The anti-allergic compound tranilast attenuates inflammation and inhibits bone destruction in collagen-induced arthritis in mice.

BACKGROUND AND PURPOSE: Recent findings suggest the importance of mast cells in the pathogenesis of rheumatoid arthritis and their potential as a therapeutic target. Tranilast is an anti-allergic compound with a potent membrane-stabilizing effect on mast cells and a wide range of anti-inflammatory effects, thus may be advantageous in the treatment of arthritis. Here, we have evaluated the effects of tranilast on the progression of collagen-induced arthritis in mice. EXPERIMENTAL APPROACH: Tranilast (400 mg.kg(-1).day(-1)) was orally administered for 8 weeks to mice with established collagen-induced arthritis. Arthritis was assessed by clinical signs and X-ray scores. In paw tissue, the numbers of mast cells and osteoclasts were measured by histological analysis, and several inflammatory factors were assessed by RT-PCR and Western blot analysis.* KEY RESULTS: TNF-alpha-positive mast cells were present extensively throughout the inflamed synovium of vehicle-treated arthritic mice, with some mast cells in close proximity to osteoclasts in areas of marked bone and cartilage destruction. Tranilast significantly reduced clinical and X-ray scores of arthritis and decreased numbers of TNF-alpha-positive mast cells and mRNA levels of TNF-alpha, chymase (mouse mast cell protease 4), tryptase (mouse mast cell protease 6), stem cell factor, interleukin-6, cathepsin-K, receptor activator of nuclear factor-kappaB, and of receptor activator of nuclear factor-kappaB-ligand, but increased interleukin-10 mRNA level in paws of arthritic mice. Osteoclast numbers were decreased by treatment with tranilast. CONCLUSIONS AND IMPLICATIONS: Tranilast possesses significant anti-rheumatic efficacy and, probably, this therapeutic effect is partly mediated by inhibition of mast cell activation and osteoclastogenesis.

A strategy in wastewater treatment process for significant reduction of excess sludge production.

A novel wastewater treatment process (S-TE PROCESS) with significantly reduced production of excess sludge has been developed. The process consists of two different stages, one for a biological wastewater treatment and the other for a thermophilic aerobic digestion of the resulting sludge. A portion of return sludge from the wastewater treatment step is injected into a thermophilic aerobic sludge digester (TASD), in which the injected sludge is solubilized by the action of thermophilic aerobic bacteria. The solubilized sludge is returned to the aeration tank in the wastewater treatment step for its further degradation. Pilot-scale facilities of the S-TE process and the conventional activated sludge process as a control, both treating the same industrial wastewater, were comparatively operated for totally 270 days. As a result, 93% reduction in overall excess sludge production was achieved in the S-TE operation. The SS solubilization rate in TASD was stable at around 30%. Only a slight increase in the effluent SS and TOC concentrations was observed compared with those of the control facility. Otherwise the removal efficiency of TOC was approximately 95% for both plants. A full-scale plant treating domestic sewage was operated for three years, showing 75% reduction of overall excess sludge production. It was concluded that the new process was feasible.

Enhancement of metabolizing herbicides in young tubers of transgenic potato plants with the rat CYP1A1 gene.

A rat P450 monooxygenase gene ( CYP1A1) was introduced into potato plants to enhance the metabolism of the environmental contaminants in subterranean organs. The CYP1A1 gene was kept under the control of the potato patatin promoter to enhance tuber-specific expression. A total of 106 transgenic plants (PAT1A1 plants) were obtained following selection by a resistance test to kanamycin and PCR analysis. PAT1A1 plants treated with 10% exogenous sucrose showed a higher activity of monooxgenase in the leaves than the non-transgenic plants. This indicated that the activity enhanced by 10% sucrose was due to the patatin promoter containing the sucrose-inducted elements. One representative transgenic plant, Ag2197, was selected on the basis of monooxgenase activity in the leaves and Western blot analysis. Ag2197 was found to accumulate a large amount of CYP1A1 mRNA and protein in the developing tuber but not in the mature tuber. The residual herbicides, atrazine and chlortoluron, were analyzed in the micro-tubers of Ag2197 and non-transgenic plants. The amount of residual herbicides in Ag2197 was much lower than that in the non-transgenic plant, indicating that the transgenic plant metabolized the herbicides to a detoxified form. The transgenic plants produced in this study might be useful for the phytoremediation of chemical pollution in the soil.

Inducible cross-tolerance to herbicides in transgenic potato plants with the rat CYP1A1 gene.

A gene of the enzyme involved in xenobiotic metabolism in mammalian liver was introduced into potato to confer inducible herbicide tolerance. A rat cytochrome P450 monooxygenase, CYP1A1 cDNA, was kept under the control of the tobacco PR1a promoter in order to apply the system of chemical inducible expression using the plant activator Benzothiadiazole (BTH). Transgenic plants were obtained based on the kanamycin resistance test and PCR analysis. Northern-blot analysis revealed the accumulation of mRNA corresponding to rat CYP1A1 in the transgenic plants treated with BTH (3.0 micro mol/pot), whereas no accumulation of the corresponding mRNA occurred without BTH treatment. These transgenic plants also produced a protein corresponding to CYP1A1 in the leaves by BTH treatment. The transgenic plants with BTH application showed a much-higher tolerance to the phenylurea herbicides chlortoluron and methabenzthiazuron than non-transgenic plants. These findings indicated that the ability of metabolizing the two herbicides to less-toxic derivatives was displayed in the transgenic plants after BTH treatment. Transgenic plants harboring the CYP1A1 cDNA fused with the yeast P450 reductase (YR) gene under the control of PR1a were also produced. Although the plants showed a lower expression level of the fused gene than transgenic plants with CYP1A1 cDNA alone, they were tolerant to herbicides. These facts suggested that the CYP1A1 enzyme fused with YR showed a higher specific activity than CYP1A1 alone. This study demonstrated that the mammalian cDNA for the de-toxification enzyme of herbicides under the control of the PR1a promoter conferred chemical-inducible herbicide tolerance on potato.

Mast cell chymase inhibits smooth muscle cell growth and collagen expression in vitro: transforming growth factor-beta1-dependent and -independent effects.

In the vulnerable areas of fibrous caps of advanced atherosclerotic lesions, chymase-containing mast cells are present. In such areas, the numbers of smooth muscle cells (SMCs) and the content of collagen are reduced. In this in vitro study, we found that the addition of chymase, isolated and purified from rat serosal mast cells, to cultured rat aortic SMCs of the synthetic phenotype (s-SMCs) inhibited their proliferation by blocking the G(0)/G(1)-->S transition in the cell cycle. Rat chymase and recombinant human chymase inhibited the expression of collagen type I and type III mRNA in s-SMCs and in human coronary arterial SMCs. The growth-inhibitory effect of chymase was partially reversed by addition to the culture medium of an antibody capable of neutralizing the activity of transforming growth factor-beta1 (TGF-beta1). Immunocytochemistry showed that the s-SMCs expressed and synthesized extracellular matrix-associated TGF-beta1. On exposure to mast cell chymase, the extracellular matrix-associated latent TGF-beta1 was released and activated, as demonstrated by immunoblotting and by an ELISA with TGF-beta1 type II receptor for capture. When added to s-SMCs, such chymase-released TGF-beta1 was capable of inhibiting their growth. In contrast, the inhibitory effect of chymase on collagen synthesis by s-SMCs did not depend on TGF-beta1. Taken together, the findings support the hypothesis that chymase released from activated mast cells in atherosclerotic plaques contributes to cap remodeling.

Activation of paracrine TGF-beta1 signaling upon stimulation and degranulation of rat serosal mast cells: a novel function for chymase.

As a source of transforming growth factor beta1 (TGF-beta1), mast cells have been implicated as potential effector cells in many pathological processes. However, the mechanisms by which mast cells express, secrete, and activate TGF-beta1 have remained vague. We show here by means of RT-PCR, immunoblotting, and immunocytochemistry that isolated rat peritoneal mast cells synthesize and store large latent TGF-beta1 in their chymase 1-containing secretory granules. Mast cell stimulation and degranulation results in rapid secretion of the latent TGF-beta1, which is converted by chymase 1 into an active form recognized by the type II TGF-beta serine/threonine kinase receptor (TbetaRII). Thus, mast cells secrete active TGF-beta1 by a unique secretory mechanism in which latent TGF-beta1 and the activating enzyme chymase 1 are coreleased. The activation of latent TGF-beta1 specifically by chymase was verified using recombinant human latent TGF-beta1 and recombinant human chymase. In isolated TbetaRI- and TbetaRII-expressing peritoneal macrophages, the activated TGF-beta1 induces the expression of the plasminogen activator inhibitor 1 (PAI-1), whereas in the mast cells, the levels of TbetaRI, TbetaRII, and PAI-1 expression were below detection. Selective stimulation of mast cells in vivo in the rat peritoneal cavity leads to rapid overexpression of TGF-beta1 in peritoneal mast cells and of TbetaRs in peritoneal macrophages. These data strongly suggest that mast cells can act as potent paracrine effector cells both by secreting active TGF-beta1 and by enhancing its response in target cells.

Isoform-selective upregulation of mast cell chymase in the development of skin fibrosis in scleroderma model mice.

The involvement of connective-tissue-type mast cells and chymase, a protease unique to their secretory granules, has been implicated in fibrotic diseases. To elucidate the role of chymase in fibroproliferative inflammation, in this study we examined the enzymatic activity and mRNA expression of chymase in the sclerotic skin of tight-skin mice; syngeneic Pallid mice served as the control. Dorsal skin specimens from mice aged 5, 10, and 20 wk were evaluated by morphometric and biochemical analyses. At ages 10 and 20 wk, the hydroxyproline concentration in tight-skin dermis was higher than that in Pallid. At any age, the subcutaneous fibrous layer was thicker in tight-skin than in Pallid. In accordance with these fibrous changes, both connective-tissue-type mast cell counts and chymase activity were higher in tight-skin skin than in Pallid skin up to 20 wk of age. Age-matched (10-wk-old) tight-skin and Pallid were quantified for their mRNA of connective-tissue-type mast-cell-specific chymase, mouse mast cell protease-4, by the competitive reverse transcriptase polymerase chain reaction technique, which revealed its higher level in tight-skin than Pallid. In contrast, the mRNA level of mouse mast cell protease-5, the chymase isoform of undifferentiated mast cells, in tight-skin skin was only a tenth that of mouse mast cell protease-4 and no different from the mouse mast cell protease-5 mRNA level of Pallid mice. An in situ hybridization study confirmed the higher expression of mouse mast cell protease-4 by connective-tissue-type mast cells in tight-skin skin than Pallid skin. These results strongly support the contention that the connective-tissue-type mast cell chymase plays a crucial role in fibroproliferative remodeling of the skin.

Expression of human cytochromes P450 1A1 and P450 1A2 as fused enzymes with yeast NADPH-cytochrome P450 oxidoreductase in transgenic tobacco plants.

Among 11 isoforms of the human cytochrome P450 enzymes metabolizing xenobiotics, CYP 1A1 and CYP 1A2 were major P450 species in the metabolism of the herbicides chlortoluron and atrazine in a yeast expression system. CYP1A2 was more active in the metabolism of both herbicides than CYP1A1. The fused enzymes of CYP1A1 and CYP1A2 with yeast NADPH-cytochrome P450 oxidoreductase were functionally active in the microsomal fraction of the yeast Saccharomyces cerevisiae and showed increased specific activity towards 7-ethoxyresorufin as compared to CYP1A1 and CYP1A2 alone. Then, both fused enzymes were each expressed in the microsomes of tobacco (Nicotiana tabacum cv. Samsun NN) plants. The transgenic plants expressing the CYP1A2 fusion enzyme had higher resistance to the herbicide chlortoluron than the plants expressing the CYP1A1 fusion enzyme did. The transgenic plants expressing the CYP1A2 fused enzyme metabolized chlortoluron to a larger extent to its non-phytotoxic metabolites through N-demethylation and ring-methyl hydroxylation as compared to the plants expressing the CYP1A1 fused enzyme. Thus, the possibility of increasing the herbicide resistance in the transgenic plants by the selection of P450 species and the fusion with P450 reductase is discussed.

Functional reconstitution of an active recombinant human chymase from Pichia pastoris cell lysate.

We have previously reported efficient production of mature human chymase (h-chymase) using an original system of expression in Pichia pastoris (Nakakubo et al., 2000), whereby recombinant h-chymase (rh-chymase) was secreted as a mature form with the correct N-terminal amino acid sequence and was easily purified. In the course of investigation of secretory rh-chymase, we also found large amounts of chymase to be present in insoluble form in the transformant cell. Although the cellular rh-chymase had no proteolytic activity, its chymotryptic activity was restored in a reconstitution process utilizing guanidine and glutathione. As with secretory rh-chymase, efficient purification was possible by heparin affinity chromatography. The purified cellular rh-chymase showed the same mobility as secretory rh-chymase in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) before and after deglycosylation. N-terminal amino acid sequence analysis revealed that the signal peptide had been correctly removed. K(m) value (5.93 mM), as well as pH profile and inhibition profile toward protease inhibitors of reconstituted cellular rh-chymase, indicated that the rh-chymase enzymatically closely resembles native h-chymase. Furthermore, it showed a greatly restricted proteolytic activity towards Ang I, and formed Ang II without the further cleavage which is a feature of h-chymase. It was thus found that the insoluble rh-chymase stored in the cells could be solubilized and reconstituted to give the same structure as h-chymase, not only in terms of enzyme active site but also of substrate recognition site.

Secretory production of recombinant human chymase as an active form in Pichia pastoris.

We succeeded in expressing in a Pichia pastoris (P. pastoris) host a cDNA encoding a mature human chymase (h-chymase) which was secreted directly into the culture medium. Recombinant human heart chymase (rh-chymase) was purified from the culture medium via a single one-step heparin-agarose column chromatography tracing, using succinyl-Ala-Ala-Pro-Phe-para-nitroanilide (Suc-AAPF-pNA) hydrolysing activity. On SDS-polyacrylamide gel electrophoresis (SDS-PAGE), the rh-chymase showed a diffused protein band with molecular weight of 32-37 kDa. After deglycosylation, however, rh-chymase changed to a sharp protein band with molecular weight 28 kDa, which is equal in size to deglycosylated h-chymase. The rh-chymase had an activity to convert one of the natural substrates, angiotensin I, to angiotensin II. Double reciprocal plot analysis revealed that the K(m) value ofrh-chymase against Suc-AAPF-pNA was approximately 5.1 mM, which is close to that of purified h-chymase.

Solubilization of organic sludge by thermophilic aerobic bacteria as a pretreatment for anaerobic digestion.

Solubilization of organic sludge by thermophilic aerobic bacteria as a pretreatment for anaerobic digestion was investigated. The thermophilic bacteria which solubilized organic sludge were isolated from the thermophilic aerobic digestion reactor. The bacterium type SPT2-1 could grow at pH ranging from 5.0 to 8.5 with optimal temperature at 60-70 degrees C. In batch experiments, 25-30% of volatile suspended solids (VSS) in the pre-heated sludge were solubilized on inoculating with the isolated bacteria although little was solubilized without inoculation. The isolated bacteria appeared to secret the extracellular enzymes including proteases and amylases. In continuous flow experiments, sludge solubilization rate (VSS removal) was around 40% under aerobic as well as microaerobic conditions. No accumulation of volatile fatty acids in the treated sludge was observed under aerobic conditions while significant amounts of them were accumulated under microaerobic conditions. Production of biogas on anaerobic digestion of the microaerobically-pretreated sludge was increased by 1.5 when compared with the sludge without pretreatment.

Angiotensin II type 1 receptor antagonist, TCV-116, prevents neointima formation in injured arteries in the dog.

We investigated the effect of an angiotensin (Ang) II antagonist, (+/-)-1-(cyclohexyloxycarbonyloxy)-ethyl 2-ethoxy- 1- [[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]- 1H-benzimidazole-7-carboxylate (TCV-116), on neointima formation in dog artery injured by a balloon catheter. Dogs were orally treated with 10 mg/kg TCV-116 or placebo twice a day for 5 weeks. After treatment with these drugs for 1 week, the right carotid artery was injured by a balloon catheter. The left carotid artery was regarded as the control. In the group treated with placebo, neointima formation in the injured arteries was observed. The activities of angiotensin converting enzyme (ACE) and chymase in the injured carotid arteries were increased 2.56- and 3.26-fold compared with those in the non-injured arteries, respectively. The neointimal area in dogs treated with placebo and TCV-116 were 0.51 +/-0.07 and 0.21 +/-0.07 mm2, respectively, and this difference was significant. In conclusion, an Ang II antagonist, TCV-116, prevented neointima formation by blocking the action of Ang II generated by both ACE and chymase in the injured arteries.

Inhibition of the angiotensin II Type 1 receptor by TCV-116: quantitation by in vitro autoradiography.

Inhibition of angiotensin (Ang) II type 1 (AT1) receptors in various target tissues of adult Sprague-Dawley rats was studied after single oral administration of TCV-116. The effects of TCV-116 on Ang II-receptor binding were assessed by quantitative in vitro autoradiography using 125I-[Sar1,Ile8]Ang II as a ligand. Four hours after the administration of TCV-116 (1 mg/kg), Ang II-receptor binding was markedly inhibited in the kidney (20% of control), adrenal cortex (27%), thoracic aorta (57%), heart (55%) and testis (76%) where AT1 receptors predominate. In the brain, orally administered TCV-116 produced a significant inhibition of binding both to the circumventricular organs (38%), which are devoid of the blood-brain barrier (BBB), and to the discrete regions within the BBB such as the paraventricular hypothalamic nucleus (48%), nucleus of the solitary tract (60%). Twenty-four hours after the administration, Ang II-receptor binding had partly recovered to approximately 50-85% of control levels. In contrast, throughout the experimental period, Ang II-receptor binding was little affected in sites where Ang II type 2 (AT2) receptors predominate such as the adrenal medulla and the nucleus of the inferior olive. These data indicate that orally administered TCV-116 specifically binds to AT1 receptors both in peripheral tissues and the central nervous system.

Tranilast suppresses vascular chymase expression and neointima formation in balloon-injured dog carotid artery.

BACKGROUND: Activation of vascular chymase plays a major role in myointimal hypertrophy after vascular injury by augmenting the production of angiotensin (ANG) II. Because chymase is synthesized mainly in mast cells, we assumed that the chymase-dependent ANG II formation could be downregulated by tranilast, a mast cell-stabilizing antiallergic agent. We have assessed inhibitory effects of tranilast on neointima formation after balloon injury in the carotid artery of dogs, which share a similar ANG II-forming chymase with humans, and further explored the pathophysiological significance of vascular chymase. METHODS AND RESULTS: Either tranilast (50 mg/kg BID) or vehicle was orally administered to beagles for 2 weeks before and 4 weeks after balloon injury. Four weeks after the injury, remarkable neointima was formed in the carotid arteries of vehicle-treated dogs. Chymase mRNA levels and chymaselike activity of vehicle-treated injured arteries were increased 10.2- and 4.8-fold, respectively, those of uninjured arteries. Angiotensin-converting enzyme (ACE) activity was slightly increased in the injured arteries, whereas ACE mRNA levels were not. Tranilast treatment completely prevented the increase in chymaselike activity, reduced the chymase mRNA levels by 43%, and decreased the carotid intima/media ratio by 63%. In vehicle-treated injured arteries, mast cell count in the adventitia showed a great increase, which was completely prevented by the tranilast treatment. Vascular ACE activity and mRNA levels were unaffected by tranilast. CONCLUSIONS: Tranilast suppressed chymase gene expression, which was specifically activated in the injured arteries, and prevented neointima formation. Suppression of the chymase-dependent ANG II-forming pathway may contribute to the beneficial effects of tranilast.

Lack of effect of carbohydrate depletion on some properties of human mast cell chymase.

Human chymase from vascular tissues was purified to homogeneity by heparin affinity and gel filtration chromatography. Treatment of human chymase with endoglycosidase F resulted in cleavage of the carbohydrate moiety yielding a deglycosylation product that did not lose its catalytic activity. This enzymatic deglycosylation product was enough to explore possibilities that N-glycan might modify some properties of human chymase. Substrate specificity, optimum pH and the elution profile from the heparin affinity gel were not affected by the deglycosylation. Only a slight but significant difference was observed in the Km value for conversion of angiotensin I to angiotensin II. Other kinetic constants such as kcat were not influenced. The kinetics of conversion of big endothelin-1 to endothelin-1(1-31) were not significantly affected. The deglycosylated human chymase was more susceptible to deactivation under alkaline pH and thermal stress. Even at physiological temperature and pH, the activity of glycosylated human chymase was more stable. From these results, it appears that the N-glycan of human chymase contributes to the stability of this enzyme but not to its functional properties.

Effect of an angiotensin II receptor antagonist, candesartan cilexetil, on canine intima hyperplasia after balloon injury.

The roles of angiotensin (Ang) II as produced by two different enzymes, angiotensin-converting enzyme (ACE) and chymase, were investigated in a canine experimental model where intima hyperplasia was induced by balloon catheterization in the common carotid and femoral arteries. The animals received oral candesartan cilexetil (3 mg/kg) or enalapril (10 mg/kg) twice a day for 5 weeks. After 1 week of active drug therapy, the common carotid and femoral arteries were unilaterally injured by balloon catheterization. In the common carotid arteries, both ACE and chymase activities were increased by the injury, with the increase in chymase activities being greater than that in ACE activities. In the femoral arteries, ACE, but not chymase, activities were significantly increased by the injury. Both candesartan cilexetil and enalapril reduced blood pressure almost equally. Enalapril increased plasma renin activity more strongly than did candesartan cilexetil, and significantly decreased vascular and plasma ACE activities. Candesartan cilexetil significantly suppressed the formation of intima hyperplasia in both the carotid and femoral arteries, while enalapril significantly suppressed intima hyperplasia in the femoral, but not in the carotid arteries. These results indicate that local Ang II production by ACE and chymase is involved in the hyperplasia seen in injured intima, and the difference in the inhibitory action of candesartan and enalapril reflects the extent of contribution of each enzyme. The effect of the ACE inhibitor, enalapril, depended on the activity of ACE, whereas that of the Ang II receptor antagonist, candesartan, was independent of ACE activity.