Photodynamic therapy (PDT) generates free radicals through the absorption of light by photosensitizers. PDT shows promise in the treatment of intimal hyperplasia, which contributes to restenosis, by completely eradicating cells in the vessel wall. This study investigates the mechanisms of PDT-induced cell death. PDT, using the photosensitizer chloroaluminum-sulfonated phthalocyanine (1 mg/kg) and laser light ( ‫؍‬ 675 nm) 100 J/cm 2 was administered to rat carotid arteries after balloon injury-induced intimal hyperplasia. Apoptosis was determined by cell morphology with light microscopy and transmission electron microscopy, DNA cleavage by terminal dUTP nick-end labeling staining, and nucleosomal fragmentation (ladder pattern) by DNA agarose gel electrophoresis. Four hours after PDT, apoptosis was observed in vascular cells, as evidenced by terminal dUTP nick-end labeling staining and transmission electron microscopy. Within 24 hours no cells were present in the neointima and media. Immunofluorescence using an ␣-smooth muscle cell actin antibody confirmed the disappearance of all neointimal and medial cells within 24 hours. No inflammatory cell infiltrate was observed during this time frame. Apoptosis was sharply confined to the PDT treatment field. These data demonstrate that vascular PDT induces apoptosis as a mechanism of rapid, complete, and precise cell eradication in the artery wall. These findings and the lack of inflammatory reaction provide the basis for understanding and developing PDT for a successful clinical application in the treatment of hyperplastic conditions such as restenosis.

Abstract-␥-Irradiation (␥-RT) and photodynamic therapy (PDT) are known to inhibit intimal hyperplasia. The common mechanism is that both modalities produce free radicals, but unlike ␥-RT, PDT generates them through the absorption of light by photosensitizers. The purpose of this in vitro study was to assess the differences that PDT and ␥-RT have on the fibroproliferative response after vascular injury by comparing their effects on vascular smooth muscle cells (SMCs) and on the extracellular matrix (ECM). Mitochondrial activity (tetrazolium salt), proliferation ([ 3 H]thymidine incorporation), and the mechanisms of cell death (terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labeling [TUNEL] staining) were used to assess differences between PDT (100 J/cm 2 ) and ␥-RT (10 or 20 Gy) on SMC injury. The different effects on bioregulatory molecules were investigated by quantitating the proliferation of SMCs cultured with conditioned medium and on treated ECM. PDT of SMCs reduced proliferation and mitochondrial activity (0.5Ϯ0.75% and 1.7Ϯ4.25%, respectively, PϽ0.0001), whereas ␥-RT of SMCs decreased cell proliferation but did not affect metabolic activity. Stimulation with calf serum of ␥-RT-treated SMCs did not affect proliferation but increased mitochondrial enzyme activity (160Ϯ11%, PϽ0.0005). The conditioned medium, derived from PDT-but not ␥-RT-treated SMCs, did not stimulate effector SMC proliferation compared with ␥-RT-treated SMCs (16Ϯ4.1% versus 80Ϯ16.8%, PϽ0.0001). Apoptosis was the principle cytotoxic mechanism after PDT, whereas ␥-RT cells were growth arrested but viable. PDT of the ECM reduced effector SMC proliferation compared with controls and ␥-RT cells (18Ϯ6.5% versus 100Ϯ17.7% and 84Ϯ8.9%, respectively, PϽ0.0001). These data suggest that ␥-RT and PDT may inhibit restenosis but by different mechanisms. The effects of PDT are more diverse and may result in improved outcome while avoiding the teratogenic exposure due to ionizing irradiation. (Arterioscler Thromb Vasc Biol.

these findings indicate that PDT can interfere with factors that lead to the vascular fibrotic response. In this way, PDT, with its cytotoxic and extracellular effects, can promote healing of the vessel wall without the stimulus of fibrosis that can lead to restenosis.