title: The Antiproliferative Effect of Bevacizumab on Human Tenon Fibroblasts Is Not Mediated by Vascular Endothelial Growth Factor Inhibition.
abstract: OBJECTIVE
  Vascular endothelial growth factor-signaling in human tenon fibroblasts (hTFs) has recently become a target for antifibrotic treatment in glaucoma filtration surgery. The anti-VEGF antibody bevacizumab (BVC) has been shown to increase filtration bleb size. Given the relatively high concentration of BVC needed to obtain an effect, we investigated whether BVC acts through VEGF inhibition or via non-antigen-dependent ways.
  
  
  METHODS
  Human tenon fibroblast primary cultures were obtained from strabismus surgery subjects. Under low (0.2%) and high (10%) serum conditions, cells were incubated with BVC, ranibizumab (RNB), aflibercept (AFB), or rituximab (RTX) at different concentrations. Total number of cells and number of dead or proliferating (5-bromo-2-deoxy-uridine-positive) cells were assessed after 24 hours. Concentrations of VEGF-A in cell culture media was measured with ELISA. Intracellular IgG was detected with immunostaining and Western blot analysis.
  
  
  RESULTS
  In quiescent hTF culture (0.2% serum) the addition of 5 mg/mL BVC induced widespread cell death. Under proliferative conditions (10% serum), BVC reduced the number of proliferating cells. No such effect was observed with 2.5 mg/mL BVC or with 10 mg/mL AFB or 2.5 mg/mL RNB, although they were equally effective in binding free VEGF-A in the culture media. Instead, the CD20 antibody RTX, which did not bind VEGF, induced hTF death and inhibited proliferation in a BVC-comparable fashion. Bevacizumab, AFB, and RTX were detected intracellularly in a concentration-dependent manner.
  
  
  CONCLUSIONS
  The cell death-inducing and antiproliferative effect of 5 mg/mL BVC appeared not to depend on VEGF inhibition. Our data question a direct role of VEGF for hTF survival and proliferation.
authors.affiliation: Department of Ophthalmology, Georg-August University Medical Center Göttingen, Göttingen, Germany.
authors.forename: Charlotte V
authors.initials: CV
authors.lastname: Fischer
authors.affiliation: Department of Ophthalmology, Georg-August University Medical Center Göttingen, Göttingen, Germany.
authors.forename: Viktoria
authors.initials: V
authors.lastname: Mans
authors.affiliation: Department of Ophthalmology, Georg-August University Medical Center Göttingen, Göttingen, Germany.
authors.forename: Maren
authors.initials: M
authors.lastname: Horn
authors.affiliation: Department of Ophthalmology, Georg-August University Medical Center Göttingen, Göttingen, Germany.
authors.forename: Sabine
authors.initials: S
authors.lastname: Naxer
authors.affiliation: Department of Ophthalmology, University Medical Center Kiel, University of Kiel, Kiel, Germany.
authors.forename: Alexa
authors.initials: A
authors.lastname: Klettner
authors.affiliation: Department of Ophthalmology, Georg-August University Medical Center Göttingen, Göttingen, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: van Oterendorp