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

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.

scholarly journals Effects of paeonol on the expression of NF-κB pathways in human umbilical veins endothelial cells induced by homocysteine

2015 ◽  
Vol 10 (3) ◽  
pp. 604
Author(s):  
Qian Xu ◽  
Kai Cao ◽  
Yan-Hong Xiao ◽  
Chao Du ◽  
Xian-Hui Dong ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Protein Expression ◽  
Cell Viability ◽  
Protein Degradation ◽  
Mrna Expression ◽  
Umbilical Vein ◽  
Model Group ◽  
Human Umbilical Vein ◽  
Umbilical Vein Endothelial Cells

<p class="Abstract">The aim of this study was to investigate the effects of paeonol on the expression of NF-κB pathway induced by homocysteine. After Human umbilical vein endothelial cells exposed to homocysteine for 24 hours,  paeonol (0.15-0.6 mmol/L) improved the cell viability (p&lt;0.05). NF-κB p65 mRNA expression was reduced largely (p&lt;0.05) and IκB-α protein expression increased significantly (p&lt;0.01). The staining of NF-κB p65 in nucleus was not as much as those in homocysteine injured model group (p&lt;0.01). Therefore, paeonol can inhibit IκB-α protein degradation and suppress NF-κB transferred into nuclear in order to inhibit the activation of NF-κB.</p><p> </p>

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

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.

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

2002 ◽  
Vol 11 (4) ◽  
pp. 369-377 ◽  
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.

The functional thrombin receptor is associated with the plasmalemma and a large endosomal network in cultured human umbilical vein endothelial cells

1995 ◽  
Vol 108 (3) ◽  
pp. 1155-1164 ◽  
Author(s):  
R. Horvat ◽  
G.E. Palade
Keyword(s):  
Endothelial Cells ◽  
Umbilical Vein ◽  
Morphological Evidence ◽  
Thrombin Receptor ◽  
Human Umbilical Vein ◽  
Cultured Endothelial Cells ◽  
Human Thrombin ◽  
Membrane Spanning ◽  
Umbilical Vein Endothelial Cells ◽  
G Protein Coupled

The functional thrombin receptor, normally expressed by endothelial cells and platelets, is a member of the G protein-coupled, seven membrane-spanning-domain receptor family and is thought to be responsible for most, if not all, the cell stimulatory effects of thrombin. Upon binding, thrombin cleaves the receptor's N-terminal ectodomain, unmasking a new N terminus, which by itself activates the receptor. Using antibodies to different domains of the human thrombin receptor, we have localized the receptor in cultured human umbilical vein endothelial cells by indirect immunofluorescence and immunoelectron microscopy. We found the receptor expressed on the plasmalemma of cultured endothelial cells in individual units rather than in clusters, at lower concentration than, and at different sites from, thrombomodulin. We also found the receptor associated with a distinct, intracellular, transferrin receptor-containing, tubulovesicular network. The thrombin receptor-positive structure spread from the perinuclear region to the periphery of the cells, exhibiting a number of varicosities interconnected by branching tubular elements, strikingly similar to an image recently described for a continuous endosomal reticulum. Our results provide morphological evidence for the presence of the functional thrombin receptor at relative low density on the surface of cultured endothelial cells (compared to thrombomodulin) and in relatively large quantities inside the cells, associated with an endosomal compartment.

Hypoxia-induced expression of complement receptor type 1 (CR1, CD35) in human vascular endothelial cells

1999 ◽  
Vol 276 (2) ◽  
pp. C450-C458 ◽  
Author(s):  
Charles D. Collard ◽  
Cuneyt Bukusoglu ◽  
Azin Agah ◽  
Sean P. Colgan ◽  
Wende R. Reenstra ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Protein Expression ◽  
Vascular Endothelial Cells ◽  
Umbilical Vein ◽  
Receptor Type ◽  
Complement Receptor ◽  
Complement Receptor Type ◽  
Umbilical Vein Endothelial Cells ◽  
Complement Receptor Type 1

Reoxygenation of hypoxic human umbilical vein endothelial cells (HUVECs) increases protein expression of the complement regulators CD46 and CD55. As the receptor for C3b is known to be present on injured bovine endothelial cells, we investigated whether hypoxia or inflammatory mediators induce complement receptor type 1 (CR1; CD35) expression on HUVECs. CR1 protein expression increased 3.7 ± 0.6-fold as measured by ELISA on HUVECs following hypoxia (48 h, 1% O2). Colocalization of CD35 and von Willebrand factor by confocal microscopy confirmed that CD35 was predominantly intracellular. Lipopolysaccharide or tumor necrosis factor-α also significantly increased HUVEC CR1 protein expression. Western blot analysis of neutrophil or hypoxic HUVEC lysates revealed a 221-kDa CR1 band under nonreducing conditions. RT-PCR of hypoxic HUVEC mRNA revealed a single band that, after sequencing, was identified as CD35. In situ hybridization of hypoxic HUVECs, but not normoxic HUVECs or fibroblasts, demonstrated increased CD35 mRNA. Hypoxic HUVECs bound immune complexes and acted as a cofactor for factor I-mediated cleavage of C3b. Thus hypoxia induces functional HUVEC CR1 expression.

Lysophosphatidylcholine Decreases the Synthesis of Tissue Factor Pathway Inhibitor in Human Umbilical Vein Endothelial Cells

1998 ◽  
Vol 79 (01) ◽  
pp. 217-221 ◽  
Author(s):  
Koichi Kokame ◽  
Toshiyuki Miyata ◽  
Naoaki Sato ◽  
Hisao Kato
Keyword(s):  
Endothelial Cells ◽  
Mrna Expression ◽  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Umbilical Vein ◽  
Mrna Levels ◽  
Down Regulation ◽  
Human Umbilical Vein ◽  
Tissue Factor Pathway ◽  
Umbilical Vein Endothelial Cells

SummaryThrombotic complications are frequently associated with atherosclerosis. Lysophosphatidylcholine (LPC), a component accumulated in oxidatively modified LDL (ox-LDL), is known to play a crucial role in the initiation and progression of atherosclerotic vascular lesions. Since a vascular anticoagulant, tissue factor pathway inhibitor (TFPI), has the function of regulating the initial reaction of tissue factor (TF)-induced coagulation, we investigated the effect of LPC on TFPI synthesis in cultured human umbilical vein endothelial cells (HUVEC). The treatment of HUVEC with LPC for 24 h decreased TFPI antigen levels in both the culture medium and the cell lysate in a dose-dependent manner. Northern blot analysis revealed that LPC caused a time-dependent decrease in the TFPI mRNA levels. The levels of TFPI antigen and mRNA were decreased to 72% and 38%, respectively, by the incubation with 50 μM LPC for 24 h. The down-regulation by LPC of TFPI mRNA expression was not observed in the presence of cycloheximide, suggesting that protein synthesis was involved in the suppression of TFPI mRNA expression. The TFPI mRNA levels in actinomycin D-treated cells were relatively stable, indicating that the down-regulation of TFPI mRNA by LPC would be partly explained by the enhanced mRNA destabilization. In contrast to the significant down-regulatory effects of LPC on TFPI expression, LPC did not induce TF mRNA expression in HUVEC. These results indicate that LPC accumulated in the atherosclerotic vascular wall would suppress endothelial TFPI synthesis, reducing the antithrombotic property of endothelial cells.

Estradiol-17β regulates the induction of VCAM-1 mRNA expression by interleukin-1β in human umbilical vein endothelial cells

Life Sciences ◽  
1994 ◽  
Vol 54 (13) ◽  
pp. PL221-PL227 ◽  
Author(s):  
Kenji Nakai ◽  
Chuichi Itoh ◽  
Kazuhiko Hotta ◽  
Tomonori Itoh ◽  
Masao Yoshizumi ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Mrna Expression ◽  
Umbilical Vein ◽  
Interleukin 1Β ◽  
Human Umbilical Vein ◽  
Umbilical Vein Endothelial Cells ◽  
Estradiol 17Β

scholarly journals Soluble CD14 participates in the response of cells to lipopolysaccharide.

1992 ◽  
Vol 176 (6) ◽  
pp. 1665-1671 ◽  
Author(s):  
E A Frey ◽  
D S Miller ◽  
T G Jahr ◽  
A Sundan ◽  
V Bazil ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Leukocyte Adhesion ◽  
Umbilical Vein ◽  
Cell Types ◽  
Mononuclear Phagocytes ◽  
Soluble Cd14 ◽  
Bovine Endothelial Cells ◽  
Endothelial Leukocyte Adhesion Molecule ◽  
Umbilical Vein Endothelial Cells ◽  
Serum Dependent

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.

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

1995 ◽  
Vol 311 (2) ◽  
pp. 589-594 ◽  
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.

Sign in / Sign up

Export Citation Format

Share Document