Overexpression of heme oxygenase-1 protects smooth muscle cells against oxidative injury and inhibits cell proliferation

To investigate whether the expression of exogenous heme oxygenase-1 (HO-1) gene within vascular smooth muscle cells (VSMC) could protect the cells from free radical attack and inhibit cell proliferation, we established an in vitro transfection of human HO-1 gene into rat VSMC mediated by a retroviral vector. The results showed that the profound expression of HO-1 protein as well as HO activity was 1.8- and 2.0-fold increased respectively in the transfected cells compared to the non-transfected ones. The treatment of VSMC with different concentrations of H2O2 led to the remarkable cell damage as indicated by survival rate and LDH leakage. However, the resistance of the HO-1 transfected VSMC against H2O2 was significantly raised. This protective effect was dramatically diminished when the transfected VSMC were pretreated with ZnPP-IX, a specific inhibitor of HO, for 24 h. In addition, we found that the growth potential of the transfected cells was significantly inhibited directly by increased activity of HO-1, and this effect might be related to decreased phosphorylation of MAPK. These results suggest that the overexpression of introduced hHO-1 is potentially able to reduce the risk factors of atherosclerosis, partially due to its cellular protection against oxidative injury and to its inhibitory effect on cellular proliferation.

Differences in the primary culture, purification and biological characteristics between endothelial cells and smooth muscle cells from rat aorta

Objective： To investigate the differences of primary culture, purification and biological characteristics between endothelial cells and smooth muscle cells from rat aorta. Methods： Endothelial cells were obtained using the vascular ring adherence, collagenase digestion method and an improved vascular ring adherence method, while smooth muscle cells were separated from tissue sections of rat aorta. Clones of endothelial cells were selected by limiting dilution assay. Both cell types were identified using specific cell immunofluorescent markers, and phase contrast microscopy was used to observe the morphological disparity between endothelial cells and smooth muscle cells at the single cell and colony level. Cell proliferation was determined by the cell counting kit-8. Differences between endothelial cells and smooth muscle cells were evaluated in trypsin digestion time, attachment time and recovery after cryopreservation. Results： Endothelial cells were obtained by all three methods. The improved vascular ring method provided the most reproducible results. Cells were in good condition, and of high purity. Smooth muscle cells were cultured successfully by the tissue fragment culture method. Clonal expansion of single endothelial cells was attained. The two cell types expressed their respective specific markers, and the rate of proliferation of smooth muscle cells exceeded that of endothelial cells. Endothelial cells were more sensitive to trypsin digestion than smooth muscle cells. In addition, they had a shorter adherence time and better recovery following cryopreservation than smooth muscle cells. Conclusion： The improved vascular ring method was optimal for yielding endothelial cells. Limiting dilution is a novel and valid method for purifying primary endothelial cells from rat aorta. Primary rat endothelial cell and vascular smooth muscle cell cultures exhibited different morphological characteristics, proliferation rate, adherence time, susceptibility to trypsin digestion and recovery after cryopreservation. Our research can be a good foundation for further application in the regeneration of blood vessel.

Inhibitory Effects of Roscovitine on Proliferation and Migration of Vascular Smooth Muscle Cells In Vitro

Abnormal proliferation and migration of vascular smooth muscle cells （VSMCs） are the major cause of in-stent restenosis （ISR）. Intervention proliferation and migration of VSMCs is an im- portant strategy for antirestenotic therapy. Roscovitine, a second-generation cyclin-dependent kinase in- hibitor, can inhibit cell cycle of multiple cell types. We studied the effects of roscovitine on cell cycle distribution, proliferation and migration of VSMCs in vitro by flow cytometry, BrdU incorporation and wound healing assay, respectively. Our results showed that roscovitine increased the proportion of Go/G1 phase cells after 12 h （69.57±3.65 vs. 92.50±1.68, P=0.000）, 24 h （80.87±2.24 vs. 90.25±0.79, P=0.000） and 48 h （88.08±3.86 vs. 88.87±2.43, P=-0.427） as compared with control group. Roscovifine inhibited proliferation and migration of VSMCs in a concentration-dependent way. With the increase of concen- tration, roscovitine showed increased capacity for growth and migration inhibition. Roscovitine （30 μmol/L） led to an almost complete VSMCs growth and migration arrest. Combined with its low toxicity and selective inhibition to ISR-VSMCs, roscovitine may be a potential drug in the treatment of vascular stenosis diseases and particularly useful in the prevention and treatment of ISR.

Morphological Characteristics of Smooth Muscle Cells Isolated from the Rat Ductus Deferens

The aim of this study was to establish a method of isolating and culturing smooth muscle cells from the ductus deferens of rats. Smooth muscle cells were prepared from ductus deferens by explanting technique after dissection of adventitia and intimae, and cultured in vitro. The identification of the smooth muscle cells were verified by using anti u-smooth muscle actin （a-SMA） immunohistochemistry studies. The result suggested that the cells are multi-morphous, showing long fusiform or star shapes. The apophysis of cells contacted and coalesced to each other, in some regions the cells overlapped in multilayer, while in the other regions they formed monolayer that fluctuated and showed a ＂peak-valley＂ shape. They presented a positive reaction through immunohistochemistry studies. The purity of the cells was more than 99% through this method. The culturing of smooth muscle cells by explanting technique is simple and stable.

Influence of Angiotensin II on α1-Adrenergic Receptors Function in Rat Aorta and Expression in Vascular Smooth Muscle Cells

Angiotensin II (Ang II) is the main mediator of the Renin-Angiotensin-System acting on AT1 and other AT receptors. It is regarded as a pleiotropic agent that induces many actions, including functioning as a growth factor, and as a contractile hormone, among others. The aim of this work was to examine the impact of Ang II on the expression and function of α1-adrenergic receptors (α1-ARs) in cultured rat aorta, and aorta-derived smooth muscle cells. Isolated Wistar rat aorta was incubated for 24 h in DMEM at 37˚C, then subjected to isometric tension and to the action of added norepinephrine, in concentration-response curves. Ang II was added (1 × 10−5 M), and in some experiments, 5-Methylurapidil (α1A-AR antagonist), AH11110A (α1B-AR antagonist), or BMY-7378 (α1D-AR antagonist), were used to identify the α1-AR involved in the response. Desensitization of the contractile response to norepinephrine was observed due to incubation time, and by the Ang II action. α1D-AR was protected from desensitization by BMY-7378;while RS-100329 and prazosin partially mitigated desensitization. In another set of experiments, isolated aorta-derived smooth muscle cells were exposed to Ang II and α1-ARs proteins were evaluated. α1D-AR increased at 30 and 60 min post Ang II exposure, the α1A-AR diminished from 1 to 4 h, while α1B-AR remained unchanged over 24 h of Ang II exposure. Ang II induced an increase of α1D-AR at short times, and BMY-7378 protected α1D-AR from desensitization.

Effect of TRPV1 Channel on Proliferation and Apoptosis of Airway Smooth Muscle Cells of Rats

Airway remodeling is an important pathological feature of asthma and the basis of severe asthma. Proliferation of airway smooth muscle cells （ASMCs） is a major contributor to airway remod- eling. As an important Ca2＋ channel, transient receptor potential vanilloid 1 （TRPV1） plays the key role in the cell pathological and physiological processes. This study investigated the expression and activity of TRPV1 channel, and further clarified the effect of TRPV1 channel on the ASMCs proliferation and apoptosis in order to provide the scientific basis to treat asthmatic airway remodeling in clinical practice Immunofluorescence staining and reverse transcription polymerase chain reaction （RT-PCR） were used to detect the expression of TRPVI in rat ASMCs. Intracellular Ca2＋ was detected using the single cell confocal fluorescence microscopy measurement loaded with Fluo-4/AM. The cell cycles were observed by flow cytometry. MTT assay and Hoechst 33258 staining were used to detect the proliferation and apoptosis of ASMCs in rats respectively. The data showed that： （1） TRPV1 channel was present in rat ASMCs. （2） TRPV1 channel agonist, capsaicin, increased the Ca2~ influx in a concentration-dependent manner （EC50=284.3＋58 nmol/L）. TRPV1 channel antagonist, capsazepine, inhibited Ca2＋ influx in rat ASMCs. （3） Capsaicin significantly increased the percentage of S＋G2M ASMCs and the absorbance of MTT assay. Capsazepine had the opposite effect. （4） Capsaicin significantly inhibited the apoptosis, whereas capsazepine had the opposite effect. These results suggest that TRPV1 is present and mediates Ca2＋ influx in rat ASMCs. TRPV1 activity stimulates proliferation of ASMCs in rats.

Effects of Total Flavonoids ofHippophae RhamnoidesL.on Intracellular Free Calciumin Cultured Vascular Smooth Muscle Cells of Spontaneously Hypertensive Rats and Wistar-Kyoto Rats

To explore the effects of total flavonoids of Hippophae rhamnoides L. （TFH） quercetin （Que） and isorhamnetin （Isor） on the intracellular free calcium （[Ca^2＋]） in vascular smooth muscle cells （VSMC） of spontaneously hypertensive rats （SHR） and Wistar-Kyoto rats （WKY）. Metheds： Fluo 3-acetoxymethylester（Fluo-3/AM） was used to observe the effects of TFH （100mg/L） and its essential monomers, namely Que （10^-4mol/L） and Isor （10^-4mol/L） on changes of [Ca^2＋]1 in cultured SHR and WKY VSMC （abbr. to Ca-SHR ＆ Ca-WKY） following exposure to high K^＋, norepinephrine （NE） and angiotensin Ⅱ （AngⅡ）, and to compare with the effects of verapamil （Ver）. Results： （1） TFH, Que and Isor had inhibitory effects on resting Ca-SHR （P〈0.05）, but had no significant effects on Ca-WKY （P〉0.05）. （2） High K^＋ could increase Ca-SHR more significantly than Ca-WKY （P〈0.05）; TFH, Que and Isor could inhibit the elevation of [Ca^2＋]1 induced by high K^＋ -depolarization, with the effects similar to that of Ver, and the effect on Ca-SHR was more significant than that on Ca-WKY （P〈0.05）. （3） NE and Ang Ⅱ could increase Ca-SHR more significantly than Ca-WKY （P〈0.05）, TFH, Que and Isor had remarkably inhibitory effect on the elevation of Ca-SHR and Ca-WKY induced by NE or Ang Ⅱ. （4） In the absence of extracellular Ca^2＋ , TFH, Que and Isor also had certain inhibitory effect on Ca-SHR and Ca-WKY induced by NE, and the effect on the former was more significant than that on the latter（P〈0.05）. Ceaclusiea： TFH, Que and Isor might decrease the levels of [Ca^2＋], in VSMCs by blocking both voltage-dependent calcium channels （VDC） and receptoroperated calcium channels （ROC） in physiological or pathological state, which may be one of the important mechanisms of their hypotensive and protective effects on target organs in patients with hypertension.

Different Responses of Cell Cycle between Rat Vascular Smooth Muscle Cells and Vascular Endothelial Cells to Paclitaxel

Summary： Although previous reports showed dmg-eluting stent （DES） could effectively inhibit neointima formation, in-stent restenosis （ISR） remains an important obstacle. The purpose of this study was to investigate different effects of paclitaxel on proliferation and cell cycle regulators between vascular smooth muscle cells （VSMCs） and vascular endothelial cells （VECs） of rats in vitro. The cultured VSMCs and VECs of rats from the same tissues were examined by using immunohistochemistry, flow cytometry and Western blotting in control and paclitaxel-treated groups. The results showed paclitaxel could effectively inhibit proliferation of VSMCs and VECs. However, as compared with VECs, prolif- eration of VSMCs in paclitaxel-treated group decreased less rapidly. The percentage of cells in G0-G1 and G2-M phases was reduced, and that in S phase increased after treatment for 72 h. The expression of cyclin D1 and B1, p27 and PCNA in VSMCs of paclitaxel-treated group was up-regulated, but that of p21 down-regulated as compared with VECs. It is concluded that there are significant differences in the expression of cell cycle regulators and proliferation rate between paclitaxel-treated VSMCs and paclitaxel-treated VECs, suggesting that the G1 S checkpoint regulated by paclitaxel may play a critical role in the development of complications of DES, which provides new strategies for treatments of ISR.

Different distributions of interstitial cells of Cajal and platelet-derived growth factor receptor-α positive cells in colonic smooth muscle cell/interstitial cell of Cajal/plateletderived growth factor receptor-α positive cell syncytium in mice

AIM To investigate the distribution and function of interstitialcells of Cajal(ICCs) and platelet-derived growth factor receptor-α positive(PDGFRα+) cells in the proximal and distal colon.METHODS The comparison of colonic transit in the proximal and distal ends was performed by colonic migrating motor complexes(CMMCs). The tension of the colonic smooth muscle was examined by smooth muscle spontaneous contractile experiments with both ends of the smooth muscle strip tied with a silk thread. Intracellular recordings were used to assess electrical field stimulation(EFS)-induced inhibitory junction potentials(IJP) on the colonic smooth muscle. Western blot analysis was used to examine the expression levels of ICCs and PDGFRα in the colonic smooth muscle.RESULTS Treatment with NG-nitro-L-arginine methyl ester hydrochloride(L-NAME) significantly increased the CMMC frequency and spontaneous contractions, especially in the proximal colon, while treatment with MRS2500 increased only distal CMMC activity and smooth muscle contractions. Both CMMCs and spontaneous contractions were markedly inhibited by NPPB, especially in the proximal colon. Accordingly, CyPPA sharply inhibited the distal contraction of both CMMCs and spontaneous contractions. Additionally, the amplitude of stimulationinduced nitric oxide(NO)/ICC-dependent slow IJPs(sIJPs) by intracellular recordings from the smooth muscles in the proximal colon was larger than that in the distal colon, while the amplitude of electric field stimulationinduced purinergic/PDGFRα-dependent fast IJPs(fIJPs) in the distal colon was larger than that in the proximal colon. Consistently, protein expression levels of c-Kit and anoctamin-1(ANO1) in the proximal colon were much higher, while protein expression levels of PDGFRα and small conductance calcium-activated potassium channel 3(SK3) in the distal colon were much higher.CONCLUSION The ICCs are mainly distributed in the proximal colon and there are more PDGFRα+ cells are in the distal colon, which generates a pressure gradient between the two ends of the colon to propel the feces to the anus.

Urocortin, the neuropeptide, inhibits the viability of ECV304 cells and rat vascular smooth muscle cells

Objective: This study aims to investigate the effects of urocortin (Ucn) on the viability of endothelial cells (ECV304) and rat vascular muscle cells (VSMC). Methods: Rat aortic VSMC were isolated from the rats' thoracic aorta. We studied the effect of Ucn on the viability of ECV304 cells and VSMC by using a tetrazolium (MTT) assay.Results: Ucn (10 -7 mol/L) inhibited the viability of ECV304 cells and VSMC. Inhibition rates are 13% and 15%, respectively(P<0.05, compared with Control). This inhibition was not dependent on the affecting time and was not affected by the addition of ATP-sensitive potassium channel (KATP channel) blocker, glybenclamide (Gly, 10 mol/L). Conclusion: Ucn inhibits the viability of ECV304 and VSMC. Our results suggest that Ucn may be a new vasoactive agent and may have a beneficial effect in the process of vascular remodeling (VR).

Resveratrol inhibits angiotensin II-induced ERK1/2 activation by downregulating quinone reductase 2 in rat vascular smooth muscle cells

Our previous studies showed that resveratrol could inhibit the proliferation of vascular smooth muscle cells （VSMCs） and repress mRNA and protein expression of quinone reductase 2 （NQO2）. This study further explored the potential mechanisms whereby resveratrol inhibits the proliferation of rat VSMCs. Lentiviral vectors that incorporated NQO2 small interfering RNA （siRNA） were constructed and transduced into rat VSMCs. The cell proliferation was detected using the bromodeoxyuridine （BrdU） assay. Cultured rat VSMCs were stimulated with angiotensin II and the level of reactive oxygen species （ROS） was measured using a ROS assay kit. A realtime quantitative PCR was used to detect NQO2 mRNA levels. Extracellular signal-regulated kinase （ERK1/2） and NQO2 protein expression were determined by Western blotting analysis. The inhibitory effect of resveratrol （10 and 50 μmol/L） on the proliferation of rat VSMCs in the NQO2 siRNA group was significantly weaker than that in the normal and scrambled siRNA group （P 〈 0.01）. The ROS level in the NQO2 siRNA and resveratrol （50 μmol/L） treatment groups were lower than that in the normal and scrambled siRNA groups （P 〈 0.01 in both）. Compared with the normal and scrambled siRNA group, the phosphorylation of ERK1/2 was significantly decreased in the NQO2 siRNA and resveratrol （50 μmol/L） treatment group （P 〈 0.01 in both）. In conclusion, high concentration of resveratrol inhibits angiotensin II-induced ERK1/2 phosphorylation and subsequent proliferation by down-regulation of NQO2 in cultured rat VSMCs.

MicroRNA-146a Promotes Embryonic Stem Cell Differentiation towards Vascular Smooth Muscle Cells through Regulation of Kruppel-like Factor 4

Objective Vascular smooth muscle cell(VSMC)differentiation from stem cells is one source of the increasing number of VSMCs that are involved in vascular remodeling-related diseases such as hypertension,atherosclerosis,and restenosis.MicroRNA-146a(miR-146a)has been proven to be involved in cell proliferation,migration,and tumor metabolism.However,little is known about the functional role of miR-146a in VSMC differentiation from embryonic stem cells(ESCs).This study aimed to determine the role of miR-146a in VSMC differentiation from ESCs.Methods Mouse ESCs were differentiated into VSMCs,and the cell extracts were analyzed by Western blotting and RT-qPCR.In addition,luciferase reporter assays using ESCs transfected with miR-146a/mimic and plasmids were performed.Finally,C57BL/6J female mice were injected with mimic or miR-146a-overexpressing ESCs,and immunohistochemistry,Western blotting,and RT-qPCR assays were carried out on tissue samples from these mice.Results miR-146a was significantly upregulated during VSMC differentiation,accompanied with the VSMC-specific marker genes smooth muscle-alpha-actin(SMαA),smooth muscle 22(SM22),smooth muscle myosin heavy chain(SMMHC),and h1-calponin.Furthermore,overexpression of miR-146a enhanced the differentiation process in vitro and in vivo.Concurrently,the expression of Kruppel-like factor 4(KLF4),predicted as one of the top targets of miR-146a,was sharply decreased in miR-146a-overexpressing ESCs.Importantly,inhibiting KLF4 expression enhanced the VSMC-specific gene expression induced by miR-146a overexpression in differentiating ESCs.In addition,miR-146a upregulated the mRNA expression levels and transcriptional activity of VSMC differentiation-related transcription factors,including serum response factor(SRF)and myocyte enhancer factor 2c(MEF-2c).Conclusion Our data support that miR-146a promotes ESC-VSMC differentiation through regulating KLF4 and modulating the transcription factor activity of VSMCs.

Eukaryotic Expression of Human Arresten Gene and Its Effect on the Proliferation of Vascular Smooth Muscle Cells

The eukaryotic expression of human arresten gene and its effect on the proliferation of in vitro cultured vascular smooth cells （VSMCs） in vitro were investigated. COS-7 cells were transfected with recombinant eukaryotic expression plasmid pSecTag2-AT or control plasmid pSecTag2 mediated by liposome. Forty-eight h after transfection, reverse transcription-polymerase chain reaction （RT-PCR） was used to detect the expression of arresten mRNA in the cells, while Western blot assay was applied to detect the expression of arresten protein in concentrated supernatant. Primary VSMCs from thoracic aorta of male Sprague-Dawley rats were cultured using the tissue explant method, and identified by immunohistochemical staining with a smooth muscle-specific anti-α- actin monoclonal antibody before serial subcuhivation. VSMCs were then co-cultured with the concentrated supernatant and their proliferation was detected using Cell Counting Kit-8 （CCK-8） in vitro. The results showed that RT-PCR revealed that the genome of arresten-transfected cells contained a 449 bp specific fragment of arresten gene, suggesting the successful transfection. Success- ful protein expression in supernatants was confirmed by Western blot. CCK-8 assay showed that the proliferation of VSMCs were inhibited significantly by arresten protein as compared with control cells （F=40. 154, P〈0.01）. It was concluded that arresten protein expressed in eukaryotic cells can inhibit proliferation of VSMCs effectively in vitro, which would provide possibility to the animal experiments.

Effects of calcium-activated chloride channels on proliferation of pulmonary artery smooth muscle cells in rats under chronic hypoxic condition

Objective：To investigate the effects of calcium-activated chloride （ClCa） channels on proliferation of pulmonary artery smooth muscle cells（PASMCs） in rats under chronic hypoxic condition. Methods：The cultured PASMCs were placed under normoxic and chronic hypoxic conditions：The cells were observed by light and electron microscope; The cell cycles were observed by flow-cytometry; Immunocytochemistry staining was used to detect the expressions of PCNA, c-fos and c-jun of PASMCs; Cytoplasmic free Ca^2＋ concentration （[Ca^2＋]i） in PASMCs was investigated by fluorescent quantitation using fluorospectrophotometer. Results：The PASMCs were contractile phenotype under normoxic conditions. Observation by transmission electron microscope： In kytoplasm of contractile phenotype cells, myofilament bundles were abundant and the content of cell organs such as Golgi＇s bodies were rare. The PASMCs were synthetic phenotype under chronic hypoxic condition. There were increased free ribosomes, dilated rough endoplasmic reticulums, highly developed Golgi complexes, decreased or disappeared thick filaments and dense body in kytoplasm of synthetic phenotype cells. After NFA and IAA-94, the situations were reversed The number of S ＋G2M PASMCs were significantly increased in chronic hypoxic condition; The NFA and IAA-94 were shown to significantly decrease them from （28.6±1.0）% to （16.0±1.6）% and the number of G0G1 PASMCs significantly increased from （71.4± 1.9）% to （83.9 ± 1.6）% （P〈 0.01）. In chronic hypoxic conditions, the expression of proliferating cell nucleus antigen was significantly increased; The NFA and IAA-94 were shown to significantly decrease it from （81 ± 6）% to （27 ± 7）%（P 〈 0.01）. The expression of c-fos and c-jun were significantly increased in＇chronic hypoxic conditions; The NFA and IAA-94 were shown to significantly decrease them from 0.15 ±0.02, 0.32 ± 0.05 to 0.05 ± 0.01, 0.12 ± 0.05, respectively （P〈 0.01）; Under chronic hypoxic conditions, [Ca^2＋]i was increased; The NFA and IAA-94 decreased it from （281.8±16,5）nmol/L to （117.7 ± 15.4）nmol/L（P 〈 0.01）. Conclusion：Hypoxia initiated the change of PASMCs from contractile to synthetic phenotype and increased proliferation of PASMCs. NFA and IAA-94 depressed cell proliferation by blocking ClCa channels in hypoxic condition. These may play an important role in proliferation of PASMCs under chronic hypoxic conditions.

Effects of caffeic acid phenethyl ester on proliferation of vascular smooth muscle cells in rats

Objective： To investigate the inhibitory effect of caffeic acid phenethyl ester（CAPE） on the proliferation of vascular smooth muscle cells （VSMC） activated by lipopolysaccharide （LPS） and to clarify its mechanism. Methods： VSMC activated by LPS （1 mg-L^-1） were treated with CAPE at different concentrations. The inhibitory effecfs of CAPE on the proliferation of VSMC were determined by methabenzthiazuron（MTT） colorimetry. The effects of CAPE on the expression of proliferating cell nuclear antigen （PCNA） and Survivin protein in VSMC were evaluated by immunocytochemistry staining technique （SABC method）. Cell cycle was analyzed by flow cytometry（FCM） with propidiumiodide （PI） labeling method. The relative expression level of Survivin mRNA was measured with real-time quantified RT-PCR technique. Results. CAPE exerted significant inhibitory effects on. proliferation of VSMC at concentrations ranging from 5 mg·L^-1 to 80 mg·L^-1, decreased the rate of cells positive for PCNA and Survivin protein and repressed the expressioh of Survivin mRNA in a dose- and time-dependent manner （P 〈 0.05）. FCM analysis displayed that CAPE up-regulated the ratio of G0/G1 stages and reduced the percentage of VSMC in S stage （P 〈 0.05）. Conclusion： CAPE can significantly inhibit the proliferation of VSMC activated by LPS in a dose- and time-dependent manner, which may be carded out through regulating cell cycle and repressing the expression of PCNA and Survivin.

Effects of Tetrandrine on Cytosolic Free Calcium in Cultured Bovine Aortic Smooth Muscle Cells

\ The effects of tetrandrine (Tet) on cytosolic free calcium ([Ca2+]i) in subcultured bovine aortic smooth muscle cells (SMC) were studied by Fura2 and ARCMMIC cation measurement system. Tet (1～100 μmol·L-1) had no effect on the resting [Ca2+]i, but had inhibitory effects on [Ca2+]i elevation induced by high K+, 5HT, ATP, Ang II and NE in the presence of extracellular Ca2+. High concentration of Tet also inhibited Pheinduced [Ca2+]i elevation in absence of extracellular Ca2+. Tet (1～100 μmol·L-1) inhibited KCl (60 mmol·L-1) induced [Ca2+]i elevation in dosedependent manner, the IC50 value was 9.2 (95% confidence limits: 5.7～14.9) mmol·L-1. The results suggested that Tet had blocking effects on both VOC and ROC in bovine aortic SMC. It appears that the mechanisms of blocking effect of Tet on ROC might be primarily due to its Ca2+ entry blocking effects.

OVER-EXPRESSION OF EXTRACELLULAR SIGNAL-REGULATED KINASE IN VASCULAR SMOOTH MUSCLE CELL OF HYPERTENSIVE RATS

Objective To investigate whether extracellular signal-regulated kinase (ERK1/2) was involved in changes of vascular smooth muscle cell (VSMC) under hypertension.Methods Two-kidney one clip Wistar hypertensive rats (WHR) were sacrificed and their right kidneys were harvested 4 weeks after surgery.The spontaneously hypertensive rats (SHR) were divided into 4, 8, and 16 weeks old groups (SHR4w, SHR8w, and SHR16w), respectively.The control group were sham operated age-matched Wistar rats.Immunohistochemical technique and Western blotting were applied to study ERK1/2 protein expression in VSMC of the renal vascular trees in WHR, SHR, and control rats.Results Blood pressure in two-kidney one clip WHR obviously increased at one week after surgery, and reached to 198.00±33.00 mm Hg at the end of experiment, significantly higher than that in the control rats (P<0.01).Blood pressure in SHR4w (108.00±11.25 mm Hg) was similar to that in the controls.However, it rose to 122.25±21.75 mm Hg in SHR8w, and even up to 201.75±18.00 mm Hg in SHR16w, which were significantly higher than that of both the SHR4w and the controls (P<0.01).The rate and degree of glomerular fibrosis in WHR were significantly higher than controls (P<0.05).Hyaline degeneration of the afferent arterioles was found in WHR.In contrast, either fibrosis of glomerulus or hyaline degeneration of the arterioles or protein casts was not observed in SHR4w, SHR8w, and SHR16w.Immunohistochemical staining results showed expression of ERK1 was similar to that of ERK2.The positive rates of ERK2 staining in VSMC of afferent arterioles, interlobular, interlobar, and arcuate arteries in two-kidney one clip WHR were significantly higher (7.09%±1.75%, 14.57%±4.58%, 29.44%±7.35%, and 13.63%±3.85%, respectively) than that of the controls(P<0.01).The positive rates of ERK2 staining in VSMC at afferent arterioles, interlobular, interlobar, and arcuate arteries in SHR16w were significantly higher (12.09%±1.40%, 24.17%±6.92%, 32.44%±4.05%, and 18.61%±3.35%, respectively) than that of the controls (P<0.01), too.The expression of ERK1/2 protein of kidney in WHR and SHR16w was significantly higher than that in the controls by Western blotting assay (P<0.01).Conclusion Extracellular signal transduction system are highly expressed in kidney VSMC of two-kidney one clip WHR and SHR.Phospho-ERK1/2 may play an important role in VSMC hypertrophy and hyperplasia under hypertension.

Vascular smooth muscle cell differentiation－2010

Vascular smooth muscle cells have attracted considerable interest as a model for a flexible program of gene expression.This cell type arises throughout the embryo body plan via poorly understood signaling cascades that direct the expression of transcription factors and microRNAs which,in turn,orchestrate the activation of contractile genes collectively defining this cell lineage.The discovery of myocardin and its close association with serum response factor has represented a major break-through for the molecular understanding of vascular smooth muscle cell differentiation.Retinoids have been shown to improve the outcome of vessel wall remodeling following injury and have provided further insights into the molecular circuitry that defines the vascular smooth muscle cell phenotype.This review summarizes the progress to date in each of these areas of vascular smooth muscle cell biology.

Both Hypoxic Endothelial Cell Conditioned Medium and Hypoxia Elevate Intracellular Free Calcium in Pulmonary Artery Smooth Muscle Cells

By using Ca2+ -sensitive fluorescent probe, Fura-2 , the effects of endothelial cell-conditioned medium and hypoxia on intracellular free calcium ( [Ca2+]i) in cultured pulmonary artery smooth muscle cell (PASMC) were studied. Normoxic porcine pulmonary artery endothelial cell-conditioned medium (NPAECCM) obviously elevated [Ca2+]i in PASMC,whereas the hypoxic porcine pulmonary artery endothelial cell conditioned medium (HPAECCM)significantly elevated [Ca2+]i in PASMC much more than NPAECCM. Both the effects of NPAECCM and HPAECCM were dependent on the cultured endothelial cell extracellular calcium concentrations, ranged from 1.8 mmol/L to 2. 4 mmol/L.Meanwhile, hypoxia directly increased, which was partially inhibited by verapamil,[Ca2+]i in PASMC through Ca2+ influx pathway.The data suggest that the augmented regulation of endothelial cell on PASMC via Ca2+ second messenger system and the hypoxia-induced Ca2+ influx into PASMC,particularly the former, may be components of mechanisms underlying hypoxic pulmonary vasoconstriction and chronic pulmonary hypertension.

Oxidative modification of high density lipoprotein induced by cultured human arterial smooth muscle cells

Objective: To observe the oxidative modification of high density lipoprotein (HDL) induced by cultured human arterial smooth muscle cells (SMCs). Methods: HDL cocultured with SMCs at 37℃ in 48 h was subjected, and native HDL (N-HDL) served as control. Oxidative modification of HDL was identified by using agarose gel electrophoresis. Absorbances of conjugated diene (CD) and lipid hydroperoxide (LOOH) were measured with ultraviolet spectrophotometry at 234 and 560 nm respectively, and fluorescence intensity of thiobarbuturic acid reaction substance (TBARS) with fluorescence spectrophotometry at 550 nm emission wavelength with excitation at 515 nm. Results: In comparison with N-HDL, the electrophoretic mobility of SMCs-cocultured HDL was increased, and the contents of CD, LOOH and TBARS HDL were very significantly higher than those of the control HDL (P<0.01). Conclusion: Oxidative modification of HDL can be induced by human arterial SMCs.