Enhancement of tumor necrosis factor-induced endothelial cell injury by cycloheximide

1990 ◽  
Vol 259 (2) ◽  
pp. L123-L129
Author(s):  
K. B. Nolop ◽  
U. S. Ryan
Keyword(s):  
Endothelial Cells ◽  
Protein Synthesis ◽  
Endothelial Cell ◽  
Tumor Necrosis Factor ◽  
Tumor Necrosis ◽  
Morphological Evidence ◽  
Human Aorta ◽  
Dependent Manner ◽  
Inhibitor Of Protein Synthesis ◽  
Necrosis Factor

Tumor necrosis factor (TNF), a potent polypeptide mediator released by activated monocytes and macrophages, has a number of proinflammatory effects on endothelial cells. TNF is cytotoxic to tumor cells in vivo and in vitro, but TNF-induced toxicity to endothelial cells is less well established. We now report that cycloheximide (CHX), an inhibitor of protein synthesis, renders endothelial cells highly susceptible to TNF-induced lysis. TNF alone did not change the overall rate of protein synthesis by endothelial cells, whereas the addition of CHX completely abolished protein synthesis. Endothelial cells incubated in TNF alone in high concentrations (up to 1,000 U/ml) showed minimal rounding up and release of 51Cr. Likewise, CHX alone (5 micrograms/ml) had no significant effect on endothelial cell morphology and release of 51Cr. However, incubation of endothelial cells in both CHX and TNF caused injury in a dose-dependent manner. Morphological evidence of cell retraction, rounding, and detachment began within 2 h, but specific 51Cr release did not begin to rise until after 4 h. These changes were not observed when endothelial cells were incubated with TNF/CHX at 4 degrees C. The combination of TNF/CHX was lethal to all endothelial cells tested (bovine pulmonary artery, human umbilical vein, and human aorta), with human aortic cells showing the most pronounced changes. We conclude that healthy endothelial cells are resistant to TNF-induced lysis, but inhibition of their ability to make protein renders them highly susceptible.

scholarly journals Tumor necrosis factor/cachectin interacts with endothelial cell receptors to induce release of interleukin 1.

1986 ◽  
Vol 163 (6) ◽  
pp. 1363-1375 ◽  
Author(s):  
P P Nawroth ◽  
I Bank ◽  
D Handley ◽  
J Cassimeris ◽  
L Chess ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Tumor Necrosis Factor ◽  
Host Response ◽  
Tumor Necrosis ◽  
Time Dependent ◽  
Target Tissue ◽  
Dependent Manner ◽  
Heat Treated ◽  
Necrosis Factor

Tumor necrosis factor/cachectin (TNF) has been implicated as a mediator of the host response in sepsis and neoplasia. Recent work has shown that TNF can modulate endothelial cell hemostatic properties, suggesting that endothelium is a target tissue for TNF. This led us to examine whether endothelial cells have specific binding sites for TNF and augment the biological response to TNF by elaborating the inflammatory mediator, IL-1. Incubation of 125I-recombinant human TNF with confluent, cultured human umbilical vein endothelial cells resulted in time-dependent, reversible, and saturable binding. Binding was half-maximal at a TNF concentration of 105 +/- 40 pM, and at saturation 1,500 molecules were bound per cell. Heat-treated TNF, which is biologically inactive, did not bind to endothelium. In addition to surface binding, TNF induced the elaboration of IL-1 activity by endothelial cells in a time-dependent manner. Generation of IL-1 activity required protein synthesis and was half-maximal at a TNF concentration of 50 +/- 20 pM. IL-1 activity from TNF-treated endothelium could be adsorbed by an immobilized antibody to IL-1. Heat-treated TNF was ineffective in eliciting endothelial cell IL-1. These data indicate that TNF can bind specifically to endothelium and initiate a cascade of inflammatory and coagulant events on the vessel surface potentially central to the host response to neoplasia and sepsis.

scholarly journals Intracellular NAD levels regulate tumor necrosis factor protein synthesis in a sirtuin-dependent manner

2009 ◽  
Vol 15 (2) ◽  
pp. 206-210 ◽  
Author(s):  
Frédéric Van Gool ◽  
Mara Gallí ◽  
Cyril Gueydan ◽  
Véronique Kruys ◽  
Pierre-Paul Prevot ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Tumor Necrosis Factor ◽  
Tumor Necrosis ◽  
Dependent Manner ◽  
Necrosis Factor

scholarly journals Modulation of endothelial cell hemostatic properties by tumor necrosis factor.

1986 ◽  
Vol 163 (3) ◽  
pp. 740-745 ◽  
Author(s):  
P P Nawroth ◽  
D M Stern
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Tumor Necrosis Factor ◽  
Cell Surface ◽  
Protein C ◽  
Tumor Necrosis ◽  
Protein S ◽  
Active Role ◽  
Procoagulant Activity ◽  
Necrosis Factor

Tumor necrosis factor/cachectin (TNF) is a mediator of the septic state, which involves diffuse abnormalities of coagulation throughout the vasculature. Since previous studies have shown that endothelial cells can play an active role in coagulation, we wished to determine whether TNF could modulate endothelial cell hemostatic properties. Incubation of purified recombinant TNF with cultured endothelial cells resulted in a time- and dose-dependent acquisition of tissue factor procoagulant activity. Concomitant with enhanced procoagulant activity, TNF also suppressed endothelial cell cofactor activity for the anticoagulant protein C pathway; both thrombin-mediated protein C activation and formation of functional activated protein C-protein S complex on the cell surface were considerably attenuated. Comparable concentrations of TNF (half-maximal affect at approximately 50 pM) and incubation times (half-maximal affect by 4 h after addition to cultures) were required for each of these changes in endothelial cell coagulant properties. This unidirectional shift in cell surface hemostatic properties favoring promotion of clot formation indicates that, in addition to leukocyte procoagulants, endothelium can potentially be instrumental in the pathogenesis of the thrombotic state associated with inflammatory and malignant disorders.

scholarly journals Tumor necrosis factor alpha induces endothelial galactosyl transferase activity and verocytotoxin receptors. Role of specific tumor necrosis factor receptors and protein kinase C

Blood ◽  
1995 ◽  
Vol 85 (3) ◽  
pp. 734-743 ◽  
Author(s):  
NC van de Kar ◽  
T Kooistra ◽  
M Vermeer ◽  
W Lesslauer ◽  
LA Monnens ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Protein Synthesis ◽  
Protein Kinase ◽  
Tumor Necrosis Factor ◽  
Tumor Necrosis ◽  
Tnf Alpha ◽  
Transferase Activity ◽  
Necrosis Factor Alpha ◽  
Galactosyl Transferase ◽  
Necrosis Factor

Infections with verocytotoxin (VT) producing Escherichia coli have been strongly implicated in the epidemic form of hemolytic uremic syndrome (HUS). Endothelial damage plays a central role in the pathogenesis of HUS. In vitro studies have shown that VT can damage endothelial cells after interaction with its cellular receptor globotriaosylceramide (GbOse3cer). Cytokines, such as tumor necrosis factor alpha (TNF alpha) and interleukin-1 (IL-1) can potentiate the toxic effect of VT by inducing a protein-synthesis dependent increase in VT receptors on endothelial cells. In this study, the mechanisms underlying the increase in endothelial VT receptors induced by TNF alpha were studied in more detail. To investigate which proteins were involved in this induction, endothelial cells were incubated with and without TNF alpha in the presence of 14C-galactose or 14C-glucose. Thin-layer chromatography (TLC) analysis of the glycolipid extracts of these cells demonstrated a markedly enhanced incorporation of 14C-galactose in GbOse3cer and other galactose-containing glycolipids, suggesting that TNF alpha enhanced galactosyl-transferase activity. To examine the role of the two recently cloned TNF-receptors (TNFR-p75 and TNFR-p55) in the TNF alpha-induced increase in GbOse3cer in human endothelial cells, cells were incubated with TNF alpha, the TNFR-p55 selective R32W-S86T- TNF alpha-mutant, or the TNFR-p75 selective D143N-A145R-TNF alpha- mutant. The effect of TNF alpha activation, determined by binding- experiments with 125I-VT-1, could be largely, but not completely mimicked by R32W-S86T-TNF alpha. Although incubation of cells with D143N-A145R-TNF alpha did not show an increase in VT-1 binding, the monoclonal antibody utr-1, which prevents binding to TNFR-p75, decreased the TNF alpha-induced VT-1 binding. Activation of protein kinase C (PKC) by phorbol ester increases the expression of VT-1 receptors; this effect was prevented by the PKC inhibitor Ro31–8220 and by homologous desensitization by pretreatment with phorbol ester. In contrast, the presence of the protein kinase inhibitor Ro31–8220 or desensitization of PKC activity reduced the TNF alpha-induced increase in VT-1 receptors maximally by 50% and 24%, respectively. Comparable reductions in overall protein synthesis and the synthesis of E-selectin and plasminogen activator inhibitor-1 (PAI-1) were observed. This suggests an effect on general protein synthesis rather than a specific effect of PKC in the signal transduction pathway, by which TNF alpha induces VT-1 receptors. Our results indicate that TNF alpha can increase the VT-1 receptors on endothelial cells by inducing galactosyl- transferase activity, that this action of TNF alpha mainly occurs via the TNFR-p55; and that PKC activation increases expression of VT-1 receptors by a separate mechanism that acts additively to the TNF alpha- induced increase in VT-1 receptors.

Fluid Shear Stress Attenuates Tumor Necrosis Factor-α–Induced Tissue Factor Expression in Cultured Human Endothelial Cells

Blood ◽  
1998 ◽  
Vol 91 (11) ◽  
pp. 4164-4172 ◽  
Author(s):  
Yutaka Matsumoto ◽  
Yohko Kawai ◽  
Kiyoaki Watanabe ◽  
Kazuo Sakai ◽  
Mitsuru Murata ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Tumor Necrosis Factor ◽  
Tissue Factor ◽  
Shear Force ◽  
Tumor Necrosis ◽  
Shear Stresses ◽  
Dependent Manner ◽  
Tnf Α ◽  
Necrosis Factor

Abstract Hemodynamic forces modulate various endothelial cell functions under gene regulation. Previously, we have shown that fibrinolytic activity of endothelial cells is enhanced by the synergistic effects of shear stress and cytokines. In this study, we investigated the effect of shear stress on tumor necrosis factor (TNF)-α–induced tissue factor (TF) expression in cultured human umbilical vein endothelial cells (HUVECs), using a modified cone-plate viscometer. Shear stresses at physiological levels reduced TNF-α (100 U/mL)–induced TF expression at both mRNA and antigen levels, in a shear-intensity and exposure-time dependent manner, whereas shear stress itself did not induce TF expression in HUVECs. TF expressed on the cell surfaces measured by flow cytometry using an anti-TF monoclonal antibody (HTF-K180) was also decreased to one third by shear force applied at 18 dynes/cm2 for 15 hours before and 6 hours after TNF-α stimulation. Furthermore, functional activity of TF, as assessed by the activation of factor X in the presence of FVIIa and Ca2+, was also decreased by shear application. However, the stability of TF mRNA was not decreased in the presence of shear stress. These results suggest that shear force acts as an important regulator of TF expression in endothelium at the transcriptional level.

scholarly journals Tumor necrosis factor and interleukin-1 induce expression of the verocytotoxin receptor globotriaosylceramide on human endothelial cells: implications for the pathogenesis of the hemolytic uremic syndrome

Blood ◽  
1992 ◽  
Vol 80 (11) ◽  
pp. 2755-2764 ◽  
Author(s):  
NC van de Kar ◽  
LA Monnens ◽  
MA Karmali ◽  
VW van Hinsbergh
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Tumor Necrosis Factor ◽  
Tumor Necrosis ◽  
Interleukin 1 ◽  
Tnf Alpha ◽  
Human Endothelial Cells ◽  
Uremic Syndrome ◽  
Necrosis Factor

Abstract The epidemic form of the hemolytic uremic syndrome (HUS), beginning with an acute gastroenteritis, has been associated with a verocytotoxin- producing Escherichia coli infection. The endothelial cell is believed to play an important role in the pathogenesis of HUS. Endothelial cell damage by verocytotoxin-1 (VT-1) in vitro is potentiated by the additional exposure of inflammatory mediators, such as tumor necrosis factor-alpha (TNF-alpha). Preincubation of human umbilical vein endothelial cells (HUVEC) with TNF-alpha resulted in a 10- to 100-fold increase of specific binding sites for 125I-VT-1. Furthermore, interleukin-1 (IL-1), lymphotoxin (TNF-beta), and lipopolysaccharide (LPS) also markedly increase VT-1 binding. Several hours' exposure to TNF-alpha was enough to enhance the number of VT-1 receptors on the endothelial cells for 2 days. The TNF-alpha-induced increase in VT-1 binding could be inhibited by simultaneous addition of the protein synthesis inhibitor cycloheximide. Glycolipid extracts of TNF-alpha- treated cells tested on thin-layer chromatography demonstrated an increase of globotriaosylceramide (GbOse3cer), a functional receptor for VT-1, which suggests that preincubation of human endothelial cells with TNF-alpha leads to an increase in GbOse3cer synthesis in these cells. We conclude from this study that TNF-alpha and IL-1 induce one (or more) enzyme(s) that is (are) rate-limiting in the synthesis of the glycolipid VT-1 receptor, GbOse3cer. These in vitro studies suggest that, in addition to VT-1, inflammatory mediators play an important role in the pathogenesis of HUS.

Fluid Shear Stress Attenuates Tumor Necrosis Factor-α–Induced Tissue Factor Expression in Cultured Human Endothelial Cells

Blood ◽  
1998 ◽  
Vol 91 (11) ◽  
pp. 4164-4172
Author(s):  
Yutaka Matsumoto ◽  
Yohko Kawai ◽  
Kiyoaki Watanabe ◽  
Kazuo Sakai ◽  
Mitsuru Murata ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Tumor Necrosis Factor ◽  
Tissue Factor ◽  
Shear Force ◽  
Tumor Necrosis ◽  
Shear Stresses ◽  
Dependent Manner ◽  
Tnf Α ◽  
Necrosis Factor

Hemodynamic forces modulate various endothelial cell functions under gene regulation. Previously, we have shown that fibrinolytic activity of endothelial cells is enhanced by the synergistic effects of shear stress and cytokines. In this study, we investigated the effect of shear stress on tumor necrosis factor (TNF)-α–induced tissue factor (TF) expression in cultured human umbilical vein endothelial cells (HUVECs), using a modified cone-plate viscometer. Shear stresses at physiological levels reduced TNF-α (100 U/mL)–induced TF expression at both mRNA and antigen levels, in a shear-intensity and exposure-time dependent manner, whereas shear stress itself did not induce TF expression in HUVECs. TF expressed on the cell surfaces measured by flow cytometry using an anti-TF monoclonal antibody (HTF-K180) was also decreased to one third by shear force applied at 18 dynes/cm2 for 15 hours before and 6 hours after TNF-α stimulation. Furthermore, functional activity of TF, as assessed by the activation of factor X in the presence of FVIIa and Ca2+, was also decreased by shear application. However, the stability of TF mRNA was not decreased in the presence of shear stress. These results suggest that shear force acts as an important regulator of TF expression in endothelium at the transcriptional level.

scholarly journals CytochromeP-450 epoxygenases protect endothelial cells from apoptosis induced by tumor necrosis factor-α via MAPK and PI3K/Akt signaling pathways

2007 ◽  
Vol 293 (1) ◽  
pp. H142-H151 ◽  
Author(s):  
Shilin Yang ◽  
Li Lin ◽  
Ji-Xiong Chen ◽  
Craig R. Lee ◽  
John M. Seubert ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Tumor Necrosis Factor ◽  
Signaling Pathways ◽  
Tumor Necrosis ◽  
Tumor Necrosis Factor Α ◽  
Akt Signaling ◽  
Tnf Α ◽  
Factor Α ◽  
Necrosis Factor

Endothelial cells play a vital role in the maintenance of cardiovascular homeostasis. Epoxyeicosatrienoic acids (EETs), cytochrome P-450 (CYP) epoxygenase metabolites of arachidonic acid in endothelial cells, possess potent and diverse biological effects within the vasculature. We evaluated the effects of overexpression of CYP epoxygenases on tumor necrosis factor-α (TNF-α)-induced apoptosis in bovine aortic endothelial cells. CYP epoxygenase overexpression significantly increased endothelial cell viability and inhibited TNF-α induction of endothelial cell apoptosis as evaluated by morphological analysis of nuclear condensation, DNA laddering, and fluorescent-activated cell sorting (FACS) analysis. CYP epoxygenase overexpression also significantly inhibited caspase-3 activity and downregulation of Bcl-2 expression induced by TNF-α. The antiapoptotic effects of CYP epoxygenase overexpression were significantly attenuated by inhibition of the phosphatidylinositol 3-kinase (PI3K)/Akt and MAPK signaling pathways; however, inhibition of endothelial nitric oxide synthase activity had no effect. Furthermore, CYP epoxygenase overexpression significantly attenuated the extent of TNF-α-induced ERK1/2 dephosphorylation in a time-dependent manner and significantly increased PI3K expression and Akt phosphorylation in both the presence and absence of TNF-α. Collectively, these results suggest that CYP epoxygenase overexpression, which is known to increase EET biosynthesis, significantly protects endothelial cells from apoptosis induced by TNF-α. This effect is mediated, at least in part, through inhibition of ERK dephosphorylation and activation of PI3K/Akt signaling.

scholarly journals The Role of Osteoprotegerin and Tumor Necrosis Factor-related Apoptosis-inducing Ligand in Human Microvascular Endothelial Cell Survival

2004 ◽  
Vol 15 (6) ◽  
pp. 2834-2841 ◽  
Author(s):  
L. B. Pritzker ◽  
M. Scatena ◽  
C. M. Giachelli
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Tumor Necrosis Factor ◽  
Cell Survival ◽  
Tumor Necrosis ◽  
Microvascular Endothelial Cell ◽  
Endothelial Cell Survival ◽  
Trail Receptors ◽  
Microvascular Endothelial ◽  
Necrosis Factor

Endothelial cell survival and antiapoptotic pathways, including those stimulated by extracellular matrix, are critical regulators of vasculogenesis, angiogenesis, endothelial repair, and shear-stress-induced endothelial activation. One of these pathways is mediated by αvβ3 integrin ligation, downstream activation of nuclear factor-κB, and subsequent up-regulation of osteoprotegerin (OPG). In this study, the mechanism by which OPG protects endothelial cells from death was examined. Serum-starved human microvascular endothelial cells (HMECs) plated on the αvβ3 ligand osteopontin were protected from cell death. Immunoprecipitation experiments indicated that OPG formed a complex with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in HMECs under these conditions. Furthermore, inhibitors of TRAIL, including recombinant soluble TRAIL receptors and a neutralizing antibody against TRAIL, blocked apoptosis of serum-starved HMECs plated on the nonintegrin attachment factor poly-d-lysine. Whereas TRAIL was unable to induce apoptosis in HMECs plated on osteopontin, the addition of recombinant TRAIL did increase the percentage of apoptotic HMECs plated on poly-d-lysine. This evidence indicates that OPG blocks endothelial cell apoptosis through binding TRAIL and preventing its interaction with death-inducing TRAIL-receptors

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