIF-2 predicts that inhibition of HIF PHD will not drive pathological angiogenesis.
Pediatric research, 2014
Hypoxic-ischemic insults to the neonatal brain may cause neurodevelopmental disorders. Vulnerability of different areas of the neural tissue to hypoxic-ischemic stress might be explained by either heterogeneous sensitivity to oxygen or neuroprotective capability. Our understanding of regional heterogeneity is still incomplete in terms of metabolic reconfiguration and/or activation of neuroprotective mechanisms. We studied, by western blotting, reverse-transcriptase PCR, and tandem mass spectrometry, the response of retina and choroid at protein, gene, and metabolic levels during hypoxia in a piglet model of acute postnatal hypoxia. We evidenced a metabolic shift towards glycolysis in choroid after hypoxia while retina experienced a dramatic energy stress with decreased mitochondrial metabolites. Hypoxia-inducible transcription factor-1α (HIF-1α) was not stabilized in retina during hypoxia, supported by a deficient signaling from v-akt murine thymoma viral oncogene (AKT) and ERK1/2, ...