Hidenori Wake

Anti-high mobility group box 1 monoclonal antibody ameliorates brain infarction induced by transient ischemia in rats

The high mobility group box‐1 (HMGB1), originally identified as an architectural nuclear protein, exhibits an inflammatory cytokine‐like activity in the extracellular space. Here we show that treatment with neutralizing anti‐HMGB1 monoclonal antibody (mAb; 200 μg, twice) remarkably ameliorated brain infarction induced by 2‐h occlusion of the middle cerebral artery in rats, even when the mAb was administered after the start of reperfusion. Consistent with the 90% reduction in infarct size, the accompanying neurological deficits in locomotor function were significantly improved. Anti‐HMGB1 mAb inhibited the increased permeability of the blood‐brain barrier, the activation of microglia, the expression of TNF‐α and iNOS, and suppressed the activity of MMP‐9, whereas it had little effect on blood flow. Intracerebroventricular injection of HMGB1 increased the severity of infarction. Immunohistochemi‐cal studyrevealed that HMGB1 immunoreactivityin the cell nuclei decreased or disappeared in the affected areas, suggesting the release of HMGB1 into the extracellular space. These results indicate that HMGB1 plays a critical role in the development of brain infarction through the amplification of plural inflammatory responses in the ischemic region and could be an out‐standingly suitable target for the treatment. Intravenous injection of neutralizing anti‐HMGB1 mAb provides a novel therapeutic strategy for ischemic stroke.— Liu, K., Mori, S., Takahashi, H. K., Tomono, Y., Wake, H., Kanke, T., Sato, Y., Hiraga, N., Adachi, N., Yoshino, T., Nishibori, M. Anti‐high mobility group box 1 monoclonal antibody ameliorates brain infarction induced by transient ischemia in rats. FASEB J. 21, 3904–3916 (2007)

Anti-high Mobility Group Box-1 Monoclonal Antibody Protects the Blood–Brain Barrier From Ischemia-Induced Disruption in Rats

Background and Purpose— High mobility group box-1 (HMGB1) exhibits inflammatory cytokine-like activity in the extracellular space. We previously demonstrated that intravenous injection of anti-HMGB1 monoclonal antibody (mAb) remarkably ameliorated brain infarction induced by middle cerebral artery occlusion in rats. In the present study, we focused on the protective effects of the mAb on the marked translocation of HMGB1 in the brain, the disruption of the blood–brain barrier (BBB), and the resultant brain edema. Methods— Middle cerebral artery occlusion in the rat was used as the ischemia model. Rats were treated with anti-HMGB1 mAb or control IgG intravenously. BBB permeability was measured by MRI. Ultrastructure of the BBB unit was observed by transmission electron microscope. The in vitro BBB system was used to study the direct effects of HMGB1 in BBB components. Results— HMGB1 was time-dependently translocated and released from neurons in the ischemic rat brain. The mAb reduced the edematous area on T2-weighted MRI. Transmission electron microscope observation revealed that the mAb strongly inhibited astrocyte end feet swelling, the end feet detachment from the basement membrane, and the opening of the tight junction between endothelial cells. In the in vitro reconstituted BBB system, recombinant HMGB1 increased the permeability of the BBB with morphological changes in endothelial cells and pericytes, which were inhibited by the mAb. Moreover, the anti-HMGB1 mAb facilitated the clearance of serum HMGB1. Conclusions— These results indicated that the anti-HMGB1 mAb could be an effective therapy for brain ischemia by inhibiting the development of brain edema through the protection of the BBB and the efficient clearance of circulating HMGB1.

Anti–high mobility group box-1 antibody therapy for traumatic brain injury

High mobility group box‐1 (HMGB1) plays an important role in triggering inflammatory responses in many types of diseases. In this study, we examined the involvement of HMGB1 in traumatic brain injury (TBI) and evaluated the ability of intravenously administered neutralizing anti‐HMGB1 monoclonal antibody (mAb) to attenuate brain injury.

Advanced Glycation End Products Subspecies-Selectively Induce Adhesion Molecule Expression and Cytokine Production in Human Peripheral Blood Mononuclear Cells

Advanced glycation end products (AGEs) are proteins or lipids that become glycated after exposure to diverse reducing sugars. Accumulation of AGEs induces diabetes complications. Microinflammation is a common major mechanism in the pathogenesis of diabetic vascular complications. Activation of monocytes/macrophages and T cells plays roles in the pathogenesis of atherosclerosis. The activation of T cells requires the enhanced expression of adhesion molecules on monocytes. AGEs activate monocytes by engaging the receptor for AGE (RAGE); however, little is known about the profile of agonist activity of diverse AGE moieties on monocytes. We investigated the effect of four distinct AGE subtypes (AGE-modified bovine serum albumin; AGE-2, AGE-3, AGE-4, and AGE-5) at concentrations ranging from 0.1 to 100 μg/ml on the expression of intercellular adhesion molecule-1, B7.1, B7.2, and CD40 on monocytes and its impact on the production of interferon-γ and tumor necrosis factor-α in human peripheral blood mononuclear cells. Among the AGEs examined, AGE-2 and AGE-3 selectively induced adhesion molecule expression and cytokine production. Antagonism experiments using antibodies against adhesion molecules demonstrated that cell-to-cell interaction between monocytes and T/natural killer cells was involved in AGE-2- and AGE-3-induced cytokine production. AGE-2 and AGE-3 up-regulated the expression of RAGE on monocytes. The effects of AGE-2 and AGE-3 were inhibited by nuclear factor-κB and p38 mitogen-activated protein kinase inhibitors. These results indicated that AGE-2 and AGE-3 activated monocytes via RAGE, leading to the up-regulation of adhesion molecule expression and cytokine production.

Effects of adenosine on adhesion molecule expression and cytokine production in human PBMC depend on the receptor subtype activated

Adenosine suppresses immune responses through adenosine2A (A2A) receptors, by raising intracellular cAMP. Interleukin (IL)‐18 up‐regulates the expression of intercellular adhesion molecule (ICAM)‐1 on monocytes, leading to production of pro‐inflammatory cytokines such as IL‐12, interferon (IFN)‐γ and tumor necrosis factor (TNF)‐α by human peripheral blood mononuclear cells (PBMC). We have previously demonstrated that elevation of cAMP inhibits this IL‐18‐induced expression of adhesion molecules. In the present study, we examined the effect of adenosine on the IL‐18‐induced up‐regulation of ICAM‐1 on human monocytes and production of IL‐12, IFN‐γ and TNF‐α by PBMC.

Histidine-rich glycoprotein inhibited high mobility group box 1 in complex with heparin-induced angiogenesis in matrigel plug assay

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.

Voluntary exercise induces neurogenesis in the hypothalamus and ependymal lining of the third ventricle.

In the adult hypothalamus and ependymal lining of the third ventricle, tanycytes function as multipotential progenitor cells that enable continuous neurogenesis, suggesting that tanycytes may be able to mediate the restoration of homeostatic function after stroke. Voluntary wheel running has been shown to alter neurochemistry and neuronal function and to increase neurogenesis in rodents. In the present study, we found that voluntary exercise improved the survival rate and energy balance of stroke-prone spontaneously hypertensive rats (SHRSP/Kpo). We also investigated the effect of exercise on the proliferation and differentiation of hypothalamic cells using immunoreactivity for tanycytes and neural markers. The proliferation of elongated cells, which may be the tanycytes, was enhanced in exercising SHRSP compared to sedentary rats before and after stroke. In addition, the proliferation of cells was correlated with the induction of fibroblast growth factor-2 in the subependymal cells of the third ventricle and in the cerebrospinal fluid. Some of the newborn cells of exercising SHRSP showed differentiation into mature neurons after stroke. Our results suggest that voluntary exercise correlates with hypothalamic neurogenesis, leading to recovery of homeostatic functions in the adult brain after stroke.

Advanced glycation end products enhance monocyte activation during human mixed lymphocyte reaction.

Posttransplant diabetes mellitus (PTDM) is a frequent complication among transplant recipients. Ligation of advanced glycation end products (AGEs) with their receptor (RAGE) on monocytes/macrophages plays roles in the diabetes complications. The enhancement of adhesion molecule expression on monocytes/macrophages activates T-cells, leading to reduced allograft survival. We investigated the effect of four distinct AGE subtypes (AGE-2/AGE-3/AGE-4/AGE-5) on the expressions of intracellular adhesion molecule (ICAM)-1, B7.1, B7.2 and CD40 on monocytes, the production of interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha and the proliferation of T-cells during human mixed lymphocyte reaction (MLR). AGE-2 and AGE-3 selectively induced the adhesion molecule expression, cytokine production and T-cell proliferation. The AGE-induced up-regulation of adhesion molecule expression was involved in the cytokine production and T-cell proliferation. AGE-2 and AGE-3 up-regulated the expression of RAGE on monocytes; therefore, the AGEs may activate monocytes, leading to the up-regulation of adhesion molecule expression, cytokine production and T-cell proliferation.

Anti-high mobility group box-1 (HMGB1) antibody inhibits hemorrhage-induced brain injury and improved neurological deficits in rats

As one of the most lethal stroke subtypes, intracerebral hemorrhage (ICH) is acknowledged as a serious clinical problem lacking effective treatment. Available evidence from preclinical and clinical studies suggests that inflammatory mechanisms are involved in the progression of ICH-induced secondary brain injury. High mobility group box-1 (HMGB1) is a ubiquitous and abundant nonhistone DNA-binding protein, and is also an important proinflammatory molecule once released into the extracellular space from the nuclei. Here, we show that treatment with neutralizing anti-HMGB1 mAb (1 mg/kg, i.v. twice) remarkably ameliorated ICH-injury induced by local injection of collagenase IV in the striatum of rats. Administration of anti-HMGB1 mAb inhibited the release of HMGB1 into the extracellular space in the peri-hematomal region, reduced serum HMGB1 levels and decreased brain edema by protecting blood-brain barrier integrity, in association with decreased activated microglia and the expression of inflammation-related factors at 24 h after ICH. Consequently, anti-HMGB1 mAb reduced the oxidative stress and improved the behavioral performance of rats. These results strongly indicate that HMGB1 plays a critical role in the development of ICH-induced secondary injury through the amplification of plural inflammatory responses. Intravenous injection of neutralizing anti-HMGB1 mAb has potential as a novel therapeutic strategy for ICH.

Anti-high mobility group box-1 (HMGB1) antibody attenuates delayed cerebral vasospasm and brain injury after subarachnoid hemorrhage in rats

Although delayed cerebral vasospasm (DCV) following subarachnoid hemorrhage (SAH) is closely related to the progression of brain damage, little is known about the molecular mechanism underlying its development. High mobility group box-1 (HMGB1) plays an important role as an initial inflammatory mediator in SAH. In this study, an SAH rat model was employed to evaluate the effects of anti-HMGB1 monoclonal antibody (mAb) on DCV after SAH. A vasoconstriction of the basilar artery (BA) associated with a reduction of nuclear HMGB1 and its translocation in vascular smooth muscle cells were observed in SAH rats, and anti-HMGB1 mAb administration significantly suppressed these effects. Up-regulations of inflammation-related molecules and vasoconstriction-mediating receptors in the BA of SAH rats were inhibited by anti-HMGB1 mAb treatment. Anti-HMGB1 mAb attenuated the enhanced vasocontractile response to thrombin of the isolated BA from SAH rats and prevented activation of cerebrocortical microglia. Moreover, locomotor activity and weight loss recovery were also enhanced by anti-HMGB1 mAb administration. The vasocontractile response of the BA under SAH may be induced by events that are downstream of responses to HMGB1-induced inflammation and inhibited by anti-HMGB1 mAb. Anti-HMGB1 mAb treatment may provide a novel therapeutic strategy for DCV and early brain injury after SAH.