SPARC Inhibition Reduces Wound Healing Activity in Human Tenons Fibroblasts in an in vitro Model of Subconjunctival Fibrosis: A Comparison to Mitomycin-C Treatment

Abstract

Purpose: : To evaluate the wound healing functions of SPARC (secreted protein, acidic, rich in cysteine) in vitro and compare its anti-fibrotic effects to Mitomycin-C (MMC)

Methods: : Cell-mediated collagen gel contraction, cell proliferation, adhesion (xCELLigence system) and migration assays were used. Markers of fibrosis were determined with western blot and ELISA in human Tenons fibroblasts, SPARC siRNA knockdown fibroblasts, SPARC knock-out and wild type mouse fibroblasts. The anti-fibrotic effects of SPARC inhibition were compared with MMC treated cells.

Results: : Inhibition of cell mediated contraction was comparable between SPARC siRNA and MMC treated cells. Collagen I expression was reduced in siRNA knockdown compared to MMC treatment (0.4mg/ml). Loss of SPARC delayed cell migration in the knockdown cells but to a lesser extent than with MMC treatment. Matrix metalloproteinase activity was not affected by SPARC inhibition but proteolytic activity increased in MMC treated cells. SPARC inhibition reduced cell adhesion compared to MMC treatment. TGF-β treatment increased collagen I expression by fivefold in MMC-treated cells compared to siRNA knockdown. Fibronectin and alpha smooth muscle actin (ASMA) protein expression were both markedly increased in MMC treated cells exposed to TGF-β compared to all other treatment conditions. The antifibrotic effects in the SPARC knockout mouse cells showed similar results to the human knockdown.

Conclusions: : SPARC inhibition allowed wound healing to occur in vitro without causing extensive cell death which is commonly associated with MMC. More importantly, the absence of SPARC prevented the development of a TGF-β induced fibrotic phenotype, which was present in MMC treated fibroblasts. Modulating SPARC expression has the potential to replace MMC as an anti-fibrotic strategy for treating subconjunctival scarring following trabeculectomy surgery.

Keywords: wound healing • extracellular matrix