title: In vitro analyses of the anti-fibrotic effect of SPARC silencing in human Tenon's fibroblasts: comparisons with mitomycin C.
abstract: Failure of glaucoma filtration surgery (GFS) is commonly attributed to scarring at the surgical site. The human Tenon's fibroblasts (HTFs) are considered the major cell type contributing to the fibrotic response. We previously showed that SPARC (secreted protein, acidic, rich in cysteine) knockout mice had improved surgical success in a murine model of GFS. To understand the mechanisms of SPARC deficiency in delaying subconjunctival fibrosis, we used the gene silencing approach to reduce SPARC expression in HTFs and examined parameters important for wound repair and fibrosis. Mitomycin C-treated HTFs were used for comparison. We demonstrate that SPARC-silenced HTFs showed normal proliferation and negligible cellular necrosis but were impaired in motility and collagen gel contraction. The expression of pro-fibrotic genes including collagen I, MMP-2, MMP-9, MMP-14, IL-8, MCP-1 and TGF-β(2)  were also reduced. Importantly, TGF-β(2)  failed to induce significant collagen I and fibronectin expressions in the SPARC-silenced HTFs. Together, these data demonstrate that SPARC knockdown in HTFs modulates fibroblast functions important for wound fibrosis and is therefore a promising strategy in the development of anti-scarring therapeutics.
authors.affiliation: Ocular Wound Healing and Therapeutics, Singapore Eye Research Institute, Singapore. seet.li.fong@seri.com.sg
authors.forename: Li-Fong
authors.initials: LF
authors.lastname: Seet
authors.forename: Roseline
authors.initials: R
authors.lastname: Su
authors.forename: Li Zhen
authors.initials: LZ
authors.lastname: Toh
authors.forename: Tina T
authors.initials: TT
authors.lastname: Wong
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D018932:Chemokine CCL2 / Q000235:genetics / Q000378:metabolism; D002921:Cicatrix / Q000378:metabolism; D024042:Collagen Type I / Q000235:genetics / Q000378:metabolism; D005347:Fibroblasts / Q000187:drug effects* / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism; D005355:Fibrosis / Q000188:drug therapy*; D055785:Gene Knockdown Techniques; D020868:Gene Silencing; D006801:Humans; D020287:In Situ Nick-End Labeling; D016209:Interleukin-8 / Q000235:genetics / Q000378:metabolism; D053511:Matrix Metalloproteinase 14 / Q000235:genetics / Q000378:metabolism; D020778:Matrix Metalloproteinase 2 / Q000235:genetics / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000235:genetics / Q000378:metabolism; D016685:Mitomycin / Q000494:pharmacology*; D015676:Osteonectin / Q000235:genetics* / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D058475:Tenon Capsule / Q000166:cytology / Q000378:metabolism; D053781:Transforming Growth Factor beta2 / Q000235:genetics / Q000378:metabolism; D014945:Wound Healing / Q000187:drug effects
chemical_list: C111835:CCL2 protein, human; D018932:Chemokine CCL2; D024042:Collagen Type I; D005353:Fibronectins; D016209:Interleukin-8; D015676:Osteonectin; D034741:RNA, Small Interfering; D053781:Transforming Growth Factor beta2; D016685:Mitomycin; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9; D053511:Matrix Metalloproteinase 14