Methylglyoxal (MGO), a byproduct produced in the process of glycolysis, has cytotoxicity and forms advanced glycation endproducts (AGEs), which cause cell failure in several tissues. Because MGO is mainly removed by the action of glyoxalase I (GLO1), the activity of this enzyme contributes to the accumulation of MGO. We recently found that quinetazone, a diuretic pharmaceutical agent, has the potential to inhibit GLO1 activity. Therefore, we explored whether diuretics that have a similar structure to quinetazone inhibit GLO1. The inhibitory characteristics of diuretics with recombinant GLO1 were spectrophotometrically determined. Cell proliferation and accumulation of MGO-derived AGEs were evaluated by MTT assay and Western blotting. Among the thiazide, thiazide-like, and loop diuretics, metolazone and azosemide were found to inhibit GLO1 activity by 97% at 100 µM. Furthermore, we examined whether the substructures of these diuretics have inhibitory activity, quinazolinone or phenyltetrazole were determined to be the minimal structures of metolazone or azosemide required for inhibition of GLO1, respectively. In proximal renal tubule-like HK-2 and vascular endothelial cell-like EA.hy926 cells, these diuretics were shown to inhibit cell proliferation and induce accumulation of MGO-derived AGEs. In contrast, the substructures of these diuretics that did not affect GLO1 activity did not cause these changes. Metolazone and azosemide have inhibitory effects against GLO1. Considering that these diuretics are clinically employed as pharmaceutical agents, high or prolonged dosages may contribute to pathogenesis through GLO1 inhibition, followed by MGO and/or AGE accumulation.

INTRODUCTION: Tumor necrosis factor-α (TNF-α)-induced angiogenesis plays a critical role in tumor progression and metastasis, making it an important therapeutic target in cancer treatment. Suppressing angiogenesis can effectively limit tumor growth and metastasis. However, despite advancements in understanding angiogenic pathways, effective strategies to inhibit TNF-α-mediated angiogenesis remain limited. METHODS: This study investigates the antiangiogenic effects of histidine-rich glycoprotein (HRG), a multifunctional plasma protein with potent antiangiogenic properties, on TNF-α-stimulated human endothelial cells (EA.hy926). Tube formation assays were performed to assess angiogenesis, and gene/protein expression analyses were conducted to evaluate HRG's effects on integrins αV and β8. The role of nuclear factor erythroid 2-related factor 2 (NRF2) in HRG-mediated antiangiogenic activity was also examined through nuclear translocation assays and NRF2 activation studies. RESULTS: At physiological concentrations, HRG effectively suppressed TNF-α-induced tube formation in vitro and downregulated TNF-α-induced expression of integrins αV and β8 at both the mRNA and protein levels. HRG treatment promoted NRF2 nuclear translocation in a time-dependent manner. Furthermore, activation of NRF2 significantly reduced TNF-α-induced tube formation and integrin expression, suggesting that NRF2 plays a key role in HRG-mediated antiangiogenic effects. DISCUSSION AND CONCLUSION: Our findings indicate that HRG suppresses TNF-α-induced angiogenesis by promoting NRF2 nuclear translocation and transcriptional activation, which in turn inhibits integrin αV and β8 expression. Given the essential role of angiogenesis in tumor progression, HRG's ability to regulate this process presents a promising therapeutic strategy for cancer treatment.

We previously found that ribosomal protein L9 (RPL9) is a novel advanced glycation end product (AGE)-binding protein that can decrease pro-inflammatory TNF-α expression stimulated by lipopolysaccharide (LPS) plus high-mobility group box 1 (HMGB1), suggesting that RPL9 has a role in regulating LPS+HMGB1-stimulated inflammatory reactions. Among the various ribosomal proteins, it was found that RPS5 reproduced the regulatory activity of RPL9 on LPS+HMGB1-stimulated TNF-α expression in macrophage-like RAW264.7 cells. RPL9 and RPS5 share a common feature as cationic proteins. Polylysine, a cationic polypeptide, and a synthetic peptide of the cationic region from RPL9 also exhibited reducing activity on LPS+HMGB1-induced TNF-α expression. By pull-down assay, RPL9 and RPS5 were confirmed to interact with AGEs. When AGEs coexisted with LPS, HMGB1, plus RPL9 or RPS5, the reducing effect of TNF-α expression by these cationic ribosomal proteins was shown to be abrogated. The results suggest that cationic ribosomal proteins have a regulatory role in the pro-inflammatory response induced by LPS+HMGB1, and in the pathophysiological condition of accumulating AGEs, this regulatory effect is abolished, which exacerbates inflammation.

Advanced glycation end products (AGEs) are a heterogeneous group of compounds that are non-enzymatically produced by reactions between carbonyl compounds and proteins. Many types of AGEs are produced according to the type or concentration of the reacting carbonyl compound. We have previously demonstrated that a glycolaldehyde-derived AGE suppresses stimulator of interferon gene (STING)/TANK-binding kinase 1 (TBK1)/interferon regulatory transcription factor 3 (IRF3), which is a component of the innate immune system. In this report, we investigated the effects of AGEs prepared by several carbonyl compounds on STING/TBK1/IRF3 signaling. AGEs used in the present study were numbered based on the carbonyl compound type: AGE1, derived from glucose; AGE2, derived from glyceraldehyde; AGE3, derived from glycolaldehyde; AGE4, derived from methylglyoxal; and AGE5, derived from glyoxal. AGEs derived from aldehyde (AGE2 and AGE3) and dicarbonyl compounds (AGE4 and AGE5) suppressed cyclic GMP-AMP (cGAMP)-induced activation of STING/TBK1/IRF3 signaling, with different suppression efficiencies observed. Lysine modification by carbonyl compounds was related to the efficiency of the suppressive effect on STING/TBK1/IRF3 signaling. Among the AGEs used, only AGE1 enhanced cGAMP-induced activation of STING/TBK1/IRF3 signaling. Enhancing the modulation of STING/TBK1/IRF3 signaling by AGE1 was mediated by toll-like receptor 4. These results indicated that modulation of STING/TBK1/IRF3 signaling by prepared AGEs is dependent on the type and concentration of the carbonyl compound present. Modulating STING/TBK1/IRF3 signaling by AGEs may involve modification of lysine residues in proteins.

BACKGROUND: Advanced glycation end products (AGEs) are heterogeneous proinflammatory molecules produced by a non-enzymatic glycation reaction between reducing sugars (and their metabolites) and biomolecules with amino groups, such as proteins. Although increases in and the accumulation of AGEs have been implicated in the onset and exacerbation of lifestyle- or age-related diseases, including diabetes, their physiological functions have not yet been elucidated in detail. METHODS AND RESULTS: The present study investigated the cellular responses of the macrophage cell line RAW264.7 stimulated by glycolaldehyde-derived AGEs (Glycol-AGEs) known as representative toxic AGEs. The results obtained showed that Glycol-AGEs significantly promoted the proliferation of RAW264.7 cells at a low concentration range (1-10 µg/mL) in a concentration-dependent manner. On the other hand, neither TNF-α production nor cytotoxicity were induced by the same concentrations of Glycol-AGEs. The increases observed in cell proliferation by low concentrations of Glycol-AGEs were also detected in receptor triple knockout (RAGE-TLR4-TLR2 KO) cells as well as in wild-type cells. Increases in cell proliferation were not affected by various kinase inhibitors, including MAP kinase inhibitors, but were significantly suppressed by JAK2 and STAT5 inhibitors. In addition, the expression of some cell cycle-related genes was up-regulated by the stimulation with Glycol-AGEs. CONCLUSIONS: These results suggest a novel physiological role for AGEs in the promotion of cell proliferation via the JAK-STAT pathway.

Histamine is a well-known inflammatory mediator, but how histamine induces angiogenesis remains poorly understood. In the present study, we demonstrated a dose-dependent dynamic tube formation in the human endothelial cell line EA.hy926 in the presence of histamine that was completely blocked by histamine H1 receptor (H1R) and protein kinase C (PKC) inhibitors. However, histamine H2, H3, and H4 receptor inhibitors did not inhibit tube formation, suggesting that H1R-PKC signaling is involved in histamine-induced tube formation. Moreover, we found an H1-specific induction of vascular endothelial growth factor (VEGF) expression. Inhibition of VEGF receptor 2 (VEGFR2) suppressed the histamine-induced tube formation, indicating that VEGF is downstream of histamine signaling. Additionally, we demonstrated that histamine stimulation induces the expression of critical regulators of angiogenesis such as matrix metalloproteinase (MMP)-9 and MMP-14 metalloproteases, as histamine-induced tube formation is blocked by MMP inhibitors. In summary, our study indicates that histamine can activate the H1R in human endothelial cells and thereby promote tube formation through the PKC, MMP, and VEGF signaling pathways.

AIMS: We have previously reported that advanced glycation end products derived from incubation of albumin with glycolaldehyde (glycol-AGE), lead to suppression of the toll-like receptor 4 (TLR4) signaling response to lipopolysaccharide. Glycol-AGE-induced suppression of TLR4 signaling is involved in the downregulation of CD14, which is an adaptor protein necessary for transferring lipopolysaccharide to TLR4. Therefore, glycol-AGEs impair the innate immune response through suppression of the upstream process in TLR4 signaling. However, the effect of glycol-AGEs on intracellular signaling related to the innate immune response remains unclear. This study aimed to examined the effect of glycol-AGEs on stimulator of interferon gene (STING) signaling in macrophages. MAIN METHODS: In differentiated THP-1 cells, which are a human monocytic leukemia cell line, cyclic GMP-AMP (cGAMP) transfection was used to activate STING signaling. The phosphorylation levels of TANK-binding kinase 1 (TBK1)/interferon regulatory transcription factor 3 (IRF3) were evaluated by western blot analysis. Downstream cytokine levels were evaluated by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assays. KEY FINDINGS: Glycol-AGEs suppressed cGAMP-induced phosphorylation of TBK1 and IRF3, as well as the production of cytokines regulated by IRF3. There was no effect of glycol-AGEs on the efficacy of cGAMP transfection. Treatment of a neutralizing antibody against CD36 prevented cGAMP-induced phosphorylation of TBK1 and IRF3, and also upregulation of interferon-β and C-X-C motif chemokine ligand 10 in glycol-AGE-treated cells. SIGNIFICANCE: Glycol-AGEs negatively regulate cGAMP-induced activation of STING/TBK1/IRF3 signaling via CD36. Our findings suggest that glycol-AGEs lead to impairment of the innate immune response by suppressing intracellular signaling.

BACKGROUND: Methylglyoxal (MGO) is a known toxic byproduct of glycolysis, with MGO-induced cytotoxicity believed to contribute to the pathogenesis of several diseases. Glyoxalase I (GLO1) is a key enzyme for eliminating MGO in mammalian cells, therefore, compounds affecting GLO1 activity are potential therapeutic agents for MGO-induced disorders. Previously, we found nordihydroguaiaretic acid (NDGA) as a potent GLO1 inhibitor. METHODS: The inhibitory characteristics of NDGA were determined spectrophotometrically with recombinant GLO1. NDGA-induced growth-inhibition and accumulation of MGO-derived advanced glycation end products (AGEs) were examined in EA.hy926 cells. RESULTS: NDGA showed significant inhibition of GLO1 enzymatic activity in a dose-dependent manner. Its Ki value was estimated to be 146-fold lower than that of myricetin, a known GLO1 inhibitor. The co-addition of MGO with NDGA to the cells resulted in significant growth inhibition, suggesting that MGO accumulation, sufficient to affect cell growth, was caused by NDGA inhibiting GLO1. These findings were supported by the observations that the addition of aminoguanidine, a typical MGO scavenger, significantly reversed cell-growth inhibition by co-addition of MGO with NDGA, and that an increase in intracellular MGO-derived AGEs was observed during incubation with the co-addition of MGO with NDGA. CONCLUSION: NDGA was found to be a novel and potent inhibitor of GLO1. The co-addition of NDGA with MGO to the cells resulted in increased intracellular MGO accumulation followed by enhanced cell-growth inhibition.

BACKGROUND: We previously reported that advanced glycation endproducts (AGEs) increase the proinflammatory activity of high mobility group box-1 (HMGB1), a representative damage-associated molecular pattern molecule (DAMP), through their direct interaction. This suggested that AGEs activate other DAMPs and led us to search for novel DAMPs capable of interacting with AGEs. METHODS AND RESULTS: The chromatographic analysis using AGE-immobilized gel revealed the ribosomal protein family to be a factor with binding activity to AGEs. Ribosomal protein L9 (RPL9), a member of the ribosomal protein family, was found in the centrifugal supernatant of ruptured cells and in the serum of lipopolysaccharide (LPS)-stimulated sepsis model mice, exhibiting similar characteristic properties to HMGB1. Although HMGB1 potentiated LPS-stimulated TNF-α expression in macrophage-like RAW264.7 cells, RPL9 hardly exhibited this activity. Of note, RPL9 significantly suppressed the potentiated mRNA expression and protein production of TNF-α by HMGB1 plus LPS stimulation, suggesting its regulatory roles in DAMP-induced proinflammatory activity. Based on the differential scanning fluorimetric analysis, the direct interaction between RPL9 and HMGB1 may play a role in the suppressive effects of RPL9. CONCLUSIONS: This study suggested that RPL9 is a novel type of DAMP with a regulatory role in the proinflammatory response and provided insight into the pathophysiology of inflammatory diseases.

Toxic advanced glycation end products (toxic AGEs) derived from glycolaldehyde (AGE3) have been implicated in the development of diabetic vascular complications such as retinopathy characterised by excessive angiogenesis. Different receptor types, such as receptor for AGEs (RAGE), Toll like receptor-4 and scavenger receptors, are expressed in endothelial cells and contribute to AGE-elicited alteration of cell function. In the present study, we examined the involvement of AGE-related receptors on AGE-induced angiogenesis in endothelial cells. The effects of pharmacological inhibitors or receptor neutralizing antibodies on AGE3-induced tube formation were investigated using the in vitro Matrigel tube formation assay in b.End5 cells (mouse endothelial cells). AGE3-induced signalling pathways and receptor expression changes were analysed by Western blot analysis and flow cytometry, respectively. Both FPS-ZM1, a RAGE inhibitor, and fucoidan, a ligand for scavenger receptors, suppressed AGE3-induced tube formation. Cocktails of neutralizing antibodies against the scavenger receptors CD36, CD163 and LOX-1 prevented AGE3-induced tube formation. AGE3 activated mTOR signalling, resulting in facilitation of tube formation. Activation of the AGE-RAGE pathway also led to the upregulation of scavenger receptors. Taken together, our findings suggest that the scavenger receptors CD36, CD163 and LOX-1 in conjunction with the RAGE receptor work together to mediate toxic AGE-induced facilitation of angiogenesis.

Idiopathic pulmonary fibrosis (IPF) is the most common and most deadly form of interstitial lung disease. Osteopontin (OPN), a matricellular protein with proinflammatory and profibrotic properties, plays a major role in several fibrotic diseases, including IPF; OPN is highly upregulated in patients' lung samples. In this study, we knocked down OPN in a bleomycin (BLM)-induced pulmonary fibrosis (PF) mouse model using small interfering RNA (siRNA) to determine whether the use of OPN siRNA is an effective therapeutic strategy for IPF. We found that fibrosing areas were significantly smaller in specimens from OPN siRNA-treated mice. The number of alveolar macrophages, neutrophils, and lymphocytes in bronchoalveolar lavage fluid was also reduced in OPN siRNA-treated mice. Regarding the expression of epithelial-mesenchymal transition (EMT)-related proteins, the administration of OPN-siRNA to BLM-treated mice upregulated E-cadherin expression and downregulated vimentin expression. Moreover, in vitro, we incubated the human alveolar adenocarcinoma cell line A549 with transforming growth factor (TGF)-β1 and subsequently transfected the cells with OPN siRNA. We found a significant upregulation of Col1A1, fibronectin, and vimentin after TGF-β1 stimulation in A549 cells. In contrast, a downregulation of Col1A1, fibronectin, and vimentin mRNA levels was observed in TGF-β1-stimulated OPN knockdown A549 cells. Therefore, the downregulation of OPN effectively reduced pulmonary fibrotic and EMT changes both in vitro and in vivo. Altogether, our results indicate that OPN siRNA exerts a protective effect on BLM-induced PF in mice. Our results provide a basis for the development of novel targeted therapeutic strategies for IPF.

Hyperglycaemia provides a suitable environment for infections and the mechanisms of glucose toxicity include the formation of advanced glycation end-products (AGEs), which comprise non-enzymatically glycosylated proteins, lipids, and nucleic acid amino groups. Among AGE-associated phenotypes, glycolaldehyde-derived toxic AGE (AGE-3) is involved in the pathogenesis of diabetic complications. Internalisation of endotoxin by various cell types contributes to innate immune responses against bacterial infection. An endotoxin derived from Gram-negative bacteria, lipopolysaccharide (LPS), was reported to enhance its own uptake by RAW264.7 mouse macrophage-like cells, and an LPS binding protein, CD14, was involved in the LPS uptake. The LPS uptake induced the activation of RAW264.7 leading to the production of chemokine CXC motif ligand (CXCL) 10, which promotes T helper cell type 1 responses. Previously, we reported that AGE-3 was internalised into RAW264.7 cells through scavenger receptor-1 Class A. We hypothesized that AGEs uptake interrupt LPS uptake and impair innate immune response to LPS in RAW264.7 cells. In the present study, we found that AGE-3 attenuated CD14 expression, LPS uptake, and CXCL10 production, which was concentration-dependent, whereas LPS did not affect AGE uptake. AGEs were reported to stimulate the receptor for AGEs and Toll-like receptor 4, which cause inflammatory reactions. We found that inhibitors for RAGE, but not Toll-like receptor 4, restored the AGE-induced suppression of CD14 expression, LPS uptake, and CXCL10 production. These results indicate that the receptor for the AGE-initiated pathway partially impairs the immune response in diabetes patients.

High-mobility group box-1 (HMGB1) protein has been postulated to play a pathogenic role in severe sepsis. Histidine-rich glycoprotein (HRG), a 75 kDa plasma protein, was demonstrated to improve the survival rate of septic mice through the regulation of neutrophils and endothelium barrier function. As the relationship of HRG and HMGB1 remains poorly understood, we investigated the effects of HRG on HMGB1-mediated pathway in endothelial cells, focusing on the involvement of specific receptors for HRG. HRG potently inhibited the HMGB1 mobilization and effectively suppressed rHMGB1-induced inflammatory responses and expression of all three HMGB1 receptors in endothelial cells. Moreover, we first clarified that these protective effects of HRG on endothelial cells were mediated through C-type lectin domain family 1 member A (CLEC-1A) receptor. Thus, current study elucidates protective effects of HRG on vascular endothelial cells through inhibition of HMGB1-mediated pathways may contribute to the therapeutic effects of HRG on severe sepsis.

Background: Cerebral small vessel disease (CSVD) is associated with future stroke. Although pathological alteration in small vessels of patients with CSVD can be detected by neuroimaging, diagnosis of CSVD is delayed because it is an asymptomatic disease. The stroke-prone spontaneously hypertensive rat (SHRSP) show similar pathological features to human CSVD and develop stroke-related symptoms with advancing age.Objective: We investigated the time course of haematological parameters in Wistar rats and SHRSP.Material and Methods: Blood cells were analysed using an automated haematological analyser.Results: SHRSP develop stroke-related symptoms including onset of neurological symptoms, decreased body weight and blood brain barrier leakage between 12 and 14 weeks of age. Lymphocyte counts were gradually decreased at 3 weeks before development of stoke-related symptoms and then were further decreased after the development of stroke-related symptoms. The both mean platelet volume and large platelet ratio gradually increased at 3 weeks before the development of stoke-related symptoms. However, although SHRSP showed more microcytic red cells than Wistar rats, the trajectories of change in erythrocyte-related parameters were similar between Wistar rats and SHRSP.Conclusion: Our pilot study suggests that alterations of lymphocyte count and platelet volume predictive indicators for asymptomatic CSVD and symptomatic stroke in SHRSP.

Advanced glycation end products (AGEs) are considered to be related to the pathogenesis of some inflammatory diseases. AGEs were reported to stimulate the receptor for AGEs (RAGE), which causes inflammatory reactions. However, recently, toll-like receptors (TLRs), in addition to RAGE, have been reported to be related to AGE-mediated cellular responses, and it remains unclear which receptor is responsible for AGE recognition. To reveal the role of pattern-recognition receptors, including TLRs and/or RAGE, in AGE-mediated cellular responses, we generated macrophage-like RAW264.7 knockout (KO) cells lacking these receptors by genome editing using the CRISPR/Cas9 system and assessed AGE-stimulated changes in these cells. Comparison of the established clones suggested that RAGE partially affects the expression of TLRs. In the KO clone lacking TLR4 and TLR2, AGE-stimulated tumor necrosis factor alpha (TNF-α) expression and phosphorylation of IκBα, p38, and extracellular signal-regulated kinase (ERK) were significantly attenuated, suggesting that AGE-mediated responses are largely dependent on TLRs. On the other hand, on comparison of the AGE-stimulated responses between the KO clone lacking TLR4 and TLR2, and the clone lacking TLR4, TLR2, and RAGE, RAGE played little role in AGE-stimulated TNF-α transcription and ERK phosphorylation. Taken together, this study suggested that AGE-stimulated inflammatory responses occur mainly through TLRs rather than RAGE.

Advanced glycation end-products, especially toxic advanced glycation end-products derived from glyceraldehyde (advanced glycation end-product-2) and glycolaldehyde (advanced glycation end-product-3), are biologically reactive compounds associated with diabetic complications. We previously demonstrated that toxic advanced glycation end-products were internalised into macrophage-like RAW264.7 cells through scavenger receptor-1 class A (CD204). Toxic advanced glycation end-product uptake was inhibited by fucoidan, a sulphated polysaccharide and antagonistic ligand for scavenger receptors, suggesting that sulphated polysaccharides are emerging candidates for treatment of advanced glycation end-product–related diseases. In this study, we compared the effects of six types of sulphated and non-sulphated polysaccharides on toxic advanced glycation end-product uptake in RAW264.7 cells. Fucoidan, carrageenan and dextran sulphate attenuated toxic advanced glycation end-product uptake. Fucoidan and carrageenan inhibited advanced glycation end-product-2–induced upregulation of SR-A, while advanced glycation end-product-3–induced upregulation of scavenger receptor-1 class A was only suppressed by fucoidan. Dextran sulphate did not affect scavenger receptor-1 class A levels in toxic advanced glycation end-product–treated cells. Chondroitin sulphate, heparin and hyaluronic acid failed to attenuate toxic advanced glycation end-product uptake. Heparin and hyaluronic acid had no effect on scavenger receptor-1 class A levels, while chondroitin sulphate inhibited advanced glycation end-product-3–induced upregulation of scavenger receptor-1 class A. Taken together, fucoidan and carrageenan, but not the other sulphated polysaccharides examined, had inhibitory activities on toxic advanced glycation end-product uptake and toxic advanced glycation end-product–induced upregulation of scavenger receptor-1 class A, possibly because of structural differences among sulphated polysaccharides.

BACKGROUND AND PURPOSE: Microvascular barrier breakdown is a hallmark of sepsis that is associated with sepsis-induced multiorgan failure. Histidine-rich glycoprotein (HRG) is a 75-kDa plasma protein that was demonstrated to improve the survival of septic mice through regulation of cell shape, spontaneous ROS production in neutrophils, and adhesion of neutrophils to vascular endothelial cells. We investigated HRG's role in the LPS/TNF-α-induced barrier dysfunction of endothelial cells in vitro and in vivo and the possible mechanism, to clarify the definitive roles of HRG in sepsis. EXPERIMENTAL APPROACH: EA.hy 926 endothelial cells were pretreated with HRG or human serum albumin before stimulation with LPS/TNF-α. A variety of biochemical assays were applied to explore the underlying molecular mechanisms on how HRG protected the barrier function of vascular endothelium. KEY RESULTS: Immunostaining results showed that HRG maintains the endothelial monolayer integrity by inhibiting cytoskeleton reorganization, losses of VE-cadherin and β-catenin, focal adhesion kinase degradation, and cell detachment induced by LPS/TNF-α. HRG also inhibited the cytokine secretion from endothelial cells induced by LPS/TNF-α, which was associated with reduced NF-κB activation. Moreover, HRG effectively prevented the LPS/TNF-α-induced increase in capillary permeability in vitro and in vivo. Finally, Western blot results demonstrated that HRG prevented the phosphorylation of MAPK family and RhoA activation, which are involved mainly in the regulation of cytoskeleton reorganization and barrier permeability. CONCLUSIONS AND IMPLICATIONS: Taken together, our results demonstrate that HRG has protective effects on vascular barrier function in vitro and in vivo, which may be due to the inhibition of MAPK family and Rho activation.

High mobility group box-1 (HMGB1), a representative damage associated-molecular pattern (DAMP), has been reported to be involved in many inflammatory diseases. Several drugs are thought to have potential to control the translocation and secretion of HMGB1, or to neutralize extracellular HMGB1 by binding to it. One of these drugs, anti-HMGB1 monoclonal antibody (mAb), is highly specific for HMGB1 and has been shown to be effective for the treatment of a wide range of CNS diseases when modeled in animals, including stroke, traumatic brain injury, Parkinson's disease, epilepsy and Alzheimer's disease. Thus, anti-HMGB1 mAb not only is useful for target validation but also has extensive potential for the treatment of the above-mentioned diseases. In this review, we summarize existing knowledge on the effects of anti-HMGB1 mAb on CNS and PNS diseases, the common features of translocation and secretion of HMGB1 and the functional roles of HMGB1 in these diseases. The existing literature suggests that anti-HMGB1 mAb therapy would be effective for a wide range of CNS and PNS diseases.

BACKGROUND: High mobility group box 1 (HMGB1) protein plays a key role in triggering inflammatory responses in many diseases. Our previous study showed that HMGB1 is found upstream of secondary damage in traumatic brain injury (TBI). We found that anti-HMGB1 monoclonal antibody (mAb) effectively decreased acute brain damage, including the disruption of the blood-brain barrier, brain edema, and neurologic dysfunction. This effect of anti-HMGB1 mAb lasts for at least 1 week. In this study, we explored subacute effects of anti-HMGB1 mAb after TBI. METHODS: TBI was induced in rats by fluid percussion. Anti-HMGB1 mAb or control mAb was given intravenously after TBI. Histochemical staining, plasma levels of HMGB1, motor activity and memory, and video electroencephalography monitoring were evaluated 2 weeks after fluid percussion injury. RESULTS: Anti-HMGB1 mAb remarkably attenuated accumulation of activated microglia in the rat cortex in the ipsilateral hemisphere after TBI. Anti-HMGB1 mAb also prevented neuronal death in the hippocampus in the ipsilateral hemisphere after TBI. Treatment of rats with anti-HMGB1 mAb inhibited HMGB1 translocation and suppressed impairment of motor function. The beneficial effects of anti-HMGB1 mAb on motor and cognitive function persisted for 14 days after injury. Treatment with anti-HMGB1 mAb also had positive effects on electroencephalography activity. CONCLUSIONS: The beneficial effects of anti-HMGB1 mAb continued during the subacute postinjury phase, suggesting that anti-HMGB1 mAb may prevent cognitive dysfunction after TBI.

High mobility group box protein1 (HMGB1), a ubiquitous chromatin component, is released by necrotic cells, apoptotic cells, and cells in profound distress. HMGB1 plays a critical role as a proinflammatory mediator. HMGB1 represents an important new target for drug development in a variety of inflammatory disorders, including stroke, brain injury, arteriosclerosis, and cancer. The antibodies against HMGB1 and its receptors ar hopeful candidates for immunotherapeutic strategy for treating patients with these diseases. HMGB1 forms immunostimulatory complexes by interaction with cytokines and other endogenous or exogenous factors. The HMGB1-partner molecule complexes can enhance the immune response induced by the ligand alone. The current status of HMGB1 works is summarized and future prospects will be provided in this review.

When monocytes, macrophages, and vascular endothelial cells are stimulated by RAGE ligands such as HMGB1 and AGEs, NF-κB is activated through activation of MAP kinases, increased ROS production, and activation of small G proteins. phosphorylation of transcription factors is induced. As a result, various inflammation-related factors such as inflammatory cytokines (IL-1, IL-6, TNF-α, etc.), chemokines (MCP-1, etc.), cell adhesion factors (ICAM-1, etc.), matrix metalloproteinases, etc. It has been suggested that this is the molecular basis of various pathogenesis including inflammation, diabetic complications, and tumor growth. In this paper, we discuss various strategies for drug discovery targeting HMGB1 and AGE-RAGE system, and as the first step in developing new molecular-targeted drugs that can suppress the AGEs-RAGE reaction, We describe the construction of an AGEs-RAGE binding assay based on the solid-phase method and the search for drug seeds that exhibit binding blocking activity.

High Mobility Group Box-1 (HMGB1) has been identified as a chromatin-binding non-histone protein localized in the cell nucleus, and is thought to play an important role in maintaining chromatin structure, regulating transcriptional activity, and DNA repair. However, in 1999, a research group led by Kevin Tracey et al. rediscovered it as a delayed mediator of septic shock. Blood-brain barrier breakdown in the acute phase of ischemic stroke is thought to be brought about by activation of vascular endothelial cells, microglia, astroglia, and circulating leukocytes, and their interactions, resulting in intracerebral inflammatory reactions. The release of HMGB1 from necrotic cells during such processes may play an important role in brain inflammation. The dynamics of HMGB1 in the brain during the acute phase of cerebral ischemia, which have recently been reported one after another, and treatment using shRNAs targeting HMGB1 and anti-HMGB1 antibodies will be reviewed, focusing on the findings of the authors.

Cross-linking of the B cell antigen receptor (BCR) with an anti-IgM antibody has been shown to induce dramatic apoptosis in type I Burkitt's lymphoma (BL) cells. However, the apoptotic mechanism triggered via BCR remains unknown. Here we reports a mechanism of BCR ligation-induced apoptosis involving protein phosphatase calcineurin and its specific substrate, transcriptional factor NF-AT. In response to BCR cross-linking, endogenous calcineurin was rapidly activated, and this facilitated nuclear translocation of NF-ATc2, a subtype of NF-AT members. Interestingly, nuclear-imported NF-ATc2 functioned pro-apoptotically in BL cells. The effect of NF-ATc2 was efficiently blocked with FK506, which prevented its nuclear translocation through inactivation of calcineurin. In addition, TR3 induction during BCR cross-linking was reduced by FK506 and the VIVIT peptide, which is a highly selective inhibitor for NF-AT. This strongly suggests that activation of NF-ATc2 by calcineurin is essential for TR3 recruitment, and that TR3 can be considered as a candidate for death effector in BCR-mediated apoptosis. Therefore, NF-ATc2 plays a crucial role in BCR-mediated apoptosis in type IBL, providing greater insight into unique BL characteristics through BCR signaling.

Damage-Associated Molecular Patterns(DAMPs)は細胞ストレスに伴い放出される内因性免疫応答調節因子である。過剰なDAMPsの生成は、炎症の遷延化や増悪を促す。慢性炎症性疾患の病変部位では、血管新生を伴うリモデリングが誘導され、病態増悪の本態として考えられている。その機序として、DAMPsと炎症性メディエーターの相互作用によるマクロファージ(MΦ)の過剰な活性化が関与することが示唆されている。本研究では、慢性炎症性病態であるアレルギー性炎症に焦点をあて、その病態形成に関与するDAMPsのInterleukin-33(IL-33)と炎症性メディエーターのヒスタミンによる血管新生に対する影響について解析を行った。 ヒト血管内皮細胞株EA.hy926細胞を用いたin vitro実験系において、ヒスタミンはIL-33の発現量を増加させることを確認した。ヒスタミンによるIL-33発現量の増加作用は、ヒスタミンH1受容体が関与することを明らかにした。 さらに、マトリゲルを用いたin vitro血管新生実験モデルにおいて、ヒスタミンはヒスタミンH1受容体を介して管腔形成を促進させることを見出した。ヒスタミンにより血管内皮増殖因子(vascular endothelial growth factor、VEGF)の発現上昇が認められたが、ヒスタミンによる管腔形成促進作用はVEGF受容体の阻害剤では抑制されなかった。したがって、ヒスタミンはVEGFとは異なる機序を介して血管新生を促進する可能性が示唆された。

恒常性維持機構(ホメオスタシス)を経年変化するホメオダイナミクスとして解析した知見を基に、自家組織による再生・修復促進法の開発が期待される。この開発は、様々な医療の分野にに影響を及ばすのみならず、老年期のQOLの向上といった人類の希求するテーマを解決するものである。一般に、適度な運動負荷が良好な結果をもたらすことは知られているが、これを様々な方法で解析して、根拠あるものとして証明することが大切である。まず、申請者らはそのための実験系を確立できた。適度な運動負荷という曖昧なものではなく、運動の代わりとなる、治療薬候補探索と提案をすることができた。治療薬候補の病態改善効果を検討できた。

脳卒中を自然発症する重症高血圧症動物モデル(SHRSP)を用いて病態進行と骨髄血管性ニッチの動的変化を検討し、骨髄造血系の制御が血管病の治療標的となり得るか検討した。また運動介入が血管性ニッチ改善に寄与するか検討した。発症期の血管性ニッチでは内皮細胞が障害され、幹細胞の生存・保持に重要なSDF-1産生能や低酸素環境の劣化、骨髄球増加とCD34+血球・血管系幹（前駆）細胞の減少を認めた。運動は骨髄内皮細胞と間葉系細胞を維持し、全身性炎症反応を改善し、CD34+細胞の増殖・骨髄内保持と動員を促進した。発症後ニッチ細胞のFGF-2産生が増加し、血管性ニッチ再構築と造血系制御に関与する可能性を示した。

血漿高ヒスチジン糖タンパク（HRG）の炎症病態における肝HRG遺伝子発現、及び、血漿HRGタンパクの抗炎症作用の解析をおこなった。複数種の慢性・急性炎症モデルを用いて肝遺伝子発現を網羅的に解析したところ、HRG遺伝子の発現変動と相関・逆相関する核内タンパク遺伝子を計14種同定した。これら候補遺伝子には、HRGの遺伝子発現を制御する因子が含まれる可能性が高い。また、敗血症モデルの血漿を用いたプロテオーム解析によって、HRGと結合親和性をもつ血漿タンパクを計3種同定した。これら同定タンパクは、すべて炎症病態に関与する因子であり、その炎症応答機序はHRGによって制御される可能性が示唆された。

本研究では、生体内において酸化ストレス除去を担う分子経路のマスター転写因子であるNrf2に着目し、運動器において最も含有量の多い幹細胞である間葉系幹細胞においてその発現調節機序と機能を明らかにすることを目的に研究を行った。加齢組織においては慢性炎症、あるいは転写因子Notch1の減少によりmiR-155が誘導され、C/ebpβの発現が抑制されることでNrf2の発現抑制が生じ、抗ストレス経路の破綻が生じることが明らかとなった。Nrf2には幹細胞の自己複製、エピジェネティック修飾の維持に関わる機能を有することも示唆されたことから、Nrf2は運動機能維持を目指した標的分子になりうると考えられた。

ホメオダイナミクスは、マクロファージの分化・活性化を介する血管内皮細胞の障害と再生・修復で表現される。担い手のDAMPsの内、IL-18やadvanced glycation end product(AGE)、histidine-rich glycoprotein(HRG)、high mobility group box1(HMGB1)に着目し、ホメオダイナミクス破綻状態である組織障害効果と、再生誘導活性を検証した結果、マクロファージの分化・活性化の決定因子ではなく、増強因子であることがわかった。DAMPs濃度は障害範囲に比例し、この活性を制御することはホメオダイナミクス回復に有効である。

敗血症は感染症に伴う重篤な臓器障害の病態であり、世界的に死因の上位を占め続けている。敗血症の新規開発薬物は過去数十年継続されてきたが、有効な薬物は何一つ見出されることがなかった。本研究では、申請者が見出した敗血症時の血漿タンパクHistidine-rich glycoprotein (HRG) の低下を原因とする免疫血栓の形成の機序を解明し、HRGの補充療法が如何にしてその病態を改善するかを明らかにした。敗血症病態に関与する細胞として、好中球や血管内皮細胞に加え、赤血球の重要性を実験データに基づき指摘した。マウスモデルにおいてHRG補充療法は、敗血症性ARDS病態に対し有効である。

第3脳室を囲む視床下部は細分化された核の神経活動を介して全身の生理的恒常性維持と組織再生に関わる。継続的な運動を脳卒中発症ラットにさせると脳傷害後の神経再生に重要な側脳室下帯の神経新生・分化・成熟に加え、第3脳室神経幹細胞候補のtanycyteの自己複製と神経新生・成熟の活性化を認めた。また脳室域のFGF-2とEGF産生が増加した。 脳組織再生には傷害部の骨髄由来血管系幹細胞による血管網の構築が重要である。骨髄幹細胞は骨髄の間葉系細胞と血管内皮細胞やSDF-1などにより維持されるが、脳血管障害時にはこれらの細胞極性と増殖因子の産生が低下した。運動は幹細胞の微小環境を改善し脳傷害の改善に関与する。

外科侵襲に伴う臓器障害は時に致死的になる。この一因として外科侵襲に対する過剰な免疫応答が挙げられている。本研究ではこの免疫応答システムの解析を行い、その制御によって臓器障害の抑制、ひいては予後の改善を目的として研究を行った。 外科侵襲による免疫応答の原因物質であるDAMPsの中のHMGB1に注目した。HMGB1はマクロファージを介して血管新生の制御に関与していると考えられた。このHMGB1の制御により、肝阻血再灌流モデルの予後改善を認めた。これは、肝再生が肝細胞の肥大変化ではなく、肝細胞増殖にシフトすることによるものと考えられた。 HMGB1の制御は術後の阻血再灌流障害を改善させると考えられる。

High mobility group box1(HMGB1)は、免疫応答に大きく関与している。申請者らは、HMGB1が脳梗塞、アテローム性動脈硬化症などの原因因子で、抗HMGB1抗体でこれらを抑制できることを報告した。HMGB1の高血圧症の病態生理への関与はまだ明らかではないので、脳卒中易発症高血圧症自然発症ラット (SHRSP)を用いて検討してきた。血管内皮細胞障害が高血圧症の原因であるが,HMGB1の血管内皮細胞障害は確認出来なかったが、好中球活性化が血管内皮細胞障害を誘導する機序を解明することが出来た。好中球活性化制御因子を特定したので、高血圧症新規治療薬の開発に向けた研究を継続する。

ラット脳硬膜上から加えた液体衝撃により作製した脳外傷では、大脳半球の広範な領域に脳血管の透過性亢進を生じ、脳浮腫が惹起された。受傷局所では神経細胞の核内に局在しているHMGB1が細胞質領域から細胞外に放出された。同領域では、血液脳関門の構造破綻とミクログリア細胞の活性化像ならびに細胞数の増加が認められ、炎症性サイトカインの発現が上昇していた。以上の外傷誘発性の炎症応答は、抗HMGB1抗体の受傷後投与により85％抑制された。HMGB1と結合する生薬甘草中活性成分グリチルリチンは、抗体と類似の作用を示した。同様に、マウスの脊髄損傷モデルで抗HMGB１抗体は運動麻痺症状を著明に改善することができた。

腫瘍細胞株をマウスの背部皮下に移植した担癌マウスに浸透圧ポンプを用いて、抗HMGB1モノクローナル抗体を持続投与したところ、コントロールと比較して抗HMGB1抗体投与群において、有意に腫瘍増殖の抑制が見られた。また、抗HMGB1抗体投与群の腫瘍組織中の新生血管密度がコントロールと比較して有意に減少していた。しかしながら、免疫不全マウスを用いた担癌マウスモデルでは、抗HMGB1抗体に抗腫瘍効果は認められなかったため、抗HMGB1抗体の抗腫瘍効果は直接的な作用ではなく、リンパ球等を介した免疫反応による効果である可能性も示唆された。

本研究では生活習慣病の病態形成に関与するAdvanced glycation end products (AGEs)やHigh mobility group box-1 (HMGB-1)等のReceptor for AGE (RAGE) リガンドが臓器移植における拒絶反応や虚血再灌流障害に及ぼす影響・メカニズムをIn vitroおよびVivoの両面から解析した。その結果、臓器移植におけるRAGEリガンドを介した自然免疫機構による拒絶反応や虚血再灌流障害の制御に向けた研究データの集積を達成し得た。

成体脳の神経幹細胞分化の条件は極めて難しい。運動療法（習慣）の有効性を受け、それを決定する要因として(1)神経幹(前駆）細胞自体の生存と活性、(2)幹（前駆）細胞の成長や分化に関与する炎症性分子や神経栄養因子などの脳内微小環境、(3)傷害後の時間的因子を考え、重症高血圧と脳卒中自然発症性のラットに長期の自発運動をさせ検討した。生存率の延長、神経機能改善は発症後2日目から運動を開始したラットにも認められた。運動により骨髄細胞老化が抑制され、内在性神経幹(前駆）細胞の増加と成長分化が促進された。全身性及び脳局所の炎症反応が抑制され、血管内皮細胞由来NO産生と傷害後早期に神経栄養因子が誘導された。

High mobility group box1(HMGB1)は、壊死細胞核から細胞外へ放出される新規サイトカイン分子である。申請者は、HMGB1を標的とする抗体医薬を発想し、すでにラット中大脳動脈2時間閉塞・再灌流モデルを用いて、抗HMGB1単クローン抗体の劇的な脳梗塞の改善、脳浮腫の抑制作用を明らかにしてきた。虚血再灌流の脳障害に対して、抗HMGB1抗体はBBB構造・機能の保護を中心として多数の炎症応答反応を抑制することで、脳梗塞の進行を止め、脳を保護したことを解明した。

Damage-associated molecular patternであるHigh mobility groupbox-1(HMGB1)は、虚血脳神経細胞や脳外傷局所の神経細胞核から細胞質を経て細胞外へ放出された。放出されたHMGB1は、血管内皮細胞、周皮細胞に働き、収縮性反応を誘導して脳血管透過性を亢進させた。HMGB1由来ペプチドに、低血圧誘導その他の活性を見出した。抗HMGB1単クローン抗体は脳外傷時の脳血管透過性亢進と炎症反応を抑制した。

ラット脳血管内皮細胞、血管周皮細胞、アストログリア細胞からなる試験管内血液-脳関門培養系を用いて、血管透過性に対するHMGB1の効果を検討した。この実験系において、脳側から、昆虫細胞Sf9を使って作製したヒスタグ付きヒト組換え体HMGB1を、1あるいは10・g/ml添加し、上下室間の電気抵抗の変化とエバンスブルー標識ウシ血清アルブミンの漏出を測定したところ、組換え体HMGB1は、濃度依存的に血管透過性を亢進させた。HMGB1のC末端配列を認識する単クローン抗体で予め組換え体HMGB1を処理すると、HMGB1の血管透過性亢進は抑制された。このとき、それぞれの細胞層の構造変化を観察したところ、血管内皮細胞と周皮細胞に収縮性の変化が認められ、細胞間隙の形成が生じていた。一方、底面のアストログリアには著明な形態変化は認められなかった。以上の結果から、in vivoで観察されたHMGB1による脳血管の透過性亢進は、HMGB1が脳血管内皮細胞と周皮細胞に直接作用した結果であることが強く示唆された。脳梗塞とならび脳血管の透過性が亢進し脳浮腫を招来する病態として、脳外傷を挙げることができる。そこで、ラットにfluid percussionによる脳外傷を作成し、受傷局所におけるHMGB1局在の変化を調べた。Percussion injury局所の大脳皮質錐体神経細胞では、脳虚血部位で観察されたのと同様のHMGB1細胞内局在のトランスロケーションが観察された。つまり、正常脳部位では神経細胞核内に局在していたHMGB1が受傷部位では細胞質内に拡散している像や、細胞質内で顆粒状に集積している像が観察された。以上の結果から、脳外傷時には脳虚血と類似するHMGB1トランスロケーションの存在が明らかとなった。今後、動員されたHMGB1による脳血管透過性亢進について検討する必要がある。

RAGE ligandであるHMGB1は、壊死組織から、もしくはある種の刺激で単球・マクロファージからサイトカインのごとく分泌され、免疫応答に関与している。B細胞リンパ腫の濾胞はHMGB1を強発現していた。HMGB1は、ヒト悪性リンパ腫B細胞cell lineの増殖を誘導した。B細胞リンパ腫の発生原因は、単球・マクロファージによるB細胞の増殖と分化異常である。RAGE ligandである、HMGB1やAGEは単球を活性化した。故に、抗HMGB1抗体はB細胞リンパ腫の増殖を抑制すると考えた。

自然炎症という概念が提唱されてきた。これまでの自然免疫の延長線上に存在するものと、私共は考えている。私共は、受容体リガンドに注目して研究を進めている。

脳障害時のヌクレオカインHMGB1の脳内動態が詳細に検討された。つまり、正常脳細胞においては、殆どのHMGB1は神経細胞あるいはグリア細胞の核内、特に核小体に局在していること、一定時間の脳虚血によって大きく、4つのパターンの局在変化を示すことが明らかになった。一つ目は、核内においてほぼ均質に分布していたHMGB1が、レンコンの断面様に非存在部位を作って再分布する様式を示すタイプ、二つ目は、核膜部位に凝集塊を作る形で集積するタイプ、三つ目は、虚血の極く早期から細胞質内に顆粒状に分布移動するタイプ、四つ目は、均一な細胞質分布変化と細胞収縮を伴うタイプであった。これらの四つのタイプは、脳神経核の特定の部位に見られる傾向があった。上記の変化は中大脳動脈2時間閉塞後に再灌流し、その2時間後に得られた所見である。上記のHMGB1局在変化を生じた細胞の細胞外部位には小か顆粒状のHMGB1陽性構造共焦点レーザー顕微鏡による観察では多数見出され、細胞外放出像をとらえたものと考えられていた。Macintosh tissue chopperで作製されたラット海馬スライスを用いて、シェイファー側枝の高頻度刺激により、CA1領域にLTPを誘導した。この時に回収された灌流液中のHMGB1をElisaとウエスタンブロット法で測定を試みたが、両方とも検出感度以下のレベルであった。従って、LTP時にHMGB1の放出が顆亢進する実験的証拠は、今回の実験条件では、得られなかった。

虚血性心疾患と脳血管疾患は死因の上位を占める疾患群で、有効な治療法開発が待望されている。組換え型プラスミノーゲンアクチベータによる血栓溶解療法が梗塞の急性期治療法として導入されてきたが、治療有効時間帯の制限や、出血等の副作用のため有効性に限界があることも明らかになってきた。申請者は最近数年間、生体機能を調節する分子群のうち、特に単球/マクロファージの制御に関与する因子についての研究を進めてきた。単球/マクロファージの調節因子に注目した大きな理由は、このグループの分子群の中に各種疾患の炎症病態形成に関与する重要な因子が存在すると考えたからである。 申請者らは病巣指向性の抗ヌクレオカイン単クローン抗体による脳梗塞、心筋梗塞およびクモ膜下出血後脳血管攣縮の治療法開発を行ってきた。その結果、脳梗塞およびクモ膜下出血後脳血管攣縮に対してはこの中和抗体が著効し、壊死範囲が減少して後遺症が減少することがわかり、それぞれ特許第3876325号、3882090号という成果を得た。また、脳梗塞に対するに抗ヌクレオカイン単クローン抗体の治療効果ついてはFASEB Journalに『Anti-high mobility group box l monoclonal antibody ameliorates brain infarction induced by transient ischemia in rats.』として報告した。しかし、心筋梗塞モデルラットを用いた中和抗体の梗塞巣縮小効果の検討を行ったところ、左前下降枝下部の結紮による梗塞モデルにおいて、中和抗体は逆に心筋壊死範囲を増大した。この事は、治療薬として中和抗体の使用を考える上で、心機能障害者に対する使用の注意事項として確認できた。これらの一連の研究に対して、平成18年度武田科学振興財団報彰基金研究奨励と両備てい園記念財団研究助成を得ることができた。また、この研究に関連して、Mol Pharmacol.2006,70:450-453,FASEB J2007,21;3904-3916,Circulation J in pressなどの論文報告をした。

臨床的に敗血症と診断された下部消化管穿孔を中心とした患者に使用されたポリミキシンB固相化カラムを回収し、氷冷生食1500mlで洗浄後、カラムを開封しフィルターを取り出した。生食中でシェアストレスをかけることによってフィルター付着細胞を除去した後、フィルターに結合しているタンパク因子を飽和硫酸アンモニウム、グリシン-塩酸緩衝液(pH4.0)で連続して溶出した。溶出分画を回収後、バッファー交換し濃縮した。 回収された分画におけるヌクレオサイトカインを特異抗体を用いて検出したところ、飽和硫安による回収分画にヌクレオサイトカインが存在することが明らかにされた。カラムからは大量の白血球細胞が回収された。これらの白血球をFCS不含RPMI1640でインキュベートすることによってインキュベーション上清を得た。タンパク成分を硫安沈殿で回収し、透析後ヌクレオサイトカインの存在をウエスタンブロットで確認した。抗ヌクレオサイトカイン抗体をアガロースビーズに共有結合させ、このアフィニティ担体に上記のポリミキシンフィルターからの回収タンパク質ならびに白血球細胞のインキュベーション上清をアプライした。 標準品ヌクレオカインは、この抗体アフィニティカラムに結合したが、インキュベーション上清中のヌクレオカインは結合性を示さなかった。このことからヌクレオカインの翻訳後化学修飾、複合体形成、結合阻害物質の存在等の可能性が強く示唆させた。

炎症や組織再生の過程は,様々な生理活性因子群による極めて精巧な生命活動であり,その破綻は連鎖的な組織リモデリング(病的な組織構造や機能障害)に続く重篤な難治性疾患(動脈硬化や喘息など)を引き起こす。従って,これに寄与する因子群の同定と制御機構の解明は,病態の未然防止や治療法開発に通ずる最重要課題と言える。研究代表者らは,組織リモデリングに働く可能性を秘めた因子群がヒト血液中に存在する事を認め,血漿因子の一つである高ヒスチジン糖タンパク質(HRG)や細胞表面接着因子を介した細胞間相互作用が炎症や組織再生にとって重要な役割を担っている事を見出した。HRGについては高いヒスチジン含有量に着目した特異的精製法を既に開発しているので,これを用いて高純度のHRGを調製し,組織炎症時の重要な生体現象とされる血管新生系並びに好中球走化性に及ぼす役割について検討した。マトリゲルを用いたプラグアッセイを用いて血管新生に及ぼす影響を検討した所,bFGFとヘパリン混合液による血管新生反応に対してHRGが極めて強い阻害作用を発揮する事を見出した。この作用はHRGの血中濃度に相当する1μMで観察され,HRG分子中のヒスチジンリッチドメインが機能を担う事も明らかとなった。走化性については,HRGが好中球に対し有意な遊走活性を示す事が明らかとなった。一方,単球細胞などの免疫担当細胞の機能解析研究の中で,細胞間相互作用を担う細胞表面接着因子群の発現変化が,生体防御の観点から極めて重要な役割を果たしている事やサイトカインなどの様々な因子によって発現が影響を受ける事も明らかとなった。これらの知見からヒト血漿中には炎症や組織再生に働く極めて多彩な生理活性を示す因子群が存在し,複雑な制御の下でそれらの機能を発揮している事が明らかとなった。

申請者は、これまでインターロイキン18(IL-18)の種々の免疫応答調節機能について研究を行い、その作用機序として、免疫担当細胞の膜抗原蛋白である接着分子ICAM-1、B7、CD40を介する免疫細胞間相互作用の亢進が関与することを報告した。一方、炎症・免疫担当細胞で産生放出される内因性誘導型ヒスタミンの機能解析を行った結果、ヒスタミンは抗原提示細胞上の接着分子の発現抑制を介して免疫応答を制御することを見い出した(1)。このような、IL-18によって増幅される免疫炎症反応応答は、実験的肝炎モデルマウスにおいても認められ、さらに内因性誘導型ヒスタミンがH2受容体を介して肝炎を抑制することを報告した(2)。また、他のオータコイドのプロスタグランディン(PG)E1、PGE2、アドレナリンがヒスタミンの効果を模倣することを見い出した(3-8)。 1. J.Pharmacological.Sci.,92:7-12,2003.,review. 2. Gastroenterology,127:892-902,2004. 3. J.Pharmacol.Exp.Ther.,304:634-642,2003. 4. Transplantation,75:2100-2015,2003. 5. J.Pharmacol.Exp.Ther.,307:1188-1195,2003. 6. J.Neuroimmunol.,151:137-47,2004. 7. J.Pharmacol.Exp.Ther.,309:1213-1220,2004. 8. Transplantation,77:293-301,2004.

生体防御反応は、白血球を中心とする細胞間相互作用によって営まれ、それら細胞の活性化は液性因子ならびに接着分子を介して制御されている。液性因子としてすでに多くのサイトカイン、ケモカイン分子が同定されているものの、敗血症に代表されるSIRSあるいはCARS病態形成に関与するような血中因子の解析は殆ど進んでいない。このような背景に立って本研究では、以下の諸点について明らかにした。 1)現在我が国の敗血症治療には、血中LPSの除去を目的としてポリミキシンB結合カラム(PMX)への直接血液還流法が用いられている。本研究では、LPSの吸着除去に加え、液性タンパク因子の除去仮説の下に、網羅的、系統的な吸着タンパクプロファイリングとそれらの白血球細胞機能に対する効果の評価をおこなった。 2)群特異的リガンド(ポリミキシンB)でアフィニティ精製された血漿蛋白複合体プロテオミクスの解析により、S100A8,S100A12などのEFハンドカルシウム結合タンパクと、MIFならびにHMGB1を同定した。精製されたS100A12を用いてIL-18による刺激・非刺激時の単球/マクロファージ上のICAM-1、B7.1、B7.2、CD40、CD29、CD62L、CD31などの接着分子群の発現に対する効果を明らかにした。同様に、HMGB1の作用特性について解析した。 3)同定された因子の中で、HMGB1はエンドトキシン血症の致死応答に関与するメディエターと考えられている。組み換え体ヒトHMGB1タンパクを大腸菌に発現させることに成功した。大量精製法を目下検討しているが、この標品を得て敗血症における役割の研究に発展させる計画である。

近年、難治性疾患の原因として、HMGB1をはじめとしたRAGE ligandsが挙げられている。RAGE ligandとその受容体のRAGEとの結合に注目し、病態のメカニズムを検討し、抗体医薬や低分子化合物を開発する。