Requirement for binding of catalytically active factor VIIa in tissue factor-dependent experimental metastasis

Tissue factor (TF), the initiating cell surface receptor of the coagulation cascade, plays important roles in embryogenesis, angiogenesis, and tumor cell metastasis. It is controversial whether proteolytic function of TF complexed with its serine protease ligand VIIa is required for metastatic tumor dissemination. We show here in a model for TF-dependent experimental hematogenous metastasis, that TF supports metastasis by both proteolytic activity of the TF-VIIa complex and currently undefined functions of the cytoplasmic domain. We demonstrate that ligand binding of VIIa to TF is required for metastasis. Antimetastatic properties of covalently inactivated VIIa provide evidence that ligand binding is insufficient per se to support metastasis, emphasizing that proteolytic activity is necessary for the metastatic process. Ala or Asp mutations of cytoplasmic serine residues were introduced to preclude or mimic phosphorylation. In vivo analysis of these mutants suggests that local protease generation on the tumor cell surface does not serve simply to activate the cytoplasmic domain of TF by serine phosphorylation. Thus, extracellular functions of the catalytically active TF-VIIa complex cooperate with specific functions of the TF cytoplasmic domain to support the complex process of hematogenous tumor cell dissemination.     

Tissue-factor antigen and activity in human coronary atherosclerotic plaques

BACKGROUND: Coronary atherosclerotic-plaque thrombosis is a key event in the pathogenesis of unstable angina and myocardial infarction. Although plaque rupture or fissuring frequently occurs in atherosclerosis, only a small proportion of ruptured plaques develop thromboses. METHODS: Tissue-factor antigen and activity were measured in atherectomy samples from 50 consecutive patients with coronary artery disease (stable angina n = 19, unstable angina n = 24, and myocardial infarction n = 7). FINDINGS: Median tissue-factor antigen and activity concentrations were significantly higher in plaques from patients with unstable angina and myocardial infarction than in those from patients with stable angina (antigen: 66.1 pg/mg [interquartile range 43.8-82.5] vs 32.4 pg/mg [9.8-43.4], p = 0.0001; activity: 0.22 mU/mg [0.17-0.41] vs 0.13 mU/mg [0.05-0.16], p = 0.0004). INTERPRETATION: Tissue-factor, an initiator of the coagulation cascade, may account for the different thrombotic responses to the rupture of human coronary atherosclerotic plaques.     

Apoptosis and related proteins in different stages of human atherosclerotic plaques

BACKGROUND: The transition of a fatty streak into an atherosclerotic plaque is characterized by the appearance of focal and diffuse regions of cell death. We have investigated the distribution of apoptotic cell death and apoptosis-related proteins in early and advanced atherosclerotic lesions. METHODS AND RESULTS: Human atherosclerotic plaques were studied by whole-mount carotid endarterectomy specimens (n=18). This approach allowed comparison of adaptive intimal thickenings, fatty streaks, and advanced atherosclerotic plaques of the same patient. The fatty streaks differed from adaptive intimal thickenings by the presence of BAX (P<0.01), a proapoptotic protein of the BCL-2 family. Both regions were composed mainly of smooth muscle cells (SMCs), and macrophage infiltration was low and not different. Apoptosis, as detected by DNA in situ end labeling (terminal deoxynucleotidyl transferase end labeling [TUNEL] and in situ nick translation) was not present in these regions. Apoptosis of SMCs and macrophages, however, was present in advanced atherosclerotic plaques that were present mainly in the carotid sinus. A dense infiltration of macrophages (5.8+/-3% surface area) was present in these advanced atherosclerotic plaques. Cytoplasmic remnants of apoptotic SMCs, enclosed by a cage of thickened basal lamina, were TUNEL negative and remained present in the plaques as matrix vesicles. CONCLUSIONS: We conclude that SMCs within human fatty streaks express BAX, which increases the susceptibility of these cells to undergo apoptosis. The localization of these susceptible SMCs in the deep layer of the fatty streaks could be important in our understanding of the transition of fatty streaks into atherosclerotic plaques, which are characterized by regions of cell death. Matrix vesicles are BAX-immunoreactive cytoplasmic remnants of fragmented SMCs that can calcify and may be considered the graves of SMCs that have died in the plaques.     

Colocalization of CPP-32 with apoptotic cells in human atherosclerotic plaques

BACKGROUND: Apoptosis that has been reported in human atherosclerosis may contribute to the remodeling of atherosclerotic plaques. The identification of specific markers for apoptosis in these plaques would permit the development of specific therapeutic strategies to limit their progression. Cysteine protease CPP-32 is essential for apoptotic death in mammalian cells and appears to be an attractive candidate. METHODS AND RESULTS: We studied 12 atherosclerotic plaques from 12 patients who underwent carotid endarterectomy. Apoptosis was analyzed by in situ end labeling of fragmented DNA (TUNEL method) and corroborated by the presence of DNA fragmentation in agarose gel electrophoresis. CPP-32 was detected with the use of a specific monoclonal antibody, and its expression was compared with that of interleukin-1beta-converting enzyme (ICE). We showed that CPP-32 was highly expressed in 10 of 12 atherosclerotic plaques and that it colocalized with apoptotic cells. Expression of ICE generally paralleled that of CPP-32, but ICE was also detected in plaques negative for CPP-32 and showing no apoptosis. CONCLUSIONS: CPP-32 is highly expressed within human atherosclerotic plaques and is closely related to apoptosis. This finding suggests that CPP-32 may be the ICE-like enzyme responsible for apoptosis in human atherosclerosis and opens new perspectives for the development of therapeutic strategies to alter the progression of this disease.     

The solution structure of human coagulation factor VIIa in its complex with tissue factor is similar to free factor VIIa: a study of a heterodimeric receptor-ligand complex by X-ray and neutron scattering and computational modeling

Factor VIIa (FVIIa) is a soluble four-domain plasma serine protease coagulation factor that forms a tight complex with the two extracellular domains of the transmembrane protein tissue factor in the initiating step of blood coagulation. To date, there is no crystal structure for free FVIIa. X-ray and neutron scattering data in solution for free FVIIa and the complex between FVIIa and soluble tissue factor (sTF) had been obtained for comparison with crystal structures of the FVIIa-sTF complex and of free factor IXa (FIXa). The solution structure of free FVIIa as derived from scattering data is consistent with the extended domain arrangement of FVIIa seen in the crystal structure of its complex with sTF, but is incompatible with the bent, less extended domain conformation seen in the FIXa crystal structure. The FVIIa scattering curve is also compatible with a subset of 317 possible extended structures derived from a constrained automated conformational search of 15 625 FVIIa domain models. Thus, the scattering data support extended domain models for FVIIa free in solution. Similar analyses showed that the solution scattering derived and crystal structures of the FVIIa-sTF complex were in good agreement. An automated constrained search for allowed structures for the complex in solution based on scattering curves showed that only a small family of compact models gave good agreement, namely those in which FVIIa and sTF interact closely over a large surface area. The general utility of this approach for structural analysis of heterodimeric complexes in solution is discussed. Analytical ultracentrifugation data and the modeling of these data were consistent with the scattering results. It is concluded that in solution FVIIa has an extended or elongated domain structure, which allows rapid interaction with sTF over a large surface area to form a high-affinity complex.     

Cellular localization of insulin-like growth factor II mRNA in the human fetus and the placenta: detection with a digoxigenin-labeled cRNA probe and immunocytochemistry

IGF-II plays a major role in the regulation of human fetal growth and development. However, more extensive information on the cellular sites of IGF-II synthesis in the fetus would provide more insight into its role in fetal organogenesis. Thus we have determined the sites of IGF-II synthesis in 18-26-wk gestation human fetal tissues using in situ hybridization with a digoxigenin-labeled cRNA probe to localize IGF-II mRNA in fetal liver, kidney, adrenal gland, cerebral cortex, costal cartilage, skeletal muscle, and lung, and in placental tissue. In human fetal tissues it has to date been impossible to clearly assign IGF-II mRNA to epithelial cells of entodermal origin. Besides their already known localization in cell matrix and a variety of mesodermal cell types, strong IGF-II mRNA-positive signals were detected in epithelial cells in the liver (hepatocytes), bronchial and bronchiolar epithelium, undifferentiated renal tubular epithelium, mature glomerular epithelium, pelvic urothelium, and adrenal epithelial cells of the zona persistens. To identify the cellular location of immunoreactive IGF-II, we also performed immunocytochemical studies in tissues of the same fetuses. Every tissue studied except the cerebral cortex contained immunoreactive cells; however, immunostaining was generally weaker than in situ hybridization signals. Our data show that the distribution of IGF-II in human fetal tissue is much more widespread than hitherto thought. A digoxigenin-labeled detection system for IGF-II is more capable of detecting the cellular expression pattern of IGF-II than radioactive probes and is suitable for analysis of routinely prepared paraffin-embedded material.     

Studies of the mechanism for enhanced cell surface factor VIIa/tissue factor activation of factor X on fibroblast monolayers after their exposure to N-ethylmaleimide

Fibroblast monolayers constitutively expressing surface membrane tissue factor (TF) were treated with 0.1 mM N-ethylmaleimide (NEM) for 1 min to inhibit aminophospholipid translocase activity without inducing general cell damage. This resulted in increased anionic phospholipid in the outer leaflet of the cell surface membrane as measured by the binding of 125I-annexin V and by the ability of the monolayers to support the generation of prothrombinase. Specific binding of 125I-rVIIa to TF on NEM-treated monolayers was increased 3- to 4-fold over control monolayers after only brief exposure to 125I-rVIIa, but this difference progressively diminished with longer exposure times. A brief exposure of NEM-treated monolayers to rVIIa led to a maximum 3- to 4-fold enhancement of VIIa/TF catalytic activity towards factor X over control monolayers, but, in contrast to the binding studies, this 3- to 4-fold difference persisted despite increasing time of exposure to rVIIa. Adding prothrombin fragment 1 failed to diminish the enhanced VIIa/TF activation of factor X of NEM-treated monolayers. Moreover, adding annexin V, which was shown to abolish the ability of NEM to enhance factor X binding to the fibroblast monolayers, also failed to diminish the enhanced VIIa/TF activation of factor X. These data provide new evidence for a possible mechanism by which availability of anionic phospholipid in the outer layer of the cell membrane limits formation of functional VIIa/TF complexes on cell surfaces.     

In situ detection of tissue factor within the coronary intima in rat cardiac allograft vasculopathy

Proteasomes are multicatalytic complexes that function as the major proteolytic machinery in regulated protein degradation. The eukaryotic 20S proteasome proteolytic core structure comprises 14 different subunits: 7 alpha-type and 7 beta-type. DTS7 is a dominant temperature-sensitive (DTS) lethal mutation at 29 degrees that also acts as a recessive lethal at ambient temperatures. DTS7 maps to cytological position 71AB. Molecular characterization of DTS7 reveals that this is caused by a missense mutation in a beta-type subunit gene, beta2. A previously characterized DTS mutant, l(3)73Ai1, results from a missense mutation in another beta-type subunit gene, beta6. These two mutants share a very similar phenotype, show a strong allele-specific genetic interaction, and are rescued by the same extragenic suppressor, Su(DTS)-1. We propose that these mutants might act as "poison subunits," disrupting proteasome function in a dosage-dependent manner, and suggest how they may interact on the basis of the structure of the yeast 20S proteasome.     

Complex-dependent inhibition of factor VIIa by antithrombin III and heparin

The regulation of the factor VIIa-tissue factor complex is essential for control of the hemostatic response. However, the role of the inhibitor antithrombin III in the regulation of factor VIIa has remained in question. The inhibition of factor VIIa activity by antithrombin III and heparin in the presence and absence of tissue factor was evaluated using the fluorescent substrate m-LGR-nds. Our data show that the activity of recombinant human factor VIIa is inhibited by antithrombin III in the presence of heparin at a rate of 1.7 x 10(2) M-1 s-1. In the presence of tissue factor, the rate constant for this reaction increases to 5.6 x 10(3) M-1 s-1. A 1:1 stoichiometric complex between factor VIIa and antithrombin III, with an apparent molecular weight of 110,000, was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A heterogeneous mixture of factor VIIa products with molecular weights between 50,000 and 80,000, most likely representing proteolytically degraded factor VIIa-antithrombin III complexes, was also observed.     

In situ non-radioactive detection of nuclear factors in paraffin sections by Southwestern histochemistry

CD8+ T cells often differentiate into highly cytotoxic cells, secreting a Th1-like or type 1 cytokine pattern characterized by the production of IFN-gamma. However, cytotoxic, and in some reports, noncytotoxic, type 2 cells that secrete IL-4, IL-5, or IL-10 instead of IFN-gamma, can be generated when CD8+ T cells are primed in the presence of IL-4. Here, we show that IL-4 can also generate typical CD8 type 1 responses. Indeed, while presence of TGF-beta biases the development of CD8 T cells that, then, produce little cytolytic activity and IFN-gamma, addition of IL-4 results in the recovery of cytotoxicity and IFN-gamma production. The cooperative effects of TGF-beta and IL-4 imply dual functions, not only for IL-4, but also for TGF-beta. Indeed, depending on the presence or absence of IL-4, TGF-beta either inhibits or induces the generation of type 1 CD8+ T cells. Physiologically, the ratio of local IL-4/TGF-beta concentration may therefore be a critical element in determining the outcome of T cell responses to pathogen and autoantigens. It allows CD8 T cells to switch from an immunotolerant state in the presence of only TGF-beta or IL-4, to an immunocompetent proinflammatory type 1 state in the absence or presence of both cytokines.     

In situ localization of ATPase activity in cells of plants infected by maize dwarf mosaic potyvirus

Cells of healthy maize plants as well as those infected by maize dwarf mosaic potyvirus were examined by electron microscopy for the location of ATPase activity. In healthy and virus infected plants, ATPase activity was found in plasma membranes, chloroplast thylakoid membranes, nuclear membranes and in mitochondria. In virus-infected cells, ATPase activity was also observed in cytoplasmic vesicles which were found in close proximity to the virus-specific cytoplasmic inclusion bodies (CI), at the ends of the arms of the CI and in plasmodesmata.     

In situ visualization of intratumor growth factor signaling: immunohistochemical localization of activated ERK/MAP kinase in glial neoplasms

Abnormal growth factor signaling is implicated in the pathogenesis of gliomas. The extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway is a likely target, linking receptor tyrosine kinase activation to downstream serine/threonine phosphorylation events regulating proliferation and differentiation. Signaling within heterogeneous cell populations of gliomas cannot be adequately assessed by traditional biochemical enzyme assays. Immunohistochemical detection of doubly phosphorylated (activated) ERK/MAPK permitted visualization of spatially discrete cellular patterns of ERK/MAPK activation, compared with the relatively uniform expression of total ERK/MAPK protein. The astrocytic tumors, regardless of grade, had the highest overall degree of enzyme activation, whereas oligodendrogliomas had the least. Anaplastic progression in oligodendrogliomas resulted in a larger number of cells with active ERK/MAPK. Within glioblastomas, microvascular hyperplasia and necrosis were associated with ERK/MAPK activation in adjacent tumor cells. In addition to spatial patterns of intratumor paracrine signaling, a possible cell-cycle-associated regulation was detected: mitotic and actively cycling tumor cells showed diminished activation relative to cells in G0. Although ERK/MAPK activation was not restricted to neoplastic glia, consistent patterns of selective activation in tumor cells suggests that sustained activation may contribute to the neoplastic glial phenotype.     

In situ evaluation of esterase stereoselectivity in two-dimensional electropherograms and tissue sections

We have constructed an epitope-tagging vector, pCMV-Tag1, for gene expression in mammalian cells. This vector, which allows for N-terminal, C-terminal and internal tagging of the gene product of interest with the FLAG and/or c-myc epitopes, enables researchers to rapidly and efficiently characterize gene products in vivo.     

The inhibition of human factor VIIa-tissue factor by antithrombin III-heparin is enhanced by factor X on a human bladder carcinoma cell line

The effects of influenza A and B and RSV on mortality in England and Wales were assessed by regression analysis for the period 1975-90. Morbidity data from sentinel practices were used to calculate 4-weekly rates of aggregated upper respiratory tract infections (URTI); PHLS laboratory reports were used as indices of infection, and 4-weekly death rates from all causes, excluding childbirths, were used to study relationships with mortality. Deaths correlated strongly with influenza A and B reports, temperature, and interactions between aggregated URTI and temperature, and RSV outbreaks and temperature. Estimates of 'seasonal' 4-weekly mortality associated with URTI were made by substituting into primary regression models the mean of annual trough consultation rates for aggregated URTI and baseline values for RSV and influenza. Peak 4-weekly mortality associated with URTIs was estimated at c. 24000 and c. 28000 during combined influenza and RSV epidemics of 1975-6 and 1989-90 respectively. Secondary regression analysis was carried out with the estimated 'seasonal' 4-weekly deaths associated with URTI as dependent variable and laboratory data as regressors. Estimated excess mortality associated with influenza was considerable even during years without major epidemics. Overall during the 15 winters the estimated mortality associated with RSV was 60-80% more than that associated with influenza. The modelling permits only a crude estimate of RSV associated mortality. None the less it suggests that RSV is an important cause of winter mortality.