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: It has been proposed that MMC improves surgical outcome at least in part through the induction of apoptosis, which leads to early termination of the wound healing response . To determine whether the induction of apoptosis or cell necrosis plays a role in the anti-fibrotic effect of SPARC knockdown, we analysed annexin V expression on the cell surface of si-SPARC-transfected HTFs using flow cytometry and compared that against MMC-treated cells. Surprisingly, MMC treatment resulted in 3.5-fold less early apoptotic cells compared to untreated HTFs at 72 hrs (Fig. 3A, upper panel, P ∇ 9.6χ10−6). However, MMC did cause a significant 12-fold increase in late apoptotic or necrotic cells compared to control (Fig. 3A, upper panel, P ∇ 6.1χ10−4). In comparison, SPARC knockdown did not result in a significant change in early apoptosis compared to si-Scram-transfected HTFs (Fig. 3A, lower panel, P ∇ 0.8). Instead, SPARC silencing appeared to suppress necrotic cell death compared to control si-Scram-transfected HTFs by 1.6-fold (Fig. 3A, lower panel, P ∇ 0.03). Analyses of the cells by TUNEL assay confirmed the flow cytometry results. As TUNEL preferentially labels cell apoptosis over necrosis, the number of TUNEL-positive cells upon MMC treatment was not observed to be greater than untreated HTFs (Fig. 3B). A dramatic difference between si-SPARC- and si-Scram-transfected HTFs was also not observed by TUNEL staining, in agreement with the flow cytometry results (Fig. 3B). Hence, the anti-fibrotic effect of SPARC depletion is not likely to be associated with the induction of apoptosis in HTFs.
Affiliation: Ocular Wound Healing and Therapeutics, Singapore Eye Research Institute, Singapore. firstname.lastname@example.org