In vitro analyses of the anti-fibrotic effect of SPARC silencing in human Tenon's fibroblasts: comparisons with mitomycin C.
Bottom Line: We previously showed that SPARC (secreted protein, acidic, rich in cysteine) knockout mice had improved surgical success in a murine model of GFS.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.
Affiliation: Ocular Wound Healing and Therapeutics, Singapore Eye Research Institute, Singapore. email@example.comShow MeSH
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Mentions: We next examined whether silencing of SPARC or MMC treatment affect the expression of pro-fibrotic genes as well as their up-regulation by TGF-β, a cytokine viewed to play a master role during wound healing. The HTFs were analysed for the expression of collagen Iα1, fibronectin and α-SMA mRNAs in the absence or presence of TGF-β2 by qPCR. Collagen I mRNA expression was significantly reduced in both MMC-treated and SPARC-silenced HTFs relative to their respective controls (Table 1). MMC treatment appeared to be significantly more effective than SPARC silencing, although this is only a marginal 1.3-fold (Table 1). Incubation with TGF-β2 resulted in marked up-regulation of collagen I mRNA, but to a significantly lesser extent in both SPARC-silenced and MMC-treated HTFs (Table 2). In fact, both SPARC-silenced and MMC-treated HTFs repressed collagen I gene expression induced by TGF-β2 by the same magnitude, at 0.6-fold that of their respective TGF-treated controls (Table 2). In this capacity, there was no significant difference between SPARC-silenced and MMC-treated HTFs. These observations were verified at the protein level which showed that collagen I production was not significantly induced either in MMC-treated or in si-SPARC-transfected HTFs by TGF-β2 (Fig. 6A and B).
Affiliation: Ocular Wound Healing and Therapeutics, Singapore Eye Research Institute, Singapore. firstname.lastname@example.org