Proteoglycans from human umbilical vein endothelial cells

Human umbilical vein endothelial cells were incubated with [35S]sulphate and investigated for their proteoglycan production. By gel chromatography, ion-exchange chromatography and CsCl density-gradient centrifugation we obtained preparative amounts of the endothelial proteoheparan sulphate HSI and of proteochondroitin sulphate from the conditioned medium of mass-cultured human umbilical vein endothelial cells. Approximately 90% of the 35S-labeled material in the endothelial cell conditioned medium was proteochondroitin sulphate. This molecule, with a molecular mass of 180-200 kDa, contains four side-chains of 35-40 kDa and a core protein of 35-40 kDa. Two proteoheparan sulphate forms (HSI and HSII) from the conditioned medium were distinguished by molecular mass and transport kinetics from the cell layer to the medium in pulse-chase experiments. One major form (HSI), with an approximate molecular mass of 160-200 kDa a core protein of 55-60 kDa and three to four polysaccharide side-chains of 35 kDa each, was found enriched in the cellular membrane pellet. Another proteoheparan sulphate (HSII), with polysaccharide moieties of 20 kDa, is enriched in the subendothelial matrix (substratum).     

Possible mechanisms underlying the cholesterol-raising effect of the coffee diterpene cafestol

Cafestol, a coffee diterpene present in unfiltered coffee brews, potently raises serum lipids in humans. The mechanism through which this dietary compound influences liporotein metabolism is largely unknown. Unravelling the mechanism of action might lead to new insights into the regulation of serum cholesterol levels in humans. This review summaries ways in which cafestol may act on serum lipids.     

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.     

Surface glycoproteins of cultured human umbilical vein endothelial cells

Radioactive surface-specific and metabolic labeling techniques were used to characterize the surface glycoprotein pattern of cultured human endothelial cells. Electrophoretic analysis of whole cells, surface labeled either by the galactose oxidase/sodium borotritide or the periodate/sodium borotritide method, revealed several major polypeptides in the Mr region of ca 40-220. During primary culture, the surface labeling pattern showed no changes related to cell density or to the establishment of confluence. A slightly different polypeptide profile was, however, seen when primary culture cells were labeled as an intact monolayer and not in suspension. On the other hand, in cells from later passages, when compared to their parental cells of early passages, there was a distinct intensification of polypeptides with Mr 155 and 90.     

Proteomic study of human umbilical vein endothelial cells in culture

The endothelium is a single layer of cells lining the inside face of all blood vessels. It constitutes a major metabolic organ which is critically involved in the generation and the regulation of multiple physiological and pathological processes such as coagulation, hemostasis, inflammation, atherosclerosis, angiogenesis and cancerous metastasis dissemination. In order to increase our knowledge about the protein content and the main biological pathways of human vascular endothelial cells, we have undertaken the proteomic analysis of the most explored present endothelial cell model, i.e. primocultures of human umbilical vein endothelial cells (HUVECs). Using low levels of protein loads (~ 30 nug), the association of two-dimensional electrophoresis with matrix-assisted laser desorption/ionization-time of flight mass spectrometry, liquid chromatography-tandem mass spectrometry and database interrogations allowed us to identify 53 proteins of suspected endothelial origin in quiescent HUVECs. Beside cytoskeletal proteins such as actin, tubulin, tropomyosin and vimentin, we identified various proteins more especially implicated in cellular motility and plasticity (e.g. cofilin, F-actin capping protein and prefoldin), in regulation of apoptosis and senescence (protease inhibitor 9, glucose related proteins, heat shock proteins, thioredoxin peroxidase, nucleophosmin) as well as other proteins implicated in coagulation (annexin V, high mobility group protein), antigen presentation (valosin containing protein and ubiquitin carboxyl terminal hydrolase isozyme L1) and enzymatic capabilities (glutathione-S-transferase, protein disulfide isomerases, lactate deshydrogenase). The presented annotated 2-D maps of HUVECs will be soon available on the web at http://www. huvec.com.     

Antiangiogenic effect of rosiglitazone is mediated via peroxisome proliferator-activated receptor gamma-activated maxi-K channel opening in human umbilical vein endothelial cells

Recent evidence shows that peroxisome proliferator-activated receptor gamma (PPARgamma) ligands induce the antiangiogenic effect in endothelial cells and tumors. In the present study, we elucidated the involvement of maxi-K channel activation in the antiangiogenic effect of rosiglitazone, a well known PPARgamma ligand in human umbilical vein endothelial cells. We found that the antiangiogenic effects of rosiglitazone were reversed by either bisphenol A diaglycidyl ether, a PPARgamma antagonist, or iberiotoxin, a maxi-K channel blocker. Knockdown of maxi-K channel expression also reversed the antiangiogenic effects. Iberiotoxin reversed the rosiglitazone-induced hyperpolarization while having no effect on the endogenous PPARgamma activation, suggesting that rosiglitazone activates maxi-K channel via PPARgamma. In the rosiglitazone-induced antiangiogenic process, endothelial nitric-oxide synthase-Ser1179 phosphorylation and NO production were significantly elevated, and treatment with the NOS inhibitor N(G)-monomethyl-L-arginine acetate abolished the antiangiogenic and apoptotic effects of rosiglitazone, indicating NO as a key mediator of the rosiglitazone actions. In conclusion, rosiglitazone significantly inhibited VEGF165-induced angiogenesis by a proapoptotic mechanism via PPARgamma-mediated NO production, followed by maxi-K channel opening.     

Myricetin ameliorates high glucose-induced endothelial dysfunction in human umbilical vein endothelial cells

Endothelial dysfunction is recognized as the initial detectable stage of cardiovascular disease, a serious complication of diabetes. In this study, we evaluated effects of myricetin on high glucose (HG)-elicited oxidative damage in human umbilical vein endothelial cells (HUVECs). The cells were pre-incubated with myricetin and then treated with HG to induce apoptosis. The effect of myricetin on viability was investigated by MTT assay. The levels of lipid peroxidation (LPO) were determined by thiobarbituric acid (TBA) method. The protein expression of Bax, Bcl-2 and caspase-3 was measured by western blot analysis. Moreover, the effect of myricetin on total antioxidant capacity (TAC) and total thiol molecules was also determined. Our results showed that myricetin was able to markedly restore the viability of endothelial cells under oxidative stress. Myricetin reduced HG-caused increase in LPO levels. Also, TAC and total thiol molecules were notably elevated by myricetin. Incubation with myricetin decreased the protein expression levels of Bax, whereas it increased the expression levels of the Bcl-2, compared with HG treatment alone. Furthermore, myricetin significantly decreased cleaved caspase-3 protein expression. It is concluded that myricetin may protect HUVECs from oxidative stress induced by HG via increasing cell TAC and reducing Bax/Bcl-2 protein ratio, and caspase-3 expression.     

Vascular endothelial growth factor upregulates follistatin in human umbilical vein endothelial cells

Vascular endothelial growth factor (VEGF), plays a key role in angiogenesis. Many endogenous factors can affect angiogenesis in endothelial cells. VEGF is known to be a strong migration, sprouting, survival, and proliferation factor for endothelial cells during angiogenesis in endothelial cells. Searching for novel genes, involved in VEGF signaling during angiogenesis, we carried out differential display polymerase chain reaction on RNA from VEGF-stimulated human umbilical vein endothelial cells (HUVECs). In this study, follistatin (FS) differentially expressed in VEGF-treated HUVECs, compared with controls. Addition of VEGF (10 ng/mL) produced an approximately 11.8-fold increase of FS mRNA. FS or VEGF produced approximately 1.8- or 2.9-fold increases, respectively, in matrix metalloproteinase-2 (MMP-2) secretion for 12 h, compared to the addition of a control buffer. We suggest that VEGF may affect the angiogenic effect of HUVECs, through a combination of the direct effects of VEGF itself, and the indirect effects mediated via induction of FSin vitro.     

Thrombin-induced ATP release from human umbilical vein endothelial cells

ATP and its degradation products play an important role as signaling molecules in the vascular system, and endothelial cells are considered to be an important source of nucleotide release. To investigate the mechanism and physiological significance of endothelial ATP release, we compared different pharmacological stimuli for their ability to evoke ATP release from first passage cultivated human umbilical vein endothelial cells (HUVECs). Agonists known to increase intracellular Ca(2+) levels (A23187, histamine, thrombin) induced a stable, non-lytic ATP release. Since thrombin proved to be the most robust and reproducible stimulus, the molecular mechanism of thrombin-mediated ATP release from HUVECs was further investigated. ATP rapidly increased with thrombin (1 U/ml) and reached a steady-state level after 4 min. Loading the cells with BAPTA-AM to capture intracellular calcium suppressed ATP release. The thrombin-specific, protease-activated receptor 1 (PAR-1)-specific agonist peptide TFLLRN (10 μM) fully mimicked thrombin action on ATP release. To identify the nature of the ATP-permeable pathway, we tested various inhibitors of potential ATP channels for their ability to inhibit the thrombin response. Carbenoxolone, an inhibitor of connexin hemichannels and pannexin channels, as well as Gd(3+) were highly effective in blocking the thrombin-mediated ATP release. Specifically targeting connexin43 (Cx43) and pannexin1 (Panx1) revealed that reducing Panx1 expression significantly reduced ATP release, while downregulating Cx43 was ineffective. Our study demonstrates that thrombin at physiological concentrations is a potent stimulus of endothelial ATP release involving PAR-1 receptor activation and intracellular calcium mobilization. ATP is released by a carbenoxolone- and Gd(3+)- sensitive pathway, most likely involving Panx1 channels.     

Homocysteine uptake by human umbilical vein endothelial cells in culture

The characteristics of the uptake of L-homocysteine by cultures of human umbilical vein endothelial cells have been examined. Uptake occurred by Na(+)-dependent and Na(+)-independent systems, but was essentially independent of the pH of the uptake medium. The Na(+)-independent system corresponded to system L, being totally inhibited by the presence of beta-2-aminobicyclo(2,2,1)heptane-2-carboxylic acid (BCH) a system L analogue. It was concluded on the basis of starvation experiments coupled with failure to detect any inhibition in the presence of 2-methylaminoisobutyric acid (MeAIB), a system A analogue, that the Na(+)-dependent uptake was wholly accounted for by system ASC. The kinetic properties of systems L and ASC were determined by omitting Na+ from the uptake medium and incorporating BCH in the medium, respectively. It has been concluded on the basis of the inhibitory effects of a number of amino acids that uptake of homocysteine occurs by those systems which transport cysteine.     

Thrombin-induced vimentin phosphorylation in cultured human umbilical vein endothelial cells

Cultured human umbilical vein endothelial cells were stimulated with thrombin (1 unit/ml) for 15-30 s and then lysed with a solution of Triton X-100 containing [gamma-32P]adenosine triphosphate. Thrombin-stimulated human umbilical vein endothelial cells showed an enhanced incorporation of 32P into at least 12 different proteins as compared to control cells treated similarly. The observed enhanced phosphorylation required the active site of thrombin because diisopropylphosphoryl-thrombin had no effect on the level of phosphorylation. The molecular weight of one of the phosphoproteins was similar to that of the intermediate filament protein vimentin (55-60 kDa), a major protein in endothelial cells. This 59-kDa protein was Triton X-100-insoluble and reacted on a Western blot with antibody raised in guinea pig against Chinese hamster ovary cell vimentin. Addition of the anti-vimentin antibody to the thrombin-stimulated, phosphorylated lysate immuno-precipitated a single 32P-labeled protein (59 kDa). These results demonstrate that thrombin rapidly stimulates the phosphorylation of vimentin in cultured endothelial cells and links thrombin stimulation to the phosphorylation of a cytoskeletal protein.     

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.     

Transport of L-cystine in human umbilical vein endothelial cells in culture

The uptake of L-cystine into cultured human umbilical vein endothelial cells has been shown to occur by a Na⁺-independent system which is inhibited by L-glutamate and L-homocysteine, but not by other amino acids. It is likely that the system transporting L-cystine is shared by L-glutamate. Thiol groups associated with membrane bound components appear to be essential for L-cystine uptake but it is not yet evident whether these constitute an integral part of the transporterper se.     

Up-regulation of thrombomodulin in human umbilical vein endothelial cells in vitro

Previous studies have shown that thrombomodulin (TM) on endothelial cells is down-regulated by endotoxin, interleukin-1 beta (IL-1 beta), and tumor necrosis factor (TNF). This loss of anti-coagulant potential is thought to be related to the hypercoagulable state in sepsis, inflammation, and cancer. The current studies describe up-regulation of TM in human umbilical vein endothelial cells (HUVECs) by several compounds as judged by increased surface cofactor activity, surface TM antigen, and TM mRNA levels. Surface TM activity was increased by active phorbol esters (10(-8) M, 24-48 h), analogs of cAMP (1-10 mM, 4 h), and forskolin (10(-5) M, 24-48 h). Up-regulation of TM in HUVECs by 4 beta-phorbol 12-myristate 13-acetate (PMA) and dibutyryl cAMP (dBcAMP) was due to de novo synthesis of TM protein resulting from increased TM mRNA levels. The results suggest that protein kinase C and protein kinase A may be involved in cellular regulatory mechanisms for TM expression. In addition, PMA effects on surface TM activity are biphasic, with an initial reduction followed by a significant enhancement. Hence, we propose that compounds capable of increasing intracellular cAMP concentrations in HUVECs may be useful in preventing thrombosis by increasing the anti-thrombotic properties of endothelial cells.     

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.     

Transglutaminase-mediated cross-linking of fibrinogen by human umbilical vein endothelial cells

The interaction of endothelial cells with soluble or substrate-immobilized 125I-labeled fibrinogen (125I-FGN) was analyzed. Binding experiments involved incubation of 125I-FGN with cell suspensions at 4 degrees C. Bound ligand was quantitated by centrifugation of cells through silicone oil followed by scintillation analysis of the cell pellet. Calcium-dependent binding of 125I-FGN reached a maximum after 3 h and represented about 60% of the total. Half-maximal saturation occurred at 60 nM, and about 9 x 10(4) molecules were bound/cell at saturation (approximately 100 nM). Calcium-dependent binding was completely inhibited by unlabeled fibrinogen, partially inhibited by a monoclonal antibody (7E3) against glycoprotein IIb-IIIa, but not inhibited by fibrinogen fragments D or E, an anti-glycoprotein IIIa polyclonal antibody, or the Arg-Gly-Asp-Ser tetrapeptide. In contrast, the Arg-Gly-Asp-Ser tetrapeptide as well as the monoclonal antibody 7E3 markedly inhibited attachment of endothelial cells to substrate-immobilized fibrinogen, whereas fragment D or E did not. Both in suspension and monolayer, the 125I-FGN underwent cross-linking involving principally the A alpha chain. The transglutaminase inhibitors putrescine, histamine, and cystamine interfered with 125I-FGN binding and cross-linking by suspended cells. Since cross-linking in suspension was limited to bound 125I-FGN and since transglutaminase activity was not detectable in the binding buffer, cross-linking may have been mediated by a cell-associated transglutaminase.     

Isolation of human umbilical vein endothelial cells (HUVEC)

Angiogenesis is a complex multi-step process, where in response to angiogenic stimuli, new vessels are created from the existing vasculature. These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, lumen formation, anastomosis, and formation of a new basement membrane. Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels often do not resemble vessels in vivo. Here we demonstrate an optimized in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts. This model recapitulates all of the key early stages of angiogenesis, and importantly the vessels display patent intercellular lumens surrounded by polarized EC. Vessels can be easily observed by phase-contrast and time-lapse microscopy, and recovered in pure form for downstream applications.     

Three electrophysiological phenotypes of cultured human umbilical vein endothelial cells

The conventional whole cell patch-clamp technique was used to measure the resting membrane conductance and membrane currents of nonstimulated cultured human umbilical vein endothelial cells (HUVECs) in different ionic conditions. Three electrophysiological phenotypes of cultured HUVECs (n = 122) were determined: first, 20% of cells as type I mainly displaying the inwardly rectifying potassium current (IKi); second, 38% of cells as type II in which IKi was super-posed on a TEA-sensitive, delayed rectifying current; third, 27% of cells as type III predominantly displaying the outwardly rectifying current which was sensitive to TEA and slightly inhibited by a chloride channel blocker niflumic acid (N.A.). In cells of type I, the mean zero-current potential (V0) was dependent on extracellular K+ ([K+]o) but not on Cl-, indicating major permeability to K+. Whereas V0 of type II was also affected by extracellular Cl- ([Cl-]o), indicating the contribution of an outward Cl- current in setting V0. The cells of type III were not sensitive to decrease of [Cl-]o and the outward current was activated in a relative stable voltage range. This varying phenotypic expression and multipotential behavior of HUVECs suggests that the electrical features of HUVEC may be primarily determined by embryonic origin and local effect of the microenvironment. This research provided the detailed electrophysiological knowledge of the endothelial cells.     

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.