Kazuhiro Abeyama

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.

Extracellular high mobility group box chromosomal protein 1 is a coupling factor for hypoxia and inflammation in arthritis

OBJECTIVE Tissue hypoxia is closely associated with arthritis pathogenesis, and extracellular high mobility group box chromosomal protein 1 (HMGB-1) released from injured cells also has a role in arthritis development. This study was thus undertaken to investigate the hypothesis that extracellular HMGB-1 may be a coupling factor between hypoxia and inflammation in arthritis. METHODS Concentrations of tumor necrosis factor alpha, interleukin-6, vascular endothelial growth factor, lactic acid, lactate dehydrogenase, and HMGB-1 were measured in synovial fluid (SF) samples from patients with inflammatory arthropathy (rheumatoid arthritis and pseudogout) and patients with noninflammatory arthropathy (osteoarthritis). The localization of tissue hypoxia and HMGB-1 was also examined in animal models of collagen-induced arthritis (CIA). In cell-based experiments, the effects of hypoxia on HMGB-1 release and its associated cellular events (i.e., protein distribution and cell viability) were studied. RESULTS In SF samples from patients with HMGB-1-associated inflammatory arthropathy (i.e., samples with HMGB-1 levels >2 SD above the mean level in samples from patients with noninflammatory arthropathy), concentrations of HMGB-1 were significantly correlated with those of lactic acid, a marker of tissue hypoxia. In CIA models in which the pathologic phenotype could be attenuated by HMGB-1 neutralization, colocalization of HMGB-1 with tissue hypoxia in arthritis lesions was also observed. In cell-based experiments, hypoxia induced significantly increased levels of extracellular HMGB-1 by the cellular processes of secretion and/or apoptosis-associated release, which was much more prominent than the protein release in necrotic cell injury potentiated by oxidative stress. CONCLUSION These findings indicate that tissue hypoxia and its resultant extracellular HMGB-1 might play an important role in the development of arthritis.

The Role of Vascular Endothelial Growth Factor in Human Dental Pulp Cells: Induction of Chemotaxis, Proliferation, and Differentiation and Activation of the AP-1-dependent Signaling Pathway

Vascular endothelial growth factor (VEGF) is a potent mitogen in endothelial cells, but little is known about its activity in other cell types. To clarify the role of VEGF in human dental pulp cells and pulp tissue, we investigated the effects of VEGF on the chemotaxis, proliferation, and differentiation of human dental pulp cells. VEGF induced a strong chemotactic response in human dental pulp cells in a dose-dependent manner. VEGF also marginally enhanced the proliferation of human dental pulp cells and induced an increase in alkaline phosphatase in human dental pulp cells. However, these effects of VEGF were not observed in reference to human skin fibroblasts. Analyses by the reverse-transcription/polymerase-chain-reaction method and flow cytometry showed that the mRNAs of two VEGF receptors, fms-like tyrosine kinase and kinase insert domain-containing receptor, were expressed in human dental pulp cells, whereas only fms-like tyrosine kinase mRNA was expressed in human skin fibroblasts. VEGF induced the activation of activator protein I (AP-1) and c-fos mRNA expression in human dental pulp cells. The AP-1 inhibitor curcumin strongly inhibited VEGF-induced alkaline phosphatase production in human dental pulp cells. In addition, VEGF antisense oligonucleotide suppressed the production of VEGF and alkaline phosphatase in human dental pulp cells. These results suggest that VEGF produced by human dental pulp cells acts directly upon human dental pulp cells in an autocrine manner, and may promote the chemotaxis, proliferation, and/or differentiation of human dental pulp cells via the utilization of kinase insert domain-containing receptor and in part through AP-1 by increasing c-fos.

Stage-Specific Secretion of HMGB1 in Cartilage Regulates Endochondral Ossification

ABSTRACT High mobility group box 1 protein (HMGB1) is a chromatin protein that has a dual function as a nuclear factor and as an extracellular factor. Extracellular HMGB1 released by damaged cells acts as a chemoattractant, as well as a proinflammatory cytokine, suggesting that HMGB1 is tightly connected to the process of tissue organization. However, the role of HMGB1 in bone and cartilage that undergo remodeling during embryogenesis, tissue repair, and disease is largely unknown. We show here that the stage-specific secretion of HMGB1 in cartilage regulates endochondral ossification. We analyzed the skeletal development of Hmgb1−/− mice during embryogenesis and found that endochondral ossification is significantly impaired due to the delay of cartilage invasion by osteoclasts, osteoblasts, and blood vessels. Immunohistochemical analysis revealed that HMGB1 protein accumulated in the cytosol of hypertrophic chondrocytes at growth plates, and its extracellular release from the chondrocytes was verified by organ culture. Furthermore, we demonstrated that the chondrocyte-secreted HMGB1 functions as a chemoattractant for osteoclasts and osteoblasts, as well as for endothelial cells, further supporting the conclusion that Hmgb1−/− mice are defective in cell invasion. Collectively, these findings suggest that HMGB1 released from differentiating chondrocytes acts, at least in part, as a regulator of endochondral ossification during osteogenesis.

A novel function of the receptor for advanced glycation end-products (RAGE) in association with tumorigenesis and tumor differentiation of HCC.

BackgroundThe expression of the receptor for advanced glycation end products (RAGE) has an impact on the mechanisms giving rise to characteristic features of various cancer cells. The purpose of this study was to elucidate the clinicopathological relevance of the level of RAGE expression in patients with hepatocellular carcinoma (HCC) and to explore the effect of RAGE expression on the characteristic features of HCC.MethodsThe expression of RAGE was assessed in paired cancer and noncancerous tissues with HCC, using reverse-transcription polymerase chain reaction (RT-PCR), and immunohistochemistry. The quantitative RT-PCR data were analyzed in association with the clinicopathological factors of the patients with HCC. In in vitro experiments, the survival of RAGE-transfected Cos7 and mock-transfected Cos7 cells was compared under hypoxic conditions. In addition, after reducing RAGE levels in RAGE-transfected Cos7 cells by siRNA, similar experiments were performed.ResultsThe expression of RAGE mRNA was lower in normal liver than in hepatitis and highest in HCC. Furthermore, in HCC, it was high in well- and moderately differentiated tumors but declined as tumors dedifferentiated to poorly differentiated HCC. Furthermore, HCC lines resistant to hypoxia were found to have higher levels of RAGE expression, and RAGE transfectant also showed significantly prolonged survival under hypoxia.ConclusionsOur results suggest that HCC during the early stage of tumorigenesis with less blood supply may acquire resistance to stringent hypoxic milieu by hypoxia-induced RAGE expression.

Enhanced production of vascular endothelial growth factor by human monocytic cells stimulated with endotoxin through transcription factor SP-1

The effect of endotoxin on the regulation of vascular endothelial growth factor (VEGF) mRNA expression in human monocytic (THP-1) cells was examined. Endotoxic lipopolysaccharide (LPS) from Escherichia coli and synthetic E. coli-type lipid A (LA-15-PP) enhanced VEGF mRNA expression. LPS-induced VEGF mRNA accumulation was regulated, at least in part, at the transcriptional level. Enhancement of VEGF gene expression by LPS was shown by gel shift analysis and use of transcription factor inhibitors to be mediated via the activation of SP-1.

Temporomandibular disorders: MR assessment of inflammatory changes in the posterior disk attachment during the menstrual cycle.

Purpose Our goal was to correlate the menstrual cycle with joint pain, MR evidence of the disk, and posterior disk attachment in patients with temporomandibular disorders. Method Forty-two women underwent MRI involving conventional T1-and T2-weighted gadolinium-enhanced fat-suppressed SE imaging sequences. Results There was a strong statistical difference in the degree of joint pain between proliferated phase and secretory phase groups (p < 0.005). Joint pain had a tendency to increase at the secretory phase. Significantly less contrast enhancement of the posterior disk attachment was observed in the proliferated phase than in the secretory phase (p < 0.001) or menstrual phase (p < 0.01). In addition, anterior disk displacement without reduction of the temporomandibular joint was closely associated with joint pain. Conclusion Our results suggest that positional changes of the disk and the menstrual cycle may play a role in the degree of joint pain and inflammatory pathology of the posterior disk attachment.

Interleukin 6 mediated differentiation and rescue of cell redox in PC 12 cells exposed to ionizing radiation

The differentiation of PC12 cells to a neuron‐like morphology was induced by ionizing radiation in the presence of serum. This effect was detectable at 5 grays (Gy) and reached a maximum at 10–20 Gy. Increases in the DNA binding activity of nuclear factor κB (NF‐κB) and increased Interleukin 6 (IL‐6) mRNA levels were observed at a dose of 15 Gy. Neutralization of supernatant IL‐6 by the addition of anti‐IL‐6 antibody inhibited the neuronal differentiation and decreased cellular redox. Ionizing radiation and serum may act synergistically as neurotropic factors.

Antibiotic cyclic AMP signaling by “primed” leukocytes confers anti-inflammatory cytoprotection

The mechanism underlying anti‐inflammatory effects of macrolide antibiotics remains uncertain. In this study, we first show the evidences concerning the possible link between leukocytic cyclic adenosine monophosphate (cAMP) signaling and the mechanism of anti‐inflammatory, cytoprotective actions of macrolides. The clinical range of macrolides (i.e., erythromycin, roxithromycin, and clarithromycin) preferentially inhibited nuclear factor‐κB activation mediated by reactive oxygen intermediates, inducing cAMP‐dependent signaling [i.e., cAMP and cAMP‐responsive element‐binding protein (CREB)] by “primed” but not “resting” leukocytes. In this context, cAMP/CREB inhibition with adenosine 3′:5′‐cyclic monophosphothioate, rp‐isomer (rp‐cAMPs) and CREB decoy oligonucleotides reduced the anti‐inflammatory actions of macrolides. These results thus indicate that macrolide‐induced cAMP/CREB signaling, selectively by primed leukocytes, plays a major role in the mechanism of anti‐inflammatory actions of macrolides.

Inflammatory Cytokine Production and Specific Antibody Responses against Possible Causative Bacteria in Patients with Multilesional Periapical Periodontitis

We examined the induction of inflammatory cytokines (including interleukin-1, interleukin-6, interleukin-8, and tumor necrosis factor-alpha) by several species of possible causative bacteria in periapical periodontitis. Assays were done on human whole blood cultures from patients with differing numbers of periapical lesions; those having radiographically clear periapical lesions in 10 or more teeth (high lesion group), in one or two teeth (low lesion group), and healthy volunteers having no periapical lesions (no lesion group). Prevotella melaninogenica ATCC 25845 induced interleukin-6 more strongly in subjects from the high lesion group than in the other groups. To ascertain the degree of sensitization by test bacteria, we examined the reactivities of antibodies in serum and saliva from the six subjects to different bacterial species. Porphylomonas gingivalis cells reacted strongly with sera from the high lesion group. Thus, Prevotella melaninogenica and Porphylomonas gingivalis may be involved in multilesional periapical periodontitis by inducing specific cytokines and/or humoral immune responses.