[AIM] M1 microglia influence astrocytic neuroprotective functions, including engulfment of cell debris. Recently, extracellular zinc has been shown to aggravate M1 phenotype in microglia through intracellular zinc accumulation and reactive oxygen species (ROS) generation. Here, we investigated whether zinc-enhanced M1 microglia affects the astrocytic engulfing activity.

[METHODS] Mouse primary astrocytes were preincubated with microglial-conditioned medium (MCM) collected from M1 microglia induced by lipopolysaccharide (LPS) after ZnCl₂ treatment in the presence of TPEN, a membrane permeable zinc chelator, or Trolox, a ROS scavenger, and then incubated with fluorescent latex beads. P2X7 receptors (P2X7R) mRNA level in astrocytes was measured by real-time PCR.

[RESULTS] MCM from M1 microglia increased the astrocytes bead uptake. This increased uptake activity was suppressed when MCM from LPS-induced M1 microglia pretreated with ZnCl₂ was applied to astrocytes, which was further abolished by TPEN and Trolox. In addition, P2X7R mRNA level was increased in astrocytes treated with MCM from M1 microglia, but not in the M1 microglia pretreated with ZnCl₂.

[CONCLUSION] These results suggest zinc pretreatment abolishes the ability of M1 microglia to increase the engulfing activity in astrocytes via alteration of astrocytic P2X7R.

The spinal dorsal horn (SDH) receives somatosensory inputs from the periphery and descending pain modulatory inputs from several brain regions including the locus coeruleus (LC). Recent progress has been made in understanding neuronal circuits in the SDH, but the role of astrocytes, one type of glial cells, in somatosensory information processing and behavior under physiological conditions is entirely unknown. Here, by establishing a method to monitor SDH astrocytic activities using an in vivo Ca²⁺ imaging technique, we revealed that superficial SDH astrocytes were activated following noxious stimulation by intraplantar capsaicin injection and that the astrocytic responses required activation of a_1A-adrenergic receptors (a_1A-AR) through descending noradrenergic signaling from the LC. Pharmacological inhibition of LC–SDH noradrenergic pathway and selective knockdown of a_1A-AR in superficial SDH astrocytes prevented capsaicin-induced pain hypersensitivity to light mechanical stimulation. Moreover, pharmacological activation of a₁-AR in superficial SDH astrocytes was sufficient to induce mechanical pain hypersensitivity. Our findings demonstrate for the first time the potential ability of superficial SDH astrocytes to modulate mechanosensory behavior as a non-neuronal gate for the descending noradrenergic pathway from the brain.

Amyotrophic lateral sclerosis (ALS) is a motor neuron specific neurodegenerative disease. Accumulation of mutant Cu/Zn-superoxide dismutase (SOD1) protein aggregate in the spinal motor neurons is a common pathological hallmark in several types of ALS animal models and patients. The glymphatic system is a waste clearance system in the central nervous system: the directional flow of the cerebrospinal fluid (CSF) through the perivascular into interstitial spaces and the perivascular localization of aquaporin-4 (AQP4) promote its directional flow. Previously we reported that the AQP4 localization is aberrant and its expression is highly upregulated in SOD1-ALS mice during the progression of ALS symptoms (Watanabe et al., Neurosci Res, 133, 48-57, 2017). In the present study, we found the increase in the abnormal SOD1 protein deposition in SOD1-ALS/AQP4 knockout mice and the clearance of the protein from the spinal cord was slowed in AQP4 knockout mice. When we injected fluorescent labeled ovalbumin into the cisterna magna, the solute accumulation was greater in the SOD1-ALS mice than that in the wild-type mice. Our study suggests that the aberrant AQP4 distribution in the ALS model mice disrupts directional CSF flow and accelerates accumulation of toxic proteins in the spinal cord.

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.

Rapid intraperitoneal water injection induces acute hyponatremia that creates an osmotic gradient driving for water entry into the brain, leading to subsequent cerebral edema. Paravascular spaces, which are covered by astrocyte end-feet, have been suggested to participate in the fluid circulation in cerebral cortex, however, it has not been clarified whether they morphologically change during the edema formation. Here we have established an in vivo imaging method with a closed cranial window under isoflurane anesthesia to observe paravascular spaces and astrocytes using CAG-GFP transgenic mice. We simultaneously monitored electro-corticogram (ECoG) and other physiological parameters, such as cerebral blood flow (CBF), heart rate, and arterial blood pressure, to examine their responses up to 40 min after the bolus injection of distilled water equal to 10% of body weight. We first confirmed that water injection indeed increased brain tissue water content, which was alleviated in aquaporin-4 (AQP4) knockout mice. While control and AQP4 knockout mice did not differ in the cell swelling of astrocyte, even AQP4 are expressed in the astrocyte end-feet, paravascular space closure was prevented in AQP4 knockout mice. Furthermore, the ECoG power reduction in AQP4 knockout mice was less than that in control mice. These results implicate that the regulation of paravascular spaces may play roles in modulating brain water circulation and brain edema formation, which might be controlled by AQP4.

Although prostaglandin D₂ (PGD₂) represents anti-angiogenic role in tumor model, its role in physiological and pathological angiogenesis still remain unknown. We here evaluated the role of PGD₂ on retinal angiogenesis using genetically modified mice. In postnatal 8th day retina of WT, lipocalin-type PGD synthase (L-PGDS) was expressed in endothelial cells. Gene deficiency of L-PGDS impaired the physiological angiogenesis of retina, accompanied with increased mRNA expression of pro-angiogenic factor VEGF. In vitro study showed that L-PGDS inhibition elevated the hypoxia-induced VEGF expression, which was inhibited by treatment of a PGD₂ metabolite 15d-PGJ₂. We next generated a pericyte deficiency-induced retinal angiogenesis model by injection of anti-PDGFRβ antibody. In P8 retina of WT, the injection of antibody induces inflammation in retina, and infiltrating macrophages expressed hematopoietic PGD synthase (H-PGDS). Gene deficiency of H-PGDS or PGD receptor DP accelerated the angiogenesis. This phenomenon was accompanied with increased mRNA expression of one of the chemokines, Stromal derived factor 1α. In isolated macrophage, hypoxia increased the expression of cytokines, wich was inhibited by adding receptor inhibitor. Taken together, L-PGDS promotes physiological angiogenesis and H-PGDS attenuate pathological angiogenesis in mouse retina.

Myocardial atrophy, characterized by the decreases in size and contractility of cardiomyocytes, is caused by severe malnutrition and/or mechanical unloading. Extracellular adenosine 5'-triphosphate (ATP), known as a danger signal, is recognized to negatively regulate cell volume. However, it is obscure whether extracellular ATP contributes to cardiomyocyte atrophy. Here, we report that ATP induces atrophy of neonatal rat cardiomyocytes (NRCMs) without cell death through P2Y2 receptors. ATP led to overproduction of reactive oxygen species (ROS) through increased amount of NADPH oxidase (Nox) 2 proteins, due to increased physical interaction between Nox2 and canonical transient receptor potential 3 (TRPC3). This ATP-mediated formation of TRPC3-Nox2 complex was also pathophysiologically involved in nutritional deficiency-induced NRCM atrophy. Strikingly, knockdown of either TRPC3 or Nox2 suppressed nutritional deficiency-induced ATP release, as well as ROS production and NRCM atrophy. Taken together, we propose that TRPC3-Nox2 axis, activated by extracellular ATP, is the key component that mediates nutritional deficiency-induced cardiomyocyte atrophy.

Heparan sulfate（HS）is a sulfated linear polysaccharide at the cell surface and in the extracellular matrix. HS plays an important role in various physiological and pathophysiological processes. Although previous studies showed the existence of HS in skeletal muscles, the roles of HS in these tissues remain unclear.

　First, we examined the role of HS in the differentiation of muscle cells using C2C12 cells, a mouse myoblast cell line. CRISPR/CAS9 technology was used to delete Ext1, which encodes a heparan sulfate synthase. HS deletion dramatically impaired myoblast differentiation, demonstrating the essential role of HS in vitro. In order to confirm the importance of HS in vivo, we created skeletal muscle specific Ext1 knockout mice by Cre-loxP system (cKO). Muscle weakness of cKO was observed in treadmill tests and wire hang tests. Contraction of isolated soleus muscles from cKO was also impaired. Histological observation revealed that the cross sectional areas of various muscles were smaller in cKO. Electromicroscopic observation showed that myofibrils were thinner in cKO. Finally, we examined muscle differentiation after muscle injury by BaCl₂ injection to tibialis anterior muscle (TA). We showed the reduced expression level of myosin heavy chain and the increased number of centronucleated cells in cKO TA, indicating that the muscle regeneration after injury was attenuated in cKO.

　These results demonstrate that HS plays an important role in skeletal muscle, especially in differentiation.

Mesenchymal stem cells are defined in vitro by the ability to form fibroblastic colony and differentiate into adipocytes, osteocytes, and chondrocytes. Although PDGFRα⁺ cells are thought to be the origin of mesenchymal stem cells in various tissues, their roles in each organ have not been elucidated. Here, we compared characters of PDGFRα⁺ cells derived from several organs such as lung, liver, small intestine, heart, subcutaneous fat, and skeletal muscle to clarify their specific functions in each organ.

We first compared differentiation potentials of PDGFRα⁺ cells residing in various tissues. We cultured PDGFRα⁺ cells isolated from each tissue by FACS and induced them to differentiate into several mesenchymal lineages. Consequently, each PDGFRα⁺ population showed distinct differentiation potential. To investigate their roles in respective organs, we performed RNA-Seq and revealed that PDGFRα⁺ cells have gene expression patterns unique to their original organ, suggesting that they have specific functions depending on the tissue where they reside. Among these tissues, we focused on skeletal muscle because PDGFRα⁺ cells in muscle have been shown to be essential for homeostatic muscle maintenance. Using Using RNA-seq data of PDGFRα⁺ cells from various tissues of young and aged mice, we identified several genes that are specifically expressed in PDGFRα⁺ cells derived from young muscles. We expect that these genes play important roles to maintain muscle integrity and we will pursue a study to elucidate their functions.

Preclinical predictions using cell assay system is a major issue in drug development. With advances in iPS cell technology, human iPS cell-derived cardiomyocytes (hiPSC-CMs) are a valuable tool to characterize the pharmacological effects of drugs on heart cells. However, current approaches to evaluate cardiac contractile function in vitro are limited to low-throughput methods. We here test middle-through put and noninvasive assay system with motion field imaging (SI8000 system, Sony corporation) using high speed video image of hiPSC-CMs.

Human iPSC-CMs were kept at 37°C, 5% CO₂ and beating cells were recorded as sequential phase-contrast images. Motion vectors of hiPSC-CMs were analyzed by the SI8000 system. After the measurement, tissue-types (atrial or ventricular) were determined by immunostaining using anti-MLC2a and anti-MLC2v, respectively, and compared the motion vector traces. Contraction and relaxation velocities in atrial-like myocytes were faster than those in ventricular-like myocytes. Application of 100 nM isoproterenol induced the same trends on contractile functions in each cell-type of hiPSC-CMs, but beta2-antagonist blocked the effects only in atrial-like myocytes, indicating that the statistical comparison of these data allows us to identify tissue-types of hiPSC-CMs. Our results suggest a substantial potential to increase accuracy of pharmacological assessment.

Introduction: Anti-influenza drug oseltamivir delayed the atrial conduction and prolonged the atrial effective refractory period (AERP) in guinea pig hearts, and reduced the inducibility of burst pacing-induced atrial fibrillation (Af) in Langendorff-perfused rabbit hearts.

Methods: The canine persistent Af model (n=6) was prepared for the further in vivo characterization of the antiarrhythmic effect of oseltamivir. Moreover, we evaluated electropharmacological effect of oseltamivir on atria using the halothane-anesthetized dog (n=4). These results were compared with those of pure Na⁺ channel blocker pilsicainide (n=6 and n=4, respectively). Furthermore, we evaluated the action of oseltamivir on ion channels expressed in HEK293 and CHO cells using the whole-cell patch-clamp technique (n=3). 

Results: Oseltamivir (3 and 30 mg/kg) terminated the Af in 1 and 5 out of 6 animals, respectively, whereas pilsicainide (3 mg/kg) did it in 2 out of 6. Oseltamivir (0.3, 3 and 30 mg/kg) and pilsicainide (1 and 3 mg/kg) delayed the inter-atrial conduction in a dose- and frequency-dependent manner. Oseltamivir prolonged the AERP in a dose-dependent but frequency-independent manner, whereas pilsicainide did it in a dose- and frequency-dependent manner. IC₅₀ values of oseltamivir against I_K,ACh, I_Kr, I_Na, I_CaL and I_Kur were 179, 225, >1000, >1000 and >1000 μM, respectively. 

Conclusion: Oseltamivir can exert potent anti-Af effect through multi-channel inhibitory action, of which electrophysiological profile may be different from that of pilsicainide.

Goreisan is often used for gastrointestinal symptoms associated with bacterial and viral infections, to care diarrhea and to prevent general dehydration. Although several clinical reports have shown the effectiveness of Goreisan, pharmacological properties and underlining mechanism of Goreisan has not been clear. In this study, therefore, we investigated the antidiarrheic effect of Goreisan using a mouse model of enterocolitis induced by Lipopolysaccharide (LPS). Goreisan did not affect TNF-α mRNA expression, but markedly improved tissue injury and diarrhea scores. On the other hand, aquaporin-3 (AQP3) is expressed in the intestinal epithelium, and responsible for the absorption of water in the intestinal tract. Interestingly, both AQP3 mRNA and protein expression in the intestinal epithelium in LPS-treated group were significantly reduced, and Goreisan inhibited this decrease in AQP3. Decrease in AQP3 is thought to be associated with development of diarrhea, and therefore, Goreisan is estimated to have improved diarrhea symptoms by regulating the expression of AQP3. These results confirmed the effectiveness of Goreisan for infectious gastroenteritis, and it is also suggested a new effect of Goreisan.

Chronic inflammation of the retina involves in the etiology of choroidal neovascularization (CNV), but the mechanisms are still not fully understood in detail. Progranulin is a growth factor secreted from myeloid cells and the deficiency of that results in aberrant inflammation in the central nerve system. The purpose of this study was to investigate the role of progranulin in the pathology of CNV.

By using grn knockout (Grn^−/−) and wild-type (Grn^+/+) mice with laser-induced CNV model, we evaluated the area of CNV and the accumulation of macrophages around CNV. To evaluate inflammation of macrophages, we constructed macrophage cell lines (RAW264.7) in which the expression of progranulin was knocked-down by RNA interference. Expression level of VEGF-A, IL-1β and C3 were evaluated by Western blotting.

At 14 days after laser injury, average of CNV area and number of Iba-1⁺ cells around CNV in the Grn^−/− mice significantly increased compared with those in Grn^+/+. When progranulin was knocked down, the expression level of VEGF-A, IL-1β and C3 were increased in RAW264.7 cells.

These findings indicate that progranulin deficiency might promote the progression of CNV via aberrant activation of macrophages and microglial cells.

Macrophage plasticity is essential for liver wound healing; however, the mechanisms of macrophage phenotype switch are largely unknown. Dendritic cells (DCs) are critical initiators of innate immune responses and orchestrate inflammation following hepatic injury. We have shown that PGE₂/EP3 promotes liver repair after hepatic ischemia-reperfusion (I/R). The present study examined whether signaling via EP3 in DCs regulates macrophage plasticity during liver repair by subjecting EP3-deficient (EP3^-/-) and wild-type (WT) mice to hepatic I/R. Compared with WT mice, EP3^-/- mice showed delayed liver repair as indicated by increased levels of ALT and hepatic necrosis, which accompanied by reduced expression of hepatic growth factors. Flow cytometry analysis revealed that accumulation of Ly6C^low reparative macrophages and monocyte-derived DCs (moDCs) was suppressed in EP3^-/- livers. Adoptive transfer of moDCs from EP3^-/- mice resulted in impaired repair, along with increased Ly6C^high inflammatory macrophages. When bone marrow macrophages (BMMs) co-cultured with moDCs, BMMs from WT mice, but not from EP3^-/- mice up-regulated expression of genes related to a reparative macrophage phenotype. In the presence of an EP3 agonist, interleukin (IL)-13 derived from moDCs drove BMMs to increase expression of genes characteristic of a reparative macrophage phenotype. The results suggest that EP3 signaling in moDCs facilitates liver repair by inducing IL-13-mediated switching of macrophage phenotype from pro-inflammatory to pro-reparative.

Sepsis is systemic inflammatory response syndrome caused by invasive infection. Although it is known that prostaglandin (PG)F_2α level is elevated in the plasma of the patients with sepsis, its role in the sepsis remains unclear. We aimed to investigate the role of PGF_2α receptor (FP) signaling in lipopolysaccharide (LPS)-induced sepsis using FP receptor antagonist AL8810 in mice. Sepsis was induced by intraperitoneal injection of LPS (5 mg/kg). AL8810 (10 mg/kg) was intraperitoneally administered at 30 min before LPS injection. Mice were monitored to detect the response to LPS for 24 hours. LPS administration promoted PGF_2α production in peritoneal lavage fluid (PLF). At 6 hours after LPS administration, the number of macrophages and neutrophils in PLF was increased, as compared with naïve mice. AL8810 administration enhanced neutrophil migration, but not macrophage migration, in PLF. At 24 hours after injection, there was no difference in number of these cells between LPS and/or AL8810-administered mice. At 24 hours after LPS administration, the mRNA expression of proinflammatory cytokines such as IL-6, TNF-α, IL-1β, and CXCL2 in lung and liver was elevated. Conversely, they were decreased in AL8810-administered mice. It is known that IL-10 decreased excessive inflammatory responses in the acute phase of sepsis. At 3-6 hours after LPS administration, IL-10 levels in PLF were increased, as compared with naïve mice. AL8810 administration enhanced IL-10 production further. In addition, immunostaining showed that Gr-1-positive neutrophils in PLF expressed IL-10. Then, anti-IL-10 antibody administration increased LPS-induced IL-6 and CXCL-2 expression as well as AL8810-decreased these gene expressions. The findings suggest that FP receptor antagonist attenuated LPS-induced sepsis by increasing neutrophil-derived anti-inflammatory cytokine IL-10 production.

[Background] Macrophage (Mφ) plays crucial roles in innate immunity and its dysfunction is involved in the pathogenesis of chronic inflammatory diseases such as arteriosclerosis and diabetes. Cytokine secretion and phagocytosis are main functions of Mφ and modulated by the activity of ion channel, ionotropic purinergic P2X7 receptor.

Caveolin-1 (Cav-1) enables effective intracellular Ca²⁺ signaling by accumulating Ca²⁺ channels and their associated proteins within caveolae structure. In this study, the functional coupling between Cav-1 and P2X7 receptor was analyzed using Cav-1 knockout (Cav-1 KO) mice.

[Methods] In murine bone marrow-derived Mφ (BMDM), expression of Cav-1 was analyzed by real-time PCR and Western Blotting. Localization of Cav-1 and P2X7 receptor was analyzed with total internal reflection fluorescence microscope (TIRFM). Ca²⁺ influx and K⁺ efflux through P2X7 receptor were measured with Fluo-4 AM and APG-2, respectively. Furthermore, activation of P2X7 receptor was measured by nuclear dye (TOPRO-3) uptake.

[Results] The expression of Cav-1 was increased by LPS (lipopolysaccharide, 1 μg/mL)-induced inflammatory stimulation in BMDM. Thereafter, Cav-1 was co-localized with P2X7 receptor on the cell membrane. ATP (1 mM)-evoked TOPRO-3 uptake was increased in BMDM derived from Cav-1 KO mice compared to WT. Furthermore, Ca²⁺ influx and K⁺ efflux following ATP stimulation were increased in Cav-1 KO compared to WT. These results suggest that the activity of P2X7 receptor is enhanced and thus Ca²⁺ influx and K⁺ efflux are facilitated in BMDM derived from Cav-1 KO mice.

[Conclusion] Cav-1 negatively regulates the activation of P2X7 receptor and modulates immune responses in Mφ. This study may contribute to the development of novel drugs for chronic inflammatory diseases.

The primary cilium is a microtubule-based sensory organelle that transduces its signals through specifically distributed receptors and ion channels on the ciliary membrane. The proximal region of the ciliary axoneme is surrounded by an invaginated membrane, called ciliary pocket. Primary cilium is formed during the G0/G1 phase in many cell types, including neural progenitor cells, and is resorbed as the cells re-enter cell cycle. Dysregulation of the ciliary dynamics is associated with hereditary disorders, such as microcephaly. Tctex-1, a cytoplasmic dynein light chain, has a dynein-independent role when it is phosphorylated at Thr94. We have shown that (T94)Tctex-1 phosphorylated by the action of insulin-like growth factor 1 accelerates branched actin organization and clathrin-dependent endocytosis at the ciliary pocket. The machinery was critical for ciliary resorption, cell cycle re-entry, and self-renewal of the neural progenitor cells in the developing neocortex. However, it remains unclear how Tctex-1 regulates the endocytosis. In the present study, we identified microtubule-associated serine/threonine kinase 4 (MAST4), a function-unknown protein, as a binding protein to Tctex-1. In retinal pigmented epithelial cells (RPE-1), a model cell line for cilia researches, we found that knockdown of MAST4 suppressed endocytosis, ciliary resorption, and cell cycle re-entry, emphasizing on the significance of phospho-(T94)Tctex-1-MAST4 pathway as a part of such biological events.

Endoplasmic reticulum (ER) is responsible for protein biosynthesis and folding, but accumulation of unfolded proteins leads to disturbance of ER proteostatis and subsequent clinical pathologies including diabetes, neurodegenerative disease and cancer. Chemical chaperones are chemical compounds that help protein folding and suppress aggregation, and receiving increased attention as potential therapeutic approaches for ER stress-related diseases. In this study, we established a novel ER stress reporter cell line and identified compound X as a chemical chaperone from the 217,765-compound chemical library. Compound X directly binds to secreted or membrane proteins and inhibits protein aggregation during tunicamycin induced ER stress. Furthermore, compound X significantly prevented cell death caused by chemically induced ER stress and by an aggression-prone mutant prion protein. These results show the therapeutic potential of compound X as a chemical chaperone that can ameliorate ER stress-related diseases.

Vacuolar-type H⁺- ATPase (V-ATPase), a multi-subunit protein complex, has two distinct functions on lysosomes: acidifying the lysosomal lumen and controlling mTOR-S6K (mTORC1) signaling via Ragulator. Both functions are crucial for several biological processes. However, little is known about how the functions are coordinated and whether V-ATPase also regulates mTOR-AKT (mTORC2) signaling. We found that knocking down (KD) of a subunit of V-ATPase in human induced pluripotent stem cells (hiPSCs) impaired its functions: increasing lysosomal pH and decreasing mTORC1 signaling. Unexpectedly, the KD also attenuated mTORC2-AKT signaling. Treatment of hiPSCs with bafilomycin A1, a specific inhibitor of V-ATPase proton pump activity, increased lysosomal pH as expected, and decreased both mTORC1 and mTORC2 signaling activities. Therefore, in addition to mTORC1, V-ATPase seemingly regulates the mTORC2-AKT. We are now investigating how V-ATPase regulates mTORC2. Furthermore, we are examining the effects of V-ATPase inhibition on the mTOR signaling in vivo. We will discuss our results in this meeting.

Mucociliary transport, which is a host-defense mechanism of the airway, consists of the mucous layer and the beating cilia lining on the airway surface. Although beating of cilia is the most important in this system, the regulation of beating is not fully understood. Among a few pharmacological stimuli which has been known to increase the ciliary beating, ATP is one of the most effective. However, ATP is not useful expectorant, because of its wide-spread pharmacological activity. In the present study, therefore, we have examined the purinergic receptor, which is involved in the increase in ciliary beating by ATP, in isolated mouse airway cilia. ATP significantly increased both ciliary beat frequency (CBF) and ciliary bend angle (CBA), whereas ADP increased only CBF. In contrast, adenosine and UTP did not increase CBF and CBA. Interestingly, increase in CBF by ATP was abolished by BAPTA-AM, but CBA was not affected, suggesting that ATP differently regulates CBF and CBA. Finally, increase in CBF by ATP was completely inhibited by MRS2179, a P2Y₁ receptor antagonist. Therefore, we may propose P2Y₁ receptor agonist as a new airway clearance stimulator, which increases ciliary beating.

Not published

Schizophrenia is a severe mental illness that affects about 1% of the population. Genetic and environmental factors contribute to the development of schizophrenia. However, the exact pathoetiology remains unclear. We generated Rho GTPase-activating protein 10 (ARHGAP10) mutant mice carrying similar variations found in Japanese schizophrenia patients. In the present study, we examined spatiotemporal expression of ARHGAP10 mRNA in the brain of mice. The expression levels of ARHGAP10 mRNA were higher in the striatum (ST) and nucleus accumbens (NAc) than those in other brain regions. We performed a series of behavior test to evaluate cognitive and emotional function in ARHGAP10 mutant mice. They showed an increase in anxiety level, and manifested potentiation of methamphetamine-induced hyperlocomotion and visual discrimination task. Morphological analysis revealed that methamphetamine-treated ARHGAP10 mutant mice showed an increase in the number of c-Fos-positive-cells in the dorsal medial striatum (dmST) and NAc core than those in wild-type littermates. Golgi staining indicated that ARHGAP10 mutant mice showed an increase in neuronal complexity and spine density in the same brain regions compared to the wild-type mice. These results suggest that ARHGAP10 gene variations may lead to the development of cognitive and emotional deficits with morphological abnormality in the dmST and NAc core neurons.

Role of central nervous systems in regulation of energy homeostasis including feeding behavior has paid much attention these days, but their mechanisms are still unclear. Since the hypothalamus is a key regulator in feeding behavior, we investigated the role of dopaminergic functions in the lateral hypothalamus (LH) in feeding behavior. Both food intake and glucose injection increased dopamine levels in the LH. When retrograde tracer Fluoro-Gold (FG) was injected to the LH, the FG-positive cells were present in the ventral tegmental area (VTA) and the substantia nigra pars compacta (SNC), which were tyrosine hydroxylase-positive. Injections of both dopamine D₁ (SKF 38393) and D₂ (quinpirole) receptor agonists into the LH decreased food intake, which were antagonized by respective antagonist. When the dopaminergic activity in the LH was inhibited by a Ca²⁺ channel inhibitor pregabalin, pregabalin inhibited the increase of dopamine levels induced by glucose injection, and it also increased food intake. These results have indicated that food intake activates dopamine neurons projecting from the VTA and the SNC to the LH through increase in the blood glucose levels. Moreover, it is suggested that the promotion of dopaminergic functions in the LH terminates feeding behavior by the stimulation of dopamine D₁ and D₂ receptors.

Temporal lobe epilepsy (TLE) is the most common form of epilepsy. The hippocampus, located in the mesial temporal lobe, is implicated in the development of TLE. However, mechanisms underlying hippocampal epileptogenesis in TLE remain unclear. Here, we investigated whether ocular albinism 1 gene product (GPR143), which is highly expressed in the hippocampus, is involved in hippocampal epileptogenesis in TLE. We induced limbic seizures by administration of kainic acid. We found that seizure scores reduced in Gpr143-gene deficient (GPR143-KO) mice compared to wild-type (wt) mice. Next, we performed histological examination. To evaluate granule cell reorganization, we measured the width of the granule cell layer 6 days after seizure induction. The granule cell layer dispersed less in GPR143-KO mice than wt mice. We further found that an increased number of survival neurons and a morphological change of microglia in the CA3 region and its surrounding area in GPR143-KO mice, respectively. Thirty days after seizure induction, we observed aberrant sprouting of granule cell axons in the molecular layer. We immunohistochemically assessed the distribution of synaptoporin, a protein that is often present in the mossy fiber boutons, in the molecular layer. The intensity of ectopic synaptoporin signals decreased in GPR143-KO mice, suggesting that mossy fiber sprouting occured less compared to wt mice. Thus, our findings indicate that GPR143 is involved in the modulation of seizure phenotype and hippocampal epileptogenesis in TLE.

[Background]

Histidine-rich glycoprotein (HRG) is 75 kDa plasma glycoprotein produced from the liver. In previous study, we reported that HRG treatment prevents lethality of sepsis model mice and HRG regulated spherical shape change, passage of microcapillary and production of extracellular ROS on the human neutrophils. Next, we analyzed functional evaluation of neutrophils spheroidized by HRG.

[Method]

Phagocytosis analysis: We quantified the area of fluorescence-labeled bacteria by pHrodo in the neutrophils. Viability analysis: The number of intact neutrophils were counted by the staining with calcein-AM. Determination of extracellular ROS production: After adding isoluminol, HRP and each test reagent to neutrophils, the intensity of luminescence at 30 minutes were measured.

[Result]

Neutrophils treated with HRG showed increased activity of phagocytosis in a dose-dependent manner. HRG also induced high survival rate. When Zymosan A was added to neutrophils, the increased ROS production was observed in the presence of HRG.

[Discussion]

The neutrophils treated with HBSS or HSA are firmly attached to the bottom of the plate and being stimulated with regard to ROS production. In contrast, HRG maintained the spherical shape of neutrophils, phagocytic activity and responsiveness to Zymosan A. These results suggested that HRG may act on neutrophils to suppress excessive adhesion to vascular endothelium under normal condition and induce the functional activation when neutrophils meet bacteria.

Lymphangiogenesis is associated with the growth of endometriosis. In this study, we examined the role of vascular endothelial growth factor (VEGF) receptor 1 (VEGFR1) signaling in lymphangiogenesis and tissue growth in an endometriosis model. Using wild-type (WT) and VEGFR1 tyrosine kinase (TK) deficient mice, endometrial fragments were implanted into the peritoneal wall of mice. Endometrial tissue growth and lymphangiogenesis as indicated by lymphatic vessel density were determined. Endometrial fragments from wild-type (WT) mice transplanted into in host WT mice (WT→WT) grew with increased lymphangiogenesis accompanied by increases in pro-lymphangiogenic factors, VEGF-C and VEGF-D. The implant size and lymphangiogenesis were reduced in the TK^-/-→TK^-/-. Immunofluorescence demonstrated that both VEGF-C and VEGF-D were expressed in both CD11b⁺ and S100A4⁺ cells. When cultured bone marrow-derived macrophages and fibroblasts were stimulated with placental growth factor (PlGF), a specific agonist for VEGFR1, the mRNA levels of VEGF-C and VEGF-D were increased in a VEGFR1 dependent manner. A VEGFR3 kinase inhibitor significantly suppressed the size of implants, lymphangiogenesis, pro-lymphangiogenic factors, and accumulation of CD11b⁺ and S100A4⁺ cells. These results suggest that VEGFR1 signaling in macrophages and fibroblasts promote the growth and lymphangiogenesis in endometrial tissue. Therefore, VEGFR1 blockade is a potential treatment for endometriosis.

Lymphangiogenesis has functional consequences not only for lymphatic transport, but also for inflammation resolution. Thromboxane A₂ (TxA₂) has been suggested to involve not only in induction of inflammation, but also in resolution of inflammation. We investigated the functional role of TxA₂ receptor (TP) signaling in inflammation-associated formation of newly lymphatic vessels. Lymphangiogenesis in the diaphragm of TP knockout mice (TPKO) or their wild-type (WT) counterparts was induced by repeated intraperitoneal injection of LPS. Compared with WT, LPS-induced lymphangiogenesis in TPKO was suppressed, which was accompanied by reduced expression of vascular endothelial growth factor (VEGF)-C and VEGF-D in CD11b+ and CD4+ cells in diaphragm tissue. TP was expressed in CD11b+ and CD4+ cells, but not in LYVE-1+ cells (lymphatic vessels). U46619, an agonist for TxA₂, did not proliferate cultured lymphatic endothelial cells. As compared with controls, mice with macrophage TP receptor deletion showed attenuation of lymphangiogenesis with reduced expression of VEGF-C and VEGF-D. When fluorescein isothiocyanate (FITC)-dextran was injected into the peritoneal cavity, the amount of residual FITC-dextran in macrophage-specific deletion of TP receptor was greater than that in controls. The same was true for mice with T cell TP receptor deletion. The present results suggest that TP signaling in macrophages and T cells plays a critical role in inflammation-related lymphangiogenesis and drainage function of lymphatics in the diaphragm.

Newly formation of blood and lymphatic vessels is involved in the development of endometriosis. We have demonstrated that calcitonin gene-related peptide (CGRP) promotes wound healing and wound-associated formation of blood and lymphatic vessels via receptor activity-modifying protein 1 (RAMP1), a subunit of the CGRP receptor. In the present study, using wild-type (WT) mice and RAMP1 deficient (RAMP1^-/-) mice, we examined whether RAMP1 plays a role in the growth of endometriosis by angiogenic responses. Ectopic endometriosis model was created by transplantation of endometrial tissue fragments from donor mice into the peritoneal wall of host mice. The sizes and density of blood and lymphatic vessels in the RAMP1^-/- implants from host RAMP1^-/- mice (RAMP1^-/-→RAMP1^-/-)  were reduced as compared with the WT→WT. The mRNA levels of markers for blood and lymphatic vessels as well as growth factors for angiogenesis and lymphangiogenesis in the RAMP1^-/-→RAMP1^-/- were lower than those in the WT→WT. Immunofluorescence demonstrated that RAMP1 was expressed in CD11b⁺ and S100A4⁺ cells, and these cells also co-localized with VEGF-A, VEGF-C, and VEGF-D. Cultured macrophages and fibroblasts increased the mRNA levels of VEGF-A, VEGF-C, and VEGF-D in a RAMP1 dependent manner. These results suggest that RAMP1 signaling in macrophages and fibroblasts is critical for the growth of endometriosis by promoting angiogenesis and lymphangiogenesis.

Central post-stroke pain (CPSP) is one of the secondary diseases of cerebral stroke. However, the detailed mechanism remains unclear. Recently, it is reported that the ablation of supraspinal orexin neurons induced mechanical allodynia in mice. In this study, we tested the involvement of supraspinal orexin system in the regulation of CPSP. Male ddY mice (5 weeks old) were subjected to 30 min of bilateral carotid artery occlusion (BCAO). Colocalization of fluorogold (a retrogradely transported tracer) with orexin-A were determined by double-immunofluorescence. Mechanical allodynia was measured by von Frey filament test. Intrathecal (i.t.) injection of fluorogold was colocalized with orexin-A positive cells in the hypothalamus. On day 3 after BCAO, the withdrawal responses to mechanical stimuli were significantly increased and prepro-orexin was decreased as compared with sham. The BCAO-induced mechanical allodynia suppressed by the intracerebroventricular injection of orexin-A. This effect of orexin-A was significantly inhibited by the i.t. injection of an orexin 1 or 2 receptor antagonist. These results suggest that the supraspinal orexin-A system mediated by the OX1R and OX2R play an important role in the regulation of CPSP.

Cyclic phosphatidic acid (cPA) is a naturally occurring phospholipid mediator found in mammalian tissues and cells. It has been reported that cPA has novel biological activities; attenuates traumatic brain injury-mediated neuronal cell death and inhibits chronic and acute inflammation. However, there are some seemingly unanswerable questions. In this study, our lab has successfully used Click chemistry approach to derivatize azide-tagged metabolically stabilized cPA analogue, 2-carba-cPA (2ccPA). Using affinity chromatography with 2ccPA beads, we successfully captured a potential target protein (30k) from the mouse microglial cell (SIM-A9). We then analyzed by LC-MS/MS and identified adenine nucleotide translocase 2 (ANT2) as a 2ccPA binding protein. We would like to discuss on potential roles of 2ccPA-ANT2 axis regulating microglial mediated neuroinflammation and related diseases.

This work was supported by the Japan Society for the Promotion of Science KAKENHI [grant number 19K07322] and by the Ito Foundation [grant number ken-1].

G-protein coupled receptors (GPCRs) are one of the largest membrane protein families in eukaryotes and mediates various important function of the cells. Recently, there is increasing evidence that several GPCRs may form complexes, and complexed GPCRs may mutually regulate their function. Previous reports including ours unveiled the interaction between two GPCRs expressed in neurons, type 1 metabotropic glutamate receptor (mGluR1) and gamma-aminobutyric acid B receptor (GABA_BR). mGluR1 is expressed particularly in cerebellum and known to play crucial roles in synaptic plasticity and motor learning. GABA_BR is widely expressed in central nervous system and regulates neuronal excitability. Our previous study suggested that GABA_BR modulates mGluR1-mediated synaptic plasticity in cerebellar Purkinje cell. In this study, we showed the mechanism underlying modulation between mGluR1 and GABA_BR. Biochemical analysis and molecular imaging assay showed that mGluR1 form complexes with GABA_BR at the cell membrane. Moreover, an assay monitored intracellular signaling transduction revealed functional interaction between these GPCRs. Our findings provide a novel insight into the regulatory mechanism of synaptic plasticity and motor learning, and indicate that complex formation and functional interaction between distinct GPCRs might have significant roles under physiological and pathophysiological conditions.

The vagus nerve is a parasympathetic nerve and plays a critical role in the regulation of autonomic functions to maintain homeostasis including heart rate, respiratory rhythms and gastric motility. It remains largely unknown about vagal physiological functions in naïve behavior due to technical limitations of vagal spike recordings. Here, we developed a vagal spike recording technique with a cuff-shaped electrode in a freely moving rodent animal while simultaneously monitoring central and peripheral bioelectrical signals. The spike rates of both afferent and efferent fiber types increased simultaneously following increased locomotion and were higher in novel environment than in familiar one. The electrode contains multiple recording sites to identify afferent and efferent vagal spikes. For manipulating vagus nerve spikes, we used two strains of transgenic mice expressing channelrhodopsin2, a photosensitive protein, in afferent and efferent vagal fibers. These results provide novel insights into vagal physiological function and make a new step forward to uncover a neurophysiological basis underpinning the brain-body axis.

Loss of orexin-producing neurons in the lateral hypothalamus causes the chronic sleep disorder narcolepsy-cataplexy. Narcoleptic humans suffer from two major symptoms, excessive sleepiness and cataplexy in the active phase, and these symptoms in mouse models are manifested as sleep/wakefulness fragmentation and SOREMs (direct transitions from wakefulness to REM sleep), respectively. The neuropeptides orexin-A (OXA) and orexin-B (OXB) act on two receptors orexin type-1 receptor (OX1R) and orexin type-2 receptor (OX2R). Orexin receptor agonists are expected to be of potential value for treating human narcolepsy. Here, to confirm the fundamental strategy aimed at improving narcoleptic symptoms, we examined the association between orexin receptor subtypes and these symptoms by intracerebroventricular (ICV) administration of the OX2R-selective agonist [Ala¹¹, _D-Leu¹⁵]-OXB in orexin knockout mice. OXA and [Ala¹¹, _D-Leu¹⁵]-OXB similarly decreased the number of SOREMs. Further, transition frequencies between NREM sleep and wake states in narcoleptic model mice were similarly decreased. We confirmed in vivo that [Ala¹¹, _D-Leu¹⁵]-OXB did not activate OX1R-expressing LC noradrenergic neurons by Fos staining. Therefore, OX2R-selective agonism is sufficient to ameliorate narcoleptic symptoms, both cataplexy and fragmentation of wakefulness in model mice. Activation of LC noradrenaline neurons expressing OX1R are not essential for suppression of these symptoms.

Microparticles (MPs) which are micro vesicles shed from the membrane of vascular and blood cells are considered as one of the causes of endothelial dysfunction in the diabetic vascular complications. In this study, we examined the effect of MPs derived from diabetes mice on vascular function, focusing on extracellular signal-regulated kinases (ERK)1/2-conteining MPs. MPs were prepared from streptozotocin (STZ)-induced diabetic mice (STZ), controls (Cont) and STZ and Cont mice treated with ERK1/2 inhibitor, PD98059 (PD). Vascular reactions and protein expressions were examined. STZ-derived MPs (STZ MPs) were found to have increased amounts of MP and to be attached to the endothelial cells as compared to Cont-derived MPs (Cont MPs). Furthermore, we found that ERK1/2 was contained in the MPs, especially STZ MPs. In addition, ERK1/2 activity and expression were increased in Cont vessels treated with STZ MPs. STZ PDMPs (PD-treated STZ derived MPs) improved the attenuated endothelial dependent relaxation in aortic rings. On the other hand, direct treatment of PD in STZ aortic rings did not improve the attenuated endothelial dependent relaxation. These results suggested that ERK1/2-containing MPs regulate ERK1/2 activity in blood vessels and cause endothelial dysfunction during diabetes.

Although there are several reports suggested that advanced glycation end-products (AGEs) cause vascular endothelial dysfunction, the direct relationship between AGEs and smooth muscle contractile function remains unclear. Therefore, we investigated the long-term effects of AGE-bovine serum albumin (AGE-BSA) on contractile responses in rat carotid arterial rings using organ-culture technique. After exposure of AGE-BSA (0.001-0.1 mg/mL) for approximately 1 day in carotid artery, concentration–response curves were investigated under endothelium denuded artery. Contractile responses of high K⁺ or serotonin did not alter among groups treated with and without AGE-BSA. Treatment with AGE-BSA (0.1 mg/mL) (vs. control; PBS) increased thromboxane A₂ analog-induced contraction, whereas decreased noradrenaline-induced contraction. The decreased noradrenaline-induced contraction by AGE-BSA was prevented by co-treated with organic cation transporter-3 (OCT-3) inhibitor corticosterone. The protein expression of OCT-3 in endothelium-denuded carotid artery was similar between control and AGE-BSA groups. These results suggest that ligand specific alterations of contractile responses by AGE-BSA exposure were seen in carotid arteries, and that decreased noradrenaline-induced contraction by AGE-BSA may be partly due to increased OCT-3 activity rather than the expression.

Advanced glycation end-products (AGEs) play a pivotal role in vascular function in various (patho)physiological conditions. Although uridine diphosphate (UDP) is an important extracellular nucleotide, the direct relationship between AGEs and UDP regarding their effects on vascular functions remain unclear. Therefore, we investigated the effects of AGE-bovine serum albumin (AGE-BSA) on UDP-mediated responses in rat carotid arterial rings. Concentration–dependent contraction but not relaxation was obtained following UDP application to carotid arteries with and without endothelia; the contraction was greater in the AGE-BSA-treated (0.1 mg/mL for 60 min) group than the control (1.0 v/v% PBS) group. The difference in UDP-induced contraction between the control and AGE-BSA-treated groups was not abolished by L-NNA [a nitric oxide synthase (NOS) inhibitor], whereas the difference was abolished by indomethacin [a cyclooxygenase (COX) inhibitor], ozagrel [a thromboxane synthase (TXS) inhibitor], and by SQ29548 [a thromboxane-prostanoid (TP) receptor antagonist]. The release of TXB₂, a metabolite of TXA₂, was increased by UDP in both groups, whereas the levels were similar between two groups. The release of PGE₂, other vasoconstrictor prostanoid, was similar among the groups (UDP-stimulated or -unstimulated control/AGE-BSA-treated groups). The contraction induced by U46619, a TP receptor agonist, in the presence of L-NNA was increased in the AGE-BSA-treated group compared with the control group. We conclude that the increase in UDP-induced contraction in the presence of AGE-BSA can be attributed to an increase in the COX/TXS/TP receptor pathway, in particular, TP receptor signaling.

[Background and Aim]

We previously reported that resveratrol, an activator of an NAD⁺-dependent deacetylase SIRT1, improves exercise tolerance with upregulation oxidative type of muscle fibers in a mouse model of Duchenne muscular dystrophy. SIRT1 deacetylates and activates PGC-1α, a co-activator to promote expression of oxidative muscle fibers. Here, we examined whether SIRT1 maintains oxidative muscle fibers for exercise capacity in the skeletal muscle.

[Methods and Results]

We first compared the expression level of SIRT1 in several types of skeletal muscles in wild-type mouse (WT). The SIRT1 protein level was the highest in the soleus muscle (+3.5-fold), which is mainly made up of oxidative fibers, compared to the other glycolytic muscles such as quadriceps, gastrocnemius, tibial anterior, and extensor digitorum longus muscles. The SIRT1 mRNA level was most abundant also in the soleus muscle. Immunohistological analysis using soleus muscle sections showed that the percentage of type IIa, one of oxidative muscle fibers, was significantly lower in the skeletal muscle-specific SIRT1 knockout mouse (SIRT1MKO) than that in WT (42% vs. 56%) at 79-98 weeks of age. In contrast, the percentage of glycolytic type IIx+IIb fibers was higher in the SIRT1MKO (15%) compared to WT (11%). Treadmill running distance at 15 weeks of age was significantly shorter in SIRT1MKO (158±10 m) than that in WT (1088±33 m).

[Conclusion]

These results suggest that SIRT1 maintains exercise capacity by preserving oxidative muscle fibers in the skeletal muscle.

The diameter of sinusoid has been shown to be changed by perfusion of adrenaline and acetylcholine. Thus, the liver blood flow is suggested to be regulated by the autonomic nervous systems in the sinusoid. However, it remains to be elucidated which cells in the sinusoid are involved in the response. The present study focused on hepatic stellate cells (HSCs) encircling the sinusoid and aimed to determine 1) whether HSCs constrict in response to noradrenaline (NAd) and 2) whether HSCs regulate liver blood flow. To measure HSC constriction quantitatively, we developed a novel method using fluorescent beads. We observed that HSCs constricted in response to NAd, which was suppressed by the α₁-adrenoceptor inhibitor bunazosin and the non-muscle myosin II inhibitor blebbistatin (Bleb;1 µM). In contrast, Bleb (1 µM) had no effect on the contraction of isolated portal veins. The NAd-induced constriction of HSCs was also suppressed by xestospongin C, YM-58483, W-7, ML-9, and H-1152. In addition, Bleb (1 µM) decreased the perfusion pressure of the liver increased by NAd. This response appears to be due to HSC relaxation, since Bleb had the inhibitory effect on HSCs but not on portal veins. These results suggest that NAd induces constriction of HSCs via increasing Ca²⁺ influx and Ca²⁺ sensitization, thereby regulating liver blood flow.

Differentiation of human endometrial stromal cells into decidual cells is indispensable for the embryo implantation during the mid-secretory phase of the menstrual cycle. After the implantation, placental villus cytotrophoblasts differentiate into syncytiotrophoblasts to form placenta. These differentiation events of ESCs and trophoblasts cells are induced by the activation of cyclic AMP (cAMP) signaling pathway. Progesterone (P4) receptor membrane component 1 (PGRMC1) is a member of a P4 binding complex implicated in female reproduction, however, its role in ESCs and trophoblast cells differentiation has not been examined. In this study, we explored the significance of PGRMC1 in cAMP-induced differentiation of ESCs and trophoblast cells. Treatment of ESCs with the cAMP analog dibutyryl (db)-cAMP repressed PGRMC1 expression. Both knockdown and inhibition of PGRMC1 significantly promoted the expression of differentiation markers such as IGF-binding protein 1 and prolactin in db-cAMP-stimulated ESCs. Furthermore, inhibition of PGRMC1 facilitated the production of human chorionic gonadotropin (HCG), which is the differentiation marker of syncytiotrophoblast in placental choriocarcinoma BeWo cells. These findings suggest the significance of P4-independent inhibitory action of PGRMC1 in cAMP-induced ESCs and trophoblast differentiation.

Endometriosis is characterized by the ectopic inflammation, growth, and fibrotic changes in the lesion. Previous study indicates that peritoneal bleeding may accelerate inflammation partially through the activation of protease-activated receptor (PAR) and the prostaglandin (PG) EP2 receptor in endometriosis-like graft of the mouse model. To explore the involvement of hypoxia and/or the inflammatory mediators in epithelial-mesenchymal transition (EMT) of endometrial cells, we examined the effects of thrombin, a PAR agonist and PGE2 on EMT marker expression and cell migration in human endometrial stromal (EtsT499) and epithelial (EM-1) cells. The endometrial cells in 3D culture system incubated for 18 h under hypoxia were cultured in the presence or absence of the combined treatment of thrombin and PGE2 (Throm/PG) for 72 h. Hypoxic conditions increased expression of CXCR4, an EMT marker in EM-1, but not in EtsT499, whereas Throm/PG did not affect CXCR4 in both cells. Throm/PG treatment promoted the migration of EM-1 under hypoxia. Thus, Throm/PG stimulation under hypoxia enhanced CXCR4 expression and accelerated migration of the endometrial epithelial cells. Our data suggests that the inflammatory mediators in retrograde menstrual fluid may be associated with the pathophysiology of ectopic endometrial EMT and migration.

The chronic hyperglycemia that occurs in type 2 diabetes causes deterioration of pancreatic β-cell dysfunction which involves a decrease in insulin secretory response and a decrease in β-cell mass. Thus, to promote β-cell function and survival would provide therapeutic approaches to prevent the onset and development of type 2 diabetes. Citrus flavonoids are known to have health benefits, especially those related to improvement of type 2 diabetes. However, little is known about the effects of these flavonoids on pancreatic β-cell functions. We have previously demonstrated that nobiletin has anti-diabetic effects on β-cell functions. Tangeretin and sudachitin are polymethoxy flavonoids (PMF) contained in citrus peel and have a similar structure to nobiletin. In the present study, we investigated the effects of the PMFs on glucose-induced insulin secretion (GSIS) and β-cell apoptosis in the β-cell line INS-1 and compared these effects with those of nobiletin. Tangeretin significantly increased GSIS at 10 μM and inhibited thapsigargin-induced apoptosis. Sudachitin also significantly increased GSIS at 100 μM but did not affect β-cell apoptosis. The anti-diabetic effects of tangeretin on β-cell functions were more potent than those of sudachitin, but they were less potent than those of nobiletin. These results suggest that nobiletin has more remarkable anti-diabetic effects on β-cells, i.e., more potent insulinotropic and anti-apoptotic effects, than tangeretin and sudachitin.

Human endometrial stromal cells (ESCs) differentiate into decidual cells during the mid-secretory phase of the menstrual cycle following the postovulatory rise in progesterone (P4). Progesterone (P4) is a predominant inducer of the differentiation which is essential for the establishment of pregnancy. In this study, we explored the roles of 5α-reductases-mediated P4 metabolism in the differentiation of ESCs induced by P4 and dibutyryl cAMP (P4/db-cAMP) treatment. The ability of P4 metabolism in differentiated ESCs was compared with that in undifferentiated cells. The residual P4 level in media was much higher in the differentiated ESCs than in control cells, whereas the amount of the P4 metabolite allopregnanolone was less in the differentiated cells. Treatment of ESCs and endometrial epithelial cells with the 5α-reductase inhibitors dutasteride and finasteride repressed P4 metabolism. Furthermore, inhibition of 5α-reductase facilitated expression of differentiation markers, IGF-binding protein 1 and prolactin in P4/db-cAMP-stimulated ESCs. The expression of SRD5A1, which encodes 5α-reductases type 1, was reduced in differentiated ESCs and epithelial cells. These data suggests endometrial 5α-reductase metabolizes P4 and the enzyme-mediated metabolizing pathway maybe involved in the increase in P4 level for promoting ESC differentiation.

Testosterone, primarily produced in Leydig cells, is essential for a variety of systemic functions, and deficiency of this hormone results in late-onset hypogonadism (LOH) in climacteric male. In the present study, we prepared the extract of fruit body of Cordyceps militaris parasitized in Samia Cynthia ricini, designated as Ryukyu-Kasou (RK), as a novel candidate for ameliorating male menopause symptom. To explore the effect of RK on LOH, we have investigated the testosterone dynamics using castrated rats and isolated primary rat Leydig cells. Testosterone propionate (TP) and the extract of RK were administered to castrated rats for 12 days. The serum levels of testosterone and dihydrotestosterone (DHT) were maintained highly in the RK-treated group, compared with control. In addition, RK reduced TP-stimulated increases in the weight of prostate gland. When cultured testicular cells were stimulated with luteinizing hormone (LH) or dibutyryl-cyclic AMP (cAMP) in the presence or absence of RK, LH- or cAMP-induced testosterone secretion in the media was enhanced by the presence of RK with no changes in the mRNA expression of steroidogenic enzymes. Thus, RK-derived unknown compounds suppressed the testosterone catabolism in vivo and stimulated testosterone secretion in vitro, suggesting the possibility of RK for improving LOH.

It is known that there are species differences in the purine metabolic system between humans and rodents (e.g. urate oxidase (Uox), and hypoxanthine phosphoribosyltransferase (HPRT), etc.). URAT1 (SLC22A12) is renal urate (UA) reabsorption transporter and the target for UA-lowering therapies. In humans, URAT1 deficiency has a significant UA-lowering effect (ULE), but not in Urat1-knokout (KO) mice. The aim of this study is the establishment and urate kinetic profiling of high HPRT activity Urat1-Uox double knockout (DKO) mice and the investigation of the effect of xanthine oxidoreductase inhibitor (XOI), topiroxostat in this model mice. Topiroxostat 1 mg/kg (Top) was administered to DKO mice for 7 days by feeding diet. Oxypurines (UA, hypoxanthine and xanthine) and creatinine in plasma and urine were measured by HPLC. DKO mice showed a significant decrease in plasma UA levels, increased fractional excretion of UA (FE_UA), and enhanced Top-induced ULEs, compared with Uox-KO only. Thus, high HPRT activity seems to be important for producing ULE by URAT1 inhibition. The combination therapy of URAT1 inhibition and XOI showed an effective ULEs, suggesting that it is useful for the treatment of hyperuricemia.

Amyloid-β (Aβ) accumulation in the brain is the first trigger for the onset of Alzheimer's disease (AD), and its prevention and elimination are promising strategies for AD therapy. Previously, we demonstrated that injection of mouse bone marrow (BM)–derived microglia-like (BMDML) cells into the brain decreases Aβ and ameliorates cognitive impairment in a mouse model of AD. In this study, considering majority of AD patients are elderly and less invasive ways for preparing autologous microglia-like cells are needed, we focused on hematopoietic stem cells (HSCs) in peripheral blood (PB). Mouse HSCs were mobilized from BM to PB by administration of granulocyte colony-stimulating factor (G-CSF) and CXCR4 antagonist and were collected from PB. Collected HSCs were subsequently differentiated into microglia-like cells upon stimulation with colony- stimulating factor 1 (CSF-1) and interleukin-34. The PB-derived microglia-like (PBDML) cells expressed macrophage/microglia markers and effectively phagocytosed Aβ. We further found that PBDML cells injected into the hippocampi of AD model mice diffused in the brain with phagocytosing Aβ, and contributed to the reduction of brain Aβ and improvement of cognitive impairment. These results suggest that PBDML cells could be promising candidate source for the development of cell therapy against AD.

Glaucoma is second leading cause of blindness worldwide which is characterized by progressive degeneration of retinal ganglion cells (RGCs). Elevated intraocular pressure (IOP) is one of the highest risk factors and IOP-lowering agents are used to prevent glaucoma. New molecular target is required because of the side effects, drug resistance, and insufficiency for IOP reduction by a part of pre-existing agents. Here, we report that P2Y₁ receptor (P2Y₁R) activation induces IOP reduction and knock out of P2Y₁R (P2Y₁KO) causes sustained IOP elevation associated with age-dependent RGC degeneration. Topical application of MRS2365, selective agonist for P2Y₁R, caused significant reduction in IOP in wild-type (WT) mice but not in P2Y₁KO mice. We also found that P2Y₁KO mice showed significantly higher IOP level than that in WT mice. Because sustained IOP elevation is one feature of hypertensive glaucoma, we checked RCG damages and found that the number of RGCs in P2Y₁KO mice was comparable at 3 months old but significantly smaller at 12 months old. Furthermore, optical coherence tomography (OCT) revealed that 12-month-old P2Y₁KO mice showed thinner ganglion cell and inner plexiform layers, general diagnostic feature of glaucoma patients. Taken together, our results demonstrated that (1) P2Y₁R activation reduces IOP; (2) loss-of-function of P2Y₁R causes sustained elevation in IOP and (3) hypertensive glaucoma-like phenotypes in middle-aged mice.

HYBID (hyaluronan binding protein involved in hyaluronan depolymerization) is a novel factor associated with hyaluronan depolymerization. HYBID facilitates the several tumor progression and the expression level of HYBID is helpful as a predictor of tumor progression including colon and pancreatic tumor. Though HYBID is important for hyaluronan metabolism in brain, there is no report on glioma. Therefore, we evaluated the role of HYBID and hyaluronan on glioma using in vitro and in vivo glioma models.

First, we evaluated the cell proliferation, migration, and the expression of some related proteins after knock of hybid by using siRNA in U251 human glioma cell. Moreover, we evaluated the tumor size by using the in vivo glioma model with HYBID KO and WT mice. Murine glioma model was estimated by hematoxylin and eosin staining.

Hybid knock down suppressed the glioma cell proliferation, migration and Wnt/β-catenin signal related protein. HYBID may promote the glioma progression via Wnt/β-catenin signal. Moreover, tumor size in HYBID KO mice were smaller than that in HYBID WT mice. This result indicates that host derived HYBID is contributed to glioma progression.  

In conclusion, these findings indicate that HYBID was an important factor for glioma progression.

Intracerebral hemorrhage (ICH), a bleeding into the brain parenchyma, is a devastating neurologic disease with the highest mortality among all stroke subtypes. In ICH brain, thrombin induces activation of microglia/macrophages followed by neuroinflammation. Furthermore, ICH leads to infiltration of numerous leukocytes. Recent report shows the arachidonic acid metabolite, leukotriene B₄ (LTB₄), participates pathological progression of ICH (Hijioka et al., 2017). In this study, we focused on lipoxin A₄ (LXA₄), synthesized from arachidonic acid as same as LTB₄. Treatment of murine microglial cell line BV-2 cells with thrombin (30 U/mL) increased mRNA expression level of inducible NO synthase (iNOS) and interleukin-6 (IL-6). Pretreatment with LXA₄ (100 µM) suppressed thrombin-induced increases in iNOS and IL-6 mRNA expression. Moreover, immunocytochemical analysis revealed the translocation of nuclear factor-κB (NF-κB) into the nucleus induced by thrombin, and thrombin-induced nuclear translocation of NF-κB was suppressed by LXA₄. Finally, daily intravenous administration of LXA₄ receptor agonist, BML-111 (1 mg/kg) attenuated the motor dysfunction of mouse model of ICH. These data suggest that LXA₄ may be the novel therapeutic agent for ICH.

Anthocyanins possess high antioxidant activity and are the major group of polyphenols in blackcurrant, a regional specialty in Aomori prefecture. In this study, we investigated the effect of black currant extracts on cognitive and emotional abnormalities in the senescence-accelerated mouse prone 8 (SAMP8). Four month-old SAMP8 mice were fed a basal diet supplemented with 3% blackcurrant extracts for 2 months, and then behavioral experiments were conducted. In the novel object recognition test, treatment with blackcurrant extracts improved the memory impairment in SAMP8 mice. In addition, reduced anxiety-like behavior in SAMP8 mice was reversed by the extracts in the elevated plus maze test. These results suggest that supplementation of blackcurrant extracts has the potential to improve cognitive and emotional abnormalities in aging as well as age-related neurodegenerative diseases such as Alzheimer's disease.