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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.

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

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Collagen I and fibronectin protein expressions were not induced in SPARC knockdown HTFs in response to TGF-β2 but α-SMA expression remained inducible. (A) HTFs were treated with MMC (left panel) or transfected with siRNAs (right panel), with or without TGF-β2, for 72 hrs before being analysed for collagen I protein abundance by immunoblotting for collagen Iα1 and GAPDH (loading control). The data shown are representative of three independent experiments. (B) Densitometric analysis of immunoblots from three independent experiments represented in (A). Data are presented as mean fold induction ± S.D. relative to their respective controls (untreated: left panel; si-Scram–transfected HTFs: right panel) from three independent experiments. The GAPDH level was used for normalization. The P value for each comparison is indicated above the bars. (C) HTFs were subjected to the indicated treatments as in (A) and analysed for fibronectin expression. The data shown are representative of three independent experiments. (D) Densitometric analysis of immunoblots from three independent experiments represented in (C). (E) HTFs were subjected to the indicated treatments as in (A) and analysed for α-SMA protein expression. The data shown are representative of three independent experiments. (F) Densitometric analysis of immunoblots from three independent experiments represented in (E).
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fig06: Collagen I and fibronectin protein expressions were not induced in SPARC knockdown HTFs in response to TGF-β2 but α-SMA expression remained inducible. (A) HTFs were treated with MMC (left panel) or transfected with siRNAs (right panel), with or without TGF-β2, for 72 hrs before being analysed for collagen I protein abundance by immunoblotting for collagen Iα1 and GAPDH (loading control). The data shown are representative of three independent experiments. (B) Densitometric analysis of immunoblots from three independent experiments represented in (A). Data are presented as mean fold induction ± S.D. relative to their respective controls (untreated: left panel; si-Scram–transfected HTFs: right panel) from three independent experiments. The GAPDH level was used for normalization. The P value for each comparison is indicated above the bars. (C) HTFs were subjected to the indicated treatments as in (A) and analysed for fibronectin expression. The data shown are representative of three independent experiments. (D) Densitometric analysis of immunoblots from three independent experiments represented in (C). (E) HTFs were subjected to the indicated treatments as in (A) and analysed for α-SMA protein expression. The data shown are representative of three independent experiments. (F) Densitometric analysis of immunoblots from three independent experiments represented in (E).

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).


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)

Collagen I and fibronectin protein expressions were not induced in SPARC knockdown HTFs in response to TGF-β2 but α-SMA expression remained inducible. (A) HTFs were treated with MMC (left panel) or transfected with siRNAs (right panel), with or without TGF-β2, for 72 hrs before being analysed for collagen I protein abundance by immunoblotting for collagen Iα1 and GAPDH (loading control). The data shown are representative of three independent experiments. (B) Densitometric analysis of immunoblots from three independent experiments represented in (A). Data are presented as mean fold induction ± S.D. relative to their respective controls (untreated: left panel; si-Scram–transfected HTFs: right panel) from three independent experiments. The GAPDH level was used for normalization. The P value for each comparison is indicated above the bars. (C) HTFs were subjected to the indicated treatments as in (A) and analysed for fibronectin expression. The data shown are representative of three independent experiments. (D) Densitometric analysis of immunoblots from three independent experiments represented in (C). (E) HTFs were subjected to the indicated treatments as in (A) and analysed for α-SMA protein expression. The data shown are representative of three independent experiments. (F) Densitometric analysis of immunoblots from three independent experiments represented in (E).
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fig06: Collagen I and fibronectin protein expressions were not induced in SPARC knockdown HTFs in response to TGF-β2 but α-SMA expression remained inducible. (A) HTFs were treated with MMC (left panel) or transfected with siRNAs (right panel), with or without TGF-β2, for 72 hrs before being analysed for collagen I protein abundance by immunoblotting for collagen Iα1 and GAPDH (loading control). The data shown are representative of three independent experiments. (B) Densitometric analysis of immunoblots from three independent experiments represented in (A). Data are presented as mean fold induction ± S.D. relative to their respective controls (untreated: left panel; si-Scram–transfected HTFs: right panel) from three independent experiments. The GAPDH level was used for normalization. The P value for each comparison is indicated above the bars. (C) HTFs were subjected to the indicated treatments as in (A) and analysed for fibronectin expression. The data shown are representative of three independent experiments. (D) Densitometric analysis of immunoblots from three independent experiments represented in (C). (E) HTFs were subjected to the indicated treatments as in (A) and analysed for α-SMA protein expression. The data shown are representative of three independent experiments. (F) Densitometric analysis of immunoblots from three independent experiments represented in (E).
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).

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