Tissue factor pathway inhibitor attenuates the progression of malignant pleural mesothelioma in nude mice

Malignant pleural mesothelioma (MPM) is a rare cancer that is refractory to current treatments. It is characterized by a robust deposition of transitional fibrin that is in part promoted by tumor cells. MPM cells express tissue factor (TF) and the tissue factor pathway inhibitor (TFPI), but their contribution to the pathogenesis of MPM has been unclear. We found that REN MPM cells fail to express TFPI. Based on the tumor growth-promoting properties of TF, we hypothesized that the stable transfection of TFPI into REN MPM cells would decrease their aggressiveness. We tested our hypothesis using in vitro, in vivo, and ex vivo analyses. TFPI knock-in decreased the proliferation, invasion, and TF activity of REN cells in vitro. REN TFPI knock-in cells, empty vector, and naive control cells were next injected intrapleurally into nude mice. The expression of TFPI significantly decreased tissue invasion, inflammation, and the deposition of fibrin and collagen associated with tumor tissue, pleural effusions, and tumor burden. In ex vivo analyses, REN cells were cultured from harvested tumors. The overexpression of TFPI was maintained in cells propagated from TFPI knock-in tumors, and attenuated the activation of Factor X and the invasiveness of tumor cells. These analyses demonstrate that TFPI reduces the aggressiveness of MPM in vitro and in vivo, and that its effect involves the inhibition of TF procoagulant activity. These observations suggest that the interactions of TF and TFPI represent a novel therapeutic target in the treatment of MPM.     

Abnormalities of pathways of fibrin turnover in lung lavage of rats with oleic acid and bleomycin-induced lung injury support alveolar fibrin deposition.

Alveolar fibrin deposition commonly accompanies acute lung injury, but the nature of the local abnormalities of coagulation and fibrinolysis that support pathologic fibrin deposition are not well understood. The trended abnormalities of procoagulant and fibrinolytic activities occurring in lung lavage fluids of Fischer 344 rats after lung injury induced by intravenous oleic acid (OA) or intratracheal bleomycin were studied. After injury by either agent, bronchoalveolar lavage (BAL) contained increased procoagulant activity and decreased fibrinolytic activity. Lavage procoagulant activity was mainly due to an activator of Factor X attributable to the extrinsic coagulation pathway, and fibrinolytic activity was almost completely plasminogen dependent. Major mechanisms of inhibition of fibrinolytic activity involved both the inhibition of the plasminogen activator (PA) and plasmin. These abnormalities were temporally associated with prominent alveolar fibrin deposition in both models. In OA-treated animals, lavage fibrinolytic activity was absent or profoundly decreased, and antiplasmin and procoagulant activities were increased within 4 hours after the induction of acute lung injury. By 24 hours after OA, lavage PA inhibitor (PAI) activity was elevated with sustained antiplasmin activity. By 3 days after OA, these abnormalities had resolved in association with almost complete resolution of alveolar fibrin deposits. Within 3 days after bleomycin-induced lung injury, lavage procoagulant activity was increased and fibrinolytic activity was depressed due to increased antiplasmin and PAI activities. These conditions persisted for 2 weeks, during which time alveolar fibrin deposition was associated with the development of pulmonary fibrosis. These data indicate that a disruption of the normal balance between procoagulant and fibrinolytic activities occurs in alveolar lining fluids of rats with alveolitis induced by either OA or bleomycin, and that concurrent abnormalities of pathways of fibrin turnover that occur in alveolar lining fluids promote the alveolar fibrin deposition associated with these lung injuries.     

Immunological characterization of plasminogen activators in human mixed saliva

In mixed saliva obtained from six healthy volunteers, the plasminogen activators were characterized immunologically using antibodies specific for human tissue plasminogen activator and urokinase, which were raised in goats immunized with low molecular weight urokinase and tissue-type activator from melanoma cells. The fibrinolytic activity in mixed saliva upon stimulation was assayed on fibrin plates containing plasminogen after preincubation with immunoglobulins with and without specific antibodies. In both centrifuged and uncentrifuged saliva, antibodies against tissue plasminogen activator completely quenched the fibrinolytic activity. By contrast, antibodies against urokinase had no suppressive effect, neither did non-immunized goat serum influence the fibrinolytic activity in mixed saliva. In conclusion, during physiological conditions tissue plasminogen activator appears to regulate fibrinolytic activity in mixed saliva, in which no activity of urokinase-like plasminogen activators could be demonstrated.     

Method for measuring fibrinolytic activity in a single layer of cells.

A method has been developed for measuring fibrinolytic activity in a single layer of cells--for example, venous endothelium or peritoneal mesothelium. A single layer of cells was collected on a gelatin disc, incubated on a fibrin plate for 24 h, and the resulting area of lysis measured. This was converted to a measure of fibrinolytic activity expressed in Ploug units of urokinase by reference to areas of lysis created by standard amounts of urokinase placed on similar fibrin plates. The method has been used for measuring fibrinolytic activity in venous endothelium and peritoneal mesothelium and has demonstrated that the activity in a single layer of endothelial cells is only about one-quarter of that in an equivalent area of whole vein wall. It has also shown that peritoneal fibrinolytic activity is reduced after peritoneal trauma. This method maybe useful in the investigation of the fibrinolytic system in a variety of pathological conditions--for example, thrombosis and intraperitoneal adhesions.     

Pathways of fibrin turnover of human pleural mesothelial cells in vitro.

The mesothelium contains both procoagulant and fibrinolytic activities. An imbalance between these activities could account for the abnormal fibrin turnover and pleural fibrin deposition that is characteristic of pleural inflammation. Procoagulant activity of human pleural mesothelial cells (HPMC) is in part due to tissue factor, and the prothrombinase complex can also assemble at the HPMC surface. HPMC express tissue plasminogen activator (tPA) but no detectable fibrinolytic activity in a fibrin plate assay. Inhibition of HPMC fibrinolytic activity is due, in part, to elaboration of plasminogen activator inhibitors-1 and -2 (PAI-1 and PAI-2) as well as antiplasmins. Synthesis of PAI-1 and PAI-2 is inhibited by actinomycin D and cyclohexamide. HPMC PAI-1 is increased by transforming growth factor-beta (TGF-beta) and tumor necrosis factor-alpha (TNF-alpha), as is tPA release, while PAI-1 mRNA is unchanged and tPA mRNA is increased. PAI-2 release is induced by TNF-alpha and TGF-beta. Because they are a rich source of PAI-1 and PAI-2, HPMC may contribute to the high levels of these inhibitors in pleural exudates. Stimulation of HPMC by TNF-alpha or TGF-beta in vitro did not alter HPMC procoagulant activity nor the balance of elevated PAI and antiplasmins relative to PA, changes that collectively favor formation and persistence of pericellular fibrin.     

Malignant melanoma. Interaction with coagulation and fibrinolysis pathways in situ.

Immunohistochemical techniques applied to fresh frozen sections of metastatic malignant melanoma tissue revealed abundant fibrinogen (or fibrin I) in perivascular areas throughout the tumor connective tissue stroma. Fibrin was readily detected in a focal distribution in the connective tissue around nodules of viable tumor. Staining for D-dimer of cross-linked fibrin (using an antibody that cross-reacted with fragment D of fibrinogen) coincided with staining for fibrin. Diffuse staining of tumor cell bodies was observed for Factor X, and Factor XIII ("a" subunit) was detected in scattered areas of connective tissue throughout the tumors. Factor VII was not detected, and only rare tumor cells stained for tissue factor. These results support the concept that a tumor cell-associated, thrombin-generating pathway exists in situ in malignant melanoma tissue that includes Factor X but neither tissue factor nor Factor VII. By contrast, tumor cell staining was observed rarely for urokinase and to a variable extent for tissue plasminogen activator.     

Depressed bronchoalveolar urokinase activity in patients with adult respiratory distress syndrome

Abundant deposition of bronchoalveolar fibrin and fibronectin occurs during the exudative phase of the adult respiratory distress syndrome (ARDS), promoting hyaline-membrane formation and subsequent alveolar fibrosis. To explore the mechanisms that account for the persistence of bronchoalveolar fibrin and fibronectin, we compared the activity of urokinase, which is necessary for plasminogen activation and fibrin degradation, in cell-free bronchoalveolar-lavage fluid from 8 patients with ARDS, 9 patients with acute pulmonary diseases other than ARDS, and 10 normal subjects. The mean level of urokinase activity in the lavage fluid from the patients with ARDS was 0.003 IU per milliliter of fluid (range, 0 to 0.008), which was significantly lower (P = 0.001) than the level in the fluid from either the patients with pulmonary diseases other than ARDS (0.118 IU per milliliter [range, 0.032 to 0.295]) or the normal subjects (0.129 IU per milliliter [range, 0.045 to 0.198]). The lavage fluid from all the patients with ARDS also had antiplasmin activity, which would promote the persistence of fibrin. A true decrease in urokinase activity was confirmed by the failure of the lavage fluid from the patients with ARDS to convert [125I]plasminogen to plasmin. Despite the low urokinase activity, immunochemical assays revealed normal levels of urokinase antigen in the fluid from the patients with ARDS, suggesting the presence of urokinase inhibitors. Inhibitors were demonstrated directly by a fibrin gel-underlay assay that detects complexes of urokinase with inhibitors. Plasminogen-activator inhibitor type 1 was the principal inhibitor identified. We conclude that increased antifibrinolytic activity due to both urokinase inhibitors and antiplasmins in the bronchoalveolar compartment of patients with ARDS contributes to the formation and persistence of hyaline membranes, a key component of alveolar histopathology in ARDS.     

A novel in vitro model of human mesothelioma for studying tumor biology and apoptotic resistance

Like many tumors, malignant mesothelioma exhibits significant chemoresistance and resistance to apoptosis in vivo that is not seen in current in vitro models. To study the mechanisms of this multicellular resistance, biologically relevant in vitro models are necessary. Therefore, we characterized and tested human mesothelioma tissue grown in vitro as tumor fragment spheroids. After 5-10 d in culture, fragments from each of 15 human mesothelioma tumors rounded into spheroids. The tumor fragment spheroids maintained multiple characteristics of the original tumors for up to 3 mo including the presence of viable mesothelioma cells, macrophages, and a collagen-rich stroma. In 14-d-old spheroids, mesothelioma cells showed the same proliferation rate and expression of a death receptor, DR5, as in the original tumor. To determine responses to treatment, we treated tumor fragment spheroids grown from three separate tumors with agents, TNF-related apoptosis-inducing ligand (TRAIL) plus cycloheximide, that induced near total apoptosis in three human mesothelioma cell lines (M28, REN, MS-1) grown as monolayers (94 +/- 6% apoptosis; mean +/- SEM). Compared with mesothelioma cells in monolayers, mesothelioma cells in the spheroids were resistant to TRAIL plus cycloheximide (32 +/- 4% apoptosis; mean +/- SEM). Apoptotic resistance of mesothelioma cells was significantly reduced by inhibiting either the PI3K/Akt pathway with LY294002 (47 +/- 6% apoptosis) or the mTOR pathway with rapamycin (50 +/- 17% apoptosis). We conclude that human mesothelioma can be maintained in vitro in a biologically relevant model that exhibits apoptotic resistance, thereby permitting study of its tumor biology and of novel approaches to therapy.     

The origin of fibrin breakdown products and the interpretation of their appearance in the circulation

It has been shown that the incubation of human plasma with urokinase at a concentration sufficient to cause rapid lysis of the clots formed on the addition of thrombin does not give rise to the production of measurable concentrations of non-clottable fibrinogen breakdown products. Also, breakdown products could not be detected in the course of experiments in vivo when urokinase was administered to monkeys and only in very low concentrations when a fibrinolytic state was induced by exercise in three healthy human volunteers. In contrast, high concentrations of breakdown products were found after thrombin infusion into monkeys.It is concluded that circulating fibrinogen is not readily broken down into non-clottable products by the fibrinolytic enzymes, and that normal animals and healthy human subjects do not have substantial deposits of fibrin that are available for breakdown during a fibrinolytic episode. The presence of breakdown products in the circulation is therefore likely to be indicative of the fibrinolytic response to an initial coagulation event.     

Malignant mesothelioma of the pericardium: case reports and immunohistochemical studies including Ki-67 expression

Pericardial malignant mesothelioma (PMM) is extremely rare compared with pleural cases of mesothelioma. We present the clinical and pathological features of three autopsy cases with PMM. All three cases showed rapid progress and died of heart failure. Detailed examination was obtained from the autopsy. Macroscopic appearances of each case showed a thickened pericardium due to tumor invasion. Microscopic observations of all cases led to a diagnosis of epithelial-type malignant mesothelioma (MM). The results of immunohistochemical examinations were similar to the previous published work on pleural mesothelioma. To disclose the pathological characteristics of PMM, we analyzed Ki-67 labeling index (LI) of three cases of PMM and five cases of pleural MM that died within 2 years. The difference of Ki-67 LI between PMM and pleural MM was not significant (P > 0.05). The poor prognosis of patients with PMM must be caused by restricted cardiac wall motion due to tumor involvement, in addition to the tumor proliferation itself.     

Immunological comparison between human and rat plasminogen activators in blood and the vessel wall.

To evaluate the rat as an experimental model for plasminogen activator research, the ability of antibodies specific for human tissue type plasminogen activator and urokinase to suppress the plasminogen activator activity in whole plasma and in the vessel wall was studied in both rat and man. Plasminogen activator activity in plasma was assayed on fibrin plates containing plasminogen. Plasminogen activator in the vessel wall was shown by the fibrin side technique. Antibodies against human tissue type melanoma cell activator and urokinase were raised in goats and mixed into the fibrin film or the fibrin plates. In both species antibodies to melanoma cell activator were able to suppress the plasminogen activator activity completely in plasma and in the vessel wall. Anti-urokinase, however, had no suppressing effect. In rat plasma the inhibitory effect on the fibrinolytic activity was seen only with high concentrations of antibodies against melanoma cell activator, which suggests that rat plasminogen activator in plasma and vessel walls is similar to, but not identical with, human tissue type plasminogen activator.     

Pathways of coagulation activation in situ in rheumatoid synovial tissue.

Immunohistochemical techniques were applied to rheumatoid synovium in order to detect components of coagulation and fibrinolysis pathways within these tissues. These techniques revealed an intact coagulation pathway and plasminogen activator inhibitor-2 associated with macrophage-like cells that were present throughout these tissues, especially in subsurface areas. Cell-associated thrombin generation appeared to account for conversion of abundant fibrinogen to fibrin. Occasional macrophage-like cells also stained for urokinase but tissue-type plasminogen activator and plasminogen activator inhibitor-1 were restricted to vascular endothelium. Intense synovial fibrin deposition (with the limited evidence for associated fibrinolysis) may contribute to local inflammation and explain certain clinical features of rheumatoid arthritis. These findings suggest novel treatment hypotheses for this disease.     

Presence of fibrin prolongs the retainment of locally injected tissue plasminogen activator.

Tissue plasminogen activator, injected intradermally or intramuscularly into rats, remained at the site of injection for longer periods when injected together with particles of plasminogen-free fibrin than after injection without fibrin. The results show that fibrin deposits can enhance the local effects of fibrinolytic agents under physiological conditions. They substantiate the role of fibrin in the regulation of focal fibrinolytic activity in tissue repair, which was previously supported by in vitro experiments.     

Thromboplastic and fibrinolytic activities of V2 and V7 carcinomas of rabbit, with special reference to fibrin deposition and thrombus formation in the tumors

Thromboplastic and fibrinolytic activities of V2 and V7 carcinomas, the two transplantable rabbit tumors of the same viral origin, were studied in relation to fibrin deposition and thrombus formation in the tumors. Thromboplastic activity of V7 carcinoma was comparatively high, while that of V2 carcinoma was as low as that of muscle tissue. More fibrin deposits in the stroma and more thrombi in the small vessels were found at the advancing border of V7 carcinoma than that of V2 carcinoma. These differences might be associated with higher thromboplastic activity of V7 carcinoma than that of V2 carcinoma. Fibrinolytic activity of both tumors was high and it was confirmed to be localized in the tumor cells by Todd's method. Fibrin deposits in the stroma were found more abundantly somewhat apart from the advancing border of the tumor nests of both tumors. It was suggested that plasmin activated by plasminogen activator released locally from the tumor cells might digest fibrin deposited in the stroma just close to the tumor nests.     

THROMBOPLASTIC AND FIBRINOLYTIC ACTIVITIES OF V2 AND V7 CARCINOMAS OF RABBIT, WITH SPECIAL REFERENCE TO FIBRIN DEPOSITION AND THROMBUS FORMATION IN THE TUMORS

Thromboplastic and fibrinolytic activities of V2 and V7 carcinomas, the two transplantable rabbit tumors of the same viral origin, were studied in relation to fibrin deposition and thrombus formation in the tumors. Thromboplastic activity of V7 carcinoma was comparatively high, while that of V2 carcinoma was as low as that of muscle tissue. More fibrin deposits in the stroma and more thrombi in the small vessels were found at the advancing border of V7 carcinoma than that of V2 carcinoma. These differences might be associated with higher thromoboplastic activity of V7 carcinoma than that of V2 carcinoma. Fibrinolytic activity of both tumors was high and it was confirmed to be localized in the tumor cells by Todd's method. Fibrin deposits in the stroma were found more abundantly somewhat apart from the advancing border of the tumor nests of both tumors. It was suggested that plasmin activated by plasminogen activator released locally from the tumor cells might digest fibrin deposited in the stroma just close to the tumor nests.     

The plasminogen activator inhibitor "paradox" in cancer

Proteolysis in general and specifically the plasminogen activating system regulated by urokinase (uPA) its specific receptor, the GPI membrane anchored urokinase receptor (uPAR) and the specific plasminogen activator inhibitor 1 (PAI-1) plays a major role in tumorigenesis, tumor progression, tumor invasion and metastasis formation. This is exemplified by a body of published work showing a positive correlation between the expression of uPA or uPAR in several tumors and their malignancy. It is generally assumed that such a "pro-malignant" effect of the uPA-uPAR system is mediated by increased local proteolysis thus favoring tumor invasion, by a pro-angiogenic effect of this system and also by uPA-uPAR signaling towards the tumor thereby shifting the tumor phenotype to a more "malignant" one. However, when tumor patients are analyzed for long term survival, those with high levels of the inhibitor of the system, PAI-1 have a much worse prognosis than those with lower PAI-1 levels. This indicates that increased overall proteolysis alone cannot be made responsible for the adverse effects of the plasminogen activating system in tumors. Moreover, it becomes increasingly evident that components of the fibrinolytic system secreted by the tumor cells themselves are not solely responsible for a correlation between the plasminogen activating system and tumor malignancy; components of the plasminogen activating system secreted by stroma cells or cells of the immune system such as macrophages contribute also to the impact of fibrinolysis on malignancy. This review summarizes the evidence for the role of plasminogen activator inhibitor-1 in mediating the malignant phenotype and possible mechanism thereby trying to explain the "PAI-1 paradox in cancer" on a molecular level.     

Tissue- and Urokinase-Type Plasminogen Activators and Type 1 Plasminogen Activator Inhibitor in Melanomas and Benign Skin Pigment Neoplasms

The content of urokinase- and tissue-type plasminogen activators and plasminogen activator inhibitor PAI-1 in the cytosol of primary and metastatic melanomas and benign skin pigment neoplasms was estimated by enzyme immunoassay. It was shown that local growth and invasion of melanomas are related to suppressed expression of tissue plasminogen activator. The content of urokinase plasminogen activator increases in patients with distant metastases and large thickness of the primary tumor.     

The role of leukocytes in the pathogenesis of fibrin deposition in bovine acute lung injury.

The peculiarly fibrinous nature of bovine acute lung injury due to infection with Pasteurella haemolytica A1 suggests an imbalance between leukocyte-directed procoagulant and profibrinolytic influences in the inflamed bovine lung. Calves with experimental pneumonia produced by intratracheal inoculation with P. haemolytica A1 developed acute locally extensive cranioventral fibrinopurulent bronchopneumonia. Pulmonary alveolar macrophages (PAM) recovered by segmental lavage from affected lung lobes were 30 times more procoagulant than PAM obtained from unaffected lung lobes and 37-fold more procoagulant than PAM from control calf lungs. Unlike the enhancement of procoagulant activity, profibrinolytic activity (plasminogen activator amidolysis) of total lung leukocytes (PAM and plasminogen activator neutrophils [PMN]) was decreased 23 times in cells obtained from affected lung lobes and also was decreased four times in cells obtained from unaffected lobes of infected animals. This marked imbalance in cellular procoagulant and fibrinolytic activity probably contributes significantly to enhanced fibrin deposition and retarded fibrin removal. In addition, PAM from inflamed lungs were strongly positive for bovine tissue factor antigen as demonstrated by immunocytochemistry. Intensely tissue factor-positive PAM enmeshed in fibrinocellular exudates and positive alveolar walls were situated such that they were likely to have, in concert, initiated extrinsic activation of coagulation in the acutely inflamed lung. These data collectively suggest that enhanced PAM-directed procoagulant activity and diminished PAM- and PMN-directed profibrinolytic activity represent important modifications of local leukocyte function in bovine acute lung injury that are central to the pathogenesis of lesion development with extensive fibrin deposition and retarded fibrin removal.     

The activation of plasminogen by tissue plasminogen activator is not enhanced by different heparin species as assessed in vitro with a solid-phase fibrin method

The aim of this study was to evaluate the effect of several heparin species (standard heparin, heparin of low molecular weight: IC 831422, heparin of high affinity for antithrombin III: IC 831435, and heparin of low affinity for antithrombin III: IC 831436) on the different steps of the fibrinolytic mechanism i.e., interaction of tissue-type plasminogen activator (t-PA) with plasminogen activator inhibitor-1 (PAI-1), binding of t-PA and plasmin(ogen) to fibrin and activation of plasminogen on the fibrin surface, in the presence of plasma proteins and factors that modulate fibrinolysis. Fibrinolytic and plasmin amidolytic activities were measured in the presence and absence of heparin. The spectrophotometric assays were performed in the presence and absence of solid-phase fibrin using selective chromogenic substrates for plasmin and saturating concentrations of plasminogen.The overall fibrinolytic activity, the amidolytic activity of mixtures of t-PA or urokinase with plasminogen in the absence of fibrin, the binding of t-PA and plasmin(ogen) to fibrin and the t-PA/PAI-1 interaction were not modified by heparin. In contrast, the activation of plasminogen by fibrin-bound t-PA decreased as a function of the concentration of heparin. Although this effect was not significant at concentrations usually used in routine heparin therapy (0.1 to 1 iu/ml) it might have some relevance when heparin is injected in bolus doses.In conclusion, by using a solid-phase fibrin method which allows the analysis of t-PA/PAI-1 balance, data were obtained indicating that the heparin species tested here neither competed with fibrin for the binding of t-PA, nor potentiated the activation of plasminogen at the fibrin surface.     

Resistance of cancer cells to immune recognition and killing

It is well recognized that, in order for a wound to heal, the fibrin clot must be eliminated by fibrinolytic enzymes. In certain instances, however, fibrin is ineffectively degraded or even not degraded. For example, in pregnancy, the placenta contains a layer of fibrin (Nitabuck's layer) which presents as 'self' to the immune system. Similar situations have been observed in many solid tumors. A hypothesis is presented according to which tumor cells can escape detection and attack by the immune system in most cancer patients. The tumor dons a 'coat' of the host's own protein on its cell surface. The coat is composed of fibrin and of a polymeric form of human serum albumin (HSA) which, by contrast to pure fibrin, is resistant to fibrinolytic degradation. Such a coated tumor appears as 'self' to the immune system, and thus is not detected as a tumor by the immune system (i.e. natural killer cells). When tumors are prepared for in vitro assays against drugs, they are routinely treated with proteolytic enzymes (e.g. pepsin, or chymotrypsin, etc.) which dissolve the protein coat, exposing the tumor cell surface to the drug. Thus, the in vivo existence of a coat on the tumor surface may explain why some drugs have little or no effect in vivo, while the same drugs are active in vitro. Ltd Copyright 2000 Harcourt Publishers Ltd.