Post-transcriptional silencing of TRPC1 ion channel gene by RNA interference upregulates TRPC6 expression and store-operated Ca entry in A7r5 vascular smooth muscle cells

This study investigates functional consequences of TRPC1 ion channel downregulation observed in aging rat aorta by employing RNA interference in cultured vascular smooth muscle cells. For this purpose, A7r5 aortic smooth muscle cells were used in quantitative gene and protein expression as well as in functional analyses. According to quantitative RT-PCR results, TRPC3, TRPC4 and TRPC5 mRNAs were not at detectable levels. In siTRPC1-transfected cells, TRPC1 mRNA and protein levels were decreased by 40% and 64%; however, those of TRPC6 were drastically increased by 100% and 200%, respectively. In fura-2-loaded TRPC1 knockdown cells, despite the decreased TRPC1 levels, cyclopiazonic acid-induced Ca²⁺ entry and store-operated Ca²⁺ entry following Ca²⁺ addition were elevated by 77% and 135%, respectively. Results suggest that decrease in TRPC1 may be compensated by upregulated TRPC6 that possibly takes part in store-operated Ca²⁺ entry in vascular smooth muscle cells.     

Store-operated calcium entry in vascular smooth muscle

In non-excitable cells, activation of G-protein-coupled phospholipase C (PLC)-linked receptors causes the release of Ca(2+) from intracellular stores, which is followed by transmembrane Ca(2+) entry. This Ca(2+) entry underlies a small and sustained phase of the cellular [Ca(2+)](i) increases and is important for several cellular functions including gene expression, secretion and cell proliferation. This form of transmembrane Ca(2+) entry is supported by agonist-activated Ca(2+)-permeable ion channels that are activated by store depletion and is referred to as store-operated Ca(2+) entry (SOCE) and represents a major pathway for agonist-induced Ca(2+) entry. In excitable cells such as smooth muscle cells, Ca(2+) entry mechanisms responsible for sustained cellular activation are normally considered to be mediated via either voltage-operated or receptor-operated Ca(2+) channels. Although SOCE occurs following agonist activation of smooth muscle, this was thought to be more important in replenishing Ca(2+) stores rather than acting as a source of activator Ca(2+) for the contractile process. This review summarizes our current knowledge of SOCE as a regulator of vascular smooth muscle tone and discusses its possible role in the cardiovascular function and disease. We propose a possible hypothesis for its activation and suggest that SOCE may represent a novel target for pharmacological therapeutic intervention.     

高血压患者动脉平滑肌细胞培养的探讨

目的对高血压患者动脉平滑肌细胞培养方法进行探讨。方法运用植块贴壁法对高血压患者动脉平滑肌细胞进行体外培养,并用倒置显微镜进行形态学观察和用免疫组化对培养细胞进行鉴定。结果运用植块贴壁法成功培养了高血压患者动脉平滑肌细胞,培养的细胞呈典型“峰谷”样生长,免疫组化S-P法检测α-平滑肌肌动蛋白单克隆抗体(α-SMActin)表达,确认为人血管平滑肌细胞(hVSMC)。经传代培养至3～5代,细胞生长特性未见异常改变。结论植块贴壁法培养的高血压患者动脉平滑肌细胞生长稳定性好,且培养与纯化能同步进行。     

依泽替米贝通过小凹蛋白1调节大鼠血管平滑肌细胞内脂质蓄积

目的:观察依泽替米贝(ezeti mibe)对大鼠血管平滑肌细胞内胆固醇蓄积的影响以及相关的作用机制。方法:以原代培养大鼠血管平滑肌细胞(rat VSMCs)为研究对象,以20 mg/L胆固醇:甲基β环糊精复合物(Chol:MβCD)孵育细胞48 h形成荷脂细胞模型。不同浓度的依泽替米贝(3、10、30μmol/L)处理细胞24 h,或以30μmol/L依泽替米贝分别处理细胞不同时间(0、6、12、24、48 h),HPLC检测细胞内TC、游离胆固醇(FC)的含量,Western blotting检测小凹蛋白1(caveolin-1)蛋白的表达。结果:不同浓度依泽替米贝(3、10、30μmol/L)作用于VSMCs源性荷脂细胞不同时间,细胞内TC、FC的含量呈浓度依赖性减少,以30μmol/L浓度孵育24 h作用最强。Chol:MβCD明显减少细胞caveolin-1蛋白表达水平,依泽替米贝能够逆转这种作用。结论:依泽替米贝抑制Chol:MβCD诱导的大鼠平滑肌细胞中胆固醇蓄积作用可能与caveolin-1有关。     

HMGB1 increases RAGE expression in vascular smooth muscle cells via ERK and p-38 MAPK-dependent pathways

The increased expression of receptors for advanced glycation end-product (RAGE) is known as a key player in the progression of vascular remodeling. However, the precise signal pathways regulating RAGE expression in vascular smooth muscle cells (VSMCs) in the injured vasculatures are unclear. Given the importance of mitogen-activated protein kinase (MAPK) signaling in cell proliferation, we investigated the importance of MAPK signaling in high-mobility group box 1 (HMGB1)-induced RAGE expression in VSMCs. In HMGB1 (100 ng/ml)-stimulated human VSMCs, the expression of RAGE mRNA and protein was increased in association with an increase in AGE-induced VSMC proliferation. The HMGB1-induced RAGE expression was attenuated in cells pretreated with inhibitors for ERK (PD98059, 10 μM) and p38 MAPK (SB203580, 10 μM) as well as in cells deficient in ERK and p38 MAPK using siRNAs, but not in cells deficient of JNK signaling. In cells stimulated with HMGB1, the phosphorylation of ERK, JNK, and p38 MAPK was increased. This increase in ERK and p38 MAPK phosphorylation was inhibited by p38 MAPK and ERK inhibitors, respectively, but not by JNK inhibitor. Moreover, AGE-induced VSMC proliferation in HMGB1-stimulated cells was attenuated in cells treated with ERK and p38 MAPK inhibitors. Taken together, our results indicate that ERK and p38 MAPK signaling are involved in RAGE expression in HMGB1-stimulated VSMCs. Thus, the ERK/p38 MAPK-RAGE signaling axis in VSMCs was suggested as a potential therapeutic target for vascular remodeling in the injured vasculatures.     

虎杖苷对VSMC内钙信号的调节机制初探

目的　观察虎杖苷对大鼠主动脉平滑肌细胞(VSMC)内游离钙浓度的变化 ,以探讨虎杖苷对血管平滑肌细胞的调节机制。方法　用荧光染料Fluo 3 AM标记细胞 ,在粘附式细胞仪上测定细胞内游离钙的变化。结果　给予虎杖苷 5min后 ,VSMC内游离钙浓度升高。当虎杖苷加入前 10min用河豚毒素和优降糖分别预处理后 ,其VSMC内游离钙都不再明显升高 ;而给予普萘洛尔、甲氰咪胍及亚甲蓝分别预处理时 ,细胞内钙浓度反而升得更高 ,与单纯虎杖苷作用相比差异都有显著性。结论　虎杖苷可增加细胞内游离钙浓度 ,其作用可能与钠、钾通道开放有关 ,并且受 β肾上腺素能受体、H2 受体系统及鸟苷酸环化酶系统的反向调节。     

p38 MAPK activation is required for esculetin-induced inhibition of vascular smooth muscle cells proliferation

The phenolic compound esculetin is known to inhibit the proliferation of vascular smooth muscle cells (VSMC). However, the signaling pathway by which esculetin mediates its molecular effects in VSMC remains to be identified. The present results suggest an unexpected role of the p38 MAPK signaling pathway in esculetin-induced inhibition of VSMC growth. Treatment of VSMC with esculetin resulted in significant growth inhibition and G1-phase cell-cycle arrest, which was followed by down-regulation of cyclins and cyclin-dependent kinase (CDK) expression. This G1-phase cell-cycle arrest was due to up-regulation of p21WAF1 expression. In addition, esculetin treatment activated p38 MAPK and ERK1/2. Pretreatment with SB203580, which is a p38 MAPK specific inhibitor, or expression of the dominant negative p38 MAPK (DN p38 MAPK) gene blocked esculetin-induced p38 MAPK activation and p21WAF1 expression. Finally, both the growth inhibition and the down-regulation of CDKs induced by esculetin were suppressed by either SB203580 or the DN p38 MAPK mutant gene. In conclusion, these results demonstrate that activation of p38 MAPK contributes to esculetin-induced p21WAF1 expression in VSMC by decreasing both the cyclin D1/CDK4 and cyclin E/CDK2 complexes. These novel results regarding the molecular mechanism of esculetin action suggest new preventive and therapeutic treatments for atherosclerosis.     

血管内皮生长因子受体-3在血管内皮细胞和平滑肌细胞中的表达

目的探讨血管内皮细胞生长因子受体-3（VEGFR-3）在血管内皮细胞和平滑肌细胞中的表达。方法分离人脐带中的脐静脉和脐动脉,冰冻切片后以免疫荧光法检测VEGFR-3的表达。结果VEGFR-3荧光信号表达于脐静脉内皮细胞和平滑肌细胞,以及脐动脉内皮细胞、平滑肌细胞和外周细胞。结论血管内皮细胞和平滑肌细胞均表达VEGFR-3。     

缬沙坦对球囊损伤后血管平滑肌细胞增生的影响

目的 探讨缬沙坦因子防治人类血管成形术后再狭窄的可行性方法观察新的非肽类血管紧张素Ⅱ的Ⅰ型(AT_1)受体拮抗剂,缬沙坦(Valsartan),对血管平滑肌细胞(VSMC)增生的影响。对大鼠髂动脉球囊内皮剥脱术后过度增生模型采用~3H-TdR和~3H-Leu掺入,免疫组化染色检测VSMC中增殖细胞核抗原(PCNA)的表达及图象分析血管壁形态学变化。结果 缬沙坦显著减少~3H-TdR和~3H-Leu的掺入量,减少新生内膜面积和PCNA阳性细胞数。结论 缬沙坦能抑制大鼠动脉的球褒内皮损伤后的VSMC增生,可能它是防治血管成形术再狭窄的有效药物。     

半胱氨酸对人血管平滑肌细胞增殖和凋亡的影响

目的 观察半胱氨酸(Cys)对人血管平滑肌细胞(VSMCs)增殖和凋亡的影响.方法 采用组织贴块法体外培养人VSMCs,用不同浓度的Cys培养液孵育细胞24h,用MTT法检测VSMCs增殖,流式细胞术检测VSMCs凋亡.RT-PCR法检测bax mRNA的表达.结果 体外培养的人VSMCs在终浓度为100、200、500和10001μmol/L Cys的培养液中孵育24h后的吸光度(A)值均高于对照组(P<0.05),表明Cys可诱导人VSMCs增殖;但同时.VSMCs凋亡细胞数明显高于对照组(P<0.05),bax的表达增强,表明Cys亦诱导了人VSMCs凋亡.结论 浓度较高时半胱氨酸可同时诱导VSMCs增殖和凋亡.     

Naringin-induced p21WAF1-mediated G1-phase cell cycle arrest via activation of the Ras/Raf/ERK signaling pathway in vascular smooth muscle cells

The flavonoid naringin has been shown to play a role in preventing the development of cardiovascular disease. However, the exact molecular mechanisms underlying the roles of integrated cell cycle regulation and MAPK signaling pathways in the regulation of naringin-induced inhibition of cell proliferation in vascular smooth muscle cells (VSMCs) remain to be identified. Naringin treatment resulted in significant growth inhibition and G₁-phase cell cycle arrest mediated by induction of p53-independent p21WAF1 expression; expression of cyclins and CDKs in VSMCs was also down-regulated. In addition, among the pathways examined, blockade of ERK function inhibited naringin-dependent p21WAF1 expression, reversed naringin-mediated inhibition of cell proliferation and decreased cell cycle proteins. Moreover, naringin treatment increased both Ras and Raf activations. Transfection of cells with dominant negative Ras (RasN17) and Raf (RafS621A) mutant genes suppressed naringin-induced ERK activity and p21WAF1 expression. Finally, naringin-induced reduction in cell proliferation and cell cycle protein was abolished in the presence of RasN17 and RafS621A mutant genes. The Ras/Raf/ERK pathway participates in p21WAF1 induction, leading to a decrease in cyclin D1/CDK4 and cyclin E/CDK2 complexes and in naringin-dependent inhibition of cell growth. These novel and unexpected findings provide a theoretical basis for preventive use of flavonoids to the atherosclerosis disease.     

自发性高血压大鼠左室心肌壁内小动脉平滑肌瞬时受体电位通道的表达

目的:探索自发性高血压大鼠(Spontaneously hypertensive rats,SHR)心肌壁内小动脉重构与瞬时受体电位通道(Transient receptor potential channel,TRPC)蛋白表达的相关性.方法:24周龄雄性SHR(n＝8)为实验组,设同周龄同性别正常血压大鼠为对照组(WKY,n＝8).计算大鼠左室质量指数(Left ventficular mass index,LVMI)评价左室肥厚情况.采用HE染色观察管腔直径为10～69 μm的左室心肌壁内小动脉结构特点并测定管腔面积(Lumen area,LA)、管壁面积(Wall area,WA)、管壁面积/管腔面积(WA/LA).a-平滑肌肌动蛋白(Alphasmooth muscle actin,α-SMA)免疫荧光染色鉴定血管平滑肌细胞(Vascular smooth muscle cell,VSMC).免疫荧光染色检测平滑肌细胞TR-PC1、TRPC3、TRPC5、TRPC6蛋白表达强度.结果:SHR组SBP及LVMI均明显高于WKY组[(200±4) vs (118±9)mm Hg,1 mm Hg=0.133 kPa;(3.11±0.03) vs (2.42±0.10) mg/g,均P＜0.05];SHR组WA及WA/LA均比WKY组增加[(2951±224) vs (2654±190) μm2;(2.01±0.04) vs(1.53±0.03),均P＜0.05],而LA比WKY组减少[(1469±117) vs(1730±98) μm2,P＜0.05],α-SMA免疫荧光染色阳性表明增厚的血管中层为平滑肌细胞.SHR组心肌壁内小动脉平滑肌细胞TRPC1、TRPC3蛋白表达均明显高于WKY组(分别为65±8 vs 28±3和60±4vs 26±8,均P＜0.05),TRPC5蛋白表达与WKY组无统计学差异(28±7 vs 28±7,P＞0.05);SHR组与WKY组均未表达TRPC6.结论:24周SHR左室心肌壁内小动脉平滑肌表达TRPC1、TRPC3、TRPC5而未表达TRPC6.24周SHR左室心肌壁内小动脉重构与平滑肌TRPC1、TRPC3表达上调相关.     

Arsenic alters vascular smooth muscle cell focal adhesion complexes leading to activation of FAK-src mediated pathways

Chronic exposure to arsenic has been linked to tumorigenesis, cardiovascular disease, hypertension, atherosclerosis, and peripheral vascular disease; however, the molecular mechanisms underlying its pathological effects remain elusive. In this study, we investigated arsenic-induced alteration of focal adhesion protein complexes in normal, primary vascular smooth muscle cells. We demonstrate that exposure to environmentally relevant concentrations of arsenic (50 ppb As³⁺) can alter focal adhesion protein co-association leading to activation of downstream pathways. Co-associated proteins were identified and quantitated via co-immunoprecipitation, SDS-PAGE, and Western blot analysis followed by scanning densitometry. Activation of MAPK pathways in total cell lysates was evaluated using phosphor-specific antibodies. In our model, arsenic treatment caused a sustained increase in FAK-src association and activation, and induced the formation of unique signaling complexes (beginning after 3-hour As³⁺ exposure and continuing throughout the 12-hour time course studied). The effects of these alterations were manifested as chronic stimulation of downstream PAK, ERK and JNK pathways. Past studies have demonstrated that these pathways are involved in cellular survival, growth, proliferation, and migration in VSMCs.     

Preconditioning with low concentration NO attenuates subsequent NO-induced apoptosis in vascular smooth muscle cells via HO-1-dependent mitochondrial death pathway

Nitric oxide (NO) signaling pathways are important in both the maintenance of vascular homeostasis and disease progression. Overproduction of NO has been associated with ischemia/reperfusion (I/R) injury. Growing evidences suggest that NO preconditioning has cytoprotective effects against I/R injury. However, the mechanism with which NO mediates these effects remains to be elucidated. The purpose of this study was to examine the mechanism of how NO preconditioning inhibits subsequent NO-induced apoptosis in vascular smooth muscle cells (VSMC), specifically focusing on heme oxygenase-1 (HO-1). According to our data, sodium nitroprusside (SNP) increased HO-1 expression in a concentration dependent manner. Preconditioning with low concentration SNP (0.3mM) inhibited subsequent high concentration SNP (1.5mM)-induced apoptosis, and this effect was reversed by the HO-1 inhibitor SnPP. Low concentration SNP-mediated protection involved p38 kinase inactivation and increased Bcl-2 expression. Furthermore, mitochondrial membrane potential was concomitantly increased with decreased expressions of Bax, Apaf-1, and activity of caspase-3, which was reversed by SnPP treatment. Our results show that low concentration SNP preconditioning suppresses subsequent high concentration SNP-induced apoptosis by inhibiting p38 kinase and mitochondrial death pathway via HO-1-dependent mechanisms in VSMC.     

肝素十二糖对血管平滑肌细胞增殖作用的研究

目的:研究肝素十二糖(dp12)对血管平滑肌细胞(vascular smooth muscle cells,VSMCs)增殖作用的影响,并分析其分子水平的作用机制。方法:通过以10%新生牛血清诱导牛胸主动脉血管平滑肌细胞增殖建立模型,然后考察实验室精制的不同浓度的肝素寡糖对血管平滑肌细胞增殖作用的影响;四甲基偶氮唑盐法(MTT)检测dp12对VSMCs增殖的影响;流式细胞仪检测细胞周期分布;RT-PCR法检测ERK1/2转录变化情况;Western Blotting法检测ERK1/2和p-ERK1/2的表达。结果:MTT结果显示不同浓度的dp12可以明显抑制由10%新生牛血清诱导的血管平滑肌细胞的增殖;细胞周期实验揭示dp12抑制血清诱导的VSMCs从G1期进入S期,影响细胞周期;dp12通过下调ERK基因的转录进而下调ERK1/2的表达,此外dp12抑制ERK1/2的磷酸化进而影响细胞增殖。结论:dp12使VSMCs在G1期/S期阻滞,通过抑制ERK基因的转录和ERK蛋白的磷酸化抑制VSMCs增殖,可能是dp12抗VSMCs增殖的机制之一。     

Docetaxel inhibits cyclooxygenase-2 induction in vascular smooth muscle cells

The aim of this study was to determine whether docetaxel affects expression of cyclooxygenase-2 (COX-2) in vascular smooth muscle cells. Cultured rat aortic smooth muscle cells (RASMCs) were stimulated with interleukin-1b (IL-1β). COX-2 expression level and ERK activity were evaluated by Western blot analysis. COX-2 expression as well as tubulin formation was also evaluated by immunocytochemical analysis. IL-1β induced COX-2 expression in RASMCs, which was inhibited by docetaxel (5–20 µg/ml) in a concentration-dependent manner. IL-1β increased ERK activity, which was not affected by docetaxel. IL-1β-induced COX-2 expression level was markedly augmented at 24 h after washing out docetaxel from the culture medium. Immunocytochemical analysis revealed that COX-2 immunoreactivity in RASMCs stimulated with IL-1β was decreased in the presence of docetaxel but was recovered at 24 h after washing out docetaxel, while docetaxel-induced change in tubulin formation, namely, polymerization of α-tubulin fibers, remained at 24 h after washing out docetaxel. The results suggest that docetaxel inhibits COX-2 induction, and this action of docetaxel is reversible and ERK-independent.     

Calcium and the replication of human vascular smooth muscle cells: studies on the activation and translocation of extracellular signal regulated kinase (ERK) and cyclin D1 expression

Since the precise role of sarcoplasmic reticular Ca2+ in mediating vascular smooth muscle cells (VSMC) proliferation is unknown, the effect of pre-incubation with thapsigargin on extracellular signal regulated kinase (ERK) activation, the translocation of activated of ERK 1/2 to the nucleus, cyclin D1 expression, the onset of S phase and cytosolic Ca2+ levels were studied. Human saphenous vein VSMCs (hVSMC) were incubated with 10 nM thapsigargin for 24 h followed by stimulation with fetal calf serum and the activation of ERK1/2 and cyclin D1 assessed by western blotting, the intracellular distribution of ERK1/2 using indirect immunofluorescence, the onset of S-phase with the incorporation of bromodeoxyuridine and sarcoplasmic reticular Ca2+ status using FURA-2. Thapsigargin had a marginal effect on ERK1/2 activation only at 5 min and 10 min after stimulation with fetal calf serum. In contrast, the rapid translocation of ERK1/2 to the nucleus was completely blocked by thapsigargin. S phase was delayed by 8 h by thapsigargin which co-incided with the recovery of cytosolic Ca2+ levels and cyclin D1 expression. It is concluded that the inhibitory effect of thapsigargin (depletion of Ca2+ pools) on hVSMC replication is mediated through the inhibition of translocation of activated ERK1/2 to the nucleus and not to the phosphorylation of ERK, per se, which in turn prevents cyclin D1 expression and thus progression of the cell cycle.     

Stim, ORAI and TRPC channels in the control of calcium entry signals in smooth muscle

Ca2+ entry signals are crucial in the control of smooth muscle contraction. Smooth muscle cells are unusual in containing plasma membrane (PM) Ca2+ entry channels that respond to voltage changes, receptor activation and Ca2+ store depletion. Activation of these channel subtypes is highly coordinated. The TRPC6 channel, widely expressed in most smooth muscle cell types, is largely non-selective to cations and is activated by diacylglycerol arising from receptor-induced phosholipase C activation. Receptor activation results largely in Na+ ion movement through TRPC6 channels, depolarization and subsequent activation of voltage-dependent L-type Ca2+ channels. The TRPC6 channels also appear to be activated by mechanical stretch, resulting again in depolarization and L-type Ca2+ channel activation. Such a coupling may be crucial in mediating the myogenic tone response in vascular smooth muscle. The emptying of stores mediated by inositol 1,4,5-trisphosphate receptors triggers the endoplasmic reticulum (ER) Ca2+ sensing protein stromal-interacting molecule (STIM) 1 to translocate into defined ER-PM junctional areas in which coupling occurs to Orai proteins, which serve as highly Ca2+-selective low-conductance Ca2+ entry channels. These ER-PM junctional domains may serve as crucial sites of interaction and integration between the function of store-operated, receptor-operated and voltage-operated Ca2+ channels. The STIM, Orai and TRPC channels represent highly promising new pharmacological targets through which such control may be induced.