Expression modification of uncoupling proteins and MnSOD in retinal endothelial cells and pericytes induced by high glucose: the role of reactive oxygen species in diabetic retinopathy.
Uncoupling proteins (UCPs) are mitochondrial transporters present in the inner membrane of mitochondria. They belong to the family of anion mitochondrial carriers. UCPs could act as proton carriers activated by metabolites and create a shunt between complexes of the respiratory chain and ATP synthase. The increased leakiness of the mitochondrial inner membrane to protons may be to minimize superoxide production by limiting the maximum Deltamu(H+). The purpose of this study was to detect UCP expression in retinal capillary cells and their modification in high levels of glucose. The role of reactive oxygen species (ROS) of mitochondria and UCPs in pathogenesis of diabetic retinopathy was investigated. Bovine retinal capillary endothelial cells and pericytes were cultured with selective culture media, respectively. Passage cells were cultured in three different glucose concentrations (5, 23, 30 mM) until passage four. ROS changes in mitochondria of these cells in different glucose concentrations were detected with scanning laser confocal microscopy (SLCM). The mitochondria membrane potential (Deltapsi), cell death rate and apoptosis rate were measured with flowing cytometry. UCP expression in retinal capillary cells was detected by immunocytochemistry. Expression and modification of MnSOD and uncoupling proteins (UCPs) in different concentrations of glucose were detected by means of semi-quantitative RT-PCR. ROS in mitochondria of both endothelial cells and pericytes increased as the glucose concentration of media increased. Deltapsi and cell death rate of endothelial cells increased also. ROS was correlated to Deltapsi and cell death rate positively in endothelial cells. No difference in Deltapsi and cell death rate among different glucose levels was found in pericytes. Apoptosis rate of endothelial cells and pericytes in high glucose levels was higher than that in lower glucose levels. UCP1 and UCP2 were expressed in cultured retinal capillary cells whereas UCP3 was not. At high levels of glucose, expression of UCP1, UCP2 and MnSOD increased to accommodate ROS production compensatively. The compensative mechanism disappeared when glucose concentration was too high (30 mM). The results of this study showed that increasing mitochondrial ROS could be induced by high glucose concentration. Those proteins related to antioxidation mechanism, such as MnSOD and UCPs, could exert compensative action to a certain extent. This compensative action was insufficient when the glucose concentration was too high.
Cui
Yan
Y
Department of Ophthalmology, Shanghai First People's Hospital, 85 Wu Jin Road, Shanghai 200080, PR China.
Xu
Xun
X
Bi
Hongsheng
H
Zhu
Qi
Q
Wu
Jianfeng
J
Xia
Xin
X
Qiushi Ren
Ho
Patrick C P
PC
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002417:Cattle; D016923:Cell Death / Q000187:drug effects; D002478:Cells, Cultured; D003930:Diabetic Retinopathy / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000187:drug effects; D005786:Gene Expression Regulation / Q000187:drug effects; D005947:Glucose / Q000494:pharmacology*; D007473:Ion Channels / Q000096:biosynthesis / Q000235:genetics; D008564:Membrane Potentials / Q000187:drug effects; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000096:biosynthesis* / Q000235:genetics; D020286:Pericytes / Q000187:drug effects* / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D012160:Retina / Q000187:drug effects* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D013482:Superoxide Dismutase / Q000096:biosynthesis / Q000235:genetics; D000071256:Uncoupling Protein 1; D000071246:Uncoupling Protein 2
D007473:Ion Channels; D024101:Mitochondrial Proteins; D017382:Reactive Oxygen Species; D000071256:Uncoupling Protein 1; D000071246:Uncoupling Protein 2; D013482:Superoxide Dismutase; D005947:Glucose