过氧化氢对血管平滑肌细胞的作用

目的　研究过氧化氢 (H2 O2 )对兔血管平滑肌细胞的作用机制。方法　兔血管平滑肌细胞 (VSMCs)原代培养 ,微量细胞培养四甲基偶氮唑蓝法 (MTT)检测不同浓度H2 O2 在不同时段对其增殖影响 ,确定进一步研究的H2 O2 浓度 ,并应用流式细胞仪检测此浓度H2 O2 对VSMCs细胞周期和细胞凋亡的作用。结果　MTT法检测 ,H2 O2 浓度≥ 10 0 μmol/L(10 0～ 14 0 0 μmol/L)时 ,与VSMCs作用仅 1h ,即出现吸光度值显著降低 (P <0 .0 1) ;流式细胞仪细胞周期检测 ,H2 O2 浓度≥ 10 0 μmol/L时 ,与VSMCs作用 2 4h后 ,其G1期细胞数显著高于对照组 (94.65 %和 79.97% ) (P <0 .0 1) ,以H2 O2浓度在 10 0 μmol/L时尤为明显 ;流式细胞仪AnnexinV PI双标细胞凋亡检测 ,10 0 μmol/LH2 O2 有促进VSMCs凋亡作用 ,并在作用 48h左右达到高峰 ,占 2 6.76% ,与对照组相比 ,差异有显著性 (P <0 .0 1)。结论　H2 O2 浓度≥ 10 0 μmol/L时 ,可显著抑制VSMCs增殖 ;当H2 O2 浓度为 10 0 μmol/L时 ,有促进VSMCs凋亡作用。     

VEGF inhibits PDGF-stimulated calcium signaling independent of phospholipase C and protein kinase C

INTRODUCTION: Despite advances in both open and endovascular techniques for treatment of arterial occlusive disease, restenosis because of neointimal hyperplasia continues to be a major cause of graft failure and restenosis. This phenomenon has been attributed to vascular smooth muscle cell (VSMC) activation by several potent mitogens including platelet derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) released at the site of injury. PDGF is known to stimulate calcium influx in VSMC that has been shown to be critical for VSMC migration and proliferation. We have previously shown that VEGF inhibits PDGF-stimulated VSMC proliferation. The objective of this set of experiments was to investigate whether VEGF modulated PDGF-stimulated Ca2+ influx in VSMC. MATERIALS AND METHODS: Primary cultured human aortic SMC were grown to subconfluency and assigned to the following groups: no stimulation, stimulation with PDGF-BB (20 ng/ml), stimulation with VEGF165 (40 ng/ml), or a combination of PDGF-BB + VEGF165. Ca2+ influx was measured using a Fura-2 fluorescence assay. The intracellular Ca2+ fraction was assayed with the Fura-2 assay by using Ca2+-free media. Phospholipase Cgamma1 (PLCgamma1), protein kinase C (PKC), and Akt phosphorylation was assessed with standard immunoblotting techniques at 1, 5, and 10 min time points. Ca2+-calmodulin kinase II (CaMKII) activity was extrapolated from the phosphorylation of Phospholamban B (PLB), a well-known protein substrate, at 1, 5, and 10 min time points. RESULTS: PDGF stimulation resulted in a 328 +/- 9 nm total calcium influx in VSMC. The combination of VEGF + PDGF resulted in a 273 +/- 21 nm total calcium influx, an amount significantly less than with PDGF alone (P < 0.04). PDGF stimulation resulted in a 72 +/- 35 nm intracellular calcium release. The addition of VEGF to PDGF resulted in an intracellular calcium release of only 15 +/- 11 nm, a significant decrease compared to PDGF alone (P < 0.01). The phosphorylation of PLCgamma1, PKC, and Akt was equivalent at 1, 5, and 10 min between the PDGF and the PDGF + VEGF treatment groups. There was an increase in CaMKII activity at 1 and 5 min time points in both the PDGF and PDGF + VEGF treatment groups suggesting that extracellular calcium influx is sufficient for CaMKII activation. CONCLUSION: VEGF inhibits PDGF-stimulated total calcium influx and, in particular, PDGF-stimulated intracellular calcium release in VSMC. The equivalent phosphorylation of PLCgamma1, PKC, and Akt suggests that the inhibitory mechanism by VEGF on calcium influx occurs downstream of these proximal mediators. The inhibition of intracellular calcium release did not inhibit CaMKII activity. VEGF may play an important role in modulating PDGF induced VSMC proliferation by specifically inhibiting intracellular calcium release in response to PDGF.     

人脂肪干细胞向血管平滑肌细胞诱导分化的实验研究

目的：体外诱导培养人脂肪干细胞（adi pose-der i ved st em cel l s,ADSCs）,观察是否可以分化形成血管平滑肌细胞（vascul ar smoot h muscl e cel l s,VSMCs）,为构建组织工程化血管寻找新的种子细胞来源。方法：免疫磁珠法分选收集原代脂肪干细胞,加入PDGF-BB、TGF-β1诱导14天后进行检测。结果：实验组细胞生长呈现血管平滑肌细胞所特有的峰谷样生长,血管平滑肌细胞特有的表面抗原标记物表达阳性。结论：脂肪干细胞定向诱导培养后,具有血管平滑肌细胞的特性,有可能成为构建组织工程化血管新的种子细胞来源。     

血管平滑肌细胞中睾酮膜受体的研究

目的:观察血管平滑肌细胞(VSMCs)胞膜有无睾酮(T)特异性结合位点(膜受体),并对其与经典的细胞内雄激素受体(iAR)的关系作初步探讨。方法:贴块法培养雄性SD大鼠胸主动脉VSMCs,给以异硫氰酸荧光素(FITC)标记的牛血清白蛋白-睾酮复合物(T-BSA-FITC)作用,流式细胞仪检测VSMCs荧光标记情况,以及iAR拮抗剂氟他胺对T-BSA-FITC与VSMCs结合的抑制作用;用免疫荧光法标记胞膜完整和TritonX-100处理后胞膜透化VSMCs表达的iAR,流式细胞术检测细胞平均荧光强度,进一步转换为相对荧光强度进行统计学分析。结果:与0作用时间点相比,T-BSA-FITC与VSMCs作用10s,细胞相对荧光强度即显著增加(P<0.01);VSMCs与T-BSA-FITC作用10min,细胞相对荧光强度显著高于对应摩尔浓度的BSA-FITC、BSA-FITC+BSA、BSA-FITC+BSA+T对照(P值分别为0.357,0.832和0.823)。与单纯T-BSA-FITC作用相比,预先给以iAR拮抗剂氟他胺对VSMCs荧光强度没有显著影响(P=0.318)。胞膜完整VSMCs与iAR抗体作用后,细胞相对荧光强度较单纯加入FITC标记二抗无明显增加(P=0.733);而胞膜透化VSMCs与iAR抗体作用后,细胞荧光强度较单纯FITC标记二抗处理明显增加(P=0.024)。结论:VSMCs中存在睾酮膜受体,其性质不同于经典的iAR。     

兔血管平滑肌细胞和聚羟基丁酯(PHB)的细胞相容性实验研究

目的：探索血管平滑肌细胞和新型可降解材料聚羟基丁酯（PHB）的细胞相容性，为组织工程血管的构建寻找理想的支架材料。方法：将组织块法体上培养与兔血管平滑肌细胞种植在PHB膜片和PHB三维微孔支架上，在相差显微镜下观察细胞的粘附和生长情况。用MTT法测定细胞粘附率和细胞增殖指数，复合培养7天后进行扫描电镜观察并用流式细胞仪（FCM）的测定细胞周期，DNA指数。结果：兔血管平滑肌细胞在PHB膜片上粘附率为77％，细胞增殖符合细胞的生长曲线，在PHB三维微孔支架上生长情况良好，并被证实为二倍体细胞。结论：兔血管平滑肌细胞和聚羟基丁酯（PHB）的细胞相容性较好，但细胞与材料间的粘附有待进一步改善。     

改良组织块法成人主动脉血管平滑肌细胞的培养

目的 建立成人主动脉血管平滑肌细胞体外培养的有效方法.方法 无菌条件下分离成人主动脉的平滑肌层,剪成1 mm3的碎片,0.1％ Ⅰ型胶原酶预处理1h,组织块贴壁法,以含胎牛血清20％的DMEM低糖培养基培养.倒置显微镜观察培养细胞的形态学特点,免疫荧光法检测平滑肌细胞肌动蛋白(α-SMA)的表达.结果 培养至4～5d组织块边缘即有少量细胞爬出,呈长梭形,胞质丰富;培养至1～2周组织块边缘细胞融合,呈典型的“峰谷”样表现.免疫荧光染色结果显示胞质内α-SMA的表达丰富,荧光强度在(++)～ (+++).结论 胶原酶预处理组织的改良组织块贴壁法培养人主动脉血管平滑肌细胞是一种可行有效的实验方法.     

诱导骨髓基质细胞成血管平滑肌细胞的研究

目的 探讨体外诱导犬骨髓基质细胞(SMSC)为血管平滑肌细胞(VSMC)及其成血管的能力.方法 用含血小板源性生长因子(PDGF-BB)10ìg/L的内皮细胞培养液-2(EGM-2)诱导,无PDGF-BB的EGM-2培养液为对照,检测a-平滑肌肌动蛋白(a-SM actin)与肌钙结合蛋白(calponin)的表达并接种于聚羟基乙酸(PGA)培养4周后取材.结果 诱导组(n=5)细胞逐步呈平滑肌样转变,表达a-SM actin与calponin,流式细胞仪阳性率分别为(45.16±0.97)%、(37.54 4±1.15)%,可成血管样结构,对照组(n=5)变化不明显,阳性率(7.92±1.04)%、(6.37±0.83)%,成血管样结构能力差.结论 体外可诱导犬BMSC为VSMC并有成血管样结构的能力.     

Protein kinase C delta activated adhesion regulates vascular smooth muscle cell migration

BACKGROUND: Vascular smooth muscle cell (VSMC) migration, fundamental in the pathophysiology of atherogenesis and restenosis, is a coordinated process governed by the formation and disassembly of focal adhesions. Previous studies have demonstrated that VSMC migration is regulated via a signaling network involving protein kinase C delta (PKCdelta). In these studies, we test the hypothesis that PKCdelta regulates VSMC migration through modulation of cell adhesion. MATERIALS AND METHODS: Using primary VSMCs isolated from PKCdelta wild type (+/+) and knock-out (-/-) mice, the effects of PKCdelta on VSMC migration and adhesion were assessed by chemotaxis and cell adhesion. RESULTS: In evaluating cell migration, we found a decrease in platelet-derived growth factor-BB (PDGF-BB; 5 ng/mL x 6 h) stimulated migration of PKCdelta-/-VSMCs as compared to PKCdelta+/+VSMCs, by 59.4 +/- 5.9% (P < 0.01). A similar reduction in migration of PKCdelta-/-VSMCs (66.5 +/- 5.7%, P < 0.01) was also observed on collagen-coated (COL) membranes. Next, we examined cell attachment, a critical step of migration. PKCdelta-/-VSMCs exhibited significantly reduced adherence by 50.3 +/- 1.8% (P < 0.01). A similar defect of PKCdelta-/-VSMCs was also observed on the COL surface, 30.7 +/- 2.3% (P < 0.01). Interestingly, PDGF-BB did not stimulate attachment of VSMCs of either genotype. Consistent with these results, Rottlerin (2 microM), a selective inhibitor of PKCdelta, blocked migration and attachment of VSMCs by 56.8 +/- 3.4% (P < 0.01) and 37.7 +/- 1.9% (P < 0.01), respectively. CONCLUSIONS: Taken together, our data indicate that PKCdelta activation is necessary for VSMC adhesion, which could, at least in part, contribute to the regulatory function of this kinase in cell migration thus pathogenesis of vascular lesions.     

Human prostatic smooth muscle cells in culture: Estradiol enhances expression of smooth muscle cell-specific markers

Smooth muscle cells (SMCs) constitute a major cellular component of prostatic stroma. SMC tension plays an important role in urethral obstruction secondary to benign prostatic hyperplasia (BPH). We have developed an in vitro procedure for the propagation of human prostatic SMCs. Tissue specimens from patients undergoing radical prostatectomy or cystectomy were enzymatically disaggregated and cultured in MCDB-131 medium supplemented with horse serum, insulin, conditioned medium from the tumor cell line CRL-5813, and steroid hormones. The medium was assembled on the basis of the effects these supplements have on the growth of SMC cultures and on the expression of the two markers desmin and smooth muscle myosin. Addition of 0.1 μM of estradiol to the growth medium dramatically increased expression of these SMC-specific markers. Dihydrotestosterone (DHT) and hydrocortisone had a similar, albeit less pronounced effect. At three to five passages, about two thirds of the cells were immunohistologically positive for smooth muscle myosin or desmin. Almost all cells were positive for the myofibroblast marker smooth muscle α-actin throughout 10 passages and more. In SMC cultures, cells staining for smooth muscle myosin and desmin were found to seek direct contact to myofibroblasts. They grew in aggregates on a layer of myofibroblasts which adhered to the surface of the culture vessel. As revealed by transmission electron microscopy the cultured cells exhibited morphological features of myofibroblasts. Characteristics of smooth muscle cells, such as prominent bundles of microfilaments associated with dense bodies, basal laminae investing the cells, and numerous caveolae at the cell surfaces were regularly observed in cultures of low passages. After several passages, these features were markedly decreased and organelles of the biosynthetic system became more prominent. In summary, we present an in vitro model of prostatic SMCs and demonstrate that steroid hormones have characteristic effects on these cells. SMC cultures are expected to facilitate investigation of the functions and properties of human prostatic SMCs. Prostate 30:117–129, 1997. © 1997 Wiley-Liss, Inc.     

An IkappaB-alpha mutant inhibits cytokine gene expression and proliferation in human vascular smooth muscle cells.

BACKGROUND: Inflammatory reaction and intimal proliferation of smooth muscle cells are characteristics of vascular stenotic lesions. Nuclear factor kappaB (NF-kappaB) is involved in regulation of inflammation and cell survival in a variety of cell types. We tested a hypothesis that selective inhibition of NF-kappaB by expression of a mutated, nondegradable inhibitor of NF-kappaB, IkappaB-alphaM, would inhibit proinflammatory cytokine expression and proliferation in human vascular smooth muscle cell. MATERIALS AND METHODS: Smooth muscle cells were cultured from internal mammary artery and infected with recombinant adenovirus vectors. RESULTS: Adenoviral expression of IkappaB-alphaM inhibited diverse signal-triggered cellular IkappaB-alpha degradation, subsequent NF-kappaB activation, and transactivation of proinflammatory cytokine genes. Expression of IkappaB-alphaM in low-density VSMC led to a 60% reduction in serum-stimulated cell growth and a 10% increment in apoptotic incidence but was without effect in high-density cultures. Coexpression of NF-kappaB p65 attenuated apoptosis in low-density cells induced by IkappaB-alphaM. Therefore, the susceptibility to apoptosis induction in the low-density cells correlated with lower constitutive NF-kappaB activity. The induction of apoptosis by IkappaB-alphaM and the rescue by NF-kappaB p65 might be explained by mutual control of NF-kappaB p65 and IkappaB-alphaM access to the nucleus. CONCLUSION: Our results suggest that expression of nondegradable IkappaB-alpha might have therapeutic potential in both vascular inflammatory reaction and smooth muscle cell proliferation.     

体外高磷环境下血管平滑肌细胞转分化过程的研究

目的 观察体外高磷培养条件下,血管平滑肌细胞（VSMC）向类似成骨样细胞转分化的动态变化特点。 方法 Western印迹和免疫荧光法观察不同磷浓度（1.0、2.5、3.5 mmol/L）培养条件下,人VSMC的标志蛋白α平滑肌肌动蛋白（α-SMA）以及成骨细胞特异性的核转录因子Cbfα1、相关骨基质蛋白（骨桥蛋白、I型胶原和骨钙素）的动态变化过程。实时荧光定量PCR法分析上述指标的基因表达水平。在此基础上,以硝酸银染色和茜素红染色了解细胞的钙盐沉积状况,最后通过电镜观察不同培养条件下细胞超微结构的改变。 结果 在无血清的培养条件下,高磷（2.5、3.5 mmol/L）刺激细胞12 h时,胞内 Cbfα1的表达就明显增加（P ＜ 0.05）;3 d后高磷组（2.5、3.5 mmol/L）细胞中I型胶原和骨桥蛋白的含量显著上调（均P ＜ 0.05）;第6天骨钙素的产生开始增加;15 d时高磷组（2.5、3.5 mmol/L）α-SMA的含量才显著下降（P ＜ 0.05）。实时荧光定量PCR结果显示,高磷环境培养1 d时,细胞中骨桥蛋白、I型胶原的mRNA水平显著升高（均P ＜ 0.05）;5 d时骨钙素的mRNA表达显著增加（P ＜ 0.05）;10 d时α-SMA的mRNA含量显著减少（P ＜ 0.05）。在高磷营养液中培养15 d时,细胞出现多处斑片状的钙盐沉积。电镜可见胞质内产生大量胶原纤维和钙化的基质囊泡。 结论 高磷可诱导VSMC向类似成骨样细胞转分化,这是一个复杂的、有多个环节、有序的动态变化过程。     

硫酸吲哚酚对大鼠主动脉平滑肌细胞增生影响的机制研究

目的：探讨硫酸吲哚酚（indoxyl sulfate,IS）对大鼠血管平滑肌细胞（vascular smooth muscle cell,VSMC）增生的影响以及这种变化和氧化应激的关系。方法实验均分为8组：正常组、IS 100μM组、IS 300μM组、IS 500μM组、辛伐他汀10μM＋正常组、辛伐他汀10μM＋IS 100μM组、辛伐他汀10μM＋IS 300μM组、辛伐他汀10μM＋IS 500μM组。采用WST-1法检测VSMC增殖情况;硫代巴比妥酸法检测培养基中丙二醛（malondialdehyde,MDA）含量;ELISA方法测定晚期氧化蛋白产物（advanced oxidation protein products,AOPP）。结果与正常组比较,IS 300μM组和 IS 500μM 组上清液的 WST-1（OD 值）[（1.55±0.27）比（1.18±0.25）与（1.73±0.30）比（1.18±0.25）,P〈0.01]含量、MDA含量[（2.60±0.47）μg/ml比（1.59±0.21）μg/ml与（2.82±0.54）μg/ml比（1.59±0.21）μg/ml,P〈0.01]和 AOPP 含量[（67.94±8.58）μmol/L 比（54.97±8.46）μmol/L与（72.09±9.49）μmol/L比（54.97±8.46）μmol/L,P〈0.01]均明显增高。与同浓度 IS 组相比,辛伐他汀10μM＋IS 300μM 组和辛伐他汀10μM＋IS 500μM 组上清液 WST-1（OD 值）[（1.22±0.24）比（1.55±0.27）与（1.32±0.30）比（1.73±0.30）,P〈0.05]、MDA[（1.64±0.38）μg/mL 比（2.60±0.47）μg/ml 与（1.70±0.40）μg/ml 比（2.82±0.54）μg/ml,P〈0.01]含量和 AOPP 含量[（55.56±7.41）μmol/L比（67.94±8.58）μmol/L 与（55.54±6.80）μmol/L 比（72.09±9.49）μmol/L,P〈0.05]均明显降低。大鼠 VSMC 上清液的 WST-1含量与 MDA 含量呈正相关（r=0.621,P〈0.01）,大鼠VSMC上清液的WST-1含量与AOPP含量呈正相关（r=0.581,P〈0.01）。结论 IS可浓度依赖性地促进大鼠 VSMC增生,其作用可能与 IS增加 VSMC的氧化应激有关;辛伐他汀可抑制此作用。     

Characterization of the control of intracellular [Ca2+] and the contractile phenotype of cultured human detrusor smooth muscle cells

PURPOSE: We measured the functional properties of cultured human detrusor myocytes with respect to their ability to regulate their intracellular [Ca2+] and generate force in collagen matrices. MATERIALS AND METHODS: Human detrusor biopsies were dissociated into single cells by collagenase treatment and used immediately or cultured in D-valine medium and subsequently used after culture trypsinization. Intracellular [Ca2+] was measured in Fura-2 loaded myocytes. Cell force development was measured by incorporating cells into a collagen gel and attaching it to an isometric strain gauge. RESULTS: Carbachol was equally effective in generating Ca transients in freshly isolated and cultured cells. Carbachol potency (pEC50) and the magnitude of Ca2+ transients were similar. Adenosine triphosphate potency was decreased in cultured cells and Ca2+ transients showed properties consistent with a purinoceptor shift from a purinergic subtype. Temporal restitution of Ca2+ transients was similar in the 2 groups, indicative of retained intracellular Ca2+ stores in cultured cells. Cultured cells (approximately 10(6)) embedded in collagen gel generated a force about 10 times greater than that generated by gel alone. The cell dependent force could be further increased by adding carbachol. CONCLUSIONS: Cultured cells retain the ability to generate agonist induced intracellular Ca2+ transients. There was no evidence that the cell culture altered the properties of muscarinic receptors, although purinoceptor mediated properties were altered. Restitution experiments indicated that functional intracellular Ca2+ stores were retained in cultured cells. Cultured cells also retained a contractile phenotype, especially in response to carbachol. The magnitude of force was attenuated, which may be a function of the biomechanical properties of the gel used to embed the cells.     

Molecular mechanisms mediating vascular calcification: role of matrix Gla protein

Patients with chronic kidney disease (CKD) have a higher incidence of vascular calcification and a greatly increased risk of cardiovascular death. The mechanisms involved in the accelerated vascular calcification observed in CKD have recently become clearer, leading to the hypothesis that a lack of natural inhibitors of calcification may trigger calcium deposition. One of these inhibitory factors, matrix Gla protein (MGP), is the focus of the present review. MGP, originally isolated from bone, is a vitamin K-dependent protein that is also highly expressed by vascular smooth muscle cells. MGP has been confirmed as a calcification-inhibitor in numerous studies; however, its mechanism of action is not completely understood. It potentially acts in several ways to regulate calcium deposition including: (i) binding calcium ions and crystals; (ii) antagonizing bone morphogenetic protein and altering cell differentiation; (iii) binding to extracellular matrix components; and (iv) regulating apoptosis. Its expression is regulated by several factors including retinoic acid, vitamin D and extracellular calcium ions, and a reduced form of vitamin K (KH2) is important in maintaining MGP in an active form. Therefore, strategies aimed at increasing its expression and activity may be beneficial in tipping the balance in favour of inhibition of calcification in CKD.     

Methylmethacrylate monomer produces direct relaxation of vascular smooth muscle in vitro

Methylmethacrylate bone cement is associated with severe hypotensive reactions during surgery and anesthesia. The purpose of this in vitro study was to determine if methylmethacrylate monomer could produce hypotension by acting directly on vascular smooth muscle.
Segments of human saphenous vein or rabbit thoracic aorta were cut into rings. The rings were mounted in isolated tissue chambers in order to measure isometric tension development.
Methylmethacrylate monomer (methylmethacrylic acid ester) produced direct relaxation of venous or aortic rings preconstricted with either potassium ion or noradrenaline. The relaxation was concentration-dependent, occurring at concentrations from 10^-1 to 10^-1 M. The relaxation of rabbit aortic rings {preconstricted with noradrenaline) was unaffected by pre-treatment with atropine, propranolol, cimetidine, indomethacin, or methylene blue. Endothelial stripping with Triton X-100, sufficient to completely abolish acetylcholine-induced relaxation, also had little effect on methylmethacrylate-induced relaxation. Methylmethacrylate produced direct relaxation of rabbit aortic rings constricted with either potassium or noradrenaline in calcium-deficient media, and inhibited subsequent calcium-induced constriction.
These results suggest that methylmethacrylate monomer may interfere with intracellular and extracellular calcium mobilization and excitation/contraction coupling in vascular smooth muscle. The direct relaxation of venous and arterial smooth muscle produced by methylmethacrylate monomer may contribute in part to the hypotension that can occur when acrylic bone cement is employed during orthopedic procedures.     

微小RNA-17-5p下调同源盒蛋白HOXB13抑制血管平滑肌细胞增殖的研究

目的:探讨微小RNA(miR)-17-5p调控同源盒蛋白(HOXB13)的分子机制。方法:从SD大鼠胸主动脉分离和培养血管平滑肌细胞(VSMCs);利用细胞计数试剂盒检测细胞增殖;反转录定量聚合酶链反应检测miRNA和miR-17-5p的表达;细胞转染miR-17-5p mimics、anti-miR-17-5p和小干...     

Chronic high pressure potentiates the antiproliferative effect and abolishes contractile phenotypic changes caused by endothelial cells in cocultured smooth muscle cells

High in vitro pressures have been reported to alter smooth muscle cell (SMC) and endothelial cell (EC) phenotype, while endothelial cells (ECs) can influence the proliferation, phenotype, and contractile features of smooth muscle cells (SMC) in coculture systems. However, little is known about the in vitro effects of pressure on EC/SMC cocultures. We therefore sought to compare SMC proliferation in independent and EC coculture under ambient and high pressure, and identify changes in the contractile phenotype of SMCs by measuring levels of the L-type Ca(2+) channel a(1) subunit (dihydropyridine-DHP receptor) which is critical for Ca(2+) transients, differentiation and contractility in SMC. METHODS: Rat aortic SMCs in independent culture (SMC/0) and coculture with ECs (SMC/EC) were maintained in 5% CO(2) under either atmospheric or high pressure (130 mmHg). SMC were counted at 0, 1, 3, and 5 days and compared to initial cell counts of day 0 before the exposure to experimental conditions. DHP receptor levels were quantitated by Western blotting (three similar studies). RESULTS: ECs suppressed SMC proliferation on day 1 of coculture in both atmospheric and high pressure (20% inhibition vs independent culture, P < or = 0.05). By day 3, cocultured SMC under atmospheric pressure displayed no EC-mediated inhibition, and at day 5, atmospheric cocultured SMCs revealed statistically significant enhanced proliferation as compared with SMCs in independent cultures. However, cocultured SMCs exposed to 130 mmHg pressure displayed sustained sensitivity to EC growth inhibition at both days 3 and 5 of the experiment. Coculture decreased SMC DHP-receptor levels under atmospheric pressure. However, this effect was abolished in cocultures exposed to high pressure. CONCLUSIONS: High pressure substantially alters the regulatory influence of EC on SMC proliferation and contractile potential. This pressure/coculture model should increase our understanding of cellular interaction in hypertensive vasculopathy.     

Sphingosine-1-phosphate induces G(alphai)-coupled,PI3K/ras-dependent smooth muscle cell migration

BACKGROUND: Sphingolipids such as sphingosine-1-phosphate (S-1-P) are potent extracellular mediators released in response to vessel injury. S-1-P binds to G-protein-coupled receptors, which can be either G(alphai) or G(alphaq) linked. This study examines the signaling pathways involved in vascular smooth muscle cell migration after stimulation by S-1-P. We hypothesized that S-1-P stimulates migration of smooth muscle cells that is dependent upon a G(alphai)-coupled receptor, ras, phosphoinositol 3-kinase (PI3-K), and ERK 1/2. METHODS: Vascular smooth muscle cells were cultured in vitro. A linear wound assay and Boyden chamber assay of migration were employed in the presence of S-1-P and inhibitors of G(alphai) [pertussis toxin (PTx), 100 ng/ml], G(alphaq) (GP-2A, 10 microM), ras [manumycin A (MA), 10 microM], PI3-K [Wortmannin (Wn), 10 microM], and MEK1 [PD98059 (PD), 25 microM]. Western blotting was performed separately to examine p42/p44 MAP kinase (ERK 1/2) activation in response to S-1-P with these inhibitors. RESULTS: S-1-P induced vascular smooth muscle cell migration. This response was decreased by preincubation with PTx, suggesting a receptor linked, G(alphai)-mediated response. Application of a G(alphaq) inhibitor did not affect this response. S-1-P induced ERK 1/2 phosphorylation in a time-dependent manner. This S-1-P-induced cell migration was PD-sensitive in the Boyden chamber assay, confirming that it is MEK1- and ERK1/2-dependent. Inhibition of ras with MA and PI3-K with Wn also reduced ERK phosphorylation and smooth muscle cell migration in response to S-1-P. CONCLUSIONS: S-1-P induces smooth muscle cell migration through a G(alphai)-linked, ras- and PI3-K-coupled, ERK 1/2-dependent process. Understanding signal transduction will allow targeted molecular interventions to treat the response of a vessel to injury.