Limits...
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 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.

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

Human ASM cell proliferation after stimulated by CTGF.A. Results from the MTT assay after ASM cells were incubated in CTGF (0–30 ng/mL) for 1 day. B. Results from the MTT assay after ASM cells were incubated in CTGF (0–30 ng/mL) for 3 days. C. Results from the MTT assay after ASM cells were incubated in CTGF (0–30 ng/mL) for 5 days. D. The results from the MTT assay after ASM cells were incubated for 1–5 days in CTGF (10 ng/mL) with or without the scFv monomer, scFv dimer, or LY294002. E. The results from EdU incorporation assay after ASM cells were incubated for 1–5 days in CTGF (10 ng/mL) with or without the scFv monomer, scFv dimer, or LY294002. * p<0.05, ** p<0.01, *** p<0.005, **** p<0.001.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4257608&req=5

pone-0113980-g005: Human ASM cell proliferation after stimulated by CTGF.A. Results from the MTT assay after ASM cells were incubated in CTGF (0–30 ng/mL) for 1 day. B. Results from the MTT assay after ASM cells were incubated in CTGF (0–30 ng/mL) for 3 days. C. Results from the MTT assay after ASM cells were incubated in CTGF (0–30 ng/mL) for 5 days. D. The results from the MTT assay after ASM cells were incubated for 1–5 days in CTGF (10 ng/mL) with or without the scFv monomer, scFv dimer, or LY294002. E. The results from EdU incorporation assay after ASM cells were incubated for 1–5 days in CTGF (10 ng/mL) with or without the scFv monomer, scFv dimer, or LY294002. * p<0.05, ** p<0.01, *** p<0.005, **** p<0.001.

Mentions: To investigate the effect of CTGF on ASM cell proliferation, the cells were exposed to various concentrations of CTGF (0, 10, 20, 30 and 40 ng/mL) for 1–5 days, and the amount of cell growth was determined using a MTT assay. There was no statistically significant difference in growth among the various concentration groups when the ASM cells were incubated in CTGF for 1 day (p>0.05, Figure 5A). There was, however, a significant increase in cell growth in the CTGF group (≥10 ng/mL) compared with the control group when the cells were cultured for 3 or 5 days (p<0.05, Figure 5B and C). The cell proliferation induced by 10 ng/mL CTGF after a 3 or 5-day incubation period was significantly inhibited in the presence of the scFv monomer, scFv dimer, or LY294002 (Figure 5D). Similar results were observed in EdU incorporation assay (Figure 5E).


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)

Human ASM cell proliferation after stimulated by CTGF.A. Results from the MTT assay after ASM cells were incubated in CTGF (0–30 ng/mL) for 1 day. B. Results from the MTT assay after ASM cells were incubated in CTGF (0–30 ng/mL) for 3 days. C. Results from the MTT assay after ASM cells were incubated in CTGF (0–30 ng/mL) for 5 days. D. The results from the MTT assay after ASM cells were incubated for 1–5 days in CTGF (10 ng/mL) with or without the scFv monomer, scFv dimer, or LY294002. E. The results from EdU incorporation assay after ASM cells were incubated for 1–5 days in CTGF (10 ng/mL) with or without the scFv monomer, scFv dimer, or LY294002. * p<0.05, ** p<0.01, *** p<0.005, **** p<0.001.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0113980-g005: Human ASM cell proliferation after stimulated by CTGF.A. Results from the MTT assay after ASM cells were incubated in CTGF (0–30 ng/mL) for 1 day. B. Results from the MTT assay after ASM cells were incubated in CTGF (0–30 ng/mL) for 3 days. C. Results from the MTT assay after ASM cells were incubated in CTGF (0–30 ng/mL) for 5 days. D. The results from the MTT assay after ASM cells were incubated for 1–5 days in CTGF (10 ng/mL) with or without the scFv monomer, scFv dimer, or LY294002. E. The results from EdU incorporation assay after ASM cells were incubated for 1–5 days in CTGF (10 ng/mL) with or without the scFv monomer, scFv dimer, or LY294002. * p<0.05, ** p<0.01, *** p<0.005, **** p<0.001.
Mentions: To investigate the effect of CTGF on ASM cell proliferation, the cells were exposed to various concentrations of CTGF (0, 10, 20, 30 and 40 ng/mL) for 1–5 days, and the amount of cell growth was determined using a MTT assay. There was no statistically significant difference in growth among the various concentration groups when the ASM cells were incubated in CTGF for 1 day (p>0.05, Figure 5A). There was, however, a significant increase in cell growth in the CTGF group (≥10 ng/mL) compared with the control group when the cells were cultured for 3 or 5 days (p<0.05, Figure 5B and C). The cell proliferation induced by 10 ng/mL CTGF after a 3 or 5-day incubation period was significantly inhibited in the presence of the scFv monomer, scFv dimer, or LY294002 (Figure 5D). Similar results were observed in EdU incorporation assay (Figure 5E).

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 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.

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