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.     

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.     

Expression of ERK and MKP-1 in vascular smooth muscle of spontaneously hypertensive rats with different ages

AIM: To investigate the expression of mitogen- activated protein kinase and mitogen-activated protein kinase phosphatase-1 in thoracic aorta smooth muscles of spontaneously hypertensive rats (SHR) and Wistar Kyoto rats (WKY) with different ages and the relationship between those and hypertension. METHODS: The caudal arterial pressure was measured by tail-cuff. Protein expression of p-ERK was detected by Western blotting, and MKP-1 mRNA in thoracic aorta smooth muscle was examined by RT-PCR. RESULTS: (1) The blood pressure of SHR was obviously higher than that of age-matched WKY (P<0.01), elevated with age (P<0.05) and became stable from 14-week-old. (2) The expression of p-ERK and MKP-1 in SHR was higher than that in WKY in 5-week-old rats, and the expression of p-ERK increased with age, while the expression of MKP-1 decreased with age (P<0.05). CONCLUSION: MKP-1 may play an important role in the development of hypertension in SHR. The decrease in the expression of MKP-1 that resulted in the activation of MAPK may induce vascular smooth muscle proliferation and hypertrophy.     

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 effect of hydrogen sulfide (H2S) on cultured rat aortic smooth muscle cells

AIM:To explore the effects of hydrogen sulfide (H₂S) on proliferation of vascular smooth muscle cells (VSMC) stimulated by endothelin (ET-1, 10^-7mol/L) and mitrogen-activated protein kinase (MAPK) activity in VSMCs.METHODS:Cultured VSMCs were divided into six groups: (1) control group, (2) serum group, (3) endothelin group, (4) NaHS groups, (5) serum+NaHS group, and (6) endothelin+NaHS group. VSMC proliferation was measured by[³H]-TdR incorporation and MAPK activity in VSMC was determined by radioactivity assay.RESULTS:ET-1 increased VSMC[³H]-TdR incorporation by 2.39 times (P＜0.01) and MAPK activity by 1.62 times(P＜0.01), as compared with control. H₂S (5×10^-5-5×10^-4mol/L) decreased VSMC[³H]-TdR incorporation and MAPK activity by 16.8%-37.4% and 7.4%-33.6%, respectively (P<0.05 or P＜0.01).CONCLUSION:This study demonstrates that H₂S inhibits ET-1-induced proliferation of VSMC, which might be mediated by the inhibition of MAPK.     

Effect of PP60c-Src on Ang II-induced signal transduction in rat vascular smooth muscle cells

AIM: The aim of the present study was to clarify the mechanism of intracellular signal transduction in Ang II-induced proliferation of vascular smooth muscular cells (VSMC) by observing the effect of c-Src on Ang II-mediated mitogen-activated protein kinase (MAPK) activation and c-Fos protein expression in cultured VSMC of rats. METHODS: Cultured aortic VSMCs from SD rats were transfected with anti-sense c-Src oligodeoxynucleotides (ODNs) wrapt with lipofectin to inhibit c-Src activity and protein production. Untransfected VSMCs were used as control. We observed the role of Ang II stimulation in MAPK activation and c-Fos protein expression. c-Src kinase activity was measured by protein immunoprecipitation and kinase autophosphorylation. The phosphorylation rate of the substrate myelin basic protein (MBP) was employed to assess MAPK activity. Western immunoblot was used to detect protein expression of c-Src and c-Fos. RESULTS: c-Src protein expression in VSMC transfected with different concentrations of anti-sense ODNs significantly decreased in a negative dose-effect manner. c-Src kinase activity was also markedly inhibited . Following the stimulation of Ang II on transfected VSMCs with anti-sense ODNs, the increase rate of c-Src activity was 8.7% of that in control, the activity of MAPK was 1.6% compared with control and c-Fos protein expression was as 30.0% as that of control. CONCLUSION: Ang II induces c-Src activation. MAPK activation and c-Fos protein expression by Ang II is dependent on c-Src activation. These findings indicate that c-Src is an important signal factor in Ang II -induced VSMC proliferation.     

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.     

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.     

Inducing vascular smooth muscle cell proliferation by insulin involves in phosphatidylinositol 3-kinase and ERK1/2

AIM: To investigate the effect of insulin on vascular smooth muscle cells (VSMCs) proliferation and to evaluate the intracellular signaling pathways involved.METHODS: VSMCs separated from Sprague-Dawley rats were used in this study. The proliferation of VSMCs induced by insulin was assayed by ［3H］-thymidin incorporation. The protein expression and activity of p-ERK1/2 were determined by immunblot and ［γ-32P］ATP incorporation.RESULTS: Insulin induced cell proliferation in a concentration-dependent manner. The proliferative effect of insulin on VSMCs was inhibited partly by LY294002 (48.8%), an inhibitor of PI-3 kinase, and the ERK1/2 inhibitor PD98059 (43.6%), respectively. Moreover, phosphorylation of ERK1/2 and activity of ERK1/2 induced by insulin were also inhibited partly by LY294002.CONCLUSION: PI-3 kinase and ERK1/2 are involved in insulin induced VSMCs proliferation.     

Effects of angiotensin II receptor antagonist on remodeling of renal arterioles in hypertension

AIM: To investigate the effects of angiotensin II receptor antagonist on remodeling of renal arterioles in hypertension. METHODS: Eighteen 4 weeks old male rats were divided into three groups: Wistar-Kyoto rats (WKY) for normotensive group, and spontaneously hypertensive rats (SHR) for hypertensive group, and SHR treated with losartan orally (15 mg·kg^-1·d^-1). The rats were raised to 16 weeks old. The morphometric parameters of the renal arterioles, and the widths of vascular smooth muscle cells (VSMC) and intercellular space were studied on kidney slices by light microscope and electromicroscope respectively, combined with computer-assistant image analysis system. The minimal renal vascular resistance (RVR_min) was studied by isolated kidney perfusion system. RESULTS: The systolic blood pressure of the tail artery, wall thickness, wall area, ratio of wall thickness to inner diameter, width of VSMC of renal arterioles and RVR_min were all smaller or lower in losartan group than those of SHR.     

Effects of probucol on the proliferation of rat vascular smooth muscle cells stimulated by basic fibroblast growth factor and hydrogen peroxide

AIM: To investigate the effect of probucol on proliferation of rat vascular smooth muscle cells(VSMC) stimulated by basic fibroblast growth factor (bFGF) and/or hydrogen peroxide(H₂O₂). METHODS: Effects of probucol on VSMC proliferation and DNA synthesis stimulated by bFGF and/or H₂O₂ were observed by means of MTT test, cell number count and [3H]-TdR incorporation. RESULTS: ①Probucol significantly inhibited proliferation and DNA synthesis in VSMC stimulated by bFGF and/or H₂O₂, with dosage-dependent manner. Cell number, A value and [3H]-TdR incorporation in group probucol+bFGF and group probucol+H₂O₂ were reduced by 40.0%, 39.1%, 45.5% and 46.9%, 45.0%, 39.5%, respectively, compared with group bFGF and group H₂O₂ (P＜0.05, P＜0.01, respectively). ②Pretreatment of VSMC with probucol for 24 h prior to bFGF and/or H₂O₂ stimulation exhibited significant inhibiton of VSMC proliferation and DNA synthesis, but after prestimulation by bFGF and/or H₂O₂ for 24 h, probucol had no influence on VSMC proliferation and DNA synthesis (P＞0.05). CONCLUSION: Probucol dramatically inhibited proliferation and DNA synthesis in VSMC stimulated by bFGF and/or H₂O₂, but had no inhibitory effect on the cell proliferation prestimulated by bFGF and /or H₂O₂.     

Inhibitory effect of canstatin RNA transfection on the growth of cultured rabbit vascular smooth muscle cells

AIM: To investigate the effect of canstatin on cultured rabbit vascular smooth muscle cells(VSMC). METHODS: By means of cationic liposome mediated method, canstatin RNA was transferred into cultured VSMC. The proliferation quantity of VSMC were determined by the cell counting method and thymidine(-TdR) incorporation. RESULTS: Canstatin RNA could be effectively transferred into cultured primary rabbit aortic smooth muscle cells by the cationic liposome-Dosper and could markedly inhibit VSMC proliferation. CONCLUSION: Transfection of canstatin RNA could inhibit the growth of VSMC in vitro.     

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.     

Effect of fibronectin on proliferation and collagen synthesis in cardiac fibroblasts from spontaneously hypertensive rats

AIM:To investigate the effects of fibronectin (FN) on the proliferation and collagen synthesis in cultured rat cardiac fibroblasts (CFb) derived from SHR (CFb_SHR) and WKY (CFb_WKY). METHODS:CFb derived from 12-week-old spontaneously hypertensive rat (SHR) and WKY was cultured by outgrowth of tissue block. Cell proliferation of CFb was measured by cell number counting and[³H]-TdR incorporation using 24-well plates pre-coated with 5 μg/cm² of FN. Collagen synthesis was determined by [³H]-proline incorporation. RESULTS:As compared with control, the cell number of fibroblasts derived from SHR and WKY were significantly increased to 163.75% and 170.42% respectively after 72 h incubation with FN in the presence of 0.4% FCS from a intial cell density of 1×10⁴ cells/mL. DNA synthesis of CFb was markedly promoted by FN. FN induced an increased in [³H]-proline incorporation in both CFb_SHR and CFb_WKY. CONCLUSION:FN is able to promote cell proliferation and collagen synthesis of CFb derived both from SHR and WKY.     

Retinoid X receptor agonists antagonize high-glucose-induced proliferation of rat vascular smooth muscle cells by inactivation of protein kinase C

AIM: To explore the effect of retinoid X receptor (RXR) agonists on high-glucose-induced proliferation of rat aortic smooth muscle cells (RASMCs). METHODS: RASMCs were cultured in DMEM containing glucose at normal concentration (5.5 mmol/L). For high glucose treatment, glucose solution was added up to a final concentration of 25 mmol/L. The proliferation of RASMCs was detected by WST-1 assay. DNA synthesis was measured by the method of BrdU incorporation. Cell cycle progression was determined by flow cytometry. Phosphorylated protein kinase C (PKC) and the expression levels of cyclin-dependent kinase 2(CDK2) and p27Kip1 were detected by immunoblotting. RESULTS: High glucose increased DNA synthesis, cell cycle progression, the expression of CDK2 and the proliferation of RASMCs. Meanwhile, the expression of p27Kip1 was decreased by high glucose. Treatment of RASMCs with RXR natural ligand 9-cis-retinoic acid (9-cis-RA) resulted in significant inhibition of high-glucose-induced proliferation, DNA synthesis, cell cycle progression and the expression of CDK2 in a concentration-dependent manner. 9-cis-RA also reversed the effect of high glucose on the expression of p27Kip1. RXR specific ligand SR11237 demonstrated the same effect as the effect of 9-cis-RA at the same concentration. PKC inhibitor showed the similar effect on high-glucose-induced proliferation and the expression of CDK2 and p27Kip1 as the RXR agonists did. Furthermore, 9-cis-RA and SR11237 rapidly inhibited high-glucose-induced activation of PKC. CONCLUSION: PKC is involved in high-glucose-induced proliferation of RASMCs. RXR agonists inhibit high-glucose-induced proliferation by depressing PKC activation in vascular smooth muscle cells.     

Interleukin-6 induced upregulation of basic fibroblast growth factor isoform through phorbol ester-sensitive protein kinase C in rat vascular smooth muscle cells

AIM: The role of protein kinase C(PKC) in the effect of Interleukin-6(IL-6) on basic fibroblast growth factor(bFGF) expression was investigated in rat vascular smooth muscle cells(VSMC). METHODS: Western-blotting was adopted to observe the variation of bFGF and its receptor type I isoforms expression. RESULTS: IL-6 increased all the three basic fibroblast growth factor isoforms in a dose-dependent(0-10.0 μg/L) manner. The upregulatory activities peaked at 24 h as demonstrated. In addition, after exhaustion of intracellular phorbol ester-sensitive PKC, the upregulatory effects of IL-6 on bFGF exprssion in VSMC declined( P <0.01). CONCLUSION: IL-6 increased bFGF expression in a phorbol ester-sensitive PKC-dependent manner in rat VSMC.     

《中国科学技术专家传略》农学编.园艺卷2出版发行

AIM: To make hyperglycemia models and observe the effects of hyperglycemia on expressive type transformation of vascular smooth muscle cell (VSMC) in rats. METHODS: Using tissue culture and radioactivity analysis methods.RESULTS:①Hyperglycemia group was lower than control group in NO₂^- content, nitric oxide synthase activity and cGMP content;②Hyperglycemia group was higher than control group in endothelin, insulin, total chdesterol and [³H]-TdR penetrated rate of VSMC. CONCLUSION: It was possible that hyperglycemia reduces the expressive type of VSMC to change and promotes VSMC proliferation.