Inhibitory Effect of Photodynamic Therapy with Indocyanine Green on Rat Smooth Muscle Cells

Abstract

Background: Vascular smooth muscle cells play a critical role in the intimal hyperplasia of restenosis. A previous study of a rat balloon injury model demonstrated that photodynamic therapy (PDT) using indocyanine green (ICG) and near-infrared (NIR) light irradiation reduced intimal hyperplasia in carotid arteries. However, the effect of ICG-PDT on smooth muscle cells remains unclear. This study aimed to evaluate the effects of PDT with ICG and NIR irradiation on the viability of vascular smooth muscle (A-10) cells.

Methods: A-10 cells were incubated with ICG at different concentrations for different time intervals. Intracellular accumulation of ICG inside the cells was observed by light microscopy, ultraviolet-visible (UV-VIS) spectrophotometry and spectrofluorometry. Cell viability and cell death after ICG-PDT were assessed by 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide assay and lactate dehydrogenase release assay. Changes in nuclear morphology and cell cycle distribution were evaluated to determine the possible cell death mechanism mediated by ICG-PDT.

Results: ICG uptake in A-10 cells increased with the amount of ICG in the culture media. The intracellular accumulation of ICG reached a maximum at 8 h. After ICG-PDT, cell viability decreased and cell death increased in a concentration- dependent manner. The half maximal inhibitory concentration of ICG was 8.3 μM with 4 J/cm² NIR irradiation. Membrane blebbing and chromatin condensation were observed, and the percentage of cells in the sub-G₁ phase increased after ICG-PDT. Thus, apoptosis might be responsible for decreasing the viability of A-10 cells by ICG-PDT.

Conclusions: This study demonstrated that ICG-PDT had an inhibitory effect on smooth muscle cells, possibly via an apoptosis pathway.

Keywords: Cell viability; Indocyanine green; Near-infrared; Photodynamic therapy; Smooth muscle cell.

Figure 1

Phase-contrast micrographs of rat smooth muscle (A-10) cells after 24-h incubation with indocyanine green (ICG). A-10 cells were exposed to ICG for 24 h and observed under a phase-contrast microscope. There were more cells with green-color staining in cultures exposed to higher concentration of ICG. ICG concentration: 0 μM (A), 1 μM (B), 10 μM (C) and 50 μM (D). Original magnification 100x.

Figure 2

Intracellular uptake of indocyanine green (ICG) in rat smooth muscle (A-10) cells as a function of incubation time. A-10 cells were exposed to 20 μM ICG for up to 24 h. The ICG uptake was measured by using the optical absorption of ICG at 780 nm (black square) and fluorescence emission at 830 nm (white square). Intracellular ICG level was expressed as normalized absorption intensity or fluorescence intensity per mg protein measured in cell extract. Values represent mean ± standard error of three separate experiments.

Figure 3

Cell viability of rat smooth muscle (A-10) cells after photoactivation of indocyanine green (ICG). A-10 cells were incubated for 8 h in the presence of different concentrations of ICG and then exposed to 780 nm light irradiation at a light fluence of 4 J/cm². Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay 24 h after light exposure. Data are mean ± standard error of three replicates of each treatment.

Figure 4

Cytoxicity of rat smooth muscle (A-10) cells after photoactivation of indocyanine green (ICG). A-10 cells were incubated for 8 h in the presence of different concentrations of ICG and then exposed to 780 nm light irradiation at a light fluence of 4 J/cm². Cytotoxicity was measured by lactate dehydrogenase release assay 24 h after light exposure. Data are mean ± standard error of three replicates of each treatment.

Figure 5

Changes in nucleus morphology of rat smooth muscle (A-10) cells after photodynamic therapy (PDT) of indocyanine green (ICG). A-10 cells were incubated with 9 μM ICG for 8 h and then exposed to 780 nm light irradiation at a light fluence of 4 J/cm². The nucleus was stained with Hoechst 33258 (blue) 45 min after PDT and observed under a fluorescence microscope. (A) Representative image of control group (untreated) under bright-field. (B) Fluorescence image of control group. (C) Phase-contrast image of A-10 cells received PDT. Blebbing of cells was shown here. (D) Fluorescence image of A-10 cells received PDT. Perinuclear condensation was seen. Original magnification 400x.

Figure 6

Cell cycle analysis of rat smooth muscle (A-10) cells after photodynamic therapy (PDT) of indocyanine green (ICG). A-10 cells were incubated with 6 μM ICG for 8 h and then exposed to 780 nm light irradiation at a light fluence of 2 J/cm². Quantification of cell distribution in individual cell cycle phase was analyzed 12 h after PDT by flow cytometry. Bars represent mean ± standard error of three independent experiments. (*) signifies significant difference between medians of all groups (* p < 0.05).

Supplementary figure

Nuclear morphology analyses of rat smooth muscle (A-10) cells after ICG-PDT.