Metallothionein inhibits homocysteine-induced proliferation of rat vascular smooth muscle cells

AIM:To investigate the effect of metallothionein(MT) on proliferation of rat vascular smooth muscle cells (VSMCs) stimulated by homocysteine and its mechanism. METHODS:VSMCs proliferation was measured by [³-H]-TdR incorporation, mitogen-activated protein kinase(MAPK)activity were determined by immunoprecipitation method, the intracellular contents of MT and malondialdehyde (MDA)were assayed by -hemoglobin saturation method and TBA reaction, respectively, and lactate dehydrogenase (LDH) leakage was measured by NADH oxidation. RESULTS:Hcy(10^-6-10^-4 mmol/L) stimulated [³-H]-TdR incorporation by the VSMCs in a concentration-dependent manner. Compared with control, [³-H]-TdR incorporation in VSMCs treated with 0.1 mmol/L Hcy was increased by 4.2 fold (P＜0.01). Meanwhile, Hcy enhanced MAPK activity, MDA formation and LDH release (P＜0.01)in a concentration-dependent manner. Treatment of VSMCs with MT alone did not change above parameters, compared with control. However, MT (10^-6-10^-4 mol/L)attenuated significantly Hcy-stimulated proliferation of VSMCs (P＜0.01)in a concentration-dependent manner. And MT inhibited obviously Hcy-induced activation of MAPK activity, MDA formation and LDH release. Preincubation of VSMCs with 0.5 mmol/L ZnCl₂ for 6 h induced an increase cellular MT content by 5.7-fold (P＜0.01). The MT-overexpressed VSMCs resisted Hcy-stimulating action on MAPK activity, MDA formation and LDH leakage (P＜0.01). CONCLUSION:These results show that MT has an inhibitory effect on Hcy-induced VSMCs proliferation, and that MT could inhibit Hcy-stimulated MAPK activity and lipid peroxidation.     

Inhibitory effects of Lobelia Chinensis Lour alkaloid on the proliferation of human umbilical artery vascular smooth muscle induced by endothelin-1

AIM: To investigate the effects of Lobelia Chinensis Lour Alkaloid (LCLA) on the proliferation of cultured vascular smooth muscle cells (VSMC) induced by ET-1. METHODS: Human umbilical artery VSMC was cultured and divided into five groups: ET group, ET+LCLA group, ET+BQ-123 group，ET+ staurosporine （ST） group and control group. The cell proliferation activity was subsequently quantified by cell counting kit-8 (CCK-8) and ［3H］-TdR incorporation. Flow cytometry was used to examine cell cycle. Quantitative immunohistochemical technique was used to investigate the expression of proliferating cell nuclear antigen (PCNA) and confocal microscope was used to measure the fluorescent intensity of Ca2+. Cytotoxicity was measured by Trypan blue exclusion and LDH colorimetry tests. RESULTS: BQ-123 (10-6mol/L), ST (10-7mol/L) and LCLA (100, 200 and 400 mg/L) inhibited the increase in cell number, ［3H］-TdR incorporation, the percentage of the S phase and markedly decreased the expression of PCNA and fluorescent intensity of Ca2+ in response to ET-1 of VSMC (P<0.05). CONCLUSION: LCLA (100-400 mg/L) inhibits ET-1-induced proliferation of VSMC in a dose-dependent manner and the anti-proliferative effect is realized by reducing the Ca2+ concentration in VSMC.     

Effect of xeroderma pigmentosum group D gene on proliferation of human umbilical arterial smooth muscle cells induced by Ox-LDL

AIM: To investigate the effects of xeroderma pigmentosum group D (XPD) gene on the proliferation of human umbilical arterial smooth muscle cells (HUASMCs) induced by oxidized low-density lipoprotein (Ox-LDL). METHODS: The recombinant plasmid pEGFP-N2/XPD was transfected into HUASMCs by liposome. The cells were divided into blank control group, pEGFP-N2 group, pEGFP-N2/XPD group, Ox-LDL group, Ox-LDL+pEGFP-N2 group and Ox-LDL+pEGFP-N2/XPD group. The proliferation rate of the cells was detected by MTT and EdU assays. The apoptotic rate and cell cycle distribution were analyzed by flow cytometry. The protein levels of XPD, caspase-3, Bcl-2 and Bax were determined by Western blot. RESULTS: Compared with blank control group, the expression of XPD was increased in pEGFP-N2/XPD group (P<0.05). According to the results of MTT and EdU assays, the cell proliferation in pEGFP-N2/XPD group was reduced compared with blank control group (P<0.05). Compared with Ox-LDL group, the cell proliferation in Ox-LDL+pEGFP-N2/XPD group was significantly inhibited (P<0.05). According to the results of flow cytometry, the cell proportion of S phase decreased and the G₀/G₁-phase cell proportion increased significantly in pEGFP-N2/XPD group and Ox-LDL+pEGFP-N2/XPD group compared with blank control group and Ox-LDL group, repectively (P<0.05). Compared with blank control group and Ox-LDL group, the protein level of Bcl-2 decreased and the protein levels of Bax and cleaved caspase-3 increased in pEGFP-N2/XPD group and Ox-LDL+pEGFP-N2/XPD group, respectively (P<0.05). CONCLUSION: XPD inhibits the proliferation of HUASMCs and promotes their apoptosis, and reduces the promoting effect of Ox-LDL on the proliferation of HUVSMCs. XPD may be the target for treatment of atherosclerosis.     

Effects of She Xiang Bao Xin Wan (SXBXW) on the proliferation of primary cultured human umbilical artery vascular smooth muscle cells induced by endothelin-1

AIM: To observe the effects of She Xiang Bao Xin Wan (SXBXW) of Chinese patent medicine on the proliferation of primary cultured vascular smooth muscle cells (VSMCs) from human umbilical artery and stimulated by endothelin-1 (ET-1) in vitro. METHODS: The proliferation cell models of primary cultured VSMCs were established by ET-1 stimulation. Six groups in the experiment were divided into control group; ET-1 group; ET-1+SXBXW 0.25 g/L; ET-1+SXBXW 0.5 g/L; ET-1+SXBXW 1.0 g/L and ET-1+SXBXW 2.0 g/L groups, respectively. The proliferation induced by ET-1 and the suppression mediated by SXBXW on VSMCs were measured by MTT method. The inhibitory rate and the cytotoxicity of SXBXW were detected by lactate dehydrogenase colorimetry and trypan blue exclusion tests. The effect of ET-1 and SXBXW on the cell proliferation cycle was analyzed by flow cytometry. RESULTS: Compared to control group, ET-1 significantly enhanced the proliferation of VSMCs (P<0.01). However, a certain dose of SXBXW inhibited effectively the proliferation of VSMCs induced by ET-1 in a dose-dependent manner (P<0.01). Meanwhile, SXBXW showed no influenced on both the number of living cells and the release of lactate dehydrogenase, although it inhibited the proliferation of VSMCs, indicating that SXBXW was no cytotoxicitic effect on VSMCs. ET-1 enhanced the proliferation of VSMCs by means of promoting the transition of the cell cycle from G1 phase to S phase. However SXBXW significantly inhibited the proliferation mediated by ET-1. CONCLUSION: SXBXW plays the role in suppressing VSMCs proliferation induced by ET-1. The mechanism may be involved in blocking the cell cycle from G1 phase into S phase.     

Probucol inhibits proliferation of rat aortic smooth muscle cells stimulated with oxidized low-density lipoprotein

AIM: To investigate the relationships between antiproliferative mechanisms of probucol and protein expressions of signaling molecules ERK1/2, MKP-1, HO-1 and Trx-1 in rat aortic smooth muscle cells (RASMCs) stimulated with ox-LDL. METHODS: The effects of probucol on cell cycle, cell proliferation and the expressions of ERK1/2, MKP-1, HO-1 and Trx-1 in the presence of ox-LDL were observed by means of MTT test, FCM and Western blotting. RESULTS: (1) Probucol significantly inhibited the proliferation of RASMCs stimulated with ox-LDL. A value in 100 μmol/L probucol+35 mg/L ox-LDL group was reduced by 34.9% as compared to ox-LDL group (P<0.01). (2) Probucol protected against ox-LDL-induced RASMCs proliferation through inducing cell growth arrest at G0/G1 phase and cell apoptosis. (3) ox-LDL increased the expression of p-ERK1/2 by 34.7% (P<0.01) and decreased MKP-1 by 60.0% (P<0.01), respectively, as compared to control. Probucol attenuated the increase in ox-LDL-stimulated p-ERK1/2 level by 15.7%, but increased MKP-1 expression by 2 times (P<0.01). (4)ox-LDL at concentration of 35 mg/L decreased the intracellular Trx-1 expression by 28.9% (P<0.05), and slightly increased the level of HO-1 expression as compared to control (P<0.05). Probucol enhanced the expression of Trx-1 by 91.6% (P<0.01) and HO-1 by 31.9% (P<0.01), respectively as compared to ox-LDL group. CONCLUSION: Probucol inhibits ox-LDL-stimulated the proliferation of RASMCs through increases in MKP-1/HO-1 expression, suppression of cell cycle progression and induction of cell apoptosis.     

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.     

Quercetin inhibits endothelin-1-induced T-type Ca2+ channel expression in human umbilical arterial smooth muscle cells

AIM: To investigate the effect of quercetin on endothelin-1-induced T-type calcium channel(TCC) expression in primary cultured human umbilical arterial smooth muscle cells for exploring the protective role of quercetin in cardiovascular system. METHODS: Human umbilical arterial smooth muscle cells were verified by immunocytochemistry. The cells in 2-3 passages were used and randomly divided into control group, quercetin alone group, model group and experimental group. The cells in control group were cultured without any drugs for 24 h. The cells in quercetin alone group were cultured with 80 μmol/L quercetin for 24 h. The cells in model group were cultured with ET-1 at the concentration of 100 nmol/L for 24 h. The cells in experimental groups were pretreated with quercetin for 1 h, then coincubated with 100 nmol/L ET-1 for 24 h. The concentrations of quercetin used in this study were 20, 40and 80 μmol/L, respectively. The expression of α_1G, a TCC major subunit, was assayed at mRNA and protein levels by RT-PCR and Western blotting, respectively. The TCC currents(I_caT) were detected by the technique of whole-cell patch-clamp. RESULTS: Compared with control and experimental group, I_CaT density (P<0.01) and the expression of α_1G at mRNA (P<0.05) and protein (P<0.01) levels in model group were significantly increased. No significant difference in the results of quercetin alone group and control group was observed. CONCLUSION: The protective roles of quercetin in cardiovascular functions are related to the depressive effects of quercetin on ET-1-induced increase in both I_CaT density and the expression of α_1G at mRNA and protein levels in cultured human vascular smooth muscle cells.     

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.     

Fluvastatin inhibits migration of rat vascular smooth muscle cells

AIM: To investigate the inhibitory effects of fluvastatin on the migration of rat vascular smooth muscle cells (VSMCs) induced by angiotensin II (AngⅡ) and platelet derived growth factor-BB (PDGF-BB). METHODS: Cultured VSMCs derived from rat thoracic aorta were used. The activity of heat shock protein 27 (HSP27) was evaluated by Western blotting with specific phospho-HSP27 antibody. The effect of F-actin polymerization was detected by FITC-phalloidine staining and examined by confocal microscopy. Modified Boyden chamber technique was employed for VSMCs migration assessment. RESULTS: The phosphorylation of HSP27 in VSMCs was increased by the stimulation of AngⅡ and PDGF-BB in a concentration-dependent manner. Treatment with AngⅡ and PDGF-BB resulted in a substantial increase in the number of stress fibers and rearrangement of these structures into ordered parallel arrays. The migration of VSMCs was promoted by AngⅡ and PDGF-BB. Reorganization of actin cytoskeleton stimulated with AngⅡ and PDGF-BB was inhibited by a specific HSP27 inhibitor quercetin (100 μmol/L) pretreatment. The inhibitory rates of 100 μmol/L quercetin on the migration of VSMCs induced by AngⅡ and PDGF-BB were 55.3% and 53.6%,respectively (P<0.01). The phosphorylation of HSP27 in response to AngⅡ and PDGF-BB was suppressed by fluvastatin in a dose-dependent manner, and maximal inhibitory rates were between 42.1% and 58.5% with 10^-5 mol/L fluvastatin,respectively (P<0.01).CONCLUSION: Fluvastatin influences the migration of VSMCs in part by inhibiting HSP27 phosphorylation.     

Influence of Sini decoction on the proliferation and apoptosis of artery smooth muscle cells after balloon injury

AIM: To investigate the influence of Sini decoction (SND) on the proliferation and apoptosis of rabbit abdominal aorta smooth muscle cells after ballon injury and discuss the effect of vascular smooth muscle cell's (VSMCs) proliferation and apoptosis in post-percutaneous coronary intervention (PCI) restenosis (RS) and the feasibility of SND preventing post-PCI RS. METHODS: The animal model of rabbit abdominal aorta ballon injury was set up and the therapertic group was treated with SND. The shape of proliferative and apoptotic cell were investigated by electron microscope. Immunohistochemistry staining was performed using α-actin,PCNA and Cyclin E monoclonal antibodies. In situ Cell Death Detection Kit was used to identify apoptotic cells. Abdomial aorta angiography was operated in the 84th day subgroup and the stenosis degree was evalued by quantitative angiographic analysis. RESULTS: As compared with the control group, the therapeutic group displayed a lower proliferative percentage and a higher apoptosic percentage (P<0.05). Moreover, the apoptosic peek time was on the 14th day after operation,which was longer than the control group. CONCLUSION: SND effectly inhibited the proliferation of VSMCs and iuduced apoptosis in VSMCs.     

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.     

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.     

CDK and JNK mediate FAK to enhance proliferation in human pulmonary artery smooth cells

AIM:To find out the mechanism of focal adhesion kinase (FAK) facilitating human pulmonary artery smooth muscle cells (HPASMCs) proliferation.METHODS:HPASMCs were isolated from normal part of lungs of two carcinoma patients who undergone lung partial resection. Cultured HPASMCs stimulated by fibronection(40 mg/L) were passively transfected with ODNs, sense focal adhesion kinase (FAK), mismatch sense and antisense-FAK, respectively. Expression of FAK, Jun NH2-terminal kinase (JNK) and cyclin-dependent kinase2 (CDK2) proteins were detected by immunoprecipitation and Western blotting. Cell cycle and cell apoptosis were analyzed by flow cytometry. In addition, cytoplasma FAK expression was detected by immunohistochemistry staining.RESULTS:The protein expressions of FAK, JNK and CDK2 in HPASMCs decreased in FAK ASODNs group and increased in FAK SODNs group. Meanwhile, the proportion of cells at G1 phase decreased significantly in FAK SODNs group, while the cells at S phase increased significantly. In contrast, the proportion of cells at G1 phase was increased significantly in FAK ASODNs group. The level of cell apoptosis in FAK ASODNs group was higher. FAK expression in FAK SODNs group was strongly stained by immunocytochemistry, whereas that in FAK ASODNs group was weakly stained. CONCLUSION:The results suggest that FAK via JNK, CDK2 signaling pathway enhances HPASMCs proliferation.     

Recombinant human interleukin-10 inhibits vascular smooth muscle cell proliferation by TNF-α and PDGF-BB in vitro

AIM: To determine the effects of recombinant human interleukin-10(rhIL-10) on proliferation of vascular smooth muscle cells(VSMC) by TNF-α and PDGF-BB and neointimal hyperplasia after rat carotid arterial injury.METHODS: Rat aortic VSMC was cultured and treated with rhIL-10 with or without tumor necrosis factor-α(TNF-α) and platelet-derived growth factor-BB (PDGF-BB), respectively.Proliferation of VSMC was quantified by colormetric assay.Cell cycle analysis was performed by flow cytomertry.SD rats were treated with recombinant human IL-10(rhIL-10) for 3 days after carotid arteries injury.Neointima to media area ratio at the site of arterial injury was measured at 28 days after balloon injury.RESULTS: Compared to control,both TNF-α and PDGF-BB stimulated VSMC proliferation. rhIL-10 alone had no effect on VSMC growth.With TNF-α or PDGF-BB stimulation,rhIL-10,at dose as low as 10 μg/L,inhibited VSMC growth( P <0.05) for both cases.Cell number in G 0/G 1 phase of PDGF-BB and rhIL-10 co-treatment group was higher than those of PDGF-BB treatment alone( P <0.01) by flow cytometry analysis.The same results were observed in TNF-α and rhIL-10 co-treatment group( P <0.01).Compared with the arterial injury group,neotima/media area ratio of recombinant human IL-10 group was reduced at 45%( P <0.01).CONCLUSIONS: The anti-inflammatory cytokine rhIL-10 inhibits TNF-α and PDGF-BB-induced VSMC proliferation,respectively.These results suggest the possibility that recombinant human IL-10 as a potential therapeutic approach prevents neointimal hyperplasia.     

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.     

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.     

PAF promotes proliferation of smooth muscle cells of rat airway

AIM: To study the effect of platelet-activating factor(PAF) on proliferation of cultured rat airway smooth muscle cells(ASMCs).METHODS: The cells were divided into control group and PAF group. The cells in PAF group were subdivided into four small groups by concentrations of PAF 10-6, 10-7, 10-8, 10-9 mol·L-1, MTT assay was used not only to investigate the effects of PAF on proliferation of ASMC but also to confirm the optimal concentration. Flow cytometry and immuneohistochemistry for proliferating cell nuclear antigen (PCNA) were also used to analyse its function on proliferation of ASMC.RESULTS: PAF (10-6-10-9mol·L-1) stimulated the cell proliferation and 10-7mol·L-1 PAF reached the maximal effect. The cell percentage of the ASMCs of 107 mol·L-1 PAF subgroup at G0/1 phase (68.67%) was much lower than that of control group (85.57%, P<0.01), in this subgroup, the percentage of expression of PCNA at 48 h (71.05%±1.22%) was significantly increased compared with the control group (53.27%±2.56%, P<0.05).CONCLUSION: PAF can stimulate the proliferation of cultured ASMC in a time-dependent, but not dose-dependent manner.     

Calcium-sensing receptor mediates hypoxia-induced proliferation of rat pulmonary artery smooth muscle cells through PI3K pathways

AIM:To explore the signal transduction pathways of calcium-sensing receptor(CaSR) that mediates hypoxia-induced proliferation of rat pulmonary artery smooth muscle cells(PASMCs). METHODS:The expression of cyclin D1 and phosphorylated protein kinase B(p-Akt) was analyzed by Western blotting. Cell proliferation was tested using a BrdU incorporation assay, and cell cycle analysis was carried out using a flow cytometric assay. RESULTS:Hypoxia significantly increased the expression of cyclin D1 and p-Akt, the BrdU incorporation and the cell proliferation index. GdCl₃, an agonist of CaSR, amplified the effect of hypoxia. LY294002,a PI3K inhibitor, decreased the up-regulation of cyclin D1 expression and the BrdU incorporation, and also inhibited the increase in the cell proliferation index induced by hypoxia and GdCl₃ in PASMCs. CONCLUSION: The CaSR mediates hypoxia-induced proliferation of rat PASMCs through PI3K pathways.     

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.     

Focal adhesion kinase antisense oligodeoxynucleotide promotes apoptosis in human pulmonary artery smooth muscle cells

AIM: To investigate whether focal adhesion kinase (FAK) takes part in the intracellular signaling pathway involved in apoptosis of human pulmonary artery smooth muscle cells (HPASMCs). METHODS: Cultured HPASMCs stimulated by fibronection (40 mg/L) were passively transfected with antisense-FAK oligodeoxynucleotides (ODNs). Expression of FAK and caspase-3 proteins was detected by immunoprecipitation and Western blotting. In addition, apoptosis were measured by TUNEL. RESULTS: The protein expression of FAK in HPASMCs decreased and caspase-3 expression upregulated in HPASMCs in antisense-FAK ODNs group. At the same time, antisense-FAK ODNs transfection increased the rate of cell apoptosis. CONCLUSION: These results suggest that FAK may be related with the apoptosis of HPASMCs. Antisense-FAK ODNs inhibit HPASMC proliferation and may facilitate their apoptosis via caspase-3 signaling pathway.