Effect of estrogen on the gene expression of caveolin-1 in rat vascular smooth muscle cells

AIM: To assess the effect of estrogen on the gene expression of caveolin-1 in rat vascular smooth muscle cells (VSMCs). METHODS: Wistar rats were ovariectomized and subjected to subcutaneous implantation of placebo pellets (OVX+V group) or estradiol pellets (OVX+E group). 2 weeks after implantation, the expression of caveolin-1 gene in endothelium-denuded aortic tissue was examined by RT-PCR. Furthermore, Northern blotting was used to analyze the mRNA expression of caveolin-1 in cultured rat VSMCs. RESULTS: RT-PCR showed that expression of caveolin-1 gene was significantly higher in OVX+E group than that in OVX+V group. Northern blot analysis showed that the mRNA expression of caveolin-1 was higher in VSMCs pretreated with 17β-estradiol (17β-E₂) than that in VSMCs without 17β-E₂ pretreatment. CONCLUSION: Estrogen up-regulates the gene expression of caveolin-1 in the vascular wall, partially indicating the cardiovascular effect of estrogen.     

Role of nucleolin in Ang II-induced phenotypic transformation of vascular smooth muscle cells

AIM: To investigate the effect of nucleolin on angiotensin II (Ang II)-induced phenotypic transformation of vascular smooth muscle cells (VSMCs). METHODS: Ang II was used to induce the phenotypic transformation of VSMCs. The spatial and temporal expression patterns of nucleolin, and the effects of Ang II on the expression of VSMC phenotypic transformation markers α-smooth muscle actin (α-SMA), calponin, smooth muscle protein 22 α (SM22α) and osteopontin (OPN) were investigated. The techniques of gene over-expression and RNA interference were used to assess the effect of nucleolin on the expression of Ang II-mediated VSMC phenotypic transformation markers. RESULTS: The expression of α-SMA, SM22α and calponin at the mRNA and protein levels was gradually decreased by Ang II stimulation, while the expression of OPN at mRNA and protein levels was gradually increased. The expression of nucleolin was gradually up-regulated in the VSMCs treated with Ang II at different concentrations for various duration (P<0.05). Ang II induced nucleolin translocation from the nucleus to cytoplasm. Over-expression of nucleolin promoted the VSMC phenotypic transformation induced by Ang II. Down-regulation of nucleolin suppressed the promotion of phenotypic transformation. CONCLUSION: Nucleolin promotes Ang II-induced phenotypic transformation of VSMCs, and its mechanism may be related to its function of cytoplasmic translocation.     

Effect of calcitonin gene-related peptide on myocardin expression and phenotypic switch in vascular smooth muscle cells

AIM: To investigate the effects of calcitonin gene-related peptide (CGRP) on myocardin expression and phenotypic switch in vascular smooth muscle cells (VSMCs). METHODS: VSMCs were obtained by aortic tissue adherent culture and treated with angiotensin Ⅱ (AngⅡ), AngⅡ + CGRP or AngⅡ + CGRP + CGRP_8-37. The protein expression of myocardin and the phenotypic proteins of the VSMCs was detected by Western blot. RESULTS: The expression of myocardin in cultured VSMCs showed downregulation along with time expansion. The protein level of myocardin was higher at 48 h and 72 h than that at baseline in the cultured VSMCs (P<0.05). However, the myocardin was lower at 48 h and 72 h than that at baseline after treatment with CGRP in cultured VSMCs (P<0.05). Furthermore, at 48 h in cultured VSMCs, the myocardin decreased along with α-smooth muscle actin (α-SMA) (P<0.05), and osteopontin (OPN) increased (P<0.05) in AngⅡ group compared with control group. After treatment with CGRP, the levels of myocardin and α-SMA become higher (P<0.05) but OPN was lower (P<0.05) in CGRP group than those in AngⅡ group. CGRP_8-37 abrogated CGRP-induced increase in myocardin and α-SMA and decrease in OPN in CGRP_8-37 group compared with CGRP group. CONCLUSION: CGRP may regulate the phenotypic switch of the VSMCs and maintain the cells in contractile phenotype through the upregulation of myocardin protein, which may be accomplished by the combination of CGRP and its receptor.     

Eukaryotic expression of human Arresten gene and its effect on the proliferation and migration of vascular smooth muscle cells

AIM: To express human Arresten gene in eukaryotic cell,and to investigate its effect on the proliferation and migration in vitro of rat primary cultured thoracic aortic vascular smooth cells (VSMCs).METHODS: COS-7 cells were transfected with recombinant eukaryotic expression plasmid pSecTag2-AT or control plasmid pSecTag2 mediated by liposome.48 hours after transfection,polymerase chain reaction (RT-PCR) was used to detect the expression of Arresten mRNA in the cells,while Western blotting assay was applied to detect expressed Arresten protein in concentrated supernatants.VSMCs were then co-cultured with the concentrated supernatants;and its proliferation was detected using cell counting kit-8 (CCK-8) in vitro.Migration of VSMCs was assayed by a microchemotaxis chamber and a polycarbonate filter (Transwell's chamber) with pores of 8 μm in diameter.RESULTS: RT-PCR revealed that the genome of Arresten-transferred cells contained a 449bp specific fragment of Arresten gene.Successful protein expression in supernatants was confirmed by Western blotting.CCK-8 assay showed that the proliferation of VSMCs was inhibited significantly by Arresten protein as compared with control group (P<0.01).Transwell's chamber showed that the number of control group,pSecTag2 transfected group and pSecTag2-AT transfected group were 28.70±3.97,26.10±4.53 and 14.00±3.33 (P<0.01).CONCLUSION: Arresten protein expressed in eukaryotic cells inhibits the proliferation and migration of VSMCs effectively in vitro.     

Effect of fibrinogen,fibrin and fibrin degradation products on the proliferation and migration of human vascular smooth muscle cells

AIM: To investigate the effect of fibrinogen (Fg), fibrin (Fb) and fibrin degradation products (FDPs) on the proliferation and migration of human vascular smooth muscle cells (VSMC).METHODS: The effects of Fg, Fb and FDPs on the proliferation of VSMC were observed by means of cell counting and MTT test, migration assays were performed using the wounding model and the transwell cell culture apparatus.RESULTS: Fg itself did not stimulate the proliferation of VSMC, but stimulated VSMC migration. Fb and FDPs both stimulated the proliferation and migration of VSMC, meanwhile the effect of Fb was in a dose-dependent manner.CONCLUSION: Fb, in particular FDPs, may play an important role by stimulating the proliferation and migration of VSMC in restenosis and atherogenesis.     

Effects of p204 expression on expression of p21 and proliferation of vascular smooth muscle cells in rats

AIM: To observe the effect of interferon-inducible protein 204 (p204) on the expression of p21 and proliferation of vascular smooth muscle cells (VSMCs) in rats. METHODS: Interferon alpha (IFN-α) and small interference RNA (siRNA) targeting p204 gene ( Ifi204 ) was used to intervene cultured VSMCs in vitro instantaneously, then the cell vitality was determined by MTT assay to reflect the cell proliferation. The cell cycle was analyzed by flow cytometry. The expression of p204 and p21 at mRNA and protein levels was determined by semi-quantitative RT-PCR and Western blotting. RESULTS: In rat VSMCs, IFN-α induced the increase in the expression of p204 at mRNA and protein levels, reduced the cell vitality and the G₁/S phase transition, and up-regulated the expression of p21 at mRNA and protein levels. Transfection of Ifi204 siRNA restrained the expression of p204 and p21, increased the cell vitality and promoted the G₁/S phase transition. CONCLUSION: The expression of p204 restrains the proliferation of rat VSMCs, probably by activating the expression of p21.     

miR-17 regulates senescence of rat vascular smooth muscle cells

AIM: To investigate the effect of microRNA-17 (miR-17) on the senescence of vascular smooth muscle cells (VSMCs) and the underlying mechanism.METHODS: The medial layer of the thoracic aorta was collected from the SD rats and isolated for primary culture. VSMCs were identified by immunofluorescence staining. The VSMCs were collected at the 4th~6th generations, and then the miR-17 mimics and miR-17 inhibitor were transfected into the VSMCs by liposome method. After 24 h, the cell senescence was induced by D-galactose. The VSMCs were divided into the following 6 groups:aging induction+miR-17 mimics (A-miR-17) group, aging induction+miR-17 inhibitor (A-anti-miR-17) group, A-control group, normal (N)+miR-17-mimics (N-miR-17) group, N-anti-miR-17 group, and N-control group. On day 3 after the addition of D-galactose, the senescence of VSMCs was observed with β-galactosidase staining. The expression of miR-17, p16 and p21 was detected by RT-qPCR and immunohistochemistry. RESULTS: miR-17 expression in the VSMCs was significantly lower in A-control group than that in N-control group (P<0.01). Compared with A-control group, the expression of miR-17 in the VSMCs was significantly increased in A-miR-17 group (P<0.01), while that was significantly decreased in A-anti-miR-17 group (P<0.01). The number of β-galactosidase positive staining cells in A-anti-miR-17 group was significantly higher than that in A-miR-17 group (P<0.01). The expression of p21 at mRNA and protein levels in the VSMCs was significantly lower in A-miR-17 group than that in A-control group (P<0.01), and the expressions of p21 at mRNA and protein levels was significantly higher in A-anti-miR-17 group than that in A-miR-17 group (P<0.01). CONCLUSION: miR-17 inhibits rat VSMCs senescence induced by D-galactose, the underlying mechanism is associated with the inhibition of p21 expression.     

Chemerin promotes proliferation of mouse vascular smooth muscle cells by up-regulating p-JNK

AIM: To investigate the proliferation property of stable chemerin gene knockdown vascular smooth muscle cells (VSMCs) and to explore its mechanism. METHODS: The normal VSMCs, chemerin gene interfering control VSMCs and stable chemerin gene knockdown VSMCs were divided into normal group, PDGF group, control group and knockdown group. The VSMCs in PDGF group were given platelet-derived growth factor-BB (PDGF-BB) to initiate proli-feration. The cell counting and BrdU assay were employed to investigate the proliferation property of VSMCs. The mitogen-activated protein kinase (MAPK) signal pathway was determined by Western blot. RESULTS: The cell number and BrdU A value in PDGF-BB-treated VSMCs significantly increased as compared with the normal VSMCs(CONCLUSION: Chemerin promotes the proliferation of mouse vascular smooth muscle cells by up-regulating p-JNK production.     

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.     

Effects of Chlamydia pneumoniae infection on adhesion and migration of vascular smooth muscle cells

AIM: To investigate the effects of Chlamydia pneumoniae (C.pn) infection on the adhesion and migration of vascular smooth muscle cells (VSMCs). METHODS: Primary VSMCs, isolated from the aorta of SD rats, was infected with C.pn after the culture and propagation of C.pn in the HEp-2 cells. The morphological characteristics of C.pn inclusions in VSMCs were examined under the fluorescence microscope with acridine orange (AO) staining. The specific DNA fragment of C.pn was identified by polymerase chain reaction (PCR). Cell adhesion assay was performed to investigate the effect of C.pn infection on the adhesion of VSMCs to collagen I. Wound-healing assay and Transwell assay were performed to detect the effects of C.pn infection on VSMCs migration. RESULTS: The C.pn inclusions and infection spots were observed in VSMCs under fluorescence microscope with AO staining. The inclusions were larger than infection spots in volume, but were much lower in count. A 437 bp-specific fragment of C.pn DNA was detected in C.pn-infected VSMCs by PCR. In the cell adhesion assay, the absorbance values in C.pn infection group were far higher than those in control group 2 h after infection (P<0.01). The cell adhesion ratio in C.pn infection group was 134.38%. The migration distance of VSMCs infected with C.pn was significantly longer than that of the control cells 24 h after infection by a wound-healing assay (P<0.05). More migratory VSMCs infected with C.pn for 24 h were found in a Transwell assay than those in control group (P<0.01). CONCLUSION: C.pn infection significantly promotes the adhesion and migration of VSMCs.     

Proliferative effect of PDGF and anti-proliferative activity of AMPK on vascular smooth muscle cells

AIM: To investigate the proliferative effect of platelet-derived growth factor (PDGF) and anti-proliferative activity of AMP-activated protein kinase (AMPK) on vascular smooth muscle cells (VSMCs). METHODS: The proliferation of VSMCs cultured with PDGF and activation of AMPK were observed. VSMCs were divided in 4 groups: control group; PDGF group; 5-aminoimidazole-4 -carboxamide-1-β-D-riboside (AICAR) group and AICAR+PDGF group. The time course of AMPK activation was determined. The protein level of mTOR was also measured. RESULTS: Compared with control group, the proliferative rate in PDGF group was significantly increased. The growth of VSMCs was inhibited in a time-dependent manner and the activity of p-mTOR was significantly decreased in AICAR group. Compared with control group, the expression of p-AMPK in PDGF group was significantly decreased, and that in AICAR group and AICAR+PDGF group was significantly increased. The expression of p-AMPK in AICAR+PDGF group was higher than that in PDGF group. The activity of p-mTOR in PDGF group was significantly higher than that in control group, while that of AICAR group and AICAR+PDGF group was significantly decreased. The expression of p-mTOR in AICAR+PDGF group was lower than that in PDGF group. CONCLUSION: Stimulation of VSMCs with PDGF promotes the cell proliferation, which can be inhibited by AICAR. The proliferation of VSMCs activated by AMPK is probably correlated with the down-regulation of mTOR expression.     

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.     

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.     

Effects of rat mesenchymal stem cells modified by CGRP on proliferation and phenotype transformation of vascular smooth muscle cells in vitro

AIM：To explore the effects of rat mesenchymal stem cells (MSCs) modified by calcitonin gene-related peptide (CGRP) on the proliferation and phenotype transformation of rat vascular smooth muscle cells (VSMCs) in vitro. METHODS：Rat MSCs and VSMCs were isolated, cultured and identified. The MSCs were infected by lentivirus which carried genes encoding enhanced green fluorescent protein (EGFP) and CGRP. The expression levels of CGRP in CGRP-modified MSCs were detected using real-time PCR and enzyme-linked immunosorbent assay (ELISA). The prolife-ration and migratory abilities of VSMCs were evaluated by MTT assay, Trypan blue staining and scratch test. The expression levels of α-smooth muscle actin (α-SMA) and osteopontin (OPN) were assessed by Western blotting. RESULTS：Compared with MSCs and MSCs-EGFP groups, the expression levels of CGRP in MSCs-CGRP group were markedly increased (P<0.01). The results of MTT assay and scratch test demonstrated that the proliferation and migratory abilities of VSMCs in MSCs-CGRP group were significantly inhibited compared with MSCs and MSCs-EGFP groups (P<0.05). Furthermore, cell viability was >90% shown by Typan blue staining. Western blotting showed that the expression of α-SMA was increased and that of OPN was decreased in MSCs-CGRP group compared with MSCs and MSCs-EGFP groups (P<005). CONCLUSION：CGRP-modified MSCs could secrete CGRP protein and inhibit the proliferation and migration of VSMCs, which may be associated with deterring the phenotype transformation from contractile type to synthetic type. These results lay a foundation for gene therapy in vivo.     

Role of caveolin-1 on down-regulation of LPS-induced monocyte chemotactic protein 1 by 17β-estradiol in vascular smooth muscle cells

AIM: To elucidate the effect of caveolin-1 on the down-regulation of LPS-induced monocyte chemotactic protein 1 (MCP-1) by 17β-estradiol (E₂) in vascular smooth muscle cells (VSMCs).METHODS: The primary-cultured VSMCs were exposed to E₂ at concentrations of 10^-9-10^-6 mol/L. LPS-induced MCP-1 production was assayed by ELISA. The protein expression of caveolin-1 was determined by Western blotting and was silenced by β-methyl cyclodextrin(β-MCD) or caveolin-1 specific siRNA. RESULTS: LPS significantly enhanced MCP-1 production. E₂ at concentrations of 10^-9-10^-6 mol/L inhibited LPS-induced MCP-1 production. The use of caveolin-1 inhibitor β-MCD or silencing the protein expression of caveolin-1 by specific siRNA largely impaired LPS-enhanced MCP-1 production, while E₂ markedly inhibited caveolin-1 expression. CONCLUSION: Inhibition of LPS-induced MCP-1 production by E₂ is related to the suppression of caveolin-1.