Comparative study of plasminogen activators in cancers and normal mucosae of human urinary bladder.

We have developed a highly sensitive sandwich enzyme immunoassay for determination of urokinase-type plasminogen activator (u-PA) and tissue-type plasminogen activator (t-PA) antigen levels in extracts of human tissues. We determined antigen levels of PAs in extracts of 31 primary cancers and 15 normal mucosal tissues of the urinary bladder using this method. U-PA antigen levels in extracts of bladder cancers were significantly higher than those in normal tissues (p less than 0.005). U-PA antigen levels significantly increased as histological grading of malignancy advanced. There was no correlation between t-PA antigen level and malignancy. These results indicate that an increase of u-PA antigen level may be a parameter of malignant transformation and may play an important role in invasiveness of cancer.

Suramin inhibits intimal thickening following intimal injury in the rabbit aorta in vivo.

OBJECTIVE: Proliferation of vascular smooth muscle cells is a major event in atherogenesis. Several growth factors have been well documented to control this proliferation. Inhibition by suramin of the binding of some growth factors to their receptors has recently been reported. The aim of this study was to examine the effects of this agent on neointimal thickening following intimal mechanical injury, as well as on platelet function. METHODS: Intimal thickening was induced by indwelling of polyethylene tubing for 24 h in the rabbit aorta. Rabbits were killed 10 d after drawing out the tubing. Throughout the experiment, suramin (15 mg.kg-1) was injected intravenously every 24 h. Morphological and morphometrical studies were performed in the suramin treated group (n = 6) and in a control group (n = 6). Platelet-rich plasma was prepared from animals before and 3 h after injection of suramin. Platelet aggregation and ATP release induced by collagen were examined. Platelet adhesion on the de-endothelialised area of the rabbit aorta was also examined in the two groups. RESULTS: The mean intimal thickening in the suramin treated group was significantly less than in the control group. Smooth muscle cell replication and cell density in the thickened intima of the suramin treated group were less than in control. Suramin did not affect collagen induced platelet aggregation, ATP release, or platelet adhesion. CONCLUSIONS: Suramin has an inhibitory effect on the neointimal thickening and intimal smooth muscle cell proliferation after intimal injury in the rabbit aorta, but has no effect on platelet function.

Ultrastructural study of TPA-induced cell motility: human well-differentiated rectal adenocarcinoma cells move as coherent sheets via localized modulation of cell-cell adhesion.

We previously found that 12-O-tetradecanoylphorbol-13-acetate (TPA)-enhanced invasion of Matrigel was associated with augmentation of cell motility but not with metalloproteinase activity in a highly metastatic variant (L-10) of human rectal adenocarcinoma cell line RCM-1. In a two-dimensional cell motility assay, TPA induced active L-10 cell locomotion with characteristic morphology; the cells moved outwards from the cell islands mainly as a localized coherent sheet of cells. The leading cells showed locomotor morphologies with fan-shaped leading lamellae while the following cells had cell contacts on all sides and appeared to lack leading lamellae. In the present ultrastructural study, the following cells frequently showed tapering cytoplasmic protrusions and leading lamella-like processes underlapping a preceding cell, indicating that the locomotion mechanism is almost the same for both the leading and following cells. For this type of locomotion as a coherent sheet we propose that localized modulation of cell-cell adhesion was induced such that wide intercellular gaps occurred at the lower portion of the cells to allow the cells to extend the tapering cytoplasmic processes and leading lamellae while close cell-cell contacts remained at the upper portion of the cells. These TPA-induced changes took place predominantly in the cells at the periphery of the cell islands, while the cells in the middle of the cell islands maintained close cell-cell contacts including complex interdigitation all around the cells, suggesting the modulation of TPA action by cell-cell interaction. Additionally, consistent with the evidence for junctional complexes between the cells moving outwards, the Lucifer-yellow dye transfer studies showed some, limited cell-cell coupling, suggesting the presence of at least some gap junctional intercellular communication in the moving cell sheets.

Effects of suramin on metastatic ability, proliferation, and production of urokinase-type plasminogen activator and plasminogen activator inhibitor type 2 in human renal cell carcinoma cell line SN12C-PM6.

Effects of suramin, a polysulfonated naphthylurea compound, on metastatic ability, proliferation, and production of plasminogen activators and plasminogen activator inhibitors were studied using the highly metastatic human renal cell carcinoma cell line, SN12C-PM6. After renal subscapular implantation of tumor cells in nude mice, suramin significantly inhibited metastasis of tumor cells to the lungs and liver. In vitro growth of tumour cells was inhibited by suramin in a dose-dependent manner, at relatively low doses (ID50 = 105 micrograms/ml). Plasminogen activator inhibitor type 2 (PAI-2) production by tumor cells was enhanced by suramin (100 micrograms/ml), whereas urokinase-type plasminogen activator (uPA) production was suppressed. Thus, the increase in PAI-2 and the decrease in uPA production correlated with the inhibitory effects on tumour growth and metastasis by suramin. Therefore suramin may be beneficial for the treatment of patients with an early stage of renal cancer with potential risk of metastasis.

Vascular injuries induced by materials released from platelet-rich thrombus in vivo.

Polyethylene tubing was inserted into the ascending aorta of rabbits via the right common carotid artery and placed for one or four weeks continuously to induce vessel wall injury and thrombotic events, and then the direct non-injured segments from the descending thoracic and abdominal aorta were examined morphologically and for [3H]thymidine incorporation into endothelial cells and smooth muscle cells. The descending aortas of experimental rabbits showed endothelial damage and increased mitosis of endothelial cells. [3H]Thymidine incorporation into the intima and media was significantly increased in the experimental group. This experiment indicates that materials released from activated platelets and/or thrombi into the circulation can cause endothelial damage and smooth muscle cell proliferation at downstream and remote aortic segments.

Vascular contraction in perfused carotid arteries of cholesterol-fed rabbits.

The purpose of the present study was to investigate the effects of hypercholesterolemia on sympathetic vascular responsiveness in the perfused rabbit carotid artery. Two groups of rabbit carotid arteries were evaluated for the simultaneous measurement of noradrenaline (NA) release and vasoconstrictor response induced by electric nerve stimulation and for exogenous NA-induced vasoconstriction in vitro. One group of rabbits was fed a diet containing 0.5% cholesterol for 2 weeks and the other group was fed standard rabbit chow. By scanning electron microscopy, monocytes adhering to the endothelial cells and penetrating into the subendothelium were observed. Neither endothelial denudation nor platelet adhesion could be detected. Rabbit carotid arteries were cannulated and perfused with a physiological solution at a constant flow rate. The vessels were subjected to both transmural field stimulation (TFS; 1.5-24 Hz) and exogenous NA administration. TFS caused a frequency-dependent increase in endogenous NA release with subsequent pressor responses in both groups. Exogenous NA also induced a dose-dependent pressor response, but a significant reduction was observed in the cholesterol-fed group. Methoxamine induced a similar response in both groups. It was concluded that hypercholesterolemia decreased the sensitivity of extrajunctional alpha-receptors in the perfused rabbit carotid artery.

Aortic endothelial cell damage induced by beta-VLDL and macrophages in vitro.

We performed an in vitro study to assess damage to swine aortic endothelial cells by rabbit beta-VLDL and/or rabbit peritoneal macrophages. Incubation of cultured aortic endothelial cells with beta-VLDL, macrophages, or macrophage lysate induced endothelial cell damage time- and dose-dependently as estimated by [3H]adenine release. Incubation of endothelial cells with both beta-VLDL and macrophages produced a synergistic effect on the increase of [3H]adenine release. Pretreatment of the endothelial cells with some kinds of antioxidants (probucol 50 micrograms/ml, vitamin E 50 microM, superoxide dismutase-polyethylene glycol 0.5 mg/ml, or catalase-polyethylene glycol 0.5-1.0 mg/ml) significantly prevented the endothelial damage by beta-VLDL or macrophage lysate. We conclude that beta-VLDL and/or macrophages could induce endothelial cell damage and that some kinds of antioxidants could prevent it.

Localization and activity of tissue factor in human aortic atherosclerotic lesions.

Tissue factor (TF) is a transmembrane protein that serves as the major initiator of the blood coagulation cascade. The overexpression of TF antigen and mRNA has previously been reported in advanced atherosclerotic lesions. Recently TF procoagulant activity has also been identified in these lesions. However, localization and activity of TF in various stages of atherosclerosis have not yet been reported. We studied TF localization and its activity in three stages of the human atherosclerotic lesions (diffuse intimal thickening, fatty streak, and atheromatous plaque). The thoracic aortas were obtained from 23 autopsy cases and were examined immunohistochemically using an anti-human TF polyclonal antibody and biotinylated factor VIIa (FVIIa) as a probe to test the FVIIa-binding ability of TF. In addition, the TF-mediated activation of factor X (FX) was quantitatively assessed using a chromogenic assay. In lesions of the diffuse intimal thickening and the fatty streak, almost all of intimal smooth muscle cells (SMCs), macrophages, and endothelial cells were positive for TF. In the atheromatous plaques, TF antigen was detected extensively in the extracellular matrix as well as in the intimal cells. TF in all stages of atherosclerotic lesions had the ability to bind biotinylated FVIIa. TF activity was detected in each lesion and was more prominent in fatty streaks and atheromatous plaques than in the diffuse intimal thickening. These results indicate that active TF is expressed in the early stage of atherosclerotic lesions as well as in the advanced stage, and it contributes to the thrombotic property of human atherosclerotic lesions.

Effects of inflation pressure of balloon catheter on vascular injuries and subsequent development of intimal hyperplasia in rabbit aorta.

Balloon catheter de-endothelialization is the most popular means of arterial injury in experimental animals and has been used as the model system to investigate atherogenesis and restenosis after percutaneous transluminal coronary angioplasty (PTCA). The aim of this study was to examine the relationship between balloon inflation pressure and vascular damage and also subsequent intimal hyperplasia. Retrograde pullback balloon injury of rabbit aortas was made at three different balloon pressures (1.5, 1.75, and 2.0 atm). The medial injuries, such as necrosis of smooth muscle cells and disruption of elastic lamina, were occasionally found in the injured segment of the aorta by balloon catheter at 1.75 atm and more frequently at 2.0 atm. No prominent medial injury was observed in the aortic segment to balloon catheter injury at 1.5 atm; Intimal hyperplasia developed in each animal and increased with time, 2, 4, and 8 weeks after injury. The intimal hyperplasia followed by balloon injury at 1.75 and 2.0 atm was more prominent than that at 1.5 atm, however, the development of the intimal hyperplasia was not parallel to the degree of inflation pressure. On the other hand, decrease of DNA content of the media and reduction of norepinephrine-induced vasoconstriction were observed in a pressure-dependent manner after balloon injury. These findings indicate that intimal hyperplasia is not proportionally correlated to the severity of the vascular injury. The control of inflation pressure is very important in order to examine vascular injuries, subsequent intimal hyperplasia and vasomotor responses in animal models of balloon catheter injury.

Expression of tissue factor in the rabbit aorta after balloon injury.

Tissue factor (TF) is a primary initiator of the extrinsic pathway of blood coagulation. Recently TF has been shown to be overexpressed in atherosclerotic lesions and it is thought to contribute to the thrombogenicity of the plaques. We studied TF expression in the media and the neointima of rabbit aortas at various intervals after balloon injury. TF protein was immunohistochemically detected in smooth muscle cells (SMCs) of the inner layer of the media at 2 h after injury and was subsequently detected in SMCs in the neointima, whereas no TF expression was detected in the uninjured aortas except for the adventitia. Immunohistochemical and immunoelectron microscopic studies revealed that TF-positive SMCs were of an immature or synthetic phenotype and TF protein was detected in the rough endoplasmic reticulum in SMCs. TF mRNA in the intima and media increased at 2 h after injury and returned to near baseline levels at 12-24 h, whereas TF activity also increased at 2 h and continued at similar levels over the next 72 h. TF mRNA and activity increased markedly at 2-8 weeks after injury. These data suggest that TF is rapidly induced in the medial SMCs and hereafter is constitutively expressed in the neointima. TF expressed in the neointima may contribute to hypercoagulable properties of injured arteries.

Second nation-wide study of atherosclerosis in infants, children and young adults in Japan.

This paper reports the results of the second nation-wide cooperative study of atherosclerosis in young Japanese, aged from 1 month to 39 years, who were autopsied between 1991 and 1995. Atherosclerotic lesions in 1066 aortas and 974 coronary arteries were classified into fatty streaks, fibrous plaques and complicated lesions and quantificated with the point-counting method. The results of this study were compared with those of the former study, which was conducted 13 years earlier in almost the same fashion as this study. Atherosclerosis of aorta, which was determined by surface involvement (SI) of atherosclerotic lesions and atherosclerotic index (AI), increased with age in both sexes of the former and the present studies and their tendency for the progression of the extent of atherosclerotic lesions appeared to be similar. In the coronary arteries, the mean values of SI and AI in the males of the present study were greater significantly than those in the male of the former studies and in the female of the both studies in the third and fourth decades. This difference suggests that atherosclerotic lesions are increasing in young Japanese males. It also suggests that these subjects may be increasingly susceptible to atherosclerotic cardiovascular disease with increasing age.

Tissue factor induces migration of cultured aortic smooth muscle cells.

Tissue factor (TF) plays a key role as a primary initiator on the extrinsic coagulation cascade. Recently, upregulation of TF has been reported in human atherosclerotic lesions. We investigated the effects of TF on migration and proliferation of cultured smooth muscle cells (SMCs) from rabbit aortas. We tested three kinds of recombinant human TF (L-TF: the full length of TF with relipidation, NL-TF: the full length of TF without relipidation, and S-TF: a soluble form of TF1-219). Only L-TF had coagulant activity. All kinds of TF showed the chemotactic migration activity for SMCs. The migration ability of TFs was comparable to those of platelet-derived growth factor (PDGF)-BB and basic fibroblast-growth factor (bFGF), and was inhibited by anti-TF polyclonal and monoclonal antibodies. On the other hand, none of the forms of TF induced SMC proliferation. These results indicate that TF is not only a coagulation factor but also a strong chemotactic factor for vascular SMCs, and suggest that TF could play an important role in atherogenesis as well as in hemostasis and thrombosis.

Tissue factor pathway inhibitor inhibits aortic smooth muscle cell migration induced by tissue factor/factor VIIa complex.

Tissue factor (TF), a transmembrane glycoprotein, forms a high affinity complex with factor VII/VIIa (FVIIa) and thereby initiates blood coagulation. Tissue factor pathway inhibitor (TFPI) is an endogenous protease inhibitor of TF/FVIIa-initiated coagulation. We previously reported that TF was a strong chemotactic factor for cultured vascular smooth muscle cells (SMCs). In this study, we examined the contribution of FVIIa and the effect of TFPI to TF-induced cultured SMC migration. TF/FVIIa complex showed a strong migration ability, however, neither TF alone nor FVIIa induced SMC migration. TF/FVIIa treated by a serine protease inhibitor and the complex of TF and inactivated FVIIa (DEGR-FVIIa) did not stimulate SMC migration. Pretreatment with hirudin and the antibodies to alpha-thrombin and factor X had no effect on TF/FVIIa-induced SMC migration, although alpha-thrombin and factor Xa also induced SMC migration respectively. TFPI markedly inhibited TF/FVIIa-induced SMC migration in a concentration-dependent manner, but did not affect the SMC migration induced by platelet-derived growth factor (PDGF)-BB, basic fibroblast-growth factor (bFGF), or alpha-thrombin. These results indicate that the catalytic activity of TF/FVIIa complex is important on SMC migration, and TFPI can reduce SMC migration as well as thrombosis.

Fibrin-rich and platelet-rich thrombus formation on neointima: recombinant tissue factor pathway inhibitor prevents fibrin formation and neointimal development following repeated balloon injury of rabbit aorta.

Thrombus formation and neointimal growth are the critical events in restenosis after balloon angioplasty. However, the responses of diseased vessels to injuries caused by balloon angioplasty have not been well examined. We investigated the thrombus formation and neointimal development following the balloon injury to the previously induced neointima in the rabbit aorta and the effects of recombinant tissue factor pathway inhibitor (rTFPI) on these responses. Rabbit thoracic aortas were subjected to injury with a Fogarty 4F balloon catheter at 1.75 atm (first injury), and 4 weeks later the same vessels were subjected to the second injury with a Swan-Ganz 5F balloon catheter at 1.4 atm (mild-injury group) or 1.8 atm (severe-injury group), and immediately after that a retrograde bolus injection of rTFPI (100 microg/kg body weight) or saline was performed into the injured segments via the central tube of the Swan-Ganz catheter. Twenty minutes after the second injury, the injured surfaces were covered with platelet-rich thrombi in the mild-injury group and with fibrin-rich thrombi in the severe-injury group. Damaged intimal smooth muscle cells, which were immunohistochemically positive for tissue factor (TF), were observed beneath the fibrin-rich thrombi. The neointima 4 weeks after the second injury was significantly thicker in the severe-injury group than in the mild-injury group. The bolus infusion of rTFPI markedly inhibited fibrin formation on the injured surfaces, and significantly reduced the neointimal development in the severe-injury group at 4 weeks after the second injury. These results indicate that TF-dependent coagulation pathway is primarily responsible for fibrin-rich thrombus formation and may play an important role in neointimal development following the balloon injury to the rabbit aortic neointima. Additionally the bolus administration of rTFPI to the injured vessels could prevent mural thrombus formation and neointimal growth after balloon angioplasty.

Miliary tuberculosis presenting as fever and jaundice with hepatic failure.

A 58-year-old male patient with miliary tuberculosis presenting as jaundice and hepatic dysfunction was reported. He was admitted to the Miyazaki Medical College Hospital, Miyazaki, Japan, because of fever and jaundice. Chest x-ray revealed a calcified primary affect of tuberculosis in the left upper lung field and miliary shadows throughout both lung fields. Liver function tests showed indications of obstructive jaundice and hepatic dysfunction. He rapidly deteriorated and died 3 days after admission. Autopsy revealed disseminated miliary tuberculosis in all major organs. Many miliary tubercles were densely distributed in the liver, especially in and near the portal tracts. The intestine was free from tuberculous lesions. Miliary tuberculosis with jaundice is rare and its pathogenesis is discussed.

Ganglioneuroblastoma of the thymus: an adult case with the syndrome of inappropriate secretion of antidiuretic hormone.

A 61-year-old woman was admitted to the hospital because of general fatigue. Laboratory examinations showed hyponatremia, plasma hypo-osmolarity, and inappropriate increased concentration of the plasma antidiuretic hormone (ADH) in the presence of concentrated urine. Magnetic resonance imaging revealed a mass lesion in the anterior mediastinum. An extended thymectomy was performed under the diagnosis of thymoma with the syndrome of inappropriate secretion of antidiuretic hormone (SIADH). Histologically the tumor was located in the thymic tissue and was diagnosed as ganglioneuroblastoma. Immunohistochemical studies showed the existence of ADH in the tumor cells. To the knowledge of the authors, this is the first case of ganglioneuroblastoma of the thymus with SIADH.

Scanning and transmission electron microscopic study of human pulmonary pneumocystosis.

Histopathologic examinations were performed in 13 autopsy cases with Pneumocystis pneumonia, five of whom were examined with scanning as well as transmission electron microscope. Findings by scanning electron microscopy were compared with those by light microscopy and transmission electron microscopy. By scanning electron microscopy, two principal structural varieties of Pneumocystis organisms, smooth-surfaced organisms and rough-surfaced organisms, could be classified. It was indicated that rough-surfaced organisms and smooth-surfaced organisms correspond respectively to trophozoite-type organisms and thick-walled cysts, recognized with transmission electron microscope. Intraalveolar honeycombed material was mainly composed of dilated trophozoite-type organisms, containing thick-walled cysts scatteredly.

Magnetic resonance imaging of bone marrow changes after irradiation.

RATIONALE AND OBJECTIVES: Signal changes of irradiated bone marrow are thought to represent fatty change. However, more recent studies have indicated that other factors may contribute to these signal changes. We performed animal studies to investigate magnetic resonance (MR) signal change factors in correlation with histologic findings. MATERIALS AND METHODS: Forty-two male Wistar rats were given single doses (3, 8, and 20 Gy) of irradiation. Magnetic resonance imaging (1.5 T) was performed using spin-echo technique. RESULTS: In the acute phase (days 1-3), the T1 time was prolonged, probably secondary to decreased cellularity and edema. After this phase, the irradiated marrow showed marked T1 and T2 shortening that correlated with hemorrhage. In the chronic phase (after day 10), fatty replacement, fibrosis, and regeneration were observed. T2 times were stable in spite of regeneration because of the susceptibility effect of hemosiderin. CONCLUSION: The MR signal changes of irradiated marrow reflect not only fatty replacement, but hemorrhage as well.

Herpes simplex esophagitis--a study in autopsy series.

Autopsy cases were reviewed in order to determine the incidence and underlying diseases of herpes esophagitis. Review of both autopsy records and the preserved viscera of 145 cases disclosed 9 cases (6%) of herpes esophagitis. Herpes simplex virus type I specific antigen was detected in all of these nine cases by immunohistochemical method. Eight of nine cases had malignant diseases. Review only of autopsy records of other 254 cases disclosed 5 cases (2%) of herpes esophagitis. Thus, herpes esophagitis is thought to be more common than has previously been appreciated and may be overlooked at autopsy. Although antemortem diagnosis of this condition has been very rare, clinicians should have an accurate knowledge of this disease because herpetic esophageal ulcer may act as a portal of entry for generalized dissemination of virus and other pathogens, and effective antiviral agents are becoming available.

Vascular injuries induced by materials released from white mural thrombus and hypercholesterolemia in vivo.

Materials released from platelet-rich white mural thrombi into the arterial circulation and dietary hypercholesterolemia could cause endothelial injury and regeneration, and also smooth muscle cell migration and proliferation in vivo in the intima. In addition, a combination of these two factors showed an additive effect on the endothelial injury in vivo. Further experiments are in progress to clarify how high a level of serum cholesterol and/or oxidized LDL may be sufficient for endothelial injury using this in vivo model for the better understanding of detailed pathogenesis of atherosclerosis.