title: Effects of ranibizumab on the extracellular matrix production by human Tenon's fibroblast.
abstract: Anti-Vascular Endothelial Growth Factors (Anti-VEGF) agents have received recent interest as potential anti-fibrotic agents for their concurrent use with trabeculectomy. Preliminary cohort studies have revealed improved bleb morphology following trabeculectomy augmented with ranibizumab. The effects of this humanized monoclonal antibody on human Tenon's fibroblast (HTF), the key player of post trabeculectomy scar formation, are not fully understood. This study was conducted to understand the effects of ranibizumab on extracellular matrix production by HTF. The effect of ranibizumab on HTF proliferation and cell viability was determined using MTT assay (3-(4,5-dimethylthiazone-2-yl)-2,5-diphenyl tetrazolium). Ranibizumab at concentrations ranging from 0.01 to 0.5 mg/mL were administered for 24, 48 and 72 h in serum and serum free conditions. Supernatants and cell lysates from samples were assessed for collagen type 1 alpha 1 and fibronectin mRNA and protein level using quantitative real time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA). After 48-h, ranibizumab at 0.5 mg/mL, significantly induced cell death under serum-free culture conditions (p < 0.05). Ranibizumab caused significant reduction of collagen type 1 alpha 1 (COL1A1) mRNA, but not for fibronectin (FN). Meanwhile, COL1A1 and FN protein levels were found upregulated in treated monolayers compared to control monolayers. Ranibizumab at 0.5 mg/mL significantly reduced cell viability in cultured HTF. From this study, we found that single application of ranibizumab is inadequate to induce the anti-fibrotic effects on HTF, suggesting the importance of adjunctive therapy. Further studies are underway to understand mechanism of actions of ranibizumab on HTF.
authors.affiliation: Faculty of Medicine, Brain and Neuroscience Communities of Research, Universiti Teknologi MARA (UiTM) Shah Alam, Selangor, Malaysia.
authors.forename: Siti Munirah
authors.initials: SM
authors.lastname: Md Noh
authors.affiliation: Faculty of Medicine, Brain and Neuroscience Communities of Research, Universiti Teknologi MARA (UiTM) Shah Alam, Selangor, Malaysia.
authors.forename: Siti H
authors.initials: SH
authors.lastname: Sheikh Abdul Kadir
authors.affiliation: Faculty of Medicine, Brain and Neuroscience Communities of Research, Universiti Teknologi MARA (UiTM) Shah Alam, Selangor, Malaysia.
authors.forename: Zakaria M
authors.initials: ZM
authors.lastname: Bannur
authors.affiliation: Faculty of Medicine, Brain and Neuroscience Communities of Research, Universiti Teknologi MARA (UiTM) Shah Alam, Selangor, Malaysia.
authors.forename: Gabriele Anisah
authors.initials: GA
authors.lastname: Froemming
authors.affiliation: Faculty of Medicine, Brain and Neuroscience Communities of Research, Universiti Teknologi MARA (UiTM) Shah Alam, Selangor, Malaysia.
authors.forename: Narimah
authors.initials: N
authors.lastname: Abdul Hamid Hasani
authors.affiliation: Faculty of Medicine, Brain and Neuroscience Communities of Research, Universiti Teknologi MARA (UiTM) Shah Alam, Selangor, Malaysia.
authors.forename: Hapizah
authors.initials: H
authors.lastname: Mohd Nawawi
authors.affiliation: Centre for Eye Research Australia, The University of Melbourne, Melbourne, VIC, Australia.
authors.forename: Jonathan G
authors.initials: JG
authors.lastname: Crowston
authors.affiliation: Faculty of Medicine, Brain and Neuroscience Communities of Research, Universiti Teknologi MARA (UiTM) Shah Alam, Selangor, Malaysia. Electronic address: sushilvasudevan@yahoo.com.
authors.forename: Sushil
authors.initials: S
authors.lastname: Vasudevan
mesh_terms: D020533:Angiogenesis Inhibitors / Q000494:pharmacology*; D061067:Antibodies, Monoclonal, Humanized / Q000494:pharmacology*; D018929:Cell Culture Techniques; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D024042:Collagen Type I / Q000235:genetics* / Q000378:metabolism; D000090602:Collagen Type I, alpha 1 Chain; D004797:Enzyme-Linked Immunosorbent Assay; D005347:Fibroblasts / Q000187:drug effects* / Q000378:metabolism; D005353:Fibronectins / Q000235:genetics* / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D005786:Gene Expression Regulation / Q000502:physiology*; D005902:Glaucoma, Open-Angle / Q000601:surgery; D006801:Humans; D012333:RNA, Messenger / Q000235:genetics; D000069579:Ranibizumab; D060888:Real-Time Polymerase Chain Reaction; D058475:Tenon Capsule / Q000166:cytology*; D014130:Trabeculectomy; D042461:Vascular Endothelial Growth Factor A / Q000037:antagonists & inhibitors; D014746:Vimentin / Q000378:metabolism
chemical_list: D020533:Angiogenesis Inhibitors; D061067:Antibodies, Monoclonal, Humanized; D024042:Collagen Type I; D000090602:Collagen Type I, alpha 1 Chain; D005353:Fibronectins; D012333:RNA, Messenger; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D014746:Vimentin; D000069579:Ranibizumab
keywords: collagen type 1 alpha 1; enzyme-linked immunosorbent assay; fibronectin; human Tenon's fibroblast; quantitative real time polymerase chain reaction; ranibizumab; trabeculectomy