Early mitochondrial fragmentation in retinal endothelial cells and vascular dysfunction in ocular hypertension glaucoma.

Abstract

Purpose : Vascular dysfunction has been proposed to contribute to the pathogenesis of glaucoma.
Early metabolic stress in retinal endothelial cells is predicted to have harmful effects on the integrity of the neurovascular unit and might compromise vascular homeostasis. Here, we tested the hypothesis that ocular hypertension triggers early mitochondrial alterations in endothelial cells impairing the integrity of the blood-retinal-barrier (BRB) in glaucoma.

Methods : Ocular hypertension was induced by injection of magnetic microbeads into the anterior chamber of EndoMito-EGFP mice, a strain expressing GFP selectively in the mitochondria of endothelial cells. Capillary density, mitochondrial volume, and the number of mitochondrial components from glaucomatous and control retinas were 3D-reconstructed and quantified using Imaris (Bitplane). The expression of dynamin-related protein (DRP-1), mitofusin-2 (MFN-2) and optic atrophy-1 (OPA-1) was assessed by western blot analysis of primary endothelial cells. Mitochondrial structure was evaluated by transmission electron microscopy (TEM) and their oxygen consumption rate was monitored by Seahorse analysis. The integrity of the BRB was quantified using Evans blue.

Results : Our data demonstrate a significant decrease in total endothelial cell mitochondrial volume at two and three weeks after induction of ocular hypertension (108 ± 5 µm³ or 93 ± 7 µm³, respectively) relative to non-injured naïve retinas (140 ± 2 µm³) (mean ± S.E.M x 10^-3, ANOVA p <0.001, N=6/group). Frequency distribution analysis showed a substantial increase of smaller mitochondrial complexes (<0.5 µm³) in endothelial cells from glaucomatous retinas. Significant upregulation of DRP-1 was found in vessels isolated from hypertensive retinas compared to naïve controls, while MFN-2 and OPA-1 remained unchanged. Pathological alterations in the mitochondrial outer membrane and cristae were confirmed by TEM, and were accompanied by a 2-fold reduction in the oxygen consumption rate (n=3-6/group). Structural changes in endothelial cell mitochondria correlated with a 2.6-fold increase in BRB leakage in glaucoma relative to controls (N=4/group, P<0.01).

Conclusions : Our data demonstrate that ocular hypertension triggers early structural and molecular alterations in endothelial cell mitochondria leading to vascular dysfunction in glaucoma.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.