title: Potential apoptotic effect of ultraviolet-A irradiation during cross-linking: a study on ex vivo cultivated limbal epithelial cells.
abstract: OBJECTIVE
  To study the effects of ultraviolet-A (UV-A) irradiation, in the presence or absence of riboflavin, on ex vivo cultured limbal epithelial cells (LECs).
  
  
  METHODS
  The study was carried out in a super specialty ophthalmic hospital. Ex vivo cultured LECs were grown on denuded amniotic membranes and exposed to similar levels of UV-A radiation used during corneal cross-linking (CXL), in the presence or absence of the photosensitiser, riboflavin. These cells were then used for extraction of RNA, cDNA conversion, and antibody staining. Quantitative PCR and immunofluorescence staining were performed to evaluate the apoptotic state of treated and non-treated LECs. Statistical analyses were evaluated using a Student's t test.
  
  
  RESULTS
  We found that bcl-2, an antiapoptotic gene, was downregulated, whereas, bax, a proapoptotic gene, was upregulated. After LECs were exposed to UV-A radiation, a significant upregulation of both caspase 3 and caspase 9 was observed in treated cells when compared with untreated LECs.
  
  
  CONCLUSIONS
  These results indicate that exposure of LECs to UV-A dosages similar to those used in the CXL procedure promotes the expression of genes known to promote apoptosis. In the presence of riboflavin, the damage caused by UV-A treatment was marginalised, but not totally blocked.
authors.affiliation: Stem Cell Research Laboratory, Narayana Nethralaya, Bangalore, Karnataka, India.
authors.forename: Himanshu
authors.initials: H
authors.lastname: Matalia
authors.forename: Rohit
authors.initials: R
authors.lastname: Shetty
authors.forename: Kamesh
authors.initials: K
authors.lastname: Dhamodaran
authors.forename: Murali
authors.initials: M
authors.lastname: Subramani
authors.forename: Vincent
authors.initials: V
authors.lastname: Arokiaraj
authors.forename: Debashish
authors.initials: D
authors.lastname: Das
mesh_terms: D017209:Apoptosis / Q000235:genetics / Q000528:radiation effects*; D053148:Caspase 3 / Q000378:metabolism; D053453:Caspase 9 / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000166:cytology* / Q000502:physiology / Q000528:radiation effects*; D015870:Gene Expression / Q000528:radiation effects; D006801:Humans; D007640:Keratoconus / Q000532:radiotherapy; D016850:Limbus Corneae / Q000166:cytology*; D017319:Photosensitizing Agents / Q000494:pharmacology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics; D012256:Riboflavin / Q000494:pharmacology; D014466:Ultraviolet Rays / Q000009:adverse effects*; D014467:Ultraviolet Therapy / Q000009:adverse effects; D051028:bcl-2-Associated X Protein / Q000235:genetics
chemical_list: C494826:BAX protein, human; D017319:Photosensitizing Agents; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; C505407:CASP3 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053453:Caspase 9; D012256:Riboflavin