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Histidine-rich glycoprotein (HRG) is a heparin-binding glycoprotein present in plasma at 100microg/ml. A recent study revealed that HRG suppressed heparin-dependent basic fibroblast growth factor (bFGF)-induced angiogenesis. Additionally, we reported that high mobility group box 1 (HMGB1) in complex with heparin induces angiogenesis; therefore, we examined the effect of HRG on heparin-dependent HMGB1-induced angiogenesis in the present study. HRG completely inhibited angiogenesis induced by HMGB1 in complex with heparin. HRG inhibited the diffusion of a complex of HMGB1 with heparin from matrigel into surrounding tissue. HRG also competed with HMGB1 for heparin binding in vitro. Moreover, HRG inhibited heparin-dependent vascular endothelial growth factor-A(165) (VEGF-A(165))-induced angiogenesis. These results strongly suggested that HRG might be an inhibitor of angiogenesis induced by growth factors with heparin binding activity and that HRG may be a potential drug for angiogenic diseases, including tumor growth.

Angiogenesis involves complex processes mediated by several factors and is associated with inflammation and wound healing. High mobility group box 1 (HMGB1) is released from necrotic cells as well as macrophages and plays proinflammatory roles. In the present study, we examined whether HMGB1 would exhibit angiogenic activity in a matrigel plug assay in mice. HMGB1 in combination with heparin strongly induced angiogenesis, whereas neither HMGB1 nor heparin alone showed such angiogenic activity. The heparin-dependent induction of angiogenesis by HMGB1 was accompanied by increases in the expression of tumor necrosis factor-alpha (TNF-alpha) and vascular endothelial growth factor-A120 (VEGF-A120). It is likely that the dependence of the angiogenic activity of HMGB1 on heparin was due to the efficiency of the diffusion of the HMGB1-heparin complex from matrigel to the surrounding areas. VEGF-A165 possessing a heparin-binding domain showed a pattern of heparin-dependent angiogenic activity similar to that of HMGB1. The presence of heparin also inhibited the degradation of HMGB1 by plasmin in vitro. Taken together, these results suggested that HMGB1 in complex with heparin possesses remarkable angiogenic activity, probably through the induction of TNF-alpha and VEGF-A120.

Interaction between the receptor for advanced glycation end products (RAGE) and its ligands has been implicated in the pathogenesis of various inflammatory disorders. In this study, we establish an in vitro binding assay in which recombinant human high-mobility group box 1 (rhHMGB1) or recombinant human S100A12 (rhS100A12) immobilized on the microplate binds to recombinant soluble RAGE (rsRAGE). The rsRAGE binding to both rhHMGB1 and rhS100A12 was saturable and dependent on the immobilized ligands. The binding of rsRAGE to rhS100A12 depended on Ca2+ and Zn2+, whereas that to rhHMGB1 was not. Scatchard plot analysis showed that rsRAGE had higher affinity for rhHMGB1 than for rhS100A12. rsRAGE was demonstrated to bind to heparin, and rhS100A12, in the presence of Ca2+, was also found to bind to heparin. We examined the effects of heparin preparations with different molecular sizes - unfractionated native heparin (UFH), low molecular weight heparin (LMWH) 5000Da, and LMWH 3000Da - on the binding of rsRAGE to rhHMGB1 and rhS100A12. All 3 preparations concentration-dependently inhibited the binding of rsRAGE to rhHMGB1 to a greater extent than did rhS100A12. These results suggested that heparin's anti-inflammatory effects can be partly explained by its blocking of the interaction between HMGB1 or S100A12 and RAGE. On the other hand, heparin would be a promising effective remedy against RAGE-related inflammatory disorders.

We have reported that facilitation of central histaminergic activity prevents the development of ischemia-induced brain injury. Since cerebral edema is a major cause of brain damage, we studied effects on brain edema of postischemic administration of L-histidine, a precursor of histamine, and thioperamide, a histamine H(3)-receptor antagonist, both of which enhance central histaminergic activity. Focal cerebral ischemia for 2 h was provoked by transient occlusion of the right middle cerebral artery in rats, and the water content and infarct size were determined 24 h after reperfusion. Changes in the extracellular concentration of histamine were examined in the striatum by a microdialysis procedure, and effects of these compounds were evaluated. Repeated administration of L-histidine (1000 mg/kg x 2, i.p.), immediately and 6 h after reperfusion, reduced the increase in the water contents in ischemic regions. Simultaneous administration of thioperamide (5 mg/kg, s.c.) with L-histidine (1000 mg/kg, i.p.) completely prevented edema formation and alleviated brain infarction, although a single dose of L-histidine, immediately after reperfusion, showed no benefits. The striatal histamine level was gradually increased after reperfusion as well as during ischemia. Simultaneous administration of thioperamide with L-histidine markedly increased the brain histamine concentration, and the value increased up to 230% of that in the saline group 5 - 6 h after reperfusion. L-Histidine alone did not affect the increase in the histamine output after ischemia. These findings suggest that further activation of the central histaminergic system after initiation of cerebral ischemia prevents development of ischemia-induced brain edema.

Inflammatory reactions play an important role in ischaemia/reperfusion injury in various organs. Since histamine H(4) action has been shown to prevent the development of ischaemia/reperfusion liver injury, we examined the effects of dimaprit, a histamine H(2)/H(4) receptor agonist, on ischaemia-induced cytokine release and liver damage. Male Wistar rats (300 g) were subjected to warm ischaemia for 30 min. by occlusion of the left portal vein and hepatic artery under halothane anaesthesia. Saline or dimaprit (20 mg/kg, subcutaneously) was injected immediately after reperfusion of blood flow. Transient ischaemia provoked severe liver damage 24 hr after reperfusion, and the plasma concentrations of alanine transaminase and aspartate transaminase were 4600 IU/l and 13,200 IU/l, respectively. The values in the dimaprit group were 55% and 46% of those in control animals, respectively. Dimaprit also reduced the infarct size to 50%. Liver ischaemia markedly increased interleukin-12 levels 2-24 hr after reperfusion. The dimaprit treatment depressed the values to 40-64% of those in the corresponding control group 4-24 hr after reperfusion. Since interleukin-12 facilitates cell-mediated cytotoxicity, the protective effect of dimaprit may be attributed to regulation of cytokine release during reperfusion.

LPS stimulates CD14/Toll-like receptor (TLR) 4, leading to induce TNF-alpha production. Cell-to-cell interaction through the engagement between intercellular adhesion molecule (ICAM) 1 on monocytes and its ligand on T cells has been suggested to play a role in the TNF-alpha production by LPS-treated human peripheral blood mononuclear cells (PBMCs). Adenosine is reported to inhibit LPS-induced TNF-alpha production. However, little is known about the mechanism of the inhibitory effects induced by adenosine on the LPS-induced immune responses. We found that adenosine inhibited the expression of ICAM-1 and the production of TNF-alpha by human PBMC via adenosine A2A receptor in the presence of LPS. However, the stimulation of A1R or A3R enhanced the actions of adenosine. Adenosine had no effect on the expression of CD14 and TLR-4, suggesting that the inhibitory effects of adenosine on the LPS actions might be independent of the expression of CD14 and TLR-4. Thus, adenosine differentially regulates the expression of ICAM-1 and the production of TNF-alpha through plural subtypes of receptors.

Adenosine inhibited interleukin (IL)-18 production in lipopolysaccharide (LPS)-stimulated monocytes. The action of adenosine was antagonized by an adenosine A2A-receptor (A2AR) antagonist and was mimicked by an A2AR agonist, suggesting that the stimulation of A2AR may be involved in the actions of adenosine. On the other hand, the stimulation of A1R and A3R inhibited the actions of A2AR stimulation, whereas the stimulation of A2BR had no effect on them. Activation of A2AR is known to increase cyclic adenosine monophosphate (cAMP) levels and to activate protein kinase A (PKA). A PKA inhibitor prevented the actions of A2AR stimulation, indicating that the action mechanism of A2AR stimulation may be via the activation of the cAMP/PKA pathway.

Background and Objectives: Because the histaminergic system in the brain is involved in regulation of pain, the relationship between central histaminergic activity and neuropathic pain is of interest., Methods: Neuropathic pain was induced in rats by partial ligation of the left sciatic nerve, and changes in the extracellular concentration of histamine in the right striatum were examined by a microdialysis procedure 2 weeks later. The nociceptive threshold was determined with von Frey tests, and effects of histaminergic ligands were examined., Results: Although the extracellular concentration of histamine did not differ between the sham-operated and ligated groups, histaminergic activity assessed by metoprine-induced accumulation of histamine was facilitated in ligated animals. The metoprine treatment ameliorated neuropathic pain in ligated animals, although the agent did not affect the threshold in sham-operated rats. Either intracerebroventricular (ICV) administration of histamine (30 microg) or intraperitoneal (IP) administration of L-histidine (370 mg/kg) decreased the nociceptive threshold in ligated rats. However, a high dose of histamine (180 microg ICV) increased the nociceptive threshold. Ranitidine (100 microg ICV), an H2 antagonist, increased the threshold, whereas pyrilamine (15 microg ICV), an H1 antagonist, showed no remarkable change. Administration of thioperamide (30 microg ICV), an H3 antagonist, increased the threshold, although systemic administration of the agent (3.6 mg/kg IP) decreased it., Conclusions: Blockade of supraspinal histamine H2 receptors or stimulation of spinal H3 receptors may contribute to alleviation of neuropathic pain.

Inflammation is a crucial factor in the development of ischemia-induced brain injury. Since facilitation of central histaminergic activity ameliorates reperfusion injury, effects of postischemic administration of L-histidine, a precursor of histamine, and thioperamide, a histamine H3 receptor antagonist, on inflammatory cell infiltration were evaluated in a rat model of transient occlusion of the middle cerebral artery. After reperfusion for 12, 24, or 72 h following 2 h of occlusion, brain slices were immunohistochemically stained with antibodies against myeloperoxidase and CD68, which were markers of polymorphonuclear leukocytes and macrophages/microglia, respectively. After reperfusion for 12-24 h, the number of neutrophils on the ischemic side increased markedly, whereas the increase was not observed on the contralateral side. Administration of L-histidine (1000 mg/kg x 2, i.p.), immediately and 6 h after reperfusion, reduced the number of neutrophils to 52%. Simultaneous administration of thioperamide (5 mg/kg, s.c.) further decreased the number of neutrophils to 32%. Likewise, the ischemia induced increase in the number of CD68-positive cells after 24 h was suppressed by L-histidine injections. The L-histidine administration decreased the number of CD4+ T lymphocytes on both ischemic and contralateral sides after 12 h, and concurrent administration of thioperamide prolonged the effect. Although administration of mepyramine (3 nmol, i.c.v.) did not affect suppression of leukocyte infiltration, ranitidine tended to reverse the effect of L-histidine. These data suggest that enhancement of central histaminergic activity suppresses inflammatory cell recruitment after ischemic events through histamine H2 receptors, which may be a mechanism underlying the protective effect of L-histidine.