Role of calcium-sensing receptor in hypoxia-induced proliferation of rat pulmonary artery smooth muscle cells

AIM: To explore the role of calcium-sensing receptor (CaSR) in rat pulmonary artery smooth muscle cells (PASMCs) and its effect on hypoxia-induced proliferation of PASMCs. METHODS: The expression and distribution of CaSRs were detected by Western blotting and immunofluorescence observation. The intracellular concentration of free calcium ([Ca]_i) was determined by confocal laser scanning microscopy. The cell viability was analyzed by MTT assay. The expression of PCNA and CaSRs was determined by Western blotting. RESULTS: CaSR protein was expressed in rat PASMCs. Hypoxia significantly increased the expression of CaSR and PCNA,[Ca]_i and the cell viability. GdCl₃ (an agonist of CaSR) or NPS2390 (an antagonist of CaSR) amplified or weakened the effect of hypoxia, respectively.CONCLUSION: CaSR is expressed in rat PASMCs. The activation of CaSR is involved in the proliferation of PASMCs induced by hypoxia.     

Role of α1 adrenoceptor in proliferation of hypoxic pulmonary artery smooth muscle cells

AIM: To investigate the role of α₁ and β₂ adrenoceptors(α₁AR and β₂AR) in the proliferation of hypoxic pulmonary artery smooth muscle cells (PASMCs).METHODS: PASMCs were isolated by an explant method from neonatal bovine pulmonary arteries. The cultured PASMCs were exposed to 6.6% O₂ for 6 h, 12 h and 24 h. The method of -TdR incorporation was used to measure the proliferation of PASMCs. _i was assayed with Fura-2/AM. The mRNA expression of α₁AR, β₂AR, c-fos and c-myc was determined by Northern blotting. The effects of activation of α₁AR and β₂AR, and inhibition of α₁AR on the above indexes were observed by treating PASMCs with different AR agonists and antagonists under hypoxic condition.RESULTS: Significant increase in TdR incorporation in hypoxic PASMCs with α₁AR activation was observed, and marked decrease in that was induced by α₁AR inhibition. However, no significant change was found after β₂AR activation. _i , the mRNA expression of c-fos, c-myc, α₁AR and β₂AR in PASMCs were increased after hypoxia.CONCLUSION: Hypoxia induces the increase in _i and mRNA expression of c-fos and c-myc, leading to the proliferation of PASMCs. The hypoxic proliferation of PASMCs is intervened by α₁AR, but not β₂AR. The remodeling of pulmonary arteriole and pulmonary hypertension may be involved in the processes of pulmonary arteriole constriction and proliferation induced by hypoxia through up-regulation of α₁AR.     

Effects of FHL1 on proliferation and migration of rat pulmonary arterial smooth muscle cells stimulated by cigarette smoke

AIM:To explore the role of four-and-a-half LIM domain 1 (FHL1) in the proliferation and migration of rat distal pulmonary arterial smooth muscle cells (PASMCs) stimulated by cigarette smoke extract (CSE). METHODS:Rat distal PASMCs were isolated and primarily cultured. The cells were divided into 6 groups: blank control group, negative transfection group, FHL1 siRNA transfection group, CSE group, CSE + negative transfection group and CSE + FHL1 siRNA transfection group. The mRNA and protein expression of FHL1 was detected by real-time PCR and Western blotting, respectively. Cell proliferation and migration were determined by CCK-8 and Transwell assays, respectively. RESULTS:CSE promoted the proliferation and migration of PASMCs, and increased the expression of FHL1 protein (P<0.01), but did not change the expression of FHL1 mRNA (P>0.05). FHL1 siRNA transfection attenuated the proliferation and migration of PASMCs induced by CSE (P<0.01). CONCLUSION: FHL1 protein is involved in CSE-induced proliferation and migration of rat PASMCs.     

Effects of docosahexaenoic acid on large-conductance calcium-activated potassium channels in rat pulmonary artery smooth muscle cells

AIM：To investigate the effects of docosahexaenoic acid (DHA) on large-conductance calcium-activated potassium channels (BKCa) in rat pulmonary artery smooth muscle cells (PASMCs)．METHODS：BKCa currents in individual PASMCs were recorded by patch-clamp technique in whole-cell configuration．Calcium sparks in PASMCs caused by DHA were recorded by confocal microscopy. RESULTS：DHA activated BKCa . BKCa current densities were (30.5±6.5)pA/pF,(59.4±5.8)pA/pF, (87.2±4.3)pA/pF and (117.3±7.1) pA/pF (P<0.01) with the addition of DHA at concentrations of 0, 0.1, 1 and 10 μmol/L, respectively. Hypoxia inhibited BKCa currents in PASMCs, but this inhibition was reversed by DHA (10 μmol/L). DHA (10 μmol/L) induced an increase in ［Ca2+］i with a maximal increase rate of (71.9±4.1)%. CONCLUSION：DHA activates BKCa in rat PASMCs, leading to the vasodilation of pulmonary arteries.     

Effects of EGLN1 siRNA on growth of rat pulmonary artery smooth muscle cells under hypoxic condition

AIM: To observe whether EGLN1 gene is involved in the growth of pulmonary arterial smooth muscle cells (PASMCs) during hypoxia when EGLN1 gene expression was interference by siRNA. METHODS: The rat primary pulmonary arterial smooth muscle cells were cultured, and the specific lipidosome of EGLN1 siRNA was constructed and transfected into the PASMCs. The transfected PASMCs were cultured under hypoxia or normoxia conditions, respectively. The viability of the PASMCs was detected by CCK-8 assay. The protein expression of EGLN1 and vascular endothelial growth factor (VEGF) was determined by Western blot. RESULTS: The viability of the PASMCs was increased and the protein expression of VEGF was up-regulated in the PASMCs under hypoxic condition in a time-dependent manner. In hypoxia or normoxia condition, the viability and VEGF protein expression of the PASMCs were suppressed by EGLN1 siRNA. CONCLUSION: EGLN1 gene may involve in the growth of rat PASMCs by regulating VEGF protein level under hypoxic condition.     

Effects of bradykinin on proliferation of porcine pulmonary artery smooth muscle cells induced by TGF-β1

AIM: To investigate the effects of bradykinin (BK) on the proliferation of pulmonary artery smooth muscle cells (PASMCs) induced by transforming growth factor beta 1 (TGF-β1) and its possible mechanisms. METHODS: Primary porcine PASMCs were isolated, cultured and identified, and the cells at passages 2~6 were used in this study. The viability of PASMCs was determined by Cell Counting Kit-8 assay. The protein expression of phosphatidylinositol 3-kinase (PI3K), phosphorylated Akt (p-Akt) and phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) was detected by Western blotting. RESULTS: TGF-β1 promoted the proliferation of PASMCs in a dose-dependent manner (P<005). BK significantly inhibited the proliferation of PASMCs induced by TGF-β1 (P<005), and attenuated the elevated expression of PI3K, p-Akt and p-ERK1/2 proteins (P<005). HOE-140, a BK type 2 receptor (B2R) inhibitor, reversed the effects of BK (P<005). CONCLUSION: BK inhibits TGF-β1-induced proliferation of PASMCs, which may be associated with inactivation of PI3K/Akt and ERK1/2 signaling pathways.     

Calcium-sensing receptor modulates pulmonary artery tension through G-protein-PLC-IP3 pathways

AIM: To observe the role of calcium-sensing receptor (CaSR) in the regulation of pulmonary artery tension. METHODS: The intracellular calcium concentration ([Ca²⁺]_i) was detected by laser-scanning confocal microscopy, and the pulmonary artery tension was determined by the pulmonary arterial ring technique. RESULTS: Increased levels of [Ca²⁺]_o or Gd³⁺ (an agonist of CaSR) induced the increase in [Ca²⁺]_i and pulmonary artery constriction in a concentration-dependent manner. Additionally, the effects of Ca²⁺ and Gd³⁺ were inhibited by U73122 and D609 (specific inhibitor of PLC), and 2-APB and heparin (specific antagonist of IP₃ receptor). However, U73343 (U73122 inactive analogue) did not take effect. CONCLUSION: CaSR may be involved in the regulation of pulmonary artery tension by increasing [Ca²⁺]_i through G-protein-PLC-IP₃ pathway.     

Smad pathway participates the process of proliferation of vascular smooth muscle cells induced by extracellular signal regulated kinase pathway

AIM: To investigate whether Smad pathway participates the process of extracellular signal regulated kinase (ERK) induced the proliferation of vascular smooth muscle cells (VSMCs). METHODS: Human umbilical artery smooth muscle cells (hUASMCs) were divided into four groups: control group, PDGF (platelet derived growth factor) group, ERK blocking agent group and PDGF+ERK blocking agent group. MTT assay was used to detect the proliferation of hUASMCs (A value). Immunohistochemical technique was used to detect the expression of PCNA, phosphorylated ERK (p-ERK) and phosphorylated Smad2/3 (p-Smad2/3) protein in hUASMCs. The expression of Smad2/3 mRNA in hUASMCs was detected by RT-PCR. RESULTS: The proliferation of hUASMCs and the expression of PCNA, p-ERK and p-Smad2/3 proteins in hUASMCs in PDGF group were increased obviously than those in other groups (P<0.01). No difference in the expression of Smad2/3 mRNA in hUASMCs among groups was observed. CONCLUSION: Smad pathway participates the process of ERK pathway that induces the proliferation of hUASMCs at the level of protein.     

Effect of diazoxide on change of H2O2 in rat pulmonary artery smooth muscle cells and proliferation of hypoxic rat pulmonary artery smooth muscle cells

AIM: To investigate the contribution of diazoxide,an opener of mitochondrial ATP-sensitive K+ channel (MitoK_ATP),and mitochondrial membrane potential (ΔΨm) to change of H₂O₂ in rat pulmonary artery smooth muscle cells (PASMCs) and to unbalance between cell proliferation and apoptosis of PASMCs induced by hypoxia.METHODS: The rat PASMCs were isolated from fresh normal lung tissues and cultured,which were divided into 6 groups,as follows: ① control group;② diazoxide group;③ 5-HD group;④chronic hypoxia group;⑤ chronic hypoxia+diazoxide group;⑥ chronic hypoxia +5-HD group.The relative change in mitochondrial potential was detected with rhodamine fluorescence (R-123) technique.The level of H₂O₂ in rat PASMCs was detected with chemiluminescence method.The proliferation of rat PASMCs was examined by cell cycle analysis and MTT colorimetric assay.RESULTS: After exposed to diazoxide for 24 h,the intensity of R-123 fluorescence,the level of H₂O₂ and the A value in normoxic rat PASMCs were significantly increased,and the apoptosis of rat PASMCs was significantly decreased as compared with control group (P<0.05).However,there were no significant changes in these tests after the rat PASMCs had been exposed to 5-HD for 24 h.Chronic hypoxia or chronic hypoxia+diazoxide markedly increased the intensity of R-123 fluorescence,the level of H₂O₂ and the A value in rat PASMCs,and also markedly decreased the apoptosis of rat PASMCs as compared with control group (P<0.05),and these changes were more significant in chronic hypoxia +diazoxide group than those in chronic hypoxia group (P<0.05).5-HD partly weakened the effect of hypoxia on the intensity of R-123 fluorescence,the level of H₂O₂,the A value and the apoptosis of rat PASMCs (P<0.05).Significant and positive correlations were found between the intracellular H₂O₂ and the R-123 fluorescence or the A value.Significant and negative correlation was found between the intracellular H₂O₂ and the apoptosis of rat PASMCs.CONCLUSION: The results suggest that the opening of MitoK_ATP followed by a depolarization of ΔΨm can contribute to the increase in the level of H₂O₂ in rat PASMCs and to the proliferation of rat PASMCs induced by hypoxia.This might be a mechanism of the development of hypoxic pulmonary hypertension.     

Response of mitochondrial reactive oxygen species in pulmonary arterial smooth muscle cells under acute hypoxic condition

AIM:To observe the response of mitochondrial reactive oxygen species (ROS) in rat pulmonary artery smooth muscle cells (PASMCs) under acute hypoxic condition. METHODS:The cultured PASMCs were under normoxic (35 ℃, 5% CO2, 21% O2, 74% N2) or acute hypoxic (35℃, 5% CO2, 1% O2, 94% N2) condition. The cells were incubated with molecular probes chloromethyl dichlorodihydrofluorescein diacetate (CM-H2DCF/DA) and RedoxSensor Red CC-1 to detect the ROS generation by laser scanning confocal microscopy. The mitochondria were isolated and mitochondrial inhibitors were used to detect the ROS generation functional unit sites by spectrophotometry under acute hypoxic condition. RESULTS:Under acute hypoxic condition, the intracellular ROS was significantly increased in hypoxia group with 3.35 folds higher of H2O2 than that in normoxia group. The contents of H2O2 and O-·2 in hypoxia group were 1.61 folds higher than those in normoxia group. Compare with hypoxia goup, pretreatment with the mitochondrial electron transport chain (ETC) complex I inhibitor MPP, the complex II inhibitors NPA and TTFA as well as the complex III pre-ubisemiquinone site inhibitor myxothiazol all remarkably reduced hypoxia-induced increase in ROS generation in PASMCs (reduced by 60%, 73%, 75% and 61%, respectively, P<0.01), whereas the complex III postubisemiquinone site inhibitor antimycin A and the complex IV inhibitor NaN3 had no effect on hypoxia-induced increase in ROS generation (increased by 13% and 9.1%, respectively, P>0.05). Direct detection of mitochondrial ROS showed the same results as the intracellular ROS. CONCLUSION: The intracellular ROS increases significantly in rat PASMCs under acute hypoxic condition. The mitochondrial ETC complex I, complex II and complex III pre-ubisemiquinone sites increase ROS generation, whereas the complex III postubisemiquinone site and complex IV do not produce this effect under acute hypoxic condition.     

Correlative cell signaling pathway for expression of heme oxygenase-1 induced by ginkgo biloba extract in rat vascular smooth muscle cells

AIM: To explore the effects of heme oxygenase-1(HO-1) protein expression induced by ginkgo biloba extract (EGB761) in rat vascular smooth muscle cells (RVSMC) and the correlative cell signaling pathway.METHODS: The RVSMC lines were revived.Serial passage to 6 generation was carried out and divided into different groups.The cells were treated respectively with vehicle,purely EGB761,EGB761 plus zinc protoporphyrin IX or other specific inhibitors of cell signaling pathway.Western blotting method was used to detect the expression of HO-1 in RVSMC.RESULTS: EGB761 induced HO-1 protein expression in a dose dependent manner.ZnPPⅨ and genitein significantly inhibited HO-1 protein expression induced by EGB761 (0.10±0.01,0.07±0.01 vs 0.61±0.07,P<0.01,respectively).However,calphostin-C,LY294002,Bay11- 7082 had no apparent effects on HO-1 protein expression induced by EGB761 (0.63±0.07,0.65±0.07,0.64±0.06 vs 0.61±0.07,P>0.05,respectively).CONCLUSION: (1) EGB761 significantly induces HO-1 protein expression in RVSMC,and the effect can be inhibited by a specific HO inhibitor ZnPPⅨ.(2) The HO-1 protein expression induced by EGB761 in RVSMC is mediated by tyrosine protein kinase pathway.     

Effects of hypoxia-inducible factor-1 signal pathway on proliferation in hypoxic pulmonary artery smooth muscle cells

AIM: To investigate the effect of hypoxia on the proliferation and apoptosis of pulmonary artery smooth muscle cells (PASMC); and to evaluate the role of hypoxia-inducible factor-1α(HIF-1α), iNOS, P-ERK1/2 protein expression in hypoxic pulmonary hypertension (HPH) pathogenesis.METHODS: Cultured rat PASMC were divided into normoxic group; hypoxic group; hypoxia+ADM(adrenomedulin) group, hypoxia+L-NAME(iNOS inhibitor) group; hypoxia+PD98059 group. Proliferation was investigated by MTT and PCNA. Apoptosis was examined by flow-cytometry. Westen blotting was used to measure protein expression of HIF-1α, P-ERK1/2 and iNOS. RESULTS: (1) A value of 24 h-hypoxia was significantly higher than that in the normoxic group (P<0.01). In the hypoxia+PD98059 group, ADM was significantly lower than that in hypoxia group, whereas A value of the hypoxia+L-NAME was significantly higher than that in hypoxic group and normoxic group (P<0.01). (2) PCNA was positive in PASMC after 24 h hypoxia (P<0.01). PD98059, ADM inhibited the expression of PCNA significantly (P<0.01), whereas L-NAME increased the expression of PCNA significantly (P<0.01). (3) Apoptosis index was not significantly difference among the different groups (P>0.05). (4) HIF-1α, iNOS and P-ERK1/2 expression was poorly positive in normoxic group, positive after hypoxia for 4h (P<0.01), reaching its peak at 8 h hypoxia (P<0.01), HIF-1α, P-ERK1/2 expression declined after 24 h hypoxia. L-NAME promoted the expression of HIF-1α, PD98059 inhibited the expression of HIF-1α, iNOS and P-ERK1/2 partly. ADM inhibited the expression of HIF-1α partly, promoted the expression of iNOS. CONCLUSION: (1) Hypoxia stimulates the proliferation of PASMC, and has no obvious effects on the apoptosis of PASMC. (2) HIF-1 plays an importent role in the proliferation of hypoxic PASMC.     

STAT3 mediates PDGF-BB-induced Pim-1 expression in rat vascular smooth muscle cells

AIM: To study the expression of Pim-1 in vascular smooth muscle cells (VSMCs) induced by platelet-derived growth factor BB (PDGF-BB). METHODS: VSMCs isolated from rats were treated with different concentrations of PDGF-BB for different time. The proliferation of VSMCs was detected by cell counting. The mRNA expression of Pim-1 was measured by real-time RT-PCR. The STAT3 activity was determined by Western blotting. Actinomycin D, AG490, and small interfering RNA (siRNA) for Pim-1 or STAT3 were used to investigate the underlying mechanisms. RESULTS: Pim-1 gene silencing attenuated the proliferation of VSMCs in response to PDGF-BB. The mRNA expression of Pim-1 was up-regulated by PDGF-BB at concentrations of 10 μg/L~50 μg/L for 1 h, and was maximally induced at the concentration of 20 μg/L. The time of Pim-1 mRNA expression maximally occurred 30 min after PDGF-BB exposure. Incubation of VSMCs with PDGF-BB resulted in a significant activation of STAT3. VSMCs pretreated with actinomycin D showed a significant decrease in the mRNA expression of Pim-1. Treatment with AG490 or knockdown of STAT3 in VSMCs resulted in inactivation of STAT3, and significantly suppressed the mRNA expression of Pim-1. CONCLUSION: PDGF-BB-induced VSMC proliferation is partly attributed to Pim-1. VSMCs strongly increase Pim-1 mRNA upon stimulation with PDGF-BB, and STAT3 signaling pathway appears to be efficient for regulation of Pim-1 expression. This process may play a critical role in development of vascular remodeling.     

Farrerol inhibits nicotine-induced proliferation of pulmonary vascular smooth muscle cells by enhancing Kv1.5 and Kv2.1 expression

AIM:To study the effect of farrerol (Far) on nicotine-induced proliferation of rat pulmonary smooth muscle cells (PASMCs), and further to explore its relationship with voltage-dependent potassium channels (Kv) 1.5 and Kv2.1. METHODS:Firstly, the effect of nicotine on the proliferation of PASMCs was detected by cell counting method, and the optimal concentration of nicotine was selected. Primary cultured PASMCs were randomly divided into 5 groups:normal control group, nicotine (1 μmol/L)group, nicotine (1 μmol/L) + Far (10^-6 mol/L, 10^-5 mol/L and 10^-4 mol/L) Far group. The activity of caspase-3 was measured by apoptosis kit, the cell viability was measured by CCK-8 assay, the apoptotic rate was analyzed by flow cytometry. The expression of Kv1.5 and Kv2.1, and apoptosis-related factors Bcl-2 and Bax at mRNA and protein levels was determined by RT-qPCR and Western blot respectively. RESULTS:Nicotine at 1 μmol/L increased the number of PASMCs to the maximum extent (P<0.01). Nicotine at 1 μmol/L significantly reduced the caspase-3 activity and enhanced the cell viability of the PASMCs (P<0.01). Farrerol at 10^-6~10^-4 mol/L eliminated the effect of PASMCs induced by nicotine in a concentration dependent manner. Compared with control group, nicotine at 1 μmol/L significantly increased the proliferation and inhibited the apoptotic rate of rat PASMCs (P<0.01). The apoptotic rate of PASMCs in farrerol intervention group was significantly higher than that in nicotine group (P<0.01). Nicotine at 1 μmol/L significantly inhibited the expression of Kv1.5, Kv2.1 and Bax but increased the expression of Bcl-2 in PASMCs (P<0.01). Farrerol at 10^-5 mol/L obviously inhibited the effect of PASMCs induced by nicotine. CONCLUSION:Farrerol eliminates nicotine-induced inhibition of caspase-3 and Bax, and enhancement of Bcl-2 in PASMCs by enhancing Kv1.5 and Kv2.1 expression.     

Inhibitory effect of Salidroside on the proliferation of rabbit pulmonary artery smooth muscle cells under hypoxia

AIM: To investigate the effect of Salidroside on the proliferation, DNA synthesis, intracellular Ca²⁺ content of rabbit PASMC (pulmonary artery smooth muscle cells) under hypoxia. METHODS: Techniques of cell culture, MTT test, [³H][³H][³H]-TdR incorporation, fluo-3 and confocal laser scanning microscopy were used. RESULTS: The A value of MTT and [³H][³H]-TdR incorporation of PASMC increased significantly by 62% (P＜0.05) and 138% (P＜0.01) after 24 h hypoxia. Salidroside (32×10^-5 mol/L) inhibited the action of hypoxia on the proliferation of PASMC, the A value of MTT and [³H][³H]-TdR incorporation declined significantly by 29% (P＜0.05) and 37% (P＜0.01) compared with hypoxia group. A calcium channel blocker, verapamil could also inhibit the accelerative effect of hypoxia on the proliferation of PASMC. The intracelluler Ca²⁺ content of PASMC raised markedly under hypoxia, but the effect of hypoxia on the intracelluler Ca²⁺ content could be inhibited by Salidroside. CONCLUSION: Salidroside inhibited the proliferation, DNA synthesis of PASMC induced by hypoxia. The inhibitory action of Salidroside on the increase in intracellular Ca²⁺ concentration under hypoxia might be one of the mechenisms.     

Effects of Rho kinase on p-Smad1 nuclear translocation in rat pulmonary artery smooth muscle cells

AIM: To explore the mechanism of bone morphogenetic protein (BMP) and Rho kinase signal pathways on the proliferation of pulmonary artery smooth muscle cells. METHODS: Pulmonary smooth muscle cells were isolated from the rat distal pulmonary artery and cultured. BMP and Rho kinase pathways were activated by BMP-2 and platelet-derived growth factor BB(PDGF-BB),respectively. Rho kinase inhibitor Y-27632 and MEK inhibitor U0126 were also used. Immunofluorescent staining was applied to observe p-Smad1 distribution across the nucleus, and the cells with positive p-Smad1 nuclear accumulation were counted and the nuclear translocation rate was calculated. The total p-Smad1 and its distribution across the nucleus were quantitatively determined by Western blotting. The cell proliferation was analyzed by CCK-8 assay. RESULTS: Exposure to BMP-2 significantly increased both the total amount of p-Smad1 and its nuclear accumulation in pulmonary smooth muscle cells. Pretreatment with PDGF-BB significantly decreased the nuclear accumulation of p-Smad1 induced by BMP-2 without decrease of total p-Smad1. However, pretreatment with Y-27632 or U0126 reversed the inhibitory effect of PDGF-BB on p-Smad1 nuclear accumulation. BMP-2 significantly inhibited the cell proliferation, but PDGF-BB blocked the effect of BMP-2 and significantly increased the cell proliferation. After pretreated with Y-27632 or U0126, the PDGF-BB-activated cell proliferation was suppressed.CONCLUSION: PDGF-BB-activated Rho kinase inhibits BMP-2-induced p-Smad1 nuclear translocation via MEK/ERK1/2, and increases pulmonary artery smooth muscle cell proliferation.     

Chlamydia pneumoniae infection induces vascular smooth muscle cell migration through stimulating Rac1 activity via PI3K activation

AIM: To determine the role of Rac1 activation in the migration of vascular smooth muscle cells (VSMCs) induced by Chlamydia pneumoniae (C.pn) infection and to investigate the effect of phosphatidylinositol 3-kinase (PI3K) on Rac1 activation during the process. METHODS: The recombinant plasmid encoding glutathione S-transferase (GST)-p21-binding domain of p21-activated kinase 1(PBD) fusion protein was transformed into the competent bacteria to induce the expression of the fusion protein. The fusion protein was purified. GST-pull down assay was performed to evaluate the activity of Rac1 in C.pn-infected VSMCs pretreated with or without PI3K inhibitor LY294002 (25 μmol/L). Wound-healing assay and Transwell assay were performed to observe the changes of C.pn infection-induced VSMCs migration with or without pre-incubation of Rac1 inhibitor NSC23766 (50 μmol/L). RESULTS: Enough and biologically active GST-PBD fusion protein was obtained after purification. The activity of Rac1 in VSMCs infected with C.pn for 24 h increased significantly and was higher than that in control group. Rac1 activation induced by C.pn infection was inhibited by the pretreatment of VSMCs with LY294002. The migration ability of C.pn-infected VSMCs pre-incubated with NSC23766 was significantly reduced and was lower than that in C.pn infection group. CONCLUSION: C.pn infection induces the migration of VSMCs possibly through stimulating Rac1 activity via PI3K activation.     

Effect of TRPC6 on PDGF-induced proliferation of rat airway smooth muscle cells

AIM:To investigate the role of canonical transient receptor potential channel 6 (TRPC6) in the proliferation of airway smooth muscle cells (ASMCs) induced by platelet-derived growth factor (PDGF). METHODS:Rat ASMCs were cultured by enzyme digestion and tissue adhesion. The method of indirect immunofluorescence was applied to identify the ASMCs and to detect the expression of TRPC6 in ASMCs. The proliferation of ASMCs was determined by CCK-8 assay. The mRNA expression of TRPC6 was tested by real-time PCR. The protein expression of TRPC6 was analyzed by Western blotting. The influence of TRPC6 blocker at different concentrations on the proliferation of ASMCs was measured by CCK-8 assay. RESULTS:The results of immunofluorescence indicated that TRPC6 expression in ASMCs was positive. PDGF at concentration of 20 μg/L induced the proliferation of ASMCs compared with control group (P<0.05). When ASMCs were treated with both PDGF and different concentrations of TRPC6 blocker SKF96365, the proliferation of ASMCs was attenuated in dose-dependent and time-dependent manners as compared with the cells treated with PDGF alone (P<0.05). The mRNA expression of TRPC6 in PDGF group was significantly increased (P<0.05) after ASMCs were treated with PDGF for 12 h, 24 h and 48 h. The protein level of TRPC6 in PDGF group was significantly increased after ASMCs were treated with PDGF for 24 h and 48 h compared with control group (P<0.05). CONCLUSION: Up-regulation of TRPC6 at mRNA and protein levels is most possibly related to the proliferation of ASMCs induced by PDGF. Therefore, TRPC6 is involved in the proliferation of ASMCs.     

Inhibitory effect of carbon monoxide on proliferation of rat pulmonary artery smooth muscle cells under anoxia

AIM: To investigate the effect of endogenous and exogenous carbon monoxide on the proliferation of pulmonary artery smooth muscle cells under anoxic condition. METHODS: Primary culture of rat PASMCs were passed every 3 days, the 3-5 passages were used. PASMCs were divided into 5 groups, cultured under normoxia and hypoxia and treated with HO inducer hemin, CO scavenger bovine hemoglobin (Hb) and exogenous carbon monoxide (CO), respectively. After 48 hours incubation under the conditions mentioned above, the following assay were carried out: 1) the MTT colorimetric assay and immunocytochemical staining were used to study the energy metabolism and the expression of proliferating cell nuclear antigen (PCNA) in PASMCs. 2) flow cytometry was used to analyze the cell cycle of PASMCs. RESULTS: In comparison with the control group, the value of MTT colorimetric assay was higher, the immunocytochemical staining of PCNA was stronger and the percentages of PASMCs in S and G₂M phases in the anoxia group were higher (P＜0.01). After treatment with hemin and CO, the above indexes were decreased (P＜0.01 or P＜0.05). But treatment with Hb made the above indexes increased (P＜0.01 or P＜0.05). CONCLUSION: The endogenous CO suppress the proliferation of PASMC in an autocrine way. Both the induction of endogenous CO by hemin and the treatment with exogenous CO could suppress the rat PASMCs' proliferation under anoxic condition.