Y Deng

Meth-A sarcoma cells were stable transfected to overexpress (sense construct) or underexpress (antisense construct) tissue factor. In vitro, there was no difference in plating efficiency or growth between these cell lines. In vivo, tumor cells transfected to overexpress tissue factor grew more rapidly, and established larger and more vascularized tumors than control transfectants. Antisense transfectants grew the slowest and were the least vascularized. Anticoagulation of mice with warfarin did not alter the difference between these tumor lines. Tumor cells over-expressing tissue factor released more (compared with control transfectants) mitogenic activity for endothelial cells in parallel with enhanced transcription of vascular permeability factor/vascular endothelial cell growth factor (VEGF/VPF), and diminished transcription of thrombospondin (TSP2), a molecule with anti-angiogenic properties. Antisense tissue factor transfectants, while releasing the lowest amount of mitogenic activity, had increased thrombospondin and decreased VEGF/VPF transcription compared with control transfectants or wild-type cells. Experiments with these sense, antisense, truncated sense, or vector tumor lines gave comparable results in complete medium, serum free medium or in the presence of hirudin, indicating that the activation of the coagulation mechanism was not likely to be responsible for changes in tumor cell properties. These results suggest that tissue factor regulates angiogenic properties of tumor cells by altering the production of growth regulatory molecules of endothelium by a mechanism distinct from tissue factor activation of the coagulation mechanism.

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.

Thrombomodulin (TM), recognized as an essential vessel wall cofactor of the antithrombotic mechanism, is also expressed by a wide range of tumor cells. Tumor cell lines subcloned from four patients with malignant melanoma displayed a negative correlation between TM expression and cell proliferation in vitro and in vivo. Overexpression of wild-type TM decreased cell proliferation in vitro and tumor growth in vivo. TM mutants with altered protein C activation capacity lead to a similar effect. In contrast, transfection of melanoma cells with mutant TM constructs, in which a portion of the cytoplasmic or lectin domain was deleted, abrogated the antiproliferative effect associated with overexpression of wild-type TM. Experiments performed with either peptide agonists/antagonists of the thrombin receptor, with hirudin, or with inhibitors of thrombin-TM interaction did not alter the growth inhibitory effect of TM overexpression. These data suggest that TM exerts an effect on cell proliferation independent of thrombin and the thrombin receptor, possibly related to the binding of novel ligands to determinants in the lectin domain which might trigger signal transduction pathways dependent on the cytoplasmic domain.

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.

BACKGROUND: Hydronephrosis is associated with interstitial fibrosis and occlusion of renal capillaries by fibrin. However, the mechanisms leading to fibrin formation is unknown. METHODS AND RESULTS: Twenty days after unilateral ligation of the ureter, interstitial fibrosis occurred in the ligated kidney. Fibrosis was preceded by infiltration of inflammatory cells (macrophages, B and T lymphocytes). Staining with an antibody against von Willebrand factor demonstrated newly formed capillaries in the fibrosing tissue as well as prominent fibrin deposition. Fibrin staining was found around vessels, in the interstitium, the glomeruli, and tubuli. Fibrin deposition was less prominent in the non-ligated kidney and almost absent in sham-operated animals. The expression of tissue factor, the central initiator of coagulation, was induced within 5 days after ligation in the operated kidney but not in the sham-operated animals. Tissue factor positivity was observed by immunohistochemistry in vascular endothelial cells, the vessel wall, tubular epithelial cells, glomerular capsular cells, Bowman's space and in the interstitium. Tissue factor induction was due to increased transcription, since in-situ hybridization showed increased levels of mRNA in the ligated kidney compared to sham-operated rats. The tissue factor gene is under control of the transcription factors activator protein-1 (AP-1) and nuclear factor-kappa B (NF-kappa B). When extracts of operated organs were compared with kidneys of sham-operated rats or contralateral kidneys in electrophoretic mobility shift assays, an increase in AP-1 and NF-kappa B binding activity to their respective binding sites in the tissue factor gene was observed in the operated, but not in the contralateral kidney or kidneys of sham-operated animals. CONCLUSION: Ureteral ligation leads to infiltration of inflammatory cells, increased AP-1 and NF-kappa B expression in the kidney, resulting in increased tissue factor transcription and translation, and ultimately in increased fibrin deposition.