title: Simultaneous application of bevacizumab and anti-CTGF antibody effectively suppresses proangiogenic and profibrotic factors in human RPE cells.
abstract: OBJECTIVE
  Retinal pigment epithelial (RPE) cells play key roles in the development of choroidal neovascularization and subsequent fibrosis. We investigated the impact of bevacizumab, antihuman vascular endothelial growth factor (VEGF) antibody, and anticonnective tissue growth factor (anti-CTGF) neutralizing antibody, individually or in combination, on proangiogenic and profibrotic properties of RPE cells.
  
  
  METHODS
  Primary cultures of human RPE cells were incubated with different concentrations of bevacizumab (0.25, 0.5, and 0.8 mg/ml) and/or anti-CTGF (10 μg/ml), and cell proliferation and apoptosis were determined. Expression and activity of proangiogenic and profibrotic genes including matrix metalloproteinases (MMP)-2 and 9, VEGFA, CTGF, vascular endothelial growth factor receptor-1 (VEGFR-1), cathepsin D, tissue inhibitor of metalloproteinases (TIMP) -1 and -2, and alpha smooth muscle actin (α-SMA) were assessed with slot blot, real-time RT-PCR, and zymography.
  
  
  RESULTS
  Bevacizumab alone inhibited proliferation of RPE cells while anti-CTGF or bevacizumab and anti-CTGF combined had no inhibitory effect in this regard. Bevacizumab increased MMP-2, MMP-9, and cathepsin D but decreased VEGFA and VEGFR-1 expression. The CTGF level was increased by using 0.25 mg/ml bevacizumab but decreased at the 0.8 mg/ml concentration of bevacizumab. Treatment with anti-CTGF antibody decreased MMP-2 expression whereas combined treatment with bevacizumab and anti-CTGF resulted in decreased expression of MMP-2, TIMP-1, cathepsin D, VEGFA, CTGF, and α-SMA in the treated cultures.
  
  
  CONCLUSIONS
  Treatment of RPE cells with the combination of bevacizumab and anti-CTGF could effectively suppress the proangiogenic and profibrotic activity of RPE cells.
authors.affiliation: National Institute of Genetic Engineering and Biotechnology, Tehran, Iran.
authors.forename: Abouzar
authors.initials: A
authors.lastname: Bagheri
authors.affiliation: National Institute of Genetic Engineering and Biotechnology, Tehran, Iran.
authors.forename: Zahra-Soheila
authors.initials: ZS
authors.lastname: Soheili
authors.affiliation: Ophthalmic Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
authors.forename: Hamid
authors.initials: H
authors.lastname: Ahmadieh
authors.affiliation: Blood Transfusion Research Center High Institute for Research and Education in Transfusion Medicine, Tehran, Iran.
authors.forename: Shahram
authors.initials: S
authors.lastname: Samiei
authors.affiliation: Department of Ophthalmology and Visual Sciences, University of Wisconsin School of Medicine and Public Health, Madison, WI.
authors.forename: Nader
authors.initials: N
authors.lastname: Sheibani
authors.affiliation: National Institute of Genetic Engineering and Biotechnology, Tehran, Iran.
authors.forename: Shamila Darvishalipour
authors.initials: SD
authors.lastname: Astaneh
authors.affiliation: Ocular Tissue Engineering Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
authors.forename: Mozhgan Rezaei
authors.initials: MR
authors.lastname: Kanavi
authors.affiliation: National Institute of Genetic Engineering and Biotechnology, Tehran, Iran.
authors.forename: Azam
authors.initials: A
authors.lastname: Mohammadian
mesh_terms: D000199:Actins / Q000235:genetics / Q000378:metabolism; D020533:Angiogenesis Inhibitors / Q000494:pharmacology*; D061067:Antibodies, Monoclonal, Humanized / Q000494:pharmacology*; D057134:Antibodies, Neutralizing / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D000068258:Bevacizumab; D002402:Cathepsin D / Q000235:genetics / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D055513:Connective Tissue Growth Factor / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D004357:Drug Synergism; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D020778:Matrix Metalloproteinase 2 / Q000235:genetics / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000235:genetics / Q000378:metabolism; D061251:Primary Cell Culture; D055213:Retinal Pigment Epithelium / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D015398:Signal Transduction; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000235:genetics / Q000378:metabolism; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic; D042461:Vascular Endothelial Growth Factor A / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D040281:Vascular Endothelial Growth Factor Receptor-1 / Q000235:genetics / Q000378:metabolism
chemical_list: C541116:ACTA2 protein, human; D000199:Actins; D020533:Angiogenesis Inhibitors; D061067:Antibodies, Monoclonal, Humanized; D057134:Antibodies, Neutralizing; C526317:CCN2 protein, human; D019715:Tissue Inhibitor of Metalloproteinase-1; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D019716:Tissue Inhibitor of Metalloproteinase-2; D055513:Connective Tissue Growth Factor; D000068258:Bevacizumab; D040281:Vascular Endothelial Growth Factor Receptor-1; D002402:Cathepsin D; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9