Correction of diabetic retinopathy by mitochondrial transfer

Abstract

Purpose : Mitochondrial (Mt) damage precedes histopathological abnormalities in diabetic retinopathy (DR). Recent studies show that Mt transfer can rescue cells from bioenergetic abnormalities and cell death. This study in DR cell culture and animal models examines whether i) Mt transfer can normalize bioenergetics of retinal cells and ii) if bone marrow derived mesenchymal stem cells (MSC) represent a source of Mt for transfer to damaged retinal endothelial cells (REC).

Methods : BREC, hMSC, and ARPE19 cells were cultured using established protocols. ARPE19 cells were depleted of mitochondria by culturing in media containing 50 ng/ml ethidium bromide and 50 µg/ml uridine for up to six passages. Mt were labeled using rLV-EF1-EGFP infection. Mt were isolated using differential centrifugation protocols. Mt haplogroups were analyzed at the sequence depth ID 30,000; range 1,000 to 100,000. Respiratory activity was assessed using a custom microfluidic respirometer. Retinal ischemia-reperfusion (I/R) mouse model was used to assess Mt transfer in vivo.

Results : The effect of Mt transfer on bioenergetics was assessed in Mt depleted Rho^o ARPE19 cells. ARPE19 cells have U5 mitochondrial haplogroup with high respiratory activity, however adherent Rho^o ARPE19 cells had no significant respiratory activity (0.2±0.08 pmol O₂/sec/10⁶ cells). Overnight incubation of adherent Rho^o ARPE19 cells with Mt isolated from control ARPE19 cells led to progressive increase in the respiratory activity over 10 days up to 3.4±0.7 pmol O₂/sec/10⁶ cells, which is equivalent to the activity of the wild type ARPE19 cells at day 10 (4.2±1.7 pmol O₂/sec/10⁶ cells). Moreover, normalization of respiratory activity due to Mt transfer improved viability and increased growth rate of Rho^o ARPE19 cells. We next determined if Mt transfer occurs between vascular progenitor cells and REC in DR cell culture and animal models. BREC treated with TNFα (10 ng/ml) for 24 hrs induced tunnelling nanotube (TNT) formation and Mt transfer by non-diabetic hMSC. No Mt transfer was observed in control BREC. 4 days following injection of MSC, TNT formation and Mt transfer was observed using IHC in I/R injured, but not in the uninjured mouse eye when injected with hMSC.

Conclusions : Exogenous Mt incorporate into cellular mitochondrial networks and normalize bioenergetics of damaged retinal cells. Mt transfer is a mechanism that provides paracrine support to damaged retinal vasculature.

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