Inhibition of NR2F2 Attenuates Vascular Dysfunction in Diabetic Retinopathy by Alleviating Endoplasmic Reticulum Stress in Retinal Microvascular Endothelial Cells

Diabetic retinopathy (DR), the leading cause of adult blindness, has its risk increased by excessive endoplasmic reticulum stress (ERS). Nuclear receptor subfamily 2 group f member 2 (NR2F2) is an orphan nuclear receptor with essential roles in angiogenesis. However, its roles in DR remain unknown. Notably, NR2F2 protein expression was upregulated in streptozotocin-induced diabetic mice retina and the retinal endothelial cells characterized by endothelial marker CD31. Retinal NR2F2 expression was knocked down in diabetic mice using adeno-associated virus serotype 2. NR2F2 knockdown increased retinal thickness, decreased acellular capillary formation and FITC-labeled dextran leakage, and increased tight junction (TJ) protein ZO-1 expression in vivo. Additionally, NR2F2 knockdown inhibited reactive oxygen species generation and the ERS marker CHOP expression in vivo. In high glucose (HG)-induced human retinal microvascular endothelial cell (HRMEC) monolayers, NR2F2 knockdown inhibited FITC-dextran flux, increased transendothelial electrical resistance, reduced VEGFA secretion and restored expression and continuous distribution of TJ proteins. NR2F2 knockdown also reduced ERS markers expression and inhibited ERS-related signaling pathways in vitro. mRNA-seq analysis in NR2F2-knockdown HRMECs exposed to HG showed that NR2F2 inhibition upregulated CYB5R2 mRNA level. NR2F2 was bound directly to the CYB5R2 promoter for transcriptional repression in HRMECs. CYB5R2 overexpression inhibited ERS-related protein expressions and barrier dysfunction in HRMEC monolayers, and CYB5R2 knockdown reversed the endothelial protective effect of NR2F2 inhibition. In conclusion, NR2F2 knockdown attenuates vascular dysfunction by alleviating ERS in retinal microvascular endothelial cells and alleviates disease progression in DR mice. This may be achieved by reducing CYB5R2 transcriptional repression. Graphical Nuclear receptor subfamily 2 group f member 2 (NR2F2) knockdown increased the NADH-Cytochrome b5 reductase 2 (CYB5R2) expression in human retinal microvascular endothelial cells by reducing CYB5R2 transcriptional repression. The upregulation of CYB5R2 attenuated vascular dysfunction by inhibiting endoplasmic reticulum stress (ER Stress)-related pathways including PERK, IRE1, and ATF6 pathways in retinal microvascular endothelial cells, thereby inhibiting disease progression in diabetic retinopathy mice.