Characteristics of tumour vessels in cytological squash smears of astrocytic tumours.

OBJECTIVE: Smear preparations are useful tools from which to diagnose brain tumours intraoperatively. Although vascular proliferation is histologically a key feature of high-grade astrocytoma, the characteristics of tumour vessels in smear preparations have not been determined. METHODS: We examined the density and morphological parameters (area, width, nuclear layer and branches of vessel wall) of tumour vessels in squash smears of 43 primary astrocytomas (grade II diffuse astrocytomas, n=9; grade III anaplastic astrocytomas, n=13; grade IV glioblastomas, n=21) and normal brain tissues (n=11). RESULTS: Vessel density and all morphological parameters were significantly higher in grade IV than in the other grades of tumours and in normal brain tissue. Vessel area, width and nuclear layer were greater in grade III than in normal brain tissue. The sensitivity and specificity of these vessel parameters for astrocytomas were 75-100% and 82-100%, respectively. CONCLUSIONS: Tumour vessel evaluations from squash smears provide useful information for the intraoperative diagnosis and grading of astrocytic tumours.

EMMPRIN (CD147) expression and differentiation of papillary thyroid carcinoma: implications for immunocytochemistry in FNA cytology.

OBJECTIVE: Extracellular matrix metalloproteinase inducer (EMMPRIN) and its induced matrix metalloproteinases (MMPs) play a crucial role in tumour progression, invasion and metastasis. EMMPRIN expression has been demonstrated in several tumours, but its expression profile in thyroid cancer remains unclear. METHODS: We evaluated the expression profile of EMMPRIN at various stages of differentiation of thyroid carcinoma, including 20 cases of well-differentiated papillary carcinoma (WDPC), 15 cases of papillary carcinoma with a poorly differentiated carcinoma component (PC/PDC) and four cases with an undifferentiated carcinoma (UDC) component, using paraffin-embedded sections for immunohistochemical stains. Also, we used 32 fine needle aspiration cytology and imprint smears from the same cases for immunocytochemical stains. The staining results were evaluated with a scoring system. RESULTS: Immunohistochemical staining showed that EMMPRIN expression was absent or weak in almost all WDPC specimens, whereas it was moderate or strong in PDC and UDC components. In tumours that showed a gradual morphological transformation from WDPC to PDC components, the expression of EMMPRIN was progressively stronger from the areas of WDPC to those of PDC. WDPC, PC/PDC and UDC had expression scores of 4.9, 45.0 and 245.7, respectively. Results of immunocytochemical staining showed almost the same staining profile as those of immunohistochemical staining. The cytological atypia of EMMPRIN-positive cells was greater than that of negative cells. CONCLUSION: These results indicated that EMMPRIN expression correlates significantly with the degree of dedifferentiation of thyroid carcinoma. This study demonstrates the feasibility of expression of EMMPRIN using fine needle aspiration samples. Therefore, immunocytochemical analysis of EMMPRIN may be a novel aid to evaluate the differentiation of thyroid carcinoma.

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.

Combination of a brief irrigation with tissue factor pathway inhibitor (TFPI) and adenovirus-mediated local TFPI gene transfer additively reduces neointima formation in balloon-injured rabbit carotid arteries.

BACKGROUND: Tissue factor pathway inhibitor (TFPI) is a physiological antagonist of TF. We tested whether a brief irrigation with TFPI protein (rTFPI) or TFPI gene transfer into injured arteries would suppress TF activity and reduce fibroproliferative changes and investigated whether a combination of these methods would show an additive effect. METHODS AND RESULTS: We prepared adenoviruses expressing either TFPI (AdTFPI) or bacterial ss-galactosidase (AdLacZ). Rabbit carotid arteries were balloon-injured and either infected with AdTFPI (or AdLacZ) or irrigated briefly with rTFPI (or saline). After injury, TF activity in arteries increased and was sustained; however, it was suppressed during the initial 24 hours by rTFPI irrigation (but not by gene transfer) and for a substantial period of time by TFPI gene transfer (but not by rTFPI irrigation). Four weeks later, the ratio of the intimal to medial areas was 34.3+/-8.7% (mean+/-SD, n=14) in saline-treated arteries and 33.3+/-4.2% in AdLacZ-infected arteries (P:=NS versus saline). However, it was reduced to 25.5+/-8.5% in rTFPI-irrigated arteries (P:<0.01 versus saline) and to 20.7+/-5.3% in AdTFPI-infected arteries (P:<0.01 versus AdLacZ). With a combination of irrigation and gene transfer, the ratio was further reduced to 12.6+/-4.7% (P:<0.01 versus rTFPI, P:<0.05 versus AdTFPI). Systemic coagulation status was not affected in these animals. CONCLUSIONS: A combination of rTFPI irrigation and TFPI gene transfer overcomes the shortcomings shown by each method when used alone and achieves a full coverage of TF activity suppression, thereby enhancing their therapeutic effects without systemic side effects. This combination may be an effective strategy for the prevention of thrombosis and proliferative changes after angioplasty in humans.

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.

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.

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.

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.

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.

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.

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.

Cavitating Pneumocystis pneumonia in an autopsied case of adult T-cell leukemia.

Cavitating necrosis is rare in Pneumocystis carinii pneumonia. In this report, we describe an autopsy patient with adult T-cell leukemia associated with cavitating Pneumocystis carinii pneumonia. The patient, a 61-year-old woman, died of an acute crisis of adult T-cell leukemia associated with diffuse pulmonary infection of Pneumocystis carinii. Postmortem examination revealed necrotic foci in both lungs, one of which, in the left lower lobe, had a central cavitation. Microscopically, leukemic cell infiltration was abundant in the lung parenchyma but not in the necrotic lesions. Pneumocystis carinii organisms were distributed diffusely in the alveoli and also in the cavity wall. Intranuclear and intracytoplasmic inclusion bodies were scattered in the lung indicating cytomegalovirus infection. However, no bacterial or fungal infection was detected in the lungs, even in the necrotic lesions. Cavitating Pneumocystis carinii pneumonia occurs in other immunodeficiency diseases apart from AIDS. To our knowledge, this report is the first case of cavitating Pneumocystis carinii pneumonia in adult T-cell leukemia.

Prognostic value of urokinase-type plasminogen activator in patients with superficial bladder cancer.

OBJECTIVES: A number of studies have demonstrated that urokinase-type plasminogen activator (u-PA) is one of the major enzymes in the development of cancer invasion and metastasis. We examined the possibility that u-PA content is an independent prognostic marker for superficial bladder cancer. METHODS: The u-PA content was estimated in superficial bladder cancer from 52 patients undergoing transurethral resection for the first time. The survival rates of the patients were calculated and the prognostic values for u-PA content (less than 8 ng/mg protein versus more than 8 ng/mg), Stage (pTa or pT1), grade (1 and 2 versus 3), number (single versus multiple), and size (less than 1 cm versus more than 1 cm) of tumor were determined by a multivariate regression model. RESULTS: Development of cancer invasion and metastasis was observed in 3 and 8 patients, respectively. The survival rate of the patients with high u-PA content was significantly lower than those with low u-PA content (P < 0.005). In the multivariate regression analysis, u-PA content was the most important risk factor for the prognosis, compared with the other factors: tumor stage, grade, multiplicity, and size. CONCLUSIONS: We have first shown that u-PA content is a new independent prognostic marker in patients with superficial bladder cancer.

Coronary atherosclerosis in youths in Kyushu Island, Japan: histological findings and stenosis.

We histologically examined the coronary arteries of 52 autopsied cases of the youths (3 to 39 years of age, mean 28.5 years) in Kyushu island, Japan, without clinical events of coronary artery diseases. The coronary artery specimens were taken from the proximal portions of the right coronary artery (Seg. 1), the left anterior descending artery (Seg. 6), and the macroscopically most stenotic region (ST). Atherosclerotic lesions were histologically classified into four types: concentric fibrous, eccentric fibrous, concentric lipid rich, or eccentric lipid-rich type. The degrees of stenosis (< 25%, 25-50%, 50 75%, > 75%) were morphometrically evaluated. The majority of coronary arteries with under 50% stenosis were of the concentric fibrous type. Lipid-rich types of coronary atherosclerosis increased in the coronary arteries with over 50% stenosis and were observed in the Seg. 6 and ST, while 70% of Seg. 1 lesions with over 50% stenosis were of a fibrous type. Serum cholesterol levels of patients with a lipid rich type of coronary atherosclerosis were significantly higher than those with a fibrous type. These results suggested that the early stage of coronary atherosclerosis in Japanese youths is mainly of a concentric fibrous type, which later develops to a lipid rich type. Hypercholesterolemia would promote the progression of atherosclerosis.

The role of tissue factor in the pathogenesis of thrombosis and atherosclerosis.

TF is a major regulator of coagulation and hemostasis. High levels of TF antigen and activity are detected in atherosclerotic lesions, particularly in the advanced lesions. When the plaques are ruptured or eroded, exposure of cellular and extracellular TF to circulating blood play a pivotal role in mediating fibrin-rich thrombus formation leading to acute coronary syndromes. On the other hand, activation of blood coagulation and deficiency of coagulation inhibitors, without endothelial cell denudation, are considered to be an important factor of thrombogenesis in the microcirculation. The imbalance between TF and TFPI seems to be important in promoting fibrin thrombus formation in the lung of endotoxin induced DIC condition.

Autopsy findings in 47 cases of adult T-cell leukemia/lymphoma in Miyazaki prefecture, Japan.

To identify factors that might improve the prognosis of adult T-cell leukemia/lymphoma (ATL), we reviewed data on 47 autopsied cases of ATL with reference to the complications and cause of death. The primary cause of death was respiratory insufficiency due to pulmonary infection. Respiratory insufficiency was also attributed to the diffuse alveolar damage and pulmonary fibrosis resulting from chemotherapy given and oxygen. About 90% of the cases had infections with one or more pathogens. Cytomegalovirus (CMV) was the most frequent pathogen involved in 35/47 (74.5%) while fungal infections were also commonly seen in 25 of the 47 cases (53.2%). Of these, 17 (70%) had pulmonary aspergillosis. Other neoplasias were present in 10 of the 47 cases, while hypercalcemia was evident in 21 patients. These findings suggest that the prevention and treatment of nosocomial infections and of drug-induced pulmonary toxicity may improve the prognosis and quality of life of ATL patients.

Intravenous injection of sonicated blood induces pulmonary microthromboembolism in rabbits with ligation of the splenic artery.

Pulmonary thromboembolism (PTE) is found in long hospitalized patients. Chronic PTE has been reported to play an important role in cardiac failure in thalassemic patients after splenectomy. However, the mechanism of PTE in these patients remains unclear. In this study, we attempted to establish an animal model of PTE. We divided New Zealand white rabbits into three groups: Group I was injected sonicated blood, II was injected non-sonicated blood after ligation of the splenic artery, and III was injected sonicated blood after ligation of the splenic artery. After injection of the sonicated blood, we examined the platelet counts every 10 minutes until 1 hour and the rabbits were sacrificed for histological examination. Platelets significantly decreased in number immediately after the injection of sonicated blood in Groups I and III. Many pulmonary thromboemboli composed mainly of platelets were found in Group III but not in other groups. These pathological changes seem to be partly similar to those of thalassemic patients after splenectomy. This animal model is thought to be useful to study the pathogenesis of pulmonary thromboembolism, especially in thalassemic patients after splenectomy.

Pathogenetic implications of hyaluronan-induced modification of vascular smooth muscle cell fibrinolysis in diabetes.

BACKGROUND: Proteolysis, modulated in part by intramural fibrinolytic system proteins and their inhibitors, appears to influence vascular smooth muscle cell (SMC) migration and proliferation and remodeling of extracellular matrix (ECM). Alterations of fibrinolysis in circulating blood and of proteolysis within vessel walls in experimental animals and patients with diabetes have been associated with accelerated vascular disease. Hyaluronan, a prominent component of ECM in normal vessels, is increased in the tunica media of macroscopically normal arterial vessels from patients with type 2 diabetes. OBJECTIVE: To determine whether hyaluronan alters the expression of the fibrinolytic system protein, plasminogen activator inhibitor type-1 (PAI-1), in human vascular SMCs, thereby potentially accelerating vascular disease in patients with type 2 diabetes. METHODS: Urokinase-type and tissue-type plasminogen activators (uPA and tPA) and PAI-1 were assayed in vascular SMC conditioned media and in cell lysates, using enzyme-linked immunosorbent assay and western blotting. RESULTS: Hyaluronan increased the 24-h release of PAI-1 into conditioned media in a concentration-dependent and time-dependent manner (1.8-fold compared with control with 1 mg/ml hyaluronan; n = 9, P < 0.01). Although the accumulation of uPA in conditioned media tended to increase also, uPA content was reduced in cell lysates (64% of control with 0.1 mg/ml hyaluronan at 24 h; n = 9, P < 0.01) without any change in PAI-1. Concentrations of tPA in conditioned media and cell lysates were unchanged. Digestion of hyaluronan with hyaluronidase (50 turbidity reducing units (TRU)/ml) or exposure of the smooth muscle cells to antihuman CD44 antibody (1 microgram/ml) that binds to the hyaluronan cell surface receptor obviated the effects of hyaluronan. CONCLUSION: Our results indicate that increases in hyaluronan increase vascular SMC expression of PAI-1, a phenomenon that may alter the balance between proteolysis and its inhibition in vessels of patients with type 2 diabetes, thereby contributing to the acceleration of macroangiopathy.