Biomodulation with low-level laser radiation induces changes in endothelial cell actin filaments and cytoskeletal organization

Abstract

The cytoskeleton is a central and vital structure of eukaryotic cells. It consists of a dynamic network of partially interconnected polymers. This extended network controls the mechanical properties of animal cells, serves as intracellular transport “pathways”, and plays a prominent role in cell motility, proliferation, and adhesion. In order to evaluate the action of laser irradiation on the cytoskeleton and proliferation of endothelial cells, rabbit aortic endothelial cells (RAEC) were irradiated with 685-nm low-level laser (20 mW output power). Fluorescent dye rhodamine-phalloidin staining was used to visualize the effect of laser irradiation on actin filaments. Irradiation with 8 J/cm² was performed four times at 12-h intervals for 24 min. Cells cultured under low fetal bovine serum condition (5% FBS) for 7 days presented actin staining predominantly in the cortical membrane region and a few actin filament stress fibers. However, the formation of stress fibers similar to those of control cells increased significantly in irradiated cells. It was concluded that laser irradiation induces changes in the cytoskeleton of endothelial cells through the reorganization of actin filaments and neo-formation of stress fibers, allowing evident cellular proliferation.

Introduction

Cell shape is governed by the cytoskeleton, which acts as a mechanical supporting framework [1], [2] and an orienting foundation for much of the cell signal transduction machinery [3], [4]. Disruption of the actin cytoskeleton has been shown to induce cell rounding and inhibit cell cycle progression in capillary endothelial cells [5], [6]. Mechanical coupling between microtubules and actin filaments has also been shown to play an important role in cell shape stability and in the control of proliferation [5]. Yet, it is not very clear whether cytoskeletal filaments contribute to shape-dependent proliferation control and, if so, how they couple to relevant biochemical transduction pathways. Currently, low-level laser therapy (LLLT) is widely applied in different branches of medicine (e.g. tissue regeneration) and several hypotheses have been proposed to explain the LLLT mechanisms [7]. In vitro studies with fibroblasts have described a proliferative effect or protein synthesis activation by low-level laser radiation dependent on irradiation wavelength, irradiance, and fluency [8], [9]. There are contradictory reports about low-level laser light stimulation of cell proliferation. A study was performed to determine the effect of wavelength on the proliferation of cultured murine cells [10]. Cultured primary cell proliferation was measured after irradiation with varying laser wavelengths. Fibroblasts proliferated faster than endothelial cells in response to laser irradiation. Maximum cell proliferation occurred at 665–675 nm, whereas irradiation at 810 nm inhibited cell division. These observations suggest that both wavelength and cell type influence cell proliferation response to low-level laser irradiation [10].

The aim of this study was to observe the effects of low-level laser irradiation on the survival threshold of cells under low serum condition and cytoskeletal organization after 7 days in the same medium.