title: Low intensity 635 nm diode laser irradiation inhibits fibroblast-myofibroblast transition reducing TRPC1 channel expression/activity: New perspectives for tissue fibrosis treatment.
abstract: OBJECTIVE
  Low-level laser therapy (LLLT) or 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 low intensity 635 ± 5 nm diode laser irradiation on fibroblast-myofibroblast transition, a key event in the onset of fibrosis, and elucidated some of the underlying molecular mechanisms.
  
  
  METHODS
  NIH/3T3 fibroblasts were cultured in a low serum medium in the presence of transforming growth factor (TGF)-β1 and irradiated with a 635 ± 5 nm diode laser (continuous wave, 89 mW, 0.3 J/cm(2) ). Fibroblast-myofibroblast differentiation was assayed by morphological, biochemical, and electrophysiological approaches. Expression of matrix metalloproteinase (MMP)-2 and MMP-9 and of Tissue inhibitor of MMPs, namely TIMP-1 and TIMP-2, after laser exposure was 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-β1/Smad3 signaling was investigated.
  
  
  RESULTS
  Diode laser treatment inhibited TGF-β1-induced fibroblast-myofibroblast transition as judged by reduction of stress fibers formation, α-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-β1-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-β1 signaling, namely reducing the expression of Smad3, the TGF-β1 downstream signaling molecule.
  
  
  CONCLUSIONS
  Low intensity irradiation with 635 ± 5 nm diode laser inhibited TGF-β1/Smad3-mediated fibroblast-myofibroblast transition and this effect involved the modulation of TRPC1 ion channels. These data contribute to support the potential anti-fibrotic effect of LLLT and may offer further informations for considering this therapy as a promising therapeutic tool for the treatment of tissue fibrosis.
authors.affiliation: Section of Anatomy and Histology, Department of Experimental and Clinical Medicine, University of Florence, Largo Brambilla 3, 50134, Florence, Italy.
authors.forename: Chiara
authors.initials: C
authors.lastname: Sassoli
authors.affiliation: Section of Anatomy and Histology, Department of Experimental and Clinical Medicine, University of Florence, Largo Brambilla 3, 50134, Florence, Italy.
authors.forename: Flaminia
authors.initials: F
authors.lastname: Chellini
authors.affiliation: Section of Physiological Sciences, Department of Experimental and Clinical Medicine, University of Florence, Viale GB Morgagni 63, 50134, Florence, Italy.
authors.forename: Roberta
authors.initials: R
authors.lastname: Squecco
authors.affiliation: Section of Anatomy and Histology, Department of Experimental and Clinical Medicine, University of Florence, Largo Brambilla 3, 50134, Florence, Italy.
authors.forename: Alessia
authors.initials: A
authors.lastname: Tani
authors.affiliation: Section of Physiological Sciences, Department of Experimental and Clinical Medicine, University of Florence, Viale GB Morgagni 63, 50134, Florence, Italy.
authors.forename: Eglantina
authors.initials: E
authors.lastname: Idrizaj
authors.affiliation: Section of Anatomy and Histology, Department of Experimental and Clinical Medicine, University of Florence, Largo Brambilla 3, 50134, Florence, Italy.
authors.forename: Daniele
authors.initials: D
authors.lastname: Nosi
authors.affiliation: Odontostomatologic Laser Therapy Center, Via dell' Olivuzzo 162, 50143, Florence, Italy.
authors.forename: Marco
authors.initials: M
authors.lastname: Giannelli
authors.affiliation: Section of Anatomy and Histology, Department of Experimental and Clinical Medicine, University of Florence, Largo Brambilla 3, 50134, Florence, Italy.
authors.forename: Sandra
authors.initials: S
authors.lastname: Zecchi-Orlandini
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D015153:Blotting, Western; D002454:Cell Differentiation / Q000502:physiology / Q000528:radiation effects*; D002478:Cells, Cultured; D005347:Fibroblasts / Q000502:physiology / Q000528:radiation effects; D005355:Fibrosis / Q000378:metabolism / Q000532:radiotherapy; D054023:Lasers, Semiconductor / Q000627:therapeutic use*; D028022:Low-Level Light Therapy / Q000379:methods*; D051379:Mice; D058628:Myofibroblasts / Q000502:physiology / Q000528:radiation effects*; D041681:NIH 3T3 Cells; D018408:Patch-Clamp Techniques; D050052:TRPC Cation Channels / Q000378:metabolism
chemical_list: D015415:Biomarkers; D050052:TRPC Cation Channels; C096351:transient receptor potential cation channel, subfamily C, member 1
keywords: low-level laser therapy (LLLT); matrix metalloproteinases (MMPs); photobiomodulation; stress fibers; stretch-activated channel (SAC); tissue inhibitor of metalloproteinase (TIMP); transforming growth factor (TGF)-β1; type-1 collagen; α-sma