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In vitro analyses of the anti-fibrotic effect of SPARC silencing in human Tenon's fibroblasts: comparisons with mitomycin C.

Seet LF, Su R, Toh LZ, Wong TT - J. Cell. Mol. Med. (2012)

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.

View Article: PubMed Central - PubMed

Affiliation: Ocular Wound Healing and Therapeutics, Singapore Eye Research Institute, Singapore. seet.li.fong@seri.com.sg

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Silencing of SPARC protected HTFs from necrosis. (A) HTFs, treated as indicated for 72 hrs, were harvested for both adherent and non-adherent cells and analysed for apoptosis and late apoptosis/necrosis. The representative scatterplots are shown. Apoptotic cells are enclosed within the blue perimeter and the mean percentage of apoptotic cells is indicated. Necrotic cells are shown within the green perimeter and the mean percentage of necrotic/late apoptotic is indicated. Data are presented as mean ± S.D. of the averages of three independent experiments, each performed in triplicate. (B) Cells were treated as in (A) and analysed by TUNEL staining (green). Nuclei were visualized by DAPI staining (blue). Cells positive for TUNEL staining are indicated by arrowheads; scale bar: 100 μm.
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fig03: Silencing of SPARC protected HTFs from necrosis. (A) HTFs, treated as indicated for 72 hrs, were harvested for both adherent and non-adherent cells and analysed for apoptosis and late apoptosis/necrosis. The representative scatterplots are shown. Apoptotic cells are enclosed within the blue perimeter and the mean percentage of apoptotic cells is indicated. Necrotic cells are shown within the green perimeter and the mean percentage of necrotic/late apoptotic is indicated. Data are presented as mean ± S.D. of the averages of three independent experiments, each performed in triplicate. (B) Cells were treated as in (A) and analysed by TUNEL staining (green). Nuclei were visualized by DAPI staining (blue). Cells positive for TUNEL staining are indicated by arrowheads; scale bar: 100 μm.

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 [12]. 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.


In vitro analyses of the anti-fibrotic effect of SPARC silencing in human Tenon's fibroblasts: comparisons with mitomycin C.

Seet LF, Su R, Toh LZ, Wong TT - J. Cell. Mol. Med. (2012)

Silencing of SPARC protected HTFs from necrosis. (A) HTFs, treated as indicated for 72 hrs, were harvested for both adherent and non-adherent cells and analysed for apoptosis and late apoptosis/necrosis. The representative scatterplots are shown. Apoptotic cells are enclosed within the blue perimeter and the mean percentage of apoptotic cells is indicated. Necrotic cells are shown within the green perimeter and the mean percentage of necrotic/late apoptotic is indicated. Data are presented as mean ± S.D. of the averages of three independent experiments, each performed in triplicate. (B) Cells were treated as in (A) and analysed by TUNEL staining (green). Nuclei were visualized by DAPI staining (blue). Cells positive for TUNEL staining are indicated by arrowheads; scale bar: 100 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3823078&req=5

fig03: Silencing of SPARC protected HTFs from necrosis. (A) HTFs, treated as indicated for 72 hrs, were harvested for both adherent and non-adherent cells and analysed for apoptosis and late apoptosis/necrosis. The representative scatterplots are shown. Apoptotic cells are enclosed within the blue perimeter and the mean percentage of apoptotic cells is indicated. Necrotic cells are shown within the green perimeter and the mean percentage of necrotic/late apoptotic is indicated. Data are presented as mean ± S.D. of the averages of three independent experiments, each performed in triplicate. (B) Cells were treated as in (A) and analysed by TUNEL staining (green). Nuclei were visualized by DAPI staining (blue). Cells positive for TUNEL staining are indicated by arrowheads; scale bar: 100 μm.
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 [12]. 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.

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.

View Article: PubMed Central - PubMed

Affiliation: Ocular Wound Healing and Therapeutics, Singapore Eye Research Institute, Singapore. seet.li.fong@seri.com.sg

Show MeSH
Related in: MedlinePlus