Abstract
Purpose: :
To explore in vitro the influence of Cyclosporin A (CsA), a powerful immunosuppressive agent, on fibrosis in term of proliferation, differentiation, ECM metabolism and contraction of primary (from n=3/biopsies and from ScienCell Res. Lab, Carlsbad, CA) cultures of conjunctival fibroblasts. CsA effects were compared with those of TGFβ1.
Methods: :
Both fibroblasts (FBs) and in vitro induced myofibroblasts (myoFBs) were used for CsA (Restasis, Allergan Inc., Irvine, CA) at scalar doses (50, 200, 400 and 800 ng/mL). Cells were also exposed to 10ng/mL TGFβ1, 800ng/mL CsA either alone or combined. Sister untreated cells were used as control. After 24 hours, cell viability and proliferation were assessed. Extracellular metabolism was investigated by measuring collagen typeI/IV (csELISA) and MMP9/TIMP1 (zymography, ssELISA) protein expression. Extracellular matrix contraction was monitored according to the 3D gel assay. Statistical analysis was assessed by parametric ANOVA-Tukey Kramer post hoc assays.
Results: :
Increasing CsA doses did not influence either proliferation nor αSMA expression in both FBs and myoFBs. CsA did not modulate significantly collagen typeI/IV nor MMP9 expression, while TGFβ1 exposure resulted in a drastic increase of collagen typeI/IV in both cell types (p<.05). A trend toward a MMP9 increase was detected upon CsA exposure while a trend to a decrease was observed for TIMP1 (p>.05). Addition of 800ng/mL CsA to 10ng/mL TGFβ1 abolished this typeI/IV increase in both FBs and myoFBs (p<.05). Merely to MMP9/TIMP1, addition of 800ng/mL CsA to 10ng/mL TGFβ1 resulted in a suppression of TGFβ1-induced MMP9/TIMP1 expression in both cell types. At low doses, CsA triggered FB mediated contraction of a 3D gel with no effects at higher doses, as compared to those of TGFβ1.
Conclusions: :
CsA eye drops are therapeutically used for severe allergic conjunctivitis, but the mechanism of action is actually unknown. Herein, CsA appears to modulate profibrogenic effects induced by TGFβ1, a well-known factor involved in FBs/myoFBs activation, suggesting new potential mechanisms of CsA in ocular tissue remodeling/fibrosis.
Keywords: conjunctiva • extracellular matrix • cyclosporine