Korean J Physiol Pharmacol.  2015 Jul;19(4):357-363. 10.4196/kjpp.2015.19.4.357.

Effect of Bevacizumab on Human Tenon's Fibroblasts Cultured from Primary and Recurrent Pterygium

• Park YM
• Kim CD
• Lee JS

• KMID: 2285579
• DOI: http://doi.org/10.4196/kjpp.2015.19.4.357

Abstract

The purpose of this study was to compare the inhibitory effect of bevacizumab on human Tenon's fibroblasts (HTFs) cultured from primary and recurrent pterygium. Cultured HTFs were exposed to 2.0, 5.0, 7.5, and 15.0 mg/mL concentration of bevacizumab for 24 hours. The 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide and lactate dehydrogenase leakage assays were then performed to assess fibroblast metabolism and viability. The matrix metalloproteinase (MMP), procollagen type I C terminal propeptide (PIP), and laminin immunoassays were performed to examine extracellular matrix production. Changes in cellular morphology were examined by phase-contrast and transmission electron microscopy. Both metabolic activity and viability of primary and recurrent pterygium HTFs were inhibited by bevacizumab in a dose-dependent manner, especially at concentrations greater than 7.5 mg/mL. Both types of HTFs had significant decreases in MMP-1, PIP, and laminin levels. Distinctly, the inhibitory effect of bevacizumab on MMP-1 level related with collagenase in primary pterygium HTFs was significantly higher than that of recurrent pterygium. Significant changes in cellular density and morphology both occurred at bevacizumab concentrations greater than 7.5 mg/mL. Only primary pterygium HTFs had a reduction in cellular density at a bevacizumab concentration of 5.0 mg/mL. Bevacizumab inhibits primary and recurrent pterygium HTFs in a dose-dependent manner, especially at concentrations greater than 7.5 mg/mL. As the primary HTFs produces larger amounts of MMP-1 compared to recurrent HTFs, significant reduction in MMP-1 level in primary pterygium HTFs after exposure to bevacizumab is likely to be related to the faster cellular density changes in primary pterygium HTFs.

Keyword

Anti-fibrotic effect; Bevacizumab; Extracellular matrix; Human Tenon's fibroblasts; Pterygium

MeSH Terms

Collagen Type I
Collagenases
Extracellular Matrix
Fibroblasts*
Humans
Immunoassay
L-Lactate Dehydrogenase
Laminin
Metabolism
Microscopy, Electron, Transmission
Pterygium*
Bevacizumab
Collagen Type I
Collagenases
L-Lactate Dehydrogenase
Laminin

Figure

• 

  Fig. 1 Absorption rate of the water-insoluble formazan dye in primary and recurrent human Tenon's fibroblasts (HTFs) exposed to bevacizumab, as determined by a scanning spectrometer (ELISA reader). After exposure to 10.0 and 15.0 mg/mL concentration of bevacizumab, the metabolic activity of primary and recurrent pterygium HTFs was significantly lower than in control HTFs.

• 

  Fig. 2 Lactate dehydrogenase (LDH) titers of primary and recurrent human Tenon's fibroblasts (HTFs) exposed to bevacizumab. Viability of cells exposed to 10.0 and 15.0 mg/mL concentration of bevacizumab was significantly less than that of control HTFs.

• 

  Fig. 3 Matrix metalloproteinase (MMP)-1 and MMP-2 activity in primary and recurrent pterygium human Tenon's fibroblasts after exposure to bevacizumab.

• 

  Fig. 4 Procollagen type 1 C terminal peptide (PIP) and laminin activity of primary and recurrent pterygium human Tenon's fibroblasts after exposure to bevacizumab.

• 

  Fig. 5 Effect of bevacizumab concentration on primary and recurrent pterygium human Tenon's fibroblast morphology (inverted phase-light microscopy and TEM images).

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