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Anti-CTGF single-chain variable fragment dimers inhibit human airway smooth muscle (ASM) cell proliferation by down-regulating p-Akt and p-mTOR levels.

Gao W, Cai L, Xu X, Fan J, Xue X, Yan X, Qu Q, Wang X, Zhang C, Wu G - PLoS ONE (2014)

Bottom Line: To further improve the bioactivity of scFv, we constructed a plasmid to express scFv-linker-matrilin-6×His fusion proteins that could self-assemble into the scFv dimers by disulfide bonds in matrilin under non-reducing conditions.The MTT and EdU assay results revealed that CTGF (≥10 ng/mL) promoted the proliferation of ASM cells, and this effect was inhibited when the cells were treated with anti-CTGF scFv dimer.The western blot analysis results showed that increased phosphorylation of Akt and mTOR induced by CTGF could be suppressed by this scFv dimer.

View Article: PubMed Central - PubMed

Affiliation: Medical School, Southeast University, Nanjing 210009, China.

ABSTRACT
Connective tissue growth factor (CTGF) contributes to airway smooth muscle (ASM) cell hyperplasia in asthma. Humanized single-chain variable fragment antibody (scFv) was well characterized as a CTGF antagonist in the differentiation of fibroblast into myofibroblast and pulmonary fibrosis in our previous studies. To further improve the bioactivity of scFv, we constructed a plasmid to express scFv-linker-matrilin-6×His fusion proteins that could self-assemble into the scFv dimers by disulfide bonds in matrilin under non-reducing conditions. An immunoreactivity assay demonstrated that the scFv dimer could highly bind to CTGF in a concentration-dependent manner. The MTT and EdU assay results revealed that CTGF (≥10 ng/mL) promoted the proliferation of ASM cells, and this effect was inhibited when the cells were treated with anti-CTGF scFv dimer. The western blot analysis results showed that increased phosphorylation of Akt and mTOR induced by CTGF could be suppressed by this scFv dimer. Based on these findings, anti-CTGF scFv dimer may be a potential agent for the prevention of airway remodeling in asthma.

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Related in: MedlinePlus

Expression levels of p-mTOR/mTOR in human ASM cells determined by Western blot analysis.The ratio was higher in cells co-cultured in CTGF (p<0.0001). The increase in the p-mTOR induced by CTGF was significantly inhibited when the cells were treated with scFv dimer, LY294002, or mAb (p<0.001). There was no significant difference between the scFv group and mAb group (p>0.05).
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pone-0113980-g007: Expression levels of p-mTOR/mTOR in human ASM cells determined by Western blot analysis.The ratio was higher in cells co-cultured in CTGF (p<0.0001). The increase in the p-mTOR induced by CTGF was significantly inhibited when the cells were treated with scFv dimer, LY294002, or mAb (p<0.001). There was no significant difference between the scFv group and mAb group (p>0.05).

Mentions: To investigate the potential mechanism involved in the anti-CTGF scFv dimer inhibition of ASM cell proliferation, Western blot analysis was used to detect the expression levels of Akt, p-Akt, mTOR, and p-mTOR. As shown in Figure 6 and 7, the p-Akt/Akt and p-mTOR/mTOR ratios significantly increased in the CTGF group compared with the control group (p<0.0001). Additionally, there were statistically significant decreases in the p-Akt/Akt and p-mTOR/mTOR ratios in the CTGF+scFv dimer, CTGF+anti-CTGF monoclonal antibody (mAb) and CTGF+LY294002 groups compared with the CTGF group (p<0.001). There was no significant difference between the CTGF+scFv dimer group and the CTGF+mAb group (p>0.05).


Anti-CTGF single-chain variable fragment dimers inhibit human airway smooth muscle (ASM) cell proliferation by down-regulating p-Akt and p-mTOR levels.

Gao W, Cai L, Xu X, Fan J, Xue X, Yan X, Qu Q, Wang X, Zhang C, Wu G - PLoS ONE (2014)

Expression levels of p-mTOR/mTOR in human ASM cells determined by Western blot analysis.The ratio was higher in cells co-cultured in CTGF (p<0.0001). The increase in the p-mTOR induced by CTGF was significantly inhibited when the cells were treated with scFv dimer, LY294002, or mAb (p<0.001). There was no significant difference between the scFv group and mAb group (p>0.05).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4257608&req=5

pone-0113980-g007: Expression levels of p-mTOR/mTOR in human ASM cells determined by Western blot analysis.The ratio was higher in cells co-cultured in CTGF (p<0.0001). The increase in the p-mTOR induced by CTGF was significantly inhibited when the cells were treated with scFv dimer, LY294002, or mAb (p<0.001). There was no significant difference between the scFv group and mAb group (p>0.05).
Mentions: To investigate the potential mechanism involved in the anti-CTGF scFv dimer inhibition of ASM cell proliferation, Western blot analysis was used to detect the expression levels of Akt, p-Akt, mTOR, and p-mTOR. As shown in Figure 6 and 7, the p-Akt/Akt and p-mTOR/mTOR ratios significantly increased in the CTGF group compared with the control group (p<0.0001). Additionally, there were statistically significant decreases in the p-Akt/Akt and p-mTOR/mTOR ratios in the CTGF+scFv dimer, CTGF+anti-CTGF monoclonal antibody (mAb) and CTGF+LY294002 groups compared with the CTGF group (p<0.001). There was no significant difference between the CTGF+scFv dimer group and the CTGF+mAb group (p>0.05).

Bottom Line: To further improve the bioactivity of scFv, we constructed a plasmid to express scFv-linker-matrilin-6×His fusion proteins that could self-assemble into the scFv dimers by disulfide bonds in matrilin under non-reducing conditions.The MTT and EdU assay results revealed that CTGF (≥10 ng/mL) promoted the proliferation of ASM cells, and this effect was inhibited when the cells were treated with anti-CTGF scFv dimer.The western blot analysis results showed that increased phosphorylation of Akt and mTOR induced by CTGF could be suppressed by this scFv dimer.

View Article: PubMed Central - PubMed

Affiliation: Medical School, Southeast University, Nanjing 210009, China.

ABSTRACT
Connective tissue growth factor (CTGF) contributes to airway smooth muscle (ASM) cell hyperplasia in asthma. Humanized single-chain variable fragment antibody (scFv) was well characterized as a CTGF antagonist in the differentiation of fibroblast into myofibroblast and pulmonary fibrosis in our previous studies. To further improve the bioactivity of scFv, we constructed a plasmid to express scFv-linker-matrilin-6×His fusion proteins that could self-assemble into the scFv dimers by disulfide bonds in matrilin under non-reducing conditions. An immunoreactivity assay demonstrated that the scFv dimer could highly bind to CTGF in a concentration-dependent manner. The MTT and EdU assay results revealed that CTGF (≥10 ng/mL) promoted the proliferation of ASM cells, and this effect was inhibited when the cells were treated with anti-CTGF scFv dimer. The western blot analysis results showed that increased phosphorylation of Akt and mTOR induced by CTGF could be suppressed by this scFv dimer. Based on these findings, anti-CTGF scFv dimer may be a potential agent for the prevention of airway remodeling in asthma.

Show MeSH
Related in: MedlinePlus