Induction Of Endothelial Tissue Factor Research Articles

Aurintricarboxylic acid inhibits endothelial activation, complement activation, and von Willebrand factor secretion in vitro and attenuates hyperacute rejection in an ex vivo model of pig‐to‐human pulmonary xenotransplantation

In the xenotransplantation of vascularized organs, such as the lung, a large area of endothelial cell layer is a big hurdle to be overcome. We investigated the potential protective effect of aurintricarboxylic acid (ATA), a known inhibitor of platelet adhesion, on endothelial damage induced by xenogeneic serum. We also assessed its role in hyperacute xenograft rejection using a porcine ex vivo lung perfusion model. Porcine endothelial cells were incubated with human serum and other inflammatory stimuli. For the evaluation of von Willebrand factor (vWF) secretion and tissue factor (TF) expression, we used human endothelial cells. E-selectin expression, complement activation, TF expression and platelet activation were investigated by flow cytometry. In an ex vivo porcine lung perfusion model, the porcine lungs were perfused with fresh human whole blood: unmodified blood (n = 5), ATA-treated blood (n = 5), and ATA and lepirudin-treated blood (n = 5). Aurintricarboxylic acid significantly inhibited TNF-alpha- or lipopolysaccharide-induced endothelial E-selectin expression in a dose-dependent manner. ATA also prevented human serum induced-E-selectin expression and human monocytic cell adhesion to porcine endothelial cells. Moreover, ATA abolished thrombin-induced vWF secretion as well as complement activation. However, ATA induced endothelial TF expression and platelet activation in vitro. In ex-vivo experiments, ATA treatment improved pulmonary function and attenuated sequestration of leukocytes. Although ATA did not influence thrombin generation, we were able to minimize its activity by adding lepirudin to the blood with ATA. Our study demonstrated in vitro protective effect of ATA on the inhibition of endothelial activation and vWF secretion and confirmed detrimental effect of ATA on induction of endothelial TF and platelet activation. The combination of ATA and lepirudin may act beneficially by preventing coagulation perturbation while maintaining improved xenograft survival.

Histamine differentially interacts with tumor necrosis factor-alpha and thrombin in endothelial tissue factor induction: the role of c-Jun NH2-terminal kinase.

Histamine plays an important role in vascular disease. Tissue factor (TF) expression is induced in vascular inflammation and acute coronary syndromes. This study examined the effect of histamine on tumor necrosis factor-alpha- (TNF-alpha-) vs. thrombin-induced endothelial TF expression. Histamine (10(-8)-10(-5) mol L-1), TNF-alpha (5 ng mL-1), and thrombin (1 U mL-1) induced TF expression in human endothelial cells. Although TF expression by TNF-alpha and thrombin was identical, histamine augmented TNF-alpha-induced expression 7.0-fold, but thrombin-induced expression only 2.6-fold. Similar responses occurred with TF activity. The H1-receptor antagonist mepyramine abrogated these effects. Differential augmentation by histamine was also observed at the mRNA level. Histamine-induced p38 activation preceded a weak second activation to both TNF-alpha and thrombin. Histamine-induced c-Jun NH2-terminal kinase (JNK) activation was followed by a strong second activation to TNF-alpha, and less to thrombin. Selective inhibition of this second JNK activation by SP600125 reduced TF induction to histamine plus TNF-alpha by 67%, but to histamine plus thrombin by only 32%. Histamine augmented TNF-alpha- and thrombin-induced vascular cell adhesion molecule 1 (VCAM-1) expression to a similar extent. Consistent with this observation, VCAM-1 induction to TNF-alpha and thrombin was mediated by p38, but not by JNK. Histamine differentially augments TNF-alpha- vs. thrombin-induced TF expression and activity, which is mediated by the H1-receptor, occurs at the mRNA level, and is related to differential JNK activation.

Interleukin-1β induced vascular permeability is dependent on induction of endothelial Tissue Factor (TF) activity

IL-1β is a pleotropic cytokine that may mediate increased procoagulant activity and permeability in endothelial tissue during inflammatory conditions. The procoagulant effects of IL-1β are mediated through induction of tissue factor (TF) but its alterations on vascular permeability are not well characterized. We found that IL-1β induced a rapid and dose-dependent increase in TF activity in human umbilical vein endothelial cells (ECs) under routine culture conditions. However, IL-1β caused a rapid and marked increase in permeability across confluent EC monolayers using a two-compartment in vitro model only in the presence of factor VIII-deficient plasma that was completely abrogated by neutralizing anti-TF antibody pre-treatment. In vitro permeability was associated with loss of EC surface expression of VE-cadherin and contraction of F-actin cytoskeletal elements that resulted in EC intercellular gap formation. These data demonstrate that IL-1β induces marked changes in permeability across activated endothelium via a TF dependent mechanism and suggest that modulation of TF activity may represent a strategy to treat various acute and chronic inflammatory conditions mediated by this cytokine.

Advanced glycation end product (AGE)-mediated induction of tissue factor in cultured endothelial cells is dependent on RAGE.

Binding of advanced glycation end products (AGEs) to the cellular surface receptor (RAGE) induces translocation of the transcription factor NF-kappaB into the nucleus and NF-kappaB-mediated gene expression. This study examines the role of RAGE in the AGE albumin-mediated induction of endothelial tissue factor, known to be partly controlled by NF-kappaB. Endothelial cells (ECs) were incubated in the presence of an 18-mer phosphorothioate oligodeoxynucleotide antisense to the 5'-coding sequence of the RAGE gene (antisense RAGE; 0.1 micromol/L). Sense oligonucleotides (sense RAGE, 0.1 micromol/L) of the same region served as control. The cellular uptake of oligonucleotides was controlled by immunofluorescence microscopy. RAGE transcription was suppressed by antisense RAGE, as demonstrated by RT-PCR reactions. AGE albumin-mediated activation of cultured ECs was studied after 48 hours of preincubation of ECs with antisense or sense RAGE. Electrophoretic mobility shift assays and Western blot analysis demonstrated that the AGE albumin-induced translocation of NF-kappaB from the cytoplasm into the nucleus was suppressed in the presence of antisense RAGE but not by sense RAGE. In parallel, AGE albumin-mediated tissue factor transcription, activity, and antigen were significantly reduced in ECs exposed to antisense RAGE, whereas sense RAGE (and nonspecific oligonucleotides) did not influence tissue factor expression. Activation of ECs and induction of tissue factor by AGE albumin in ECs is dependent on RAGE.

Synergistic induction of endothelial tissue factor by tumor necrosis factor and vascular endothelial growth factor: functional analysis of the tumor necrosis factor receptors

Tissue factor expression on the surface of endothelial cells can be induced by tumor necrosis factor (TNF) and vascular endothelial growth factor (VEGF) in a synergistic manner. We have investigated the role of the two different TNF receptors for this synergy. Firstly, stimulation of the 60 kDa TNF receptor (TNFR60) by a mutant of TNF specific for TNFR60 induced responses comparable to wild-type TNF. In contrast, stimulation of TNFR80 by a TNFR80-specific TNF mutein did not result in enhancement of tissue factor expression even in the presence of a suboptimal TNFR60 triggering. Secondly, we tested neutralizing TNF receptor antibodies for inhibition of tissue factor synthesis induced by VEGF and TNF. A TNFR60-specific antibody inhibited tissue factor production over a broad range of TNF concentrations, indicating an essential role of TNFR60 in the TNF/VEGF synergy. In contrast, blocking of TNF binding to TNFR80 strongly inhibited TNF-induced tissue factor expression at low, but less pronounced at high, TNF concentrations. In conclusion, these data are in agreement with a model in which TNFR80 participates in the synergy between VEGF and low concentrations of soluble TNF by passing the ligand to the signalling TNFR60.

Mechanism of the Tumor Necrosis Factor α-mediated Induction of Endothelial Tissue Factor

This study examines the regulation of the human tissue factor (TF) promotor in vitro and in vivo. Transient transfections were performed in bovine aortic endothelial cells to investigate the role of two fundamentally different AP-1 sites and a closely located NF-kappa B site in the human TF promoter. The NF-kappa B site is functionally active, since overexpression of NF-kappa B(p65) resulted in induction of TF mRNA and activity. Promoter analysis showed that NF-kappa B induction was dependent on the integrity of the region from base pair -188 to -181. Over-expression of Jun/Fos resulted in TF induction of transcription and protein/activity. Functional studies revealed that the proximal AP-1 site, but not the distal, was inducible by Jun/Fos heterodimers. The distal AP-1 site, which has a G-->A switch at position 4, was inductible by Jun homodimers. Electrophoretic mobility shift assays, using extracts of tumor necrosis factor alpha (TNF alpha)-stimulated bovine aortic endothelial cells, demonstrated TNF alpha-inducible binding to the proximal AP-1 site, comprising JunD/Fos heterodimers. At the distal AP-1 site, only minor induction of binding activity, characterized as proteins of the Jun and ATF family, was observed. Consistently, this site only marginally participates in TNF alpha induction. Functional studies with TF promotor plasmids confirmed that deletion of the proximal AP-1 or the NF-kappa B site decreased TNF alpha-mediated TF induction to a higher extend than loss of the distal AP-1 site. However, integrity of both AP-1 sites and the NF-kappa B site was required for optimal TNF alpha stimulation. The relevance of these in vitro data was confirmed in vivo in a mouse tumor model. Expression plasmids for a dominant negative Jun mutant or I-kappa B were packaged in liposomes. When either mutated Jun or I-kappa B were injected intravenously 48 h before TNF alpha, a reduction in TNF alpha-mediated TF expression in the tumor endothelial cells was observed. Simultaneously, fibrin/fibrinogen deposition decreased and free blood flow could be restored. Thus, TNF alpha-induced up-regulation of endothelial cell TF depends on a concerted action of members of the bZIP and NF-kappa B family.

Retinoic Acid Reduces Induction of Monocyte Tissue Factor and Tissue Factor/Factor Vila-Dependent Arterial Thrombus Formation

Agents that downregulate the induction of monocyte/macrophage tissue factor (TF) activity may attenuate the thrombotic risk associated with mechanical restoration of vessel patency or artificial arterial grafting. In such events, procoagulant macrophages in the atherosclerotic plaque and procoagulant monocytes adherent to artificial materials may be exposed to the blood stream. Ishii et al (Blood 80:2556, 1992) reported that induction of endothelial TF is downregulated by all-trans retinoic acid (ATRA), and Conese et al (Thromb Haemost 66:662, 1991) reported that retinoids downregulate monocyte procoagulant activity (PCA). These findings led us to investigate the effect of ATRA on monocyte TF expression, and to study the effect of ATRA on monocyte-induced thrombus formation in a model system of human arterial thrombogenesis. Induction of PCA in human peripheral blood monocytes by 0.5 microgram/mL lipopolysaccharide (LPS) was dose dependently reduced by ATRA, reaching a reduction of 56% at 10(-5) mol/L ATRA (P < .0001). A 38% reduction (P < .0007) in LPS-induced TF antigen expression was observed at an ATRA concentration of 10(-6) mol/L. Adherence of monocytes to plastic cover slips (Thermanox, Miles Laboratories, Naperville, IL) also triggered induction of cellular PCA, which was inhibited by more than 80% by an anti-TF monoclonal antibody (MoAb) (P < .002). Inclusion of ATRA (10(-6) mol/L) reduced this PCA by 40% (P < .03), and the TF antigen expression by 30% (P < .0001). Exposure of Thermanox adherent monocytes to flowing nonanticoagulated human blood in a parallel-plate perfusion chamber device at an arterial wall shear rate of 650 s-1 elicited significant fibrin deposition and platelet thrombus formation. Partial interruption of this thrombus formation was achieved by 10(-6) mol/L ATRA, which reduced the fibrin deposition by 80% (P < .02) and platelet thrombus formation by 50% (P < .05). In comparison, incubation of adherent monocytes with the anti-TF MoAb before the blood exposure, reduced the fibrin deposition by 83% (P < .02) and platelet thrombus volume by 75% (P < .0008). Thus, ATRA is an effective down-regulator of monocyte TF-PCA, and may reduce thrombotic complications at sites of plaque rupture, at plaque disruption after percutaneous transluminal angioplasty procedures, or on surfaces introduced by artificial arterial grafting.

Induction of Endothelial Tissue Factor by Endotoxin and Its Precursors

The structural determinants of lipopolysaccharide required for the induction of tissue factor in human umbilical vein endothelial cells were studied. Intact lipid A was essential for the induction of tissue factor whereas the incomplete lipid A precursors lipid IVA and lipid X, as well as monophosphoryl lipid A and acyloxyacyl hydrolase-treated lipopolysaccharide, were unable to induce tissue factor and tissue factor specific mRNA. However, the lipid A precursor, lipid IVA, was able to inhibit LPS-mediated induction of tissue factor; structural determinants distal to lipid A were found to be required for maximal induction of tissue factor activity and tissue factor mRNA. The presence of serum in the assay was found to amplify but was not obligate for tissue factor induction by LPS.

Synthesis and release of interleukin 1 by reoxygenated human mononuclear phagocytes.

To examine the possible involvement of cytokines in reperfusion injury, we have studied production of IL-1 by human vascular cells, including smooth muscle and mononuclear phagocytes. Exposure of cells to hypoxia (pO2 approximately 14 torr) followed by reoxygenation led to significant release of IL-1 only from the mononuclear phagocytes. Elaboration of IL-1 was dependent on the oxygen tension and duration of hypoxia (optimal at lower pO2s, approximately 14-20 torr, and after 9 h), as well as the time in reoxygenation (maximal IL-1 release at 6-9 h). Although a period of hypoxia was necessary for subsequent IL-1 production during reoxygenation of either peripheral blood monocytes or cultured monocyte-derived macrophages, no IL-1 release occurred during the hypoxic exposure. IL-1 released during reoxygenation was newly synthesized, and its production was triggered by the generation of oxygen free radicals, as it could be blocked by the addition of either allopurinol or free radical scavengers to cultures and could be stimulated in part by low concentrations of hydrogen peroxide or xanthine/xanthine oxidase. The potential pathophysiological effects of IL-1-containing supernatants from reoxygenated macrophages was shown by their induction of endothelial tissue factor and enhancement of endothelial adhesiveness for neutrophils, both of which could be blocked by anti-IL-1 antibody. The relevance of IL-1 to hypoxia/reoxygenation in vivo was suggested by the presence of circulating nanogram amounts of this cytokine in the plasma of mice during the reoxygenation period following a hypoxia.

Selective induction of endothelial cell tissue factor in the presence of a tumour‐derived mediator: A potential mechanism of flavone acetic acid action in tumour vasculature

Flavone acetic acid (FAA) is a potentially useful anti-tumour agent which has been reported to induce changes in tumour vasculature, in particular loss of bloodflow. This led us to examine whether endothelium could be a cellular target of FAA action, with resultant modulation of cell-surface coagulant properties leading to activation of coagulation and blockade of tumour blood flow. Incubation of endothelium with FAA led to the expression of functional tissue factor on the cell surface, in a time-dependent and dose-dependent (half-maximal at 0.6-0.7 mg/ml) manner. Induction of tissue-factor activity resulted from de novo translation of the tissue factor message. To explain the selectivity of FAA's action on tumour vasculature in vivo, we considered its interaction with tumour-derived factors. Starting with serum-free FO-I-melanoma cell-conditioned medium, a co-factor enhancing FAA-mediated induction of endothelial tissue factor (FO-I factor) was partially purified by sequential ion exchange and reverse phase chromatography, followed by preparative SDS-PAGE. The FO-I factor migrates with an apparent Mr of approx. 20 to 25,000 on non-reduced SDS-PAGE, is sensitive to protease K, and augments the effect of FAA on endothelial-cell-tissue factor. This activity is not found in supernatants from non-neoplastically transformed cell lines. These data lead us to hypothesize that FAA exerts its action, at least in part, by promoting activation of coagulation on the endothelial surface, and this effect is selective for the tumour bed by virtue of its interaction with a tumour-derived factor. The interaction of FAA with host factors may be important for optimizing its therapeutic efficacy for a particular tumour.