Abstract
Purpose :
In diabetic retinopathy, activated microglia migrate and release inflammatory cytokines, which initiate neuronal loss, disrupt the blood–retinal barrier, and enhance ROS production. Pericyte apoptosis is a predominant feature of early diabetic retinopathy. Reactive oxygen species (ROS) are implicated in pericyte death. However, the mechanism by which activated microglia affect retinal microvascular pericytes is unclear. We hypothesized that activated microglia may promote pericyte apoptosis by enhancing ROS production.
Methods :
We cocultured lipopolysaccharide (LPS)-activated microglia and pericytes in a Transwell system. Pericyte ROS production and the mitochondrial membrane potential (ΔΨm) were determined by flow cytometry. The pericyte protein expression levels of NADPH oxidase subunits, uncoupling protein-2, nuclear factor-κB, and caspase-3 were determined by western blotting. One-way ANOVA was used for statistical analysis.
Results :
LPS successfully activated the microglia, as demonstrated by their morphological and phenotype changes, and the significant increase in tumor necrosis factor secretion (P < 0.01). Coculture with activated microglia significantly upregulated NADPH oxidase subunits (NOX4, NOX2, and NCF1) (P < 0.01) and downregulated uncoupling protein-2 expression (P < 0.01) in pericytes. Pericyte ROS production increased by 20% in the activated microglia cocultured group, and was inhibited by pretreatment with diphenyleneiodonium, coenzyme Q10, or N-acetylcysteine. The proapoptotic pericyte changes induced by coculture with activated microglia included a decrease in pericyte ΔΨm by 9.50%, and significant increases in NF-κB–p65 nuclear translocation (P < 0.01) and activated caspase-3 (P < 0.01). These proapoptotic effects of activated microglia were inhibited by diphenyleneiodonium.
Conclusions :
Our results are consistent with our hypothesis that activated microglia may promote pericyte apoptosis by enhancing ROS production. Further studies are needed to examine retinal microglia activation and the corresponding changes in pericytes in a rat model of diabetes mellitus.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.