Teruto Hashiguchi

Proteolytic Cleavage of High Mobility Group Box 1 Protein by Thrombin-Thrombomodulin Complexes

Objective—High mobility group box 1 protein (HMGB1) was identified as a mediator of endotoxin lethality. We previously reported that thrombomodulin (TM), an endothelial thrombin-binding protein, bound to HMGB1, thereby protecting mice from lethal endotoxemia. However, the fate of HMGB1 bound to TM remains to be elucidated. Methods and Results—TM enhanced thrombin-mediated cleavage of HMGB1. N-terminal amino acid sequence analysis of the HMGB1 degradation product demonstrated that thrombin cleaved HMGB1 at the Arg10-Gly11 bond. Concomitant with the cleavage of the N-terminal domain of HMGB1, proinflammatory activity of HMGB1 was significantly decreased (P<0.01). HMGB1 degradation products were detected in the serum of endotoxemic mice and in the plasma of septic patients with disseminated intravascular coagulation (DIC), indicating that HMGB1 could be degraded under conditions in which proteases were activated in the systemic circulation. Conclusions—TM not only binds to HMGB1 but also aids the proteolytic cleavage of HMGB1 by thrombin. These findings highlight the novel antiinflammatory role of TM, in which thrombin–TM complexes degrade HMGB1 to a less proinflammatory form.

High-mobility group box 1 protein promotes development of microvascular thrombosis in rats.

Summary.  Background: Sepsis is a life‐threatening disorder resulting from systemic inflammatory and coagulatory responses to infection. High‐mobility group box 1 protein (HMGB1), an abundant intranuclear protein, was recently identified as a potent lethal mediator of sepsis. However, the precise mechanisms by which HMGB1 exerts its lethal effects in sepsis have yet to be confirmed. We recently reported that plasma HMGB1 levels correlated with disseminated intravascular coagulation (DIC) score, indicating that HMGB1 might play an important role in the pathogenesis of DIC. Objectives: To investigate the mechanisms responsible for the lethal effects of HMGB1, and more specifically, to explore the effects of HMGB1 on the coagulation system. Methods: Rats were exposed to thrombin with or without HMGB1, and a survival analysis, pathologic analyses and blood tests were conducted. The effects of HMGB1 on the coagulation cascade, anticoagulant pathways and surface expression of procoagulant or anticoagulant molecules were examined in vitro. Results: Compared to thrombin alone, combined administration of thrombin and HMGB1 resulted in excessive fibrin deposition in glomeruli, prolonged plasma clotting times, and increased mortality. In vitro, HMGB1 did not affect clotting times, but inhibited the anticoagulant protein C pathway mediated by the thrombin–thrombomodulin complex, and stimulated tissue factor expression on monocytes. Conclusions: These findings demonstrate the procoagulant role of HMGB1 in vivo and in vitro. During sepsis, massive accumulation of HMGB1 in the systemic circulation would promote the development of DIC.

Highly concentrated vascular endothelial growth factor in platelets in Crow-Fukase syndrome

We report a marked difference in concentration of vascular endothelial growth factor (VEGF) between serum and plasma in patients with Crow–Fukase syndrome (CFS). The serum/plasma VEGF levels in 4 CFS patients were 8,634/152, 5,203/176, 3,724/127, and 868/13 pg/ml, respectively. We also showed that platelets were a major source of this VEGF and that VEGF was released during platelet aggregation by physiological stimulation. It is suggested that in CFS, local VEGF concentration is markedly elevated by aggregation of platelets containing excessive VEGF and their adhesion to vascular walls, resulting in excessive physiological activities of VEGF. Our findings provide important information for developing more effective therapeutic trials.

Frameshift mutation in the collagen VI gene causes Ullrich's disease

Patients with Ullrichs disease have generalized muscle weakness, multiple contractures of the proximal joints, and hyperextensibility of the distal joints. Recently, we found a deficiency of collagen VI protein in two patients with Ullrichs disease. In this study, we detected a homozygous 26 bp deletion in exon 14 of the collagen VI alpha 2 gene (COL6A2) in one patient. This mutation causes a frameshift and a premature termination codon, and results in a truncated collagen VI alpha 2 chain. Our data suggest that at least some cases of Ullrichs disease result from recessive mutations in COL6A2.

Recombinant Thrombomodulin Protects Mice against Histone-Induced Lethal Thromboembolism

Introduction Recent studies have shown that histones, the chief protein component of chromatin, are released into the extracellular space during sepsis, trauma, and ischemia-reperfusion injury, and act as major mediators of the death of an organism. This study was designed to elucidate the cellular and molecular basis of histone-induced lethality and to assess the protective effects of recombinant thrombomodulin (rTM). rTM has been approved for the treatment of disseminated intravascular coagulation (DIC) in Japan, and is currently undergoing a phase III clinical trial in the United States. Methods Histone H3 levels in plasma of healthy volunteers and patients with sepsis and DIC were measured using enzyme-linked immunosorbent assay. Male C57BL/6 mice were injected intravenously with purified histones, and pathological examinations were performed. The protective effects of rTM against histone toxicity were analyzed both in vitro and in mice. Results Histone H3 was not detectable in plasma of healthy volunteers, but significant levels were observed in patients with sepsis and DIC. These levels were higher in non-survivors than in survivors. Extracellular histones triggered platelet aggregation, leading to thrombotic occlusion of pulmonary capillaries and subsequent right-sided heart failure in mice. These mice displayed symptoms of DIC, including thrombocytopenia, prolonged prothrombin time, decreased fibrinogen, fibrin deposition in capillaries, and bleeding. Platelet depletion protected mice from histone-induced death in the first 30 minutes, suggesting that vessel occlusion by platelet-rich thrombi might be responsible for death during the early phase. Furthermore, rTM bound to extracellular histones, suppressed histone-induced platelet aggregation, thrombotic occlusion of pulmonary capillaries, and dilatation of the right ventricle, and rescued mice from lethal thromboembolism. Conclusions Extracellular histones cause massive thromboembolism associated with consumptive coagulopathy, which is diagnostically indistinguishable from DIC. rTM binds to histones and neutralizes the prothrombotic action of histones. This may contribute to the effectiveness of rTM against DIC.

Endocannabinoid, anandamide in gingival tissue regulates the periodontal inflammation through NF-κB pathway inhibition

Anandamide (AEA) exhibits anti‐inflammatory effects. However, its role in the periodontal field remains unknown. Here, we found that gingival crevicular fluid contained a detectable level of AEA. The cannabinoid receptors CB1 and CB2 were expressed by human gingival fibroblasts (HGFs), and markedly upregulated under pathological conditions. AEA significantly reduced the production of pro‐inflammatory mediators (IL‐6, IL‐8 and MCP‐1) induced by Porphyromonas gingivalis LPS in HGFs, and this effect was attenuated by AM251 and SR144528, selective antagonists of CB1 and CB2, respectively. Moreover, AEA completely blocked LPS‐triggered NF‐κB activation, implying that AEA may regulate hyperinflammatory reactions in periodontitis.

Marked increase of matrix metalloproteinase 9 in cerebrospinal fluid of patients with fungal or tuberculous meningoencephalitis

Matrix metalloproteinases (MMPs) are believed to play an essential role in the breakdown of the extracellular matrix macromolecules in the blood-cerebrospinal fluid barrier and blood-brain barrier (BBB). In this study, the levels of MMP-2 and MMP-9 and their common tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) were measured in the cerebrospinal fluid (CSF) from patients with various meningitides including aseptic, fungal and tuberculous ones. MMP-9 production level in CSF was more increased in subacute meningitis including fungal and tuberculous meningitis than in acute aseptic meningitis and non-inflammatory neurological diseases (NIDs). Enhanced production of MMP-9 was associated with high proteolytic activity detected by gelatin zymography. The MMP-2 and TIMP-1 levels in CSF of subacute meningitis were also higher than those of NIDs. In contrast, the TIMP-2 levels in CSF of either acute aseptic or subacute meningitis were not up-regulated compared with those of NIDs. The central nervous system (CNS) complications (i.e. disturbance of consciousness, psychiatric symptoms, urinary disturbance, etc.) during the course of meningitis showed good correlation with the enhanced production of MMP-9 in CSF. Immunohistochemical studies in tuberculous meningitis demonstrated that the infiltrating mononuclear cells in the meninges were immunoreactive for both MMP-2 and MMP-9. However, the infiltrating mononuclear cells into CNS parenchyma had immunoreactivity for MMP-9, but not for MMP-2. Taken together, those data suggest that MMP-9 in CSF may be a useful marker of encephalitogenecity during the course of subacute meningitis.

Vitreous Mediators after Intravitreal Bevacizumab or Triamcinolone Acetonide in Eyes with Proliferative Diabetic Retinopathy

PURPOSE To evaluate vitreous vascular endothelial growth factor (VEGF), stromal cell-derived factor 1alpha (SDF-1alpha), interleukins (ILs), and tumor necrosis factor-alpha (TNF-alpha) after intravitreal bevacizumab or triamcinolone acetonide (TA) in eyes with proliferative diabetic retinopathy (PDR). DESIGN Interventional, consecutive, retrospective, comparative study with a historical control. PARTICIPANTS Forty-seven eyes of 47 patients affected by active PDR were investigated. Bevacizumab (1.25 mg; 19 eyes; bevacizumab group) or TA (4 mg; 10 eyes; TA group) was injected into the vitreous cavity as preoperative adjunctive therapy 7 days before vitrectomy. Eighteen eyes without injection served as controls (control group). METHODS The vitreous samples were obtained at vitrectomy and were analyzed for concentrations of total protein, VEGF, SDF-1alpha, IL-1beta, IL-6, IL-8, IL-10, IL-12p70, and TNF-alpha. MAIN OUTCOME MEASURES Vitreous concentrations of VEGF, SDF-1alpha, ILs, and TNF-alpha were compared among bevacizumab, TA, and control groups. RESULTS Vitreous concentrations of VEGF and SDF-1alpha were lower in bevacizumab and TA groups compared with the control group. The median VEGF level was 0 pg/ml (range, 0-79.2 pg/ml) in the bevacizumab group, 343.5 pg/ml (range, 0-1683.3 pg/ml) in the TA group, and 1202.5 pg/ml (range, 76-4213.9 pg/ml) in the control group. The median SDF-1alpha level was 149.2 pg/ml (range, 0-519.4 pg/ml) in the bevacizumab group, 87.5 pg/ml (range, 0-252.5 pg/ml) in the TA group, and 245.7 pg/ml (range, 0-856.8 pg/ml) in the control group. The differences in both vitreous VEGF and SDF-1alpha concentrations among 3 groups were statistically significant (P<0.001 and P = 0.010, respectively). The eyes with intravitreal bevacizumab demonstrated the lowest vitreous level of VEGF, and the level was statistically significant compared with the eyes with intravitreal TA and control eyes (P<0.001 and P<0.001, respectively). The control eyes had the highest vitreous level of SDF-1alpha, and the level was statistically significant compared with the eyes with intravitreal bevacizumab and TA (P = 0.015 and P = 0.009, respectively). There was no significant difference regarding ILs and TNF-alpha. CONCLUSIONS Intravitreal injection of bevacizumab potentially diminishes not only VEGF but also SDF-1alpha. These findings suggest that intravitreal bevacizumab may influence intraocular mediators beyond VEGF. FINANCIAL DISCLOSURE(S) The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Edaravone attenuates cerebral ischemic injury by suppressing aquaporin-4

Aquaporin-4 (AQP4) plays a role in the generation of post-ischemic edema. Pharmacological modulation of AQP4 function may thus provide a novel therapeutic strategy for the treatment of stroke, tumor-associated edema, epilepsy, traumatic brain injury, and other disorders of the central nervous system (CNS) associated with altered brain water balance. Edaravone, a free radical scavenger, is used for the treatment of acute ischemic stroke (AIS) in Japan. In this study, edaravone significantly reduced the infarct area and improved the neurological deficit scores at 24h after reperfusion in a rat transient focal ischemia model. Furthermore, edaravone markedly reduced AQP4 immunoreactivity and protein levels in the cerebral infarct area. In light of observations that edaravone specifically inhibited AQP4 in a rat transient focal ischemia model, we propose that edaravone might reduce cerebral edema through the inhibition of AQP4 expression following cerebral infarction.

Ebselen inhibits NO-induced apoptosis of differentiated PC12 cells via inhibition of ASK1-p38 MAPK-p53 and JNK signaling and activation of p44/42 MAPK and Bcl-2

Ebselen, a selenium‐containing heterocyclic compound, prevents ischemia‐induced cell death. However, the molecular mechanism through which ebselen exerts its cytoprotective effect remains to be elucidated. Using sodium nitroprusside (SNP) as a nitric oxide (NO) donor, we show here that ebselen potently inhibits NO‐induced apoptosis of differentiated PC12 cells. This was associated with inhibition of NO‐induced phosphatidyl Serine exposure, cytochrome c release, and caspase‐3 activation by ebselen. Analysis of key apoptotic regulators during NO‐induced apoptosis of differentiated PC12 cells showed that ebselen blocks the activation of the apoptosis signaling‐regulating kinase 1 (ASK1), and inhibits phosphorylation of p38 mitogen‐activated protein kinase (MAPK) and c‐jun N‐terminal protein kinase (JNK). Moreover, ebselen inhibits NO‐induced p53 phosphorylation at Ser15 and c‐Jun phosphorylation at Ser63 and Ser73. It appears that inhibition of p38 MAPK and p53 phosphorylation by ebselen occurs via a thiol‐redox‐dependent mechanism. Interestingly, ebselen also activates p44/42 MAPK, and inhibits the downregulation of the antiapoptotic protein Bcl‐2 in SNP‐treated PC12 cells. Together, these findings suggest that ebselen protects neuronal cells from NO cytotoxicity by reciprocally regulating the apoptotic and antiapoptotic signaling cascades.