A Novel Model of Tissue Contraction and Inflammation In Fibrosis After Glaucoma Filtration Surgery

Abstract

 

Purpose

 

Fibrosis plays a part in almost all blinding diseases and underlies treatment failure in many surgical procedures including glaucoma filtration surgery. Developing better medicines to modulate wound healing is challenging as in vivo fibrosis involves both inflammation and fibroblast mediated tissue contraction. Clinically, the presence of significant inflammation after glaucoma surgery indicates a poor prognosis, and scarring occurs even with higher concentrations of Mitomycin-c (MMC) (0.5 mg/ml), necessitating MMC concentrations up to 1.0 mg/ml to overcome fibrosis in these cases. We developed a novel in vitro model incorporating both aspects of fibrosis. We characterised the model and studied the effect of different doses of MMC on fibroblast contraction with or without inflammatory cells.

 

Methods

 

The U937 monocyte-like human cell line was differentiated into macrophage-like cells using phorbol 12-myristate 13-acetate (PMA). Human Tenon’s capsule fibroblasts (HTF), untreated and treated with different doses of MMC for 5 minutes, were co-cultured with macrophages in three-dimensional collagen gels. Contraction was measured using digital photography. Gelatin zymography was used to measure matrix metalloproteinase (MMP) activity.

 

Results

 

Co-culture of fibroblasts and macrophages resulted in a significant increase in collagen gel contraction. Both fibroblast protrusive activity and expression of MMP 2 and 9 were increased, compared to HTFs or macrophages alone (Fig. 1). When HTFs were treated with low doses of MMC (0.01 and 0.1 mg/ml), the inflammatory stimulus provided by macrophages was able to overcome the inhibitory effect of MMC by day 4 (Fig. 2). High dose MMC (1 mg/ml) was required to completely inhibit contraction in the co-culture model.

 

Conclusions

 

This new co-culture model for tissue contraction introduces the inflammatory element found in scarring in vivo. Higher doses of MMC are required to overcome the extra stimulatory effects of inflammatory cells. This has implications for dosing in highly inflamed eyes and this novel model may be useful in screening different combinations of anti-scarring therapies.  

 

Fig. 1. Increased expression of active MMP2 and MMP9 in co-culture at day 3 compared with HTF and macrophages alone.

 

 

Fig. 2. Higher dose of MMC (1mg/ml) is required to inhibit contraction in fibroblasts when co-cultured with macrophages. n=3, * p<0.05<br />