BACKGROUND AND PURPOSEFibroblast-to-myofibroblast differentiation is associated with scarring, an important issue in corneal surgery. Moxifloxacin (MOX), commonly applied to prevent post-operative infection, would benefit more if it modifies fibroblast-to-myofibroblast differentiation other than antimicrobial activity. Our purpose was to explore whether MOX has anti-fibrotic effect in human corneal fibroblasts (HCFs).
EXPERIMENTAL APPROACHHCFs were incubated in MOX-containing medium concurrently with TGF-b1 (co-treatment), before (pretreatment) or after (post-treatment) adding TGF-b1. HCF contractility was evaluated with a type I collagen gel contraction assay. Expression of a-smooth muscle actin (a-SMA), Smad2, phospho-Smad2-Ser467, Smad4 and Smad7 was determined by immunoblotting. Formation of a-SMA-positive filaments and distribution of active Smad2 were observed under confocal microscopy. Expression of TGF-b receptor types I (TGFBR1) and II (TGFBR2) was assessed with flow cytometry.
KEY RESULTSMOX did not affect gel contractility or a-SMA filament formation in HCFs without TGF-b1 stimulation. MOX did, however, retard HCF-containing gel contractility and a-SMA filament formation following TGF-b1 stimulation in the pretreatment and co-treatment groups but not in the post-treatment group. MOX blocked the expression of Smad2, phospho-Smad2-Ser467 and TGFBR1 under TGF-b1 incubation. Additionally, MOX enhanced Smad7 expression in TGF-b1-incubated HCFs, but did not interfere with TGF-b-triggered Smad2 nuclear translocation or Smad4 expression.
CONCLUSIONS AND IMPLICATIONSMOX inhibited TGF-b-induced fibroblast-to-myofibroblast differentiation via blocking TGFBR1 and enhancing Smad7 expression. MOX should be used before or during surgery to achieve these effects. These results suggest a de novo mechanism by which MOX participates in corneal wound healing.
AbbreviationsHCFs, human corneal fibroblasts; MOX, moxifloxacin; TGFBR1, TGF-b receptor type I; TGFBR2, TGF-b receptor type II; a-SMA, a-smooth muscle actin