Umbilical Vein Endothelial Cell Line Research Articles

Preliminary characterization of glial-secreted factors responsible for the induction of high electrical resistances across endothelial monolayers in a blood-brain barrier model.

Factors secreted by C6 glioma cells which induce electrical resistances across endothelial monolayers in an in vitro blood-brain barrier model have been partially characterised for the first time. These transendothelial electrical resistances (TEERs) were only evident when cell-free conditioned medium derived from C6 glioma cells was applied to the basolateral surfaces of confluent ECV304 or ECV304-9 cells which are both human umbilical vein endothelial cell lines (HUVEC). Electrical resistance values as high as 600 ohm. sq cm were obtained with this blood-brain barrier model and ultrafiltration techniques suggest that any factor(s) in the conditioned medium responsible for these TEERs have molecular masses of less than 1000 Da. Enzymic proteolysis and heat treatment carried out on the conditioned medium failed to inhibit its effect on the HUVEC monolayers suggesting that these C6 cell-secreted factors are unlikely to be proteins.

Modulation of mitochondrial Ca 2+ in ECV304 endothelial cells by agents which elevate CAMP

In the human umbilical vein endothelial cell-derived cell line, ECV304, we have previously shown that the elevation of [Ca 2+] m in response to agonist stimulation is dependent on Ca 2+ influx, i.e. an ATP-induced sustained increase in [Ca 2+] c results in a slow-onset, sustained elevation in [Ca 2+] m [Lawrie A.M., Rizzuto R., Pozzan T., Simpson A.W.M. A role for calcium influx in the regulation of mitochondrial calcium in endothelial cells. J Biol Chem 1996; 271: 10753–10759]. In this study, we have investigated the effect of raising CAMP on ATP-evoked elevations in both [Ca 2+] m and [Ca 2+] c by: (i) activating adenylate cyclase with the forskolin analogue — forskolin 6-[3′-(N,N-dimethylamino-propionyl)]-HCI (1 μM) (FA); (ii) addition of membrane permeable dibutyryl-CAMP (100 μM) (dbcAMP); and (iii) a combination of FA plus inhibition of CAMP phosphodiesterase using RO 20–1724 (17.5 μM) (RO);. We have found that protocols aimed at elevating CAMP significantly reduce the ATP-evoked (1–10 μM) rise in [Ca 2+] m ( n = 14); however, the [Ca 2+] c response to ATP was not affected ( n = 33). This new evidence shows that a second messenger system, other than Ca 2+ itself, may influence [Ca 2+]m changes in response to agonist stimulation.

Hypoxanthine enters human vascular endothelial cells (ECV 304) via the nitrobenzylthioinosine-insensitive equilibrative nucleoside transporter

The transport properties of the nucleobase hypoxanthine were examined in the human umbilical vein endothelial cell line ECV 304. Initial rates of hypoxanthine influx were independent of extracellular cations: replacement of Na+ with Li+, Rb+, N-methyl-D-glucamine or choline had no significant effect on hypoxanthine uptake by ECV 304 cells. Kinetic analysis demonstrated the presence of a single saturable system for the transport of hypoxanthine in ECV 304 cells with an apparent K(m) of 320 +/- 10 microM and a Vmax of 5.6 +/- 0.9 pmol/10(6) cells per s. Hypoxanthine uptake was inhibited by the nucleosides adenosine, uridine and thymidine (apparent Ki 41 +/- 6, 240 +/- 27 and 59 +/- 8 microM respectively) and the nucleoside transport inhibitors nitrobenzylthioinosine (NBMPR), dilazep and dipyridamole (apparent Ki 2.5 +/- 0.3, 11 +/- 3 and 0.16 +/- 0.006 microM respectively), whereas the nucleobases adenine, guanine and thymine had little effect (50% inhibition at > 1 mM). ECV 304 cells were also shown to transport adenosine via both the NBMPR-sensitive and -insensitive nucleoside carriers. Hypoxanthine specifically inhibited adenosine transport via the NBMPR-insensitive system in a competitive manner (apparent Ki 290 +/- 14 microM). These results indicate that hypoxanthine entry into ECV 304 endothelial cells is mediated by the NBMPR-insensitive nucleoside carrier present in these cells.

Functional Characterization of the Spontaneously Transformed Human Umbilical Vein Endothelial Cell Line ECV304: Use in anin VitroModel of Angiogenesis

To gain insight into the role of the endothelial cell during the pathophysiology of the angiogenic response, investigators have isolated micro- and macrovascular endothelial cells from a wide range of both animal and human vessels, including the umbilical vein. Human umbilical vein endothelial cells (HUVECs) isolated from umbilical cords remain a readily available and popular source of endothelial cell. However, the isolation and culture of these cells have several disadvantages, including the risk of infection, exogenous growth factor requirement, and low proliferative capacity. The heterogeneity of endothelial cells from different vascular beds as well as the heterogeneity between HUVEC isolates from different cords can make the critical interpretation of results difficult. ECV304 is a unique spontaneously transformed human umbilical vein endothelial cell line. In this report, the novel use of ECV304 cells as an alternative to HUVECs in anin vitromodel of angiogenesis using the reconstituted basement membrane extract (Matrigel) was investigated. ECV304 cells were characterized immunohistochemically and their angiogenic behavior on Matrigel was analyzed functionally by phase-contrast, electron, and time-lapse video microscopy. ECV304 cells had several practical advantages over HUVEC culture and in contrast to HUVECs, ECV304 cells exhibited an enhanced and highly reproducible capacity forin vitroangiogenesis. However, several differentiated functions were lost or reduced in the ECV304 cell line which also exhibited anomalous cytokeratin expression. ECV304 cells may provide novel insights into the mechanisms governing angiogenesis under both normal physiological and pathological conditions.

Regulation of ICAM-1 by dexamethasone in a human vascular endothelial cell line EAhy926.

Regulation by dexamethasone of intercellular adhesion molecule-1 (ICAM-1) in cultured monolayers of the human umbilical vein endothelial cell line EAhy926 was investigated. Tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) in combination or lipopolysaccharide (LPS) alone gave time- and dose-dependent increases in ICAM-1. Sustained expression of ICAM-1 was observed after short exposure (30 min) to TNF-alpha + IFN-gamma, but not to LPS. LPS-induced ICAM-1 expression was not inhibited by interleukin-1 (IL-1) receptor antagonist (0.01-100 micrograms/ml). Dexamethasone (1,000 nM) did not inhibit TNF-alpha + IFN-gamma-induced ICAM-1 expression or mRNA induction. In contrast, dexamethasone dose dependently (0.1-1,000 nM) inhibited LPS-induced ICAM-1 expression; however, its effect on mRNA was not established, because ICAM-1 mRNA induced by LPS was not detected at the time points investigated in this study (3 and 20 h). Adhesion of unstimulated human neutrophils to EAhy926 monolayers activated with TNF-alpha + IFN-gamma or LPS was increased in the presence of dexamethasone at low doses, whereas neutrophil adhesion to LPS- but not cytokine-stimulated endothelial cells was significantly reduced (P < 0.05) in the presence of a high dose of dexamethasone (1,000 nM). In conclusion, dexamethasone was demonstrated to regulate the expression and function of ICAM-1 in a stimulus-dependent manner.

Effect of Kawasaki Disease on Migration of Human Umbilical Vein Endothelial Cells

Kawasaki disease, which is characterized by systemic vasculitis causing coronary arterial involvement in childhood, shows a variety of immunoregulatory abnormalities. Especially the direct or indirect deleterious effects on endothelial cells of cytokines and anti-endothelial cell antibodies (AECA) are considered to be involved in the mechanism responsible for production of vasculitis. Intravenous administration of high doses of gamma-globulin (IVGG) has been used as an effective therapy for Kawasaki disease. To examine the behavior of endothelial cells affected by cytokines and IVGG in Kawasaki disease, we studied the effects of interferon (IFN), IL-1 beta, IL-6, and tumor necrosis factor (TNF)-alpha on the migration of human umbilical vein endothelial cell line (tHUE01) by a modified Boyden chamber method. Plasma from patients with acute Kawasaki disease markedly enhanced the migration of tHUE01 cells. Cytokines, with the exception of TNF-alpha, also enhanced the migration of tHUE01 cells in a dose-dependent manner. Anti-IFN antibody inhibited the migratory activity in response to not only IFN-gamma but also to the plasma from patients with Kawasaki disease. Rabbit AECA (rAECA) also significantly stimulated the migration of tHUE01 cells. Plasma from patients treated with IVGG did not affect the migration of tHUE01 cells. Addition of gamma-globulin significantly inhibited the migration of tHUE01 cells induced by the cytokines or rAECA. These results suggest that cytokines and AECA are important in restructuring and destroying vessel walls in Kawasaki disease by enhancing the migration of endothelial cells, and that IVGG may be therapeutically effective for this disease by suppressing this endothelial cell migration.

Interferon-β downregulates expression of VLA-4 antigen and antagonizes interferon-γ-induced expression of HLA-DQ on human peripheral blood monocytes

We have studied the effect of recombinant human IFN-β on the basal and IFN-γ-induced expression of adhesion molecules and class II MHC antigens on human peripheral blood monocytes and on ICAM-1 (intercellular adhesion molecule-1) expression of a human umbilical vein endothelial cell line (EAhy 926). We show that IFN-β downregulates both basal and IFN-γ-induced expression of VLA-4 (very late activation antigen-4) antigen on monocytes, but has no effect on the expression of CD11a, CD11b, CD11c, L-selectin, CD18, ICAM-1, β 1-integrin or CD44 on monocytes or ICAM-1 on EAhy 926 cells. We also show that IFN-β antagonizes the IFN-γ-induced expression of HLA-DQ-antigen, but not HLA-DR or HLA-DP antigens on monocyte surface. These findings may partially explain the beneficial effect of IFN-β in multiple sclerosis, since VLA-4-antigen is critical for leukocyte recruitment into inflamed brain and downregulation of HLA-class II expression diminishes antigen presenting capacity of monocytes.

Antibodies Cross-Reactive With E- and P-Selectin Block Both E- and P-Selectin Functions

E- and P-selectin are inflammation-induced cell adhesion molecules that mediate leukocyte-endothelial cell and leukocyte-platelet interactions. Monoclonal antibodies (MoAbs) specific for either E-selectin or P-selectin are protective in several animal models of inflammatory disease. To generate an MoAb with broader therapeutic potential, MoAbs that bind to both E- and P-selectin were generated by immunization of mice with mouse pre-B cell lines transfected with human E- and P-selectin. Interestingly, although the only selection criterion was the ability to bind both E- and P-selectin, all three antibodies obtained efficiently block both E- and P-selectin-mediated functions. The inhibited functions include neutrophil or HL-60 cell binding to tumor necrosis factor-alpha-activated human umbilical vein endothelial cells, E- or P-selectin transfectant cell lines, and platelet-HL-60 rosetting. These antibodies, EP-5C7, EP-2C9, and EP-1D8, recognize the same or overlapping epitope within the lectin domains of E- and P-selectin. The data suggest that functionally important epitopes of homologous proteins can be targeted by selecting for antibodies with reactivity toward both proteins. Furthermore, a potent blocking antibody specific for both E- and P-selectin may provide a more effective and broadly useful reagent for treating acute and potentially certain chronic inflammatory conditions.

Human Renal Cell Carcinoma Cells Are Sensitive to the Cytotoxic Effect of a Chimeric Protein Composed of Human Interleukin-4 and Pseudomonas Exotoxin

We have previously demonstrated that functional high-affinity interleukin-4 receptors (IL-4R) are expressed on human renal cell carcinoma (RCC) cells (N. I. Obiri et al., J. Clin. Invest. 91, 88, 1993). In the present study, we examined the cytotoxic effect (determined by inhibition of protein synthesis) of a chimeric protein composed of human IL-4 and Pseudomonas exotoxin (PE) on human RCC tumor samples obtained from patients undergoing nephrectomy. The chimeric gene encoding hIL4-PE4E was constructed by fusing a cDNA clone for human IL-4 to the 5′ end of a mutated cDNA encoding a full-length PE molecule. This gene was expressed in Escherichia coli, and large quantities of this recombinant protein were isolated to more than 95% purity. This chimeric protein, hILA-PE4E, was highly cytotoxic to all six RCC cell lines examined. The concentration of hIL4-PE4E at which 50% inhibition of protein synthesis was obtained ranged from <1 ng/ml (12 p M) to 10 ng/ml (120 p M) in five of the six isolates of RCC and 40-70 ng/ml in one other. A mutant chimeric protein which can bind to IL-4R but lacks the ADP ribosylation activity of PE was not cytotoxic to the RCC cells. The cytotoxic effect of hILA-PE4E was IL-4R mediated because a fourfold molar excess of IL-4 abrogated the cytotoxic effect of hIL4-PE4E. A neutralizing monoclonal antibody to IL-4 also abrogated the cytotoxic effect of hILA-PE4E. hIL4-PE4E showed very little cytotoxic activity to a normal human umbilical vein endothelial cell line (ID 50 = 1000 ng/ml) and a human fibroblast cell line (ID 50 ∼ 400 ng/ml). Nonactivated human peripheral blood lymphocytes (PBL) were also insensitive to hIL4-PE4E (ID 50, ∼500 ng/ml), whereas phytohemagglutinin-activated PBL were highly susceptible to the cytotoxic effect of hIL4-PE4E (ID 50, ∼4 ng/ml). These data indicate that hIL4-PE4E may be a useful agent for the treatment of human RCC without affecting normal and resting immune cells.

Cell Adhesion Activity of a 30-kDa Major Secreted Protein from Human Bladder Carcinoma Cells

Human bladder carcinoma cell line EJ-1 secreted a protein of 30 kDa as a major component. This protein was purified from the serum-free conditioned medium of EJ-1. Its N-terminal amino acid sequence was identical to that of fibroblast-derived endothelial cell growth factor (f-ECGF), but there were differences in their amino acid compositions and isoelectric points. The 30-kDa protein showed no effect on the growth of bovine capillary endothelial cells, but it promoted the attachment and spreading of rat liver cell line BRL and human umbilical vein endothelial cell line ECV-304. These results suggest that the 30-kDa protein, tentatively named "tumor-derived adhesion factor (TAF)", may be related with the aberrant cell adhesion of cancer cells.

Generation of Human Umbilical Vein Endothelial Cell Lines Which Maintain Their Differentiated Phenotype

Human umbilical vein endothelial cells have been used for the production of functional endothelial cell lines. Electroporation with the gene encoding simian virus 40 large tumour antigen under the control of the Rous sarcoma virus long terminal repeat or the human cytomegalovirus promoter has produced the cell lines, DG-RSV-LT-2 and DG-CMV-LT-4. Both cell lines have distinct cobblestone morphology, maintain organisational ability on Matrigel, express von Willebrand factor and require no supplementary growth factor apart from serum. They also retain the ability to express the endothelial markers Cadherin-5 and CD31 and regulate the expression of the adhesins intercellular adhesion molecule-1, E-Selectin and vascular cell adhesion molecule-1 in response to cytokine stimulation. The techniques described for the generation of these human umbilical vein endothelial cell lines could be used to develop alternative cell lines derived from the microvascular endothelium.

Establishment of a Human Umbilical Vein Endothelial Cell Line with Normal Functional Capabilities

Human umbilical vein endothelial cells (HUVECs) were infected with a recombinant amphotropic retrovirus containing the E6 and E7 genes of human papillomavirus type-16 (HPV-16) and the neomycin resistance gene. The cells were subsequently selected in G418 and 10 clones of resistant cells isolated and expanded for analysis. All 10 clones were of endothelial origin as determined by positive staining for Factor VIII related antigen. Functional data suggested that the clones retained the properties of normal HUVECs. All clones entered a period of crisis after about 50 generations characterised by the presence of giant, multinucleate dendritic cells. One clone emerged from this crisis period and has been established as a cell line named HPVEC4. HPVEC4 has been fully characterised indicating that it still retains all of the endothelial characteristics exhibited before entry into crisis. Specifically, HPVEC4 is able to (a) differentiate into capillaries when plated onto gels composed of basement membrane componen...

Characterization of human umbilical vein endothelial cell lines produced by transfection with the early region of SV40

Human umbilical vein endothelial cells were transfected by electroporation with the plasmid pSV3neo, containing the early region of simian virus 40. The resultant “cell lines” divide rapidly (population doubling time of 33 h) for up to 24 passages in medium supplemented with 5% (v/v) serum and 2.5 μg/ml endothelial cell growth supplement. Several of these lines express basal levels of ICAM-1 and MHC class I but not MHC class II. One cell line, designated SGHEC-7, retained a number of differentiated endothelial cell functions throughout its lifespan. These functions include increased production of tissue plasminogen activator in response to histamine, thrombin, and PMA. Stability of function and rapid growth over 24 passages endow these cells with a number of advantages over primary cultures. The homogeneous cell population and consistency of response make them ideal for biochemical and immunological studies hereto impractical with primary human endothelial cells. The success of this approach may allow the production of functional cell lines from other vascular beds.

PCNA/cyclin expression and BrdU uptake define different subpopulations in different cell lines.

Monoclonal antibodies (MAb) to a 36 KD protein, proliferating cell nuclear antigen (PCNA/cyclin), have been previously shown to be capable of identifying proliferating cells in vitro as well as in alcohol-fixed, paraffin-embedded tissue specimens. The routine use of these anti-PCNA/cyclin MAb in investigative studies and in diagnostic pathology requires a clearer understanding of the distribution of PCNA/cyclin in the different cell populations found in tissue specimens. We therefore compared the ability of MAb to three nucleus-associated proliferation markers (MAb 19A2 to PCNA/cyclin; Ki-67 to an undefined proliferation-related marker; BU-1 to 5'-bromodeoxyuridine (BrdU) incorporated into DNA) to identify the proliferating cell fraction of various cells in vitro. The cell lines were chosen to represent a spectrum of proliferation rates (high to low) and cell lineage (mesenchymal vs epithelial, non-transformed vs malignant): (a) HeLa and A-431 (two malignant carcinoma cell lines with high proliferation rates); (b) SK-5 (a non-transformed fibroblast cell line with a low proliferation rate); (c) HUVE (a non-transformed human umbilical vein endothelial cell line with a low proliferation rate). Single and double labeling immunofluorescence studies were performed after uniform 1-hr incubations with BrdU. Comparison of the overlapping distributions of detectable PCNA/cyclin expression and BrdU incorporation demonstrated substantial qualitative and quantitative differences between the different cell lines. In two of the four cell lines (HeLa, A-431) the BrdU staining distributions formed inclusive subsets of the PCNA-positive cell populations. In the HUVE cell line the two populations overlapped incompletely. In one cell line, SK-5, the two populations were mutually exclusive. MAb Ki-67 demonstrated a pattern in the SK-5 cell line that was strongly predictive of PCNA positivity, while showing no associated patterns in the other three cell lines. We conclude that PCNA/cyclin expression detected by MAb may define different cell subpopulations in different cell types relative to those incorporating BrdU or expressing the target antigen for Ki-67. This has implications for the clinical study of mixed cell populations using these antibodies.