Effect of fluvastatin on migration of vascular smooth muscle cells from rats

AIM: To investigate the effect and mechanism of fluvastatin on the migration induced by platelet derived growth factor-BB (PDGF-BB) and endothelin-1 (ET-1) in cultured vascular smooth muscle cells (VSMCs). METHODS: Cultured VSMCs derived from spontaneously hypertensive rats (SHR) were used. Cell migration was determined by modified Boyden chamber assays. Intracellular free calcium (［Ca2+］i) was measured with fluorescent Ca2+ indicator Fura-2/AM. RESULTS: PDGF-BB and ET-1 significantly induced VSMCs migration, which was inhibited by pretreatment of VSMCs with fluvastatin (10-9-10-5 mol/L) in a dose-dependent manner, and the peak inhibition rate of migration induced by PDGF-BB and ET-1 was over 86.67%. Fluvastatin also attenuated the increase in ［Ca2+］i induced by PDGF-BB and ET-1, with a peak inhibition rate of 86.76% and 65.32%, respectively. CONCLUSION: PDGF-BB and ET-1 promote migration of VSMCs from SHR．Fluvastatin may have direct inhibitory effects on cell migration induced by PDGF-BB and ET-1. The increase in ［Ca2+］i may acts as intracellular signaling in the migration in response to PDGF-BB and ET-1 in VSMCs.     

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.     

Studies of potassium channel in pulmonary artery smooth muscle cells of pulmonary hypertension rats

AIM：In this work,we investigated the difference of membrane capacitance,membrane current,current density and I-V curves between smooth muscle cells isolated from pulmonary hypertension rat (PHR) or normotensive rat (NTR) pulmonary arteries.METHODS：Thirty young male Sprague-Dawley rats,aged 8-9 weeks,were used.Body weight was (200±20)g at the start of experiments.These rats were placed into a normobaric chamber for 6 h·day-1,6 day·week-1 for 4 weeks.Hypoxic exposure was accomplished by ventilation with room air and N2 resulting in a constant O2 concentration of (10±0.5)%.Whole cell recordings were made from smooth muscle cells freshly isolated from pulmonary arteries derived from PHR or NTR.RESULTS：The membrane capacitance of pulmonary hypertension rats was larger than that in SD rats;but membrane current and current density were lower than those in SD rats (P<0.05).The I-V curves of pulmonary hypertension rat were downward shift compared with that in SD rat.Iptakalim hydrochloride 10 μmol·L-1 significantly increased potassium currents.CONCLUSIONS：Membrane capacitance,membrane current,membrane potential are decreased,I-V curves was shift downward,compared with NTR.Iptakalim hydrochloride significantly increased NTR and PHR potassium currents.     

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.     

Inhibitory effect of ferulate on attachment and migration induced by fibronectin and fibrinogen in cultured vascular smooth muscle cells from SHR

AIM: To investigate the effect of sodium ferulate (SF), one of the principal components of rhizoma ligustici wallichii, on the attachment and migration induced by fibronectin (FN) and fibrinogen (Fg) in vascular smooth muscle cells (VSMCs). METHODS: Cultured VSMCs derived from spontaneously hypertensive rats (SHR) were used. Cell attachment was conducted using a flat-bottomed 96 well polystyrene plate. Cell migration was determined by modified Boyden chamber assays. Intracellular free calcium (［Ca2+］i) was measured with fluorescent Ca2+ indicator Fura-2/AM. RESULTS: FN and Fg significantly induced the attachment and migration of VSMCs in a dose-and time-dependent manner, which was inhibited by pre-treatment of VSMCs with SF (10-7-10-3mol/L) a dose-dependentl fashion. The peak inhibition rate of attachment induced by FN and Fg was 67.12% and 70.23%, respectively, and the peak inhibition rate of SF on migration induced by FN and Fg was 69.79% and 87.06% (P<0.01). The rise of ［Ca2+］i in VSMCs provoked by FN and Fg was significantly suppressed by 10-3mol/L SF (P<0.01). CONCLUSION: The attachment, migration and increase in ［Ca2+］i induced by FN and Fg in VSMCs from SHR are suppressed by SF.     

Changes of large-conductance calcium-activated potassium channel in gastric smooth muscle cells of diabetic gastroparesis rats

AIM: To discuss the relevance between the pathogenesis of diabetic gastroparesis and the large-conductance calcium-activated potassium channels (BK_Ca) in gastric smooth muscle cells. METHODS: The SD rats were randomly divided into control group and model group. The gastric smooth muscle cells of the SD rats were enzymatically isolated in a low calcium solution containing papain. The current was recorded by patch clamp single channel recording technique. The expression of KCNMA and KCNMB1 were observed by the method of immunohistochemistry. RESULTS: The value of BK_Ca single channel conductance was (220.10±10.90) pS; the channels had distinct voltage dependent and calcium dependent characteristics. In outside-out patch (Vm =+30 mV), the activation of BK_Ca was blocked by 200 nmol/L IbTX completely. Compared with control group, the open probability and amplitude of current in model group significantly increased, while the mean open time and mean close time significantly decreased. Compared with control group, the expression of KCNMB1 in model group was significantly increased. CONCLUSION: Up-regulation of β1-subunit and increase in BK_Ca functional activities may be associated with diabetes gastroparesis in rats.     

Effect of interferon-inducible protein p204 on proliferation of vascular smooth muscle cells in rats

AIM: To study the effect of interferon-inducible protein p204 on the proliferation of vascular smooth muscle cells (VSMCs) in rats. METHODS: Cultured VSMCs were treated with interferon alpha (IFN-α) and p204 gene (Ifi204) small interfering RNA (siRNA) in vitro instantaneously. The cell vitality was detected by MTT me-thod,and the cell cycle was analyzed by flow cytometry. The expression of mRNA and proteins was determined by real-time qRT-PCR and Western blotting,respectively.RESULTS: IFN-α induced the increase in the expression of p204 at mRNA and protein levels, reduced the cell vitality, inhibited the cell cycle of G₁/S transition, and down-regulated the expression of Ras protein in VSMCs. Meanwhile, the phosphorylation levels of Raf and ERK were decreased. Transfection of Ifi204 siRNA restrained the expression of p204, increased the cell vitality and promoted the cell cycle of G₁/S transition in VSMCs. The up-regulation of Ras protein expression and the increased phosphorylation levels of Raf and ERK were also observed.CONCLUSION: The expression of p204 restrains the proliferation of VSMCs in rats by inhibiting the activation of Ras/Raf/MEK/ERK signal pathway.     

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 endogenous nitric oxide on potassium channels of bronchial smooth muscle cells from asthmatic rats

AIM: To investigate the effects of in vivo application of L-arginine on potassium channels in bronchial smooth muscle cells (BSMC) isolated from asthmatic model rats. METHODS: Male SD rats were randomly divided into control group, asthmatic group and asthmatic rats treated with L-arginine (L-Arg group). Single BSMCs were obtained by acute enzyme separation method. The resting membrane potential (Em), Ca2+-activated K+ channels (BKCa) currents and voltage-dependent K+ channel (Kv) currents in BSMCs were recorded under whole-cell patch clamp technique. RESULTS: (1) The Em of asthmatic group was significantly lower than that in control group (P<0.05). In vivo application of L-Arg significantly hyperpolarized BSMCs near to control group (P>0.05). (2) The peak current density at +50 mV of KCa: IKca in asthmatic group ［(43.8±16.5) pA/pF］ was significantly lower than that in normal group ［(72.5±19.9) pA/pF］ (P<0.01). Treatment with 300 mg/kg L-Arg significantly increased IKca in asthmatic group to (58.7±12.4) pA/pF (P<0.05). (3) The peak currents density at +50 mV of Kv: IKv in asthmatic group ［(32.4±8.7) pA/pF］ was significantly lower than that in control group ［(57.7±9.8) pA/pF］ (P<0.01). Treatment with L-Arg also significantly increased IKv in asthmatic group to (43.6±7.9) pA/pF, (P<0.05). CONCLUSION: Endogenous NO improves Em in asthmatic BSMCs, increases the activities of BKCa channels and Kv channel. These findings implicate that NO may have a potential therapeutically role in airway hypersensitivity.     

Effects of three kinds of calcium activator on the proliferation of cultured vascular smooth muscle cells in rats

AIM: To investigate the effects of intracellular free calcium ([Ca²⁺]i) from different resources on the proliferation mediated by mitogen activated protein kinase (MAPK) in vascular smooth muscle cells (VSMCs). METHODS: Cultured VSMCs were used in all experiments. Calcium influx was stimulated by angiotension Ⅱ(Ang Ⅱ). The release of intracellular calcium stores was induced by inositol trisphosphate (IP₃) and ryanodine (RY). MAPK activity was measured by [γ-³²P]-ATP incorporation MAPK protein expression by western blot, VSMCs proliferation by [³H]-Leucine ([³H]-Leu) and [³H]-Thymidine ([³H]-TdR) incorporation. RESULTS: Compared to the control VSMCs, Ang Ⅱ, IP₃ and RY significantly increased [Ca²⁺]i concentration activity of MAPK and its protein content in VSMCs. The promotion of [³H]-Leu and [³H]-TdR incorporation in VSMCs was also observed (P<0.01). CONCLUSION: The study indicated that calcium activator-induced increase in the activity and protein content of MAPK was involved in the proliferation of VSMCs, which was closely related to the [Ca²⁺]i concentration but independent to its origin.     

The intermediate phenotype of vascular smooth muscle cells in adult

The intermediate phenotype of vascular smooth muscle cell in adult is the dedifferentiation state returned from the high differentiation state, appeared on the damaged blood vessels.It is regulated by many factors.Its distribution, the characteristics of morphology and structure, the regulated transform factors and the molecular biological mechanism are introduced, and its functional significance and the role in vascular diseases are also discussed in this article.     

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.     

Lysophosphatidic acid and phenotypic modulation in vascular smooth muscle cells

Lysophosphatidic acid (LPA) is a sort of phospholipid messenger possessing diverse physiological role, which is mediated by its G protein-coupled receptors, and plays a significant role in vascular diseases. Phenotypic modulation of vascular smooth muscle cells (VSMCs) is the key initiation step of VSMCs proliferation and migration in hypertension, atherosclerosis and postangioplasty restenosis, and is the common morbility foundation of these vascular diseases. In this article we briefly review the LPA biological characteristics, its relationship with VSMCs phenotypic modulation and relative signal transduction pathway.     

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.     

Effect of cGMP on voltage-gated potassium channel in pulmonary artery smooth muscle cells from rats exposed to chronic hypoxia

AIM: To investigate the effect of cGMP on voltage-gated potassium channel in pulmonary artery smooth muscle cells (PASMCs) from rats exposed to chronic hypoxia. METHODS: (1) Wistar rats were randomly divided into control group (group A) and chronic hypoxia group (group B). Then group B received hypoxia 8 hours per day for 4 consecutive weeks. (2) Single PASMC was obtained via acute enzyme separation method. (3) Conventional whole-cell patch clamp technique was used to record resting membrane potential (Em) and ion currents of voltage-gated potassium channel. The changes of ion currents of voltage-gated potassium channel before and after applying cGMP (1 mmol/L), an agonist of protein kinase G (PKG), and cGMP plus H-8 (1 mmol/L), an inhibitor of PKG were compared between two groups. RESULTS: The Em of group B were significantly lower than that of group A. The ion currents of voltage-gated potassium channel in group A and group B were all significantly inhibited by cGMP [control group: from (118.0±5.0) pA/pF to (89.9±16.5) pA/pF, n=6, P＜0.05;chronic hypoxia group: from (81.0±5.0) pA/pF to (56.8±9.1) pA/pF, n=6, P＜0.05]and these effects were reversed by H-8 [control group: from (119.2±10.3) pA/pF to (117.8±9.1) pA/pF, n=6, P＞0.05;chronic hypoxia group: from (96.8±6.2)　pA/pF to (98.0±2.2)　pA/pF, n=6, P＞0.05]. CONCLUSIONS: The currents of voltage-gated potassium channel was inhibited by chronic hypoxic. The inhibitory effect of cGMP on currents of voltage-gated potassium channel in PASMCs from both normal and chronic hypoxic rats may be probably through the phosphorylation of voltage-gated potassium channel.     

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.     

Expression of PI3K in airway smooth muscle cell of asthmatic rats

AIM: To investigate the expression of PI3K in airway smooth muscle cell (ASMC) of asthmatic rats.METHODS: 16 Wistar rats were divided into two groups, asthma and normal control at random. After establishment of asthmatic model, flow cytometry, immunofluorescence and Western blotting were applied to detect the growth fraction of ASMC and the expression of PI3K in cultured ASMC from each rat.RESULTS: It was revealed from flow cytometry that the ratio of S + G2/M to total number of cells in asthma group ［ (27.90±3.44) % ］ was higher than that in normal control group ［ (13.00±1.56) %, P<0.05］. The expression of PI3K was observed in both asthma and normal control group. However, it was much higher in asthma group than that in normal control group. There was a positive correlation between the expression of PI3K and the growth fraction in ASMC. CONCLUSION: The increased expression of PI3K might play an important role in regulating the proliferation of ASMC in asthma.