R. Ziegler

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.

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.

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.

Clinical consequences of osteoporotic vertebral fractures, such as back pain, functional limitations, and impairment of mood, are often cited as justification for prevention and therapy. But these symptoms are poorly characterized, and a clinical grading system is not available. The aim of this study was to compare clinical measures for spinal deformation and quality of life components between patients with osteoporosis and patients with chronic low back pain (CLBP) and to determine the relationship between spinal deformation and quality of life components. A total of 130 female patients (63 osteoporotic patients, 65 +/- 7.9 years, and 77 CLBP patients, 56 +/- 6.5 years) had a standardized interview on quality of life components (pain, activities of daily life, mood) and clinical measures of spinal deformation (height reduction [HR], distance from occiput to wall [DOW], and distance from iliac crest to ribs [DIR]). Spinal X-rays were reviewed in all patients for the evidence of vertebral fractures. In osteoporotic patients, vertebral deformity was quantified by the spine deformity index (SDI) on X-rays. It was assessed whether subgroups could be identified by a combination of indices for spinal deformation (SDI, HR, DOW) using a cluster analysis. Back pain was a major complaint in both groups, without differences in pain intensity and frequency. Impairment of general well being and mood was found in about one-third of the patients in both groups. Independent of age, the disability score was significantly higher in patients with osteoporosis than in patients with CLBP. Both groups differed with respect to clinical measures of spinal deformity (HR, DOW, DIR). Among osteoporotic patients, parameters of quality of life were not linearly related to the degree of radiologically assessed vertebral deformity, but osteoporotic patients with two or more vertebral fractures tended to have more functional limitations than those with only one fracture. There was, however, a significant linear relationship between components of quality of life (disability score, pain) and clinical measures of spinal deformation (HR, DOW, DIR). The osteoporotic patients were subdivided into three clusters. The first group was characterized by low spinal deformation (decreases SDI, decreases HR, decreases DOW) and little impairment of quality of life. The second group had significantly greater spinal deformation (increases SDI, increases HR, increases DOW) and significantly more pain and functional limitations. The third group was characterized by increased kyphosis, mainly caused by nonskeletal dysfunction (decreases SDI, decreases HR, increases DOW), but pain and functional limitations were impaired to the same degree as in the second group with severe skeletal spinal deformation. We conclude that with respect to quality of life components, functional limitation is the most specific to spinal osteoporosis and is related to clinical measures of spinal deformation. Furthermore, spinal deformation and the clinical course of osteoporosis appears to be insufficiently reflected by radiological indices of vertebral deformity (such as SDI) alone. For grading the disease and for therapeutical concepts, radiological measures and clinical evaluation should be considered in combination.