title: The effect of phosphorylated Akt inhibition on posterior capsule opacification in an ex vivo canine model.
abstract: OBJECTIVE
  To evaluate whether inhibition of phosphorylated Akt (pAkt) would reduce or prevent posterior capsule opacification (PCO) in an ex vivo canine lens capsule model.
  
  
  METHODS
  Normal and cataractous lenses (n=6) were evaluated for pAkt via immunohistochemistry and immunoblotting. Primary cultures of lens epithelial cells (LEC) were exposed to ultraviolet light (UV) to induce pAkt. Cultures were then incubated in 0, 2.5, 5, or 10 µM (n=6) of a novel Akt inhibitor (AR-12) for either 8 or 24 h. Cultures were harvested and pAkt expression and telomerase activity examined by immunoblotting and telomeric repeat amplification protocol (TRAP)-enzyme linked immunosorbent assay (ELISA), respectively. Lens capsules were harvested post-sham cataract surgery and exposed to 0, 2.5, 5, 7.5, or 10 μM (n=8) of AR-12 for a total of 14 days treatment. Additional lens capsules (n=6) were exposed to 10 μM of AR-12 for 1 week followed by media alone for 1 week; or exposed to media alone for 1 week followed by 10 μM of AR-12 for 1 week. Histopathology and immunohistochemical staining were performed to evaluate PCO formation. Analysis of telomerase activity on the lens capsules was performed by TRAP-ELISA.
  
  
  RESULTS
  pAkt protein expression was increased in clinical samples of canine cataracts compared to normal lenses. Following exposure to UV, cultures of LEC significantly (p<0.05) increased expression of pAkt and telomerase activity. Treatment with AR-12 for both 8 and 24 h following UV irradiation significantly (p<0.01) decreased pAkt expression. When UV-exposed LEC were allowed to recover in the presence of either 5.0 or 10.0 µM AR-12, there was a significant (p<0.05) decrease in telomerase activity. In the ex vivo model of PCO, within the region of the capsulorhexis, PCO inhibition was maximally achieved with 10 μM of AR-12. A significant decrease in LEC was noted on the posterior capsules containing 5.0, 7.5, and 10 μM AR-12 compared to the control capsules (p<0.01). Telomerase activity decreased in a dose-dependent manner. One week of treatment with 10 μM AR-12, immediately following capsule excision, was sufficient to inhibit PCO formation, while a delay in exposure to AR-12 after 1 week of media incubation alone did not prevent PCO formation.
  
  
  CONCLUSIONS
  pAkt is known to have roles in cell survival, proliferation, and migration, and this study suggests its inhibition immediately following cataract surgery may be a useful approach to prevent PCO.
authors.affiliation: College of Optometry, The Ohio State University, Columbus, OH 43210, USA. chandler.111@osu.edu
authors.forename: Heather L
authors.initials: HL
authors.lastname: Chandler
authors.forename: Terah R
authors.initials: TR
authors.lastname: Webb
authors.forename: Curtis A
authors.initials: CA
authors.lastname: Barden
authors.forename: Mirunalni
authors.initials: M
authors.lastname: Thangavelu
authors.forename: Samuel K
authors.initials: SK
authors.lastname: Kulp
authors.forename: Ching-Shih
authors.initials: CS
authors.lastname: Chen
authors.forename: Carmen M H
authors.initials: CM
authors.lastname: Colitz
mesh_terms: D000818:Animals; D053148:Caspase 3 / Q000378:metabolism; D002386:Cataract / Q000201:enzymology* / Q000473:pathology*; D002452:Cell Count; D004195:Disease Models, Animal; D004285:Dogs; D004847:Epithelial Cells / Q000187:drug effects / Q000201:enzymology / Q000473:pathology / Q000528:radiation effects; D058750:Epithelial-Mesenchymal Transition / Q000187:drug effects / Q000528:radiation effects; D007150:Immunohistochemistry; D007903:Lens Capsule, Crystalline / Q000187:drug effects* / Q000201:enzymology* / Q000473:pathology; D010766:Phosphorylation / Q000187:drug effects / Q000528:radiation effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors* / Q000378:metabolism; D019098:Telomerase / Q000378:metabolism; D014466:Ultraviolet Rays
chemical_list: D047428:Protein Kinase Inhibitors; D051057:Proto-Oncogene Proteins c-akt; D019098:Telomerase; D053148:Caspase 3