Low intensity 635 nm diode laser irradiation inhibits fibroblast-myofibroblast transition reducing TRPC1 channel expression/activity: New perspectives for tissue fibrosis treatment

BACKGROUND AND OBJECTIVE: Photobiomodulation therapy is emerging as a promising new therapeutic option for fibrosis in different damaged and/or diseased organs. However, the anti-fibrotic potential of this treatment needs to be elucidated and the cellular and molecular targets of the laser clarified. Here we investigated the effects of a diode laser irradiation on fibroblast-myofibroblast transition, a key event in the onset of fibrosis, and elucidated some of the underlying molecular mechanisms. MATERIALS AND METHODS: NIH/3T3 fibroblasts were cultured in a low serum medium in the presence of Transforming Growth Factor (TGF)-beta1, irradiated with a 635±5 nm diode laser (continuous wave, 89 mW, 0.3 J/cm2). Fibroblast-myofibroblast differentiation was assayed by morphological, biochemical and electrophysiological approaches. Expression of matrix metalloproteinase (MMP)-2 and MMP-9 and their inhibitors TIMP-1 and TIMP-2 after laser exposure were also evaluated by confocal immunofluorescence analyses. Moreover, the effect of the diode laser on Transient Receptor Potential Canonical Channel (TRPC)1/Stretch Activated Channel (SAC) expression and activity and on TGF-beta1/Smad3 signaling was investigated. RESULTS: Diode laser treatment inhibited TGF-beta1-induced fibroblast-myofibroblast transition as judged by reduction of stress fibers formation, alfa-smooth muscle actin (sma) and type-1 collagen expression and by changes in electrophysiological properties such as resting membrane potential, cell capacitance and inwardly rectifying K+ currents. In addition, the irradiation up-regulated the expression of MMP-2 and MMP-9 and downregulated that of TIMP-1 and TIMP-2 in TGF-beta1-treated cells. This laser effect was shown to involve TRPC1/SAC channel functionality. Finally, diode laser stimulation and TRPC1 functionality negatively affected fibroblast-myofibroblast transition by interfering with TGF-beta1 signaling, namely reducing the expression of Smad3, the TGF-β1 downstream signaling molecule. CONCLUSION: Photobiomodulation with 635±5 nm diode laser inhibited TGF-beta1/Smad3-mediated fibroblast-myofibroblast transition and this effect involved the modulation of TRPC1 ion channels. These data, contributing to support the potential anti-fibrotic effect of photobiomodulation therapy , may offer clues for this therapy to be considered a new promising therapeutic tool for the treatment of tissue fibrosis.