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

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

Schematic representations of the expression vector pET28a-scFv-matrilin (A), the scFv-matrilin fusion protein sequence (B) and the scFv dimer structure (C) are shown.

<p>Schematic representations of the expression vector pET28a-scFv-matrilin (A), the scFv-matrilin fusion protein sequence (B) and the scFv dimer structure (C) are shown.</p

ScFv-matrilin analyzed using native-PAGE and Western blot.

<p>M marker, lanes 1, 2 show the fusion protein purified by nickel affinity chromatography, lanes 3, 4 show immunodotting bands with.anti-6xHis tag antibody.</p

Expression and purification of scFv-matrilin were evaluated using SDS-PAGE and Western blot.

<p>M marker, lanes 1, 2 show the fusion protein purified by nickel affinity chromatography, lanes 3, 4 show the supernatant of the cell lysate after IPTG (0.3 mM) induction at 37°C for 12 h, lane 5 shows the supernatant of the cell lysate before IPTG induction, lanes 6, 7 show immunodotting bands with.anti-6xHis tag antibody (C).</p

MTOR mRNA level in human ASM cells determined by RT-PCR.

<p>All 2^−ΔΔCT values were calculated, and the value was assumed to be 1 for the control group. The increase in the mTOR mRNA level in ASM cells was significantly inhibited when the cells were treated with scFv dimer, LY294002, or mAb (p<0.0001). There was no statistically significant difference between the scFv group and mAb group (p>0.05).</p

Human ASM cell proliferation after stimulated by CTGF.

<p>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.</p

Expression levels of p-Akt/Akt in human ASM cells determined by Western blot analysis.

<p>The ratio was higher in cells co-cultured in CTGF (p<0.0001). The increase in the p-Akt level 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).</p

Immunoreactivity assay determined by ELISA.

<p>Both the scFv dimer and scFv monomer specifically reacted with CTGF. The regression equations are Y = 0.3633+1.1273X-0.3351X² (R² = 0.938), and Y = 0.0453+0.7185X−0.1960X² (R² = 0.994) for the scFv dimer and scFv monomer, respectively. The OD450 value for the scFv dimer was two times higher than the value for scFv monomer when the concentrations were equal to the CTGF concentration.</p