Abstract
Purpose :
Corneal scarring is a leading cause of preventable worldwide blindness. The mechanisms underpinning corneal fibrosis are complex but are linked to the Transforming Growth Factor βs (TGFβ). Pirfenidone is clinically approved by the National Institute for Health and Care Excellence (UK) for the treatment of idiopathic pulmonary fibrosis through reduction in expression of several pro-fibrogenic cytokines including TGFβ.
Methods :
An immortalized human donor cornea fibroblast cell line (ABM Inc, Vancouver, CA ) were used for all experiments. Cell viabilitiy was assessed using the MTT assay with varying concentrations of TGFβ1/2/3 (1,5,10 ng/ml) and Pirfenidone (0.5,1,2 mg/ml) at 24 hours.
Cells were then incubated with TGFβ1 10ng/ml, TGFβ2 10ng/ml, TGFβ3 5ng/ml and Pirfenidone 1mg/ml in order to investigate cellular migration using scratch assays. The response was compared to cells incubated with 10% fetal calf serum containing media.
Results :
There was no significant loss of cell viability with TGFβ 1/2/3 at concentrations of 10ng/ml, 10ng/ml and 5ng/ml respectively at 24 hours. Similarly, no significant cell death was observed at 24 hours with Pirfenidone <1mg/ml. These concentrations were then used for scratch assay analysis measuring the change in wound width and % closure. TGFβ1 showed increased cell migration causing greater wound closure (p<0.05), while TGFβ2 did not reveal significant change when compared to control (p=0.32). TGFβ3 initially showed decreased cell migration for the first 24 hours but then the effects were similar to control samples (p<0.05). However, Pirfenidone 1mg/ml showed a clearly visible decrease in cell migration compared to control samples (p<0.05) and in combination with the pro-fibrotic TGFβ1 (p<0.05) which was maintained for the full duration of the experiment.
Conclusions :
Our data demonstrates that Pirfenidone antagonises TGFβ induced pro-fibrotic effects in corneal fibroblasts, and suggests that the inhibition of TGFβ1 has a critical role in the underlying mechanims of action. Topical Pirfenidone may have a protective anti-scarring role in pro-fibrotic disease states and could provide a novel therapeutic modality to attenuate scarring during wound healing.
This is a 2020 ARVO Annual Meeting abstract.