Involvement of TRPC1 and Cyclin D1 in Human Pulmonary Artery Smooth Muscle Cells Proliferation Induced by Cigarette Smoke Extract

Cigarette smoking contributes to the development of pulmonary artery hypertension(PAH).As the basic pathological change of PAH,pulmonary vascular remodeling is considered to be related to the abnormal proliferation of pulmonary artery smooth muscle cells(PASMCs).However,the molecular mechanism underlying this process remains not exactly clear.The aim of this research was to study the molecular mechanism of PASMCs proliferation induced by smoking.Human PASMCs(HPASMCs)were divided into 6 groups:0%(control group),cigarette smoking extract(CSE)-treated groups at concentrations of 0.5%,1%,2%,5%,10%CSE respectively.HPASMCs proliferation was observed after 24 h.HPASMCs were divided into two groups:0(control group),0.5%CSE group.The mRNA and protein expression levels of transient receptor potential channel 1(TRPC1)and cyclin D1 in HPASMCs after CSE treatment were respectively detected by RT-PCR and Western blotting.The intracellular calcium ion concentration was measured by the calcium probe in each group.In the negative control group and TRPC1-siRNA transfection group,the proliferation of HPASMCs and the expression of cyclin D1 mRNA and protein were detected.Data were compared with one-way ANOVA(for multiple-group comparison)and independent t-test(for two-group comparison)followed by the least significant difference(LSD)test with the computer software SPSS 17.0.It was found that 0.5%and 1%CSE could promote the proliferation of HPASMCs(P<0.05),and the former was more effective than the latter(P<0.05),while 3%and above CSE had inhibitory effect on HPASMCs(P<0.05).The mRNA and protein expression levels of TRPC1 and cyclin D1 in 0.5%and 1%CSE groups were significantly higher than those in the control group(P<0.05),while those in 3%CSE group were significantly decreased(P<0.05).Moreover,the proliferation of HPASMCs and the expression of cyclin D1 mRNA and protein in TRPC1-siRNA transfection group were significantly reduced as compared with those in the negative control group(P<0.05).It was concluded that low concentration of CSE can promote the proliferation of HPASMCs,while high concentrations of CSE inhibit HPASMCs proliferation.These findings suggested that CSE induced proliferation of HPASMCs at least in part via TRPC1-mediated cyclin D1 expression.

Fasudil inhibits platelet-derived growth factor-induced human pulmonary artery smooth muscle cell proliferation by up-regulation of p27^kipl via the ERK signal pathway

Background RhoA/ Rho kinase （ROCK） pathway is involved in pulmonary arterial hypertension （PAH） and pulmonary artery smooth muscle cell （PASMC） proliferation. Inhibition of ROCK has been proposed as a treatment for PAH. But the mechanism of RhoA/ROCK pathway and its downstream signaling in proliferation of human PASMCs is unclear. We investigated the effect of fasudil, a selective ROCK inhibitor, on platelet-derived growth factor （PDGF） induced human PASMC proliferation, and the possible association between RhoA/ROCK and extracellular signal-regulated kinase （ERK）,p27KiP1.Methods Human PASMCs were cultured with the stimulation of 10 ng/ml PDGF, and different concentrations of fasudil were added before the addition of mitogen. Cell viability and cell cycle were determined with MTT and flow cytometry respectively. ROCK activity, ERK activity and protein expression of proliferating cell nuclear angigen （PCNA） and p27Kip1 were measured by immunoblotting.Results By MTT assay, PDGF significantly increased the OD value that represented human PASMC proliferation, and pretreatment with fasudil significantly reversed this effect in a dose-dependent manner. After PDGF stimulation, the percentage of cells in S phase increased dramatically from 15.6% to 24.3%, while the percentage in G0/G1 phase was reduced from 80.6% to 59%. And pretreatment with fasudil reversed the cell cycle effect of PDGF significantly in a dose-dependent manner. PDGF markedly induced ROCK activity and ERK activity with a peak at 15 minutes, which were significantly inhibited by fasudil. In addition, fasudil significantly inhibited PDGF-induced PCNA expression and fasudil also upregulated p27Kip1 expression in human PASMCs, which decreased after PDGF stimulation.Conclusion RhoA/ROCK is vital for PDFG-induced human PASMC proliferation, and fasudil effectively inhibited PDGF-induced human PASMC proliferation by up-regulation of p27Kip1, which may be associated with inhibition of ERK activity.

Focal adhesion kinase antisense oligodeoxynucleotides inhibit human pulmonary artery smooth muscle cells proliferation and promote human pulmonary artery smooth muscle cells apoptosis

Background Pulmonary artery smooth muscle cell (PASMC) proliferation plays an important role in pulmonary vessel structural remodelling At present, the mechanisms related to proliferation of PASMCs are not clear Focal adhesion kinase (FAK) is a widely expressed nonreceptor protein tyrosine kinase Recent research indicates that FAK is implicated in signalling pathways which regulate cytoskeletal organization, adhesion, migration, survival and proliferation of cells Furthermore, there are no reports about the role of FAK in human pulmonary artery smooth muscle cells (HPASMCs) We investigated whether FAK takes part in the intracellular signalling pathway involved in HPASMCs proliferation and apoptosis, by using antisense oligodeoxynucleotides (ODNs) to selectively suppress the expression of FAK protein Methods Cultured HPASMCs stimulated by fibronectin (40 μg/ml) were passively transfected with ODNs, sense FAK, mismatch sense and antisense FAK respectively Expression of FAK, Jun NH2 terminal kinase (JNK), cyclin dependent kinase 2 (CDK 2) and caspase 3 proteins were detected by immunoprecipitation and Western blots Cell cycle and cell apoptosis were analysed by flow cytometry In addition, cytoplasmic FAK expression was detected by immunocytochemical staining Results When compared with mismatch sense group, the protein expressions of FAK, JNK and CDK 2 in HPASMCs decreased in antisense FAK ODNs group and increased in sense FAK ODNs group significantly Caspase 3 expression upregulated in HPASMCs when treated with antisense ODNs and downregulated when treated with sense ODNs When compared with mismatch sense ODNs group, the proportion of cells at G 1 phase decreased significantly in sense ODNs group, while the proportion of cells at S phase increased significantly In contrast, compared with mismatch sense ODNs group, the proportion of cells at G 1 phase was increased significantly in antisense FAK ODNs group The level of cell apoptosis in antisense FAK group was higher than in the mismatch sense group and the latter was higher than sense FAK group In addition, the sense FAK ODNs group was strongly stained by immunocytochemistry, whereas the antisense FAK ODNs group was weakly stained Conclusions The results suggest that FAK relates to the proliferation of HPASMCs Antisense FAK ODNs inhibit HPASMCs proliferation and facilitate their apoptosis It is possible that FAK via JNK, CDK 2 signalling pathways enhances HPASMCs proliferation and via caspase 3 inhibits HPASMCs apoptosis

Different effects of telmisartan and valsartan on human aortic vascular smooth muscle cell proliferation

Background Vascular smooth muscle cell proliferation is an important process in the development of atherosclerosis and is associated with other cellular processes in atherogenesis. Telmisartan is reported to have partial peroxisome proliferator-activated receptor （PPAR）-γ activating properties and has been referred to as selective PPAR modulators, but valsartan just blocks angiotensin II （Angll） type 1 （AT1） receptors. This study aimed to compare the different effects of telmisartan and valsartan on human aortic smooth muscle cells （HASMCs） proliferation. Methods Ability of telmisartan and valsartan to inhibit proliferation of HASMCs was evaluated by the Cell Counting Kit-8 （CCK-8） in continuous cell culture. Whether the antiproliferative effects of telmisartan and valsartan depend on their effects on Angll receptors or activating the peroxisome PPAR-y was also investigated in this study. Results Telmisartan inhibited proliferation of HASMCs by 52.4% （P 〈0.01） at the concentration of 25 μmol/L and the effect depended on the dose of telmisartan, but valsartan had little effect on HASMCs proliferation （P 〉0.05） and no dose response. When tested in cells stimulated with Angll, telmisartan had the same inhibition of HASMCs by 59.2% （P 〈0.05） and valsartan also inhibited it by 41.6% （P 〈0.05）. Telmisartan and valsartan had the same effect on down-regulating AT1 receptor expression and telmisartan was superior to valsartan up-regulating Angll type 2 （AT2） receptor expression. Antiproliferative effects of telmisartan were observed when HASMCs were treated with the PPAR-y antagonist GW9662 but antiproliferative effects of the PPAR-y activator pioglitazone were not observed. Conclusions Telmisartan, but not valsartan, inhibits HASMCs proliferation and has dose-dependent response without stimulation of Angll. AT2 receptor up-regulation of telmisartan contributes to its greater antiproliferative effects than valsartan. Its PPAR-y activation does not play a critical role in inhibiting HASMCs proliferation.

Contribution of protein kinase C to passively sensitized human airway smooth muscle cells proliferation

Background Airway smooth muscle proliferation plays an important role in airway remodeling in asthma. But little is known about the intracellular signal pathway in the airway smooth muscle cell proliferation in asthma. The objective of this paper is to investigate the contribution of protein kinase C (PKC) and its alpha isoform to passively sensitized human airway smooth muscle cells (HASMCs) proliferation. Methods HASMCs in culture were passively sensitized with 10% serum from asthmatic patients,with non-asthmatic human serum treated HASMCs used as the control. The proliferation of HASMCs was examined by cell cycle analysis,3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazoliumbromide (MTT) colorimetric assay and proliferating cell nuclear antigen (PCNA) immunofluorescence staining. The effect of PKC agonist phorbol 12-myristate 13-acetate (PMA) and PKC inhibitor Ro-31-8220 on the proliferation of HASMCs exposed to human asthmatic serum and non-asthmatic control serum was also examined by the same methods. The protein and mRNA expression of PKC-α in passively sensitized HASMCs were detected by immunofluorescence staining and reverse transcription-polymerase chain reaction. Results The percentage of S phase,absorbance (value A) and the positive percentage of PCNA protein expression in HASMCs passively sensitized with asthmatic serum were (16.30±2.68)%,0.430±0.060 and (63.4±7.4)% respectively,which were significantly increased compared with HASMCs treated with control serum [(10.01±1.38)%,0.328±0.034 and (37.2±4.8)%,respectively] ( P <0.05). After HASMCs were passively sensitized with asthmatic serum,they were treated with PMA,the percentage of S phase,value A and the positive percentage of PCNA protein expression were (20.33±3.39)%,0.542±0.065 and (76.0±8.7)% respectively,which were significantly increased compared with asthmatic serum sensitized HASMCs without PMA( P <0.05). After HASMCs passively sensitized with asthmatic serum were treated with Ro-31-8220,the percentage of S phase,value A and the positive percentage of PCNA protein expression were (11.21±1.56)%,0.331±0.047 and (38.8±6.0)% respectively,which were significantly decreased compared with asthmatic serum sensitized HASMCs without Ro-31-8220 ( P <0.05). The relative ratio of value A of PKC-α mRNA and the positive percentage of PKC-α protein expression in passively sensitized HASMCs were 1.23±0.10 and (61.1±9.4)% respectively, which were significantly increased compared with HASMCs treated with control serum [1.05±0.09 and (34.9±6.7)%,respectively] ( P <0.05). Conclusions The proliferation of HASMCs passively sensitized with human asthmatic serum is increased. PKC and its alpha isoform may contribute to this proliferation.