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.
Post-transplant diabetes mellitus is a frequent complication among transplant recipients. Ligation of advanced glycation end products (AGEs) with their receptor on monocytes/macrophages plays important roles in the genesis of diabetic complications. The enhancement of adhesion molecule expression on monocytes/macrophages activates T-cells, reducing allograft survival. Out of four distinct AGE subtypes (AGE-2, AGE-3, AGE-4 and AGE-5), only AGE-2 and AGE-3 induced expression of intercellular adhesion molecules (ICAMs), output of cytokines and proliferation of lymphocytes, during the mixed lymphocyte reaction (MLR). Here we have assessed the role of histamine in the actions of AGEs during the MLR.Human peripheral blood cells were used in these experiments. Flow cytometry was used to examine the expression of the ICAM-1, B7.1, B7.2 and CD40. Production of the cytokine interferon-gamma, and levels of cAMP were determined by elisa. Lymphocyte proliferation was determined by [(3)H]-thymidine uptake.Histamine concentration dependently inhibited the action of AGE-2 and AGE-3. The actions of histamine were antagonized by an H(2)-receptor antagonist, famotidine, and mimicked by H(2)/H(4)-receptor agonists, dimaprit and 4-methylhistamine. The effects of histamine were reversed by a protein kinase A (PKA) inhibitor, H89, and mimicked by dibutyryl cAMP and an adenylate cyclase activator, forskolin.Histamine down-regulated AGE-2- and AGE-3-induced expression of adhesion molecules, cytokine production and lymphocyte proliferation via histamine H(2) receptors and the cAMP/PKA pathway.
HMGB1, a nuclear protein, once released to the extracellular space, promotes somatic and visceral pain signals. We thus analyzed the role of HMGB1 in an intravesical substance P-induced bladder pain syndrome (BPS) mouse model. Intravesical administration of substance P caused referred hyperalgesia/allodynia in the lower abdomen and hindpaw without producing severe urothelial damage, which was prevented by an anti-HMGB1-neutralizing antibody, thrombomodulin α capable of inactivating HMGB1 and antagonists of RAGE or CXCR4. The HMGB1 inactivation or RAGE blockade also reversed the established bladder pain symptoms. HMGB1 and RAGE are thus considered to serve as therapeutic targets for BPS.
高ヒスチジン糖タンパク質（HRG）は主に肝臓で産生される血漿糖タンパク質である．我々は，代表的なダメージ関連分子パターン（DAMPs）の一つであるhigh mobility group box1（HMGB1）が病態進行に重要な役割を担っていることが知られている敗血症に対して，HRG補充療法が著明な治療効果を発揮することを明らかにした．その作用機序は，免疫血栓形成阻害や活性酸素産生阻害など多岐にわたる．加えて，HRGはDAMPsの一種であるヘムの毒性中和活性を有し，病原体関連分子パターン（PAMPs）の一種であるLPSの活性を中和すること，さらにHMGB1の血管内皮細胞核内から細胞外へのトランスロケーションを抑制すること等を明らかにした．これらの知見より，HRGはDAMPs/PAMPsの制御を行う血漿タンパク質であることが明らかとなった．DAMPs/PAMPsは敗血症のみならず，多くの炎症性疾患の病態進行に中心的役割を担っていることが知られているため，HRGの適応範囲は広く，創薬シーズとして非常に有望であると考えられる．
Abstract 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.
High mobility group box-1 (HMGB1) is a ubiquitous and abundant nonhistone DNA-binding protein, and is also an important proinflammatory cytokine once released into extracellular space from the nuclei. In the present study, we examined the effects of anti-HMGB1 mAb on collagenase IV-induced intracerebral hemorrhage(ICH) and autologous blood-induced subdural hemorrhage(SDH) in rats. Here, we show that treatment with neutralizing anti-HMGB1 mAb (1mg/kg, twice) remarkably ameliorated ICH- and SDH- induced brain injuries. Administration of anti-HMGB1 mAb inhibited the release of HMGB1 into the extracellular space and reduced serum HMGB1 levels, thereby decreased the number of activated microglia and the expression of inflammation-related factors including TNF-α, iNOS, IL-1β, IL-6, IL-8R, COX-2 at 24h after ICH and TNF-α, iNOS, IL-1β at 48h after SDH. In chronic phase of ICH, we found that brain tissue loss and vasospasm were apparent, which was alleviated by the treatment of anti-HMGB1 mAb. Moreover, anti-HMGB1 mAb inhibited the body weight loss and improved the behavioral performance of rats. These results strongly indicate that HMGB1 plays a critical role in the development of ICH- and SDH- induced secondary injury through the amplification of plural inflammatory responses. Intravenous injection of neutralizing anti-HMGB1 mAb provides a novel therapeutic strategy for different types of stroke.
Advanced glycation end products (AGEs) are the products of a series of nonenzymatic modifications of proteins by reducing sugars. AGEs play a pivotal role in development of diabetic complications and atherosclerosis. Accumulation of AGEs in a vessel wall may contribute to the development of vascular lesions. Although AGEs have a diverse range of bioactivities, the clearance process of AGEs from the extracellular space, including the incorporation of AGEs into specific cells, subcellular localization, and the fate of AGEs, remains unclear. In the present study, we examined the kinetics of the uptake of AGEs by mouse macrophage J774.1 cells in vitro and characterized the process. We demonstrated that AGEs bound to the surface of the cells and were also incorporated into the cytoplasm. The temperature‐ and time‐dependent uptake of AGEs was saturable with AGE concentration and was inhibited by cytochalasin D but not chlorpromazine. We also observed the granule‐like appearance of AGE immunoreactivity in subcellular localizations in macrophages. Higher concentrations of AGEs induced intracellular ROS and 4‐HNE, which were associated with activation of the NF‐ κ B pathway and caspase‐3. These results suggest that incorporation of AGEs occurred actively by endocytosis in macrophages, leading to apoptosis of these cells through NF‐ κ B activation.
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