Basic Research of Photodynamic Therapy on Vascular Endothelial Cells.

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Phototoxicity of photodynamic therapy (PDT) using ATX-S10(Na) (ATX) and Verteporfin (BPD) was evaluated by MTS Assay on vascular endothelial cells and retinal pigment epithelial cells. PDT was conducted under two kinds of treatment conditions, short dye exposure PDT in which the cells were exposed to each photosensitizer for 5 minutes followed by laser irradiation without washing out the photosensitizer in the medium, and long dye exposure PDT, in which the cells were exposed to photosensitizers for more than 60 minutes, washed with phosphate-buffered saline, followed by laser irradiation using fresh medium. A diode laser of 670 nm wavelength was used for ATX-S10(Na) excitation and that of 689 nm for BPD.The degree of phototoxicity depended on the dye concentration and radiant exposure. In short-exposure PDT on human microvascular endothelial cells (CryoHMVEC-Ad) with a laser dose of 50 J/cm^2, the ED_<90> was 6.3 μg/ml of ATX-S10(Na) and 0.04 μg/ml of BPD, while in long-exposure PDT t … More he ED_<90> was 50.0 μg/ml of ATX and 0.04 μg/ml of BPD. Laser and dye doses to obtain ED_<90> became higher when the medium was supplemented with higher concentration of serum. In short-exposure PDT, the ED_<90> of monkey choroid-retina vascular endothelial cells (CRL-1780) was 100 μg/ml of ATX-S10(Na) and 0.08 μg/ml of BPD and an radiance of 100 J/cm^2. In comparison of phototoxicity on human retinal pigment epithelial cells (ARPE-19) and vascular endothelial cells, phototoxicity on vascular endothelial cells was significantly higher than that on the retinal pigment epithelial cells with short-exposure PDT using ATX, however, long-exposure PDT with ATX and short- and long-exposure PDT with BPD failed to obtain higher phototoxicity on vascular endothelial cells than on the retinal pigment epithelial cells. The uptake aid subcellular localization of ATX and BPD was examined by measuring fluorescence of each dye. ATX was mainly localized in the lysosomes and BPD in cytoplasma diffusely.Therefore, BPD had a higher phototoxicity than ATX. Some reasons were speculated for high phototoxibly of BPD, such as different amount and speed of uptake of the photosensitizer into the cells. In short-exposure PDT with ATX, vascular endothelial cells were selectively injured without damage on the retinal pigment epithelial cells, in contrast, with other PDT conditions, it seemed difficult to destroy vascular endothelial cells without damage on the retinal pigment epithelial cells since some dye was taken up into the retinal pigment epithelial cells.The mode of cell death by PDT was observed by TUNEL staining and a laser scanning cytometer. Short-exposure PDT with ATX predominantly induced necrosis, while long-exposure PDT resulted in apoptosis. Therefore, the dye that located on or surrounding the cell membrane seems to predominantly induced necrosis of the target cell, while the dye that accumulated in lysosomes predominantly induced apoptosis.ATX-S10 had a phototoxic effect on human skin malignant melanoma cells. Most of the dead cells appeared apoptotic with weak treatment conditions that induced low cytotoxicity. In contrast, most of them appeared necrotic with treatment conditions that induced more than 90% cytotoxicity. Less