Soluble CD14 participates in the response of cells to lipopolysaccharide.

CD14 is a 55-kD protein found both as a glycosylphosphatidyl inositol-linked protein on the surface of mononuclear phagocytes and as a soluble protein in the blood. CD14 on the cell membrane (mCD14) has been shown to serve as a receptor for complexes of lipopolysaccharide (LPS) with LPS binding protein, but a function for soluble CD14 (sCD14) has not been described. Here we show that sCD14 enables responses to LPS by cells that do not express CD14. We have examined induction of endothelial-leukocyte adhesion molecule 1 expression by human umbilical vein endothelial cells, interleukin 6 secretion by U373 astrocytoma cells, and cytotoxicity of bovine endothelial cells. None of these cell types express mCD14, yet all respond to LPS in a serum-dependent fashion, and all responses are completely blocked by anti-CD14 antibodies. Immunodepletion of sCD14 from serum prevents responses to LPS, and the responses are restored by addition of sCD14. These studies suggest that a surface anchor is not needed for the function of CD14 and further imply that sCD14 must bind to additional proteins on the cell surface to associate with the cell and transduce a signal. They also indicate that sCD14 may have an important role in potentiating responses to LPS in cells lacking mCD14.

Download Full-text

INTERACTION OF S PROTEIN/VITRONECTIN WITH CULTURED ENDOTHELIAL CELLS: PROMOTION OF ATTACHMENT AND SPECIFIC BINDING

10.1055/s-0038-1643635 ◽

1987 ◽

Author(s):

K T Preissner ◽

E Anders ◽

G Müller-Berghaus

Keyword(s):

Endothelial Cells ◽

Cell Attachment ◽

Specific Binding ◽

Umbilical Vein ◽

Attachment Site ◽

Cell Types ◽

S Protein ◽

Specific Association ◽

Umbilical Vein Endothelial Cells ◽

Adhesive Proteins

The interaction of the complement inhibitor S protein, which is identical to the serum spreading factor, vitronectin, with cultured human endothelial cells of macro- and microvas- cular origin was investigated. Purified S protein, coated for 2 h on polystyrene petri dishes, induced concentration- and time-dependent attachment and spreading of human umbilical vein endothelial cells (HUVEC) as well as human omental tissqe microvasular endothelial cells (HOTMEC) at 37°C. With 3 × 105 cells/ml (final concentration) more than 50% of the cells attached within 2 h incubation at 0.3 - 3 μg/ml S protein. The effect of S protein was specific, since only monospecific antibodies against S protein prevented attachment of cells, while antibodies against fibronectin, fibrinogen or von Wille-brand factor were uneffective. The pentapeptide Gly-Arg-Gly-Asp-Ser, which contains the cell-attachment site of these adhesive proteins including S protein, inhibited the activity of S protein to promote attachment of endothelial cells in a concentration-dependent fashion; at 200 μM peptide, less than 10% of the cells became attached. Direct binding of S protein to HUVEC and HOTMEC was studied with cells in suspension at a concentration of 1 × 106 cells/ml in the presence of 1% (w/v) human serum albumin and 1 mM CaCl2 and was maximal after 120 min. Both cell types bound S protein in a concentration-dependent fashion with an estimated dissociation constant KD=0.2pM. More than 80% of bound radiolabelled S protein was displaced by unlabelled S protein, whereas binding was reduced to about 50% by the addition in excess of either fibronectin, fibrinogen, von Willebrand factor or the pentapeptide. These findings provide evidence for the specific association of S protein with endothelial cells, ultimately leading to attachment and spreading of cells. Although the promotion of attachment was highly specific for S protein, other adhesive proteins than S protein, also known to associate with endothelial cells, may in part compete with direct S protein binding.

Download Full-text

Hydrogel-Coated Textile Scaffolds as Three-Dimensional Growth Support for Human Umbilical Vein Endothelial Cells (HUVECs): Possibilities as Coculture System in Liver Tissue Engineering

Cell Transplantation ◽

10.3727/000000002783985837 ◽

2002 ◽

Vol 11 (4) ◽

pp. 369-377 ◽

Cited By ~ 24

Author(s):

Makarand V. Risbud ◽

Erdal Karamuk ◽

René Moser ◽

Joerg Mayer

Keyword(s):

Endothelial Cells ◽

Umbilical Vein ◽

Three Dimensional ◽

Mesh Size ◽

Cell Types ◽

Cell Number ◽

Human Umbilical Vein ◽

Hydrogel Matrix ◽

Umbilical Vein Endothelial Cells

Three-dimensional (3-D) scaffolds offer an exciting possibility to develop cocultures of various cell types. Here we report chitosan–collagen hydrogel-coated fabric scaffolds with defined mesh size and fiber diameter for 3-D culture of human umbilical vein endothelial cells (HUVECs). These scaffolds did not require pre-coating with fibronectin and they supported proper HUVEC attachment and growth. Scaffolds preserved endothelial cell-specific cobblestone morphology and cells were growing in compartments defined by the textile mesh. HUVECs on the scaffold maintained the property of contact inhibition and did not exhibit overgrowth until the end of in vitro culture (day 6). MTT assay showed that cells had preserved mitochondrial functionality. It was also noted that cell number on the chitosan-coated scaffold was lower than that of collagen-coated scaffolds. Calcein AM and ethidium homodimer (EtD-1) dual staining demonstrated presence of viable and metabolically active cells, indicating growth supportive properties of the scaffolds. Actin labeling revealed absence of actin stress fibers and uniform distribution of F-actin in the cells, indicating their proper attachment to the scaffold matrix. Confocal microscopic studies showed that HUVECs growing on the scaffold had preserved functionality as seen by expression of von Willebrand (vW) factor. Observations also revealed that functional HUVECs were growing at various depths in the hydrogel matrix, thus demonstrating the potential of these scaffolds to support 3-D growth of cells. We foresee the application of this scaffold system in the design of liver bioreactors wherein hepatocytes could be cocultured in parallel with endothelial cells to enhance and preserve liver-specific functions.

Download Full-text

Agonists for endothelial P2 purinoceptors trigger a signalling pathway producing Ca2+ responses in lymphocytes adherent to endothelial cells

Biochemical Journal ◽

10.1042/bj3110589 ◽

1995 ◽

Vol 311 (2) ◽

pp. 589-594 ◽

Cited By ~ 6

Author(s):

J S Wiley ◽

J R Chen ◽

G P Jamieson ◽

P J Thurlow

Keyword(s):

Endothelial Cells ◽

Lymphocytic Leukaemia ◽

Umbilical Vein ◽

Cell Types ◽

The Body ◽

Signalling Pathway ◽

Signalling Pathways ◽

Transient Rise ◽

Umbilical Vein Endothelial Cells ◽

Stimulation Of

Recirculation of lymphocytes through the body involves their frequent adhesion to endothelial cells but little is known of the signalling pathways between these two cell types. Lymphocytes from patients with chronic lymphocytic leukaemia were loaded with the Ca(2+)-sensitive indicator, fura 2, and allowed to adhere to either glass or monolayers of human umbilical-vein endothelial cells. Addition of ATP or UTP (1-10 microM) to the superfusate produced a transient rise in cytosolic Ca2+ concentration in the lymphocytes adherent to endothelium (24 of 35 cells). In contrast, ATP or UTP (1-10 microM) had no effect on the cytosolic Ca2+ of lymphocytes attached to glass. As the only lymphocyte receptor for ATP (P2Z class) requires higher ATP concentrations (> 50 microM) for Ca2+ influx and is unresponsive to UTP, the involvement of a lymphocyte P2Z purinoceptor is unlikely. Various agonists including ATP, UTP, 2-methylthioATP, ADP and histamine all stimulated increases in endothelial cytosolic Ca2+ but only ATP and UTP (both agonists for endothelial P2U purinoceptors) triggered Ca2+ transients in adherent lymphocytes. Removal of extracellular Ca2+ did not abolish the ATP-induced rise in cytosolic Ca2+ concentration in lymphocytes adherent to endothelial cells. These findings show that stimulation of endothelial P2U purinoceptors triggers an endothelial-lymphocyte signalling pathway which releases internal Ca2+ in adherent lymphocytes.

Download Full-text

Dimerization of P-selectin in platelets and endothelial cells

Blood ◽

10.1182/blood.v96.9.3070.h8003070_3070_3077 ◽

2000 ◽

Vol 96 (9) ◽

pp. 3070-3077 ◽

Cited By ~ 1

Author(s):

Fern J. Barkalow ◽

Kurt L. Barkalow ◽

Tanya N. Mayadas

Keyword(s):

Endothelial Cells ◽

Endoplasmic Reticulum ◽

Platelet Activation ◽

Cell Activation ◽

Leukocyte Adhesion ◽

Umbilical Vein ◽

Monomeric Form ◽

Intact Cells ◽

Human Umbilical Vein ◽

Umbilical Vein Endothelial Cells

P-selectin is a leukocyte adhesion receptor stored in platelets and endothelial cells and is translocated to the surface upon cell activation. Purified P-selectin is oligomeric and has increased avidity for its ligand relative to the monomeric form, but whether P-selectin self-associates in the membrane of intact cells is not known. A chemical cross-linking approach was used to show that P-selectin is present as noncovalent dimers in resting platelets, human umbilical vein endothelial cells, and heterologous RIN5F cells expressing P-selectin. The results of 2-dimensional isoelectric focusing are consistent in showing P-selectin dimers as homodimers, but they are composed of a more basic subset of P-selectin than the monomers. This suggests that the dimers are a biochemically distinct subset of P-selectin. P-selectin dimers form in the endoplasmic reticulum and Golgi compartments of human umbilical vein endothelial cells only after synthesis of the mature P-selectin subunit, and are not preferentially stored in Weibel-Palade bodies as compared with the monomeric form. Platelet activation with thrombin receptor–activating peptide leads to the presence of P-selectin monomers and homodimers on the cell surface as well as P-selectin heterodimers, which are composed of P-selectin and an unidentified protein of approximately 81 kd molecular weight. In summary, these studies demonstrate that P-selectin is homodimeric in situ and that platelet activation leads to the formation of an additional activation-specific heterodimeric species. In addition, the homodimer has unique biochemical characteristics compared with the monomeric form, and dimerization occurs in the endoplasmic reticulum and Golgi compartments of endothelial cells.

Download Full-text

Dimerization of P-selectin in platelets and endothelial cells

Blood ◽

10.1182/blood.v96.9.3070 ◽

2000 ◽

Vol 96 (9) ◽

pp. 3070-3077 ◽

Cited By ~ 37

Author(s):

Fern J. Barkalow ◽

Kurt L. Barkalow ◽

Tanya N. Mayadas

Keyword(s):

Endothelial Cells ◽

Endoplasmic Reticulum ◽

Platelet Activation ◽

Cell Activation ◽

Leukocyte Adhesion ◽

Umbilical Vein ◽

Monomeric Form ◽

Intact Cells ◽

Human Umbilical Vein ◽

Umbilical Vein Endothelial Cells

Abstract P-selectin is a leukocyte adhesion receptor stored in platelets and endothelial cells and is translocated to the surface upon cell activation. Purified P-selectin is oligomeric and has increased avidity for its ligand relative to the monomeric form, but whether P-selectin self-associates in the membrane of intact cells is not known. A chemical cross-linking approach was used to show that P-selectin is present as noncovalent dimers in resting platelets, human umbilical vein endothelial cells, and heterologous RIN5F cells expressing P-selectin. The results of 2-dimensional isoelectric focusing are consistent in showing P-selectin dimers as homodimers, but they are composed of a more basic subset of P-selectin than the monomers. This suggests that the dimers are a biochemically distinct subset of P-selectin. P-selectin dimers form in the endoplasmic reticulum and Golgi compartments of human umbilical vein endothelial cells only after synthesis of the mature P-selectin subunit, and are not preferentially stored in Weibel-Palade bodies as compared with the monomeric form. Platelet activation with thrombin receptor–activating peptide leads to the presence of P-selectin monomers and homodimers on the cell surface as well as P-selectin heterodimers, which are composed of P-selectin and an unidentified protein of approximately 81 kd molecular weight. In summary, these studies demonstrate that P-selectin is homodimeric in situ and that platelet activation leads to the formation of an additional activation-specific heterodimeric species. In addition, the homodimer has unique biochemical characteristics compared with the monomeric form, and dimerization occurs in the endoplasmic reticulum and Golgi compartments of endothelial cells.

Download Full-text

DIRECT BINDING OF SOLUBLE CD14 TO HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS

Shock ◽

10.1097/00024382-199506000-00194 ◽

1995 ◽

Vol 3 (6) ◽

pp. 56

Author(s):

J. K. Zhang ◽

T. K. Morrison ◽

M. C. Falk ◽

Y. H. Kang ◽

C. H. Lee

Keyword(s):

Endothelial Cells ◽

Umbilical Vein ◽

Soluble Cd14 ◽

Human Umbilical Vein ◽

Direct Binding ◽

Umbilical Vein Endothelial Cells

Download Full-text

Induction of stress proteins in human endothelial cells by heavy metal ions and heat shock

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1999.277.5.l1026 ◽

1999 ◽

Vol 277 (5) ◽

pp. L1026-L1033 ◽

Cited By ~ 18

Author(s):

M. Wagner ◽

I. Hermanns ◽

F. Bittinger ◽

C. J. Kirkpatrick

Keyword(s):

Heavy Metal ◽

Endothelial Cells ◽

Heat Shock ◽

Metal Ions ◽

Heavy Metal Ions ◽

Stress Proteins ◽

Umbilical Vein ◽

Cell Types ◽

Microvascular Endothelial ◽

Umbilical Vein Endothelial Cells

In the present study, we compared the induction of heat shock proteins (HSPs) by heat and heavy metal ions in three different endothelial cell types, namely, human umbilical vein endothelial cells, human pulmonary microvascular endothelial cells, and the cell line EA.hy 926. Our results show that especially Zn2+ and Cd2+ are inducers of 70-kDa (HSP70), 60-kDa (HSP60), 32-kDa (HSP32), and 27-kDa (HSP27) HSPs. The strength of inducibility is specific for each HSP. Ni2+ and Co2+ only show an inducible effect at very high concentrations, that is, in the clearly cytotoxic range. Furthermore, we investigated the time course of HSP expression and the involvement of heat shock factor-1. Our study demonstrates that the three endothelial cell types that were under investigation show comparable stress protein expression when treated with heavy metal ions or heat shock. The expression of stress proteins may be used as an early marker for the toxic damage of cells. This damage can be an inducer of acute respiratory distress syndrome in which microvascular endothelial lesions occur early. Our study provides evidence that human umbilical vein endothelial cells or EA.hy 926 cells, which are much more easily isolated and/or cultivated than pulmonary microvascular endothelial cells, could be used as alternative cell culture systems for studies on cellular dysfunction in the lung caused by toxic substances, certainly with respect to the expression of HSPs.

Download Full-text

Clot Lysis Mediated by Cultured Human Microvascular Endothelial Cells

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646991 ◽

1988 ◽

Vol 60 (03) ◽

pp. 463-467 ◽

Cited By ~ 13

Author(s):

Wolfgang Speiser ◽

Elisabeth Anders ◽

Bernd R Binder ◽

Gert Müller-Berghaus

Keyword(s):

Endothelial Cells ◽

Plasminogen Activator ◽

Shape Change ◽

Umbilical Vein ◽

Cell Types ◽

Microvascular Endothelial Cells ◽

Clot Lysis ◽

Microvascular Endothelial ◽

Umbilical Vein Endothelial Cells ◽

Fibrin Clots

SummaryThe lysis of fibrin clots on the surface of cultured human omental tissue microvascular endothelial cells (HOTMEC) and cultured human umbilical vein endothelial cells (HUVEC) was studied. Fibrin clots were made by mixing fibrinogen, plasminogen and thrombin on the surface of both cell types. Clot lysis was seen only on the surface of HOTMEC, which were found to synthesize about 100-fold more tissue plasminogen activator (tPA) antigen than HUVEC. Clot lysis of HOTMEC could be blocked by anti-tPA IgG but was not affected by the incorporation of exogenous plasminogen activator (PAI) into the clot in concentrations (75 arbitrary units) exceeding the tPA activity (21 ± 2.5 IU) of the cells. Thus, it is likely that tPA secreted by HOTMEC is protected from inhibition by PAI in the presence of fibrin and endothelial cells. The stimulation of EC to release an excess of tPA over PAI, in contrast to the secretion of an excess of PAI over tPA found in unstimulated cells in the absence of fibrin, is obviously no prerequisite for the initiation of fibrinolysis on the surface of HOTMEC. As thrombin was used for clot formation, its influence on tPA and PAI synthesis of both cell types was investigated. In contrast to HOTMEC, which were not affected by Α-thrombin, HUVEC revealed a dose-dependent increase in tPA and PAI synthesis upon incubation with the enzyme. This increase in tPA production by HUVEC was not sufficient to lyse the clots within 48 hours. Furthermore, HUVEC. behaved differently towards thrombin as these cells in contrast to HOTMEC revealed the typical shape change reaction upon incubation with the enzyme

Download Full-text

Abstract 550: Differential Regulation of CD39 Expression by Il-1ß in Leukocytes and Endothelial Cells

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.33.suppl_1.a550 ◽

2013 ◽

Vol 33 (suppl_1) ◽

Author(s):

Nadia R Sutton ◽

Danica Petrovic-Djergovic ◽

Amy Baek ◽

Yogen Kanthi ◽

Hui Liao ◽

...

Keyword(s):

Endothelial Cells ◽

Protein Expression ◽

Mrna Expression ◽

Umbilical Vein ◽

Cell Types ◽

Pathological Process ◽

Mononuclear Leukocytes ◽

Healthy Humans ◽

Membrane Spanning ◽

Umbilical Vein Endothelial Cells

Background CD39 (ENTPD-1) is a membrane-spanning ectonucleotidase that is responsible for the phosphohydrolysis of ATP to ADP and ADP to AMP. CD39 levels are modulated in multiple conditions, including pulmonary hypertension, cardiac, liver and renal injury, cancer, and autoimmune diseases. However, little is known about the upstream regulation of this molecule, or whether modulation of CD39 represents a response to inflammatory cytokines or is integral to the pathological process. Elevated levels of cytokine IL-1β are a hallmark of many inflammatory states. Objectives Objectives were to measure CD39 expression after stimulation with the IL-1β. CD39 is expressed on leukocytes and endothelial cells; therefore, CD39 expression was evaluated in these cell types. Methods Human umbilical vein endothelial cells (HUVECs) were treated with either 10 pg/ml or 1 ng/ml IL-1β for 12 hours. Cells were harvested and CD39 mRNA expression was measured using qPCR. CD39 protein expression was measured using Western Blot. Mononuclear leukocytes were isolated from whole blood of healthy humans via density gradient cell separation (n=3). Leukocytes were cultured for 12 hours with or without the addition of IL-1β (1ng/ml). Leukocytes were then assessed for expression of CD39, CD73, CD14, CD19, CD4, CD8, and CD25 by flow cytometry. Findings IL-1β (1 ng/ml) treatment resulted in decreased CD39 mRNA expression in HUVECs (1.1 vs. 3.9, p<0.01). IL-1β treatment also resulted in decreased CD39 protein expression in HUVECs (untreated, 1.56, 10pg/ml, 0.96, 1ng/ml, 0.48, untreated vs. 10pg/ml, p<0.01). IL-1β increased the percentage of T-reg CD4+CD25+ expressing CD39+ cells (treated vs untreated, 3.94 vs. 2.73%, p=0.013). There was a trend toward an increased percentage of CD4+ T cells expressing CD39+ with IL-1β treatment (13.85% vs.11.83%, p=0.25). Conclusions This is the first report of modulation of CD39 expression in response to the inflammatory cytokine IL-1β. Interestingly, IL-1β downregulated CD39 expression in HUVECs, but resulted in increased CD39 expression on a T-reg population positive for CD4 and CD25. CD39 could be differentially regulated depending on the inflammatory stimulus and target cell type.

Download Full-text

Sustained ERK phosphorylation is necessary but not sufficient for MMP-9 regulation in endothelial cells: involvement of Ras-dependent and -independent pathways

Journal of Cell Science ◽

10.1242/jcs.113.23.4319 ◽

2000 ◽

Vol 113 (23) ◽

pp. 4319-4330 ◽

Cited By ~ 1

Author(s):

E. Genersch ◽

K. Hayess ◽

Y. Neuenfeld ◽

H. Haller

Keyword(s):

Endothelial Cells ◽

Umbilical Vein ◽

Signal Transduction Pathway ◽

Cell Types ◽

Tnf Alpha ◽

Dependent Manner ◽

Necrosis Factor Alpha ◽

Type Iv ◽

Erk Phosphorylation ◽

Umbilical Vein Endothelial Cells

Endothelial expression of matrix metalloproteinase-9 (MMP-9), which degrades native type IV collagen, was implicated as a prerequisite for angiogenesis. Therefore, the aim of this study was to determine signaling requirements that regulate MMP-9 expression in endothelial cells. Both, primary and permanent human umbilical vein endothelial cells (HUVEC and ECV304, respectively) were stimulated with phorbol 12-myristate 13-acetate (PMA) and the cytokine tumor necrosis factor-(alpha) (TNF(alpha)) to induce MMP-9 expression. While both cell types responded to PMA at the protein, mRNA and promoter level by induction of MMP-9, TNF(alpha) caused this response only in ECV304. Inhibitors specific for mitogen-activated protein/ERK kinase 1/2 (MEK1/2), protein kinase C (PKC), and Ras and co-transfections of wild-type and mutant Raf were used to elucidate the signaling cascades involved. Thus, we could show that the Raf/MEK/ERK cascade is mainly responsible for MMP-9 induction in endothelial cells and that this cascade is regulated independently of PKC and Ras subsequent to TNF(alpha) stimulation and in a PKC-dependent manner as a result of PMA treatment. In addition, PMA triggers a Ras-dependent signal transduction pathway bypassing the phosphorylation of ERK. Finally, we provide evidence that sustained phosphorylation of ERK1/2 is necessary but not sufficient for expression of MMP-9.

Download Full-text