Role of protein kinase C in the expression of endothelin converting enzyme-1

Increased expression of endothelin converting enzyme-1 (ECE-1) is associated with diabetic nephropathy. The molecular mechanisms underlying this association, as yet unknown, possibly involve protein kinase C (PKC) pathways. In the present study, we examined the effects of high glucose and PKC activation on ECE-1 expression in primary human umbilical vein endothelial cells (HUVECs) and in HUVEC line (EA.hy926). Increasing glucose concentration, but not mannitol, from 5.5-22.2 mmol/liter for 3 d, enhanced prepro endothelin-1 (ET-1) mRNA expression, ET-1 levels, ECE-1 protein, and mRNA expressions by 7, 4, 20, and 2.6-fold, respectively. High glucose increased ECE-1 protein expression dose and time dependently. By Western blot analysis, PKC-beta1, -beta2, and -delta isoform levels were significantly increased relative to other isoforms when glucose level was increased. Treatment with Rottlerin, a PKC-delta isoform inhibitor, reduced significantly the glucose-induced ET-1 secretion, and ECE-1 protein expression, but (S)-13-[(dimethylamino)methyl]-10,11,14,15-tetrahydro-4,9:16,21-dimetheno 1H,1(3)H-dibenzo[e,k]pyrrolo[3,4-h] (1, 4, 3) oxadiaza-cyclohexadecene-1,3(2H)-dione or G?6976, specific PKC-beta and -alpha inhibitors, respectively, did not. Overexpression of PKC-delta but not PKC-alpha or -beta1 isoforms by adenovirus vector containing the respective cDNA in HUVECs incubated with 5.5 mmol/liter glucose, increased in parallel PKC proteins, and glucose-induced endothein-1 and ECE-1 protein expression by 4- to 6-fold. These results show that enhanced ECE-1 expression induced by hyperglycemia is partly due to activation of the PKC-delta isoform. Thus, inhibition of this PKC isoform may prevent diabetes-related increase in ET-1.     

Protein kinase C alpha promotes angiogenic activity of human endothelial cells via induction of vascular endothelial growth factor

AIMS: Protein kinase C (PKC) plays an important role in the regulation of angiogenesis. However, downstream targets of PKC in endothelial cells are poorly defined. METHODS AND RESULTS: mRNA expression of vascular endothelial growth factor (VEGF) was analysed by quantitative real-time RT-PCR in human umbilical vein endothelial cells (HUVEC) and HUVEC-derived EA.hy 926 cells. siRNA was used to knockdown PKC isoforms and VEGF. Matrigel tube formation assay was used to analyse the angiogenic activity of endothelial cells. Phorbol-12-myristate-13-acetate (PMA) enhanced the ability of HUVEC to organize into tubular networks when plated on Matrigel, a phenomenon that could be prevented by PKC inhibitors. PMA markedly increased the expression of VEGF in HUVEC and EA.hy 926 cells. The enhancement in VEGF expression was prevented by PKC inhibitors and by an inhibitor of the Erk1/2 pathway. PMA-induced tube formation was reduced by inhibition of the VEGF receptor kinase, or by VEGF knockdown. PMA led to an activation of PKC isoforms alpha, delta and epsilon in HUVEC. Knockdown of PKC alpha diminished PMA-induced VEGF expression and angiogenesis. Also endothelial progenitor cells isolated from human peripheral blood showed enhanced VEGF expression and improved angiogenic activity in response to PKC activation. Moreover, incubation of HUVEC with VEGF led to PKC alpha activation and PKC-dependent VEGF upregulation. CONCLUSIONS: PKC alpha activation promotes angiogenic activity of human endothelial cells. This is likely to be largely mediated by induction of VEGF. VEGF enhances its own expression via a PKC alpha-dependent positive feedback mechanism.     

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.     

Comparative expression profiling in primary and immortalized endothelial cells: changes in gene expression in response to hydroxy methylglutaryl-coenzyme A reductase inhibition.

Immortalized cell lines offer significant logistical advantages over primary cells when used for in-vitro studies. Immortalized cells may, however, exhibit important differences relative to their primary cell counterparts. In this study, microarrays were used to make a genome-wide comparison between primary human umbilical vein endothelial cells (HUVECs) and EA.hy926, an immortalized HUVEC cell line, in their baseline properties and in their response to inhibition of the mevalonate pathway with an inhibitor of hydroxy methylglutaryl-coenzyme A reductase (statin). HUVECs and EA.hy926 were incubated with control medium, atorvastatin, mevalonate, or a combination of atorvastatin and mevalonate for 24 h. Gene expression profiles were obtained in duplicates using Affymetrix Human Genome U133A 2.0 arrays (Santa Clara, California, USA). Probe-sets were selected according to the following criteria: a twofold or greater increase/decrease in atorvastatin-treated cells compared with untreated cells; a twofold or greater reversal of the effect of atorvastatin by combined treatment with atorvastatin and mevalonate; no significant change in gene expression in cells treated with mevalonate alone compared with untreated cells. Most genes that were expressed by untreated HUVECs, were also expressed by untreated EA.hy926 cells. EA.hy926 cells, however, constitutively expressed a large number of additional genes, many of which were related to cell cycle control and apoptosis. Atorvastatin induced differential expression (> or = twofold) of 103 genes in HUVECs (10 up, 93 down) and 466 genes in EA.hy926 cells (198 up, 268 down). Applying the above selection criteria, thrombomodulin and tissue plasminogen activator were up-regulated in both cell types, whereas, connective tissue growth factor, thrombospondin-1, and cysteine-rich angiogenic inducer 61 were down-regulated. In conclusion, EA.hy926 cells retain most of the characteristics of endothelial cells under baseline conditions as well as after treatment with atorvastatin. It is necessary, however, to carefully select and validate changes in genes that are the focus of studies when using EA.hy926 cells. While this cell line is highly useful in studies on some genes, including genes encoding molecules involved in regulating thrombohemorrhagic homeostasis, they appear to be less suited for studies focused on other genes, particularly those involved in the regulation of cell proliferation and apoptosis.     

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...     

Endothelin-2 synthesis is stimulated by the type-1 tumour necrosis factor receptor and cAMP: comparison with endothelin-converting enzyme-1 expression

ABSTRACT The synthesis of the vasoconstrictor peptide endothelin-2 (ET-2) is dependent on hydrolysis of the biologically inactive intermediate big ET-2 by an endothelin-converting enzyme (ECE). Here, mechanisms inducing ET-2 synthesis have been investigated using the human renal adenocarcinoma cell line (ACHN). Synthesis of ET-2 by ACHN cells was inhibited by phosphoramidon (IC(50( congruent with11 microM). To determine whether ET-2 synthesis occurs in parallel with the metallopeptidase ECE-1, a putative processing peptidase for big ET-2, changes in the levels of their mRNAs were compared by semi-quantitative RT-PCR under conditions causing the upregulation of ET-2 synthesis. Tumour necrosis factor-alpha (TNFalpha), forskolin and a cell-permeable cAMP analogue (dibutyryl cAMP) caused concentration-dependent increases in ET-2 synthesis. Combination of forskolin or dibutyryl cAMP with TNFalpha produced a significantly greater increase in ET-2 production than these agents alone, indicating that adenylate cyclase and TNFalpha induce ET-2 synthesis by separate signalling pathways. Studies using receptor selective TNFalpha mutants, (125(I-TNFalpha binding and TNF receptor mRNA showed that type-1 TNF receptors mediate the ET-2 response to TNFalpha. PreproET-2 mRNA levels were increased by TNFalpha at 1 h and 2 h, but returned to control levels at 4 h. Treatment with forskolin significantly increased preproET-2 mRNA levels after 1 h and 4 h. ACHN cells expressed ECE-1b and ECE-1c, but not the ECE-1a isoform of this peptidase. RT-PCR for the combined isoforms ECE-1b/c/d showed TNFalpha to increase mRNA levels at 2 h and 4 h. Forskolin had no effect on ECE-1b/c/d mRNA levels. Thus, expression of ET-2 and ECE-1b/c/d mRNAs in ACHN cells do not display the co-ordinated regulation observed with typical peptide prohormone processing enzymes and their substrates.     

Ursolic acid from the Chinese herb danshen (Salvia miltiorrhiza L.) upregulates eNOS and downregulates Nox4 expression in human endothelial cells

Danshen, the dried root of Salvia miltiorrhiza Bunge (Lamiaceae), is one of the most commonly used traditional Chinese medicines for cardiovascular indications. In EA.hy 926 cells, a cell line derived from human umbilical vein endothelial cells (HUVEC), an aqueous extract of danshen, and also a methanol extract of the plant, increased eNOS promoter activity, eNOS mRNA and protein expression, as well as endothelial NO production. A dichloromethane extract, in contrast, did not change eNOS gene expression. Thus, the active danshen constituent(s) responsible for eNOS upregulation is (are) hydrophilic and/or alcohol-soluble. One such compound is ursolic acid that significantly increased eNOS expression in EA.hy 926 cells and native HUVEC, and enhanced bioactive NO production measured in terms of its cGMP increasing activity. Other tested hydrophilic and alcohol-soluble compounds isolated from danshen had no effect on eNOS expression. Interestingly, ursolic acid also reduced the expression of the NADPH oxidase subunit Nox4 and suppressed the production of reactive oxygen species in human endothelial cells. Upregulation of eNOS and a parallel downregulation of Nox4 lead to an increase in bioactive NO. This in turn could mediate some of the beneficial effects of danshen. Ursolic acid is a prototypical compound responsible for this effect of the plant.     

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.     

Distinct cellular localization and regulation of endothelin-1 and endothelin-converting enzyme-1 expression in the bovine corpus luteum: implications for luteolysis.

Endothelin-1 (ET)-1 within the corpus luteum (CL) is rapidly up-regulated during natural or PGF(2 alpha)-induced luteolysis; however, such an increase was not observed at early luteal stage, when the CL is refractory to PGF(2 alpha). The mature and active form of ET-1 is derived from the inactive intermediate peptide, big ET-1, by ET-converting enzyme (ECE)-1. This study therefore examined the developmental and cell-specific expression of ECE-1 in bovine CL. A significant, 4-fold, elevation in ECE-1 expression (mRNA and protein levels) occurred during the transition of the CL from early to midluteal phase. Analysis using in-situ hybridization and enriched luteal cell subpopulations showed that both steroidogenic and endothelial cells of the CL expressed high levels of ECE-1 mRNA; prepro ET-1 mRNA, on the other hand, was only expressed by resident endothelial cells. These data suggest that luteal parenchymal and endothelial cells may cooperate in the biosynthesis of mature bioactive ET-1. In the mature CL, ECE-1 mRNA increase occurred both in steroidogenic and endothelial cells and was accompanied by a significant rise in ET-1 peptide. However, in contrast to ECE-1, prepro ET-1 mRNA levels were similar in early and midluteal-phase CL. Low ECE-1 levels during the early luteal phase, restricting the production of active ET-1, may explain why the immature CL is able to withstand PGF(2 alpha)-induced luteolysis.     

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.     

In situ protein Kinase C activity is increased in cultured fibroblasts from Type 1 diabetic patients with nephropathy

AIMS/HYPOTHESIS: To verify whether individual susceptibility to diabetic nephropathy resides in an intrinsic difference in Protein Kinase C (PKC) activity. METHODS: We compared the effect of different glucose concentrations on PKC activity, PKC isoform expression and diacylglycerol (DAG) content in cultured fibroblasts from 14 Type 1 diabetic patients who developed nephropathy with those in cells from 14 patients without nephropathy. We recruited 14 normal subjects as control patients. Forearm skin fibroblasts were cultured in either normal (5 mmol/l) or high (20 mmol/l) glucose concentrations. RESULTS: In normal glucose, in situ PKC activity was higher in Type 1 patients with nephropathy (10.1+/-1.4 pmol/min/mg protein; p<0.01) than in those without (6.8+/-0.8) and the normal control subjects (6.3+/-0.5). This difference was due to increased concentrations of PKCalpha isoform in the membrane fraction of fibroblasts from patients with nephropathy. DAG content was also higher in cells from Type 1 patients with nephropathy. Incubation in high glucose concentration caused a further increase in PKC activity and DAG content in quiescent fibroblasts from patients with diabetic nephropathy, with no significant changes in cells from diabetic patients without nephropathy and normal control subjects. CONCLUSION/INTERPRETATION: Differences in PKC activation could contribute to the individual susceptibility to renal damage in Type 1 diabetic patients.     

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.     

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。     

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.     

Human erythropoietin induces a pro-angiogenic phenotype in cultured endothelial cells and stimulates neovascularization in vivo

Hematopoietic and endothelial cell lineages share common progenitors. Accordingly, cytokines formerly thought to be specific for the hematopoietic system have been shown to affect several functions in endothelial cells, including angiogenesis. In this study, we investigated the angiogenic potential of erythropoietin (Epo), the main hormone regulating proliferation, differentiation, and survival of erythroid cells. Epo receptors (EpoRs) have been identified in the human EA.hy926 endothelial cell line by Western blot analysis. Also, recombinant human Epo (rHuEpo) stimulates Janus Kinase-2 (JAK-2) phosphorylation, cell proliferation, and matrix metalloproteinase-2 (MMP-2) production in EA.hy926 cells and significantly enhances their differentiation into vascular structures when seeded on Matrigel. In vivo, rHuEpo induces a potent angiogenic response in the chick embryo chorioallantoic membrane (CAM). Accordingly, endothelial cells of the CAM vasculature express EpoRs, as shown by immunostaining with an anti-EpoR antibody. The angiogenic response of CAM blood vessels to rHuEpo was comparable to that elicited by the prototypic angiogenic cytokine basic fibroblast growth factor (FGF2), it occurred in the absence of a significant mononuclear cell infiltrate, and it was not mimicked by endothelin-1 (ET-1) treatment. Taken together, these data demonstrate the ability of Epo to interact directly with endothelial cells and to elicit an angiogenic response in vitro and in vivo and thus act as a bona fide direct angiogenic factor.