Tosikazu Nakamura

Effect of Local Application of Growth Factors on Gastric Ulcer Healing and Mucosal Expression of Cyclooxygenase-1 and -2

Background/Aims: Ulcer healing involves expression of various growth factors such as epidermal growth factor (EGF), hepatocyte growth factor (HGF) and basic fibroblast growth factor (bFGF) at the ulcer margin, but the influence of EGF, HGF and bFGF applied locally with or without neutralizing anti-EGF, HGF and bFGF antibodies or cyclooxygenase (COX)-1 and COX-2 inhibitors on ulcer healing and the expression of COX-1 and COX-2 during ulcer healing have only been studied a little. Methods: Rats with gastric ulcers induced by serosal application of acetic acid (ulcer area 28 mm2) received a submucosal injection of either (1) vehicle (saline), (2) EGF, (3) HGF, and (4) bFGF with or without antibodies against EGF, HGF and bFGF or indomethacin (2 mg/kg/day i.p.), a nonspecific inhibitor of COX, or NS-398 (10 mg/kg/day i.g.) and Vioxx (5 mg/kg/day i.g.), both highly specific COX-2 inhibitors. A separate group of animals with chronic gastric fistulas was also used to assess gastric secretion during ulcer healing with and without growth factors. Each growth factor and specific antibody against EGF, HGF and bFGF (100 ng/100 µl each) were injected just around the ulcer immediately after ulcer induction and this local injection was repeated on day 2 following anesthesia and laparotomy. On days 13 and 21, the ulcer area was determined by planimetry, gastric blood flow (GBF) at the ulcer margin was examined by the H2-gas clearance technique, and mucosal generation of PGE2 and the gene expression of COX-1 and COX-2 in the non-ulcerated and ulcerated gastric mucosa were assessed. Gastric ulcers healed progressively within 21 days after induction and this effect was accompanied by a significant increase in GBF at the ulcer margin and in the expression of COX-2 in the ulcer area. Local treatment with EGF, HGF and bFGF produced a significant decrease in gastric acid secretion and significantly accelerated the rate of ulcer healing and raised GBF at the ulcer margin causing further significant upregulation of COX-2 but not COX-1 expression in the ulcerated mucosa. The acceleration of ulcer healing and hyperemia at the ulcer margin exhibited by locally applied EGF, HGF and bFGF were similar to those obtained with systemic administration of these growth factors. HGF applied submucosally, upregulated COX-2 expression and this was significantly attenuated by concurrent treatment with antibody against this peptide. Anti-EGF and anti-bFGF antibodies completely abolished the acceleration of the ulcer healing and hyperemia at the ulcer margin induced by these growth factors. Indomethacin and both COX-2 inhibitors significantly prolonged ulcer healing, while suppressing the generation of PGE2 in non-ulcerated and ulcerated gastric mucosa and GBF at the ulcer margin. The acceleration of ulcer healing by EGF, HGF and bFGF and the accompanying rise in GBF at the ulcer margin were significantly attenuated by the concurrent treatment with indomethacin or NS-398 and Vioxx. Conclusions: (1) Growth factors accelerate ulcer healing due to enhancement in the microcirculation around the ulcer and these effects are specific because they can be abolished by neutralization with antibodies; (2) COX-2-derived prostaglandins and suppression of gastric secretion may play an important role in the acceleration of ulcer healing by various growth factors, and (3) the local effects of EGF, HGF and bFGF on ulcer healing can be reproduced by their systemic application indicating the high efficacy of growth factors to accelerate this healing.

Hepatocyte growth factor in lung morphogenesis and tumor invasion: role as a mediator in epithelium-mesenchyme and tumor-stroma interactions

Abstract Hepatocyte growth factor (HGF), a ligand for Met tyrosine kinase, is a mesenchyme- or stroma-derived multipotent factor that regulates the growth, motility, and morphogenesis of various types of cells. During lung development, Met/HGF receptor mRNA was localized in lung epithelial cells, whereas HGF mRNA was localized in lung mesenchymal cells in rat embryos. Antisense HGF oligonucleotides specifically inhibited epithelial branching morphogenesis in cultured lung rudiment isolated from day-13 rat embryos, whereas recombinant HGF stimulated branching morphogenesis. Thus, HGF seems to be at least one of the mesenchyme-derived factors that support branching morphogenesis during lung development. Together with the finding that HGF plays important roles in organogenesis and morphogenesis of organs such as the liver and kidney, HGF seems to be a mediator in epithelium-mesenchyme interactions during organogenesis. Extending the conceptual framework of epithelium-mesenchyme (or epithelium-stroma) interactions, we next examined the possible involvement of HGF in tumor-stroma interactions, because the growth and motility of carcinoma cells are regulated through their interactions with host stromal cells. HGF induced in vitro migration and invasion of GB-d1 gallbladder carcinoma cells into basement membrane components and collagen-gel matrix; however, several other growth factors did not induce marked migration and invasion of the carcinoma cells. GB-d1 cells do not produce HGF, but they produce an inducing factor for HGF production in fibroblasts; the inducing molecule was identified as interleukin 1β. Cocultivation of GB-d1 cells with stromal fibroblasts embedded in a collagen-gel matrix induced invasion of GB-d1 cells into the collagen gels, but invasion was inhibited by a specific antibody against HGF. This indicates that in vitro invasion of GB-d1 cells depends on stromal fibroblasts and that the fibroblast-derived invasion factor is HGF. Since HGF stimulated in vitro migration and invasion of various carcinoma cells and several carcinoma cells produced inducing factors for HGF production in stromal fibroblasts, the looped interaction of carcinoma cells and stromal fibroblasts mediated by HGF and HGF inducers may be a mechanism responsible for acquisition of the malignant phenotype through tumor-stroma interactions.

Effects of pravastatin on plasma and urinary mevalonate concentrations in subjects with familial hypercholesterolaemia: a comparison of morning and evening administration

In order to determine whether there is a difference in the effect of the hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor pravastatin on cholesterol synthesis between the morning and the evening, we studied the 24-h profile of mevalonate in plasma and urine in 11 subjects with heterozygous familial hypercholesterolaemia. In study 1, eight subjects with familial hypercholesterolaemia took pravastatin (20 mg) once in the morning, and another 20-mg dose in the evening after a 1-week wash-out period. In study 2, five subjects with familial hypercholesterolaemia took pravastatin (20 mg per day) in the morning on 3 consecutive days and on 3 days in the evening after a 1 day wash-out. Plasma mevalonate concentrations were reduced at 9 h and 5 h after pravastatin administration in the morning and the evening, respectively. Urinary mevalonate excretion was significantly reduced at 4–8 h after pravastatin administration in the morning (51 vs 19 nmol · h−1) and at 4–16 h after pravastatin administration in the evening (56 vs 27 nmol · h−1). Daily urinary mevalonate excretion was equally and significantly reduced by pravastatin in the morning or evening. In conclusion, we found that morning and evening administration of pravastatin caused equal reductions in plasma and urinary mevalonate concentrations.

Induction of key glycolytic enzymes in mouse fetal liver cultured in circumfusion system by insulin.

Abstract Effect of insulin on key enzymes (Hexokinase, Glucokinase, Pyruvate kinase, and Glucose-6-phosphate dehydrogenase) in carbohydrate metabolism has been studied in mouse fetal liver cultured in a circumfusion system. Specific activities of GK, PK, and G-6-P DH were induced 2-or 3-fold increases in fetal liver after 12 days cultivation by a single dose of insulin. While, HK was hardly influenced by insulin. These findings suggest that mouse fetal liver cultured in a circumfusion system for 2 weeks maintains the same response to insulin as in vivo adult mouse liver.

Inhibition of HGF/SF-induced cell-matrix adhesion, invasion, migration & paxillin phosphorylation, in prostate cancer by the HGF/SF antagonist, NK4

Background and objectives Ependymomas are the third most common CNS tumours in childhood, and account for 9–12% of CNS neoplasms in all age groups. However, the prognosis for cases not completely excised is poor and the underlying biology of their development and progression is poorly understood. The few studies published to date have suggested that specific chromosomal abnormalities may be associated with the development of a significant proportion of these tumours. We set out to screen a large series of intracranial and spinal ependymomas for genetic imbalances, and to correlate these with histology and clinical behaviour. Methods Comparative genomic hybridisation (CGH) was used to detect chromosome imbalances on 86 ependymomas from 77 patients, of which 22 were children under 16, treated at one of three UK centres (Newcastle, Nottingham, Southampton). Cases were first analysed without reference to histology or clinical features, and were then divided up according to anatomical location, histology and age at presentation. Results Chromosomal imbalances were detected in tumours from 63/77 patients (82%). The majority involved entire chromosomes or chromosome arms, many showing patterns of gains suggestive of intermediate ploidy. Of previously reported abnormalities in ependymoma, the most frequent findings were gain of 1 q, seen in 13 cases (17%), and loss of 22 in 20 (26%). Whereas 1 q gain was seen mainly in posterior fossa tumours and was restricted to those with classic and anaplastic histology, loss of 22 was rarely observed in tumours from this location and their histology was predominantly classic or myxopapillary. In contrast to previous studies, loss of 6q was found in only 6 cases (8%) and in only one child. Out of 7 tumours biopsied at presentation and relapse, 4 revealed imbalances and 3 of these demonstrated progression of abnormalities at relapse. Conclusions Chromosomal imbalance is common in ependymoma and patterns of abnormality are emerging that are associated with histology or location. Further studies are needed to establish the prognostic significance of these abnormalities.Aims Endothelin, the most potent vasoconstrictor known has been implicated development and spread of malignancy. In this study, we assessed the produc endothelin-1 (ET-1) and its precursor big endothelin (big ET-1) by human cancer cel Methods Ten human cancer cell lines were cultured (lung n = 4, colorectal n = 3, gastro-oesophageal n = 2, pancreatic n = 1). The culture media were replaced wi fresh media after the cells attained confluence. After 48 hours, the conditioned were batch analysed for ET-1 and big ET-1 by using a sandwich enzyme l immunoassay (ELISA) (Biomedica, Austria). To elucidate the action of endot converting enzyme (ECE), big ET-1 was added to one of the oesophageal canc lines after they attained confluence. Similarly, the media were analysed fo presence of ET-1 and big ET-1 Results All the ten cancer cell lines produced ET-1 and nine of ten cancer cell produced big ET-1. ET-1 and big ET-1 were not produced in equimolar amounts ratio of ET-1 to big ET-1 was 0.56–11.88 (range). All three colorectal cancer cell and four of the lung cancer cell lines produced both ET-1 and Big Et-1. Interest the oesophageal cancer cell line that produced high concentrations of ET-1 d produce any measurable big ET-1. Addition of Big ET-1 into this cell line mediu assess the action of ECE, measuring ET-1 and big ET-1 after 48 hours resu complete cleavage of big ET-1 and there was no measurable big ET-1 in the me