PURPOSE: 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.
PURPOSE: 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.