Renno R Z, Delori F C, Holzer R A, Gragoudas E S, Miller J W
Laser Laboratory, Retina Service, Massachusetts Eye and Ear Infirmary. Schepens Eye Research Institute, Harvard Medical School, Boston, USA.
Invest Ophthalmol Vis Sci. 2000 Nov;41(12):3963-71.
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.
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.
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.
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.
研究将血管抑素与光动力疗法（PDT）联合应用的效果，该光动力疗法使用钆替沙林（Lu-Tex；爱尔康公司，沃思堡，德克萨斯州）作为光敏剂，作用于牛视网膜毛细血管内皮（BRCE）细胞和视网膜色素上皮（RPE）细胞，并确定在这些细胞系中PDT诱导细胞死亡的方式。
将培养的BRCE细胞和RPE细胞与血管抑素（500 ng/ml）孵育18小时，然后进行Lu-Tex/PDT处理，使用先前优化的治疗参数（3 μg/ml Lu-Tex处理30分钟，随后在732 nm下定时照射）。在细胞增殖1周后评估细胞存活率。数据采用学生t检验进行分析。使用半胱天冬酶3的荧光底物（天冬氨酸-谷氨酸-缬氨酸-天冬氨酸）-AFC（7-氨基-4-三氟甲基香豆素）[DEVD-AFC]监测PDT后细胞中的半胱天冬酶3活性。PDT后，还通过蛋白质印迹分析检测细胞裂解物中Bcl-2、Bcl-x(L)、Bax和Bak的表达。
在所有使用的光通量（5、10和20 J/cm²；P≤0.05）下，在BRCE细胞中观察到血管抑素和Lu-Tex/PDT的协同细胞毒性作用。这些发现仅在PDT之前给予血管抑素时才适用。在RPE细胞中未观察到这种相互作用的杀伤作用。在BRCE细胞和RPE细胞中，PDT后10分钟内半胱天冬酶3活性升高，且与光通量有关。PDT后在BRCE细胞和RPE细胞中观察到Bcl-2家族成员的差异调节。
血管抑素与Lu-Tex/PDT联合可增强Lu-Tex/PDT对BRCE细胞的细胞毒性作用，但对RPE细胞无此作用。这可能提供一种策略，以提高PDT在损伤毛细血管内皮细胞时对RPE细胞损伤较小的选择性。Lu-Tex/PDT在BRCE细胞和RPE细胞中诱导快速的半胱天冬酶依赖性凋亡。此外，Lu-Tex/PDT通过选择性调节Bcl-2家族成员诱导凋亡，且在BRCE细胞和RPE细胞之间存在差异。
