醛固酮对人脐血管内皮细胞凋亡的作用

目的探讨醛固酮(ALD)对人脐静脉内皮细胞(HUVEC)凋亡的作用及其可能的机制。方法 HUVEC分成3大组:正常对照组、ALD处理组(10~(-7)、10~(-6)、10~(-5)mol/L ALD)及螺内酯(10~(-5)mol/L)处理组,流式细胞术检测HUVEC凋亡,免疫印迹法检测HUVEC盐皮质激素受体(MR)蛋白水平的表达。结果 (1)与对照组比较,10~(-7)～10~(-5)mol/L ALD处理后HUVEC凋亡增加。(2)10~(-7)～10~(-5)mol/L ALD诱导的HUVEC凋亡呈剂量依赖性。(3)10~(-6)、10~(-5)mol/L ALD组加入10~(-6)mol/L螺内酯处理后凋亡下降(P<0.01),即ALD诱导的HUVEC凋亡被螺内酯部分抑制。(4)ALD上调MR蛋白表达,螺内酯可部分阻断此作用。结论 ALD可能通过上调HUVEC的MR蛋白表达而诱导细胞凋亡。     

Rosuvastatin-regulated post-translational phosphoproteome in human umbilical vein endothelial cells

Statins, 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, are widely prescribed as cholesterol-lowering drugs. Statins have recently been found to have pleiotropic effects that are independent of their lipid-lowering properties. Phosphorylation of serine, threonine, and tyrosine residues of functional proteins are considered to be important in the endothelial signaling cascade. In this study, protein phosphorylation status in human umbilical vein endothelial cells (ECs) after rosuvastatin treatment was examined. The proteins were collected from rosuvastatin-treated ECs and then the phosphorylated peptides purified by a Fe³⁺-immobilized metal-affinity chromatography bead system were examined by liquid chromatography–tandem mass spectrometry analysis. Alterations of the phosphorylation status of proteins were noticed after rosuvastatin treatment. There were 277 and 530 phosphorylated proteins identified from the control and rosuvastatin-treated ECs, respectively. Among those proteins, T78, in addition to S156 of the Ras-GTPase-activating protein, was phosphorylated after rosuvastatin treatment. Rosuvastatin reduced the phosphorylation of Y455 in HSP90 protein. Decreased phosphorylation of T211 with a concurrent increase in the T291 phosphorylation of Akt1 was observed under rosuvastatin treatment. Increased S633 phosphorylation was detected in endothelial nitric oxide synthase. Western blot analysis further showed an earlier and greater S633 phosphorylation than that of S1177 in endothelial nitric oxide synthase after rosuvastatin treatment. Changes in the phosphorylation status of these proteins may alter the protein’s function and affect endothelial physiology. The current study provides new insights leading to a better understanding of the pleiotropic effects of statins on the vascular system.     

Expression of adhesion molecules by cultured human glomerular endothelial cells in response to cytokines: comparison to human umbilical vein and dermal microvascular endothelial cells.

We investigated the expression of cell adhesion molecules on the surface of glomerular endothelial cells (GEC), dermal microvascular endothelial cells (MvE), and umbilical vein endothelial cells (HUVEC) that had or had not been stimulated by cytokines. PECAM-1 was constitutively expressed at a high level on HUVEC but its expression level decreased following stimulation by tumor necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma). PECAM-1 was also constitutively expressed on microvascular endothelial cells MvE and GEC, but at lower levels than on HUVEC, and expression by these cells also decreased in response to TNF-alpha and IFN-gamma. There was no dose-dependent effect on MvE but there was a dose-dependent effect on the level of expression of cell adhesion molecules on GEC. TNF-alpha induced the expression of VCAM-1 on HUVEC and GEC, but not MvE, while IFN-gamma induced VCAM-1 expression only on HUVEC. TNF-alpha induced the expression of E-selectin on all three kinds of endothelial cells, but IFN-gamma had no effect on E-selectin expression. GEC therefore showed expression patterns of PECAM-1, VCAM-1, and E-selectin different from those seen in HUVEC and MvE upon treatment with TNF-alpha or IFN-gamma. The use of cultured human GEC allows us to study not only the inflammatory processes, but also the pathophysiological role of GEC in hemodynamic disturbances and their interaction with intrinsic mesangial cells at the molecular and subcellular levels.     

人脐静脉内皮细胞衰老时过氧化体增殖物激活型受体α的生物活性变化

目的观察体外培养的脐静脉内皮细胞衰老时过氧化体增殖物激活型受体α(PPARα)的生物活性变化,探索致PPARα生物活性变化的可能因素。方法体外原代培养人脐静脉内皮细胞,应用β-半乳糖苷酶染色法鉴定内皮细胞衰老状态,以此分为人脐静脉内皮细胞青年组及衰老组;流式细胞技术分析细胞周期;半定量反转录-聚合酶链反应法检测内皮细胞PPARα及视黄醇类X受体α(RXRα)、核受体辅阻遏物(NCoR)mRNA表达水平;电泳迁移率变动分析检测PPARα的DNA结合活性;细胞免疫荧光组化方法检测细胞内泛素蛋白表达状况。结果与青年组内皮细胞比较,衰老组内皮细胞PPARαmRNA表达及DNA结合活性均显著降低,RXRαmRNA表达无明显差异,而NCoR mRNA表达显著增强,细胞内泛素蛋白表达显著增强。结论血管内皮细胞衰老时自身功能失调可能与PPARα生物活性降低密切相关。     

Mechanosensitive Ca2+ transients in endothelial cells from human umbilical vein.

We have investigated the changes in intracellular calcium concentration ([Ca2+]i) in human endothelial cells induced by mechanical stretch due to osmotic cell swelling. Hypotonic solutions also activate a Cl- conductance that has been described elsewhere and mainly serves to clamp the membrane potential at negative values to provide a driving force for Ca2+ influx. The increase in [Ca2+]i caused by hypotonic solutions is due to release from inositol-1,4,5-trisphosphate-sensitive Ca2+ pools and a subsequent Ca2+ influx, apparently activated by store depletion. These changes in [Ca2+]i are completely abolished if the phospholipase A2 (PLA2) activity is inhibited by either 4-bromophenacyl bromide or cyclosporin A. Arachidonic acid, applied either extracellularly or intracellularly via the patch pipette, mimics the mechanosensitive response even in cells with blocked PLA2. Metabolites of the lipo- and cyclooxygenase pathways can be excluded. Phospholipase C activation and the protein kinase A pathway are not involved in this mechanical response. Although no specific pharmacological tools for probing the role of PLA2 are available, our evidence suggests that mechanosensitivity in endothelial cells may be modulated by arachidonic acid.     

层流切应力刺激对人脐静脉血管内皮细胞Toll样受体mRNA表达的影响

目的用RT-PCR和Northern杂交技术,评价层流低切应力刺激对人脐静脉血管内皮细胞Toll样受体(TLR)表达的影响。方法将0.42 Pa切应力为低切应力组处理内皮细胞1 h,未作任何刺激的内皮细胞为对照组,从两组血管内皮细胞提取总RNA,采用RT-PCR和Northern杂交技术明确对照和切应力刺激1 h人脐静脉血管内皮细胞TLR-2和TLR-4 mRNA的表达情况。结果RT-PCR和Northern杂交均显示,层流低切应力刺激1 h后血管内皮细胞TLR-4表达增强、TLR-2几乎无变化。结论层流低切应力可引起人脐静脉内皮细胞TLR-4 mRNA表达增加,提示炎性信号受体TLR-4可能介导层流切应力诱导血管内皮细胞某些效应基因的表达。     

N-乙酰半胱氨酸对人脐静脉内皮细胞eNOS的保护作用

目的探讨N-乙酰半胱氨酸(NAC)预处理对人脐静脉内皮细胞(HUVECs)中内皮型一氧化氮合酶(e NOS)的保护作用及可能的分子机制。方法 HUVECs随机分为正常对照组、血管紧张素(Ang)Ⅱ组、单纯NAC组和NAC预处理+AngⅡ组。用Western印迹法检测e NOS蛋白、eNOS Ser1177磷酸化水平、蛋白磷酸酶(PP)2A-Cα、PP2A-cY307磷酸化水平和I_2~(PP2A)表达水平,化学比色法检测细胞组成型一氧化氮合酶(c NOS)活性及培养基中一氧化氮(NO)含量。结果 AngⅡ刺激后eNOS Ser1177、PP2A-c Y307水平和I_2~(PP2A)表达水平降低(P0.05),cNOS活性、NO含量均降低(P0.05);NAC预处理后上述变化均被逆转。结论 NAC预处理可上调PP2A-c Y307和I_2~(PP2A)水平,从而降低PP2A活性,最终保护eNOS活性。     

N-钙黏蛋白在烟曲霉黏附及侵袭内皮细胞中的作用

目的 探讨N-钙黏蛋白在内皮细胞黏附吞噬烟曲霉过程中的作用.方法 观察提取人脐静脉内皮细胞蛋白与烟曲霉的结合过程,了解N-钙黏蛋白是否可与烟曲霉结合,建立内皮细胞黏附及吞噬烟曲霉的体外模型,通过单克隆抗体阻断上皮细胞膜受体N-钙黏蛋白,再次观察脐静脉内皮细胞黏附及吞噬烟曲霉情况.结果 脐静脉内皮细胞膜蛋白N-钙黏蛋白可与烟曲霉结合,抗体阻断N-钙黏蛋白后,脐静脉内皮细胞黏附和吞噬烟曲霉能力明显下降.结论 N-钙黏蛋白是脐静脉内皮细胞黏附吞噬烟曲霉孢子的相关受体.     

骨保护素对脐静脉内皮细胞生长的影响

目的:观察骨保护素对内皮细胞数量变化及内皮型一氧化氮合酶(eNOS)的影响,探讨其对内皮细胞的作用。方法:常规培养永生化的人内皮细胞,用不同浓度(0,0·5,2·0,4·0,6·0,8·0μg/L)的骨保护素作用细胞48h后,用细胞计数试剂盒(cellcount kit,CCK-8)来检测细胞的数量;免疫细胞化学检测eNOS蛋白的表达水平,实时定量PCR检测eNOS基因的定量表达。结果:与对照组(0·936±0·146)相比,在8·0μg/L组的内皮细胞增殖量明显增加(1·291±0·200;P<0·05);eNOS蛋白表达在2·0μg/L、4·0μg/L组(P<0·05)和6·0μg/L、8·0μg/L组(P<0·01)均显著增加;而4·0μg/L、6·0μg/L、8·0μg/L组的eNOS mRNA较对照组均呈显著增高(P<0·01)。结论:骨保护素在人体的浓度波动范围内具有促进内皮细胞增殖效应,在抗动脉粥样硬化过程中,可能具有促内皮细胞修复作用,且与浓度梯度有明显的正相关性。     

瘦素对人脐静脉内皮细胞血管内皮生长因子表达的影响

目的:探讨瘦素对人脐静脉内皮细胞(HUVECs)血管内皮生长因子(VEGF)表达的影响。方法:用不同浓度的瘦素刺激原代培养的HUVECs,检测HUVECs表达VEGF的情况。结果:在相同作用时间下,随着瘦素浓度的升高,VEGF蛋白及VEGF mRNA的表达也随之升高,经统计学检验有相关性;在相同瘦素浓度下, 随着作用时间的延长,VEGF蛋白及VEGFmRNA的表达也随之升高,且有相关性。结论:瘦素可以刺激HU- VECs表达VEGF而且呈时间和剂量相关性。     

间歇低氧对人脐静脉内皮细胞中内皮素的影响

目的通过建立间歇低氧细胞模型,测定不同低氧模式下人脐静脉内皮细胞中内皮素(endothelin,ET)含量的变化,以进一步探讨ET在细胞水平对间歇低氧在阻塞性睡眠呼吸暂停综合征合并高血压患者中的作用.方法采用人脐静脉内皮细胞可传代细胞株ECV304细胞系,暴露于不同低氧条件,暴露完成后采用双抗夹心酶联免疫吸附法(ELISA法)测定培养基中ET浓度.结果间歇低氧组ET浓度[(12.86±6.68)pg/mL]与间歇正常氧组[(3.29±0.88)pg/mL]及空白对照组[(4.67±1.22)pg/mL]间差异有统计学意义(F=13.687,P<0.05).其中,间歇低氧组高于间歇正常氧组(P<0.05)及空白对照组(P<0.05),而间歇正常氧组及空白对照组间差异无统计学意义(P>0.05).相同累加低氧时间及程度的间歇低氧组ET浓度显著高于持续低氧组[(7.07±1.00)pg/mL](P<0.05).结论间歇低氧可引起ET水平升高,提示ET在间歇低氧合并高血压中可能起重要作用.     

克拉屈滨对人脐静脉内皮细胞的毒性作用及机制

目的研究克拉屈滨(2-CdA)对内皮细胞活力、迁移、周期和分泌活性的影响,以评估2-CdA在临床应用中对心血管的影响。方法 CCK-8法检测不同浓度2-CdA对内皮细胞生长的影响;划痕实验检测细胞迁移;流式细胞术检测细胞周期;吖啶橙/溴化乙啶法检测细胞凋亡;Gries法测定一氧化氮(NO)含量;ELISA法检测血管内皮生长因子(VEGF)水平。结果在0.4~40μmol/L浓度范围,2-CdA对内皮细胞有明显抑制作用,IC50为4.126μmol/L。随药物浓度升高和作用时间的延长,2-CdA的抑制作用增强;2-CdA高浓度长时间作用可导致内皮细胞凋亡。5μmol/L 2-CdA作用内皮细胞后,细胞迁移能力受抑制,细胞周期明显阻滞在S期。吖啶橙/溴化乙啶细胞染色结果显示,高浓度2-CdA可诱导内皮细胞的凋亡。5μmol/L 2-CdA作用内皮细胞后导致NO和VEGF含量减少。结论 2-CdA具有内皮细胞毒性作用,引起细胞周期阻滞,并导致细胞凋亡。     

Kinase domain insert containing receptor promotor controlled suicide gene system kills human umbilical vein endothelial cells

AIM: To evaluate the killing effect of double suicide gene mediated by adenovirus and regulated under kinase domain insert containing receptor (KDR) promoter on human umbilical vein endothelial cells. METHODS: By PCR technology, human KDR promoter gene, Escherichia coli(E. coli) cytosine deaminase (CD) gene and the herpes simple virus-thymidine kinase (TK) gene were cloned. Plasmid pKDR-CDglyTK was constructed with them. Then, a recombinant adenoviral plasmid pAdKDR CDglyTK was constructed in a "two-step transformation protocol". The newly constructed plasmids were transfected to 293 packaging cells to grow adenoviruses, which were further propagated and purified. Human umbilical vein endothelial cells (HUVEC) were infected with a different multiplicity of infection (MOI) of resultant recombinant adenovirus, the infection rate was measured with the aid of (GFP) expression. Infected cells were cultured in culture media containing different concentrations of (GCV) and/or 5-(FC), and the killing effects were measured. RESULTS: Recombinant adenoviruses AdKDR-CDglyTK were successfully constructed, and they infected HUVEC cells efficiently. Our data indicated that the infection rate was relevant to MOI of recombinant adenoviruses. HUVEC cells infected with AdKDR-CDglyTK were highly sensitive to the prodrugs, their survival rate correlated to both the concentration of the prodrugs and the MOI of recombinant adenoviruses. Our data also indicated that the two prodrugs used in combination were much more effective on killing transgeneic cells than GCV or 5-FC used alone. CONCLUSION: Prodrug/KDR-CDglyTK system is effective on killing HUVEC cells, its killing effect correlates to the concentration of prodrugs and recombinant adenovirus' MOI. Combined use of the two prodrugs confers better killing effects on transgeneic cells.     

Evidence for 15-HETE synthesis by human umbilical vein endothelial cells

Incubation of cultured human umbilical vein endothelial cells with [1-14C]-arachidonic acid, followed by RP-HPLC analysis, resulted in the appearance of two principal radioactive products besides 6-keto-PGF1 alpha. The first peak was HHT, a hydrolysis product of the prostaglandin endoperoxide. The second peak was esterified, converted to the trimethylsilyl ether derivative, and analyzed by GC/MS and was shown to be the lipoxygenase product 15-HETE. Stimulation of endothelial cells with thrombin enhanced 15-HETE synthesis from arachidonate. Subsequent experiments showed that 5-HETE and 12-HETE were also synthesized by endothelial cells, but no evidence of leukotriene synthesis was found. Incubation of the 15-HETE precursor 15-HPETE with endothelial cells resulted in the formation of four distinct ultraviolet light-absorbing peaks. Ultraviolet and GC/MS analysis showed these peaks to be 8,15-diHETEs that differed only in their hydroxyl configuration and cis-trans double-bond geometry. Formation of 8,15-diHETE molecules suggests the prior formation of the unstable epoxide molecule 14,15-LTA4 or an attack at C-10 of 15-HPETE by an enzyme with mechanistic features in common with a 12-lipoxygenase. The observation that endothelial cells can synthesize both 15-HETE and 8,15-diHETE molecules suggest that this cell type contains both a 15-lipoxygenase and a system that can synthesize 14,15-LTA4.     

Superoxide production by human umbilical vein endothelial cells in an anoxia-reoxygenation model

Ischaemia/reperfusion of cardiac tissue has been claimed to be associated with the production of oxygen free radicals, which can contribute to severe membrane damage and tissue injury. We investigated the effects of anoxia/reoxygenation treatment on superoxide radical production in an in vitro system consisting of preconfluent and confluent human endothelial cell monolayers. The influence of varying the anoxia and reoxygenation phases on superoxide production was studied. As a test of cytotoxicity, the release of the cytosolic enzyme lactate dehydrogenase in the culture medium was measured before and at 0, 24 and 48 h after anoxia-reoxygenation. Cellular damage was monitored by microscopic examination of the cultures during and after the experiments and by the expression of the von Willebrand protein and of the membrane glycoprotein IIa by indirect immunofluorescence with specific monoclonal antibodies. Our results show that the endothelial cells subjected to anoxia-reoxygenation release superoxide anions, as revealed by superoxide dismutase inhibitable cytochrome C reduction. Free radical production is dependent on cell confluent or preconfluent state and on both anoxia and reoxygenation duration. Free radical release does not seem to be accompanied by manifest cellular alteration.