A simple and biosafe method for isolation of human umbilical vein endothelial cells

Human umbilical vein endothelial cells (HUVEC_S) are used as an irreplaceable tool for the study of vascular diseases. However, the technicians who isolate HUVECs are largely exposed to potential infectious threats. Here we report the development of a specialized instrument to protect researchers from known or unknown infectious agents when they operate on human umbilical cords. This instrument can be assembled by common laboratory supplies and adapted to accommodate umbilical cords of different lengths. When the cord is enclosed within the instrument, the risk of sample contamination and operator infection is greatly reduced. Using our instrument, endothelial cells were successfully isolated from human umbilical veins without contamination. The cells were verified by their cobblestone-like morphology and by immunofluorescence staining (Factor VIII and CD31 positivity and α-SMA negativity). Our instrument simplifies and optimizes the cell extraction process, and most importantly elevates the biosafety to a higher level during the isolation of human umbilical vein endothelial cells.     

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.     

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.     

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.     

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).     

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.     

Intermittent high glucose enhances apoptosis in human umbilical vein endothelial cells in culture

To explore the effect of fluctuating glucose on endothelial cells, human umbilical vein endothelial cells were incubated for 14 days in media containing different glucose concentrations: 5 mmol/l, 20 mmol/l, or a daily alternating 5 or 20 mmol/l glucose. Apoptosis was studied by different methods: viability assay, cell cycle analysis, DNA fragmentation, and morphological analysis. Furthermore, the levels of Bcl-2 and Bax, well known proteins involved in apoptosis, were evaluated. Stable high glucose induced apoptosis in human umbilical vein endothelial cells, a phenomenon accompanied by a significant decrease of Bcl-2 and a simultaneous increase of Bax expression. However, apoptosis was enhanced in human umbilical vein endothelial cells exposed to intermittent, rather than constant, high glucose concentration. In this condition, Bcl-2 was not detectable, whereas Bax expression was significantly enhanced. These findings suggest that variability in glycemic control could be more deleterious to endothelial cells than a constant high concentration of glucose.     

Autocrine receptors for endothelins in the primary culture of endothelial cells of human umbilical vein.

Human umbilical vein endothelial cells (HUVECs) in primary culture produced and secreted endothelin 1 (ET-1) actively. Specific binding of [125I]ET-1 to these cells was not detectable because of the saturation of ET receptors with endogenously produced ET-1. However, addition of phosphoramidon, an inhibitor of ET-converting enzyme, to the medium reduced the production of ET-1 and thus the receptors on HUVECs were made available for exogenously added [125I]ET-1. Binding studies using phosphoramidon-treated HUVECs indicated the existence of a non-isopeptide-selective type (ETB) of ET receptor with a Kd of 17 pM. This receptor is thought to be involved in ET-induced vasodilation in an autocrine manner in vivo.     

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.     

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.     

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.     

Long-term serial cultivation and growth requirements for human umbilical vein endothelial cells

Summary Human umbilical vein endothelial cells (HUV-EC) grew rapidly in vitro in medium supplemented with epidermal growth factor, fetal bovine serum (FBS) and human diploid fibroblast-conditioned medium. The effect of FBS could be replaced partially by bovine serum albumin, cholesterol, and vitamin E, and completely by further addition of serum dialysate or refeeding every other day. Among these components, fibroblast-conditioned medium is essential for HUV-EC growth. The HUV-EC were cultured serially for over 50 population doublings in the 10% FBS containing fibroblast-conditioned medium and for over 40 population doublings in the serum-free medium. Mitogenic factor(s) present in the medium conditioned by fibroblasts may be related to endothelial cell growth factor and play an important role angiogenesis and regeneration of vascular endothelium in vitro.     

Expression of inducible nitric oxide synthase in human umbilical vein endothelial cells during primary culture

The adaptive response of endothelial cells to stress may lead to the upregulation of nitric oxide (NO) production. Herein, we report inducible nitric oxide synthase (iNOS) induction in primary cultures of human umbilical vein endothelial cells (HUVEC). The enzyme expression was earlier observed in 12-h cultures, reaching maximal levels after 3 days and decreasing when cells become confluent. The time course of NO production by HUVEC paralleled iNOS expression during the whole culture period, indicating that enzyme was functionally active. Conversely, iNOS induction could not be further detected in HUVEC subcultures passed once from cells presenting maximal levels of iNOS expression in the primary culture. Induction of iNOS in HUVEC was not related to lipopolysaccharide contamination, since the enzyme expression was not affected in the presence of polymyxin B added to primary cultures. Further analysis showed that aminoguanidine, a specific iNOS inhibitor, did not affect cell proliferation, suggesting that the NO produced by HUVEC may not be directly related to cell growth. Platelet endothelial cell adhesion molecule-1 expression was upregulated during cell confluence, in contrast to the decrease of iNOS expression and activity. The data suggest that iNOS expression may be a molecular mechanism mediating the adaptive response of endothelial cells to culture environment.     

Fibroblast products adsorb to the culture substrate and stimulate growth of human umbilical vein endothelial cells

Human umbilical vein endothelial cells grew with a doubling time of approximately 20 hr in medium conditioned by human diploid fibroblasts and supplemented with 10% fetal bovine serum, whereas the cells did not grow substantially in the non-conditioned serum supplemented medium. Production of the fibroblast-derived activity required the presence of insulin, EGF, or PDGF. The fibroblast derived-factor adsorbed to native culture dishes or dishes coated with gelatin and collagen. The adsorbed activity was resistant to treatment with 1% Triton X-100, and was abolished by treatment with serine proteases. Further, the extracellular matrix produced by the fibroblasts also showed growth-stimulating activity. Fibroblast-derived factors may play a role in vascularization processes during wound healing, inflammation and normal development.     

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.     

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.     

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.