Biomedical subjects

A Bierhaus

Publications and source records attributed to A Bierhaus.

At least 55 records · Page 3Linked to original sources

PubMed1998

Molecular mechanisms of diabetic angiopathy--clues for innovative therapeutic interventions.

Advanced glycation end products (AGEs), known to accumulate during aging and at accelerated rate during the course of diabetes, have been demonstrated to induce changes in endothelial properties that might contribute to the pathogenesis of micro- and macroangiopathy. Since AGE-formation not only changes the physicochemical properties of proteins, but also induces cellular signalling, activation of transcription factors and subsequent gene expression, AGEs are now regarded as important mediators of diabetic vascular disease. The growing knowledge on the molecular mechanisms underlying the AGE-dependent activation of vascular endothelial cells implicates possible new therapeutic interventions in the therapy of diabetic angiopathy.

Diabetic Angiopathies↗

PubMed1998

Hyperhomocyst(e)inemia and endothelial dysfunction in IDDM.

OBJECTIVE: Considering that elevated blood levels of homocyst(e)ine represent a known independent risk factor for macrovascular disease, we assessed the link between hyperhomocyst(e)inemia and diabetic microvascular complications. RESEARCH DESIGN AND METHODS: Homocyst(e)ine and thrombomodulin plasma levels, a marker of endothelial cell damage, were measured before and 3 h after oral methionine loading in 75 patients with stable, well-controlled IDDM and 40 healthy control subjects matched for sex and age. Exclusion criteria were hyperlipidemia, hypertension, smoking, or positive family history for cardiovascular disease. RESULTS: IDDM patients had higher pre- and postload homocyst(e)ine plasma levels than did healthy control subjects (12.0 vs. 7.7 mumol/l and 27.6 vs. 16.0 mumol/l; P < 0.001). Of 75 IDDM patients, 26 had homocyst(e)ine plasma levels above the normal range (defined as mean +2 SD of values obtained in the control group). The IDDM patients with hyperhomocyst(e)inemia had higher thrombomodulin plasma levels (62.2 vs. 38.2 ng/ml; P < 0.001), higher albumin excretion rates (485 vs. 115 mg/l; P < 0.005), and a higher prevalence of late diabetic complications (nephropathy, 76 vs. 33%; retinopathy, 69 vs. 51%; neuropathy, 57 vs. 41%; macroangiopathy, 57 vs. 33%) compared with IDDM patients with normal plasma homocyst(e)ine. In vitro experiments with human umbilical vein cells show an increased release of thrombomodulin into the culture supernatant only when endothelial cells were pretreated with advanced glycation end product (AGE)-albumin before L-homocystine was added. A synergistic action of homocyst(e)ine and AGEs might contribute to vascular complications of patients with diabetes. CONCLUSIONS: Hyperhomocyst(e)inemia is common in nephropathic diabetic patients and may contribute to the enhanced morbidity and mortality from cardiovascular diseases characteristically observed in IDDM patients with diabetic nephropathy.

Adult↗

PubMed1998

Insufficient glycemic control increases nuclear factor-kappa B binding activity in peripheral blood mononuclear cells isolated from patients with type 1 diabetes.

OBJECTIVE: The redox-sensitive transcription factor nuclear factor-kappa B (NF-kappa B) is believed to contribute to late diabetic complications. It is unknown whether NF-kappa B is influenced by glycemic control. RESEARCH DESIGN AND METHODS: To determine whether NF-kappa B is activated in patients with insufficient glycemic control (HbA1c > 10%), we developed a tissue culture-independent electrophoretic mobility shift assay (EMSA)-based semiquantitative detection system that allowed us to determine NF-kappa B activation in ex vivo-isolated peripheral blood mononuclear cells (PBMCs). We included 43 patients with type 1 diabetes in this cross-sectional study. 10 of those received the antioxidant thioctic acid (600 mg/day p.o.) for 2 weeks. RESULTS: Monocytes of patients with HbA1c levels > 10% demonstrated significantly higher NF-kappa B binding activity in an EMSA and a stronger NF-kappa B staining in immunohistochemistry than monocytes of patients with HbA1c levels of 6-8%. The increase in NF-kappa B activation correlated with an increase in plasmatic markers of lipid peroxidation. Treatment with the antioxidant thioctic acid decreased NF-kappa B binding activity. CONCLUSIONS: Hyperglycemia induces activation of the transcription factor NF-kappa B in ex vivo-isolated PBMCs of patients with type 1 diabetes. NF-kappa B activation is at least partially dependent on oxidative stress, since the antioxidant thioctic acid significantly lowered the extent of NF-kappa B binding activity.

Adult↗

PubMed1997

Cyclosporin A inhibits monocyte tissue factor activation in cardiac transplant recipients.

BACKGROUND: Fibrin deposition and thrombosis have been implicated in both allograft rejection and vasculopathy after cardiac transplantation. Because monocytes play a pivotal role in the pathophysiology of intravascular coagulation activation through their ability to synthesize tissue factor (TF), we asked (1) whether monocyte TF activation occurs in cardiac transplant recipients and (2) whether monocyte TF expression is affected by treatment with cyclosporin A (CsA). METHODS AND RESULTS: We measured levels of TF activity in peripheral blood mononuclear cells and highly purified monocytes/macrophages from 10 consecutive cardiac transplant recipients and 10 healthy control subjects. TF activity generated by both unstimulated and endotoxin-stimulated cells was significantly higher in transplant recipients than in control subjects (P<.05). Increased monocyte TF expression in transplant recipients was shown to be adversely affected by treatment with CsA: TF induction was markedly reduced by CsA serum concentrations reaching peak CsA drug levels. Inhibition of TF induction in the presence of high CsA blood concentrations was also observed when stimulation of cells was performed with interferon-gamma or interleukin-1beta. As shown by reverse transcription-polymerase chain reaction and electrophoretic mobility shift assay, respectively, treatment with CsA leads to decreased TF mRNA expression and reduced activation of the NF-kappaB transcription factor, which is known to contribute to the induction of the TF promotor in human monocytes. CONCLUSIONS: This study demonstrates that TF activation, occurring in mononuclear cells of cardiac transplant recipients, is inhibited by treatment with CsA. Inhibition of monocyte TF induction by CsA may contribute to its successful use in cardiac transplant medicine and might be useful in managing further settings of vascular pathology also known to involve TF expression and NF-kappaB activation.

Adult↗

PubMed1997

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

BACKGROUND: 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. METHODS AND RESULTS: 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. CONCLUSIONS: Activation of ECs and induction of tissue factor by AGE albumin in ECs is dependent on RAGE.

Animals↗

PubMed1997

Role of NFkappaB in the mortality of sepsis.

Binding activity for nuclear factor kappa B (NFkappaB) consensus probes was studied in nuclear extracts from peripheral blood mononuclear cells of 15 septic patients (10 surviving and 5 not surviving). Nonsurvivors could be distinguished from survivors by an increase in NFkappaB binding activity during the observation period (P < 0.001). The increase in NFkappaB binding activity was comparable to the APACHE-II score as a predictor of outcome. Intravenous somatic gene transfer with an expression plasmid coding for IkappaBalpha was used to investigate the role of members of the NFkappaB family in a mouse model of endotoxemia. In this model, increased NFkappaB binding activity was present after injection of LPS. Intravenous somatic gene transfer with IkappaBalpha given before LPS attenuated renal NFkappaB binding activity and increased survival. Endothelial cells and monocytes/macrophages were the major target cells for somatic gene transfer, transfected with an average transfection efficiency of 20-35%. Tissue factor, a gene under regulatory control of NFkappaB, was induced by LPS. Somatic gene transfer with a reporter plasmid containing the functional tissue factor promoter demonstrated NFkappaB-dependent stimulation by LPS. Intravenous somatic gene transfer with IkappaBalpha reduced LPS-induced renal tissue factor expression, activation of the plasmatic coagulation system (decrease of thrombin-antithrombin III complexes) and renal fibrin/fibrinogen deposition. Somatic gene transfer with an expression plasmid with tissue factor cDNA in the antisense direction (in contrast to sense or vector alone) also increased survival. Furthermore, antisense tissue factor decreased renal tissue factor expression and the activation of the plasmatic coagulation system.

Adult↗

PubMed1997

Differential expression of CD44s and CD44v10 proteins and syndecan in normal and irradiated mouse epidermis.

The role of the CD44s adhesion molecule, its epithelial isoforms and its relationship to epidermal proteoglycans such as syndecan was studied in normal and irradiated mouse skin. In normal mouse skin, only 10% of basal cells are strongly CD44s-immunopositive, with a cytoplasmic expression pattern. Double-label experiments with the basal cell marker keratin 14 confirmed the epithelial nature of the strongly CD44s-positive cell type in the basal layer. Some spinous keratinocytes and the majority of the remaining basal cells exhibited a weak membranous staining pattern. In contrast, the epithelial isoform, CD44v10, was strongly present in all basal and suprabasal epithelial cells of the epidermis, with a membranous staining pattern. Syndecan was found in the granular layer of the normal epidermis only. After 1 week of daily irradiation, the entire basal cell layer of the epidermis expressed CD44s in the membrane, but with a varying degree of staining intensity. This reactivity spread to the upper spinous layer after 3 weeks of treatment. In hyperproliferative epidermis, there was no difference in the staining patterns between CD44s and CD44v10. The expression of syndecan switched from the granular layer to the basal and lower spinous layers after 2 weeks of daily irradiation. Immunoreactivity for syndecan was also strongly enhanced in the dermis of irradiated samples. The results suggest an important role for syndecan and CD44 in proliferative processes during radiation-induced accelerated repopulation.

Animals↗

PubMed1997

Advanced glycation end product-induced activation of NF-kappaB is suppressed by alpha-lipoic acid in cultured endothelial cells.

Depletion of cellular antioxidant defense mechanisms and the generation of oxygen free radicals by advanced glycation end products (AGEs) have been proposed to play a major role in the pathogenesis of diabetic vascular complications. Here we demonstrate that incubation of cultured bovine aortic endothelial cells (BAECs) with AGE albumin (500 nmol/l) resulted in the impairment of reduced glutathione (GSH) and ascorbic acid levels. As a consequence, increased cellular oxidative stress led to the activation of the transcription factor NF-kappaB and thus promoted the upregulation of various NF-kappaB-controlled genes, including endothelial tissue factor. Supplementation of the cellular antioxidative defense with the natural occurring antioxidant alpha-lipoic acid before AGE albumin induction completely prevented the AGE albumin-dependent depletion of reduced glutathione and ascorbic acid. Electrophoretic mobility shift assays (EMSAs) revealed that AGE albumin-mediated NF-kappaB activation was also reduced in a time- and dose-dependent manner as long as alpha-lipoic acid was added at least 30 min before AGE albumin stimulation. Inhibition was not due to physical interactions with protein DNA binding, since alpha-lipoic acid, directly included into the binding reaction, did not prevent binding activity of recombinant NF-kappaB. Western blots further demonstrated that alpha-lipoic acid inhibited the release and translocation of NF-kappaB from the cytoplasm into the nucleus. As a consequence, alpha-lipoic acid reduced AGE albumin-induced NF-kappaB mediated transcription and expression of endothelial genes relevant in diabetes, such as tissue factor and endothelin-1. Thus, supplementation of cellular antioxidative defense mechanisms by extracellularly administered alpha-lipoic acid reduces AGE albumin-induced endothelial dysfunction in vitro.

Antioxidants↗

PubMed1997

Hyperhomocyst(e)inemia and endothelial dysfunction in IDDM.

OBJECTIVE: While elevated blood levels of homocyst(e)ine represent an independent risk factor for macrovascular disease, we assessed the link between hyperhomocyst(e)inemia and diabetic microvascular diseases. RESEARCH DESIGN AND METHODS: Plasma levels of homocyst(e)ine and thrombomodulin (TM), markers of endothelial cell damage, were measured before and 3 h after oral methionine loading in 75 patients with IDDM and 40 healthy control subjects matched for sex and age. Exclusion criteria were hyperlipidemia, hypertension, smoking, or positive family history for cardiovascular disease. RESULTS: IDDM patients had higher pre- and postload plasma levels of homocyst(e)ine than did healthy control subjects (12.0 vs. 7.7 mumol/l and 27.6 vs. 16.0 mumol/l; P < 0.001). Of 75 IDDM patients, 26 had plasma homocyst(e)ine levels above the normal range (means +/- 2 SD of values obtained in the control group). These IDDM patients with hyperhomocyst(e)inemia had higher plasma TM levels (62.2 vs. 38.2 ng/ml, P < 0.001), higher albumin excretion rates (485 vs. 115 mg/l, P < 0.005), and a higher prevalence of late diabetic complications (nephropathy, 76 vs. 33%; retinopathy, 69 vs. 51%; neuropathy, 57 vs. 41%; and macroangiopathy, 57 vs. 33%) compared with IDDM patients with normal plasma homocyst(e)ine. In vitro experiments with human umbilical vein cells showed an increased release of TM into the culture supernatant only when endothelial cells were pretreated with advanced glycation end product (AGE)-albumin before L-homocystine was added. A synergistic action of homocyst(e)ine and AGEs might contribute to vascular complications in patients with diabetes. CONCLUSIONS: Hyperhomocyst(e)inemia is common in nephropathic diabetic patients and may contribute to the enhanced morbidity and mortality from cardiovascular diseases characteristically observed in IDDM patients with diabetic nephropathy.

Administration, Oral↗

PubMed1997

Connexin 43 expression in normal and irradiated mouse skin.

The expression of connexin 43 (Cx43) in normal and irradiated mouse skin was evaluated using immunofluorescence with polyclonal antisera to Cx43 on cryostat sections. One week after the start of daily irradiation with 3 Gy, enhanced immunoreactivity in basal cells and in the lower part of the spinous layer of the epidermis was detected. In hypertrophic and hyperplastic skin, about 3 weeks after irradiation, a further increase in the expression of Cx43 was found in the epidermis as well as in the dermis. Western blot analysis of normal and irradiated skin homogenates confirmed the increased Cx43 protein concentration in the skin. In nuclear extracts of the same preparations, increased AP-1-binding activity was found. These data suggest that the gap-junction protein is expressed in response to irradiation of the skin and may be related to the subsequent hyperproliferative response which is associated with accelerated repopulation.

Animals↗

PubMed1997

The dietary pigment curcumin reduces endothelial tissue factor gene expression by inhibiting binding of AP-1 to the DNA and activation of NF-kappa B.

The natural occurring pigment curcumin, a major component of the spice tumeric, has been described to have antioxidative, anti-tumorpromoting, anti-thrombotic and anti-inflammatory properties. It appears, that the pleiotropic effects of curcumin are at least partly due to inhibition of the transcription factors NF-kappa B and AP-1. This study investigates the effect of curcumin on the TNF alpha induced expression of endothelial Tissue Factor (TF), the central mediator of coagulation known to be controlled by AP-1 and NF-kappa B. When bovine aortic endothelial cells (BAEC) were preincubated in the presence of curcumin, TNF alpha induced TF gene transcription and expression were reduced. Transient transfection studies with TF-promoter plasmids revealed that both, NF-kappa B and AP-1 dependent TF expression, were reduced by curcumin action. The observed inhibitions were due to distinct mechanisms. Curcumin inhibited TNF alpha induced I kappa B alpha degradation and the nuclear import of NF-kappa B. In contrast, inhibition of AP-1 was due to a direct interaction of curcumin with AP-1-binding to its DNA binding motif. Thus, curcumin inhibits NF-kappa B and AP-1 by two different mechanisms and reduces expression of endothelial genes controlled by both transcription factors in vitro.

Animals↗

PubMed1996

Cyclosporine a inhibits tissue factor expression in monocytes/macrophages.

Accelerated coronary atherosclerosis in cardiac allografts is the major limiting factor for long-term survival after heart transplantation. There is growing evidence that activation of the coagulation mechanism is involved in the development of transplant atherosclerosis. Tissue factor (TF) expression by cells of the monocyte/macrophage system may represent an important mechanism underlying the fibrin deposition in the affected vessels. In the present study, we investigated the effect of cyclosporine A (CsA) on the lipopolysaccharide (LPS)-induced procoagulant activity (PCA) in human monocytes/macrophages. CsA exerted a dose-dependent inhibitory effect on LPS-induced monocyte/macrophage PCA, which was identified as TF activity based on functional and immunologic characterization. As shown by reverse transcriptase-polymerase chain reaction, CsA reduced the transcription of the TF gene in LPS-stimulated monocytes/macrophages. Electrophoretic mobility shift assay showed that CsA inhibited the LPS-induced activation of the nuclear factor kappa B (NF-kappa B). As shown by Western blot analysis, CsA treatment decreased the nuclear translocation of NF-kappa B, thereby suggesting the mechanism for the inhibitory effect of CsA on TF induction. Hence, a nonimmunologic effect of CsA may contribute to its successful use in transplant medicine.

Biological Transport↗

PubMed1996

Intravenous somatic gene transfer with antisense tissue factor restores blood flow by reducing tumor necrosis factor-induced tissue factor expression and fibrin deposition in mouse meth-A sarcoma.

Fibrin is deposited on the endothelial cell surface in the vasculature of murine methylcholanthrene A-induced sarcomas after injection of tumor necrosis factor (TNF). Capillary endothelial cells of the tumor vascular bed become positive for tissue factor after TNF injection, based on immunocytochemistry and in situ hybridization. Intravascular clot formation was not dependent on tissue factor derived from tumor cells, since in vessels of tumors not expressing tissue factor, TNF also induced fibrin/fibrinogen deposition. However, the time course of fibrin/fibrinogen deposition after TNF differed in tumors expressing no, little, or greater amounts of tissue factor. Fibrin/fibrinogen deposition was more rapid in tumors in which the neoplastic cells expressed tissue factor than in tumors not expressing tissue factor. In the tumors not expressing tissue factor, activation of coagulation was dependent on TNF-induced synthesis of tissue factor by host cells, i.e., endothelium or monocytes/macrophages. Intravenous somatic gene transfer with tissue factor cDNA in the antisense orientation (but not sense or vector alone) reduced intravascular fibrin/fibrinogen deposition and restored blood flow to the tumor, showing that de novo tissue factor expression is central in TNF-induced activation of the coagulation mechanism.

Animals↗

PubMed1996

Expression of CD44 isoforms during bleomycin-or radiation-induced pulmonary fibrosis in rats and mini-pigs.

The distribution of CD44s and CD44v molecules in normal and injured lung tissue of rats and mini-pigs was studied by examining the immunohistochemical binding of monoclonal antibodies against CD44 isoforms. We showed that the expression of CD44v and CD44s varies greatly among different pulmonary fibrosis samples and that some tissues express either enhanced expression of CD44s, particularly in the interstitium and on alveolar macrophages, or very low levels of CD44v in the alveolar epithelium. Normal type II pneumocytes expressed the CD44s and CD44v molecules at the basolateral aspect of the cell. Such localisation favours a role for CD44 in epithelial cell-fibroblast interaction during lung development and repair.

Animals↗

PubMed1996

Expression of receptors for advanced glycation end-products in occlusive vascular and renal disease.

Formation and deposition of advanced glycation end-products (AGEs) has been linked to late diabetic complications. Interactions of AGEs are at least partly mediated by binding of AGEs to their cellular surface receptor RAGE. This review summarizes the immunohistological data obtained for RAGE distribution in vessel segments of diabetic and non-diabetic patients with peripheral occlusive vascular disease and in kidneys of patients with diabetic nephropathy, and inflammatory and non-inflammatory renal disease. It is demonstrated that increased RAGE expression is not restricted to diabetes mellitus but contributes to a range of vascular and renal disorders.

Diabetic Angiopathies↗

PubMed1996

Release of thrombomodulin from endothelial cells by concerted action of TNF-alpha and neutrophils: in vivo and in vitro studies.

Inflammatory cytokines decrease the expression of thrombomodulin (TM) on the endothelial cell surface by suppression of TM transcription and translation or internalization with subsequent degradation. Nevertheless, elevated serum TM levels are found in diseases associated with systemical or locally increased levels of inflammatory cytokines. To study directly the in vivo effects of tumour necrosis factor-alpha (TNF-alpha) we determined the course of serum TM after systemic recombinant human (rh)TNF-alpha therapy. The TM levels were determined by enzyme-linked immunosorbent assay (ELISA). Systemic rhTNF-alpha therapy resulted in a marked and significant increase of serum TM. Using a mouse model we studied whether increased serum TM is associated with a decreased expression of TM on the endothelial surface in vivo. The immunohistochemical staining of the vasculature of meth-A sarcoma transplanted in mice showed a loss of TM immunoreactivity 4 hr after intravenous TNF-alpha application. To study the mechanism of TNF-alpha mediated release of TM, cultured endothelial cells were incubated with neutrophils and TNF-alpha. Incubation with TNF-alpha alone did not lead to an increase of TM in vitro. However TM was released into the culture supernatant when endothelial cells pretreated with TNF-alpha were exposed to neutrophils. This was associated with morphological evidence of endothelial cell damage. Therefore, the concerted action of cytokine-stimulated endothelial cells and neutrophils results in release of TM from cultured endothelial cells after rhTNF-alpha therapy. This might explain the increased serum TM levels observed in diseases associated with increased systemic or local levels of inflammatory cytokines despite the induced internalization and the direct inhibitory effects of TNF-alpha on TM transcription and translation.

Adult↗

PubMed1996

Tissue factor expression during human and mouse development.

In the adult organism the cellular distribution of tissue factor (TF) expression corresponds to biological boundary layers forming a hemostatic barrier ready to activate blood coagulation after tissue injury. Whether TF expression might also play a role in development is unknown. To determine the significance of TF in ontogenesis, we examined the pattern of TF expression in mouse development and compared it with the distribution of TF in human post-implantation embryos and fetuses of corresponding gestational age. At early embryonic periods of murine (6.5 and 7.5 pc) and human (stage 5) development, there was strong expression of TF in both ectodermal and entodermal cells. In situ hybridization and immunohistochemistry demonstrated that TF mRNA and protein were expressed widely in epithelial areas with high levels of morphogenic activity during organogenesis. Staining for TF was seen during ontogenetic development in tissues such as epidermis, myocardium, bronchial epithelium, and hepatocytes, which express TF in the adult organism. Surprisingly, during renal development and in adults, expression of TF differed between humans and mice. In humans, maturing stage glomeruli were stained for TF whereas in mice, TF was absent from glomeruli but was present in the epithelia of tubular segments. In neuroepithelial cells, there was a substantial expression of TF. Moreover, there was robust TF expression in tissues such as skeletal muscle and pancreas, which do not express it in the adult. In contrast, expression of the physiological ligand for TF, factor VII, was not detectable during early stages of human embryogenesis using immunohistochemistry. The temporal and spatial pattern of TF expression during murine and human development supports the contention that TF serves as an important morphogenic factor during embryogenesis.

Adult↗

PubMed1995

Mechanism of the tumor necrosis factor alpha-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.

Animals↗