Regulation of extravascular coagulation by microvascular permeability.

Extravascular coagulation is a prominent feature of such important pathological processes as cellular immunity and neoplasia and has been thought to result from procoagulants associated with the inflammatory or tumor cells peculiar to these entities. It was found that increased microvascular permeability alone is sufficient to induce equivalent extravascular coagulation in several normal tissues. The results indicate that saturating levels of procoagulant are present even in normal tissues and that microvascular permeability is a rate-limiting step in extravascular coagulation.

Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid.

Tumor ascites fluids from guinea pigs, hamsters, and mice contain activity that rapidly increases microvascular permeability. Similar activity is also secreted by these tumor cells and a variety of other tumor cell lines in vitro. The permeability-increasing activity purified from either the culture medium or ascites fluid of one tumor, the guinea pig line 10 hepatocarcinoma, is a 34,000- to 42,000-dalton protein distinct from other known permeability factors.

Quantitation of fibrinogen influx and fibrin deposition and turnover in line 1 and line 10 guinea pig carcinomas.

Radiolabeled guinea pig fibrinogen (GPF) was used to measure fibrinogen influx and fibrin accumulation in line 1 and line 10 hepato- (bile duct) carcinomas growing in the s.c. space of syngeneic strain 2 guinea pigs over the course of 7 days following transplant, an interval of growth uncomplicated by immunological tumor rejection or by significant tumor necrosis. Earlier immunofluorescence studies revealed fibrin deposits in both tumors with line 1 much greater than line 10. In accord with these data, GPF accumulated in both tumors in amounts that matched or exceeded plasma fibrinogen levels. Line 1 tumor GPF content was 4-fold greater than that of line 10 tumors and 11- to 33-fold that of normal s.c. tissue. The composition of tumor fibrinogen-fibrin was investigated by aqueous and urea extraction. The fraction of total accumulated GPF that was urea insoluble, and therefore presumably cross-linked fibrin, was constant over time but strikingly different for line 1 (65%) and line 10 (48%) tumors, as compared with control s.c. tissue (18%). By 7 days, line 1 tumors (mean weight, 0.77 g) contained nearly 2 mg of fibrinogen-fibrin, and line 10 tumors (mean weight, 0.62 g) contained nearly 0.5 mg. Influx of GPF and initial clotting were constant over time and equivalent for the two tumors. Hence, the large differences in GPF accumulation observed between these tumors apparently reflect differences in fibrinolysis, not in fibrinogen influx or coagulation. The data presented indicate substantial traffic of plasma fibrinogen into and out of both tumors, as compared with control tissues, equivalent to nearly 10 and 7 ml of plasma over 7 days of growth for line 1 and line 10 tumors, respectively; comparable values for normal s.c. tissues were 1.0 to 1.4 ml plasma fibrinogen. Even in line 1 tumors with their abundant fibrin gel, only 6.3% of GPF entering tumors over 7 days was retained, as compared with 2% for line 10 tumors and approximately 1% for control tissue.

Fibrinogen influx and accumulation of cross-linked fibrin in mouse carcinomas.

125I-radiolabeled guinea pig fibrinogen was used to measure the influx (20 min) and accumulation (18 h) of fibrinogen/fibrin in three transplantable carcinomas (Lewis lung, TA3/St mammary, and MOT ovarian) growing in the subcutaneous space of syngeneic mice. Fibrinogen influx and, to an even greater extent, fibrin accumulation were substantially increased in all three tumors, as compared with normal control tissues. A significantly larger fraction of tumor-associated than control tissue radioactivity was insoluble in 3 M urea, a property of cross-linked fibrin. Positive identification of cross-linked fibrin was made by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of tumor extracts. Tumor fibrin deposits were localized by immunoperoxidase staining of tissue sections. Fibrin accumulation was also significantly increased in premalignant hyperplastic alveolar nodules that had been transplanted to cleared mammary fat pads, as compared with normal mammary tissue, and was further increased in primary mammary carcinomas that arose from hyperplastic alveolar nodules. These findings generalize to the mouse the principles that tumor vessels are hyperpermeable to plasma proteins and that fibrin accumulates in transplantable and primary tumors. Further, they demonstrate that tumor fibrin is cross-linked and therefore analogous to the fibrin deposited in thrombi, wounds, and cellular immunity.

Procoagulant activity associated with plasma membrane vesicles shed by cultured tumor cells.

Thirteen of 14 tumor cells or tumor cell lines of guinea pig, mouse, and human origin spontaneously shed procoagulant activity in short-term (4 or 14 to 22 hr) tissue culture under conditions of high cell viability. This released procoagulant activity was pelletable in the ultracentrifuge and was associated with plasma membrane-derived vesicles as determined by transmission electron microscopy and marker enzyme analysis. The procoagulant activity shed corresponded to a substantial fraction of that expressed by intact or sonicated tumor cells and was composed of activities interacting at more than a single step in the clotting sequence. One procoagulant activity associated with shed human tumor vesicles behaved as tissue factor, requiring Factor VII for activity and being inhibited by a specific anti-bovine tissue factor antibody. Guinea pig tumor vesicles also exhibited tissue factor-like activity in a two-stage assay using homologous first-stage Factor VII/X concentrate. None of the human vesicles tested expressed a direct Factor X cleaving activity, independent of Factor VII. Shed tumor vesicles also acted at a second step late in the clotting cascade at the level of prothrombinase generation, presumably by providing a phospholipid surface. Taken together, these data indicate that a wide variety of tumor cells release plasma membrane vesicles with procoagulant activity. Such vesicles, as well as intact tumor cells themselves, may play an important role in the biology of tumor growth by inducing the local fibrin deposits found in association with many solid tumors.

Enhancement of the functional repertoire of the rat parietal peritoneal mesothelium in vivo: directed expression of the anticoagulant and antiinflammatory molecule thrombomodulin.

We have used our previously described ex vivo mesothelial cell (MC)-mediated gene therapy strategy (Gene Ther. 2:393-401, 1995) to modify the functional properties of the rat parietal peritoneal mesothelium in vivo by expression of a membrane-bound recombinant protein on the MC surface. Rat primary MCs were stably transfected (using strontium phosphate DNA coprecipitation) with a plasmid containing the gene for rat thrombomodulin (TM), a transmembrane glycoprotein that functions as an essential cofactor for the physiological activation of the anticoagulant protein C by the enzyme thrombin. As demonstrated by immunohistochemistry and by direct equilibrium binding with radiolabeled thrombin, genetically modified MCs expressed high levels of TM antigen on their surface in vitro. As judged by a thrombin-dependent protein C activation assay, such MC membrane-bound TM was biologically active. Once reseeded on the denuded parietal peritoneal surface of syngeneic recipients, these TM-transfected MCs continued to express TM antigen in vivo for at least 90 days. Moreover, the recombinant TM expressed on the reconstituted parietal mesothelium retained its ability to activate protein C in a thrombin-dependent manner. Our data indicate that MC-mediated expression of TM can be used to augment the anticoagulant properties of the parietal peritoneal surface. In general, our results suggest that ex vivo MC-mediated gene therapy can be used to deliver other therapeutic transmembrane proteins to the MC surface to enhance the functional repertoire of the parietal mesothelium in vivo.

Incremental validation of situational judgment tests.

Using 3 different samples, the authors assessed the incremental validity of situational judgment inventories (SJIs), relative to job knowledge, cognitive ability, job experience, and conscientiousness, in the prediction of job performance. The SJI was a valid predictor in all 3 samples and incrementally so in 2 samples. Relative to the other predictors, SJI's partial correlation with performance, controlling for the other 4 predictors, was superior in most comparisons. Subgroup differences on the SJI also appear to be less than those for cognitive ability and job knowledge, but greater than differences in conscientiousness. The SJI should prove to be a valuable additional measure in the prediction of job performance, but several additional areas of research are suggested.

Lymphocyte migratory pathways in adjuvant disease. II. Distribution of thoracic duct lymph-borne immunoblasts.

Evidence for enhanced extravasation of thoracic duct lymph-borne immuno-blasts within joints of rats during the onset of adjuvant disease was sought by adoptive transfer of cells radiolabeled with (125I)-iodo-2-deoxyuridine. Migratory behavior of cells from normal or adjuvant disease donors, during both inductive and overt stages of the disease process, was contrasted in normal and adjuvant disease recipients. The results provided no evidence to indicate enhanced joint-seeking properties of lymph-borne immuno-blasts obtained from adjuvant disease donors, either during the period preceding overt joint involvement or during the phase of chronic inflammation. The ability of lymph-borne cells to passively transfer the disease thus appears more likely due to systemic actions of these cells, mediators produced by them, or concomitantly passaged antigen upon patterns of inflammatory cell mobilization and/or vascular endothelial cell activation.

Lymphocyte migratory pathways in adjuvant disease. I. Distribution of 51Cr-labeled thoracic duct lymph-borne.

Evidence for selective extravasation of thoracic duct lymph-borne cells, derived from rats with adjuvant disease, within joints of normal or adjuvant arthritic recipients was sough by adoptive transfer of radiolabeled cells. Control studies were carried out in parallel using thoracic ducts cells from normal donors. No increased homing of lymph-borne cells to inflamed portions of the limbs was detected when cells from adjuvant arthritic donors were compared with those of normal controls. Inflammatory changes, ie, adjuvant-induced disease, in the recipient produced a significant nonspecific enhancement of extravasation; cells from normal and adjuvant arthritic donors responded equally well. One difference in migratory behavior between lymph-borne cells from adjuvant arthritic and normal animals was the increased ability of the former to localize within certain lymph nodes. A possible association between this traffic and the development of chronic inflammatory processes within joints is discussed.

Fibrinogen influx and accumulation of cross-linked fibrin in healing wounds and in tumor stroma.

Fibrinogen enters wounds and solid tumors, where it is clotted to fibrin that may subsequently be replaced by collagenous stroma. If, as has been suggested, the pathogenesis of wound healing and tumor stroma generation is similar and dependent on fibrin deposition, then the types and amounts of fibrin deposited in wounds and tumors might also be expected to be similar. To test this hypothesis, the authors injected homologous tracer fibrinogen (125I-GPF) intravenously into guinea pigs and measured its influx and accumulation in skin wounds and syngeneic carcinomas. In support of their hypothesis, the urea-insoluble product deposited in both wounds and tumors was identified as cross-linked fibrin by gel electrophoresis. Accumulation of both total and urea-insoluble 125I-GPF was quantitatively similar in wounds and tumors. However, influx and initial clotting of 125I-GPF in tumors exceeded that in wounds; given equivalent accumulation, these data suggest that fibrin turnover is more rapid in tumors than in wounds. Fibrinogen influx and fibrin accumulation declined toward normal a few days after wounding but remained consistently elevated in tumors. Thus, the magnitude and the persistence of microvascular hyperpermeability, as well as fibrin turnover, are major points of difference that distinguish tumors from healing wounds.

Acute hemorrhagic pancreatic necrosis in mice: lack of a pathogenetic role for complement.

Feeding a choline-deficient diet containing 0.5% DL-ethionine induces an acute hemorrhagic pancreatitis in 100% of young female mice. Evidence for deposition of the third component of complement (C3) on acinar cell plasma membranes was sought, during the inductive stages, by a sandwich immunofluorescence technique. Such a localization could not be demonstrated even though the method is capable of detecting less than 8 x 10(-5) microgram of protein/mm2 of cell membrane. Artifactual binding of immunoglobulin reagents was encountered when goat antisera, with high levels of circulating immune complexes, formed the middle layer in the sandwich technique. This was attributed to the appearance of Fc receptors on the plasma membrane of degenerating acinar cells, and could be avoided by ultracentrifuging acinar cells, and could be avoided by ultracentrifuging the goat antisera prior to sue. In view of the fact that C3 cleavage represents an amplification loop in both the calssical and alternate pathways of complement activation, the lack of demonstrable C3 staining in tbe present experiments strongly suggests that complement plays no role in acinar cell necrosis in this model of pancreatitis.

Qualitative testing for circulating immune complexes by use of zone electrophoresis on agarose.

On binding of antibody to antigen an immune complex is formed that has a net surface charge different from that of either of the two components. This, together with clonal restriction of the antibody response, gives rise to distinctive patterns that are readily apparent in stained agarose gels after routine zone electrophoresis. Most circulating immune complexes appear as a rectangular pattern, with well-defined edges, located in the gamma-region. The identity of the material responsible for these patterns has been established by three different experimental approaches: analysis of tetanus/anti-tetanus complexes formed in vitro, analysis of sera from rabbits with experimental immune complex disease, and analysis of human type II and type III cryoglobulins. Studies of reproducibility, interfering substances, and correlation with other assays for detecting immune complexes indicate that zone electrophoresis in agarose gel is a sensitive, highly specific technique for immune complex detection, of potential value as a screening tool.

Accelerated cytodifferentiation of antibody-secreting cells in guinea-pig lymph nodes stimulated by sheep erythrocytes and lymphokines.

Lymphokines, produced in response to structurally unrelated antigens, altered the course of a primary anti-sheep erythrocyte plaque-forming cell response within the regional lymph nodes of normal guinea-pigs. Intralymphatic injection of a small dose of lymphokines (0-5-8 mug) 1 day after antigen priming accelerated the rate of indirect plaque-forming cell cytodifferentiation between the 5th and the 9th days of the response. This effect was not related to changes in the level of antigen trapping by lymph node macrophages, but the lymphocyte mitogenic activity may have been important for the response since there was a significant increase in [3H]thymidine incorporation within the lymphokine-treated nodes on the 3rd day following immunization.

Acute hemorrhagic pancreatic necrosis in mice: alterations of serum complement.

Induction of acute hemorrhagic pancreatic necrosis by dietary means in mice produces alterations in the serum complement system. Total hemolytic complement, i.e., CH50, and native C3 levels fall during the development of pancreatitis while, at the same time, what could be immunoreactive C3 degradation products are demonstrable both in the circulation and in the urine. No evidence of renal deposition of C3 was obtained by immunofluorescence analysis, although marked alterations in proteinuria were observed, suggesting that renal dysfunction(s) is a feature of acute hemorrhagic pancreatic necrosis. Lack of renal complement deposition, together with our earlier negative findings with respect to pancreatic localization, suggests that serum complement alterations are side effects of the pancreatitis, attributable to intravascular, pancreatic enzyme-mediated degradation of serum complement components.

Ultrastructural localization of prostaglandin endoperoxide synthase (cyclooxygenase) to isolated, purified fractions of guinea pig peritoneal macrophage and line 10 hepatocarcinoma cell lipid bodies.

Subcellular fractions of purified cytoplasmic, nonmembrane-bound lipid bodies were prepared from [3H]-arachidonic acid-labeled guinea pig peritoneal macrophages and line 10 hepatocarcinoma cells. These fractions, which contained [3H]-arachidonyl lipids, were shown to be devoid of contaminating cellular membranes by electron microscopy, and to contain prostaglandin endoperoxide (PGH) synthase by postembedding immunogold electron microscopy. These findings support a proposed role for these lipid-rich organelles in the generation of eicosanoids by oxidative metabolism of arachidonate in the cyclooxygenase pathway of inflammatory and neoplastic cells.

Fibrin containing gels induce angiogenesis. Implications for tumor stroma generation and wound healing.

Fibrin deposition is a consistent early event in solid tumors and healing wounds and precedes new blood vessel ingrowth in both. We now demonstrate that fibrin gels of themselves induce an angiogenic response in the absence of tumor cells or platelets. Angiogenesis was enhanced when certain chemoattractants or mitogens were included in the fibrin gel. Newly devised, inert plastic chambers with one porous surface were filled with varying contents and were implanted in the subcutaneous space of guinea pigs. Chambers filled with cross-linked homologous fibrin or plasma induced an angiogenic response within 4 days. Vessels entered chambers through the surface pores and flared out radially; angiogenesis was quantitated by point counting. Vessels were functional and matured along a gradient that proceeded from distal (least mature) to proximal. The intensity of the angiogenic response was enhanced when zymosan activated serum, an N-formylmethionine tripeptide, or platelet-derived growth factor was included in the fibrin matrix. Prior aldehyde fixation or boiling of fibrin-filled chambers inhibited angiogenesis, as did high concentrations of hyaluronic acid. Chambers filled with type I collagen or agarose did not induce new blood vessel formation, but addition of collagen did not reduce fibrin's capacity to initiate angiogenesis. The novel assay introduced here offers several advantages that should facilitate the study of angiogenesis. These include reproducibility, low background, objective and quantitative scoring, and the capacity to evaluate native molecules in animals of several species. Taken together, our findings strongly implicate fibrin or related proteins in the pathogenesis of angiogenesis and offer a new approach for elucidating the underlying molecular mechanisms.

Mesothelial cell-mediated gene therapy: feasibility of an ex vivo strategy.

We have developed a model system in the rat to test the feasibility of recombinant protein expression by genetically modified peritoneal mesothelial cells following autologous peritoneal implantation. Rat primary peritoneal mesothelial cells, isolated from parietal peritoneum by enzymatic digestion, were stably transduced (using a Moloney murine leukemia virus (MoMLV)-derived retroviral vector, BAG, expressing the Escherichia coli lacZ gene) to mark the cells with a reporter protein (beta-galactosidase, beta-gal). Such transduced mesothelial cells, tagged with DiO, a fluorescent lipophilic dye used for long-term tracing of transplanted cells, were then reseeded on the denuded peritoneal surface of syngeneic recipients. DiO-labeled, BAG-transduced mesothelial cells were observed to repopulate the denuded areas and remain attached there for > 90 days. Moreover, these genetically modified mesothelial cells continued to express the reporter gene product in vivo (ie beta-gal activity was present for at least 1 month). Our results demonstrate the feasibility of ex vivo gene therapy using peritoneal mesothelial cells.

Systemic delivery of a recombinant protein by genetically modified mesothelial cells reseeded on the parietal peritoneal surface.

To evaluate the ability of genetically modified peritoneal mesothelial cells to deliver recombinant proteins to the systemic circulation, we used our previously described mesothelial cell-based ex vivo gene therapy strategy. Rat primary peritoneal mesothelial cells, isolated from parietal peritoneum by enzymatic digestion, were stably transfected (using strontium phosphate DNA co-precipitation) with the plasmid pSVTKgh to express a secreted reporter gene product, human growth hormone (hgh). Such hgh-secreting mesothelial cells were reseeded on the denuded peritoneal surface of syngeneic recipients and delivery of the reporter gene product to the systemic circulation was monitored by analysis of serum samples for the presence of hgh at various times after mesothelial cell implantation. Polymerase chain reaction (PCR) analysis demonstrated that the hgh-transfected mesothelial cells repopulated the denuded areas and remained attached there for at least 12 weeks. Moreover, these genetically modified mesothelial cells continued to express the reporter gene product in vivo and secreted hgh in sufficient quantity to be detected in the systemic circulation (ie statistically significant amounts of hgh could be measured in the serum of cyclosporine A-treated rats for at least 2 months; Mann-Whitney test, P < 0.05). Our results demonstrate the successful, sustained, systemic delivery of a recombinant protein by genetically modified peritoneal mesothelial cells following their reattachment to the peritoneal surface, and suggest the potential of ex vivo mesothelial cell-mediated gene therapy for the treatment of inherited or acquired disorders requiring delivery of therapeutic proteins to the circulation.

Retinoic acid selectively inhibits the vascular permeabilizing effect of VPF/VEGF, an early step in the angiogenic cascade.

All-trans-retinoic acid (RA) and other retinoids modulate cell growth and differentiation, generally favoring terminal cell differentiation and inhibiting carcinogenesis. Retinoids are also reported to inhibit angiogenesis and endothelial cell migration, actions that are also anti-carcinogenic. Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) is a multifunctional cytokine secreted by many tumors. It renders microvessels hyperpermeable to plasma and stimulates endothelial cell migration and division. To investigate further the mechanisms by which RA inhibits angiogenesis, we evaluated the effects of RA on VPF/VEGF-induced angiogenesis and microvascular permeability. RA selectively inhibited the angiogenic response induced by VPF/VEGF, but not that induced by fibroblast growth factor-2 (FGF-2), in the CAM assay. RA and two of its isomers also inhibited the vascular permeabilizing effect of VPF/VEGF but not that induced by histamine. The vascular permeabilization induced by VPF/VEGF and blocked by RA takes place within 1-15 min, too short a time frame for RA to act by modulating transcription through classic retinoid receptors. RA also inhibited VPF/VEGF-induced phosphorylation of PLC-gamma and synthesis of cGMP but actually increased VPF/VEGF binding to cultured endothelial cells. Taken together, these findings indicate that RA selectively blocks VPF/VEGF-induced microvascular permeability and angiogenesis and also identify VPF/VEGF as a major target of RA action. The selectivity of RA's action suggests that other, RA-independent pathways must exist for the angiogenesis induced by FGF-2 and the vascular permeabilizing effect of histamine.

Lipid bodies: cytoplasmic organelles important to arachidonate metabolism in macrophages and mast cells.

Much has been learned about the biochemical nature and pharmacologic activity of the products of arachidonic acid (AA) oxidation, but relatively little is known about the structures in nucleated cells into which AA is incorporated and from which it is initially mobilized. To address this question, we administered 3H-AA or other 3H-fatty acids in vitro to human lung mast cells and alveolar macrophages as well as to mouse and guinea pig peritoneal macrophages. The subcellular distribution of 3H label was assessed by electron microscopic autoradiography, and the nature of cell-associated 3H-lipids was determined by thin layer chromatography. Autoradiographic analysis of human lung mast cells localized virtually all of the 3H-AA to cytoplasmic lipid bodies. Lipid bodies are roughly spherical, variable osmiophilic, nonmembrane-bound structures that appear in the cytoplasm of a wide variety of cells, but we have found that these lipid bodies occur with increased frequency in granulocytes, macrophages, and mast cells at sites of inflammatory, immunologic, or neoplastic processes. Macrophages also incorporated 3H-AA predominantly into cytoplasmic lipid bodies. In contrast to mast cells, however, macrophages incorporated 3H-AA into the plasma membrane as well. Stimulation of macrophage phagocytosis resulted in striking alterations of the relationships of lipid bodies to intracellular membranes, so that many lipid bodies appeared adjacent to phagolysosomes. In addition, some phagolysosomes contained 3H label, which along with other morphologic evidence suggested that lipid bodies may discharge their contents into these structures. Mast cell and macrophage cytoplasmic lipid bodies appear to represent a major site of intracellular storage and metabolism of products of AA and perhaps other fatty acids taken up from the external milieu. These heretofore neglected organelles may thus influence cellular function in a wide variety of adaptive or pathologic processes.