Cellular effects of the pulsed tunable dye laser at 577 nanometers on human endothelial cells, fibroblasts, and erythrocytes: An in vitro study

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Abstract
The 577-nm flashlamp-pumped tunable dye laser pulsed at 450 μs is rapidly becoming the treatment of choice for removal of portwine stains and other vascular ectasias. In this study, we examined the mechanisms of vessel destruction by determining the effects of laser irradiation on three types of primary target cells–erythrocytes, endothelial cells, and fibroblasts. Human endothelial cells and fibroblasts in microwell plates were irradiated at various energy densities with the laser, after which several aspects of cellular biology were determined, including (1) viability of cells by trypan blue exclusion test; (2) cell proliferation by [3H]thymidine incorporation; and (3) rate of protein synthesis using [3H]leucine incorporation as a marker. In endothelial cell cultures, both [3H]thymidine and [3H]leucine incorporations were inhibited at energy levels of 5–12 J/cm2 (P < 0.01). In fibroblast cultures, cell proliferation was similarly inhibited, while supratherapeutic energy density (⩾12 J/cm2) was required for inhibition of protein synthesis. The laser energy in the range of 5–8.5 J/cm2 had no effect on cell viability. Erythrocytes as target cells for laser energy demonstrated rapid, dose-dependent lysis, as determined by release of free hemoglobin into culture medium. Addition of erythrocytes into a coculture with endothelial cells abolished the direct inhibitory effect noted in cultures when endothelial cells were present alone. The results of the latter experiment imply that erythrocytes are the primary target cell absorbing the laser energy at 577 nm. However, direct laser effects on endothelial cells may also contribute to the mechanisms of ablation of the vascular ectasias by the tunable dye laser at 577 nm.