title: Activated microglia induce the production of reactive oxygen species and promote apoptosis of co-cultured retinal microvascular pericytes.
abstract: OBJECTIVE
  Pericyte apoptosis is a predominant feature of early diabetic retinopathy. In diabetic retinopathy, activated microglia migrate and release proinflammatory cytokines that contribute to disruption of the blood-retinal barrier, neuronal loss, and enhanced ROS production. 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
  Lipopolysaccharide (LPS)-activated microglia and pericytes were co-cultured in a cell culture 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 NF-κB-p65, and caspase-3 were determined by western blotting. One-way ANOVAs were 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). Co-culture with activated microglia significantly up-regulated NADPH oxidase subunits (NOX4, NOX2, and NCF1; P < 0.01) and down-regulated uncoupling protein 2 expression (P < 0.01) in pericytes. Pericyte ROS production increased by 20% in the activated microglia co-cultured group, and was inhibited by pretreatment with diphenyleneiodonium, coenzyme Q10, and N-acetylcysteine. The proapoptotic pericyte changes induced by co-culture with activated microglia included a 9.50% decrease in pericyte ΔΨm 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.
authors.affiliation: Department of Ophthalmology, Eye and ENT Hospital of Fudan University, 83 Fen Yang Road, Shanghai, 200031, People's Republic of China.|Institute of Eye Research, Eye and ENT Hospital of Fudan University, Shanghai, People's Republic of China.
authors.forename: Xinyi
authors.initials: X
authors.lastname: Ding
authors.affiliation: Department of Ophthalmology, Eye and ENT Hospital of Fudan University, 83 Fen Yang Road, Shanghai, 200031, People's Republic of China.|Institute of Eye Research, Eye and ENT Hospital of Fudan University, Shanghai, People's Republic of China.
authors.forename: Meng
authors.initials: M
authors.lastname: Zhang
authors.affiliation: Department of Ophthalmology, Eye and ENT Hospital of Fudan University, 83 Fen Yang Road, Shanghai, 200031, People's Republic of China.|Institute of Eye Research, Eye and ENT Hospital of Fudan University, Shanghai, People's Republic of China.
authors.forename: Ruiping
authors.initials: R
authors.lastname: Gu
authors.affiliation: Department of Ophthalmology, Eye and ENT Hospital of Fudan University, 83 Fen Yang Road, Shanghai, 200031, People's Republic of China.|Institute of Eye Research, Eye and ENT Hospital of Fudan University, Shanghai, People's Republic of China.|Institute of Brain Science, Fudan University, Shanghai, People's Republic of China.|State Key Laboratory of Medical Neurobiology, Fudan University, Shanghai, People's Republic of China.
authors.forename: Gezhi
authors.initials: G
authors.lastname: Xu
authors.affiliation: Department of Ophthalmology, Eye and ENT Hospital of Fudan University, 83 Fen Yang Road, Shanghai, 200031, People's Republic of China. whx577@163.com.|Institute of Eye Research, Eye and ENT Hospital of Fudan University, Shanghai, People's Republic of China. whx577@163.com.
authors.forename: Haixiang
authors.initials: H
authors.lastname: Wu
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis; D015153:Blotting, Western; D002417:Cattle; D002478:Cells, Cultured; D003921:Diabetes Mellitus, Experimental; D003930:Diabetic Retinopathy / Q000378:metabolism / Q000473:pathology*; D017628:Microglia / Q000378:metabolism* / Q000473:pathology; D055806:Microvessels / Q000378:metabolism / Q000473:pathology*; D018384:Oxidative Stress; D020286:Pericytes / Q000378:metabolism / Q000473:pathology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism*; D012160:Retina / Q000378:metabolism / Q000473:pathology*; D015854:Up-Regulation
chemical_list: D017382:Reactive Oxygen Species
keywords: Diabetic retinopathy (DR); Microglia activation; Pericyte apoptosis; Reactive oxygen species (ROS)