Kazuhiko Tanzawa

Matrix metalloproteinase-9 triggers the angiogenic switch during carcinogenesis.

During carcinogenesis of pancreatic islets in transgenic mice, an angiogenic switch activates the quiescent vasculature. Paradoxically, vascular endothelial growth factor (VEGF) and its receptors are expressed constitutively. Nevertheless, a synthetic inhibitor (SU5416) of VEGF signalling impairs angiogenic switching and tumour growth. Two metalloproteinases, MMP-2/gelatinase-A and MMP-9/gelatinase-B, are upregulated in angiogenic lesions. MMP-9 can render normal islets angiogenic, releasing VEGF. MMP inhibitors reduce angiogenic switching, and tumour number and growth, as does genetic ablation of MMP-9. Absence of MMP-2 does not impair induction of angiogenesis, but retards tumour growth, whereas lack of urokinase has no effect. Our results show that MMP-9 is a component of the angiogenic switch.

Endothelin-Converting Enzyme Expression in the Rat Vascular Injury Model and Human Coronary Atherosclerosis

BACKGROUND Endothelin 1 has been implicated in various human diseases, including atherosclerosis. In this study, we examined the expression and localization of endothelin-converting enzyme-1 (ECE-1), the final key enzyme of endothelin 1 processing, in rat carotid arteries after balloon injury and in human coronary atherosclerotic lesions. METHODS AND RESULTS ECE-1 mRNA levels and ECE activity in rat balloon-injured arteries started to increase between 2 and 5 days after injury. The endothelin 1 content of tissue in injured arteries was concomitantly increased. Immunohistochemical staining located ECE-1 signals in endothelial cells in uninjured arteries, whereas ECE-1 immunoreactivity was detected in neointimal smooth muscle cells in injured arteries 5 to 14 days after balloon denudation. The size of the neointima was effectively reduced by phosphoramidon, an inhibitor of neutral metalloproteases, including ECE-1. In human coronary atherosclerotic lesions, intense ECE-1 immunoreactivity was detected in subsets of cells embedded in atheromatous plaque that correspond to smooth muscle cells and macrophages, as identified by staining for smooth muscle alpha-actin and CD68 surface marker, respectively. CONCLUSIONS The present study ascertained that ECE-1 is expressed in neointimal smooth muscle cells in rat balloon-injured arteries and in both smooth muscle cells and macrophages in human coronary atherosclerotic lesions. Blockade of ECE-1 was effective in reducing neointimal formation after balloon injury. Thus, ECE-1 may contribute to the process of injury-induced neointimal formation and atherosclerosis through the autocrine/paracrine effects of endothelin 1.

Expression of endothelin-converting enzyme, endothelin-1 and endothelin receptors at the site of percutaneous coronary intervention in humans.

Objective The repair process at the site of injury after percutaneous coronary intervention (PCI) is dominated by neointimal formation composed mainly of smooth muscle cells (SMC). Endothelin-1 (ET-1) is a powerful vasoconstrictor and SMC mitogen. Endothelin-converting enzyme (ECE) is the final key enzyme of endothelin processing. The effects of ET-1 are mediated by binding to endothelin type A (ETA) and endothelin type B (ETB) receptors. The ligand/receptor/ligand-producing system (ET system) could be involved in the pathogenesis of neointimal formation in humans. Methods Fifteen post-PCI sites obtained at autopsy and eight atherectomy specimens obtained from restenotic sites were investigated using immunohistochemical single and double staining techniques. Frozen sections were stained with antibodies against ECE, ET-1, ETA and ETB receptors, SMC, macrophages and endothelial cells. Results At the early stage, less than 3 months after PCI, neointimal SMC were positive for ECE, ET-1, ETA and ETB receptors. The expression of ECE, ET-1, ETA and ETB receptors in these neointimal SMC decreased markedly from 6 months onwards. The ECE, ET-1, ETA and ETB receptor-positive cell areas were significantly (P < 0.005) greater in the first 3 months after PCI compared with 6 months or more after PCI. Atherectomy specimens also showed similar positivity. Conclusions These observations strongly suggest that the expression of ECE, ET-1, ETA and ETB receptors is enhanced in neointimal SMC at early stages after PCI injury in human coronary arteries. The increased expression of the ET system may contribute to SMC proliferation/migration and vasoconstriction in human post-PCI coronary lesions.

Synthesis and determination of the absolute configuration of matlystatin B

Abstract The title compound ( 1 ) was first synthesized and its absolute configuration was determined as shown in figure I.

Preventive effect of matrix metalloproteinase inhibitor, R-94138, in combination with mitomycin C or cisplatin on peritoneal dissemination of human gastric cancer cell line TMK-1 in nude mice.

R‐94138, a matrix metalloproteinase inhibitor, was examined for the ability to prevent peritoneal dissemination of a human gastric cancer xenograft, TMK‐1. When the supernatant of a co‐culture of TMK‐1 cells and human normal fibroblast cells was subjected to gelatin zymography, it was clear that the protein expression of MMP‐2 had been inhibited by R‐94138. When TMK‐1 was injected intraperitoneally (i.p.) into nude mice at 5×105 cells/body, the resulting peritoneal dissemination mimicked clinical carcinomatous peritonitis. When the maximum tolerated dose of mitomycin C (MMC) or cisplatin (DDP) was given 12 h after the tumor inoculation, peritoneal dissemination was completely inhibited, while the effect of R‐94138 was limited when it was given i.p. at a dose of 20 mg/kg in a schedule of q.d. ×5 starting 12 h after tumor injection. MMC and DDP also suppressed peritoneal dissemination when they were administered 1 week after the tumor inoculation at a single dose of 2 and 3 mg/kg i.p., respectively. R‐94138 inhibited peritoneal dissemination when it was administered i.p. at a dose of 30 mg/kg in a schedule of q.d. ×5 starting from 1 week after tumor injection. The combination of MMC and R‐94138 increased the preventive effect on peritoneal dissemination. R‐94138 seems to be a promising candidate to prevent peritoneal dissemination of gastric cancer.

Expression of the hepatic endothelin system in human cirrhotic livers

It is considered that endothelin‐1 participates in the development of liver cirrhosis and it has been recognized that every component of the endothelin system is upregulated in cirrhotic livers. However, the expression pattern of this system, including interaction between its components, is not fully understood in human livers. In this study, the expression pattern of the endothelin system was examined. Immunohistochemical analysis for endothelin‐1, endothelin receptors and endothelin‐converting enzyme was performed in 16 cirrhotic and 17 normal human liver tissues. Peptides, proteins, and RNAs extracted from the livers were also investigated using quantitative assays for the components of the hepatic endothelin system. Hepatic endothelin‐1 levels were significantly higher in cirrhotic livers (0.084 ± 0.052 pg/mg wet liver) than in normal livers (0.041 ± 0.032 pg/mg; p < 0.01), and were closely related to the severity of liver fibrosis and portal hypertension. Immunoreactivity for endothelin‐1, endothelin receptors, and endothelin‐converting enzyme was detected mainly in fibrous areas and in the hepatic vasculature, and was enhanced in cirrhosis. Although there was a negative correlation between the expression of receptor mRNA and the hepatic endothelin‐1 level, the amounts of the mRNAs were greater in cirrhotic livers than in normal livers. However, expression of endothelin‐converting enzyme in cirrhotic livers was increased at the protein level but was relatively reduced at the mRNA level. These findings suggest that the hepatic endothelin system is activated in human cirrhotic livers in association with worsening of the disease, but that the regulation of the components of this system in this disorder is complex. Copyright

Regulation of endothelin-converting enzyme 1 in nephrotic syndrome in rats.

Background: Nephrotic syndrome is characterized by severe proteinuria and sodium and water retention. Although endothelin (ET) 1 can cause natriuresis or antinatriuresis, the role played by ET-1 in proteinuria and in sodium retention due to nephrotic syndrome remains unclear. Methods: We investigated the role played by the ET-1 system in sodium and water retention and in proteinuria in puromycin aminonucleoside induced nephrotic syndrome in rats using microdissected nephron segments, competitive polymerase chain reaction, and Western blot. Results: The expression of prepro ET-1, ET-converting enzyme 1 (ECE-1), and ET A receptor mRNAs, but not ET B receptor mRNA, in the glomeruli was increased in rats with nephrotic syndrome. The cGMP generation in the glomeruli induced by atrial natriuretic peptide and ET-1 was decreased, whereas the ET-3-induced cGMP generation was increased in rats with nephrotic syndrome. ECE-1 mRNA expression was increased not only in the glomeruli, but also in the thick ascending limbs and collecting ducts. The protein expression of ECE-1 was increased in the membrane fraction of the cortex and in the outer and the inner medulla of nephrotic rats. Blockade of ET A and B receptors by bosentan did not inhibit the occurrence of nephrotic syndrome. However, the administration of bosentan increased the urinary sodium excretion. Conclusion: These data suggest that an activated ET-1-ET A receptor pathway in glomeruli and/or an increased ECE-1 mRNA expression in distal segments may participate in sodium and water retention, but not in the occurrence of nephrotic syndrome.

Enhanced expressions of endothelin-converting enzyme and endothelin receptors in human colonic tissues of Crohn's disease.

Endothelin-1, a powerful vasoconstrictor, forms the endothelin system together with endothelin-converting enzyme and endothelin type A and type B receptors. These endothelin system components are considered to participate in inflammatory and wound healing responses. Previous reports have suggested a role for the endothelin-1 in the pathology of Crohn’s disease. In the present study, we immunohistochemically investigated the expressions of the endothelin system components in affected human intestinal tissues of Crohn’s disease. Eighteen surgical specimens of colonic tissue obtained from patients with Crohn’s disease and 12 normal colonic tissues as controls were examined. Frozen tissue sections cut from the samples were subjected to the immunohistochemical single and double staining. The endothelin system components were expressed mainly in the muscular layers and blood vessels. In diseased colonic tissues, inflammatory infiltration and fibrotic tissue reactions with marked smooth muscle cell proliferation were frequently seen, and were closely associated with increased expressions of the endothelin system components. These results strongly suggest that endothelin-converting enzyme and endothelin type A and type B receptors collectively play a role in the inflammatory and fibrogenic processes of Crohn’s disease. Especially, submucosal smooth muscle proliferation, a histological hallmark of strictures, may be attributable to the upregulated endothelin system.

Conformational analysis of a new cyclic depsipeptide calcium blocker, leualacin, by NMR spectroscopy

Abstract The three-dimensional structure of leualacin, a novel calcium channel blocker from Hapsidospora irregularis was determined in CDCI3. Based upon the dihedral angle constraints from the analysis of 3JH,H and 3JC,H, and the distance constraints deduced from 1H-{1H} NOEs and 13C- {1H} NOES, conformers completely satisfying the NMR data were obtained by the conformational grid search program SYBYL followed by the energy minimization program XPLOR. Leualacins structure is characterized by γ- and β-turn like moieties analogous to cyclic peptides, which are fixed by trans-annular hydrogen bonds formed between L-leucine and β-alanine. L-N-methylphenylalanine and S-leucic acid were found to be connected by cis peptide bond. The RMSDs (Root Mean Square Differences) calculated for the backbone atoms among four structures are 0.6 A. The ring current effect caused by the phenylalanine moiety was reproduced by a calculation based on the resulting structures according to Bovey and Johnsons formula.