Background Increased proliferation of pulmonary vascular cells and muscularisation of pulmonary vessels arefrequently observed in human smokers and in animals exposed to cigarette smoke.To elucidate the molecularmechanisms leading to these changes,we studied the in vitro effect of cigarette smoke extract(CSE)on proliferation ofpulmonary artery smooth muscle cells(PASMCs)and activation of protein kinase C(PKC),an important kinaseimplicated in cell proliferation.Methods PASMCs cultured from 12 normal Wistar rats were studied in the following conditions:(1)PASMCs wereexposed to different concentrations of CSE for 24 hours,then MTT colorimetric assay was used for detection of cellproliferation.Cell viability was assessed by trypan blue exclusion.(2)PASMCs were pre-incubated with phorbol12-myristate 13-acetate(PMA)for 24 hours or Ro31-8220 for 30 minutes before exposure to 5% CSE for 24 hours.Cellproliferation was examined by MTT colorimetric assay,cell cycle analysis and proliferating cell nuclear antigen(PCNA)immunocytochemical staining.(3)PASMCs were exposed to 5% CSE for 24 hours.Then PKC-α mRNA expression wasdetected by reverse transcription-polymerase chain reaction(RT-PCR)and protein expression by Western blotting,whilePKC-α translocation was observed by immunofluorescence staining and confocal microscopy.(4)PASMCs weretransfected with specific antisense oligodeoxynucleotides against PKC-α 6 hours before exposure to 5% CSE for 24hours.PKC-α protein expression and cell proliferation were detected by methods described previously.Results(1)Low concentration of CSE(5%)increased proliferation of PASMCs,whereas high concentrations(20%,30%)were inhibitory as a result of cytotoxicity.(2)The value of absorbance(Value A),proliferation index(PI),S-phasecell fraction(SPF)and average optical density of PCNA staining in PASMCs from 5% CSE exposure group(0.306±0.033,0.339±0.033,0.175±0.021,0.315±0.038,respectively)were significantly increased compared with those ofcontrol group(0.249±0.018,0.177±0.055,0.092±0.023,0.187±0.022,respectively)(P<0.05).PKC down-regulationby PMA pretreatment or PKC inhibition by Ro31-8220 pre-incubation abolished the effect of 5% CSE on PASMCsproliferation.(3)After exposure to 5% CSE for 24 hours,PKC-α mRNA and protein expression in PASMCs(1.054±0.078,1.185±0.041,respectively)were much higher than in untreated cells(0.573±0.054,0.671±0.055,respectively)(P<0.01).Moreover,5% CSE induced a translocation of PKC-α from cytoplasm toward the perinuclear area and into thenucleus.(4)Specific antisense oligodeoxynucleotides against PKC-α reduced 5% CSE-induced expression of PKC-αprotein(0.713±0.047 vs 1.180±0.056),also abolished the effect of 5% CSE on PASMCs proliferation significantly.Conclusions CSE can be cytotoxic at high concentrations.But at low concentrations,it makes a mitogenic effect oncultured PASMCs.PKC,especially its alpha isozyme,seems to play an important role in CSE-induced proliferation ofPASMC.Chin Med J 2007;120(17):1523-1528 Background Increased proliferation of pulmonary vascular cells and muscularisation of pulmonary vessels are frequently observed in human smokers and in animals exposed to cigarette smoke. To elucidate the molecular mechanisms leading to these changes, we studied the in vitro effect of cigarette smoke extract (CSE) on proliferation of pulmonary tubules (PASMCs) and activation of protein kinase C (PKC), an important kinaseimplicated in cell proliferation. Methods PASMCs cultured from 12 normal Wistar rats were studied in the following conditions: (1) PASMCs wereexposed to different concentrations of CSE for 24 hours, then MTT colorimetric assay was used for detection of cell proliferation. Cell viability was assessed by trypan blue exclusion. (2) PASMCs were pre-incubated with phorbol 12-myristate 13-acetate (PMA) for 24 hours or Ro 31-8220 for 30 minutes before exposure to 5% CSE for 24 hours. Cellproliferation was examined by MTT colorimetric assay, cell cycle analysis and proliferating cell (3) PASMCs were exposed to 5% CSE for 24 hours. When PKC-α mRNA expression wasdetected by reverse transcription-polymerase chain reaction (RT-PCR) and protein expression by Western blotting, whilePKC- α translocation was observed by immunofluorescence staining and confocal microscopy. (4) PASMCs were transfected with specific antisense oligodeoxynucleotides against PKC-α 6 hours before exposure to 5% CSE for 24hours. PKC-α protein expression and cell proliferation were detected by methods described previously. The concentration of CSE (5%) increased proliferation of PASMCs, while high concentrations (20%, 30%) were inhibitory as a result of cytotoxicity. (2) The value of absorbance PI, S-phasecell fraction (SPF) and average optical density of PCNA staining in PASMCs from 5% CSE exposure group (0.306 ± 0.033,0.339 ± 0.033,0.175 ± 0.021,0.315 ± 0.038, respectively) were significantly increased compared with those ofcontro l group(0.249 ± 0.018,0.177 ± 0.055,0.092 ± 0.023,0.187 ± 0.022, respectively) (P <0.05) .PKC down-regulationby PMA pretreatment or PKC inhibition by Ro31-8220 pre-incubation abolished the effect of 5% CSE on PASMCsproliferation (3) After exposure to 5% CSE for 24 hours, PKC-α mRNA and protein expression in PASMCs (1.054 ± 0.078,1.185 ± 0.041, respectively) were much higher than in untreated cells (0.573 ± 0.054, 0.671 ± 0.055, 5% CSE induced a translocation of PKC-α from cytoplasm toward the perinuclear area and into thenucleus. (4) Specific antisense oligodeoxynucleotides against PKC-α reduced 5% CSE-induced expression of PKC- αprotein (0.713 ± 0.047 vs 1.180 ± 0.056), also abolished the effect of 5% CSE on PASMCs proliferated significantly. Conclusions CSE can be cytotoxic at high concentrations.But at low concentrations, it makes a mitogenic effect on culture pASMCs.PKC, especially its alpha isozyme, seems to play an important role in CSE-induced proliferation of PASMC. Chin Med J 2007 ; 120 (17): 1523-1528