Histamine stimulates prostacyclin synthesis in cultured human umbilical vein endothelial cells

Human venous endothelial cells synthesize prostacyclin (PGI₂) in response to treatment with histamine. The amount of PGI₂ produced is proportional to the histamine concentration over the range of 10^?7 to 10^?5 M, with a maximal response at 2–5 × 10^?6 M. PGI₂ synthesis occurs as a burst lasting less than 3 minutes after histamine addition. The H1 histamine receptor antagonist pyrilamine causes an 87% inhibition of PGI₂ synthesis, whereas the H2 antagonist cimetidine gives no significant inhibition, suggesting that PGI₂ synthesis in response to histamine is mediated by an H1 receptor.     

睾酮对人血管内皮细胞产生NO、tPA和PAI-1的影响

目的:观察不同浓度睾酮对人血管内皮细胞生长、产生舒张因子及纤溶活性的影响.方法:体外培养人脐静脉内皮细胞(HUVEC),分为五个浓度睾酮组及单纯培养基对照组.做MTT实验观察睾酮对HUVEC生长的影响;还原酶法测定各组HUVEC释放NO量;ELISA法测定各组培养基中纤溶酶原激活物(tPA)及其抑制物(PAI-1)含量.结果:3×10-10mol/L-3×10-8mol/L睾酮组与对照组相比细胞生长良好,无明显差别;而大于生理剂量的两组(3×10-6～3×10-1mol/L)3 d后细胞生长明显受到抑制(P＜0.05).各浓度睾酮组产生NO量与对照组无明显区别.而3×10-10 mol/L～3×10-8 mol/L睾酮组tPA含量明显高于对照组(P＜0.01);大剂量组tPA产生明显减少(P＜0.01).所有实验组的PAI-1含量均明显降低.结论:生理及略低于生理剂量的睾酮对HUVEC生长及释放NO无不利影响,且增加纤溶活性.说明生理剂量睾酮对血管内皮功能、心血管系统有一定的保护作用,有利于防止动脉粥样硬化的发生、发展.     

17 alpha-ethinylestradiol decreases production and release of prostacyclin in cultured human umbilical vein endothelial cells

Human umbilical vein endothelial cells (HUVEC) were incubated with the estrogen 17 alpha-Ethinylestradiol (30 ng/ml) in order to examine its regulating influence on synthesis and release of prostacyclin (PGI2). ATP (1 mg/ml) was used to stimulate PGI2-production through the purine receptors. We demonstrated that this level of estrogen decreases PGI2-synthesis by 11% and PGI2-release by 32% within 300 sec. Longer incubation times (48 hrs) resulted in the same inhibitory effect. Intracellular ATP content and methyl-3H-thymidine uptake demonstrated that the decrease of prostacyclin-concentration is not caused by reduced viability of the cells but by a direct inhibitory effect on prostacyclin synthesis and release.     

Synthesis of protein C in human umbilical vein endothelial cells.

By monitoring the activation of protein C and the regulation of factor Xa-catalyzed thrombin formation by the activated protein C (APC) on the surface of human umbilical vein endothelial cells (HUVEC), we found that functional protein C was synthesized in cultured HUVEC and expressed thereon in the presence of vitamin K. Furthermore, without exogenously added protein S, time-dependent and saturable accumulation of APC (20 fmol APC/10(5) cells) on the surface of HUVEC was observed. During prothrombin activation by the complex of membrane-bound factor Xa and endogenous factor Va formed on the surface of HUVEC, APC was generated, and the rate of thrombin formation decreased. Treatment of HUVEC with an antibody that inhibits the APC-catalyzed inactivation of endogenous factor Va clearly quenched the activity of surface-associated APC. Immunostaining of HUVEC with a horseradish peroxidase (HRP)-conjugated antibody that solely recognizes human protein C confirmed the presence of protein C on the surface of HUVEC. Northern blot analysis revealed that an about 1.8 kb mRNA species derived from HUVEC was hybridized with 32P-labeled protein C cDNA, as in the case of those from HepG2, which are known to synthesize normal protein C. The increase in the amount of protein C mRNA in HUVEC in parallel with cell growth provided supporting evidence for the synthesis of protein C during the culture of HUVEC. These results indicate that blood coagulation is regulated by endogenously generated and activated protein C, together with or without protein S, through inactivation of factor Va on the surface of endothelial cells.     

Metabolic responses induced by thrombin in human umbilical vein endothelial cells

Metabolic responses induced by thrombin in human umbilical vein endothelial cells (HUVECs) were investigated by using the cytosensor technique. Thrombin increased the extracellular acidification rate of endothelial cells, measured as an index of metabolic activity with a cytosensor microphysiometer, in a concentration-dependent fashion with an EC(50) of 1.27+/-0.59 IU/ml, which was abolished by the MAP kinase inhibitor PD98059. When intracellular Ca(2+) was chelated or PKC was inactivated, PD98059 failed to abolish the thrombin-induced acidification rate response in HUVECs. In addition, the tyrosine kinase inhibitor genistein, PKC inhibitor calphostin C, and Na(+)/H(+)exchanger antagonist MIA also partly inhibited thrombin-induced acidification rate responses. It is suggested that thrombin stimulated rapid metabolic responses via MAP kinase in HUVECs, which are calcium- and PKC-dependent.     

Lysophosphatidylglycerol stimulates chemotactic migration and tube formation in human umbilical vein endothelial cells

In this study, we observed that lysophosphatidylglycerol (LPG) stimulates extracellular signal-regulated kinase (ERK) in human umbilical vein endothelial cells (HUVECs). LPG-stimulated ERK activity was not inhibited by pertussis toxin (PTX), indicating PTX-sensitive G-proteins-independent manner. In terms of functional aspect, LPG induced chemotactic migration of HUVECs in a PTX-insensitive manner. Preincubation of HUVECs with an ERK inhibitor (PD98059) completely inhibited LPG-induced chemotactic migration, suggesting the crucial role of ERK in the process. LPG-induced ERK activation and chemotactic migration in HUVECs were not affected by an lysophosphatidic acid receptor-selective antagonist (Ki16425), indicating lysophosphatidic acid receptors-independency. We also found that LPG stimulated tube formation in HUVECs. Taken together we suggest that LPG stimulates HUVECs and result in chemotactic migration and tube formation, suggesting a new aspect of LPG as a modulator of endothelial cell functioning.     

Stimulation of cell growth and inhibition of prostacyclin production by heparin in human umbilical vein endothelial cells

We characterized human umbilical vein (HUV) endothelial cells as to cell growth and prostacyclin production to get a better understanding of the properties of endothelial cells. Endothelial cell growth supplement (ECGS) and basic fibroblast growth factor (FGF) stimulated HUV endothelial cell growth. Heparin further enhanced the cell growth stimulated by ECGS, but not the cell growth stimulated by FGF or in the absence of these growth factors. In the presence of ECGS, the prostacyclin-producing capacity of the cells was inhibited by heparin. However, in the presence of FGF of in the absence of growth factors, heparin did not inhibit prostacyclin production. Therefore, it is likely that there is a specific correlation between heparin and growth factors for endothelial cells in the blood vessel to maintain nonthrombogenicity properly. Heparin-treated cultures may not be suitable for some examinations of prostacyclin production by vascular endothelial cells.     

红杉醇对高糖诱导的人脐静脉内皮细胞NOX4、eNOS表达的影响

目的：观察红杉醇（Scq）对高糖诱导的人脐静脉内皮细胞（HUVECs）损伤的保护作用及机制。方法：原代培养HUVECs,红杉醇（0．1,1,10μmol/L）预处理1h后,30mmol/L葡萄糖诱导内皮细胞损伤。5-溴脱氧尿嘧啶核苷（BrdU）掺入法检测细胞增殖,流式细胞术检测细胞周期,2’7’-二乙酰二氯荧光素（DCFH-DA）免疫荧光法检测细胞内活性氧簇（R0s）水平,比色法检测细胞-氧化氮（NO）、丙二醛（MDA）及过氧化氢（H202）水平,real-timePCR和Westernblot检测细胞内皮型一氧化氮合酶（eNos）及NADPH氧化酶4（NOX4）mRNA和蛋白表达。结果：Seq预处理1h后能明显减轻高糖诱导的血管内皮细胞损伤,促进细胞增殖,降低胞内NOX4的表达及ROS、MDA及H202水平,上调eNOS的表达及NO水平。结论：Seq对高糖诱导的内皮细胞损伤具有一定的保护作用,其机制可能与其抗氧化、上调eNOS的表达有关。     

Histamine-induced phosphoinositide metabolism in cultured human umbilical vein endothelial cells. Association with thromboxane and prostacyclin release

Histamine stimulation of cultured human umbilical vein endothelial cells induced dose- and time-dependent increases in glycerophosphoinositol (GroPIns), inositol-1-phosphate (InsP), inositolbisphosphate (InsP2) and inositoltrisphosphate (InsP3) in addition to release of thromboxane A2 and prostacyclin. Increases in InsP2 and InsP3 were immediate while increases in GroPIns and InsP occurred only after 1 min. Thromboxane A2 and prostacyclin release paralleled GroPIns and InsP production. The data indicate that, in endothelial cells, histamine evokes early hydrolysis of polyphosphoinositides, and that subsequent mobilization of arachidonic acid for thromboxane and prostacyclin synthesis involves both deacylation and phosphodiesteratic cleavage of phosphatidylinositol.     

解脲脲原体诱导脐静脉血管内皮细胞凋亡的研究

目的通过探讨解脲脲原体(Ureaplasma urealyticum,UU)诱导人脐静脉内皮细胞(human umbilicalvein endothelial cells;HUVEC)凋亡的情况,揭示妊娠期间UU垂直传播影响胚胎发育的可能机制。方法不同剂量血清4型UU标准菌株刺激体外培养的HUVEC,通过Annexin-V.FITC/PI双染流式细胞术和DNA Ladder实验观察细胞凋亡情况。结果对照组细胞凋亡率小于各实验组(P<0.01~0.05),其凋亡率和刺激剂量、时间之间呈现一定的剂量-时间-效应关系(P>0.05)。结论UU可诱导人脐静脉内皮细胞凋亡,提示妊娠期间UU感染可能通过诱导脐静脉内皮细胞凋亡破坏胎盘屏障而影响胚胎发育。     

Organizational behavior of human umbilical vein endothelial cells

Culture conditions that favor rapid multiplication of human umbilical vein endothelial cells (HUV-EC) also support long-term serial propagation of the cells. This is routinely achieved when HUV-EC are grown in Medium 199 (M-199) supplemented with fetal bovine serum (FBS) and endothelial cell growth factor (ECGF), on a human fibronectin (HFN) matrix. The HUV-EC can shift from a proliferative to an organized state when the in vitro conditions are changed from those favoring low density proliferation to those supporting high density survival. When ECGF and HFN are omitted, cultures fail to achieve confluence beyond the first or second passage: the preconfluent cultures organize into tubular structures after 4-6 wk. Some tubes become grossly visible and float in the culture medium, remaining tethered to the plastic dish at either end of the tube. On an ultrastructural level, the tubes consist of cells, held together by junctional complexes, arranged so as to form a lumen. The smallest lumens are formed by one cell folding over to form a junction with itself. The cells contain Weibel-Palade bodies and factor VIII-related antigen. The lumens contain granular, fibrillar and amorphous debris. Predigesting the HFN matrix with trypsin (10 min, 37 degrees C) or plasmin significantly accelerates tube formation. Thrombin and plasminogen activator had no apparent effect. Disruption of the largest tubes with trypsin/EDTA permits the cells to revert to a proliferative state if plated on HFN, in M-199, FBS, and ECGF. These observations indicate that culture conditions that do not favor proliferation permit attainment of a state of nonterminal differentiation (organization) by the endothelial cell. Furthermore, proteolytic modification of the HFN matrix may play an important role in endothelial organization.     

Comparison of leukotriene A4 metabolism into leukotriene C4 by human platelets and endothelial cells.

Leukotriene (LT) A4 metabolism was studied in human platelets and endothelial cells, since both cells could be involved in transcellular formation of LTC4. Upon addition of exogenous LTA4, both cells produced LTC4 as a major metabolite at various incubation times, and no LTB4, LTD4, or LTE4 was detected. Kinetic studies revealed a higher apparent Km for LTA4 in endothelial cells as compared to platelets (5.8 microM for human umbilical vein endothelial cells (HUVEC) versus 1.3 microM for platelets); platelets were more efficient in this reaction with a higher Vmax (174 pmol/mg protein/min) versus 15 pmol/mg protein/min in HUVEC. The formation of LTC4 and corresponding kinetic parameters were not modified when platelets or endothelial cells were stimulated by thrombin prior to or simultaneously with the addition of LTA4. In both cells LTC4 synthase activity was not modified by repeated addition of LTA4 showing that it is not a suicide-inactivated enzyme. Furthermore, in platelets and endothelial cells, the enzyme activity was localized in the membrane fraction and was distinct from cytosolic glutathione-S-transferases. Platelet membrane fractions showed apparent Km values of 31 microM and 1.2 mM for LTA4 and GSH, respectively. Inhibition of LTC4 formation from platelets and endothelial cells preparations by S-substituted glutathione derivatives was correlated to the length of the S-alkyl chain. The same substances inhibited cytosolic glutathione-S-transferases with significantly lower IC50, confirming the distinct nature of the two enzymes. These results show that platelets and HUVEC possess similar enzymes for the production of LTC4 from LTA4; however, platelets seem to have a higher efficiency than HUVEC in performing this reaction.     

Antiapoptotic effect of ouabain on human umbilical vein endothelial cells

The present study investigates the effect of ouabain on caspase-3 activation in human umbilical vein endothelial cells (HUVEC). Ouabain (EC(50) 20 nM) reduced caspase-3 activity in HUVEC treated for 24h in a medium deprived of fibroblast growth factor (FGF). Incubation for 5h in the absence of both FGF and serum produced an increase in caspase-3 activity that was completely abolished by 100 nM ouabain. Pretreatment with the phosphatidylinositol 3 kinase (PI-3K) inhibitor, wortmannin, prevented the protective effect of ouabain against serum deprivation. Furthermore, Western blotting analysis revealed an increase in phosphorylation of extracellular signal-regulated kinases (ERK-1 and ERK-2) induced by 100nM ouabain in serum-deprived cells. In accord, pretreatment of HUVEC with PD98059, inhibitor of the ERK pathway, abrogated the effect of ouabain. Our results show that ouabain has an antiapoptotic effect on HUVEC through the activation of PI-3K and ERK dependent pathways.     

Differential effects of phorbol ester and diacylglycerols on inositol phosphate formation in C62B glioma cells

Application of acetylcholine (ACh) to C62B glioma cells results in a rapid release of inositol phosphates. Since this response is transient, we evaluated the possible role of protein kinase C (PKC) in its desensitization. Pretreatment with 100 nM phorbol 12,13-dibutyrate (PDBu) significantly inhibited ACh-induced accumulation of [3H]inositol mono-, bis-, and trisphosphates. However, interpretation of this result as proof of PKC involvement was complicated by the failure of 1,2-dioctanoylglycerol, 1,2-didecanoylglycerol, or 1-oleoyl-2-acetylglycerol pretreatments to mimic the phorbol ester effect. Further evidence against PKC involvement was obtained using the PKC inhibitor sphingosine; PDBu inhibition of inositol phosphate formation was not reversed by sphingosine pretreatments at concentrations which blocked ACh-stimulated PKC activation of inositol trisphosphate phosphatase activity. These results suggest that there may be phorbol effects not mediated by PKC.     

Cytotoxic and genotoxic effects of multi-wall carbon nanotubes on human umbilical vein endothelial cells in vitro

Carbon nanomaterials have multiple applications in various areas. However, it has been suggested that exposure to nanoparticles may be a risk for the development of vascular diseases due to injury and dysfunction of the vascular endothelium. Therefore, in the present study, the cytotoxic and genotoxic effects of multi-wall carbon nanotubes (MWCNTs) on human umbilical vein endothelial cells (HUVECs) were evaluated. Optical and transmission electronic microscopy (TEM) study showed that MWCNTs were able to enter cells rapidly, distribute in the cytoplasm and intracellular vesicles and induce morphological changes. Exposure to MWCNTs reduced the viability of HUVECs, and induced apoptosis in HUVECs. Furthermore, MWCNTs could cause DNA damage as indicated by the formation of γH2AX foci. MWCNTs also affected cellular redox status, e.g., increasing intracellular reactive oxygen species (ROS) and malondialdehyde (MDA) levels, as well as altering superoxide dismutase (SOD) activity and glutathione peroxidase (GSH-Px) levels. On the other hand, the free radical scavenger N-acetyl-l-cysteine (NAC) preincubation can inhibit the cytotoxic and genotoxic effects of MWCNTs. Taken together, these results demonstrated that MWCNTs could induce cytotoxic and genotoxic effects in HUVECs, probably through oxidative damage pathways.     

Glycosphingolipids of human umbilical vein endothelial cells and smooth muscle cells

Glycosphingolipids (GSLs) represent an important class of immunogens and receptors. Although cell surface antigens and receptors of endothelial cells (ECs) have been the subject of extensive biochemical investigation, no information is available about their GSLs. We report here the characterization by chromatographic and immunological techniques of GSLs of cultured human umbilical vein ECs and, for comparison, umbilical vein smooth muscle cells (SMCs). The most abundant neutral GSLs of both cell types were lactosylceramide, Gb3, and Gb4, and both cells contained complex lacto and globo series compounds. Immunostaining revealed that ECs, but not SMCs, contained long chain GSLs bearing a type 2 blood group H determinant. ECs also contained more long chain GSLs bearing an unsubstituted terminal lactosamine structure than SMCs. Labeling with galactose oxidase/NaB3H4 demonstrated that neutral glycolipids that contained three or more sugars were accessible on the cell surface. The major gangliosides of both cell types were GM3 and IV3NeuAcnLc4. Immunostaining following neuraminidase treatment revealed that most of the long chain gangliosides in both types of cells contained a lacto core structure, and that ganglio series compounds were more abundant in SMCs than ECs. Gangliosides that contain a polyfucosyllactosamine core and a globo core were also present in both cell types. These results demonstrate that endothelial and smooth muscle cells contain a large diversity of GSL structures, and provide the basis for investigation of the role of these GSLs as cell surface antigens and receptors for blood components.