Inhibitory effect of 1,25-(OH)2D3 on proliferation of airway smooth muscle cells from passively-sensitized patients

AIM: To investigate the effects of 1,25-dihydroxyvitamin D₃ on the proliferation of passively-sensitized human airway smooth muscle cells (HASMCs), and to explore its potential role in asthmatic airway remodeling.METHODS: HASMCs were passively sensitized with 10% serum from asthmatic patients.1,25-(OH)₂D₃ was used as the interventor.The effect of 1,25-(OH)₂D₃ on the cell proliferation and its optimal concentration were determined by MTT colorimetric assay.The cell cycle analysis was performed by flow cytometry.The expression of proliferating cell nuclear antigen (PCNA) was measured by the method of immunocytochemical staining.RESULTS: 1,25-(OH)₂D₃ at the concentrations of 10^-9-10^-7 mol/L markedly inhibited the cell proliferation and the maximum effect was observed at the concentration of 10^-7 mol/L.This concentration of 1,25-(OH)₂D₃ markedly suppressed the PCNA-positive rate and hampered the G₁/S transition in HASMCs passively-sensitized by asthmatic serum.CONCLUSION: 1,25-(OH)₂D₃ has direct inhibitory effects on the proliferation of passively-sensitized HASMCs in vitro, which may be concerned with the beneficial role of 1,25-(OH)₂D₃ on the prevention and therapy of asthmatic airway remodeling.     

Effect of nuclear factor-κB on proliferation of airway smooth muscle cells regulated by protein kinase C in asthmatic rats

AIM: To investigate the effect of protein kinase C (PKC)- nuclear factor-κB (NF-κB) signal pathway on proliferation of airway smooth muscle cells (ASMCs) in asthmatic rats.METHODS: (1) 16 Wistar rats were divided into asthmatic group (8 rats) and control group (8 rats).ASMCs from asthmatic group and control group were treated with PKC agonist PMA and NF-κB inhibitor PDTC.The proliferation of ASMCs was examined by cell cycle analysis,MTT colorimetric assay and proliferating cell nuclear antigen (PCNA) immunofluorescence staining,respectively.NF-κB activity was detected by NF-κB p65 immunofluorescence staining and electrophoretic mobility shift assay (EMSA),respectively.RESULTS: The percentage of S phase,A value,the positive expression rate of PCNA,the positive expression rate of NF-κB p65 and EMSA value in asthmatic ASMCs treated with PMA were higher than those in asthmatic ASMCs without treatment (P<0.05).After asthmatic ASMCs previously treated with PDTC,then with PMA,the above figures were lower than those in asthmatic ASMCs only treated with PMA and without treatment (P<0.05).The above figures in asthmatic ASMCs only treated with PDTC were lower than those in asthmatic ASMCs without treatment (P<0.05).CONCLUSION: NF-κB may contribute to the proliferation of ASMCs in asthmatic rat,in which PKC－NF-κB signal pathway is involved.     

Role of ERK on proliferation of airway smooth muscle cells in chronic asthmatic rats

AIM: To investigate the roles of extracellular signal-regulated kinase(ERK) signaling pathway on regulating proliferation of airway smooth muscle by observing the expression of ERK in airway smooth muscle(ASM) in chronic asthmatic rats.METHODS: Airway remodeling was detected in chronic asthmatic rats by using image analysis system. The expressions of ERK and proliferating cell nuclear antigen(PCNA) in lung tissue from chronic asthmatic rats were observed by immuocytochemistry staining. The expressions of ERK1/2, p ERK1/2 and PCNA were detected in airway smooth muscle (ASM) by immunofluorescence double staining with confocal microscopy, and the expressions of protein or mRNA of ERK and PCNA in ASM were also detected by immunoblotting and hybridization in situ,respectively.RESULTS: The thickening of smooth muscle and structural remodeling in airway were observed in chronic asthmatic rats by image analysis. The enhanced expressions of ERK and PCNA appeared obviously increased in same lung tissue and the expressions of protein or mRNA of ERK and PCNA were significantly increased in ASM.CONCLUSION: ERK signal pathway might be an important pathway on regulating cell proliferation of ASM resulting in asthmatic airway remodeling.     

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.     

Effects of kaempferol-3-O-rutinoside on proliferation,migration and TGFBR1 signaling pathway activation in vascular smooth muscle cells

AIM:To investigate the effects of kaempferol-3-O-rutinoside (KR) on the proliferation, migration of vascular smooth muscle cells (VSMC) and the activation of transforming growth factor β receptor 1 (TGFBR1) signaling pathway in the cells. METHODS:The viability of VSMC was detected by MTT assay. The proliferation of VSMC was measured by EdU staining. The migration ability of VSMC was examined by Transwell assay. The protein levels of the migration-associated proteins matrix metalloproteinase 2 (MMP2) and matrix metalloproteinase 9 (MMP9) were detected by Western blot. Molecular docking study was conducted to explore the interaction between KR and TGFBR1. The protein le-vels of the phosphorylated TGFBR1, Smad2 and Smad3 were determined by Western blot. RESULTS:KR inhibited the viability of VSMC in a dose-and time-dependent manner. KR reduced the ratio of EdU-positive cells in a dose-dependent manner. KR dose-dependently suppressed the migration ability of VSMC and decreased the protein levels of MMP2 and MMP9 (P<0.05). KR docked into TGFBR1 with the binding energy of -9.804 kcal/mol by forming hydrogen bonds with SER-280, ARG-215, ASP-290 and LYS-335 of TGBFR1. KR dose-dependently suppressed the activation of TGFBR1 and its downstream proteins Smad2 and Smad3 (P<0.05). CONCLUSION:KR inhibits the proliferation and migration of VSMC, possibly via blocking the TGFBR1 signaling pathway.     

Role of homocysteine in tissue factor expression in human vascular smooth muscle cells

AIM: To investigate the expression of tissue factor (TF) induced by homocysteine (Hcy) and the effect of Hcy on activation of nuclear factor-κB (NF-κB) in human vascular smooth muscle cells (VSMCs).METHODS: Human umbilical artery VSMCs were cultured by tissue explanting method,and were incubated with different dosage of Hcy/ PDTC (NF-κB inhibitor) in different time.RT-PCR was used to measure the expression of TF mRNA,and flow cytometry was used to detect the expression of TF on the surface of VSMCs.Western blotting was performed to measure the NF-κB protein level in the nuclear,flow cytometry was used to examine the expression of iNOS and the expression of TF on the surface of VSMCs.RESULTS: Hcy induced VSMCs TF mRNA expression significantly after the VSMCs were incubated with Hcy at concentrations of 10,100,500 μmol/L,respectively.There was low expression level of TF protein on the surface of the control VSMCs and Hcy also induced VSMCs TF protein expressionn on the cell surface at different concentrations.Additionally,Hcy rapidly induced the activation of NF-κB and inhibited this effect significantly by PDTC.Hcy alone did not induce the expression of iNOS in VSMCs.CONCLUSION: Hcy induces human VSMCs expression of TF in mRNA and protein.These effects were partly mediated by NF-κB.These results suggest that Hcy may play an important role in atherosclerosis and thrombosis.     

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.     

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.     

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.     

Effects of human xeroderma pigmentosum group D gene on proliferation of human vascular smooth muscle cells induced by interleukin-6

AIM: To investigate the effects of human xeroderma pigmentosum group D (XPD) gene on the proliferation of human vascular smooth muscle cells (VSMCs) induced by interleukin-6 (IL-6). METHODS: Recombinant plasmid pEGFP-N2/XPD and vacant plasmid pEGFP-N2 were transfected into VSMCs by liposome, and then these cells were incubated with IL-6 at 1×10⁵ U/L for 48 h. The cells were divided into 6 groups: blank control group; pEGFP-N2 group; pEGFP-N2/XPD group; IL-6 group; IL-6 + pEGFP-N2 group; IL-6 + pEGFP-N2/XPD group. The expression of green fluorescent protein was observed under fluorescence microscope. The cell growth was detected by MTT method. The cell cycle and apoptosis rate were examined by flow cytometre. The expression levels of XPD, Bcl-2, Bax and wild type P53 (wt-P53) were detected by RT-PCR and Western blotting.RESULTS: Green fluorescence was observed in the cells transfected with pEGFP-N2/XPD or pEGFP-N2, indicating successful transfection MTT results showed that the transfection of pEGFP-N2/XPD inhibited the cell growth, and reduced the positive effects of IL-6 on VSMCs growth. Flow cytometry results showed that the transfection of pEGFP-N2/XPD increased the apoptosis rate of VSMCs and the cell numbers in G₀/G₁ phase, decreased the cell numbers in S phase, and reduced the effects that IL-6 decreased the apoptosis rate of VSMCs and the cell numbers in G₀/G₁ phase, and increased the cell numbers in S phase. The results of RT-PCR and Western blotting showed that the transfection of pEGFP-N2/XPD increased the expression of XPD, Bax and wt-P53, decreased the expression of Bcl-2, and reduced the effects that IL-6 decreased the expression of Bax and wt-P53, and increased the expression of Bcl-2. CONCLUSION: XPD gene inhibits VSMCs proliferation, promotes VSMCs apoptosis, and reduces the effects that IL-6 promotes VSMCs proliferation and inhibits VSMCs apoptosis. Therefore, XPD gene is likely to be potential molecular target for treatment of atherosclerosis.     

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.     

Neuregulin-1 induces expression of angiogenic factors in coronary artery smooth muscle cells

ATM: To investigate the effects of neuregulin-1 (NRG-1) on the expression of angiogenic factors in human coronary artery smooth muscle cells (HCASMCs). METHODS: HCASMCs were cultured in vitro, and the cells at the 3rd passage were collected to assess the expression and phosphorylation of ErbB by Western blot. After HCASMCs were cultured under normal condition, with hypoxia and serum deprivation, or with NRG-1 treatment, the expression of vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) was determined by Western blot. RESULTS: The expression of ErbB2, ErbB3 and ErbB4 was observed in the HCASMCs, and the phosphorylation of these receptors was increased by NRG-1 treatment. Compared with control group, the expression of VEGF and Ang-1 in the HCASMCs was significantly increased in hypoxia and serum deprivation group (P<0.05﹚,while no difference in the expression of Ang-2 between the 2 groups was found.Compared with hypoxia and serum deprivation group,the expression of VEGF and Ang-1 in the H CASM Cs treated with NRG-1 was furtherincreased﹙P<0.05﹚,and no difference in the expression of Ang-2 between the 2 groups was observed.CONCLUSION: HCASMCs express ErbB2, ErbB3 and ErbB4, and the phosphorylation of the receptors is increased by NRG-1. Hypoxia, serum deprivation and NRG-1 treatment induce the increased expression of VEGF and Ang-1 significantly.     

Role of TLR4-TRPC6 in LPS-induced NF-κB P65 expression and nuclear translocation in airway epithelial 16HBE cells

AIM: To investigate the role of Toll-like receptor 4 (TLR4) and transient receptor potential channel 6 (TRPC6) signaling pathway in lipopolysaccharide (LPS)-induced nuclear factor-κB (NF-κB) P65 expression and nuclear translocation in airway epithelial cells (16HBE) for supplementing the mechanism for airway inflammation. METHODS: After stimulating the 16HBE cells with LPS at 1 mg/L for 0, 0.5, 2, 6, 12 and 24 h, the expression of NF-κB P65 at mRNA and protein levels in the 16HBE cells were determined by RT-PCR and Western blot respectively, and the nuclear translocation of NF-κB P65 was detected by immunocytochemical staining method. The effects of TLR4 inhibitor CLI-095 at 5 μmol/L and TRPC6 agonist Hyp9 at 10 μmol/L on LPS (1 mg/L)-induced NF-κB P65 expression and nuclear translocation in the 16HBE cells were determined by RT-PCR, Western blot and immunocytochemical staining. RESULTS: LPS increased the mRNA and protein expression of NF-κB P65 and nuclear translocation in the 16HBE cells(P<0.05). TLR4 inhibitor CLI-095 reduced the mRNA and protein expression of NF-κB P65 and nuclear translocation induced by LPS, while Hyp9 enhanced the mRNA and protein expression of NF-κB P65 and nuclear translocation induced by LPS in the 16HBE cells(P<0.05). CONCLUSION: LPS induces the expression and nuclear translocation of NF-κB P65 in the 16HBE cells via TLR4-TRPC6 signaling pathway.     

Effects of angiotensin-(1-7) on calcification in vascular smooth muscle cells and its signal transduction pathway

AIM: To clarify the effects of angiotensin-(1-7) on the calcification in rat vascular smooth muscle cells(VSMCs) and its signal transduction.METHODS: Calcification of cultured rat VSMCs was prepared by incubation of VSMCs with β-glycerophosphate.Calcification was confirmed by Von Kossa staining.The cells were treated with angiotensin-(1-7).The calcium content,alkaline phosphatases activity,osteocalcin and Cbfa1 mRNA expression were also measured.RESULTS: Angiotensin-(1-7) inhibited the increases of calcium content,alkaline phosphatases activity（P>0.05）,osteocalcin concentration and Cbfa1 mRNA expression in calcified VSMCs（P<0.05）,and the effects of angiotensin II on calcium content,alkaline phosphatases activity,osteocalcin concentration and Cbfa1 mRNA expression in calcified VSMCs were also inhibited （P<0.05）.Angiotensin-(1-7) increased cAMP concentration in calcified VSMCs（P<0.05）and selective PKA inhibitor blocked the effects of angiotensin-(1-7) on calcium content,alkaline phosphatases activity,osteocalcin concentration and Cbfa1 mRNA expression（P<0.05）.CONCLUSION: Angiotensin-(1-7) can inhibit beta-glycerophosphate-induced calcification in VSMCs through cAMP-PKA-Cbfa1 pathway and antagonize the effect of angiotensin II on calcification in VSMCs.     

Effect of 1α, 25-dihydroxyvitamin D3 on T helper cell 17 and expression of related cytokines in penetrating keratoplasty in mice

AIM: To evaluate the effects of 1α, 25-dihydroxyvitamin D3 on T helper cell 17 (Th17 cells) and its related cytokines in a mouse model of corneal allograft transplantation. METHODS: C57BL/6 mice were transplanted with corneal grafts from BALB/c mice and treated intraperitoneally with 1.0 μg 1α, 25-dihydroxyvitamin D3 or soybean oil every other day after operation. The transparency of the corneal grafts was evaluated for potential rejection signs by slit lamp biomicroscopy and histopathology. The expression levels of IL-17, RORγt and IFN-γ in the spleen were measured by real-time PCR. Moreover, the protein expression of RORγt and IL-17 in the peripheral blood was analyzed by Western blotting. IL-17 and IFN-γ in peripheral blood were measured by flow cytometry. RESULTS: 1α, 25-dihydroxyvitamin D3 significantly inhibited the rejection of the corneal allograft and reduced the numbers of inflammatory infiltrates in the corneal graft. In the spleen, 1α, 25-dihydroxyvitamin D3 treatment reduced the expression levels of IL-17, RORγt and IFN-γ. In the peripheral blood, 1α, 25-dihydroxyvitamin D3 treatment downregulated the expression levels of RORγt, IL-17 and IFN-γ. CONCLUSION: The effects of 1α, 25-dihydroxyvitamin D3 on suppressing corneal transplantation-induced allograft rejection in mice are closely associated with its modulation on IL-17 and related cytokine RORγt.