The receptor for advanced glycation end products mediates the chemotaxis of rabbit smooth muscle cells.

Long-term incubation of proteins with glucose leads to advanced glycation end products (AGEs) with fluorescence and a brown color. We recently demonstrated immunologically the intracellular AGE accumulation in smooth muscle cell (SMC)-derived foam cells in advanced atherosclerotic lesions. To understand the mechanism of AGE accumulation in these foam cells, we have now characterized the interaction of AGE proteins with rabbit-cultured arterial SMCs. In experiments at 4 degrees C, 125I-labeled AGE-bovine serum albumin (AGE-BSA) showed a dose-dependent saturable binding to SMCs with an apparent dissociation constant (Kd) of 4.0 microg/ml. In experiments at 37 degrees C, AGE-BSA underwent receptor-mediated endocytosis and subsequent lysosomal degradation. The endocytic uptake of 125I-AGE-BSA was effectively inhibited by unlabeled AGE proteins such as AGE-BSA and AGE-hemoglobin, but not by acetylated LDL and oxidized LDL, well-known ligands for the macrophage scavenger receptor (MSR). Moreover, the binding of 125I-AGE-BSA to SMCs was affected neither by amphoterin, a ligand for one type of the AGE receptor, named RAGE, nor by 2-(2-furoyl)-4(5)-(2-furanyl)-1H-imidazole-hexanoic acid-BSA, a ligand for the other AGE receptors, p60 and p90. This indicates that the endocytic uptake of AGE proteins by SMCs is mediated by an AGE receptor distinct from MSR, RAGE, p60, and p90. To examine the functional role of this AGE receptor, the migratory effects of AGE-BSA on these SMCs were tested. Incubation with 1-50 microg/ml of AGE-BSA for 14 h resulted in significant dose-dependent cell migration. The AGE-BSA-induced SMC migration was chemotactic in nature and was significantly inhibited (approximately 80%) by an antibody against transforming growth factor-beta (TGF-beta), and the amount of TGF-beta secreted into the culture medium from SMC by AGE-BSA was sevenfold higher than that of control, indicating that TGF-beta is involved in the AGE-induced SMC chemotaxis. These data suggest that AGE may play a role in SMC migration in advanced atherosclerotic lesions.

Closure of a gastric tube-tracheal fistula by transposition of a pedicled sternocleidomastoid muscle flap.

We treated a 65-year-old man presenting with a gastric tube-tracheal fistula, who had undergone subtotal esophagectomy. The radiological and endoscopical findings demonstrated a 4-mm gastric tube-tracheal fistula located just above the sternum. Conservative treatment using a flexible fiberscope and/or gastrofiberscope, including factor XIII with fibrinogen (Beriplast P, Tisseel, and Borheal), alpha-cyanoacrylate (Aron-alpha-A), n-butyl-2-cyanoacrylate (histoacryl), and human antihemolytic factor XIII (Fibrogammin P) in addition to total parenteral nutrition with no oral intake did not result in closure of the fistula. The fistula was therefore transected and closed through an upper median sternotomy and right partial intercostal incision followed by transposition of the sternocleidomastoid muscle flap between the gastric tube and trachea. The postoperative course was uneventful.

Advanced glycation end products and their recognition by macrophage and macrophage-derived cells.

Modification of proteins by long-term incubation with glucose leads to the formation of advanced glycation end products (AGEs). AGE proteins are taken up by macrophages via the AGE receptor, which is similar to the macrophage scavenger receptor (MSR). In the present study, we compared the ligand specificity of the AGE receptor with that of MSR by three different experiments. The endocytic uptake of 125I-acetyl-LDL by RAW cells was effectively inhibited by unlabeled AGE-bovine serum albumin (BSA), whereas the inhibitory effect of acetyl-LDL on 125I-AGE-BSA was partial. Polyanions showing an effective inhibition for endocytic uptake of AGE-BSA were not always inhibitory for endocytic degradation of acetyl-LDL. These data, together with those obtained by three-dimensional fluorescence-activated cell sorter analysis, indicate that AGE proteins are recognized by more than two receptors, of which MSR is at least one. Finally, we examined whether MSR could mediate the endocytic uptake of AGE proteins by Chinese hamster ovary cells overexpressing bovine type II MSR (CHO-SRII cells). 125I-AGE-BSA underwent endocytic degradation by CHO-SRII cells, and this was effectively inhibited by unlabeled acetyl-LDL. These results clearly show that MSR mediates the endocytic uptake of AGE proteins, suggesting a new role of MSR in biological recognition of AGE in vivo.

Advanced glycation end products stimulate plasminogen activator activity via GM-CSF in RAW 264.7 cells.

The effects of advanced glycation end products (AGE) on the plasminogen activator (PA) activity were investigated with murine macrophage cell line RAW 264.7 cells. AGE-bovine serum albumin (BSA) showed a dose-dependent induction for the urokinase-type PA (uPA) activity. The uPA induction by AGE-BSA was effectively suppressed by the antibody against granulocyte-macrophage colony-stimulating factor (GM-CSF). The uPA activity of these cells was also induced by ligands for the macrophage scavenger receptor (MSR). These data provide evidence that AGE-BSA stimulates the uPA activity via GM-CSF through MSR in RAW cells. These findings, taken together with a recent demonstration of endocytic uptake of AGE-proteins by MSR in vitro and the presence of AGE-proteins in atherosclerotic lesions, strongly suggest that the uPA induction by AGE-proteins via MSR plays an important role in human atherogenesis.

Immunoreactive hepatocyte growth factor is a strong and independent predictor of recurrence and survival in human breast cancer.

Hepatocyte growth factor (HGF) is a stromally derived modulator of epithelial cell proliferation and motility. In the present study, we have measured immunoreactive (ir)-HGF concentration in tumor extracts of 258 primary human breast cancers using an enzyme-linked immunosorbent assay and have evaluated its association with disease-free and overall survival. The median value of ir-HGF concentration was 11.0 ng/100 mg protein (range, 1.4-566.7 ng/100 mg protein). Correlation analyses between ir-HGF concentration and clinicopathological factors showed that the ir-HGF level was correlated only with tumor size (P = 0.05). No significant associations were found between ir-HGF content and age, menopausal status, nodal status, histological type, histological grade, vessel involvement, estrogen receptor, progesterone receptor, type of surgery, or postoperative adjuvant therapy. Breast cancer patients with high ir-HGF concentration had a significantly shorter relapse-free (P = 0.001) and overall survival (P = 0.001) rate when compared to those with low ir-HGF concentration at the cutoff point of 21.7 ng/100 mg protein, which was determined in another group of 82 patients. In multivariate analysis, ir-HGF level was found to be the most important independent factor in predicting relapse-free and overall survival, of greater import than lymph node involvement. The putative role of HGF in breast cancer growth and metastasis is hereby strengthened.

Production of immunoreactive polymorphonuclear leucocyte elastase in human breast cancer cells: possible role of polymorphonuclear leucocyte elastase in the progression of human breast cancer.

Breast cancer cells are known to express various proteolytic enzymes, which make them invasive and favour their dissemination to distant sites. However, it is unclear whether breast cancer cells have the ability to produce polymorphonuclear leucocyte elastase (PMN-E). We measured immunoreactive (ir) PMN-E content in the conditioned medium of two breast cancer cell lines, MCF-7 and ZR-75-1, and two normal breast epithelial cell lines, HBL-100 and Hs 578Bst, using a highly specific and sensitive enzyme immunoassay. Furthermore, ir-PMN-E content was determined in tissue extracts from 62 human breast cancers. ir-PMN-E content in the culture medium of MCF-7 cells and ZR-75-1 cells increased as a function of time, regardless of the presence or absence of oestradiol. On the other hand, no detectable ir-PMN-E was secreted into the culture medium of HBL-100 and Hs 578Bst cells. ir-PMN-E was detectable in 59 of 62 tissue extracts prepared from human breast cancers, the concentration ranging from 0.12 to 19.17 micrograms per 100 mg of protein. When 62 breast cancer specimens were categorised into four groups in terms of clinical stage, ir-PMN-E content in breast cancer tissue was significantly higher in stage III (8.90 +/- 5.13 micrograms 100 mg-1 protein) and stage IV (12.19 +/- 5.44 micrograms 100 mg-1 protein) patients than in stage I (1.64 +/- 1.54 micrograms 100 mg-1 protein) and stage II (4.23 +/- 3.74 micrograms 100 mg-1 protein) patients. Breast cancer patients with high levels of ir-PMN-E showed significantly shorter disease-free survival and overall survival than those with low levels of ir-PMN-E at the cut-off point of 8.99 micrograms 100 mg-1 protein. In the multivariate analysis, ir-PMN-E content was found to be a significant prognostic factor for disease recurrence and death in human breast cancer.

Acute suppurative thyroiditis in an asymptomatic woman: an atypical presentation simulating thyroid carcinoma.

Patients with acute suppurative thyroiditis usually have pain or tenderness in the anterior part of the neck associated with erythema and dysphagia. A 26-year-old Japanese woman with none of these symptoms presented with a left anterior cervical mass. Since physical examination and laboratory studies showed a firm and irregular tumour located in the left lobe of the thyroid without any inflammatory signs, we made a diagnosis of thyroid carcinoma. After surgery, histological examination of the thyroid specimen revealed various changes of severe inflammation, and a barium swallow demonstrated a left pyriform sinus fistula. We describe here a unique case of acute suppurative thyroiditis in an asymptomatic woman.

Differential biological significance of tissue-type and urokinase-type plasminogen activator in human breast cancer.

Plasminogen activator (PA) is a serine protease existing in two forms known as tissue-type (t-PA) and urokinase-type (u-PA). To examine whether PA is related to the postoperative clinical course of human breast cancer, total PA activity, t-PA activity, u-PA activity, and immunoreactive t-PA were determined in tissue extracts from 144 breast cancer specimens. The patients were initially divided into four groups according to the postoperative clinical course: Group I (83 patients who are disease-free), Group II (20 patients whose first metastases were found only in bone), Group III (19 patients whose first metastases were found in both bone and lung), and Group IV (22 patients whose first metastases were found only in lung). Total PA activity was significantly lower in Groups, II, III and IV than in Group I. Both t-PA activity and t-PA antigen levels were also significantly lower in Groups II, III and IV than in Group I, while no significant difference was found in u-PA activity among these groups, indicating that low activity of total PA in Groups II, III and IV was due to a decrease in t-PA but not in u-PA. In the multivariate analyses, t-PA activity was found to be an independent prognostic factor for relapse-free survival. When four groups of patients were further analysed in terms of nodal status, both t-PA activity and antigen levels were markedly decreased in the node-negative Group II compared with the node-negative Groups III and IV or with the node-positive Groups II, III and IV. Of additional interest, u-PA activity was significantly higher in node-positive patients than in node-negative patients with any group. The clinico-pathologic analyses of the patients in this series showed that node involvement and lymphatic invasion were more frequently positive in Groups III and IV than in Groups I and II. When 144 breast cancers were categorised in terms of combinations of oestrogen receptor (ER) and progesterone receptor (PgR) status, breast cancers which were positive for both receptors were found to contain the highest t-PA activity and antigen. This study provides provocative evidence suggesting a possible differential significance of t-PA and u-PA expression in human breast cancer.

Increased expression of membrane-associated phospholipase A2 shows malignant potential of human breast cancer cells.

BACKGROUND: Recently, the authors reported that membrane-associated phospholipase A2 (M-PLA2) was one of the acute phase reactants and increased in serum of patients with various malignant tumors. METHODS: M-PLA2 concentrations in tissue specimens from 78 breast cancers, 16 benign breast tumors, and 10 normal breast tissues were determined by a specific radioimmunoassay recently developed. Immunohistochemical staining was performed on all specimens by the avidin-biotin-peroxidase method. RESULTS: Tissue levels of M-PLA2 concentration were significantly higher in breast cancer than in benign breast tumor or normal breast tissue (P < 0.01). Correlation analyses between the tissue concentration of M-PLA2 and clinicopathologic factors showed that tissue M-PLA2 levels were significantly higher in patients with skin or muscle invasion, vessel involvement, and distant metastasis than in those without. In addition, this enzyme concentration was significantly greater in scirrhous carcinoma than in papillotubular or solid-tubular carcinoma. No association was found between M-PLA2 concentration and steroid hormone receptor status. Immunohistochemically, M-PLA2 was preferentially stained in the invading zone of breast cancer tissues, especially in scirrhous carcinoma. Patients with breast cancer with low levels of M-PLA2 showed significantly longer overall survival and disease-free survival compared with those with high levels of this enzyme at the cutoff point of 50 ng/100 mg protein. The combination of estrogen receptor status with M-PLA2 concentration could be a powerful prognostic factor in predicting such survival rates. CONCLUSIONS: M-PLA2 is closely related to the malignant potential of breast cancers, and the M-PLA2 contents in breast cancer tissues could be a new valuable prognostic factor, other than the hormone receptor, in delineating the status of human breast cancer.

Breast cancer prognosis is poor when total plasminogen activator activity is low.

Plasminogen activator (PA) is a serine protease which exists in two forms: tissue-type (t-PA) and urokinase-type (u-PA). The total PA activity was measured in tumour extracts of 235 breast cancer patients who were followed for a median of 8.5 years after surgery. Patients were initially divided into three groups with low (< 60 units mg-1 protein), intermediate (60-300 unit mg-1 protein), or high (> 300 unit mg-1 protein) total PA activity in tumour extracts. The PA activity was not significantly associated with the recognised prognostic factors of age, menstrual status, tumour size, lymph node involvement, histologic type, grade of anaplasia, and/or vessel involvement. A significant association was found between total PA activity and the oestrogen receptor (ER) or progesterone receptor (PgR) status. Among receptor-positive tumours, a significantly greater proportion of patients had high PA activity in their tumour extracts. Breast cancer patients with low total PA activity had a significantly shorter disease-free and overall survival rate when compared to those with intermediate or high PA activity. In univariate and multivariate analyses, total PA activity (< 60 unit mg-1 vs > or = 60 unit mg-1 protein) was found to be a significant prognostic factor for disease-free and overall survival of about the same import as lymph node involvement. Furthermore, the combination of total PA activity and nodal status could be even more precise in predicting survival times and probabilities in individual patients. This retrospective study demonstrates the total PA activity is a valuable prognostic factor in determining prognosis in human breast cancer.

Interleukin 6 stimulates the production of immunoreactive endothelin 1 in human breast cancer cells.

To investigate the potential regulation of endothelin 1 production in human breast cancer cells, we measured the release of immunoreactive endothelin 1 (ir-ET-1) from the MCF-7 and ZR-75-1 breast cancer cell lines in response to various agents including estrogen and tamoxifen as well as several cytokines. ir-ET-1 was detected in conditioned medium of MCF-7 cells and ZR-75-1 cells by specific radioimmunoassay. Among the agents tested, estrogen, tamoxifen, tumor necrosis factor, gamma-interferon, interleukin (IL) 1, and transforming growth factor beta had no effect on ir-ET-1 secretion by these breast cancer cells. However, IL-6 (20 ng/ml) treatment of MCF-7 cells and ZR-75-1 cells caused maximal increases in the amount of ir-ET-1 secreted into the culture medium to 206 and 314% of basal values after 6 h, respectively. This effect of IL-6 on ir-ET-1 secretion was inhibited by actinomycin D and cycloheximide, indicating that IL-6 stimulates de novo synthesis of ir-ET-1 at a transcriptional level. Reverse-phase high performance liquid chromatography coupled with radioimmunoassay in the conditioned medium from IL-6-treated cells revealed one major ir-ET-1 component corresponding to human standard ET-1. The present study demonstrates the potential for IL-6 to stimulate ir-ET-1 production in human breast cancer cells, which may participate in the process of acute phase reactant-like expression of this peptide and/or in the process of IL-6 enhanced breast cancer cell motility, the latter being recently clarified.