Microglia Promote Endothelial Cell Activation Through NSUN2-Mediated SQLE m5C Modification in Diabetic Retinopathy

To investigate the effect of retinal microglia on the quiescence-activation balance and angiogenesis potential of endothelial cells in diabetic retinopathy (DR). Retinal microglia from diabetic mice were isolated and cocultured with endothelial cells. The quiescence-activation status of endothelial cells was determined by flow cytometry, EdU staining and expressions of activation markers including P21 and CDK1, and the angiogenesis potential was detected by tube formation assays. RNA sequencing was performed to identify the critical gene, which was regulated by inhibiting or overexpressing lentiviruses transfection. The concentration of cholesterol and its effect on the status and function of endothelial cells, as well as the signal pathways activation, were measured. The critical m5C modification related protein was identified using Western blotting and lentiviruses transfection. Retinal microglia isolated from diabetic mice impeded quiescence but promoted the activation of endothelial cells, and then subsequently enhanced the angiogenesis potential of endothelial cells. Besides, the expression of squalene epoxidase (SQLE) was significantly increased in endothelial cells cocultured with retinal microglia isolated from diabetic mice. Inhibiting SQLE expression in endothelial cells restrained their activation levels and angiogenesis potential, and overexpressing SQLE activated endothelial cells to facilitate angiogenesis. Mechanistically, SQLE increased the concentration of cholesterol in endothelial cells, which promoted their activation via the PI3K-AKT signaling pathway. Moreover, NSUN2 increased the m5C modification level of SQLE and increased its stability, increasing the expression of SQLE in endothelial cells cocultured with retinal microglia isolated from diabetic mice. This study provides new perspectives on the interaction between microglia and endothelial cells involved in the pathogenesis of DR and elucidates the detailed mechanism of NSUN2-mediated m5C modification of SQLE to regulate cholesterol metabolism and signaling pathway activation.