Kouji Wakayama

NF-κB Is a Key Mediator of Cerebral Aneurysm Formation

Background— Subarachnoid hemorrhage caused by the rupture of cerebral aneurysm (CA) remains a life-threatening disease despite recent diagnostic and therapeutic advancements. Recent studies strongly suggest the active participation of macrophage-mediated chronic inflammatory response in the pathogenesis of CA. We examined the role of nuclear factor-&kgr;B (NF-&kgr;B) in the pathogenesis of CA formation in this study. Methods and Results— In experimentally induced CAs in rats, NF-&kgr;B was activated in cerebral arterial walls in the early stage of aneurysm formation with upregulated expression of downstream genes. NF-&kgr;B p50 subunit–deficient mice showed a decreased incidence of CA formation with less macrophage infiltration into the arterial wall. NF-&kgr;B decoy oligodeoxynucleotide also prevented CA formation when it was administered at the early stage of aneurysm formation in rats. Macrophage infiltration and expression of downstream genes were dramatically inhibited by NF-&kgr;B decoy oligodeoxynucleotide. In human CA walls, NF-&kgr;B also was activated, especially in the intima. Conclusions— Our data indicate that NF-&kgr;B plays a crucial role as a key regulator in the initiation of CA development by inducing some inflammatory genes related to macrophage recruitment and activation. NF-&kgr;B may represent a therapeutic target of a novel medical treatment for CA.

Interferon regulatory factor 8/interferon consensus sequence binding protein is a critical transcription factor for the physiological phenotype of microglia

BackgroundRecent fate-mapping studies establish that microglia, the resident mononuclear phagocytes of the CNS, are distinct in origin from the bone marrow-derived myeloid lineage. Interferon regulatory factor 8 (IRF8, also known as interferon consensus sequence binding protein) plays essential roles in development and function of the bone marrow-derived myeloid lineage. However, little is known about its roles in microglia.MethodsThe CNS tissues of IRF8-deficient mice were immunohistochemically analyzed. Pure microglia isolated from wild-type and IRF8-deficient mice were studied in vitro by proliferation, immunocytochemical and phagocytosis assays. Microglial response in vivo was compared between wild-type and IRF8-deficient mice in the cuprizon-induced demyelination model.ResultsOur analysis of IRF8-deficient mice revealed that, in contrast to compromised development of IRF8-deficient bone marrow myeloid lineage cells, development and colonization of microglia are not obviously affected by loss of IRF8. However, IRF8-deficient microglia demonstrate several defective phenotypes. In vivo, IRF8-deficient microglia have fewer elaborated processes with reduced expression of IBA1/AIF1 compared with wild-type microglia, suggesting a defective phenotype. IRF8-deficient microglia are significantly less proliferative in mixed glial cultures than wild-type microglia. Unlike IRF8-deficient bone marrow myeloid progenitors, exogenous macrophage colony stimulating factor (colony stimulating factor 1) (M-CSF (CSF1)) restores their proliferation in mixed glial cultures. In addition, IRF8-deficient microglia exhibit an exaggerated growth response to exogenous granulocyte-macrophage colony stimulating factor (colony stimulating factor 2) (GM-CSF (CSF2)) in the presence of other glial cells. IRF8-deficient microglia also demonstrate altered cytokine expressions in response to interferon-gamma and lipopolysaccharide in vitro. Moreover, the maximum phagocytic capacity of IRF8-deficient microglia is reduced, although their engulfment of zymosan particles is not overtly impaired. Defective scavenging activity of IRF8-deficient microglia was further confirmed in vivo in the cuprizone-induced demyelination model in mice.ConclusionsThis study is the first to demonstrate the essential contribution of IRF8-mediated transcription to a broad range of microglial phenotype. Microglia are distinct from the bone marrow myeloid lineage with respect to their dependence on IRF8-mediated transcription.

Amyloid β1-42 oligomer inhibits myelin sheet formation in vitro.

Accumulating evidence indicates that white matter degeneration contributes to the neural disconnections that underlie Alzheimers disease pathophysiology. Although this white matter degeneration is partly attributable to axonopathy associated with neuronal degeneration, amyloid β (Aβ) protein-mediated damage to oligodendrocytes could be another mechanism. To test this hypothesis, we studied effects of soluble Aβ in oligomeric form on survival and differentiation of cells of the oligodendroglial lineage using highly purified oligodendroglial cultures from rats at different developmental stages. Aβ oligomer at 10 μM or higher reduced survival of mature oligodendrocytes, whereas oligodendroglial progenitor cells (OPCs) were relatively resistant to the Aβ oligomer-mediated cytotoxicity. Further study revealed that Aβ oligomer even at 1 μM accelerated 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) formazan exocytosis in mature oligodendrocytes, and, more significantly, inhibited myelin sheet formation after induction of in vitro differentiation of OPCs. These results imply a novel pathogenetic mechanism underlying Aβ oligomer-mediated white matter degeneration, which could impair myelin maintenance and remyelination by adult OPCs, resulting in accumulating damage to myelinating axons thereby contributing to neural disconnections.

OPG/RANKL/RANK axis is a critical inflammatory signaling system in ischemic brain in mice

Significance Although a high-serum osteoprotegerin (OPG) level is associated with an unfavorable outcome in ischemic stroke, it is unclear whether OPG is a culprit or an innocent bystander. Here we show that the deletion of OPG and enhanced RANKL/RANK signaling contribute to the reduction of infarct volume with lower brain edema, whereas infarct volume is increased by reduced RANKL/RANK signaling in OPG−/− mice and WT mice treated with anti-RANKL neutralizing antibody. OPG, RANKL, and RANK mRNA were increased in ischemic brain and were expressed in activated microglia and macrophages. Enhanced RANKL/RANK signaling showed neuroprotective effects with reduced expression in inflammatory cytokines in LPS-stimulated neuron-glia mixed culture. Our findings propose anti-inflammatory roles for RANKL/RANK signaling in ischemic brains. Osteoprotegerin (OPG) is a soluble secreted protein and a decoy receptor, which inhibits a receptor activator of nuclear factor κB (NF-κB) ligand (RANKL)/the receptor activator of NF-κB (RANK) signaling. Recent clinical studies have shown that a high-serum-OPG level is associated with unfavorable outcome in ischemic stroke, but it is unclear whether OPG is a culprit or an innocent bystander. Here we demonstrate that enhanced RANKL/RANK signaling in OPG−/− mice or recombinant RANKL-treated mice contributed to the reduction of infarct volume and brain edema via reduced postischemic inflammation. On the contrary, infarct volume was increased by reduced RANKL/RANK signaling in OPG−/− mice and WT mice treated with anti-RANKL neutralizing antibody. OPG, RANKL, and RANK mRNA were increased in the acute stage and were expressed in activated microglia and macrophages. Although enhanced RANKL/RANK signaling had no effects in glutamate, CoCl2, or H2O2-stimulated neuronal culture, enhanced RANKL/RANK signaling showed neuroprotective effects with reduced expression in inflammatory cytokines in LPS-stimulated neuron-glia mixed culture, suggesting that RANKL/RANK signaling can attenuate inflammation through a Toll-like receptor signaling pathway in microglia. Our findings propose that increased OPG could be a causal factor of reducing RANKL/RANK signaling and increasing postischemic inflammation. Thus, the OPG/RANKL/RANK axis plays critical roles in controlling inflammation in ischemic brains.

ZPK/DLK, a Mitogen-Activated Protein Kinase Kinase Kinase, Is a Critical Mediator of Programmed Cell Death of Motoneurons

Activation of mitogen-activated protein kinase pathways is critically involved in naturally occurring programmed cell death of motoneurons during development, but the upstream mediators remain undetermined. We found that mice deficient in ZPK, also called DLK (ZPK/DLK), an upstream kinase in these pathways, have twice as many spinal motoneurons as do their wild-type littermates. Nuclear HB9/MNX1-positive motoneuron pools were generated similarly in the spinal cord of both ZPK/DLK-deficient and wild-type embryos. Thereafter, however, significantly less apoptotic motoneurons were found in ZPK/DLK-deficient embryos compared with wild-type embryos, resulting in retention of excess numbers of motoneurons after birth. Notably, these excess motoneurons remained viable without atrophic changes in the ZPK/DLK-deficient mice surviving into adulthood. Analysis of the diaphragm and the phrenic nerve revealed that clustering and innervation of neuromuscular junctions were indistinguishable between ZPK/DLK-deficient and wild-type mice, whereas the proximal portion of the phrenic nerve of ZPK/DLK-deficient mice contained significantly more axons than the distal portion. This result supports the hypothesis that some excess ZPK/DLK-deficient motoneurons survived without atrophy despite failure to establish axonal contact with their targets. This study provides compelling evidence for a critical role for ZPK/DLK in naturally occurring programmed cell death of motoneurons and suggests that ZPK/DLK could become a strategic therapeutic target in motor neuron diseases in which aberrant activation of the apoptogenic cascade is involved.

Delayed Postischemic Treatment With Fluvastatin Improved Cognitive Impairment After Stroke in Rats

Background and Purposes— Recent clinical evidences indicate that statins may have beneficial effects on the functional recovery after ischemic stroke. However, the effect of delayed postischemic treatment with statins is still unclear. In the present study, we evaluated the effects of fluvastatin in the chronic stage of cerebral infarction in a rat model. Methods— Rats exposed to permanent middle cerebral artery occlusion were treated for 3 months with fluvastatin beginning from 7 days after stroke. MRI, behavioral analysis, and immunohistochemistry were performed. Results— Two months of treatment with fluvastatin showed the significant recovery in spatial learning without the decrease in serum total cholesterol level and worsening of infarction. Microangiography showed a significant increase in capillary density in the peri-infarct region in fluvastatin-treated rats after 3 months of treatment. Consistently, BrdU/CD31-positive cells were significantly increased in fluvastatin-treated rats after 7 days of treatment. MAP1B-positive neurites were also increased in the peri-infarct region in fluvastatin-treated rats. In addition, rats treated with fluvastatin showed the reduction of superoxide anion after 7 days of treatment and the reduction of A&bgr; deposits in the thalamic nuclei after 3 months of treatment. Conclusions— Thus, delayed postischemic administration of fluvastatin had beneficial effects on the recovery of cognitive function without affecting the infarction size after ischemic stroke. Pleiotropic effects of fluvastatin, such as angiogenesis, neuritogenesis, and inhibition of superoxide production and A&bgr; deposition, might be associated with a favorable outcome.

Expression of hepatocyte growth factor and c-Met after spinal cord injury in rats.

Since hepatocyte growth factor (HGF) plays a pivotal role in the development of the central nervous system and pathological conditions, we examined the long-term changes in the mRNA and protein expression of HGF and its receptor c-Met after spinal cord injury (SCI) in rats. HGF mRNA was significantly increased from 7 days after SCI in the injured segment, and the peak was at 7 days after SCI as assessed by real-time RT-PCR. Importantly, c-met mRNA expression was up-regulated from 1 day after SCI, and reached a peak at 14 days after SCI. Although up-regulation of HGF and c-met mRNA expression in the injured segment gradually decreased, the increased expression level persisted until 56 days after SCI. Consistent with HGF mRNA expression, HGF protein level was significantly increased mainly in the injured region, which persisted until 56 days after SCI. Immunohistochemistry showed that most of GFAP-positive reactive astrocytes expressed HGF and c-Met both on 14 days and 56 days after SCI. Staining with the mitotic indicator, bromodeoxyuridine (BrdU), revealed that a small number of BrdU-incorporated cells were co-localized with HGF/GFAP-positive or c-Met/GFAP-positive cells both on 14 and 56 days. These data suggest that HGF and c-Met were up-regulated mainly in the reactive astrocytes around the injured region in the subacute to chronic stage of spinal cord injury. Since HGF plays a critical role in neurotrophic activity, activation of the HGF/c-Met signaling system might be involved in the process of post-traumatic regeneration.

PEDF is a novel oligodendrogenic morphogen acting on the adult SVZ and corpus callosum.

Pigment epithelium-derived factor (PEDF) is a serine protease inhibitor (serpin) protein with well established neuroprotective and anti-angiogenic properties. Recent studies have also shown that PEDF enhances renewal of adult subventricular zone (SVZ) neural precursors. In neurosphere cultures prepared from the SVZ of adult mice, we found that addition of recombinant PEDF to the medium enhanced expressions of oligodendroglial lineage markers (NG2 and PDGFrα) and transcription factors (Olig1, Olig2, and Sox10). Similarly, continuous PEDF administration into the lateral ventricles of adult glial fibrillary acidic protein:green fluorescent protein (GFAP:GFP) transgenic mice increased the proportions of GFAP:GFP+ and GFAP:GFP− SVZ neural precursors coexpressing oligodendroglial lineage markers and transcription factors. Notably, PEDF infusion also resulted in an induction of doublecortin- and Sox10 double-positive cells in the adult SVZ. Immunoreactive PEDF receptor was detectable in multiple cell types in both adult SVZ and corpus callosum. Furthermore, PEDF intracerebral infusion enhanced survival and maturation of newly born oligodendroglial progenitor cells in the normal corpus callosum, and accelerated oligodendroglial regeneration in lysolecithin-induced corpus callosum demyelinative lesions. Western blot analysis showed a robust upregulation of endogenous PEDF in the corpus callosum upon lysolecithin-induced demyelination. Our results document previously unrecognized oligodendrotrophic effects of recombinant PEDF on the adult SVZ and corpus callosum, demonstrate induction of endogenous CNS PEDF production following demyelination, and make PEDF a strong candidate for pharmacological intervention in demyelinative diseases.

Current therapies and investigational drugs for peripheral arterial disease

Peripheral artery disease (PAD) is associated with elevated morbidity and mortality with cardiovascular (CV) disease. The guideline recommends smoking cessation and antiplatelet/antithrombotic drugs for asymptomatic and symptomatic PAD patients. It also recommends that PAD patients with critical limb ischemia (CLI) should be considered to receive endovascular and open surgical treatment for limb salvage. Although PAD patients with CLI receive these treatments, they are sometimes unable to deliver sufficient blood flow to eliminate their symptoms. Thus specific strategies are needed to promote enough blood flow. To establish the effective method, many investigations have been performed using cell-based therapy. Endothelial progenitor cells, mononuclear cells and mesenchymal stem cells have been well investigated in clinical settings. To induce angiogenesis, vascular endothelial growth factor, fibroblast growth factor and hepatocyte growth factor (HGF) have also been transfected in PAD patients. Among them, HGF is the most promising factor because it can induce angiogenesis without the induction of vascular inflammation and increased permeability. In this review article, we summarize current treatments and investigational drugs of PAD.

Role of Central Nervous System Periostin in Cerebral Ischemia

Background and Purpose— Although periostin, an extracellular matrix glycoprotein, plays pivotal roles in survival, migration, and regeneration in various cells, its expression and function in the brain are still unknown. Here, we investigated the expression and role of periostin in the ischemic brain. Methods— Expression of full-length periostin (periostin 1 [Pn1]) and its splicing variant lacking exon 17 (periostin 2 [Pn2]) was examined by real-time reverse transcription polymerase chain reaction (RT-PCR), Western blotting, and immunohistochemical staining in male C57BL6/J mice. The actions of periostin were examined in adult primary neuronal culture and in a transient middle cerebral artery occlusion (tMCAo) model. Results— Expression of Pn2, but not of Pn1, mRNA was markedly changed after tMCAo. Pn2 mRNA was decreased in the ischemic core at 3 hours after ischemia. At 24 hours, Pn2 mRNA was significantly increased in both the peri-ischemic and ischemic regions. Periostin was mainly observed in neurons in normal brain. However, neuronal expression of periostin was decreased temporarily in the ischemic region, but increased in astrocytes and around endothelial cells at 24 hours after tMCAo. Of importance, intracerebroventricular injection of Pn2 resulted in a significant reduction in infarct volume at 24 hours after tMCAo associated with phosphorylation of Akt. Also, the Pn2-treated mice survived longer until 1 week after tMCAo. Pn2 significantly inhibited neuronal death under hypoxia and stimulated neurite outgrowth. Conclusions— Here, we demonstrated that periostin was expressed in the brain, and exogenous Pn2 exhibited neuroprotective effects and accelerated neurite outgrowth. Additional studies on periostin may provide new insights into the treatment of ischemic stroke.