Single-walled carbon nanotubes promote rat vascular adventitial fibroblasts to transform into myofibroblasts by SM₂₂-α expression

Figures & data

Figure 1 The characteristic of SWCNT and its cytotoxicity to VAFs. (A) Primary culture of rat VAFs. Morphological identification: (a) 1 day; (b) 5 days. (B) Immunological identification: (a) blank control; (b) vimentin (+); (c) desmin (−); (d) VIII F(−). (C) Particle characterization: (a) TEM of SWCNTs; (b) Raman spectra of SWCNTs. (D) Cell viability of cells incubation with SWCNTs (25, 50, 100, and 200 μg/mL) for 24, 48, and 72 hours using MTT, WST-1, and WST-8. Values are the means ± SEM of three experiments. *P < 0.05; **P < 0.01 versus M199-exposed control cells. (E) Oxidative damage including DNA-protein crosslink level and ROS production of cells after incubation with SWCNTs (0, 12.5, 25, 50, 100, and 200 μg/mL) for 72 hours.

Notes: Values are the means ± SEM of three experiments. *P < 0.05; **P < 0.01 versus M199-exposed control cells.

Abbreviations: VAF, vascular adventitial fibroblast; TEM, transmission electron microscopy; SWCNTs, single-wall carbon nanotubes; SEM, standard error of the mean; ROS, reactive oxygen species; MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; WST-1, (2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium sodium salt; WST-8, 2-(2-methoxy-4-NOxphenyl)-3-(4-NOxphenyl)-5-(2,4-methylbenzene)-2H-tetrazolim monosodium salt.

Figure 2 The immunocytochemical staining and cell ultrastructure changes assessment by TEM. (A) SWCNTs promoted VAFs transformed into MFs and immunohistochemistry of the SM₂₂-α related antigen. VAFs culture with SWCNT-free medium (a); VAFs transformed into MFs after incubation with 50 μg/mL SWCNTs for 96 hours, MFs depicted by brown staining (b). (B) Ultrastructure of rat VAFs observed by TEM: (a) typical normal cell (12,000×); (b and c) cell incubation with SWCNTs (50 μg/mL; 20 hours, 12,000×): observed shrinkage of the nucleus membrane and swelling of mitochondria; (d) typical normal cell (70,000×); (e and f) cells incubation with SWCNTs (70,000×): observed swelling of mitochondria with cristae decreasing or even disappearing, some mitochondrial transformation into little vacuoles; swelling of endoplasmic reticulum with ribosome (fine black particles) threshing; (e) observed phagocytosis phenomenon (70,000×).

Note: The arrows are SWCNT particles that entered into cells.

Abbreviations: VAF, vascular adventitial fibroblast; TEM, transmission electron microscopy; SWCNTs, single-wall carbon nanotubes; MF, myofibroblast.

Figure 3 The signal pathway of SM₂₂-α protein expression. (A) Effects of SWCNT incubation with VAFs on JNK, TGF-β1, TGF-β receptor II, and SM₂₂-α protein expression as determined by western blotting. (a) Protein levels of JNK, TGF-β1, and SM₂₂-α after cells incubation with 50 μg/mL SWCNTs for different times. (b) Protein levels of TGF-β1, TGF-β receptor II, and SM₂₂-α after cells incubation with various concentrations of SWCNTs for 96 hours. Protein levels were measured as described in the Materials and methods section. Control cells were cultured in SWCNT-free medium. Data are expressed as the mean ± SEM of three independent experiments (*P < 0.05; **P < 0.01). (B) Effects of SWCNT incubation with VAFs on pSmad2/3, Smad7, and Smad4 protein expression as determined by western blotting. (a) Protein levels of pSmad2/3, Smad7 after cells incubated with various concentrations of SWCNTs for 96 hours. (b) Protein levels of Smad4 translocated into nucleus after cell incubation with different dose of SWCNTs for 96 hours.

Notes: Protein levels were measured as described in the Materials and methods section. Control cells were cultured in SWCNT-free medium. Data are expressed as mean ± SEM of three independent experiments (*P < 0.05; **P < 0.01).

Abbreviations: SWCNTs, single-wall carbon nanotubes; VAF, vascular adventitial fibroblast; JNK, C-Jun N-terminal kinases; SEM, standard error of the mean.

Figure 4 TGF-β/Smad signal pathway.