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Biomedical subjects

Takahisa Imamura

Publications and source records attributed to Takahisa Imamura.

At least 19 recordsLinked to original sources
PubMed2006

Induction of vascular leakage and blood pressure lowering through kinin release by a serine proteinase from Aeromonas sobria.

Aeromonas sobria causes septic shock, a condition associated with high mortality. To study the mechanism of septic shock by A. sobria infection, we examined the vascular leakage (VL) activity of A. sobria serine proteinase (ASP), a serine proteinase secreted by this pathogen. Proteolytically active ASP induced VL mainly in a bradykinin (BK) B(2) receptor-, and partially in a histamine-H(1) receptor-dependent manner in guinea pig skin. The ASP VL activity peaked at 10 min to 1.8-fold of the initial activity with an increased BK B(2) receptor dependency, and attenuated almost completely within 30 min. ASP produced VL activity from human plasma apparently through kallikrein/kinin system activation, suggesting that ASP can generate kinin in humans. Consistent with the finding that a major part of the ASP-induced VL was reduced by a potent kallikrein inhibitor, soybean trypsin inhibitor that does not affect ASP enzymatic activity, ASP activated prekallikrein but not factor XII to generate kallikrein in a dose- and incubation time-dependent manner. ASP produced more VL activity directly from human low m.w. kininogen than high m.w. kininogen when both were used at their normal plasma concentrations. Intra-arterial injection of ASP into guinea pigs lowered blood pressure specifically via the BK B(2) receptor. These data suggest that ASP induces VL through prekallikrein activation and direct kinin release from kininogens, which is a previously undescribed mechanism of A. sobria virulence and could be associated with the induction of septic shock by infection with this bacterium. ASP-specific inhibitors, and kinin receptor antagonists, might prove useful for the treatment or prevention of this fatal disease.

Aeromonas↗
PubMed2006

Urokinase-immobilization suppresses inflammatory responses to polyurethane tubes implanted in rabbit muscles.

Urokinase and plasmin appear to have antiinflammatory activity in some injury models, and urokinase immobilization has been clinically used to prevent thrombus formation in various implants, including intravenous indwelling catheters and subcutaneous drainage tubes. In the present study, polyurethane tubes were embedded in rabbit muscle for 3 months and the effect of urokinase immobilization on inflammatory responses to the implanted tubes was studied at 1 week, 1 month, and 3 months. Mononuclear leukocyte accumulation occurred around implanted polyurethane tubes and peaked after 1 month, but was reduced significantly by urokinase immobilization. The treatment also lessened as well as delayed eosinophil accumulation, but did not affect fibrosis caused by implanted tubes. These results indicate suppressive effects of urokinase immobilization on polyurethane-elicited inflammatory responses and suggest that an approach to develop persistently active urokinase immobilization is rational for successful long-term device implantation.

Animals↗
PubMed2006

DX-9065a inhibits proinflammatory events induced by gingipains and factor Xa.

OBJECTIVE: Arginine-specific cysteine proteases (Rgps) from Porphyromonas gingivalis are important virulent factors of periodontal diseases. However, there is no therapeutic drug that inhibits proinflammatory events induced by these enzymes. In this study, we investigated proinflammatory activities of Rgps and activated coagulation factor X (FXa) and examined the effect of DX-9065a, a new selective inhibitor of FXa, on proinflammatory events induced by these proteinases. METHODS: Human gingival fibroblasts were stimulated with Rgps and FXa in the presence or absence of DX-9065a, and then interleukin-6 (IL-6) and matrix metalloproteinase-1 (MMP-1) release, their mRNA expression, and nuclear factor kappaB (NF-kappaB) activation were assessed using an enzyme-linked immunosorbent assay (ELISA), northern blotting, and a gel-mobility shift method, respectively. RESULTS: Rgps and FXa activated IL-6 and MMP-1 release in human gingival fibroblasts through their amidolytic activities and in mitogen-activated protein kinase (MAPK) and NF-kappaB dependent manners. DX-9065a inhibited FXa-induced IL-6 mRNA expression and NF-kappaB activation. DX-9065a inhibited amidolytic activities of FXa and Rgps in vitro and ex vivo. CONCLUSION: Rgps and FXa are potent inflammatory mediators and DX-9065a may be a useful therapeutic drug for periodontal disease.

Adhesins, Bacterial↗
PubMed2006

Selective inhibition of Porphyromonas gingivalis growth by a factor Xa inhibitor, DX-9065a.

BACKGROUND: Porphyromonas gingivalis is a causative bacterium of adult periodontitis. However, there is no drug specific for P. gingivalis and for its virulence factor. OBJECTIVES: The objective of this study was to examine the effects of a new selective inhibitor of activated factor X, DX-9065a, on growth of Porphyromonas gingivalis and other periodontopathic bacteria. METHODS: We incubated P. gingivalis and other periodontopathic bacteria in the presence or absence of DX-9065a and examined the effect of DX-9065a on bacterial growth and trypsin-like activity in its cultures. We also examined the effects of DX9065a on amidolytic activity of purified trypsin-like proteinases (gingipains RgpA and RgpB), from P. gingivalis and on trypsin-like activity in gingival crevicular fluids from patients with adult periodontitis. RESULTS: DX-9065a selectively inhibited the growth of P. gingivalis and Prevotella intermedia, and its effect on P. gingivalis was bactericidal. Trypsin-like proteinase activity was detected in P. gingivalis, and the activity was strongly inhibited by DX-9065a. DX-9065a even inhibited amidolytic activity of RgpA and RgpB from P. gingivalis. Furthermore, trypsin-like proteinase activity in gingival crevicular fluids was strongly inhibited by DX-9065a. CONCLUSIONS: DX-9065a inhibits P. gingivalis growth in part through to its ability to inhibit the trypsin-like proteinase activity in P. gingivalis and may be useful for a new drug for treatment of adult periodontitis.

Actinomyces↗
PubMed2005

Arginine-specific gingipains from Porphyromonas gingivalis stimulate production of hepatocyte growth factor (scatter factor) through protease-activated receptors in human gingival fibroblasts in culture.

Cystein proteinases (gingipains) from Porphyromonas gingivalis cleave a broad range of in-host proteins and are considered to be key virulence factors in the onset and development of adult periodontitis and host defense evasion. In periodontitis, an inflammatory disease triggered by bacterial infection, the production of hepatocyte growth factor (HGF) is induced not only by various factors derived from the host, such as inflammatory cytokines, but also by bacterial components. In this study we examined the possible enhanced production of HGF produced by human gingival fibroblasts upon stimulation with gingipains. Arginine-specific gingipain (Rgp) caused a marked production of HGF into the supernatant, the induction of HGF expression on the cell surface, and the up-regulation of HGF mRNA expression in a dose-dependent and an enzymatic activity-dependent manner. Because it has been reported that Rgp activated protease-activated receptors (PARs), we examined whether the induction of HGF triggered by Rgps on human gingival fibroblasts occurred through PARs. An RNA interference assay targeted to PAR-1 and PAR-2 mRNA revealed that gingipains-induced secretion of HGF was significantly inhibited by RNA interference targeted to PAR-1 and PAR-2. In addition, the Rgps-mediated HGF induction was completely inhibited by the inhibition of phospholipase C and was clearly inhibited by RNA interference targeted to p65, which is an NF-kappaB component. These results suggest that Rgps activated human gingival fibroblasts to secrete HGF in the inflamed sites and the mechanism(s) involved may actively participate in both inflammatory and reparative processes in periodontal diseases.

Adhesins, Bacterial↗
PubMed2005

Induction of vascular leakage through release of bradykinin and a novel kinin by cysteine proteinases from Staphylococcus aureus.

Staphylococcus aureus is a major pathogen of gram-positive septic shock and frequently is associated with consumption of plasma kininogen. We examined the vascular leakage (VL) activity of two cysteine proteinases that are secreted by S. aureus. Proteolytically active staphopain A (ScpA) induced VL in a bradykinin (BK) B(2)-receptor-dependent manner in guinea pig skin. This effect was augmented by staphopain B (SspB), which, by itself, had no VL activity. ScpA also produced VL activity from human plasma, apparently by acting directly on kininogens to release BK, which again was augmented significantly by SspB. Intravenous injection of ScpA into a guinea pig caused BK B(2)-receptor-dependent hypotension. ScpA and SspB together induced the release of leucyl-methionyl-lysyl-BK, a novel kinin with VL and blood pressure-lowering activities that are equivalent to BK. Collectively, these data suggest that production of BK and leucyl-methionyl-lysyl-BK by staphopains is a new mechanism of S. aureus virulence and bacterial shock. Therefore, staphopain-specific inhibitors and kinin-receptor antagonists could be used to treat this disease.

Animals↗
PubMed2005

Neuropeptide release from dental pulp cells by RgpB via proteinase-activated receptor-2 signaling.

Dental pulp inflammation often results from dissemination of periodontitis caused mostly by Porphyromonas gingivalis infection. Calcitonin gene-related peptide and substance P are proinflammatory neuropeptides that increase in inflamed pulp tissue. To study an involvement of the periodontitis pathogen and neuropeptides in pulp inflammation, we investigated human dental pulp cell neuropeptide release by arginine-specific cysteine protease (RgpB), a cysteine proteinase of P. gingivalis, and participating signaling pathways. RgpB induced neuropeptide release from cultured human pulp cells (HPCs) in a proteolytic activity-dependent manner at a range of 12.5-200 nM. HPCs expressed both mRNA and the products of calcitonin gene-related peptide, substance P, and proteinase-activated receptor-2 (PAR-2) that were also found in dental pulp fibroblast-like cells. The PAR-2 agonists, SLIGKV and trypsin, also induced neuropeptide release from HPCs, and HPC PAR-2 gene knockout by transfection of PAR-2 antisense oligonucleotides inhibited significantly the RgpB-elicited neuropeptide release. These results indicated that RgpB-induced neuropeptide release was dependent on PAR-2 activation. The kinase inhibitor profile on the RgpB-neuropeptide release from HPC revealed a new PAR-2 signaling pathway that was mediated by p38 MAPK and activated transcription factor-2 activation, in addition to the PAR-2-p44/42 p38MAPK and -AP-1 pathway. This new RgpB activity suggests a possible link between periodontitis and pulp inflammation, which may be modulated by neuropeptides released in the lesion.

Activating Transcription Factor 2↗
PubMed2004

Protective effect of vascular endothelial growth factor/vascular permeability factor 165 and 121 on glomerular endothelial cell injury in the rat.

Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) promotes the repair of injured vessels by stimulating angiogenesis. VEGF/VPF reportedly has cytoprotective activity but no study has shown the protective effect of VEGF/VPF on glomerular endothelial cells. We examined whether recombinant VEGF/VPF121 and VEGF/VPF165 isoforms could prevent injury of glomerular endothelial cells. Mild glomerular injury was induced in rats by an intravenous-injection of a limited dose of anti-Thy-1.1 antibody to obtain lesions similar to those found in the human disease. Recombinant VEGF/VPF165, VEGF/VPF121 or BSA was administered 4 h before the injection of the antibody, and once daily for 3 days. In the BSA-injected rats, mesangial cell lysis and endothelial cell injury in dilated capillary tufts were evident without endothelial cell apoptosis on days 1-4. Thereafter, cell proliferation and repair began and remodeling of the glomeruli was completed by day 28. Macrophages but not polymorphonuclear leukocytes accumulated significantly in the glomeruli on days 1-4. Treatment with VEGF/VPF isoform protected endothelial cells but not mesangial cells from destruction on day 1, and accelerated the repair of both types of cells, which was completed by day 18, 10 days earlier than that of the control animals. The results indicate that VEGF/VPF121 or VEGF/VPF165 can protect glomerular endothelial cells against injury, independent of apoptosis-inhibition activity, thereby promoting reconstruction of glomeruli. The protective effect of VEGF/VPF on endothelial cells suggests that it could provide therapeutic benefit for certain kidney diseases.

Animals↗
PubMed2004

Assessment of thrombin in the urine of glomerulonephritic patients by enzyme-linked immunosorbent assay.

BACKGROUND: Accumulating evidence suggests that blood clotting occurs in inflamed glomeruli, although its role in the pathophysiology of glomerulonephritis remains to be elucidated. To address this issue, a simple and reliable method for evaluating clotting in glomeruli is necessary. Here, we developed an enzyme-linked immunosorbent assay (ELISA) for thrombin in urine to evaluate the degree of clotting activation in diseased glomeruli. METHODS: Monoclonal antibodies against human alpha-thrombin were raised and used for sandwich ELISA to measure thrombin. Thrombin was measured in urine samples from normal volunteers and from patients with glomerulonephritis or disseminated intravascular coagulation (DIC). RESULTS: Thrombin antigen was not detected in the urine of healthy volunteers or of patients with DIC, but was detected in the urine from two-thirds of glomerulonephritic patients. The average concentration in positive samples was 3.79 microg/L. Urinary thrombin concentrations measured by ELISA correlated well with thrombin activities measured by hydrolysis of a synthetic substrate. CONCLUSION: We suggest that thrombin antigen in urine measured by ELISA is not affected by systemic thrombin production in the vessels, and reflects blood clotting activation in glomerulonephritic lesions. A close relationship between urinary thrombin and glomerulonephritis indicates a possible involvement of clotting in disease development, and measurement of urinary thrombin may provide a real-time marker for monitoring renal diseases.

Adult↗
PubMed2004

Activation of the kallikrein-kinin system and release of new kinins through alternative cleavage of kininogens by microbial and human cell proteinases.

Kinins are released from kininogens through the activation of the Hageman factor-prekallikrein system or by tissue kallikrein. These peptides exert various biological activities, such as vascular permeability increase, smooth muscle contraction, pain sensation and induction of hypotension. In many instances kinins are thought to be involved in the pathophysiology of various diseases. Recent studies have revealed that microbial and human cell proteinases activate Hageman factor and/or prekallikrein, or directly release kinin from kininogens. This review discusses the activation of the kinin-release system by mast-cell tryptase and microbial proteinases, including gingipains, which are cysteine proteinases from Porphyromonas gingivalis , the major pathogen of periodontal disease. Each enzyme is evaluated in the context of its association to allergy and infectious diseases, respectively. Furthermore, a novel system of kinin generation directly from kininogens by the concerted action of two proteinases is described. An interesting example of this system with implications to bacterial pathogenicity is the release of kinins from kininogens by neutrophil elastase and a synergistic action of cysteine proteinases from Staphylococcus aureus . This alternative production of kinins by proteinases present in diseased sites indicates a significant contribution of proteinases other than kallikreins in kinin generation. Therefore kinin receptor antagonists and proteinase inhibitors may be useful as therapeutic agents.

Bacterial Physiological Phenomena↗
PubMed2004

[Tissue factor expression at the site of inflammation: a cross-talk between inflammation and the blood coagulation system].

Recent studies have revealed a close association of the blood coagulation system with inflammation and immune reactions. The products of the cascade reaction of blood coagulation can work as inflammatory mediators or immune modulators, and, vice versa, some inflammatory or immune stimuli are linked to induction of blood coagulation. First, tissue factor (the blood coagulation initiator), the monocyte/macrophage tissue factor expression regulatory factors associated with inflammation and immune reactions, and the assembly of coagulation factors on leukocytes were reviewed. Second, evidence of leukocyte tissue factor expression and subsequent fibrin deposition were demonstrated at sites of infection or allergic reactions, using immunohistochemical staining. Third, the progress in the investigation of thrombin was reviewed from the viewpoint of its effects on inflammation (vascular permeability enhancement, leukocyte chemotaxis, chemical mediator release, etc.) and immune reactions (T-cell proliferation, cytokine production, etc.). The evidence presented here indicates a cross-talk between blood coagulation and inflammatory and immune reactions, suggesting that the products of the clotting reaction (e.g., thrombin) in lesions are real-time markers of inflammatory diseases.

Biomarkers↗
PubMed2004

[Evaluation of thrombin in urine as a real-time indicator of clotting activation in glomerulonephritis].

When tissues are injured and bleeding occurs, blood clotting is immediately activated and fibrin clots are formed by thrombin. Afterwards, antithrombin III promptly inactivates thrombin, which restricts the clotting to the bleeding site. In inflamed sites, tissue factor is expressed on cells in the lesion by stimulation from cytokines, and produces thrombin. In this case, thrombin may survive longer because of inefficient inactivation by antithrombin III due to dilution and less perturbation in the interstitial fluid, and therefore, has a greater chance to activate thrombin receptors (protease-activated receptors: PARs) on the cells, which induces various cellular events including proliferation, migration, and shape change. Recent studies have suggested a pathophysiological association of the PAR pathway with crescentic glomerulonephritis. However, the role of thrombin in human diseases has not been fully studied, probably because of a lack of simple and reliable methods for measuring thrombin in clinical samples. To solve this problem, we developed an ELISA system for human alpha-thrombin and applied it to the measurement of thrombin in the urine of patients with glomerulonephritis. Thrombin in urine was detected in glomerulonephritic patients but not in healthy volunteers or disseminated intravascular coagulation patients, which suggests that thrombin in urine may reflect thrombin generation by clotting activation in the glomerular lesion.

Biomarkers↗
PubMed2003

Expression of enteropeptidase in differentiated enterocytes, goblet cells, and the tumor cells in human duodenum.

Enteropeptidase (EP) is a serine proteinase and activates trypsinogen to trypsin, thus playing an important role in food digestion. Nevertheless, the localization of EP is still controversial, likely due to a lack of studies using specific antibodies against EP. The aim of this study was to define cellular localization of EP in human duodenum and expression in tumor cells at the duodenal region. Immunohistochemical staining for resected tissues was performed with two antibodies against recombinant EP light and heavy chains, respectively. In situ hybridization was done with two RNA probes that include either the light or the heavy chain sequences of proEP, respectively. The two antibodies reacted with enterocytes, accentuated on the brush border, and goblet cells, with increasing intensity from the bottom of crypts to the top of villi. Paneth cells, neuroendocrine cells, Brunner's glands, lymphocytes, smooth muscle, or connective tissue did not react with the antibodies. The two RNA probes detected EP mRNA expression only in enterocytes and goblet cells. EP is produced in enterocytes and goblet cells, and the localization on the brush border of the cells is reasonable for the physiological activation of digestive enzymes. Interestingly, the antibodies reacted with tumor cells in duodenal polyps and adenocarcinoma at the duodenum but not in Brunner's gland adenoma. EP seems to be a marker of differentiated enterocytes and goblet cells, which suggests the existence of a common progenitor of these cells. Furthermore, EP may be a useful marker of tumor cells originating from these cells.

Duodenal Neoplasms↗
PubMed2003

Enhanced expression of vascular endothelial growth factor by periodontal pathogens in gingival fibroblasts.

Vascular endothelial growth factor (VEGF) has recently attracted attention as a potent inducer of vascular permeability and angiogenesis. Aberrant angiogenesis is often associated with lesion formation in chronic periodontitis. The aim of the present study was to investigate the properties of VEGF expression in human gingival fibroblasts (HGF) culture. HGF were stimulated with lipopolysaccharide (LPS), vesicle (Ve) and outer membrane protein (OMP) from Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis. HGF constitutively produced VEGF and levels were significantly enhanced (P < 0.01) by stimulation with Ve and OMP from A. actinomycetemcomitans and P. gingivalis at concentrations of 10 microg/ml or higher. On the other hand, VEGF levels were not increased by LPS stimulation. VEGF mRNA expression was also observed in Ve- and OMP-stimulated HGF. A vascular permeability enhancement (VPE) assay was performed using guinea pigs to ascertain whether supernatant from cultures of Ve- and OMP-stimulated HGF enhance vascular permeability in vivo. Supernatant from cultures of Ve- and OMP-stimulated HGF strongly induced VPE. This was markedly suppressed upon simultaneous injection of anti-VEGF polyclonal antibodies with the supernatant. Heating and protease treatment of the stimulants reduced the VEGF enhancing levels in Ve and OMP in vitro. These results suggest that Ve and OMP may be crucial heat-labile and protease-sensitive components of periodontal pathogens that enhance VEGF expression. In addition, VEGF might be associated with the etiology of periodontitis in its early stages according to neovascularization stimulated by periodontal pathogens causing swelling and edema.

Aggregatibacter actinomycetemcomitans↗
PubMed2003

Attenuated kinin release from human neutrophil elastase-pretreated kininogens by tissue and plasma kallikreins.

Components of kinin-forming systems operating at inflammatory sites are likely to interact with elastase that is released by recruited neutrophils and may, at least temporarily, constitute the major proteolytic activity present at these sites. The aim of this work was to determine the effect of kininogen degradation by human neutrophil elastase (HNE) on kinin generation by tissue and plasma kallikreins. We show that the digestion of both low molecular mass (LK) and high molecular mass (HK) forms of human kininogen by HNE renders them essentially unsusceptible to processing by human urinary kallikrein (tissue-type) and also significantly quenches the kinin release from HK by plasma kallikrein. Studies with synthetic model heptadecapeptide substrates, ISLMKRPPGFSPFRSSR and SLMKRPPGFSPFRSSRI, confirmed the inability of tissue kallikrein to process peptides at either termini of the internal kinin sequence, while plasma kallikrein was shown to release the kinin C-terminus relatively easily. The HNE-generated fragments of kininogens were separated by HPLC and the fractions containing internal kinin sequences were identified by a kinin-specific immunoenzymatic test after trypsin digestion. These fractions were analyzed by electrospray-ionization mass spectrometry. In this way, multiple peptides containing the kinin sequence flanked by only a few amino acid residues at each terminus were identified in elastase digests of both LK and HK. These results suggest that elastase may be involved in quenching the kinin-release cascade at the late stages of the inflammatory reaction.

Amino Acid Sequence↗
PubMed2003

The role of gingipains in the pathogenesis of periodontal disease.

Gingipains are trypsin-like cysteine proteinases produced by Porphyromonas gingivalis, a major causative bacterium of adult periodontitis. HRgpA (95 kDa) and RgpB (50 kDa), products of 2 distinct but related genes, rgpA and rgpB, respectively, are specific for Arg-Xaa peptide bonds. Kgp, a product of the kgp gene, is specific for Lys-Xaa bonds. HRgpA and Kgp are non-covalent complexes containing separate catalytic and adhesion/ hemagglutinin domains, while RgpB has only a catalytic domain with a primary structure essentially identical to that of the catalytic subunit of HRgp. HRgpA and RgpB induce vascular permeability enhancement through activation of the kallikrein/kinin pathway and activate the blood coagulation system, which, respectively, are potentially associated with gingival crevicular fluid production and progression of inflammation leading to alveolar bone loss in the periodontitis site. Kgp is the most potent fibrinogen/fibrin degrading enzyme of the 3 gingipains in human plasma and is involved in the bleeding tendency at the diseased gingiva. HRgpA activates coagulation factors and degrades fibrinogen/fibrin more efficiently than RgpB due to the adhesion/hemagglutinin domains, which have affinity for phospholipids and fibrinogen. Gingipains degrade macrophage CD14, thus inhibiting activation of the leukocytes through the lipopolysaccharide (LPS) receptor, and thereby facilitating sustained colonization of P. gingivalis. Gingipains play a role in bacterial housekeeping and infection, including amino acid uptake from host proteins and fimbriae maturation. Based on the important activities of gingipains in the bacterial infection and the pathogenesis of periodontitis, the bacterial proteinases can be targets for periodontal disease therapy. Immunization with RgpB, HRgpA, or a portion of HRgpA catalytic domain attenuated P. gingivalis induced disorders in mice. In addition, a trypsin-like proteinase inhibitor retarded P. gingivalis growth specifically. Gingipains are potent virulence factors of P. gingivalis, and are likely to be associated with the development of periodontitis. It is, therefore, suggested that gingipain inhibition by vaccination and gingipain-specific inhibitors is a useful therapy for adult periodontitis caused by P. gingivalis infection.

Adhesins, Bacterial↗