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Serine and 1-carbon metabolism are required for HIF-mediated protection against retinopathy of prematurity

ABSTRACT: We determined which metabolic pathways are activated by hypoxia-inducible factor 1-mediated (HIF-1-mediated) protection against oxygen-induced retinopathy (OIR) in newborn mice, the experimental correlate to retinopathy of prematurity, a leading cause of infant blindness. HIF-1 coordinates the change from oxidative to glycolytic metabolism and mediates flux through serine and 1-carbon metabolism (1CM) in hypoxic and cancer cells. We used untargeted metabolite profiling in vivo to demonstrate that hypoxia mimesis activates serine/1CM. Both [13C6] glucose labeling of metabolites in ex vivo retinal explants as well as in vivo [13C3] serine labeling of metabolites followed in liver lysates strongly suggest that retinal serine is primarily derived from hepatic glycolytic carbon and not from retinal glycolytic carbon in newborn pups. In HIF-1α2lox/2lox albumin-Cre-knockout mice, reduced or near-0 levels of serine/glycine further demonstrate the hepatic origin of retinal serine. Furthermore, inhibition of 1CM by methotrexate blocked HIF-mediated protection against OIR. This demonstrated that 1CM participates in protection induced by HIF-1 stabilization. The urea cycle also dominated pathway enrichment analyses of plasma samples. The dependence of retinal serine on hepatic HIF-1 and the upregulation of the urea cycle emphasize the importance of the liver to remote protection of the retina.

INSTRUMENT(S): Liquid chromatography MS/MS - Negative, Liquid chromatography MS/MS -Positive, Liquid chromatography MS/MS positive

PROVIDER: MTBLS856 | MetaboLights | 2019-07-05

REPOSITORIES: MetaboLights

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Serine and 1-carbon metabolism are required for HIF-mediated protection against retinopathy of prematurity.

Singh Charandeep C Hoppe George G Tran Vincent V McCollum Leah L Bolok Youstina Y Song Weilin W Sharma Amit A Brunengraber Henri H Sears Jonathan E JE

JCI insight 20190725 14

We determined which metabolic pathways are activated by hypoxia-inducible factor 1-mediated (HIF-1-mediated) protection against oxygen-induced retinopathy (OIR) in newborn mice, the experimental correlate to retinopathy of prematurity, a leading cause of infant blindness. HIF-1 coordinates the change from oxidative to glycolytic metabolism and mediates flux through serine and 1-carbon metabolism (1CM) in hypoxic and cancer cells. We used untargeted metabolite profiling in vivo to demonstrate tha ...[more]

PMID: 31341109

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Project description:Retinopathy of prematurity (ROP) is the most common cause of childhood blindness worldwide and is caused by oxygen therapy necessary to prevent mortality after premature birth. We have previously demonstrated the efficacy of systemic hypoxia inducible factor (HIF) stabilization through HIF prolyl hydroxylase inhibition (HIF PHi) in protecting retinal vasculature from oxygen toxicity in a mouse model of ROP or oxygen induced retinopathy (OIR). We definitively demonstrated that hepatic HIF-1 can be activated to confer this protection using systemic dimethyloxalylglycine (DMOG) to prevent HIF-1α degradation. In this study we compare Roxadustat, a small molecule stabilizer of HIF-1 currently in phase 3 clinical trials for increasing erythropoiesis in adult patients with chronic kidney disease, to DMOG. We demonstrate that Roxadustat induces vascular protection during hyperoxia to induce the coordinated sequential growth of retinal vasculature with a 3-fold reduction in oxygen induced capillary loss (p-=0.001). In order to define the molecular mechanism of protection, we further compared the transcriptome of both liver and retina after systemic treatment with Roxadustat or DMOG. Similar gene expression profiles were identified in liver but very different effects on transcription were found in retinal tissues because Roxadustat, in contrast to DMOG, directly targets retina, confirmed by western blot and by rescue of the hepatic HIF-1 KO, two criteria that DMOG treatment is unable to fulfill. Systems pharmacologic analysis demonstrates that Roxadustat induces typical HIF regulated genes critical to aerobic glycolysis in liver and retinal tissues whereas DMOG, acting through either secreted hepatokines or by influence of systemic DMOG, downregulates cell adhesion/extracellular matrix interaction pathways while increasing expression of histone cluster genes. Stratification of liver transcriptomes to secreted gene products again shows close consensus of hepatic genes induced by both small molecules, and includes upregulation of a plethora of angiogenic proteins such as plasminogen activator inhibitor (PAI-1), erythropoietin (EPO), and orosomucosoid 2 (ORM2). Secondary validation of these transcripts by serum ELISA confirms secretion of EPO and PAI-1 into blood from liver. These findings definitively demonstrate that HIF stabilization 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. Systems pharmacology analysis therefore explains why intermittent, low dosage of small molecule HIF stabilizers creates a profound protective phenotype, because both pathways can take advantage of cytoprotection induced by the liver and by retina synergistically. These data provide a rationale for considering low dose, intermittent systemic administration of Roxadustat, currently in phase 3 trials in adults with chronic kidney disease, to eradicate ROP in children.

Project description:Human CD34+ stem cells are mobilized from bone marrow to sites of tissue ischemia and play an important role in tissue revascularization. This study used a murine model to test the hypothesis that intravitreal injection of human CD34+ stem cells harvested from bone marrow (BMSCs) can have protective effects in eyes with diabetic retinopathy. Streptozotocin-induced diabetic mice (C57BL/6J) were used as a model for diabetic retinopathy. Subcutaneous implantation of Alzet pump, loaded with Tacrolimus and Rapamycin, 5 days prior to intravitreal injection provided continuous systemic immunosuppression for the study duration to avoid rejection of human cells. Human CD34+ BMSCs were harvested from the mononuclear cell fraction of bone marrow from a healthy donor using magnetic beads. The CD34+ cells were labeled with enhanced green fluorescent protein (EGFP) using a lentiviral vector. The right eye of each mouse received an intravitreal injection of 50,000 EGFP-labeled CD34+ BMSCs or phosphate buffered saline (PBS). Simultaneous multimodal in vivo retinal imaging system consisting of fluorescent scanning laser ophthalmoscopy (enabling fluorescein angiography), optical coherence tomography (OCT) and OCT angiography was used to confirm the development of diabetic retinopathy and study the in vivo migration of the EGFP-labeled CD34+ BMSCs in the vitreous and retina following intravitreal injection. After imaging, the mice were euthanized, and the eyes were removed for immunohistochemistry. In addition, microarray analysis of the retina and retinal flat mount analysis of retinal vasculature were performed. The development of retinal microvascular changes consistent with diabetic retinopathy was visualized using fluorescein angiography and OCT angiography between 5 and 6 months after induction of diabetes in all diabetic mice. These retinal microvascular changes include areas of capillary nonperfusion and late leakage of fluorescein dye. Multimodal in vivo imaging and immunohistochemistry identified EGFP-labeled cells in the superficial retina and along retinal vasculature at 1 and 4 weeks following intravitreal cell injection. Microarray analysis showed changes in expression of 162 murine retinal genes following intravitreal CD34+ BMSC injection when compared to PBS-injected control. The major molecular pathways affected by intravitreal CD34+ BMSC injection in the murine retina included pathways implicated in the pathogenesis of diabetic retinopathy including Toll-like receptor, MAP kinase, oxidative stress, cellular development, assembly and organization pathways. At 4 weeks following intravitreal injection, retinal flat mount analysis showed preservation of the retinal vasculature in eyes injected with CD34+ BMSCs when compared to PBS-injected control. The study findings support the hypothesis that intravitreal injection of human CD34+ BMSCs results in retinal homing and integration of these human cells with preservation of the retinal vasculature in murine eyes with diabetic retinopathy.

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