In vitro photodynamic therapy with chlorin e6 leads to apoptosis of human vascular smooth muscle cells

Abstract

Percutaneous coronary intervention has become the most common and widely implemented method of heart revascularization. However, the development of restenosis remains the major limitation of this method. Photodynamic therapy (PDT) recently emerged as a new and promising method for the prevention of arterial restenosis. Here the efficacy of chlorin e6 in PDT was investigated in vitro using human vascular smooth muscle cells (TG/HA-VSMCs) as one of the cell types crucial in the development of restenosis. PDT-induced cell death was studied on many levels,including annexin V staining, measurement of the generation reactive oxygen species (ROS) and caspase-3 activity,and assessment of changes in mitochondrial membrane potential and fragmentation of DNA. Photosensitization of TG/HA-VSMCs with a 170 lM of chlorin e6 and subsequent illumination with the light of a 672-nm diode laser(2 J/cm2) resulted in the generation of ROS, a decrease in cell membrane polarization, caspase-3 activation, as well as DNA fragmentation. Interestingly, the latter two apoptotic events could not be observed in photosensitized and illuminated NIH3T3 fibroblasts, suggesting different outcomes of the model of PDT in various types of cells. The results obtained with human VSMCs show that chlorin e6 may be useful in the PDT of aerial restenosis, but its efficacy still needs to be established in an animal model.

Fig. 1

a Structure of chlorin e6. The presence of the chlorin ring gives the compound fluorescent and photochemical properties. b Chlorin e6 distribution in the cell may be directly observed using a fluorescence microscope equipped with a UV lamp and 605–670 nm filter. Chlorin e6 rapidly translocates through the cell membrane into the cell and accumulates in the cytoplasm. Representative microphotography is shown

Fig. 2

a Scheme of the standard in vitro photodynamic therapy experiment. Cells were plated 2 days prior to photosensitization, then incubated for 1 h in the presence of chlorin e6, washed, incubated for 30 min, and illuminated in a dark room. b The generation of reactive oxygen species (ROS) due to illumination of the photosensitized TG/HA-VSMCs. The dichlorofluorescein derivate, carboxy-H₂DCFDA was used as the ROS detection reagent. A fourfold increased in 485-/535-nm fluorescence right after illumination of TG/HA-VSMCs was detected. ch e6 170 μM chlorin e6, light 672-nm diode laser, 2 J/cm²

Fig. 3

Testing and optimization of parameters for the in vitro photodynamic therapy of TG/HA-VSMC determined by MTS assay. Toxicity of PDT induced in TG/HA-VSMC culture (MTS assay) using: a 17–170 μM concentrations of chlorin e6 (squares) and in combination with a light dose of 2 J/cm² applied using a 672-nm diode laser (triangles) or b testing the light doses of 0.6, 1.1, and 2 J/cm² in the presence (triangles) or absence (squares) of 170 μM chlorin e6

Fig. 4

Assessment of apoptosis-related events after PDT (170 μM chlorin e6 and 2-J/cm² light dose of TG/HA-VSMCs: a phosphatidylserine externalization and membrane integrity assessed by staining with Annexin V-FITC conjugate (FL-1H, green fluorescence) and propidium iodide staining (FL-3H, far red fluorescence). b Mitochondrial potential using an IC-1 probe (FL-2H, red fluorescence). c Caspase-3 activity (dark bar), the light gray bar represents caspase-3-like activity observed in the presence of the caspase-3 inhibitor Ac-DEVD-CHO. Averages from representative experiments are shown. d DNA fragmentation observed 18 h after PDT by propidium iodide (FL2H, red fluorescence)

Fig. 5

NIH3T3 response to chlorin e6 PDT. a Results of MTS assay, illumination (laser, 2 J/cm²) in the presence (triangles) or absence (squares) of the indicated concentration of chlorin e6. b Toxicity induced in the cell population after illumination with the indicated light dose in the presence (triangles) or absence (squares) of 170 μM chlorin e6; c Phosphatidylserine presentation and membrane integrity test of NIH3T3 cells subjected to PDT (170 μM chlorin e6 and 2 J/cm²). Percentages of populations are shown. A representative result of one of three independent experiments is shown. d Reactive oxygen species (ROS) as 485-/535-nm fluorescence of H₂DCFDA generated in photosensitized and/or illuminated samples. e Caspase-3 activity measured in lysates photosensitized and/or illuminated NIH3T3 cells (dark bar). The light gray bar represents caspase-3-like activity observed in the presence of the caspase-3 inhibitor Ac-DEVD-CHO