Extracellular mitochondria induce cellular damage in photoreceptors and retinal pigment epithelium in diabetic macular edema

Abstract

Purpose : The damages in photoreceptor-retinal pigment epithelium (RPE) complex are clinically known in DME. We therefore investigated the effects of extracellular microvesicles containing mitochondria on such pathological changes in this translational research.

Methods : 661W cells (mouse cone photoreceptor cell line) were cultured in DMEM containing DME sera, and the cell culture supernatants were applied to a two-step centrifugation in order to obtain extracellular microvesicles as pellets. We performed flow cytometry after staining with Mitobright and PCR was applied to characterize microvesicles derived from 661W cells. The microvesicles were administered to ARPE19 cells (human RPE cell line) and cell death was evaluated by LIVE / DEAD assay. Seven days after the subretinal injection of the microvesicles in C57BL / 6 mice, the RPE flatmount was prepared and immunostained with ZO-1 antibody.

Results : Electron microscopic analyses demonstrated mitochondria with a cristae structure in the vitreous humor of patients with diabetic retinopathy. Flow cytometry revealed mitochondria positive staining in most extracellular microvesicles, and PCR showed a significant increase in mitochondrial DNA in such microvesicles (P<0.05). Administration of the microvesicles induced cell death of ARPE19 and 661W cells in a concentration-dependent manner (P<0.001 at any concentration), but cell death was significantly suppressed by the single-stranded oligo DNAs C151 and A151 (P<0.001 in both comparisons). The aggregation, deformation and enlargement in RPE cells were observed in the RPE flatmounts of the mice administrated with the microvesicles, whereas these changes were reversed by oligo DNAs.

Conclusions : These data suggest that microvesicles containing mitochondrial DNA are involved in photoreceptor-RPE damage in DME.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.