title: SOD2 contributes to anti-oxidative capacity in rabbit corneal endothelial cells.
abstract: OBJECTIVE
  Corneal endothelial cells are rich in mitochondria, a potential source of reactive oxygen species (ROS). ROS have been implicated in endothelial cell loss during aging or in endothelial dystrophies. In this study we examined the anti-oxidative role of mitochondrial superoxide dismutase (SOD2) in corneal endothelial cells.
  
  
  METHODS
  SOD2 expression was examined by RT-PCR and western blot analysis in fresh rabbit corneal endothelium (RCE) and cell cultures. SOD2 activity, total reactive oxygen species (ROS), mitochondrial ROS, mitochondrial membrane potential (MMP), and apoptotic levels were examined in untreated, SOD2 siRNA and viral vector shRNA treated RCE cells. Scrambled siRNA and shRNA sequence targeting non-mammalian genes were used as controls.
  
  
  RESULTS
  SOD2 is expressed in both fresh and cultured rabbit corneal endothelium. SOD2 expression was reduced by ~80%-90% in cultured RCE using either siRNA or shRNA approaches. SOD2 activity was decreased by ~70%-80% for both approaches. Total cell ROS was significantly increased in shSOD2 lentivirus treated cells (9%±6%) relative to control transduction (0.4%±0.1%). MitoSOX™ staining for mitochondrial ROS in siSOD2 treated RCE cells was dramatically increased. Two minutes of UV irradiation increased total ROS levels by 15%, whereas in shSOD2 treated cells UV induced ROS was increased 29%±5% (p<0.05). MMP was reduced in shSOD2 viral treated cells by 66%±3%, significantly greater than in control transduced cells (15%±8%, p<0.05). Apoptosis increased by 1.5 fold in shSOD2 virus treated samples compared with scrambled virus and untreated cells.
  
  
  CONCLUSIONS
  SOD2 is expressed in both fresh and cultured rabbit corneal endothelium. siRNA and shRNA approaches are able to efficiently knockdown SOD2 expression and reduce enzyme activity in RCE cells. Decreased SOD2 activity causes elevated ROS production, mitochondrial membrane potential loss and early cell apoptosis. These results indicate that SOD2 is a significant anti-oxidative enzyme in RCE cells.
authors: School of Optometry, Indiana University, Bloomington, IN, USA.
authors: Cailing
authors: C
authors: Liu
authors: Diego
authors: D
authors: Ogando
authors: Joseph A
authors: JA
authors: Bonanno
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000378:metabolism*; D017209:Apoptosis / Q000528:radiation effects; D015153:Blotting, Western; D002478:Cells, Cultured; D003315:Cornea / Q000166:cytology / Q000201:enzymology* / Q000528:radiation effects; D042783:Endothelial Cells / Q000166:cytology / Q000201:enzymology* / Q000528:radiation effects; D004728:Endothelium, Corneal / Q000166:cytology / Q000201:enzymology* / Q000528:radiation effects; D015870:Gene Expression; D020868:Gene Silencing; D053078:Membrane Potential, Mitochondrial / Q000528:radiation effects; D008928:Mitochondria / Q000201:enzymology* / Q000235:genetics / Q000528:radiation effects; D018384:Oxidative Stress; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D017382:Reactive Oxygen Species / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013482:Superoxide Dismutase / Q000235:genetics / Q000378:metabolism*; D014466:Ultraviolet Rays; D000099156:Superoxide Dismutase 2
chemical_list: D000975:Antioxidants; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D013482:Superoxide Dismutase; D000099156:Superoxide Dismutase 2