Diabetic retinopathy (DR) remains one of the most common complications of diabetes despite great efforts to uncover its underlying mechanisms. The pathogenesis of DR is characterized by the deterioration of the neurovascular unit (NVU), showing damage of vascular cells, activation of glial cells and dysfunction of neurons. Activation of the hexosamine biosynthesis pathway (HBP) and increased protein O-GlcNAcylation have been evident in the initiation of DR in patients and animal models. The impairment of the NVU, in particular, damage of vascular pericytes and endothelial cells arises in hyperglycemia-independent conditions as well. Surprisingly, despite the lack of hyperglycemia, the breakdown of the NVU is similar to the pathology in DR, showing activated HBP, altered O-GlcNAc and subsequent cellular and molecular dysregulation. This review summarizes recent research evidence highlighting the significance of the HBP in the breakdown of the NVU in hyperglycemia-dependent and -independent manners, and thus identifies joint avenues leading to vascular damage as seen in DR and thus identifying novel potential targets in such retinal diseases.

The aim of this study is to investigate the vascular outcome after intravitreal mesenchymal stem cell (MSC) administration in rats without or with damage to the neurovascular unit [transgenic (TGR) rats]. Male Sprague‐Dawley (SD) and TGR rats received an intravitreal injection of 2 × 10 ⁴ rat bone marrow‐derived MSCs (BMSCs) or human adipose‐derived stem cells (ASCs) at postnatal d 30. After 4 wk, vasculature, neuronal function, and gene expression in the retinas were evaluated using retinal morphometry, electroretinography, immunofluorescence, Western blot, and quantitative PCR. Intravitreal administration of rat BMSCs and human ASCs in both SD and TGR eyes induced cataract, loss of pericytes, and increased formation of acellular capillaries. BMSCs remained in the vitreous cavity and did not migrate into the retinas. Intravitreal administration of BMSCs impacted retinal neuronal function in neither SD nor TGR rats. Retinal glial activation, elevation of IL‐1β, C3, arginase 1, and heat shock protein 90 were detected in BMSC‐injected SD rats. Intravitreal administration of MSCs induces cataract, retinal vasoregression, activation of retinal glial cells, and inflammatory response in rat eyes.—Huang, H., Kolibabka, M., Eshwaran, R., Chatterjee, A., Schlotterer, A., Willer, H., Bieback, K., Hammes, H.‐P., Feng, Y. Intravitreal injection of mesenchymal stem cells evokes retinal vascular damage in rats. FASEB J. 33, 14668‐14679 (2019). www.fasebj.org

Mitochondria are dynamic organelles regulated by fission and fusion processes. The fusion of membranes requires elaborative coordination of proteins and lipids and is particularly crucial for the function and quality control of mitochondria. Phosphatidic acid (PA) on the mitochondrial outer membrane generated by PLD6 facilitates the fusion of mitochondria. However, how PA promotes mitochondrial fusion remains unclear. Here, we show that a mitochondrial outer membrane protein, NME3, is required for PLD6-induced mitochondrial tethering or clustering. NME3 is enriched at the contact interface of two closely positioned mitochondria depending on PLD6, and NME3 binds directly to PA-exposed lipid packing defects via its N-terminal amphipathic helix. The PA binding function and hexamerization confer NME3 mitochondrial tethering activity. Importantly, nutrient starvation enhances the enrichment efficiency of NME3 at the mitochondrial contact interface, and the tethering ability of NME3 contributes to fusion efficiency. Together, our findings demonstrate NME3 as a tethering protein promoting selective fusion between PLD6-remodeled mitochondria for quality control.

Nucleoside diphosphate kinase B (NDPK-B) acts as a protective factor in the retinal vasculature. NDPK-B deficiency leads to retinal vasoregression mimicking diabetic retinopathy (DR). Angiopoetin 2 (Ang-2), an initiator of retinal vasoregression in DR, is upregulated in NDPK-B deficient retinas and in NDPK-B depleted endothelial cells (ECs) in vitro. We therefore investigated the importance of Ang-2 in NDPK-B deficient retinas and characterized the mechanisms of Ang-2 upregulation upon NDPK-B depletion in cultured ECs. The crucial role of retinal Ang-2 in the initiation of vasoregression was verified by crossing NDPK-B deficient with Ang-2 haplodeficient mice. On the molecular level, FoxO1, a transcription factor regulating Ang-2, was upregulated in NDPK-B depleted ECs. Knockdown of FoxO1 abolished the elevation of Ang-2 induced by NDPK-B depletion. Furthermore O-GlcNAcylated FoxO1 was found preferentially in the nucleus. An increased O-GlcNAcylation of FoxO1 was revealed upon NDPK-B depletion. In accordance, the inhibition of protein O-GlcNAcylation normalized NDPK-B depletion induced Ang-2 upregulation. In summary, we demonstrated that the upregulation of Ang-2 upon NDPK-B deficiency is driven by O-GlcNAcylation of FoxO1. Our data provide evidence for a central role of protein O-GlcNAcylation in NDPK-B associated vascular damage and point to the hexosamine pathway as an important target in retinal vasoregression.

Our previous studies identified that retinal endothelial damage caused by hyperglycemia or nucleoside diphosphate kinase-B (NDPK-B) deficiency is linked to elevation of angiopoietin-2 (Ang-2) and the activation of the hexosamine biosynthesis pathway (HBP). Herein, we investigated how NDPK-B is involved in the HBP in endothelial cells (ECs). The activities of NDPK-B and O-GlcNAcase (OGA) were measured by in vitro assays. Nucleotide metabolism and O-GlcNAcylated proteins were assessed by UPLC-PDA (Ultra-performance liquid chromatography with Photodiode array detection) and immunoblot, respectively. Re-expression of NDPK-B was achieved with recombinant adenoviruses. Our results show that NDPK-B depletion in ECs elevated UDP-GlcNAc levels and reduced NDPK activity, similar to high glucose (HG) treatment. Moreover, the expression and phosphorylation of glutamine:fructose-6-phosphate amidotransferase (GFAT) were induced, whereas OGA activity was suppressed. Furthermore, overall protein O-GlcNAcylation, along with O-GlcNAcylated Ang-2, was increased in NDPK-B depleted ECs. Pharmacological elevation of protein O-GlcNAcylation using Thiamet G (TMG) or OGA siRNA increased Ang-2 levels. However, the nucleoside triphosphate to diphosphate (NTP/NDP) transphosphorylase and histidine kinase activity of NDPK-B were dispensable for protein O-GlcNAcylation. NDPK-B deficiency hence results in the activation of HBP and the suppression of OGA activity, leading to increased protein O-GlcNAcylation and further upregulation of Ang-2. The data indicate a critical role of NDPK-B in endothelial damage via the modulation of the HBP.