阿托伐他汀钙对EA.hy926细胞全基因表达谱的影响及生物信息学分析

目的探讨阿托伐他汀钙对体外培养人脐静脉内皮细胞EA.hy926细胞基因表达谱的影响及作用的分子机制。方法取EA.hy926细胞分实验组和对照组,实验组采用10μmol/L阿托伐他汀钙体外干预人脐静脉内皮细胞EA.hy926 24h,用Affymetrix HG-U133plus 2.0全基因组表达芯片检测阿托伐他汀钙对EA.hy926细胞基因表达谱的影响。并运用基因富集分析(GSEA)软件、DAVID基因功能聚类分析软件及Cmap数据库对芯片数据进行分析,对相关靶基因进行实时定量PCR验证。结果与对照组比较,实验组基因芯片分析筛选出差异表达倍数>2倍的基因649个,其中上调基因295个,下调基因354个。经DAVID及GSEA基因富集分析显示,阿托伐他汀钙广泛下调了细胞周期调控相关基因,上调了Kruppel样转录因子等血管保护基因,Cmap数据库分析显示,阿托伐他汀钙与组蛋白去乙酰化酶抑制剂及白藜芦醇呈正相关的联强度高。结论阿托伐他汀钙从多角度发挥抗动脉粥样硬化作用,作用机制可能与组蛋白去乙酰化酶抑制剂及白藜芦醇相似。     

Relative contribution of different l-arginine sources to the substrate supply of endothelial nitric oxide synthase

In certain cases of endothelial dysfunction l-arginine becomes rate-limiting for NO synthesis in spite of sufficiently high plasma concentrations of the amino acid. To better understand this phenomenon, we investigated routes of substrate supply to endothelial nitric oxide synthase (eNOS). Our previous data with human umbilical vein (HUVEC) and EA.hy.926 endothelial cells demonstrated that eNOS can obtain its substrate from the conversion of l-citrulline to l-arginine and from protein breakdown. In the present study, we determined the quantitative contribution of proteasomal and lysosomal protein degradation and investigated to what extent extracellular peptides and l-citrulline can provide substrate to eNOS. The RFL-6 reporter cell assay was used to measure eNOS activity in human EA.hy926 endothelial cells. Individual proteasome and lysosome inhibition reduced eNOS activity in EA.hy926 cells only slightly. However, the combined inhibition had a pronounced reducing effect. eNOS activity was fully restored by supplementing either l-citrulline or l-arginine-containing dipeptides. Histidine prevented the restoration of eNOS activity by the dipeptide, suggesting that a transporter accepting both, peptides and histidine, mediates the uptake of the extracellular peptide. In fact, the peptide and histidine transporter PHT1 was expressed in EA.hy926 cells and HUVECs (qRT/PCR). Our study thus demonstrates that l-citrulline and l-arginine-containing peptides derived from either intracellular protein breakdown or from the extracellular space seem to be good substrate sources for eNOS.     

Increased expression of endothelin-converting enzyme-1c isoform in response to high glucose levels in endothelial cells

Endothelin-1 (ET-1) is both a potent vasoconstrictor and mitogenic factor that has been implicated as a cause of the micro- and macrovascular complications of diabetes mellitus. The pathway by which the high-glucose environment of diabetes mediates increased levels of endothelins has not been completely elucidated but appears to involve endothelin-converting enzyme (ECE-1), which converts inactive big ET-1 to active ET-1 peptide. To determine the effect of high glucose concentrations on the expression of ECE-1, hybrid endothelial cells (EA.hy926) and human umbilical vein endothelial cells (HUVEC) were both grown in various glucose concentrations. There was a 2-fold increase in ECE-1 immunoreactivity in the EA.hy926 cell line growing in medium containing 22.2 versus 5.5 mmol/l glucose after 24 h, which rose to greater than 20-fold after 5 days. Similar results were seen with HUVEC. Bradykinin or NG-nitro-L-arginine methyl ester did not change the effect of high glucose on ECE-1 protein expression. High glucose induced a 72 and 41% increase in total protein kinase C (PKC) activity in both EA.hy926 cells and HUVEC, respectively, and a 39, 49 and 109% elevation in PKC beta1, beta2 and delta expression, respectively, in EA.hy926 cells. The increase in ECE-1 expression was inhibited in both cell cultures by GF109203X (5 micromol/l), a general PKC inhibitor, while addition of 10 nmol/l phorbol myristic acid to EA.hy926 cells or HUVEC growing on medium containing 5.5 mmol/l glucose increased ECE-1 expression to a level similar to that of cells conditioned in high glucose. Human ECE-1 protein exists in four different isoforms, termed 1a, 1b, 1c and 1d. Northern blot analysis revealed that only ECE-1c isoform mRNA levels increased. Immunohistochemical staining of EA.hy926 cells grown in high glucose concentrations demonstrated an increase in the ECE-1c isoform, which occurred mainly in the plasma membrane. These results showed that the PKC pathway may play an important role in the glucose-mediated induction of ECE-1 expression. The main isoform to increase in response to high glucose was ECE-1c. This enzyme may be one of the factors contributing to the elevated ET-1 peptide levels observed in diabetes.     

Expression of ryanodine receptor type 3 and TRP channels in endothelial cells: comparison of in situ and cultured human endothelial cells

OBJECTIVE: Ca(2+) mobilization plays an important role in endothelial function by stimulating Ca(2+)-dependent synthesis of vasodilating factors. In addition to inositol-1,4,5-trisphosphate (InsP(3)) mediated Ca(2+) mobilization, Ca(2+) release from ryanodine-sensitive pools and Ca(2+)-influx through TRP channels have been suggested to be important in endothelial Ca(2+)-signaling. However, the function and molecular identity of TRP channels and ryanodine receptors in human endothelium in situ are still elusive. We hypothesized that expression of ryanodine-receptors (RyR) and TRP channels differs between human endothelium in situ and in cultured cells. METHODS: By combining single-cell RT-PCR and patch-clamp techniques, expression of RyR and TRP channels was determined in situ in endothelial cells of human mesenteric artery (HMAECs) obtained from patients undergoing bowel resection and in the endothelial cell line EA.hy926. RESULTS: At the single cell level, expression of RyR 3 was detected in 25 and 5% of HMAECs and EA.hy926 samples, respectively. Expression of the RyR 1 and 2 was not detected in either HMAECs or EA.hy926. In patch-clamp experiments in HMAECs, applications of caffeine (0.5 mM) induced sustained hyperpolarization mediated by activation of Ca(2+)-activated K channels. In EA.hy926, caffeine-induced hyperpolarization was not detected. Single HMAECs expressed the TRP genes, TRP1 and TRP3, but not TRP 4 and 6. The TRP1 was the predominantly expressed TRP gene in HMAECs in situ whereas TRP3 expression was rarely detected. EA.hy926 expressed only TRP1. In patch clamp experiments in HMAECs, Ca(2+)-store depletion activated non-selective cation currents leading to Ca(2+) entry. CONCLUSIONS: Our findings suggest that, in addition to InsP(3) mediated Ca(2+) release, Ca(2+) release from ryanodine-sensitive stores mediated by RyR3 and Ca(2+) entry through TRP1 might represent important components of endothelial Ca(2+) signaling in situ and thereby of endothelial function in intact human blood vessels.     

登革2型病毒与整合素β3相互作用机制的研究

目的 观察登革2型病毒(dengue virus+etotype 2,DV2)感染后及转染DV2病毒蛋白的质粒后,EA.hy926细胞中整合素β3表达规律及表达量的变化,以初步明确DV2与整合素β3的相互作用的可能机制.方法　经噬斑法测定DV2在EA.hy926中的增殖规律,并通过间接免疫荧光染色检测DV2感染后及病毒...     

Role of neutral amino acid transport and protein breakdown for substrate supply of nitric oxide synthase in human endothelial cells

Endothelial dysfunction is often associated with a relative substrate deficiency of the endothelial nitric oxide synthase (eNOS) in spite of apparently high intracellular arginine concentrations. For a better understanding of the underlying pathophysiological mechanisms, we aimed to characterize the intracellular arginine sources of eNOS. Our previous studies in human endothelial EA.hy926 cells suggested the existence of two arginine pools: pool I can be depleted by extracellular lysine, whereas pool II is not freely exchangeable with the extracellular space, but accessible to eNOS. In this study, we demonstrate that the eNOS accessible pool II is also present in human umbilical vein endothelial cells (HUVECs), but not in ECV bladder carcinoma cells transfected with an expression plasmid for eNOS. In the endothelial cells, one part of pool II (referred to as pool IIA) consisted of recycling of citrulline to arginine. This part could be depleted by neutral amino acids that match the substrate profile of system N transporter 1 (SN1), presumably by the removal of intracellular citrulline. SN1 was expressed in EA.hy926 cells and HUVECs as shown by real-time RT-PCR. The second part of pool II (referred to as pool IIB) could not be depleted by any of the cationic or neutral amino acids tested. Our data demonstrate that pool IIB is nourished by protein breakdown and thus represents a substrate pool likely to accumulate protein-derived endogenous inhibitors of eNOS. Preferential use of the arginine pool IIB under pathophysiological conditions might therefore explain the arginine paradox.     

Unique secretory dynamics of tissue plasminogen activator and its modulation by plasminogen activator inhibitor-1 in vascular endothelial cells

We analyzed the secretory dynamics of tissue plasminogen activator (tPA) in EA.hy926 cells, an established vascular endothelial cell (VEC) line producing GFP-tagged tPA, using total internal reflection-fluorescence (TIR-F) microscopy. tPA-GFP was detected in small granules in EA.hy926 cells, the distribution of which was indistinguishable from intrinsically expressed tPA. Its secretory dynamics were unique, with prolonged (> 5 minutes) retention of the tPA-GFP on the cell surface, appearing as fluorescent spots in two-thirds of the exocytosis events. The rapid disappearance (mostly by 250 ms) of a domain-deletion mutant of tPA-GFP possessing only the signal peptide and catalytic domain indicates that the amino-terminal heavy chain of tPA-GFP is essential for binding to the membrane surface. The addition of PAI-1 dose-dependently facilitated the dissociation of membrane-retained tPA and increased the amounts of tPA-PAI-1 high-molecular-weight complexes in the medium. Accordingly, suppression of PAI-1 synthesis in EA.hy926 cells by siRNA prolonged the dissociation of tPA-GFP, whereas a catalytically inactive mutant of tPA-GFP not forming complexes with PAI-1 remained on the membrane even after PAI-1 treatment. Our results provide new insights into the relationship between exocytosed, membrane-retained tPA and PAI-1, which would modulate cell surface-associated fibrinolytic potential.     

Prostacyclin expression by a continuous human cell line derived from vascular endothelium

Prostacyclin is primarily an endothelial cell product. It contributes to the important role of endothelium in maintaining the fluidity of blood by inhibiting platelet aggregation and by promoting vasodilation. Endothelial cells in culture tend to senesce, and the level of prostacyclin expression decreases. A permanent human cell line, EA.hy 926, derived from a fusion of primary endothelial cells with cells of a less differentiated line, has been found to sustain basal and stimulated levels of prostacyclin synthesis.     

Hyperglycemia and oxidative stress in cultured endothelial cells - a comparison of primary endothelial cells with an immortalized endothelial cell line

Diabetes mellitus is a major risk factor for the development of cardiovascular disease and oxidative stress plays an important role in this process. Therefore, we investigated the effects of hyperglycemia on the formation of reactive oxygen species (ROS) and nitric oxide/cGMP signaling in two different endothelial cell cultures. Human umbilical vein endothelial cells (HUVEC) and EA.hy 926 cells showed increased oxidative stress and impaired NO-cGMP signaling in response to hyperglycemia. The major difference between the two different cell types was the dramatic decrease in viability in HUVEC whereas EA.hy cells showed rather increased growth under hyperglycemic conditions. Starvation led to an additional substantial decrease in viability and increased superoxide formation in HUVEC. Both endothelial cell types, HUVEC and EA.hy 926, may be used as models for vascular hyperglycemia. However, high growth medium should be used to avoid starvation-induced oxidative stress and cell death.     

Altered Moesin and Actin Cytoskeleton Protein Rearrangements Affect Transendothelial Permeability in Human Endothelial Cells upon Dengue Virus Infection and TNF-α Treatment

It has been hypothesized that the host, viral factors, and secreted cytokines (especially TNF-α) play roles in the pathogenesis of secondary dengue infections. Mass spectrometry-based proteomic screening of cytoskeleton fractions isolated from human endothelial (EA.hy926) cells upon dengue virus (DENV) infection and TNF-α treatment identified 450 differentially altered proteins. Among them, decreased levels of moesin, actin stress fiber rearrangements, and dot-like formations of vinculin were observed with western blot analyses and/or immunofluorescence staining (IFA). In vitro vascular permeability assays using EA.hy926 cells, seeded on collagen-coated transwell inserts, showed low levels of transendothelial electrical resistance in treated cells. The synergistic effects of DENV infection and TNF-α treatment caused cellular permeability changes in EA.hy926 cells, which coincided with decreasing moesin levels and the production of abnormal organizations of actin stress fibers and vinculin. Functional studies demonstrated moesin overexpression restored transendothelial permeability in DENV/TNF-α-treated EA.hy926 cells. The present study improves the understanding of the disruption mechanisms of cytoskeleton proteins in enhancing vascular permeability during DENV infection and TNF-α treatment. The study also suggests that these disruption mechanisms are major factors contributing to vascular leakage in severe dengue patients.     

Fibrinolytic properties of a human endothelial hybrid cell line (Ea.hy 926)

The fibrinolytic characteristics of the endothelial hybrid cell line EA.hy 926, established by fusing a human umbilical vein endothelial cell with a human carcinoma cell line, were studied. The hybrid cell line produced large amounts of tissue-type plasminogen activator (t- PA), plasminogen activator inhibitor type 1, and a small amount of urokinase. All plasminogen activator present in conditioned medium was complexed with inhibitor because the cells secreted plasminogen activator inhibitor in excess over plasminogen activator and no activator activity was detectable in conditioned media by direct activity assays. t-PA activator activity was, however, demonstrable in conditioned media after treatment with sodium dodecyl sulfate, in agreement with t-PA antigen determinations. Increased plasminogen activator inhibitor activity could be induced by incubating the cells in the presence of endotoxin or microtubule inhibitors, whereas increased t-PA activity could be induced by microtubule inhibitors. Interleukin-1 had no effect. The fibrinolytic characteristics of the hybrid cell line were stable for at least 30 passages. The perpetual human hybrid cell line EA.hy 926 therefore may be a useful tool for the study of fibrinolysis in cultured endothelial cells.     

系统性血管炎抗内皮细胞抗体与抗中性粒细胞胞质抗体的相关性初探

目的以来源于大小血管内皮细胞的两种永生细胞株为底物,检测系统性血管炎血清中抗内皮细胞抗体(AECA),分析其与抗中性粒细胞胞质抗体(ANCA)的相关性。方法细胞酶联免疫吸附试验(Cyto-ELISA)法检测血清中AECA;间接免疫荧光法(IIF)及抗抗体结合内皮细胞表面的蛋白酶3(PR3)、抗MPO-ELISA检测血清中ANCA;IIF及抗PR3、抗MPO-ELISA检测细胞株中PR3及MPO;反转录—聚合酶链反应(RT-PCR)法检测细胞株中PR3及MPOmRNA。结果AECAEA(EA.hy926为底物所测AECA)阳性率33.6%(41/122),AECAHMEC(HMEC-1为底物所测AECA)37.7%(46/122),ANCA35.3%(43/122),AECAEA或AECAHMEC与ANCA串联诊断系统性血管炎敏感性分别为59.8%(73/122)或60.7%(74/122)。AECAEA与ANCA,AECAHMEC与ANCA分别行配对字2检验,差异均无显著性(P>0.05),符合率仅分别为49.2%及51.6%。EA.hy926和HMEC-1中蛋白水平及mRNA水平均无PR3和MPO的表达。结论EA.hy926与HMEC-1中无PR3、MPO蛋白水平的表达,ANCA与AECA可能是两种相互独立的抗体,串联检测可提高诊断敏感性。     

Drug Resistance of Endocardial Endothelial Cells is Related to Higher Endogenous ABCG2

Endocardial endothelial cells (EECs), when compared with endothelial cells of arteries and veins, possess higher resistance to apoptosis-inducing anticancer agents. The mechanism of this resistance property is unknown. We have investigated the molecular mechanism, which contributes to increased cell survival capacity in EECs. We explored whether the resistance to apoptosis is associated with the cellular expression of ATP-binding cassette transporters such as P-glycoprotein, MRP-1, and ABCG2. We used primary and immortalized porcine endocardial endothelial cells (PEECs and hTERT PEECs) and compared the results with that in porcine aortic endothelial cells (PAECs), left atrioventricular valve endothelial cells (PVECs), and human umbilical vein endothelial cell line (EA.hy926). FACS and immunoblot analysis revealed a significantly higher expression of ABCG2 in PEECs and hTERT PEECs compared to PAECs, PVECs, and EA.hy926. Using apoptosis-inducing anticancer agents such as doxorubicin and camptothecin, through chromatin condensation assay and immunoblot analysis, we demonstrated a higher resistance to apoptosis in EECs compared to PAECs, PVECs, and EA.hy926. Interestingly, resistance in EECs reversed in presence of ABCG2 specific inhibitor, fumitremorgin C. Our observations suggest that an inherently high expression of ABCG2 in EECs protects them against apoptosis in presence of anticancer agents.     

Statin-induced apoptosis of vascular endothelial cells is blocked by dexamethasone

Statins block de novo synthesis of cholesterol by inhibiting the enzyme, HMG CoA reductase. The product of this reaction, mevalonic acid, is also a precursor of isoprenoids, molecules required for the activation of signalling G-proteins, such as Ras. Signal transduction pathways involving Ras are important for cell survival and this may be why statins induce apoptotic death of several cell types. Given that statins are used to treat vascular disease, it is surprising that no studies have been conducted on vascular endothelial cells. For this reason, we have tested the effect of fluvastatin (FS) on the endothelial cell line EA.hy 926. Here we show that FS, at concentrations from 1 to 2 microM, blocks growth and induces apoptosis of the endothelial cell line, EA.hy 926. As considerable redundancy exists in cell signalling pathways for cell survival, toxicity of FS under more physiological conditions might be prevented by pathways that do not require Ras, such as those activated by adrenal or sex steroids. To test this hypothesis, first RT-PCR analysis was performed for nuclear receptor mRNA expression. This revealed the presence of mRNA for the androgen receptor (AR) and glucocorticoid receptor (GR). The effect of the AR agonist, dihydrotestosterone (DHT), and the GR agonist, dexamethasone (Dex), was then tested. Whilst DHT (100 nM) had no effect on FS-induced cell death, Dex (1 microM) blocked FS-induced apoptosis. Cell cycle analysis revealed that 24 h exposure to FS prevented cells from leaving G(1) and 24-48 h later a marked sub-G(1) peak was observed. Dex was able to reduce the sub-G(1) peak, but it failed to reduce accumulation of cells in G(1). Further studies revealed that, in addition to blocking FS-induced apoptosis, Dex was able to block apoptosis of EA.hy 926 cells induced by serum deprivation, tumour necrosis factor-alpha, oxidants, DNA damage and mitochondrial disruption. This study strongly suggests that glucocorticoids have a role to play in preventing vascular injury and they may provide a reason why statins are apparently not toxic to vascular endothelial cells in vivo.     

Permanent cell line expressing human factor VIII-related antigen established by hybridization.

A permanent human cell line, EA . hy 926, has been established that expresses at least one highly differentiated function of vascular endothelium, factor VIII-related antigen. This line was derived by fusing human umbilical vein endothelial cells with the permanent human cell line A549. Hybrid cells that survived in selective medium had more chromosomes than either progenitor cell type and included a marker chromosome from the A549 line. Factor VIII-related antigen can be identified intracellularly in the hybrids by immunofluorescence and accumulates in the culture fluid. Expression of factor VIII-related antigen by these hybrid cells has been maintained for more than 100 cumulative population doublings, including more than 50 passages and three cloning steps. This is evidence that EA . hy 926 represents a permanent line.     

Statins increase thrombomodulin expression and function in human endothelial cells by a nitric oxide-dependent mechanism and counteract tumor necrosis factor alpha-induced thrombomodulin downregulation.

Expression of functionally active thrombomodulin (TM) on the luminal surface of endothelial cells is critical for vascular thromboresistance. TM maintains thrombohemorrhagic homeostasis by forming a complex with thrombin, which subsequently loses its procoagulant properties and instead activates protein C. Acquired deficiency of endothelial TM is of particular pathophysiological significance in sepsis and related disorders. We show here that two different 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins), atorvastatin and simvastatin, strongly increase the expression and functional activity of TM in human umbilical vein endothelial cells, human coronary artery endothelial cells, and EA.hy926 endothelial cells. The increase in endothelial TM conferred by statin was prevented by the addition of mevalonic acid, geranylgeranyl-pyrophosphate, and nitric oxide scavenger, and was mimicked by the addition of a specific inhibitor of geranylgeranyl transferase, as well as by nitric oxide donors. Moreover, statin counteracted tumor necrosis factor alpha-induced downregulation of endothelial cell TM. The increase in endothelial cell TM activity in response to statin constitutes a novel pleiotropic (non-lipid-related) effect of these commonly used compounds, and may be of clinical significance in disorders where deficient endothelial TM and protein C activation play a pathophysiological role.