title: Photodynamic therapy using Lu-Tex induces apoptosis in vitro, and its effect is potentiated by angiostatin in retinal capillary endothelial cells.
abstract: OBJECTIVE
  To examine the effect of combining angiostatin with photodynamic therapy (PDT) using Lutetium Texaphyrin (Lu-Tex; Alcon, Fort Worth, TX) as a photosensitizer in bovine retinal capillary endothelial (BRCE) and retinal pigment epithelial (RPE) cells and to determine the mode of PDT-induced cell death in these cell lines.
  
  
  METHODS
  Cultured BRCE and RPE cells were incubated with angiostatin (500 ng/ml) for 18 hours and subjected to Lu-Tex/PDT, using treatment parameters previously optimized (3 microgram/ml Lu-Tex for 30 minutes followed by timed irradiation at 732 nm). Cellular survival was assessed after a 1-week cellular proliferation. Data were analyzed using Student's t-test. Caspase 3 activity was monitored in cells after PDT using a fluorogenic substrate, (Asp-Glu-Val-Asp)-AFC (7-amino-4-trifluoromethyl coumarin) [DEVD-AFC], of caspase 3. After PDT, expression of Bcl-2, Bcl-x(L), Bax, and Bak was also examined in cell lysates by Western blot analysis.
  
  
  RESULTS
  A synergistic cytotoxic effect of angiostatin and Lu-Tex/PDT was observed in BRCE cells at all fluences used (5, 10, and 20 J/cm(2); P </= 0.05). These findings applied only if angiostatin was delivered before PDT. No such interactive killing effect was observed in RPE cells. Caspase 3 activity was elevated within 10 minutes of PDT in BRCE and RPE cells and was fluence dependent. Differential modulation of Bcl-2 family members was observed after PDT in BRCE and RPE cells.
  
  
  CONCLUSIONS
  The combination of angiostatin and Lu-Tex/PDT potentiates the cytotoxic effect of Lu-Tex/PDT on BRCE but not on RPE cells. This may provide a strategy to increase the selectivity of PDT in damaging capillary endothelial cells with less damage to RPE cells. Lu-Tex/PDT induces rapid caspase-dependent apoptosis in BRCE and RPE cells. Furthermore, Lu-Tex/PDT induces apoptosis through selective modulation of members of the Bcl-2 family and differs between BRCE and RPE cells.
authors.affiliation: Laser Laboratory, Retina Service, Massachusetts Eye and Ear Infirmary. Schepens Eye Research Institute, Harvard Medical School, Boston, USA.
authors.forename: R Z
authors.initials: RZ
authors.lastname: Renno
authors.forename: F C
authors.initials: FC
authors.lastname: Delori
authors.forename: R A
authors.initials: RA
authors.lastname: Holzer
authors.forename: E S
authors.initials: ES
authors.lastname: Gragoudas
authors.forename: J W
authors.initials: JW
authors.lastname: Miller
mesh_terms: D043168:Angiostatins; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D002196:Capillaries / Q000187:drug effects / Q000378:metabolism; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002417:Cattle; D002455:Cell Division / Q000187:drug effects; D002470:Cell Survival; D002478:Cells, Cultured; D003374:Coumarins / Q000378:metabolism; D004338:Drug Combinations; D004357:Drug Synergism; D004591:Electrophoresis, Polyacrylamide Gel; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000378:metabolism; D008665:Metalloporphyrins / Q000494:pharmacology*; D009842:Oligopeptides / Q000378:metabolism; D010446:Peptide Fragments / Q000494:pharmacology*; D010778:Photochemotherapy; D017319:Photosensitizing Agents / Q000494:pharmacology*; D010857:Pigment Epithelium of Eye / Q000187:drug effects / Q000378:metabolism; D010958:Plasminogen / Q000494:pharmacology*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D012171:Retinal Vessels / Q000187:drug effects* / Q000378:metabolism
chemical_list: D003374:Coumarins; D004338:Drug Combinations; D008665:Metalloporphyrins; D009842:Oligopeptides; D010446:Peptide Fragments; D017319:Photosensitizing Agents; D019253:Proto-Oncogene Proteins c-bcl-2; C115495:aspartyl-glutamyl-valyl-aspartyl-7-amino-4-trifluoromethylcoumarin; C102825:motexafin lutetium; C437683:motexafin gadolinium; D043168:Angiostatins; D010958:Plasminogen; D053148:Caspase 3; D020169:Caspases