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Amit Sharma, Charandeep Singh, Weilin Song, Youstina Bolok, Jin Zhang, George Hoppe, Jonathan E Sears; 3-Hydroxypyruvate accumulates in the retina of newborn mice exposed to hyperoxia and may contribute to angiostasis. Invest. Ophthalmol. Vis. Sci. 2018;59(9):5462.
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© ARVO (1962-2015); The Authors (2016-present)
Oxygen therapy of preterm infants causes retinovascular growth attenuation and vascular obliteration, the basis for subsequent neovascularization in retinopathy of prematurity. We have demonstrated that metabolic reprogramming is associated with a phenotype protective against hyperoxia. To determine the molecular mechanism of oxygen toxicity, we performed comprehensive metabolomics profiling of the retina from mouse pups subjected to hyperoxia.
Newborn mouse pups were subjected to 75% oxygen on postnatal day 7 (P7) with or without IP injections of FG-4592 to stabilize hypoxia-inducible factor 1α (HIF-1α) and retinas were collected at P10. Polar metabolites were extracted by chloroform/methanol separation, dried under vacuum at -4°C, derivatized using methoxyamine/pyridine and MSTFA and analyzed using full scan GC/MS. Primary cell cultures of retinal endothelial cells (RECs) were subjected to 2% oxygen or treated with FG-4592 followed by incubation with metabolites of interest. RECs total RNA and protein extracts were analyzed for the levels of hydroxypyruvate reductase (GRHPR) and phosphoaminotransferase (PSAT1) mRNA or HIF-1α protein by RT-qPCR or western blot, respectively.
Full scan GC/MS detected significantly elevated levels of 3-hydroxypyruvate (3HP) in the hyperoxic retina otherwise undetectable in normoxia. When added directly to cultured RECs, 3HP triggered increases in GRHPR and PSAT1 mRNA—enzymes essential to the conversion of 3HP to serine—as well as increased metabolic intermediates of 3HP catabolism (glycerate, phosphoglycerate, and serine). Unexpectedly, 3HP treatment destabilized HIF-1α in RECs during hypoxia. One mM 3HP reduced while two mM 3HP completely arrested outgrowth of endothelial cell sprouts from ex vivo choroidal tissue explants without cytotoxicity; identical concentrations of pyruvate had no effect.
Hyperoxia leads to the formation and accumulation of a reactive metabolite of the serine catabolic pathway, 3HP, which destabilizes HIF in vitro. Oxygen-induced build-up of retinal 3HP may contribute to retinovascular growth attenuation and vascular obliteration in the hyperoxic retina that is reversed by systemic HIF stabilization. 3HP metabolism may provide a mechanistic basis for oxygen toxicity.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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