Effects of insulin on mitogen-activited protein kinase of vascular smooth muscle cells in spontaneously hypertensive rat

AIM:To explose the possible existing pathway of intracellular signaling transduction in hypertensive induced by insulin in rat vascular smooth muscle cells proliferation which involved mitogen-activated protein kinase. METHODS:Male spontaneously hypertensive rat (SHR) aorta and WKY(6 weeks old) were isolated and then cultured to make the purified vascular smooth muscle cells.6-8th generation of VSMC were interfered with insulin in vitro. MAPK activity was determined by myelin basic protein method and its volume was measured with Western Blot. And [³H]-TdR was used to measure DNA synthesis in VSMC proliferation. RESULTS: After the interfered with insulin the DNA synthesis was increased obviously in SHR group. MAPK activity and its contains in SHR were increased more than the control group. Protein kinase C inhibitor decreased MAPK activity induced by insulin. CONCLUSION:Proliferation of SHR VSMC in vitro was correlated with increased activity of MAPK. Insulin can affect MAPK induced activity. So an insulin-PKC-MAPK axis may exist in hypertensive VSMC.     

Effects of BTEB2 antisense gene transfection on the proliferation of vascular smooth muscle cells and its mechanism

AIM: To study the effects of BTEB2 antisense RNA on the proliferation of vascular smooth muscle cells(VSMC) and its mechanism. METHODS: After the recombinant adenovirus Ad.ASBTEB2 infected VSMC, antisense RNA and protein expression of BTEB2 were evaluated respectively by RT-PCR and Western blotting. Proliferation and cell cycle progress of VSMC were analyzed respectively by MTT test and flow cytometry. The expression of PCNA, AT1R and PDGF-BB were detected by immunocytochemistry. RESULTS: The expression of BTEB2 antisense RNA was demonstrated in VSMC after infected by recombinant adenovirus. Ad.ASBTEB2 infection significantly inhibited BTEB2 protein expression and proliferation of VSMC induced by serum, and resulted in G0/G1 blocking. The inhibitory effects of BTEB2 antisense RNA on the expression of PCNA, AT1R and PDGF-BB were demonstrated by immunohistochemistry. CONCLUSION: BTEB2 antisense RNA significantly inhibits the proliferation of VSMC, probably by suppressing the expression of VSMC proliferation related genes, such as PCNA, AT1R and PDGF-BB.     

The stimulating effects of neuropeptide Y on cultured arterial smooth muscle cell proliferation and losartan treatment

AIM:To investigate the interplay between the neuropeptide Y(NPY) and renin-angiotensin system, and relationship to the pathogenesis of hypertension. METHODS: The method of cellular culture, MTT colorimetric assay and quantitative immunocytochemistry through ACAS570 were performed for the effect of neuropeptide Y on proliferation of cultured rat vascular smooth muscle cell (VSMC) and losartan treatment. RESULTS:It was observed that exposure of VSMC to neuropeptide Y could stimulate the proliferation of VSMC and caused increase respectively in MTT OD values and expression of proliferating cell nulear antigen(PCNA) but losartan interfered with NPY stimulating effects on VSMC and decreased MTT OD values and expression of PCNA.CONCLUSION:These results demonstrated that the NPY could promote proliferation of VSMC, this effect was partly preformed through angiotensin Ⅱ receptor.     

Yangxue qingnao-containing serum inhibits rat vascular smooth muscle cell proliferation induced by lysophosphatidic acid

AIM: To observe the effects of Yangxue qingnao-containing serum on rat vascular smooth muscle cell (VSMC) proliferation induced by lysophosphatidic acid (LPA). METHODS: The [³H]-TdR incorporation and mitogen-activated protein kinasc (MAPK) activity were measured in cultured VSMC. End product of lipid peroxidation-MDA levels were also detected. RESULTS: 1×10^－9,1×10^－8 and 1×10^－7 mol/L LPA enhanced the cultured VSMC [³H]-TdR incorporation, increased MAPK activity and MDA content in a concentration-dependent manner. 5％, 10％ and 15％ Yangxue qingnao-containing serum concentration-dependently inhibited the increase in VSMC [³H]-TdR incorporation, MAPK activity and MDA content induced by LPA. CONCLUSIONS: LPA has a stimulating effect on VSMC proliferation. The LPA-induced intracellular signal transduction may be related to MAPK activity. Yangxue-qingnao can efficiently inhibit LPA -induced VSMC proliferation,MAPK activity and lipid peroxidation.     

Effect of C-reactive protein on VSMC proliferation in SHR in vitro

AIM: The purpose of this study was to investigate the effect of C-reactive protein(CRP) on vascular smooth muscle cell(VSMC) proliferation in vitro.METHODS: The aorta VSMC from spontaneous hypertension rats(SHR) was cultured and effects of the chemical intervention were observed in VSMC. First, the expressions of NF- κB and I-κB protein were detected by Western blotting after CRP and antibody of CRP receptor (CD32 antibody) were added into VSMC culture. Second, the growth rate of VSMC was calculated when angiotensin Ⅱ, pyrrolidine dithiocarbamate (PDTC) (an inhibitor of NF-κB) were added.RESULTS: The expressions of NF-κB and I-κB in VSMC of SHR were up-regulated after treated with CRP, the enhanced expressions were depressed by using CD32 antibody. The growth rate of VSMC was promoted by angiotensinⅡ and this increased proliferation was abolished by using PDTC.CONCLUSION: CRP can promote independently growth rate of VSMC in SHR by activating NF-κB.     

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.     

Intervention of resveatrol on activated nuclear factor-κB and expression of monocyte chemotactic protein-1 in cultured rabbit aortic smooth muscle cells induced by xanthine and xanthine oxidase

AIM: To investigate the role of resveatrol among red wine on the proliferation, activity of NF-κB and the expression of monocyte chemotactic protein-1 (MCP-1) induced by xanthine and xanthine oxidase in cultured rabbit aortic smooth muscle cells (SMC). METHODS: SMC proliferation was examined by 3-4, 5-dimethylthiazol-2-yl-2, 5-diphenylte tra zoliumbromide (MTT) metabolism, activity of NF-κB, the protein and mRNA expression of MCP-1 were detected by electrophoretic mobility shift assay (EMSA), immunohistochemitry and in situ hybridyzation in cultured rabbit aortic SMC. RESULTS: 100 μmol/L-200 μmol/L resveatrol (RES), an effective composition in red wine, was confirmed to inhibit metabolism and the activity of NF-κB as well as the protein and mRNA expression of MCP-1 in rabbit aortic SMC, which were promoted by the oxygen free radicals induced by xanthine and xanthine oxidase. CONCLUSION: Resveatrol may antagonist oxygen free radicals-induced proliferation and the activity of NF-κB as well as protein and mRNA expression of MCP-1 in cultured rabbit aortic SMC, which might play an important role in preventing atherosclerosis.     

Effect of neuropeptide Y on bcl-2, bax,fas expressions and proliferation in vascular smooth muscl cells

AIM: To explore the effect of neuropeptide Y on expression of apoptosis associated genes and proliferation in vascular smooth muscle cells (VSMC). METHODS: The proliferation activity of VSMC was dterminded by MTT colorimetry. The average fluorescence intensity that represented VSMC nuclear antigen (PCNA) and bcl-2, bax, fas expressions was quantitatively measured by fluorescence immunohistochemistry. RESULTS: Compared with control, the expressions of bcl-2, bax, fas, PCNA and the VSMC proliferation activity in VSMC treated with NPY were significantly increased. CONCLUSION: NPY may increase the expression of apoptosis associated genes in VMSC and promote its proliferation.     

The synergistic effect of endothelin-1 and basic fibroblast growth factor on rat vascular smooth muscle cell proliferation

AIM: The synergistic effect of basic fibroblast growth factor (bFGF) and endothelin-1 (ET-1) on rat aortic vascular smooth muscle cells (VSMC) proliferation was observed. The possible mechanism of the synergism was also investigated. METHODS: BrdU incorporation and cell counting method were adopted to value the pro-proliferative effect of VSMC. Western blotting was used to observe the variation of bFGF and FGFR-1 isoforms expression. RESULTS: bFGF and ET-1 could promote VSMC proliferation separately, and synergistically in combination. The synergism was dose- and time-dependent. ET-1 increased all the three bFGF isoforms and FGFR-1 protein level in dose- and time-dependent manner. In addition, after exhaustion of intracellular PKC, the upregulation effects of ET-1 on bFGF and FGFR-1 expressions in VSMC both inclined. CONCLUSION: bFGF and ET-1 had synergistic effect on VSMC proliferation. ET-1 may increase the responsiveness of VSMC to bFGF through modulation of bFGF isoforms together with FGFR-1, which was PKC-dependent.     

Role of angiotensin Ⅱ in the regulation of platelet-derived growth factor receptor β subunit of vascular smooth muscle

AIM:To investigate the crosstalk between angiotensin Ⅱ (AngⅡ)-mediated and platelet-derived growth factor (PDGF)-mediated signal transduction in vascular smooth muscle proliferation.METHODS:A model of renal hypertension was made by two kidney/one-clip operation. Level of PDGF receptor β subunit of aorta was measured by Western Blot analysis. The effect of Ang Ⅱ on PDGF receptor β subunit expression was investigated in culture rat aortic vascular smooth muscle cells (VSMC).RESULTS:Systolic blood pressure obviously increased at 8th week after operation, whereas the level of PDGF receptor β subunit of aorta significantly increased by 126.6% (P＜0.05) in 2K1C rats compared with control group. The expression of PDGF receptor β subunit in cultured VSMC stimulated by AngⅡ was higher than that of control by 192.74%(P＜0.01). The effect of AngⅡ was inhibited remarkably by pretreated with losartan, a kind of specific AngⅡ receptor 1 (AT₁) subtype antagonist and U73122, a kind of phospholipase C inhibitor. The effect was partly blocked by PD98059, which inhibit the activity of mitogen-activated, ERK-activating kinase (MEK).CONCLUSION:AngⅡ-induced PDGF receptor β subunit expression is regulated by the AT₁ and its downstream signal molecule-PLC and ERK, might participate in the intracellular signal transduction pathway.     

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.     

Effect of antisense oligonucleotides of c-sis on cellular cycle and proliferation of vascular smooth muscle cells in pulmonary artery

AIM:To investigate the effect of antisense oligonucleotides(ASON) of c-sis on cellular cycle and proliferation of pulmonary artery vascular smooth muscle cells(VSMC).METHODS:Tissue mass culture was done to get VSMC of pulmonary artery. Different concentrations of antisense oligonucleotides of c-sis were added into the cultures to observe the VSMC proliferation curve using MTT test. The changes of VSMC cellular cycle were also observed by flow cytometry.RESULTS:ASON with mid-to high concentrations restrained the proliferation of VSMC apparently with the peak of cell growth being attenuated or eliminated. Affected by mid-concentration ASON, PDGF-BB showed significant accelerating effect on the proliferation of VSMC. The ratio of G₀／G₁ in cellular cycle was increased significantly in VSMC culture with ASON in comparison with control. The G₀/G₁ ratio also showed significant differences among different concentration of ASON groups(P＜0.05).CONCLUSION:Mid-to high concentration of ASON was a powerful inhibitor of cellular proliferation for pulmonary artery VSMC. ASON increased the ratio of G₀／G₁ significantly and the increase seems to be ASON dosage dependent.     

Effect of calcitonin gene-related peptide on proliferation of vascular smooth muscle cells

AIM: To elucidate the effect of calcitonin gene-related peptide (CCRP) in the therapy of atherosclerosis.METHODS:Effect of CGRP on cell cycle kinetics of cultured vascular smooth muscle cells(HA-VSMC) was investigated by flow cytometry. The expression of cyclins D₁ and E required for initiation of S phase were also studied by immunochemistry method. RESULT: CGRP was shown to arrest VSMC in the G₀/G₁ phase of cell cycle and reduced expression of cyclins D₁ and E. CONCLUSION:CGRP inhibits proliferation of HA-VSMC by arresting cells in G₁ phase via limiting accumulation of cyclin D₁ and E. It might play a role in the therapy of atherosclerosis.     

Effects of adrenomedullin on proliferation of vascular smooth muscle cells induced by urotensin Ⅱ

AIM:To observe the effect of adrenomedullin(ADM)on proliferation of vascular smooth muscle cells(VSMC) induced by urotensin Ⅱ(UⅡ). METHODS:DNA synthesis of cultured rat aortic VSMC was measured by [³H]-TdR incorporation. The activities of mitogen activated protein kinase(MAPK) were determined by isotope tagged with [γ-³²P]-ATP. RESULTS:UⅡ(10^-8mol/L) significantly increased [³H]-TdR incorporation of VSMC and MAPK activities by 38%(P＜0.05) and 260%(P＜0.01) respectively compared with control group. Compared with UⅡ group, 10^-10,10^-9,10^-8mol/L ADM decreased [³H]-TdR incorporation of VSMC by 7%(P＞0.05), 32%(P＜0.05)and 41%(P＜0.01),respectively, and diminished MAPK activities by 24%(P＞0.05), 32%(P＜0.05)and 36%(P＜0.05),respectively. CONCLUSION:ADM inhibits proliferation of VSMC induced by urotensin Ⅱ through inhibiting MAPK activation.     

Jagged1 knocking down in endothelial cells accelerates PDGF induced proliferation and migration of vascular smooth muscle cells in rats

AIM: To investigate the effect of Jagged1 expression in endothelial cells (EC) on platelet derived growth factor (PDGF) induced proliferation and migration of vascular smooth muscle cells (VSMC) in rat.METHODS: Rat aorta EC was inoculated in the lower chamber and VSMC were in the upper chamber of the cell coculture system. Three groups were divided: control, sicontrol and siJagged1. The EC Jagged1 protein expression was assayed by Western blotting to evaluate small RNA interfering (RNAi) efficiency. After the cells were cocultured with PDGF for 24 h, the proliferation and migration of VSMC were respectively evaluated by ［3H］-TdR incorporation and migrating cells counting. Protein expression of α-SM-actin in VSMC was assayed by Western blotting. RESULTS: The Jagged1 protein expression in EC was significantly lower in siJagged1 group than that in control group (0.26±0.02 vs 0.67±0.02, P<0.05), and no statistic significance was observed between control and sicontrol groups. The VSMC ［3H］-TdR incorporation and migration were higher in PDGF +siJagged1 group than those in PDGF group {［3H］-TdR incorporation (23 074±2 702) counts·min-1·well-1 vs (16 442±1 803)counts·min-1·well-1, n=5, P<0.05; migration (27±4) cells/field vs (15±3)cells/field, n=5, P<0.05}. The α-SM-actin protein in VSMC was lower in PDGF + siJagged1 group than that in PDGF group (0.25±0.06 vs 0.49±0.04, n=3, P<0.05).CONCLUSION: Jagged1 knock down in rat EC accelerates PDGF induced proliferation and migration of VSMC. These results suggest that Jagged1 expression in EC plays an important role in maintaining VSMC contract phenotype and inhibiting VSMC overgrowth after arterial injury.     

Effects of the antibodies against α1- adrenergic receptor on the proliferation of vascular smooth muscle cells in rats

AIM: The autoantibodies against α1-adrenergic receptor that was found in patients with malignant hypertension, primary hypertension and refractory hypertension has the agonist activity liked the NE, and may play a role in hypertension. In this paper, the effects of this antibody on vascular smooth muscle cell (VSMC) proliferation and its mechanism were to be studied. METHODS: The cultured rat VSMC proliferation induced by the antibodies against α1- adrenergic receptor that was purified by the immune affinity chromatography, was measured by the BrdU cell proliferation assay and cell cycle distribution. The expression of c-jun and c-fos were determined by RT-PCR and Western blotting. RESULTS: Compared to the normal IgG, the antibodies against α1-adrenergic receptor promoted the VSMC proliferation and increased the mRNA and protein expression of the c-jun significantly. The role was similar to the norepinephrine, and all was blocked by prazosin, while the mRNA and protein expression of c-fos were not affected by the antibodies. CONCLUSION: The antibodies against α1-adrenergic receptor promote the rat VSMC proliferation, and increase the expression of c-jun, which maybe play a role in the vascular remodeling in hypertension.