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.

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.

Effect of Cigarette Smoke Extract on the Role of Protein Kinase C in the Proliferation of Passively Sensitized Human Airway Smooth Muscle Cells

To investigate the effect of cigarette smoke extract (CSE) on the role of protein kinase C (PKC) in the proliferation of passively sensitized human airway smooth muscle cells (HASMCs). After synchronization of cultured HASMCs, they were divided into a group A and Group B. The group A was treated with normal human serum and served as controls and the group B was treated with the serum of asthma patients. The group A was further divided into group of A_1, A_2 and A_3 and the group B was sub-divided into the group of B_1, B_2, B_3, B_4 and B_5. No other agents were added to the group A_1 and B_1. The cells of group A_2 and B_2 were stimulated with 5 % CSE for 24 h. HASMCs from group A_3 and B_3 were treated with PKC agonist PMA (10 nmol/L) and CSE (5 %) for 24 h. PKC inhibitor Ro-31-8220 (5 μmol/L) was added to the HASMCs of group B_4 for 24 h. The cells from group B_5 were stimulated with Ro-31-8220 (5 μmol/L) and CSE (5 %) for 24 h. The proliferation of HASMCs isolated from group A and B was examined by cell cycle analysis, MTT colorimetric assay and 3H-TdR incorporation test. The expression of PKC-α in each group was observed by Western blotting and RT-PCR, respectively. The results showed that the percentage of S phase, absorbance (A) value, the rate of 3H-TdR incorporation, the ratios of A value of PKC-α mRNA and the A value of PKC-α protein in HASMCs from group B_1, B_2 and B_3 were significantly increased compared to those of group A_1, A_2 and A_3 correspondingly and respectively (P<0.01). The proliferation of HASMCs of group A_2 and B_2 stimulated with CSE and group A_3 and B_3 stimulated with CSE and PMA were also significantly enhanced when group A_1, A_2 and A_3 and group B_1, B_2 and B_3 compared to each other (P<0.05, P<0.01, respectively). The percentage of S phase, absorbency (A) value, 3H-TdR incorporation rate, the ratios of A value of PKC-α mRNA and the A value of PKC-α protein in HASMCs from group B_4 treated with Ro-31-8220 and group B_5 treated with CSE and Ro-31-8220 were significantly decreased as compared to those of group B_1 and B_2 correspondingly and respectively (P<0.05, P<0.01). It was concluded that CSE can enhance the passively sensitized HASMC proliferation and the expression of PKC alpha. PKC and its alpha subtype may contribute to this process. Our results suggest cigarette may play an important role in ASMCs proliferation of asthma through PKC signal pathway.

Role of Protein Kinase C in the Activation of Store-operated Ca^(2+) Entry in Airway Smooth Muscle Cells

Store-operated Ca2＋ channels （SOCs） are plasma membrane Ca2＋ permeable channels activated by depletion of intracellular Ca2＋ store. Ca2＋ entry through SOCs is known as store-operated Ca2＋ entry （SOCE）, which plays an important role in the functional regulation of airway smooth muscle cells （ASMCs）. Protein kinase C （PKC） has been shown to have an activating or inhibiting effect on SOCE, depending on cell types and PKC isoforms that are involved. In ASMCs, the effect of PKC on SOCE has not been elucidated so far. In this study, the role of PKC in the activation of SOCE in rat ASMCs was examined by using Ca2＋ fluorescence imaging technique. The results showed that acute application of PKC activators PMA and PDBu did not affect SOCE induced by the sarcoplasmic reticulum Ca2＋-ATPase （SERCA） inhibitor thapsigargin. The non-selective PKC inhibitor chelerythrine significantly inhibited thapsigargin- and bradykinin-induced SOCE. RT-PCR assay identified PKCα, δ and ε isoforms in rat ASMCs. PKCα-selective inhibitor G6976 and PKCε-inhibiting peptide Epsilon-V1-2 had no effect on SOCE; by contrast, PKCδ-selective inhibitor rottlerin attenuated SOCE dramatically, suggesting that PKCδ was the major PKC isoform involved in the activation of SOCE in ASMCs. Moreover, PKC down-regulation by extended exposure to high doses of PMA or PDBu also reduced SOCE, confirming the essential role of PKC in the activation of SOCE in ASMCs. In addition, PKC down-regulation did not influence the expression of stromal interaction molecule 1 （STIM1） and Orai1, two elementary molecules in the regulation and activation of SOCs. These results identified PKCδ as an essential PKC isoform involved in the activation of SOCE, and confirmed that PKC regulates the function of ASMCs in a SOCE-dependent manner.

Effects of Shenmai and Aminophylline on Apoptosis of Small Airway Smooth Muscle Cells and the Expression of Relevant Genes in Rats with Emphysema

The effects of Shenmai injection (SMI) and am inophylline on apoptosis of sm all airway smooth muscle cells (SASMC) and the Fas/ Fas L expression in rats with papain- induced em physe- ma were investigated.Rat emphysema model was established by a single intratracheal instillation of papain.Apoptosis and Fas/Fas L expression of SASMC were detected by im munohistochemistry SABC and TU NEL assay at day1,3,5 ,7,15 ,30 after modeling,and the effect of SMI and am inophylline on them were observed.The results indicated that the Fas/Fas L expression positive rate in SASMC was2 .31± 0 .5 5 /1.2 8± 0 .4 7respectively.After a single intratracheal instillation of papain,the expression of Fas/Fas L positive rate in the placebo group was increased in a tim e- dependent manner.SMI could inhibit the expression of Fas/ Fas L ,but aminophylline couldn't. The positive rate of apoptosis in the control group was 0 .87± 0 .32 .After a single intratracheal in- stillation of papain,the SASMC apoptosis positive rate in the placebo group was increased in a tim e- dependent manner.The SASMC apoptosis rate in all groups was declined after treatment with SMI,but the effect of am inophylline was notobvious.Itwas dem onstrated thatin the patho- genesis of emphysem a Fas/Fas L played an important role in the regulation of SASMC apoptosis. SMI influenced the expression of Fas/ Fas L and declined SASMC apoptosis by inhibiting the releas- ing of inflamm atory m edia and played an im portant role in the therapy of em physema.

Midkine ameliorates LPS-induced apoptosis of airway smooth muscle cells via the Notch2 pathway

Objective:To evaluate the effect of midkine on lipopolysaccharide(LPS)-induced airway smooth muscle cells(ASMCs).Methods:LPS-stimulated acute lung injury model was used to analyze the effect of midkine on ASMCs in vitro.Recombinant midkine and midkine siRNA were used to investigate the role of Notch2 signaling pathway.Cell proliferation was assessed using Cell Counting Kit-8 assay.Additionally,apoptosis was measured by flow cytometry and protein and mRNA expression of midkine and Notch2 was assessed by Western blotting and qPCR,respectively.Immunofluorescence analysis was also conducted.Results:LPS increased the mRNA and protein expression of midkine and Notch2.Midkine silencing reduced LPS-induced midkine and Notch2 expression.In addition,midkine silencing further reduced the viability and increased apoptosis of ASMCs induced by LPS,which was attenuated by recombinant midkine.Conclusions:The midkine/Notch2 signaling pathway plays a regulatory role in ASMC proliferation and apoptosis in airway inflammation.

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.

Effects of Iptakalim on intracellular free calcium concentration of cultured rabbit pulmonary arterial smooth muscle cells

Objective：To explore the effects of Iptakalim on intracellular free calcium concentration and on the proliferation of cultured rabbit pulmonary arterial smooth muscle cells induced by endothelin-1 （ET-1） in vitro. Methods：A cell culture model, [^3H]-thymidine（[^3H]-TdR） incorporation test and confocal microscope were used to observe proliferation and intracellular free calcium concentration（[Ca^2±]） of rabbit PASMC induced by ET-1 in vitro. Results：The value of [^3H]-TdR incorporation in ET-1 group was increased 1.468 times higher than that in control group. Iptakalim at the concentration of 10^-7mol/L, 10^-6mol/L ,10^-5 mol/L lowered [^3H]-TdR incorporation by （19.8 ± 4.6）%, （41.2 ± 9.5）%, （54.7 ± 10.1）%, respectively, compared with the value of the cells treated with ET-1（P〈 0.01）; The intracellular fluorescence intensity of PASMC in ET-1 group was increased from 73.70 ± 10.12 to 143.84 ± 28.23, significantly higher than that in control group（P 〈 0.01）; whereas with Iptakalim,the fluorescence intensity（FI） was only increased from 74.30 ± 10.20 to 86.03 ± 9.82, significantly lower than that in ET-1 group（P 〈 0.01）. Conclusion：Iptakalim inhibited proliferation of PASMC and decreased intracellular free calcium concentration of cultured rabbit PASMC induced by ET-1.

Effects of tetrandrine on cytosolic free calcium concentration in corpus cavernosum smooth muscle cells of rabbits

Aim:To study the relaxation mechanisms of tetrandrine(Tet)on the corpus cavernosum smooth muscle.Methods: The corpus cavernosum smooth muscle cells from New Zealand white rabbits were cultured in vitro.[Ca^(2+)]_i was measured by Fluorescence Ion Digital Imaging System,using Fluo-2/AM as a Ca^(2+)-sensitive fluorescent indicator. Results:Tet(1,10 and 100 μmol/L)had no effect on the resting [Ca^(2+)]_i(P>0.05).In the presence of extracellular Ca^(2+)(2.5 mmol/L),Tet(1,10 and 100 μmol/L)inhibited [Ca^(2+)]_i elevation induced by high K^+ and phenylephrine(PE) in a concentration-dependent manner(P<0.05).In calcium free solution containing egtaic acid,Tet(1 and 10 μmol/L) had no inhibitory effects on [Ca^(2+)]_i elevation induced by PE(P>0.05).However,Tet(100μmol/L)inhibited [Ca^(2+)]_i elevation induced by PE(P<0.05).Conclusion:Tet inhibited the Ca^(2+)influx from the extracellular site via voltage- activated Ca^(2+)channel and α_1-adrenoceptor-operated Ca^(2+)channel.At a high concentration,Tet might inhibit the cytosolic calcium pool release in cultured corpus cavernosum smooth muscle cells.This inhibitory action on [Ca^(2+)]_i might be one of the relaxation mechanisms of Tet on the corpus cavernosum smooth muscle.(Asian J Androl 2006 Jul;8:405-409)

Effects of Mitochondrial ATP-sensitive K^+ Channel on Protein Kinase C Pathway and Airway Smooth Muscle Cell Proliferation in Asthma

The effects of ATP-sensitive mitochondrial K + channel(mitoK ATP) on mitochondrial membrane potential(Δψm),cell proliferation and protein kinase C alpha(PKCα) expression in airway smooth muscle cells(ASMCs) were investigated.Thirty-six Sprague-Dawley(SD) rats were immunized with saline(controls) or ovalbumin(OVA) with alum(asthma models).ASMCs were cultured from the lung of control and asthma rats.ASMCs were treated with diazoxide(the potent activator of mitoK ATP) or 5-hydroxydencanote(5-HD,the inhibitor of mitoK ATP).Rhodamine-123(R-123) was used to detect Δψm.The expression of PKCα protein was examined by using Western blotting,while PKCα mRNA expression was detected by using real-time PCR.The proliferation of ASMCs was measured by MTT assay and cell cycle analysis.In diazoxide-treated normal ASMCs,the R-123 fluorescence intensity,protein and mRNA levels of PKCα,MTT A values and percentage of cells in S phase were markedly increased as compared with untreated controls.The ratio of G 0 /G 1 cells was decreased(P<0.05) in diazoxide-treated ASMCs from normal rats.However,there were no significant differences between the ASMCs from healthy rats treated with 5-HD and the normal control group.In untreated and diazoxide-treated ASMCs of asthmatic rats,the R-123 fluorescence intensity,protein and mRNA levels of PKCα,MTT A values and the percentage of cells in S phase were increased in comparison to the normal control group.Furthermore,in comparison to ASMCs from asthmatic rats,these values were considerably increased in asthmatic group treated with diazoxide(P<0.05).After exposure to 5-HD for 24 h,these values were decreased as compared with asthma control group(P<0.05).In ASMCs of asthma,the signal transduction pathway of PKCα may be involved in cell proliferation,which is induced by the opening of mitoK ATP and the depolarization of Δψm.

K^+ Channels and Their Effects on Membrane Potential in Rat Bronchial Smooth Muscle Cells

In order to investigate the K+ channels and their effects on resting membrane potential (Em) and excitability in rat bronchial smooth muscle cells (BSMCs), the components of outward K+ channel currents and the effects of K+ channels on Em and tension in rat bronchial smooth muscle were observed by using standard whole-cell recording of patch clamp and isometric tension recording techniques. The results showed that under resting conditions, total outward K+ channel currents in freshly isolated BSMCs were unaffected by ATP-sensitive K+ channel blocker. There were two types of K+ currents: voltage-dependent delayed rectifier K+ channel (Kv) and large conductance calcium-activated K+ channel (BKc.) currents. 1 mmol/L 4-aminopyridine (4-AP, an inhibitor of Kv) caused a significant depolarization (from -8. 7±5. 9 mV to -25. 4±3. 1 mV, n=18, P<0. 001). In contrast, 1 mmol/L tetraethylammonium (TEA, an inhibitor of BKc.) had no significant effect on Em (from -37. 6±4. 8 mV to -36. 8±4.1mV, n=12, P>0. 05). 4-AP caused a concentration-dependent contraction in resting bronchial strips. TEA had no effect on resting tension, but application of 5 mmol/L TEA resulted in a left shift with bigger pD2(the negative logarithm of the drug concentration causing 50% of maximal effect) (from 6. 27±0. 38 to 6. 89±0. 54, n= 10, P<0. 05) in the concentration-effect curve of endothine-1, and a right shift with smaller pD2(from 8. 10±0. 23 to 7. 69±0. 08, n=10, P<0. 05) in the concentration-effect curve of isoprenaline. It was suggested that in rat BSMCs there may be two types of K+ channels, Kv and BKca, which serve distinct roles. Kv participates in the control of resting Em and tension. BKca is involved in the regulation of relaxation or contraction associated with excitation.

Expression of Inositol 1,4,5-trisphosphate Receptor mRNA in Myocardium of Spontaneous Hypertension Rats and Cultured Vascular Smooth Muscle Cells of Rats

Objective\ To investigate expression of inositol 1,4,5 trisphosphate receptor (IP\-3R) mRNA on sacroplasmic reticular in myocardium of spontaneous hypertension rats (SHRs) and cultured vascular smooth muscle cells (VSMC) of rats and effects of perindopril and urapidil on them. Methods\ SHRs were orally given perindopril (1.0 mg·kg\+\{ 1\}·d\+\{ 1\}) or urapidil (15 mg·kg\+\{ 1\}·d\+\{ 1\}) for 24 weeks, respectively. Expression of IP\-3R mRNA was examined by semi quantitative reverse transcription polymers chain reaction (RT PCR) using three oligonuclotide primers for each subtype of IP\-3R with β actin as internal label. Results\ All subtypes of IP\-3R were expressed in myocardium of SHR, WKY and cultured VSMC. Expression of IP\-3R mRNA in left ventricle of SHR was markedly enhanced. Urapidil could down regulate expression of IP\-3R Ⅰand IP\-3R Ⅲ, perindopril slightly increased expression of IP\-3R Ⅱ and decreased expression of IP\-3R Ⅰand IP\-3R Ⅲ in myocardium of SHR. Conclusion\ Our results suggest that expression of IP\-3R mRNA in cardiovascular system could be regulated by urapidil and perindopril.

Effect of inhibiting miR-155 expression on proliferation and migration of airway smooth muscle cell in patients with COPD

Objective:To investigate the effect of inhibiting miR-155 expression on the proliferation and migration of airway smooth muscle cells(ASMCs)in patients with chronic obstructive pulmonary disease(COPD).Methods:ASMCs were isolated and cultured from 8 patients with COPD(observation group)and 3 patients with benign lung cancer without COPD(control group).The ASMCs were transfected with miR-155 suppression expression plasmid(to detect the expression of miR-155;flow cytometry was used to detect the cell cycler of cell clones;Transwell was used to detect cell migration and invasion;Enzyme-linked immunosorbent aanti-miR-155)and the negative contre;clone formation experiment was used to detect the numbol plasmid(anti-miR-NC),and blank control group was set.Real-time quantitative PCR(RT-qPCR)was usedssay(ELISA)method was used to detect tumor necrosis factor-α(TNF-α),interleukin-6(IL-6)level.Results:The expression level of miR-155 in ASMCs of observation group was significantly higher than that in the control group(P<0.05).The miR-155 expression level in inhibited miR-155 expression group was significantly lower,compared with the negative control group and the blank group(P<0.05).In the inhibited miR-155 expression group,the proportion of G0-G1 phase cells was increased,the proportion of S phase cells was decreased,the number of cell clones,migration,and the number of invasive cells were decreased,and the levels of TNF-αand IL-6 were increased(P<0.05).Conclusions:Inhibiting the expression of miR-155 can inhibit the proliferation and migration of airway smooth muscle cells in COPD patients,and inhibit the release of proinflammatory factors.

The effect of Panton Valentin Leukocidin (PVL) on the airway smooth muscle viability

Methicillin-resistant Staphylococcus aureus (MRSA) is recognized as a worldwide pathogen, and the incidence of community-acquired infections (CA-MRSA) is increased. A virulence factor has been found in most CA-MRSA infections, the Panton-Valentin leukocidin (PVL), which causes polymorphonuclear leukocytes lysis and acute uncontrolled inflammation and tissue injury. In this study we investigated the effect of bacterial supernatant of PVL positive or negative strains on airway smooth muscle obtained from rabbit trachea. MRSA that carry the PVL-gene, confirmed by PCR, is cultured on GP agar and colonies were transferred into casein casein yeast extract medium. The culture supernatants were removed after centrifugation and the presence of PVL was confirmed using an immunochromatographic test. Rabbit tracheal ASMC were isolated and incubated with PVL positive or negative bacterial supernatant (1:20 - 1:2000) for 1 - 3 days. The effect of PVL on the ASMC morphology or viability was estimated using microscope observations or indirect immunofluores- cence with anti-Smooth muscle α-actin antibody and Dapi for DNA staining, and Trypan blue staining, respectively. ASMC incubated with PVL exhibit increased cell size, granular cytoplasm, and ruptured nuclei. Furthermore, PVL reduces cell number mainly in ASMC incubated in the presence of 10% FBS, therefore actively proliferating cells. These results show that apart from the known effect of PVL on immune cells and inflammation process, PVL has a direct toxic effect on airway smooth muscle cells.

Effects of advanced glycosylation end products on proliferation and cytosolic free calcium in cultured rat aortic smooth muscle cells

目的：研究糖基化终产物（ＡＧＥＰ）对主动脉平滑肌细胞增殖的影响及其与［Ｃａ２＋］ｉ的关系．方法：采用同位素掺入法分别测定ＤＮＡ和蛋白质合成；Ｆｕｒａ２ＡＭ测定［Ｃａ２＋］ｉ．结果：ＡＧＥＰ以浓度、时间相关的方式促进［３Ｈ］ＴｄＲ与［３Ｈ］Ｌｅｕ掺入细胞，随ＡＧＥＰ作用时间、糖化时间延长，掺入率增加明显．ＡＧＥＰ增加［Ｃａ２＋］ｉ，与时间、浓度相关，但随ＡＧＥＰ作用时间延长（４０分钟后）而有所降低，ＢＳＡ修饰中葡萄糖浓度的增加，糖基化时间延长，［Ｃａ２＋］ｉ也呈上升趋势．结论：ＡＧＥＰ刺激平滑肌细胞增殖，并与细胞［Ｃａ２＋］ｉ浓度增加有关．

Effect of basic fibroblast growth factor on the proliferation, migration and phenotypic modulation of airway smooth muscle cells

Background Proliferation, cell migration and phenotypic modulation of airway smooth muscle cells （ASMCs） are important features of airway remodelling in asthma. The precise cellular and molecular mechanisms that regulate ASMCs proliferation, migration and phenotypic modulation in the lung remain unknown. Basic fibroblast growth factor （bFGF）, a highly specific chemotactic and mitogenic factor for many cell types, appears to be involved in the development of airway remodelling. Our study assessed whether bFGF directly stimulates the proliferation, migration and phenotypic modulation of ASMCs. Methods Confluent and growth arrested human ASMCs were treated with human recombinant FGF. Proliferation was measured by BrdU incorporation and cell counting. Migration was examined using Boyden chamber apparatus. Expressions of smooth muscle （sm）-α-actin and sm-myosin heavy chain （MHC） isoform 1 were determined by RT-PCR and Westem blot analysis. Results It was found that hrbFGF （10 ng/ml）, when added to ASMCs, induced a significant increase in BrdU uptake and cell number by ASMCs as compared to controls and a significant increase in ASMCs migration with respect to controls. The mRNA and protein expressions of sm-α-actin and sm-MHC in ASMCs that were stimulated with hrbFGF decreased with respect to controls. Conclusion It appears that bFGF can directly stimulate proliferation and migration of ASMCs, however, the expressions of cells＇ contractive phenotype decreased.

Cigarette smoke extract promotes proliferation of airway smooth muscle cells in asthmatic rats via regulating cyclin D1 expression

Background Increased proliferation of airway smooth muscle cells （ASMCs） are observed in asthmatic patients and smoking can accelerate proliferation of ASMCs in asthma. To elucidate the molecular mechanisms leading to these changes, we studied in vitro the effect of cigarette smoke extract （CSE） on the proliferation of ASMCs and the expression of cyclin D1, an important regulatory protein implicated in cell cycle. Methods ASMCs cultured from 8 asthmatic Brown Norway rats were studied. Cells between passage 3 and 6 were used in the study and were divided into control group, pcDNA3.1 group, pcDNA3.1-antisense cyclin D1 （ascyclin D1） group, CSE group, CSE＋pcDNA3.1 group and CSE＋pcDNA3.1-ascyclin D1 group based on the conditions for intervention. The proliferation of ASMCs was examined with cell cycle analysis, MTT colorimetric assay and proliferating cell nuclear antigen （PCNA） immunocytochemical staining. The expression of cyclin D1 was detected by reverse transcriptase-PCR （RT-PCR） and Western blotting. Results （1） The percentage of S＋G2M phase, absorbance value at 490 nm wavelength （A490） and the expression rate of PCNA protein in CSE group were （31.22±1.17）%, 0.782±0.221, （90.2±7.0）% respectively, which were significantly increased compared with those of control group （18.36±1.02）%, 0.521±0.109, and （54.1±3.5）%, respectively） （P 〈0.01）. After the transfection with antisense cyclin D1 plasmid for 30 hours, the percentage of S＋G2M phase, A490 and the expression rate of PCNA protein in ASMCs were much lower than in untreated cells （P 〈0.01）. （2） The ratios of A490 of cyclin D1 mRNA in CSE group was 0.288±0.034, which was significantly increased compared with that of control group （0.158±0.006） （P 〈0.01）. After the transfection with antisense cyclin D1 plasmid for 30 hours, the ratios of A49o of cyclin D1 mRNA in ASMCs was much lower than in untreated cells （P 〈0.01）. （3） The ratios of A490 of cyclin D1 protein expression in CSE group was 0.375±0.008, which was significantly increased compared with that of control group （0.268±0.004） （P 〈0.01）. After the transfection with antisense cyclin D1 plasmid for 30 hours, the ratios of A490 of cyclin D1 protein expression in ASMCs was much lower than in untreated cells （P 〈0.01）. Conclusion CSE may increase the proliferation of ASMCs in asthmatic rats via regulating cyclin D1 expression.

Contribution of protein kinase C to passively sensitized human airway smooth muscle cells proliferation

Background Airway smooth muscle proliferation plays an important role in airway remodeling in asthma. But little is known about the intracellular signal pathway in the airway smooth muscle cell proliferation in asthma. The objective of this paper is to investigate the contribution of protein kinase C (PKC) and its alpha isoform to passively sensitized human airway smooth muscle cells (HASMCs) proliferation. Methods HASMCs in culture were passively sensitized with 10% serum from asthmatic patients,with non-asthmatic human serum treated HASMCs used as the control. The proliferation of HASMCs was examined by cell cycle analysis,3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazoliumbromide (MTT) colorimetric assay and proliferating cell nuclear antigen (PCNA) immunofluorescence staining. The effect of PKC agonist phorbol 12-myristate 13-acetate (PMA) and PKC inhibitor Ro-31-8220 on the proliferation of HASMCs exposed to human asthmatic serum and non-asthmatic control serum was also examined by the same methods. The protein and mRNA expression of PKC-α in passively sensitized HASMCs were detected by immunofluorescence staining and reverse transcription-polymerase chain reaction. Results The percentage of S phase,absorbance (value A) and the positive percentage of PCNA protein expression in HASMCs passively sensitized with asthmatic serum were (16.30±2.68)%,0.430±0.060 and (63.4±7.4)% respectively,which were significantly increased compared with HASMCs treated with control serum [(10.01±1.38)%,0.328±0.034 and (37.2±4.8)%,respectively] ( P <0.05). After HASMCs were passively sensitized with asthmatic serum,they were treated with PMA,the percentage of S phase,value A and the positive percentage of PCNA protein expression were (20.33±3.39)%,0.542±0.065 and (76.0±8.7)% respectively,which were significantly increased compared with asthmatic serum sensitized HASMCs without PMA( P <0.05). After HASMCs passively sensitized with asthmatic serum were treated with Ro-31-8220,the percentage of S phase,value A and the positive percentage of PCNA protein expression were (11.21±1.56)%,0.331±0.047 and (38.8±6.0)% respectively,which were significantly decreased compared with asthmatic serum sensitized HASMCs without Ro-31-8220 ( P <0.05). The relative ratio of value A of PKC-α mRNA and the positive percentage of PKC-α protein expression in passively sensitized HASMCs were 1.23±0.10 and (61.1±9.4)% respectively, which were significantly increased compared with HASMCs treated with control serum [1.05±0.09 and (34.9±6.7)%,respectively] ( P <0.05). Conclusions The proliferation of HASMCs passively sensitized with human asthmatic serum is increased. PKC and its alpha isoform may contribute to this proliferation.

Selective blockade of NF-kappa B by novel mutated I kappa B alpha suppresses CD3/CD28-induced activation of memory CD4(+) T cells in asthma

Background:Nuclear factor kappa B(NF-kappa B) overactivation plays a crucial role in T-helper 2 (Th2)-biased allergic airway inflammation by increased activation and decreased apoptosis of CD4(+) T cells. We have shown that targeted NF-kappa B suppression in dendritic cells by adenoviral gene transfer of a novel mutated inhibitor of NF-kappa B(I kappa B alpha) (AdI kappa B alpha M) contributes to T-cell tolerance, but the immunosuppressive action of AdI kappa B alpha M on memory(CD45RO(+)) CD4(+) T cells remains enigmatic. Methods:CD45RO(+) T cells from Dermatophagoides farinaei-sensitized asthmatic patients, untransfected or transfected with AdI kappa B alpha M or AdLacZ(beta-galactosidase) for 24 h, were stimulated with anti-CD3(1.0 mu g/ml) plus anti-CD28(0.5 mu g/ml) monoclonal antibody for an additional 24 h. I kappa B alpha M transgene expression and NF-kappa B activation were detected by polymerase chain reaction(PCR), reverse transcription-PCR(RT-PCR), Western blot analysis, and electrophoretic mobility shift assay. Phenotype and apoptosis were measured by flow cytometry, annexin V binding, and terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling analyses. Cytokine production and cell proliferation were determined using enzyme-linked immunosorbent assay andH-3 thymidine incorporation. Results:A unique 801-bp I kappa B alpha M cDNA and a dose-dependent increase in I kappa B alpha M transgene expression were observed in AdI kappa B alpha M-transfected CD45RO(+) T cells. Significantly, AdI kappa B alpha M inhibited CD3/CD28-mediated NF-kappa B activation in CD45RO(+) T cells, leading to evident apoptosis, reduction of eotaxin, RANTES, Th1 interferon (IFN)-gamma and interleukin (IL)-2, and Th2 (IL-4, IL-5, and IL-13 despite a slight decrease in IL-10) cytokines and secondary proliferative response. AdI kappa B alpha M also upregulated cytotoxic T lymphocyte-associated antigen 4(CTLA-4) and downregulated CD69 besides no change in CD28. Conclusion:I kappa B alpha M might be beneficial to augment memory CD4(+) T-cell tolerance through modulating B7-CD28/CTLA-4 co-stimulatory pathways and NF-kappa B-dependent cytokine profiles in allergic inflammatory diseases including asthma.