Specialized retinal endothelial cells modulates blood-retina- barrier in diabetic retinopathy
Endothelial cells (EC) play essential roles in retinal vascular homeostasis. This study aimed to characterize retinal EC heterogeneity and functional diversity using single-cell RNA sequencing (scRNA-seq). Systematic analysis of cellular compositions and cell-cell interaction networks identified a unique EC cluster with high inflammatory gene expression in the diabetic retina; sphingolipid metabolism in this cluster is a prominent aspect correlated with changes in retinal function. Among the sphingolipid-related genes, alkaline ceramidase 2 (ACER2) showed the most significant increase. We collected plasma and vitreous samples from patients with non-proliferative diabetic retinopathy (NPDR) and PDR for mass spectrometry analysis. Metabolomic profiling revealed that the ceramide levels were significantly elevated in NPDR and further increased in PDR compared with control patients. In vitro analyses showed that the ACER2 overexpression retarded endothelial barrier breakdown induced by ceramide, while silencing of ACER2 further disrupted the injury. Moreover, intravitreal injection of the recombinant ACER2 adeno-associated virus rescued diabetes-induced vessel leakiness, inflammatory response, and neurovascular disease in diabetic mouse models. Together, this study revealed a new diabetes-specific retinal EC population and a negative feedback regulation pathway that reduces ceramide content and endothelial dysfunction by upregulating ACER2 expression. These findings provide insights into cell-type targeted interventions for diabetic retinopathy. Overall design: Single live CD31+ cells from the retina were enriched using CD31 microbeads. The cell suspensions were mixed with filtered trypan blue to evaluate cell viability and concentration. Using the cell count and recommended cell concentration, we performed the following steps on a 10x Genomics chromium single-cell platform, and libraries were constructed according to the manufacturer's instructions of 10x Chromium Single Cell 3' Regent Kit v3 protocol (10X Genomics, 1000121). Briefly, single-cell suspensions were loaded to 10x Chromium. cDNA amplification and library construction were performed according to standard protocols. Libraries were sequenced using an Illumina NovaSeq 6000 sequencing system (paired-end multiplexing run, 150bp) by LC-Bio Technology Co., Ltd. (Hangzhou, China), at a minimum depth of 20,000 reads per cell.