Progranulin and granulin-like protein as novel VEGF-independent angiogenic factors derived from human mesothelioma cells.

Malignant mesothelioma is an aggressive tumor arising from the mesothelial cells of serous membranes and is associated with tumor angiogenesis, which is a prerequisite for tumor progression. Vascular endothelial growth factors (VEGFs) including VEGF-A have a crucial role in tumor angiogenesis. However, bevacizumab, a monoclonal antibody to VEGF-A, has recently been reported not to improve the progression-free survival of patients with malignant mesothelioma. Cell culture supernatant contains extracellular components such as serum, which can mask the existence of unknown cell-derived factors in the supernatant and make it difficult to detect the factors by subsequent protein analysis. We tried using serum-free culture for human mesothelioma cell lines, NCI-H28, NCI-H2452 and NCI-H2052, and only NCI-H2052 cells adapted to serum-free culture. We found that serum-free culture supernatant derived from NCI-H2052 cells induces the formation of capillary-like tube structures (tube formation) in three-dimensional culture, in which endothelial cells sandwiched between two layers of collagen or embedded in collagen are incubated with various angiogenic inducers. However, neither neutralization of VEGF-A nor RNA interference of VEGF receptor 2 (VEGFR2) suppressed the supernatant-induced tube formation. Using mass spectrometry, we identified a total of 399 proteins in the supernatant, among which interleukin-8 (IL-8), growth-regulated α-protein, midkine, IL-18, IL-6, hepatoma-derived growth factor, clusterin and granulin (GRN), also known as progranulin (PGRN), were included as a candidate protein inducing angiogenesis. Neutralizing assays and RNA interference showed that PGRN, but not the above seven candidate proteins, caused the supernatant-induced tube formation. We also found that NCI-H28 and NCI-H2452 cells express PGRN. Furthermore, we demonstrate that not only PGRN but also GRN-like protein have an important role in the supernatant-induced tube formation. Thus, mesothelioma-derived GRNs induce VEGF-independent angiogenesis.

Smoking and lipid-related indices in patients with diabetes mellitus.

AIMS: Lipid-related indices, including the ratio of LDL cholesterol to HDL cholesterol, the ratio of triglycerides to HDL cholesterol and lipid accumulation product, are known to be good discriminators for cardiovascular disease. The aim of this study was to clarify the relationships between smoking and the lipid indices in patients with diabetes. METHODS: Subjects were those who had been diagnosed as having diabetes mellitus at annual health check-ups at their places of work (n = 2563). The subjects were divided into three groups of non-smokers, light smokers (≤ 20 cigarettes/day) and heavy smokers (> 20 cigarettes/day). The relationships between smoking and the lipid indices were investigated. RESULTS: Both in all subjects and in the subjects without a habit of alcohol drinking, the LDL cholesterol:HDL cholesterol ratio and the log-transformed triglyerides:HDL cholesterol ratio tended to be higher with an increase in the amount of smoking, and the log-transformed lipid accumulation product was significantly higher in heavy smokers than in non-smokers. In the non-alcohol drinking subjects, the odds ratios of heavy smokers vs. non-smokers for high LDL cholesterol:HDL cholesterol ratio [2.32 (95% CI 1.40-3.84)], for high triglycerides:HDL cholesterol ratio [1.69 (95% CI 1.06-2.69)] and for high lipid accumulation product [1.65 (95% CI 1.02-2.67)] were significantly higher than a reference level of 1.00. The associations between smoking and the lipid indices were weaker in alcohol drinkers than in non-drinkers. CONCLUSIONS: In patients with diabetes, the levels of lipid-related indices were higher in smokers than in non-smokers, and cardiometabolic disorders, reflected by high lipid indices, are thought to be involved in the proneness of smokers to develop atherosclerotic cardiovascular disease.

Associations of alcohol drinking and cigarette smoking with serum lipid levels in healthy middle-aged men.

AIMS: The aim of this study is to determine whether influences of drinking alcohol on serum lipid levels are different in smokers and non-smokers. METHODS: Subjects were 25,689 healthy male workers aged 40 to 59 years. Serum total and HDL cholesterol and triglyceride concentrations were measured and LDL cholesterol concentrations were estimated by using the Friedewald formula. The subjects were divided into three groups by average daily consumption of cigarettes (non-smokers; light smokers, less than 20 cigarettes per day; heavy smokers, 20 or more cigarettes per day) and by average daily alcohol consumption (non-drinkers; light drinkers, less than 30 g of ethanol per day; heavy drinkers, 30 g or more of ethanol per day). RESULTS: In overall subjects, serum HDL, LDL and total cholesterol were significantly lower and triglyceride was significantly higher in heavy smokers than in non-smokers. In the smoker groups, serum total cholesterol was significantly lower in heavy drinkers than in non-drinkers, while no difference in total cholesterol was observed in non- and heavy drinkers of the non-smoker group. Both in the smoker and non-smoker groups, HDL cholesterol was higher and LDL cholesterol was lower in drinkers than in non-drinkers. The difference in LDL cholesterol between non-drinkers and drinkers was more prominent in smokers than in non-smokers. The above associations were not altered after the adjustment for age, body weight and alcohol intake. CONCLUSIONS: The results suggest that smoking increases the lowering effect of alcohol drinking on LDL cholesterol, but does not affect the relationship of alcohol drinking with HDL cholesterol.

Relation of serum sialic acid to blood coagulation activity in type 2 diabetes.

The level of serum sialic acid, which is known to reflect atherosclerotic progress and to be related to the incidence of cardiovascular diseases, is increased in patients with diabetes. To elucidate the mechanism of the relation of serum sialic acid to fibrinogen, the relationship between serum sialic acid and markers of blood coagulation activity was investigated in type 2 diabetic patients. The concentration of serum sialic acid showed significant positive correlations with blood platelet count and with plasma concentrations of fibrinogen, D-dimer, thrombin-antithrombin III complex and plasmin-alpha2 plasmin inhibitor complex. These relationships were still significant after adjustment for age, sex, smoking history, body mass index, hemoglobin A, mean arterial pressure and low-density lipoprotein cholesterol. The correlation coefficient of blood fibrinogen with serum sialic acid was still significant after adjustment for D-dimer, thrombin-antithrombin III complex or plasmin-alpha2 plasmin inhibitor complex. On the contrary, blood fibrinogen showed no significant correlation with D-dimer, thrombin-antithrombin III complex or plasmin-alpha2 plasmin inhibitor complex, although an increase in blood fibrinogen is known to be an atherosclerotic risk factor. These results suggest that the serum sialic acid level reflects blood coagulation activity in type 2 diabetic patients and is related to blood fibrinogen level independently of blood coagulation activity.

Chronic central infusion of ghrelin increases hypothalamic neuropeptide Y and Agouti-related protein mRNA levels and body weight in rats.

Ghrelin, an endogenous ligand for the growth hormone secretagogue receptor (GHS-R), was originally purified from the rat stomach. Like the synthetic growth hormone secretagogues (GHSs), ghrelin specifically releases growth hormone (GH) after intravenous administration. Also consistent with the central actions of GHSs, ghrelin-immunoreactive cells were shown to be located in the hypothalamic arcuate nucleus as well as the stomach. Recently, we showed that a single central administration of ghrelin increased food intake and hypothalamic agouti-related protein (AGRP) gene expression in rodents, and the orexigenic effect of this peptide seems to be independent of its GH-releasing activity. However, the effect of chronic infusion of ghrelin on food consumption and body weight and their possible mechanisms have not been elucidated. In this study, we determined the effects of chronic intracerebroventricular treatment with ghrelin on metabolic factors and on neuropeptide genes that are expressed in hypothalamic neurons that have been previously shown to express the GHS-R and to regulate food consumption. Chronic central administration of rat ghrelin (1 microg/rat every 12 h for 72 h) significantly increased food intake and body weight. However, it did not affect plasma insulin, glucose, leptin, or GH concentrations. We also found that chronic central administration of ghrelin increased both neuropeptide Y (NPY) mRNA levels (151.0 +/- 10.1% of saline-treated controls; P < 0.05) and AGRP mRNA levels (160.0 +/- 22.5% of saline-treated controls; P < 0.05) in the arcuate nucleus. Thus, the primary hypothalamic targets of ghrelin are NPY/AGRP-containing neurons, and ghrelin is a newly discovered orexigenic peptide in the brain and stomach.

Daunorubicin inhibits gene expression of cyclooxygenase-2 in vascular smooth muscle cells.

Daunorubicin (0.1-1 microM) concentration-dependently inhibited prostacyclin production induced by interleukin-1beta (IL-1beta, 2.5 ng/ml) in cultured aortic smooth muscle cells isolated from rats. IL-1beta stimulation caused activation of nuclear factor-kappaB (NF-kappaB) and expression of cyclooxygenase-2 (COX-2) mRNA and protein, which were inhibited by daunorubicin. However, COX activity, evaluated by conversion of exogenous arachidonic acid to prostacyclin, was not affected by daunorubicin (0.1-1 microM). Protein expression of COX-1 and NF-kappaB was not affected by daunorubicin. Daunorubicin also inhibited nitric oxide (NO) production induced by IL-1beta. These results suggest that daunorubicin attenuated prostacyclin synthesis through inhibiting expression of COX-2 mRNA, which could be explained by perturbation of NF-kappaB activation.

Range of motion after Bankart repair. Vertical compared with horizontal capsulotomy.

We studied the range of shoulder motion of patients who underwent vertical as compared with horizontal capsulotomies during open Bankart repair for recurrent anterior dislocations of the shoulder. A vertical capsulotomy was used in 10 shoulders and a horizontal capsulotomy was used in 14 shoulders. Except for the method of capsulotomy, the surgical procedure and postoperative rehabilitation were the same. The range of motion was measured at 1.5, 2, 3, 4, 5, 6, 9, and 12 months after the surgery, and at the final follow-up (average, 49 months for the vertical and 26 months for the horizontal group). No dislocations recurred, and the anterior apprehension test was negative in all of the patients in both groups. External rotation in abduction was greater in the horizontal group than in the vertical group; the differences were significantly greater at 9 months and 12 months after surgery and at the final follow-up. External rotation in adduction, flexion, and internal rotation were not significantly different between the groups. We conclude that Bankart repair through a horizontal capsulotomy preserves a better range of external rotation in abduction than does a vertical approach.

Position of immobilization after dislocation of the glenohumeral joint. A study with use of magnetic resonance imaging.

BACKGROUND: Glenohumeral dislocations often recur, probably because a Bankart lesion does not heal sufficiently during the period of immobilization. Using magnetic resonance imaging, we assessed the position of the Bankart lesion, with the arm in internal and external rotation, in shoulders that had had a dislocation. METHODS: Coaptation of a Bankart lesion was examined with use of magnetic resonance imaging, with the arm held at the side of the trunk and positioned first in internal rotation (mean, 29 degrees) and then in external rotation (mean, 35 degrees), in nineteen shoulders. Six shoulders (six patients) had had an initial anterior dislocation, and thirteen shoulders (twelve patients) had had recurrent anterior dislocation. Fast-spin-echo T2-weighted axial images were made when the dislocation had occurred less than two weeks earlier, and spin-echo T1-weighted axial images after intra-articular injection of gadolinium-diethylenetriamine pentaacetic acid were made when the dislocation had occurred more than two weeks earlier. Separation and displacement of the anteroinferior portion of the labrum from the glenoid rim were measured on the axial images, and coaptation of the anterior part of the capsule to the glenoid neck was assessed by measurement of the detached area, opening angle, and detached length. RESULTS: Separation and displacement of the labrum were both significantly less (p = 0.0047 and p = 0.0017, respectively) when the arm was in external rotation than when it was in internal rotation. The detached area and the opening angle of the anteroinferior portion of the capsule were both significantly smaller (p = 0.0003 and p < 0.0001, respectively), and the detached length was significantly shorter (p < 0.0001) with the arm in external rotation. CONCLUSION: Immobilization of the arm in external rotation better approximates the Bankart lesion to the glenoid neck than does the conventional position of internal rotation.

Estrogen receptor (ER)alpha, but not ERbeta, gene is expressed in growth hormone-releasing hormone neurons of the male rat hypothalamus.

GH synthesis and release from pituitary somatotropes is controlled by the opposing actions of the hypothalamic neuropeptides, GH-releasing hormone (GHRH), and somatostatin (SS). There is a striking sex difference in the pattern of GH secretion in rats. Early reports indicate that gonadal steroids have important imprinting effects during the neonatal period. Recently, our laboratory and others have reported that the GH secretory pattern is altered by short-term gonadal steroid treatment in adult rat, suggesting that gonadal steroids are also important determinants of the pattern of GH secretion during adult life. However, the site of action of gonadal steroids in the adult rat hypothalamus is still unknown. In this study, we used in situ hybridization in the adult male rat brain to determine whether GHRH neurons and/or SS neurons coexpress estrogen receptor alpha (ERalpha) and ERss genes. In the medial basal hypothalamus of adult male rat, the ERalpha messenger RNA (mRNA) was located in medial preoptic area (MPA) and arcuate nucleus (ARC), whereas ERss mRNA was detected in MPA, supraoptic nucleus, and paraventricular nucleus. From studies using adjacent sections, the distribution of ERalpha mRNA-containing cells appeared to overlap in part with those of GHRH and SS expressing cells only in the ARC. On the other hand, the distribution of ERss mRNA-containing cells does not appear to overlap with GHRH cells or SS cells. The double label in situ hybridization studies showed that in the ARC, 70% of GHRH neurons contain ERalpha mRNA, whereas less than 5% of SS neurons expressed the ERalpha gene. These results indicated that GHRH neurons are direct target cells for estrogens, and estrogens may act directly on GHRH neurons through ERalpha during adult life to modify GH secretory patterns.

A rare anatomic variant of the superior glenohumeral ligament.

The attachment of the superior glenohumeral ligament (SGHL) to the upper pole of the glenoid is variable and 3 types have been described. We report an anatomic variant of SGHL attachment to the upper pole of the glenoid that has not heretofore been reported in the literature. In this case, the SGHL overrode the biceps origin, continued to the superior labrum posteriorly, and had no attachment to the middle glenohumeral ligament or the anterior labrum. This variant was detected during routine arthroscopic examination undertaken before surgery on a rotator cuff tear.

Central effect of ghrelin, an endogenous growth hormone secretagogue, on hypothalamic peptide gene expression.

Ghrelin, an endogenous ligand for the growth hormone secretagogue receptor (GHS-R), was originally purified from the rat stomach. Like the synthetic GHSs, ghrelin specifically releases GH following intravenous administration. Also consistent with the central actions of GHSs, ghrelin-immunoreactive cells were shown to be located in the hypothalamic arcuate nucleus as well as the stomach. However, the central actions of ghrelin have not been elucidated. Here, we used radioactive in situ hybridization histochemistry to examine the effects of central administration of rat ghrelin on neuropeptide genes that are expressed in hypothalamic neurons that were previously shown to express GHS-R. We found that central administration of ghrelin increased both agouti-related protein (AGRP) mRNA levels (245.8 +/- 28.3% of the saline-treated controls; p < 0.01) in the hypothalamus and food intake (5.7 +/- 0.9 g ghrelin vs. 1.9 +/- 0.5 g saline; p < 0.05). On the other hand, 1 microg of rat ghrelin central administration did not alter the episodic GH release of freely moving adult male rats. Thus, ghrelin has an alternative role in stimulating food intake via an increase of AGRP rather than the release of GH from the pituitary.

A subpopulation of fibroblast growth factor-2-binding heparan sulfate is lost in human papillary thyroid carcinomas.

We hypothesized that a change in composition of proteoglycans can regulate the bioactivity of fibroblast growth factor (FGF)-2 in the thyroid. In order to test this hypothesis, we established a simple and sensitive method for detecting FGF-2-binding heparan sulfates and characterized them in papillary thyroid carcinomas and normal thyroids. The thyroid extracts were applied to a Q-Sepharose anion exchange column. After the column was washed with 10 mM of phosphate buffer, 1 microgram of human recombinant FGF-2 was added onto the column. The column was eluted with a gradient of NaCl (0.3-1.5 M). Each fraction was blotted onto nitrocellulose membrane. Immunoreactivity of heparan sulfate and FGF-2 was revealed by the incubation of membranes with the specific antibodies, and quantitatively estimated by measuring the density of the color product. In normal thyroids, immunoreactivity of heparan sulfate was detected as two peaks at 0.7 and 0.9 M of NaCl. Heparan sulfate-containing fractions also showed FGF-2 immunoreactivity, indicating the complex formation of FGF-2 and heparan sulfate. In papillary thyroid carcinomas, immunoreactivity of heparan sulfate showed various elution profiles on Q-Sepharose chromatography, including single peak at 0.7 M of NaCl and the one similar to that of the normal thyroids. However, FGF-2 immunoreactivity was detected only in the fractions eluting at 0.7 M of NaCl. This loss of a subpopulation of FGF-2-binding heparan sulfate in human papillary thyroid carcinomas may lead to the increase of free FGF-2 bioavailable in extracellular matrix.

Disturbed expression of the anti-apoptosis gene, survivin, and EPR-1 in hematological malignancies.

Survivin is a newly discovered inhibitor of the apoptosis protein, IAP, expressed during development and in human cancers. The effector cell protease receptor-1 (EPR-1) gene is oriented in the opposite direction on the same DNA double strand. Thus, the Survivin and EPR-1 (Survivin/EPR-1) genes exist in a head-to-head configuration. It is not clear whether mutual expression of the Survivin/EPR-1 genes occurs in both normal cells and cancer cells. Here, we investigated the mutual expression of the Survivin/EPR-1 genes in 12 normal peripheral blood (PB) specimens, seven normal bone marrow (BM) specimens, five lymph node (LN) specimens, and seven leukemic cell lines, and 27 patients with malignant lymphoma (ML), four with acute lymphocytic leukemia (ALL), three with acute myelocytic leukemia (AML), and four with chronic myelocytic leukemia in blastic crisis (CML-BC). Using Northern blot analysis, small amounts of EPR-1 mRNA were detected in normal PB, normal BM and LN specimens, but no Survivin mRNA was detected. However, Survivin mRNA was detected in two of the 12 normal PB, six of the seven normal BM and one of the five LN specimens using reverse transcription and polymerase chain reaction (RT-PCR). Expression of both the Survivin and EPR-1 genes was detected in six of the seven cell line samples by Northern blot, and in all of them by RT-PCR. Mutual expression of the Survivin and EPR-1 genes was detected in three of the four CML-BC samples, 15 of the 27 ML samples, two of the four ALL samples, and all three AML samples using the RT-PCR method. No EPR-1 expression with or without Survivin expression was clearly detected in eight of the nine diffuse large B-cell lymphoma (DLB) specimens, two of the six follicular center lymphoma specimens, one of the four specimens of nodular sclerosis of Hodgkin's lymphoma, two of the four ALL specimens or one of the four CML-BC specimens. The data presented here show that disrupted expression of the Survivin/EPR-1 genes occurred in many kinds of hematologically malignant cells. This may be of biological importance.

Wogonin inhibits inducible prostaglandin E(2) production in macrophages.

Effects of 5,7-dihydroxy-8-methoxyflavone (wogonin) on cyclooxygenase-2 (COX-2)-mediated prostaglandin E(2) production in macrophages were investigated. Stimulation with lipopolysaccharide (LPS; 1 microg/ml) greatly increased prostaglandin E(2) production in RAW 264.7 murine macrophages. The stimulated prostaglandin E(2) production was abolished in the presence of indomethacin (1 microM) or cycloheximide (2 microM), suggesting that the increased production of prostaglandin E(2) by LPS reflects the inducible synthesis of prostaglandin E(2) by COX-2. Wogonin (0.1-50 microM) concentration-dependently inhibited inducible prostaglandin E(2) production. Wogonin at concentrations as low as 0.5 microM directly attenuated enzymatic activity of COX-2. The protein expression of COX-2 was depressed by wogonin at concentrations of 10 microM and more. These results suggest that wogonin decreases inducible prostaglandin E(2) production in macrophages by inhibiting both COX-2 activity and COX-2 expression. The former action requires much lower doses of wogonin. These wogonin actions may explain, in part, its anti-inflammatory action.

Thickening of the roentgenometrical gastric fold in the elderly: relation to peptic ulcer incidence.

Radiographic upper gastrointestinal barium examination is commonly used to diagnose peptic ulcer. However, little attention has been paid to its thickening, except in Ménétrier disease and gastric carcinoma of Borrman type IV. The present study was undertaken to investigate the relation of age to roentgenometrical gastric fold width and history of peptic ulcer. The subjects were 724 men (35-64 years old) who participated in a periodic medical health examination and underwent radiographic upper gastrointestinal barium examination. The gastric fold width of the anterior wall in the body was evaluated by air-contrast examination and expressed in millimeters. In the group with a history of peptic ulcer, the roentgenometrical gastric fold was significantly thicker than that in the group without it. The fold width was significantly greater in the elderly group (55-64 years old) than in the young (35-44 years old) and middle-aged (45-54 years old) groups. The fold width tended to increase with age in persons with peptic ulcer history, but not in those without it. When the subjects were divided into three groups by gastric fold width, the incidence of peptic ulcer was significantly higher in the upper third group compared with the lower third group. This relationship between the gastric fold width and the incidence of peptic ulcer tends to become stronger with aging. In the heavy smoker group (> or = 10 of cigarettes per day), the gastric fold width was significantly thicker than that of non-smokers or those who smoked less. This relationship also tended to grow stronger with aging. The mean incidence of peptic ulcers was significantly higher among the heavy smokers. However, daily alcohol drinkers did not show any significant difference in gastric fold width from the other subjects. The gastric fold seems to be thicker in persons with peptic ulcer history, and the incidence of peptic ulcer is higher in persons with thicker gastric fold. These relationships tend to grow stronger with aging.a

Inhibitory effects of aclarubicin on nitric oxide production in aortic smooth muscle cells and macrophages.

The effects of aclaruhicin (ACR), an anthracycline antibiotic, on inducible nitric oxide (NO) synthesis was investigated in rat aortic smooth muscle cells (RASMCs) and RAW macrophages. ACR at concentrations as low as 0.1 microM significantly inhibited NO production induced by interleukin-1beta in RASMCs. About 5- to 10-fold higher concentrations of ACR were required for inhibition of interferon-gamma and lipopolipopolysaccharide-induced NO production in RAW cells. When ACR was subsequently administered to inducible NO synthase (iNOS) induction, the NO production was barely suppressed in RASMCs. Moreover, ACR (up to 10 microM) lacked direct inhibitory effects on iNOS activity in homogenates of these cells. ACR (0.1 microM) inhibited the expression of iNOS protein and mRNA in RASMCs without concomitant cytotoxic effects. ACR (>0.5 microM)-induced inhibition of NO production in RAW cells was associated with substantial cytotoxic effects as shown by measurement of lactate dehydrogenase release. These results suggest that ACR is a potent inhibitor of iNOS induction in vascular smooth muscle, but inhibits iNOS induction in macrophage only at high cytotoxic

Ectopic corticotroph adenoma in the cavernous sinus: case report.

OBJECTIVE AND IMPORTANCE: Adrenocorticotropin (ACTH)-secreting pituitary adenomas causing Cushing's disease are often difficult to identify because of their variable locations and their small size. This report presents histological evidence of an ectopic ACTH-secreting adenoma located entirely within the cavernous sinus. CLINICAL PRESENTATION: A 62-year-old woman presented with central obesity, hypertension, and osteoporosis. Endocrinological evaluation suggested the presence of an ACTH-secreting pituitary adenoma; however, imaging studies, including dynamic magnetic resonance imaging, did not reveal any visible lesions in the pituitary gland. Bilateral cavernous sinus sampling demonstrated a large central/peripheral ACTH gradient, with a right/left ACTH gradient. The patient was treated as having pituitary-dependent Cushing's disease, until she died suddenly as a result of acute respiratory failure. INTERVENTION: In a postmortem histological examination, an ACTH-secreting adenoma was found in the right cavernous sinus, which was completely surrounded by dura mater and had no direct connection with the pituitary gland. CONCLUSION: Although they are rare, such adenomas located in the cavernous sinus should be recognized as one of the reasons for inaccurate cavernous sinus sampling and the failure of transsphenoidal surgery for patients with ACTH-dependent Cushing's syndrome.

Intraosseous ganglion of the metatarsal bone.

We describe a rare case of intraosseous ganglion arising in the metatarsal bone. Radiographs revealed an osteolytic lesion with a fracture in the third metatarsal bone. A biopsied specimen exhibited hyaline fibrous tissue with marked myxoid change. Gadolinium-enhanced MRI, which revealed the network-like enhancement of the rim of the lesion and polycystic lesions adjacent to the joint, was helpful in making a diagnosis of intraosseous ganglion.

Effects of antitumor agents on inducible prostacyclin production in vascular smooth muscle.

The effects of various antitumor agents were examined on prostacyclin production induced by interleukin-1beta in rat aortic smooth muscle cells. Stimulation of the cells with interleukin-1beta (2.5 ng/ml) resulted in a great increase of prostacyclin production, which was abolished by indomethacin (1 microM) or cycloheximide (2 microM). Daunorubicin at 0.1-1 microM inhibited the inducible prostacyclin production in a concentration-dependent manner. However, other antitumor agents (cyclophosphamide at 1-100 microM, 5-fluorouracil at 1-100 microM and vincristine at 1-100 nM) tested did not significantly affect it. Protein expression of cyclooxygenase-2 induced by interleukin-1beta was inhibited by daunorubicin at 0.1-1 microM, but was not affected by other antitumor agents. These results suggest that daunorubicin inhibits induction of cyclooxygenase-2 and subsequent prostacyclin production in rat aortic smooth muscle cells.