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Abstract

Similar to high glucose (HG) levels, ablation of nucleoside diphosphate kinase B (NDPK-B) causes a diabetic retinopathy-like vascular pathology. NDPK-B deficient retinae and endothelial cells (ECs) showed increased levels of Angiopoietin 2 (Ang-2) and protein O-GlcNAcylation. In this study we investigated the regulation of the Ang-2 receptor, Tie-2, the Hexosamine Biosynthesis Pathway (HBP), and the protein O-GlcNAc removing enzyme O-GlcNAcase (OGA) in NDPK-B deficiency. The Ang-2 elevation in NDPK-B-/- retinae was found to be crucial for the development of vasoregression. The regulation of Tie-2 was observed in NDPK-B-/- retinae, specifically in the deep capillary layer. In vitro, the upregulation of Tie-2 was mirrored in NDPK-B-depleted ECs from different origins, whereas no alteration of Tie-2 expression was observed by HG treatment. Tie-2 enrichment occurred at the endothelial plasma membrane upon NDPK-B depletion. Similar to HG, NDPK-B depletion blunted Tie-2 receptor phosphorylation. Although both HG and NDPK-B deficiency elevated the secretion of Ang-2, intracellular and released Ang-2 was higher in NDPK-B depleted ECs. Interference with Tie-2 expression and Ang-2-binding attenuated NDPK-B depletion-induced, but not HG-induced Ang-2, upregulation. Mimicking NDPK-B depleted conditions, the overexpression of recombinant Ang-2 promoted the upregulation of Tie-2 in ECs. NDPK-B deficiency induced an increase in protein O-GlcNAcylation. Mass spectrometry measurements showed that NDPK-B depletion in ECs elevated the HBP end product, UDP-GlcNAc, without changes in glycolysis, indicating a shift from glycolysis to the HBP. Although NDPK-B accounted for about 20% of the total NDPK activity in ECs and 35% in retinae, its ablation did not alter nucleotide triphosphate levels. NDPK-B depletion led to an increase in the protein content of glutamine:fructose-6-phosphate amidotransferase (GFAT), the rate-limiting enzyme of the HBP, accompanied by an EC-specific suppression of OGA activity. Both together resulted in an increase in protein O-GlcNAcylation and Ang-2 levels which was independent of the catalytic activity of NDPK B. It was similarly rescued by adenoviral re-expression of either wild type or a catalytically inactive mutant of NDPK-B. In summary, NDPK-B depletion directly influences the participation of the HBP in glucose metabolism via alteration of GFAT levels and suppression of OGA activity, which subsequently further increases protein O-GlcNAcylation and upregulates Ang-2. NDPK-B deficiency additionally induces an increase in Tie-2 levels which further supports Ang-2 production and secretion in a positive feedback loop. As enhanced Tie-2 expression was detected on the vasculature of NDPK-B-deficient mice, showing early stages of pathological vasoregression, it likely contributes together with enhanced protein O-GlcNAcylation to this Ang-2-driven pathology leading to vasoregression.