Abstract
Purpose :
Elevated homocysteine (Hcy) levels are routinely observed in diabetic patients, and high plasma Hcy is correlated with the severity of diabetic retinopathy. In experimental models of diabetic retinopathy, supplementation with Hcy is shown to exacerbate mitochondrial damage. In a healthy cell, damaged mitochondria are selectively cleared by mitophagy, a process where they are degraded by autophago-lysosomes, but in diabetes, the removal of the damaged mitochondria is also impaired. Our aim was to investigate the effect of Hcy on the removal of the damaged mitochondria in diabetic retinopathy.
Methods :
Human retinal endothelial cells (HRECs) incubated in 5mM (NG) or 20mM D- glucose, in the absence or presence of 100µM L-Homocysteine thiolactone hydrochloride (HG and HG+Hcy groups, respectively) for 24 or 48 hours, were analyzed for mitochondrial damage (ROS by MitoSox; membrane potential by JC-I staining) and copy numbers (ratio of mtDNA-encoded CytB and nuclear DNA-encoded β-actin in the genomic DNA). Mitophagy was evaluated by quantifying mitochondrial-lysosome fusion (LysoTracker-MitoTracker staining) and mitophagy flux (Flow Cytometry).
Results :
High glucose exposure for 24 or 48 hours had no effect on mitochondrial ROS, membrane potential, mtDNA copy numbers and mitophagy. However, within 24 hours of supplementation with Hcy (HG+Hcy group), mitochondrial ROS were significantly increased and membrane potential, copy numbers and mitophagy flux were decreased, and at 48 hours, mitochondrial damage was further worsened and the mitophagy flux was reduced.
Conclusions :
Homocysteine, in a hyperglycemic milieu, both accelerates and exacerbates mitochondrial damage and compromises the removal of the damaged mitochondria. The damaged mitochondria continue to produce ROS, leading to retinal capillary cell loss and the development of retinopathy. Thus, regulation of hyperhomocysteinemia has potential of inhibiting the development of diabetic retinopathy by ameliorating mitochondrial damage and removing the damaged mitochondria.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.