Anti-VEGF Treatment on Human Tenons’ Fibroblasts Scarring Activity in vitro

Abstract

Purpose: : To assess the effects of Anti-VEGF bevacizumab (Avastin) on Human Tenons’ Fibroblast (HTF) healing activity in-vitro.

Methods: : Fibroblasts were routinely cultured in RPMI media and bevacizumab was administered at a concentration ranging from 0.025 to 12.5mg/ml. Fibroblast viability and cell death was assessed using MTT colorimetric assay, Lactate Dehydrogenase assay and live/dead assays. Fibroblast contractility was assessed using floating collagen gel assay. In addition, the mechanism of HTF cell death (post bevacizumab administration) was explored via Hoechst staining and Scanning Electron Microscopy (SEM).

Results: : Bevacizumab induced a dose dependent reduction of HTF cell numbers, (83% reduction SD +/-6%) at a concentration of 12.5mg/mL at 72 hours. The administration of bevacizumab caused a moderate inhibition (30% reduction SD +/-8%) of fibroblast gel contraction from baseline. Under serum-free conditions, bevacizumab induced fibroblast cell death at concentrations greater than 7.5mg/ml. Scanning Electron Microscopy revealed marked vacuolization in bevacizumab treated fibroblasts suggestive of cell death via autophagy.

Conclusions: : Bevacizumab (Avastin) disrupts fibroblast proliferation, inhibits contraction ability of fibroblasts and induces cell death suggestive of autophagy. Our study findings suggest anti-VEGF has a direct effect on HTF activity and viability in culture. This supports a possible role for bevacizumab in inhibiting post-operative scarring after glaucoma filtration surgery.

Keywords: wound healing • vascular endothelial growth factor • apoptosis/cell death