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Mohamed Al-Sayed Al-Shabrawey, Heba M Saleh, Khaled Elmasry, Ismail Kaddour-Dejebbar, Amany Tawfik, Ahmed Ibrahim; Hypoxia is essential for hyperglycemia to induce permeability and angiogenesis in retinal endothelial cells via GLUT1. Invest. Ophthalmol. Vis. Sci. 2017;58(8):2512. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Most diabetic patients develop diabetic retinopathy (DR) regardless tight glycemic control, suggesting other factor, in addition to hyperglycemia (HG), is essential for progression of DR. Hypoxia is the most likely factor particularly, it is followed by advanced DR. We hypothesize that in diabetes, hypoxia sensitizes retinal endothelial cells (RECs) to HG via activation of glucose transporter-1 (GLUT1) to induce intracellular glucose transport and metabolic switch from oxidative phosphorylation (OXPHOS) to glycolysis.
Human retinal endothelial cells (HRECs) were treated with HG (30 mM D-glucose) or osmotic control (5mM D-glucose+25 mM L-glucose) for 3 days with or without low oxygen (1%O2) for additional 1-2 days. Expression of GLUT1 was examined by Western blotting. Barrier function of HRECs was assessed in the presence or absence of GLUT1 inhibitor (STF31, 10 μM in DMSO) by Electric Cell-substrate Impedance Sensing (ECIS) that measures transcellular electrical resistance (TER). Expression/organization of Zonula Occluden-1 (ZO-1) was evaluated by immunofluorescence. Bioenergetic profiles of HRECs was assessed by Seahorse XF analyzers that measures oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). The length of capillary/tube formation in HRECs was measured under the joint effect of hypoxia and HG versus the effect of hypoxia or HG alone and in the presence or absence of GLUT1 inhibitor. All data were summarized as means ± SD. Group differences were evaluated by ANOVA followed by Tukey Posthoc test.
Joint treatment of HRECs with hypoxia and HG significantly upregulated GLUT1 compared to normoxia/HG (2.3±0.7 vs 0.8±0.2 respectively, P<0.05). Hypoxia/HG also decreased TER, disrupted ZO1 expression/organization and enhanced capillary formation compared to the effect of normoxia/HG (P<0.05). Effects of hypoxia were prevented by GLUT1 inhibitors. Bioenergetics profile of HRECs demonstrated higher ECAR/OCR ratio (fold change of 1.5±0.2) under joint treatment with hypoxia and HG compared to hypoxia or HG (P<0.05) alone suggesting that HRECs preferentially use glycolysis rather than OXPHOS under hypoxia/ HG condition.
Hypoxia is required to induce the permeability and angiogenic effects of hyperglycemia via GLUT1, suggesting GLUT1 as a therapeutic target to prevent progression of DR.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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