Background＆Aim: We have reported that oral pathogenic bacteria including Porphyromonas gingivalis (P.g) and Streptococcus mutans (S.m) accelerates systemic diseases. In our present study, we focused on eosinophilic esophagitis (EoE), a refractory rare disease, and analyzed oral bacteria in EoE patients to find relationship between EoE and infection of bacteria.

Method: Twenty four healthy control volunteers, 52 EoE subjects who were diagnosed by Shimane university and 22 subjects of reverse esophagitis (RE) were recruited. Oral bacteria were collected by mouse washing with 1.5 mL of distilled water. Bacterial DNA was extracted and analyzed by next generation sequencer. In addition, popular pathogenic oral bacteria including P.g and S.m were detected by PCR.

Resutls: Population of Prevotella genus was smaller in EoE and RE compared to that in healthy control. Same tendency was observed in four Prevotella spieces. On the other hand, S.m and S.m-derived collagen-binding protein (cnm), through which S.m induces dysfunction of tissues or organs, were detected more frequently both in EoE and RE than healthy control.

Conclusion: Oral bacterial flora in EoE subjects are likely to be different from that in healthy subjects, and infection to S.m is correlates with prevalence of EoE.

Purpose : The fetal liver is immature, and physiological jaundice often occurs. The symptom of jaundice will become more severe in premature infants. To investigate the maturation and the function in the liver of premature infants is necessary. Antenatal glucocorticoid (GC) administration is the standard of care for women at risk of a preterm birth. The purpose of this study was to examine whether GC administration acts on maturation factors in the fetal liver for development.

Methods : Dexamethasone were administered to pregnant rats for 2 days and the livers of 19-day-old fetuses, 21-day-old fetuses and 1-day-old neonates were analyzed. We evaluated mRNA levels of HNF4α, Ki-67 and Cyclin B as liver maturation factors and UGT1A1 as bilirubin metabolism-related factor by real-time PCR. In histochemistry, cell size of a hepatocyte was confirmed H-E staining.

Results and Discussion : The mRNA levels of HNF4α and UGT1A1 increased with growth. The mRNA expressions of HNF4a and UGT1A1 were increased in fetal liver with antenatal dexamethasone administration. Cell size of a hepatocyte was enlarged with growth, which is accelerated with antenatal GC administration. These results suggest that antenatal GC administration may accelerated maturation, and may increases bilirubin metabolism in the liver of premature infants.

Angiotensin-converting enzyme 2 (ACE2) is a negative regulator of the renin-angiotensin system, critically involved in blood pressure regulation, heart function, lung injury, or fibrotic kidney disease. Recombinant human ACE2 protein (rhACE2), currently clinically evaluated to treat acute lung failure, is a glycosylated protein, requiring time- and cost-consuming protein production in mammalian cells. Here we show that the B38-CAP, a carboxypeptidase derived from Paenibacillus sp. B38, is a novel ACE2-like enzyme to decrease angiotensin II levels in mice. Comparative analysis of protein 3D structures revealed that B38-CAP homologue shares structural similarity to mammalian ACE2 without any apparent sequence identity, containing the consensus HEXXH amino acid sequence of the M32 peptidase family. In vitro, recombinant B38-CAP protein catalyzed the conversion of angiotensin II to angiotensin 1-7, as well as other known ACE2 target peptides, with the same potency and kinetics as human ACE2. Treatment with B38-CAP reduced plasma angiotensin II levels and suppressed angiotensin II-induced hypertension, cardiac hypertrophy and fibrosis in mice. Moreover, continuous infusion of B38-CAP inhibited pressure overload-induced pathological hypertrophy, myocardial fibrosis, and cardiac dysfunction in mice, without any overt toxicity of liver and kidney. Our data identify the bacterial B38-CAP as an ACE2-like carboxypeptidase, which exhibits ACE2-like functions in vitro and in vivo. These results indicate that evolution has shaped a bacterial carboxypeptidase to a human ACE2-like enzyme. Bacterial engineering could be utilized to design improved protein drugs for hypertension and heart failure.

Pulmonary arterial hypertension (PAH) is characterized by pulmonary artery remodeling and inappropriate vasoconstriction, which results in a marked increase in pulmonary arterial pressure and right ventricular hypertrophy. Because a multiple factor participates in the pathogenesis of PAH, elucidation of the further mechanism is needed. Mitochondrial dysfunctions have been reported in the pathogenesis of PAH. In this study, to investigate the involvement of mitochondrial Na⁺/Ca²⁺ exchanger (NCLX) in the development and progression of PAH, we generated a mouse model of hypoxia-induced PAH using NCLX knockout (NCLX-KO) mice, vascular smooth muscle-specific NCLX transgenic (VSM-NCLX-Tg) mice and wild-type (WT) mice. Increase in right ventricle systolic pressure (RVSP) induced by chronic hypoxia was significantly reduced in NCLX-KO mice compared with WT mice, whereas it was markedly augmented in VSM-NCLX-Tg mice. Moreover, administration of CGP37157, a selective NCLX inhibitor, to WT mice with chronic hypoxia significantly attenuated the increase in RVSP. These results suggested that vascular smooth muscle NCLX is involved in the pathogenesis of chronic hypoxia-induced PAH.

Mast cells are not only responsible for immune functions, but are also involved in the development of various diseases. Previously, we analyzed and reported about the appearance pattern of mast cells in the early stages of development in normal Wistar rat strain. It has been also reported that chymase accumulated in granules of mast cells is involved in the development of hypertension. In this study, we analyzed the relationship between mast cell expression patterns in the early stages of development and the onset of hypertension using model animals.

We used three strains of rats, SHR/NCrj, WKY/Ncrj rats and Wistar/Slc rats. They were planned to become pregnant, and fetuses at 9.5, 11.5, 13.5, 15.5, and 18.5 days of gestation and neonates at 1, 3, 7, and 14 days after birth were obtained. mRNA was purified from whole embryos or several tissues at each stage. RT-PCR was performed using primers for six molecules, i.e., c-kit, FceRI, rMCP-I, rMC-CPA, VEGF, and TNFalpha.

As a result, there was almost no difference in the expression timing and localization of rMCP-I among strains. However, there were significant differences in the expression patterns of c-kit and VEGF during embryonal stages. The difference between Wistar and WKY was greater than Wistar and SHR. It was considered that the development of hypertension is associated with the maintenance or differentiation of blood stem cells and the development of vascular system rather than the expression pattern of mast cells. It was also suggested that WKY rats may have already been committed to the development of hypertension.

Indoxyl sulfate (IS) is a low molecular weight metabolite and a uremic toxin which induces oxidative stress in myocardial, vascular smooth muscle cells, vascular endothelial cells and also involves in cardiovascular (CV) diseases. Therefore, we investigated the association between serum IS levels and aortic stiffness in coronary artery disease (CAD) patients. A total of 144 CAD patients were recruited. Carotid-femoral pulse wave velocity (cfPWV) was measured by the SphygmoCor system and the value over 10 m/s was classified as the arterial stiffness group. Serum IS levels were determined by liquid chromatography-mass spectrometry. Fifty patients (34.7%) had arterial stiffness and higher percentages with diabetes, elderly, higher systolic blood pressure, blood urea nitrogen, creatinine, serum IS level, lower estimated glomerular filtration rate compared with the control group. After adjustment of the factors by multivariable logistic regression analysis, the serum IS levels revealed significantly correlated with arterial stiffness (odds ratio = 3.834, P = 0.031), and has potential to be an independent predictor of arterial stiffness in CAD patients. In addition, the serum IS levels (β = 0.167, adjusted R2 change: 0.026, P = 0.027) were significantly positively correlated with cfPWV values in CAD patients in multivariable forward stepwise linear regression analysis. Our results suggest that serum IS has potential as an independent biomarker for aortic stiffness in CAD patients.

Perivascular adipose tissue (PVAT) regulates vascular tone. We demonstrated that PVAT masks impaired vasodilation in the mesenteric arteries of SHRSP.Z-Lepr^fa/IzmDmcr rats (SHRSP.ZF) with metabolic syndrome (MetS); however, this enhanced vasodilation caused by PVAT disappears at around 23 weeks (wks) of age. Therefore, we investigated whether an angiotensin II type 1 receptor antagonist, azilsartan, protects against the deterioration in PVAT compensatory vasodilator function that occurs with aging in MetS.

SHRSP.ZF rats at 13 wks were orally administered azilsartan once daily for 10 wks. The vasodilation response in the superior-mesenteric arteries was determined in the presence or absence of PVAT, using organ bath methods. Azilsartan preserved both acetylcholine- and sodium nitroprusside-induced vasodilation independent of the presence or absence of PVAT, and did not improve the dysfunction in PVAT-mediated modulation of vascular tone in SHRSP.ZF rats.

This study demonstrated that the protective effect of azilsartan is mediated by restoring the endothelium- and vascular smooth muscle-mediated mechanisms, and not by improving PVAT dysfunction in MetS.

Tumor necrosis factor (TNF) is a kind of cytokine involved in infection protection and antitumor action by expressing cell adhesion molecules such as vascular cell adhesion protein-1 (VCAM-1), and by inducing apoptosis and inflammatory mediators. VCAM-1 is known to exacerbate cardiovascular disease and is a risk factor for cardiovascular disease events. Gout is not just a disease of the joints, it causes inflammation throughout the body and affects various organs. Since TNF-α is induced under inflammatory conditions such as hyperuricemia, we investigated an effect of allopurinol, a treatment agent for hyperuricemia, on VCAM-1. Human Umbilical Vein Endothelial Cells (HUVEC) were cultured confluently. Allopurinol (0.1-100 µM) was treated 20 minutes before TNF-α (10 ng/mL) exposure. The amount of VCAM-1 induced by TNF-α was evaluated using Western blotting. VCAM-1 protein levels in cultured HUVEC increased 24 hours after TNF-a exposure, which allopurinol suppressed the significantly (p < 0.05, n = 4). Allopurinol is thought to inhibit the induction of VCAM-1 by TNF-α and may decrease cardiovascular disease events.

In CARES clinical trial, febuxostat, a non purine xanthine oxidase inhibitor, was proved non-inferiority the rate of adverse cardiovascular events in patients with gout and major cardiovascular coexisting conditions. In this study, we evaluated the effect of febuxostat on the vascular cell adhesion protein 1 (VCAM-1) induction cultured Human Umbilical Vein Endothelial Cells（HUVEC）were exposed to 24-hour TNF-α (10 ng/mL) treatment. Febuxostat (0.1-100 mM) or solvent was added to the bath medium 20 minutes before TNF-α treatment. VCAM-1 protein levels in HUVEC increased after 24 hours TNF-atreatment (n = 4). Febuxostat significantly suppressed VCAM-1 induced by treatment with TNF-a in a dose-dependent manner (p < 0.05, n = 4). This finding suggests that treatment with Febuxostat on cardiovascular events may associate with the protection for the infiltration of lymphocyte or monocyte through the VCAM-1 induction in the inflamed-endothelial cells such as arterial sclerosis.

Retinopathy of prematurity (ROP) is the leading cause of childhood blindness. We reported that short-term interruption of retinal vascular development with blockade of vascular endothelial growth factor (VEGF) signaling pathway in neonatal rats induces ROP-like retinal blood vessels, such as aggressive angiogenesis and tortuous arteries. Using this ROP model rat, we examined whether a history of ROP affects retinal vasodilator responses in adulthood. ROP was induced in rats by the subcutaneous injection of the VEGF receptor tyrosine kinase inhibitor KRN633 on postnatal day (P) 7 and P8. Tortuous arteries were observed in retinas of P56 KRN633-treated (ROP) rats. Retinal vasodilator responses to endothelium-dependent vasodilators (acetylcholine and GSK1016790A [an activator of TRPV4 channels]) were smaller in P56 ROP rats than age-matched control rats. No diminishment of acetylcholine-induced retinal vasodilator response was observed in P56 ROP rats treated with L-NAME, an inhibitor of NO synthase. Retinal vasodilator responses to NOR3, an NO donor, and salbutamol, a β₂ receptor agonist, were unaltered. These results suggest that the production and release of NO in retinal blood vessels are impaired in adult rats with a history of ROP. A history of ROP may increase the risk of the onset of retinal vascular diseases in adulthood.

Recently, we confirmed that menaquinone-4 (MK-4), the most common form of vitamin K₂ in animals, induced the calcification of human aortic valve interstitial cells (HAVICs) isolated from aortic valve stenosis (AVS) patients in high inorganic phosphate (high-Pi) medium via BMP2-ALP pathway. However, the mechanism of MK-4-induced BMP2 expression is unclear. There is a report that MK-4 can enhance collagen accumulation through pregnane X receptor (PXR) resulting in bone formation. So, the involvement of PXR in MK-4-induced calcification of HAVICs and BMP2 gene expression was investigated. HAVICs from AVS patients were cultured in α-MEM containing 10% FBS, and when the cells reached 80% confluence, they were further cultured in the presence or absence of MK-4 for 7 days in high-Pi medium (3.2 mM Pi). MK-4 dose-dependently accelerated PXR activity (EC₅₀ 6.2 nM). MK-4-induced calcification was potently suppressed by two PXR inhibitors, ketoconazole and coumestrol. In physiological-Pi medium, MK-4 alone also increased BMP2 gene expression, which was significantly suppressed by coumestrol. These results suggested that MK-4 accelerates the calcification of HAVICs from AVS patients through the PXR-BMP2-ALP pathway.

Pulmonary artery smooth muscle cells (PASMCs) play an important role in a sequence of events leading to the formation of pulmonary artery hypertension (PAH). Nevertheless, little is known about the direct effects of high pressure on the function and the intercellular signaling pathways of PASMCs. The aim of this study was to evaluate the effect of pressure stress which simulates PAH on interleukin-1β (IL-1β)-induced cyclooxygenase-2 (COX-2) expression in cultured human PASMCs. To investigate the effect of PAH on PASMCs, either 20 or 60 mmHg of an atmospheric pressure was given to PASMCs by a pressure-loading apparatus. Protein expression and phosphorylation were analyzed by Western blotting. IL-1β-induced the transient COX-2 protein expression peaking at 6 h in non-pressurized cells, whereas the COX-2 expression was delayed, peaking at 12 h, in the pressurized cells. The pressure stress also delayed the peak time of IL-1β-induced mitogen-activated protein kinases (MAPKs) phosphorylation, i.e., extracellular signal-regulated kinase, p38 MAPK, and c-jun N-terminal kinase. These results suggest that the pressure stress apparatus enable to simulate PAH, and delays in IL-1β-induced COX-2 expression occurs via late activation of MAPKs in PASMC.

Cigarette smoking is known to be accompanied with a decrease in nitric oxide (NO) bioavailability in the vascular system. A shift of soluble guanylate cyclase (sGC) from the NO-sensitive form to the NO-insensitive form could decrease NO bioavailability. Therefore, this study investigated whether NO-insensitive sGC-mediated relaxation is augmented in smokers. The right gastroepiploic artery was isolated from patients undergoing gastrectomy or coronary artery bypass grafting. BAY 60-2770 (NO-insensitive sGC stimulant) evoked a concentration-dependent relaxation of the artery, which was not different between non-smokers and smokers. In addition, there was also no significant difference in the concentration-response curve for sodium nitroprusside (NO-sensitive sGC stimulant). These findings suggest that NO-insensitive sGC-mediated vascular tone regulation is not affected by cigarette smoking. It is considered that the balance between the two forms of sGC is maintained even in the blood vessels damaged by cigarette smoking.

Pulmonary veins contain a myocardial layer, whose electrical activity is considered to be involved in the genesis and maintenance of atrial fibrillation.

To obtain insight into the automaticity of the pulmonary vein myocardium, we studied　the spatio-temporal pattern of the rise in Ca²⁺ during the early phase Ca²⁺ transient of the isolated guinea pig pulmonary vein cardiomyocytes were studied with confocal microscopy.

On induction of Ca²⁺ transients by electrical field stimulation of the pulmonary vein cardiomyocytes, the rise in Ca²⁺ concentration first occurred at the subsarcolemmal region and then spread to the cell interior; this phenomenon was similar to that of atrial but not ventricular cardiomyocytes.

In pulmonary vein cardiomyocytes showing spontaneous activity, the Ca²⁺ transients were preceded by increased firing of Ca²⁺ sparks, which means Ca²⁺ release from sarcoplasmic reticulum. SEA0400, an inhibitor of the Na⁺/Ca²⁺ exchanger, decreased the frequency of the Ca²⁺ transients and eventually inhibited the Ca²⁺ transients completely without decreasing the firing of Ca²⁺ sparks.

In conclusion, the guinea-pig pulmonary vein myocardium has a tendency to show spontaneous electrical activity, which is mediated by Ca²⁺ released from the sarcoplasmic reticulum and the resulting activation of the Na⁺/Ca²⁺ exchanger.

We simultaneously assessed effects of a specific I_f channel blocker ivabradine on the atrial and ventricular automaticity in anesthetized rabbits. Under isoflurane anesthesia, the atrioventricular node of NZW rabbits (n=3) was ablated by application of radiofrequency energy, and stable idioventricular escaped rhythm was observed. The surface lead II electrocardiogram was measured to monitor changes in the atrial rate (AR) and ventricular rate (VR). The monophasic action potential (MAP) was recorded from the right ventricle to assess the MAP duration (MAP₉₀). Intravenous administrations of ivabradine hardly affected the AR or VR at 0.01 and 0.1 mg/kg. Additional administration of ivabradine at 1.0 mg/kg decreased both AR and VR by 45 and 51 beats/min, respectively. Moreover, the MAP₉₀ was prolonged with decrease of the VR, and torsade de pointes was induced in one animal. These results suggest that ivabradine affects the ventricular as well as the atrial pacemaker activity with a similar potency.

Background: Obesity is a risk factor for atrial fibrillation (AF). However, the mechanisms underlying AF in obesity remain unclear. In this study, we established the diet-induced obese-rat model with high AF inducibility and evaluated the relationship between AF inducibility and cardiac function.

Methods: Male Sprague-Dawley rats were fed with normal chow diet + normal drinking water (NCD) or high-fat diet + 30% fructose in drinking water (HFFr) for 12 weeks. These rats were subjected to hemodynamic measurements, echocardiography to assess the evaluation of the cardiac structure and function., and transesophageal burst atrial pacing for the induction of AF.

Results: HFFr-fed rats were divided into 2 groups: obese and non-obese HFFr-fed rats. Compared with NCD-fed rats, the inducibility of AF significantly increased in obese HFFr-fed rats, but not in non-obese HFFr-fed rats. On echocardiography, LVEF (an indicator of LV systolic function), and E/A ratio (a marker of LV diastolic function) didn't change among these rats. For hemodynamic measurements, LVSP, dP/dtmax, and heart rate increased in obese HFFr-fed rats.

Conclusion: We established the obese rat model with high AF inducibility while maintaining normal cardiac function. This model would be useful to elucidate the mechanisms of obesity-related AF.

Thrombospondin (TSP)-4, a matricellular protein, is highly expressed in heart tissues of various cardiac disease models. Although TSP-4 is known to regulate voltage-dependent calcium channel activity in dorsal root ganglionic neurons, it remains to be clarified whether it affects electrical activity in cardiomyocytes. We examined the effects of TSP-4 on voltage-gated ion channels in rat ventricular myocytes. Ventricular myocytes isolated from male Wistar rats were seeded on a glass plate coated with laminin. Recombinant mouse TSP-4 (5 nM) or its vehicle was treated for 4 hours. L-type calcium channel (LTCC) current, voltage-gated potassium channel (VGKC) current, and action potential duration (APD) were measured by a whole-cell patch-clamp method. TSP-4 inhibited both currents of LTCC and VGKC. TSP-4 tended to prolong APD₅₀ and APD₉₀. This study for the first time demonstrated that TSP-4 inhibits the activity of LTCC and VGKC, which consequently leads to APD prolongation. The APD prolongation might be partly due to the suppression of VGKC activity because the inhibition of LTCC should lead to an APD shortening. It is suggested that TSP-4 might be related to the ventricular arrhythmia via regulating voltage-gated ion channels.

Drug-induced arrhythmia can occur under prolonged action potential duration (APD) due to block of I_Kr. Therefore, I_Kr block and APD prolongation have been used for predicting drug induced arrhythmia.However, I_Kr blockers have difference in risk for drug-induced arrhythmia. One of the reasons is that the occurrence of drug-induced arrhythmia under bradycardia is initiated by early afterdepolarization (EAD) at the repolarization phase in prolonged action potential. For example, terfenadine, which prolongs APD and cause EAD, is considered as a drug with a high risk for drug-induced arrhythmia. On the other hand, amiodarone, which prolongs APD but does not cause EAD in clinical practice, has been considered as a relatively safe antiarrhythmic drug. Therefore, there is a possibility that EAD occurrence can account for the difference in the risks among I_Kr blockers. To study the mechanisms underlying different occurrence of EAD, we examined the effects of voltage-dependent I_CaL block property on EAD. In the present study, we used a mathematical model of human ventricular action potential. The results showed that amiodarone-like I_CaL block model suppressed EAD. But, I_CaL block models of terfenadine-like and bepridil-like increased EAD occurrence. The different effects on EAD were accounted for by difference in voltage-dependent block of I_CaL, as weak I_CaL block in hyperpolarized potential increased the occurrence of EAD. Therefore, to predict drug-induced arrhythmia, not only APD prolongation but also voltage-dependent property of I_CaL block should be checked.

We introduce a simple and universal cloning plasmid system for gene expression in prokaryotic (Escherichia coli) and mammalian cells. This novel system has two expression modes: the (subcloning) prokaryotic and mammalian modes. This system streamlines the process of producing mammalian gene expression plasmids with desired genes. The plasmid (prokaryotic mode) has an efficient selection system for DNA insertion, multiple component genes with rare restriction sites at both ends (termed "units"), and a simple transformation to mammalian expression mode utilizing rare restriction enzymes and re-ligation (deletion step). This system is highly efficient for the subcloning of blunt-end fragments, including PCR products. After the insertion of the desired gene, protein encoded by the desired gene can be detected in E. coli with IPTG induction. Then, the lac promoter and operator are readily deleted with 8-nucleotide rare-cutter blunt-end enzymes (deletion step). Following re-ligation and transformation, the plasmid is ready for mammalian expression analysis (mammalian mode). This idea (conversion from prokaryotic to mammalian mode) can be widely adapted. With pgMAX system, we made epitope-library of the calcium channel alpha1 subunit (CaV1.2) and found a novel binding site to calcium channel beta2 subunit. The pgMAX system could be widely adopted for simple expression analyses.

Nardilysin (NRDC; N-arginine dibasic convertase) is a metalloprotease of the M16 family. We reported that NRDC is a protease having localization-dependent multiple functions; an enhancer of ectodomain shedding in the extracellular space and a transcriptional coregulator in the nucleus. NRDC-deficient mice (Nrdc-/-) showed wide range of phenotypes such as hypomyelination, hypothermia, and bradycardia. In this study, we have explored a role of NRDC in the regulation of heart rate, and obtained the following results. (1) Pharmacological blocking of autonomic nervous system revealed that an intrinsic heart rate of Nrdc-/- was significantly reduced compared with that of wild-type mice. (2) In Nrdc-/- hearts, mRNA levels of Cav3.1 and HCN1/4, ion channels responsible for sinus automaticity, were significantly reduced. (3) Funny (If) current and T-type Ca current measured in the sinus node cells were markedly reduced in Nrdc-/- cells, indicating that the functions of Cav3.1 and HCN1/4 are impaired. (4) Gene knockdown of NRDC in primary rat ventricular myocyte led to the reduction of mRNA level of HCN1/4. (5) Chromatin immunoprecipitation-PCR analysis showed that NRDC binds to the promoter region of Cav3.1 and HCN1/4, suggesting the direct involvement of NRDC in transcriptional regulation of these ion channels. (6) Atrium-specific Nrdc-/- (obtained by mating Nrdc floxed mouse with Sarcolipin-Cre mouse) showed mild bradycardia and reduced Cav3.1 mRNA expression. (7) In silico simulation model of human iPS cell-derived sinus node cells recapitulated the bradycardia in NRDC-deficient cells. Together, our results indicated that NRDC in cardiomyocyte controls heart rate through the transcriptional regulation of ion channels critical for sinus automaticity.

[Background] The QT interval can be shortened by tachycardia, which may underestimate risks of the drug-induced QT-interval prolongation in the safety pharmacology studies.  To investigate utility of guinea pigs for the assessment of the QT-interval prolongation, we assessed cardiac effects of suspect drugs prolonging QT interval, sulpiride and aripiprazole, both of which exerted positive chronotropic actions in dogs.

[Methods] Under isoflurane-anesthesia, electrocardiogram and monophasic action potential (MAP) of right ventricle were continuously recorded from guinea pigs to measure the heart rate (HR) and the MAP duration (MAP₉₀), respectively.  Sulpiride (2, 20, and 60 mg/kg) or aripiprazole (0.03, 0.3, and 3 mg/kg) were administered intravenously over 10 min.

[Results] Sulpiride at 2 mg/kg did not affect HR or MAP₉₀, and increased MAP₉₀ with decrement of HR at 20 and 60 mg/kg. Aripiprazole at 0.03 and 0.3 mg/kg did not affect HR or MAP₉₀, and increased MAP₉₀ with decrement of HR at 3 mg/kg. Meanwhile, positive chronotropic actions of sulpiride and aripiprazole were not observed. 

[Conclusions] Since sulpiride and aripiprazole have been clinically reported to hardly induce tachycardia, anesthetized guinea pigs are useful for screening of drug-induced QT interval prolongation for safety pharmacology studies.

[Introduction] BCR/ABL1 tyrosine kinase inhibitors (TKIs) have improved the treatment of chronic myeloid leukemia. However, it becomes widely known that treatment with TKIs increases vascular adverse events (VAEs) and the detailed mechanism for VAEs is unknown. We hypothesized that TKIs accelerate atherosclerotic lesions and studied atherosclerotic lesions by TKIs in a familial typeⅡa model mouse.

[Methods] In order to evaluate atherosclerotic lesion by TKIs, Ldlr ^-/-and Apobec1 ^-/-(L^-/-/A^-/-) mice were used. L^-/-/A^-/- mice have a high plasma LDL levels and more pronounced development of atherosclerosis. 8-week-old male L^-/-/A^-/- mice were randomized in 4 groups (n=10 per group) and received oral gavage with DMSO, imatinib(50mg/kg), nilotinib(45mg/kg), ponatinib(10mg/kg) for 16 weeks. Thereafter, mice were sacrificed to evaluate atherosclerotic lesions and plasma cholesterol levels.

[Results] There were no significant differences in atherosclerotic lesions and plasma cholesterol levels between 4 groups.

[Discussion] We could not find an association between atherosclerosis and TKIs in this study. The onset of VAEs by TKIs is very complex and it may be difficult to explain solely by atherosclerosis.

Reactive oxygen species (ROS) play important roles in the progression of septic pathogenesis. Recent studies revealed that neutrophil adhesion on vascular wall followed by neutrophil extracellular traps (NETs) release may trigger platelet aggregation and immunothrombus formation in septic organ failure. Additionally, the adherent neutrophils-produced ROS induce the immunothrombus formation and tissue damage. Our previous study indicated that plasma histidine-rich glycoprotein (HRG) levels significantly decreased in cecal ligation and puncture (CLP) septic mice model and administration of HRG dramatically improved the survival rate of CLP mice. However, the role of HRG on neutrophil ROS production and immunothrombosis in septic condition was poorly understood. In this study, we showed that HRG inhibited immunothrombus formation in pulmonary vasculatures by keeping neutrophils quiescent morphologically and functionally using immunohistochemical staining and in vivo imaging methods and suppressed excess extracellular ROS production from neutrophils using isoluminol method. These results suggested that HRG may regulate the uncontrolled activation of circulating neutrophils in septic condition.

In Diamond-Blackfan anemia (DBA), about half of the patients have mutations in one of several ribosomal protein (RP) genes. The most frequently mutated gene (～25%) is the ribosomal protein S19 (RPS19), in which a hot spot for mutations between residues 52 and 62 has been reported. However, it is not clear why mutations in the ubiquitously expressed RPS19 gene specifically affect erythropoiesis. We previously showed in vitro that the 59th serine residue (Ser59) of RPS19 is phosphorylated by PIM1 kinase. Here we study the involvement of RPS19 and PIM1 in erythropoiesis using zebrafish to determine whether phosphorylation could affect red blood cells production. We generated the rps19 knockdown zebrafish by injection of morpholino antisense-oligo (MO) at the one-cell stage. The rps19-deficient embryos (morphants) showed abnormal morphologies and a decreased number of red blood cells. Although in vitro synthesized rps19 mRNA rescued the aberrant phenotypes in morphants, the recuperation was not shown by substitution of Ser59 residue with alanine or aspartic acid. These observations suggest that reversible phosphorylation of Ser59 is important for the function of rps19. Therefore, we injected the MO against pim1, which phosphorylates Ser59 of rps19. The pim1 morphants showed abnormal head and tail, and a decrease in the number of red blood cells. Co-injection with synthetic pim1 mRNA restored morphology and red blood cell count. These findings suggested that pim1 was related to erythropoiesis. Further consideration will be needed to yield any findings about the relationship between phosphorylation and erythropoiesis by using pim1 deficient fish.

[Introduction] Ultraviolet (UV) radiation, especially UVB (280-320 nm) from sunlight, is one of major environmental hazard to induce skin damage. A single high dose UVB exposure on the skin causes the acute inflammatory reaction which is characterized by erythema, increasing vascular permeability and edema formation. The single UVB-induced damage could be accumulated by chronic UVB irradiation. The accumulated skin damage could lead to skin aging and raise the risk of skin cancer. Therefore, the inhibition of single UVB-induced acute inflammation is important target for protecting skin from UVB exposure. Sairei-to (SRT), a traditional Japanese herbal medicine, has been used for inflammation treatment. Recently, it has been clinically evidenced to inhibit edema formation caused by radiotherapy. However, the effect of SRT on UVB-induced skin inflammation is poorly understood. In this study, we investigated the protective effect of SRT against the acute UVB-induced skin damage in hairless mice.

[Methods] Five-week-old male HR-1 hairless mice were treated with SRT suspension (625 or 1250 mg/kg orally) for 3 weeks (5 days/week). After 3 weeks administration, dorsal skins of the mice were exposed to UVB radiation at a dose of 250 mJ/cm². The change of skin erythema index (EI) and transepidermal water loss (TWEL) were measured every 24 h after UVB irradiation for 3 days. The dorsal skin tissues were collected to evaluate skin thickness, collagen fibers and infiltration of macrophage and neutrophil.

[Results] SRT significantly attenuated UVB-induced increase of EI and TWEL, and suppressed dermal thickening, collagen loss and macrophage and neutrophil infiltration, compared with UVB radiation alone group.

[Conclusion] These results indicated that SRT had preventive effect against UVB-induced skin damage and suggested that it might be a useful agent for protecting UVB-induced inflammation.

Background: Jumihaidokuto (JHT), a traditional Chinese medicine, has been clinically prescribed for the treatment of patients with skin disorder with redness and swelling, such as atopic dermatitis or acne vulgaris. However, it remains unknown how JHT ameliorates the skin disorders. In this study, we investigated the effect of JHT on UVB-induced skin inflammation model.

Methods: Male hairless mice (HR-1) were treated with 1000 mg/kg JHT for 3 weeks. After the last injection, mice were received single dose of 250mJ/cm² UVB. Before and after irradiation, we measured following factors; skin moisture content in epidermis and dermis, skin erythema dose, and transepidermal water loss (TEWL). We also evaluated the effect on skin thickness and the infiltration of neutrophil and macrophage by HE staining and immunohistochemical (IHC) analysis, respectively.

Results: UVB irradiation decreased the skin moisture content in both skin layers, and increased skin erythema dose and TEWL. Pretreatment with JHT significantly improved the skin moisture content loss and TEWL increment, but not skin erythema.

In addition, HE staining revealed UVB irradiation leaded to edema in dermis and hyperplasia in epidermis. Furthermore, IHC analysis also revealed UVB irradiation facilitated the infiltration of neutrophil and macrophage into dermis. On the other hand, pretreatment with JHT inhibited the edema, hyperplasia and the infiltration of neutrophil and macrophage induced by UVB irradiation.

Conclusion: These results suggest JHT could protect from UVB-induced skin moisture loss and inflammation.

Delayed wound healing is a major problem in patients with diabetes, which significantly impairs their quality of life. Prostaglandin (PG) D₂ is a major inflammatory lipid mediator synthesized by hematopoietic PGD₂ synthase (H-PGDS) from PGH₂, a common precursor of all of PGs. In the present study, we investigated the role of PGD₂ in cutaneous wound healing in streptozotocin (STZ)-induced diabetic mice. C57BL/6 mice were injected with 50 mg/kg of STZ intraperitoneally daily for 5 days. Four weeks after the injection of STZ, a full thickness wound was created with an 8-mm diameter biopsy punch on the dorsal of mice. Wound healing was significantly decelerated and cutaneous H-PGDS mRNA was significantly increased in diabetic mice compared with non-diabetic mice. Daily administration of H-PGDS inhibitor for 14 days was significantly promoted wound healing in diabetic mice. These results suggest that PGD₂ involved in delayed wound healing in STZ-induced diabetic mice.

【Background】

- Since dog bladder cancer is usually muscle-invasive and the malignant level is quite high, the proper treatment has not been established in a veterinary clinic. In the previous study, we generated a dog bladder cancer organoids, which could reproduce the cancer microenvironment in vivo and could be applied to the anti-cancer drug sensitivity test for each patient. However, it has not been revealed whether molecular targeting drugs are effective in the inhibition of the survival of the organoids.

【Object】

-The purpose of this study is to identify the effective molecular targeting drugs against dog bladder cancer by using dog bladder cancer organoids.

【Method】

-Dog bladder cancer organoids were treated with 14 molecular targeting drugs for 72hours. The survival rate of organoids was evaluated by an alamarblue cell viability reagent. The effects of drugs on the activation and expression of intracellular signal molecules were investigated by performing western blotting.

【Result】

-Among 14 drugs, treatment of gefitinib, erlotinib, trametinib, and afatinib inhibited the cell viability of organoids in a dose-dependent manner. Furthermore, EGFR, and ERK, and CD44 expression were suppressed by erlotinib treatment.

【Conclusion】

-These results suggest that EGFR inhibitors and a MEK inhibitor might suppress the growth of dog bladder cancer organoids through suppression of CD44 expression. This result is expected to be useful for the development of molecular targeting therapy for bladder cancer diseased dog.

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.

H₂S is endogenously produced by cystathionine-γ-lyase (CSE), cystathionine-β-synthase (CBS) or 3-mercaptopyruvate sulfurtransferase (3-MST). In the present study, we examined the role of endogenous H₂S in the survival of human multiple myeloma (MM)-derived KMS-11 cells and KMS-11/BTZ cells that acquired resistance to bortezomib (BTZ), a proteasome inhibitor. BTZ significantly decreased the viability of KMS-11 and KMS-11/BTZ cells at 10-1000 and 100-1000 nM, respectively. Both Na₂S, an H₂S donor, and GYY4173, a long-lasting H₂S releaser, slightly increased the viability of those cells. Aminooxyacetic acid (AOAA), a CBS inhibitor, strongly suppressed the viability of KMS-11 and KMS-11/BTZ cells, regardless of the presence of BTZ, and DL-propargylglycine (PPG), a CSE inhibitor, exhibited relatively minor cytotoxicity. In contrast, a 3-MST inhibitor had little or no such effect. GYY4173 significantly reversed the cell toxicity of PPG or AOAA in the presence of BTZ. BTZ treatment at 10 nM for 24 h markedly increased protein levels of CBS among three H₂S-generating enzymes in KMS-11, but not KMS-11/BTZ, cells. These data suggest that H₂S generated mainly by CBS promotes the survival of both KMS-11 and KMS-11/BTZ cells, regardless of the presence of BTZ, and that CBS inhibitors are useful to treat BTZ-resistant MM.

Immune checkpoint inhibitors such as anti-PD-1 antibody and anti-PD-L1 antibody have recently been approved for the treatment of melanoma or non-small cell lung cancer. In this study, we examined the anti-tumor effect of anti-PD-L1 antibody with low molecular weight compound.

Mice were subcutaneously inoculated with a mouse cancer cell line. They were allocated into the control，anti-PD-L1 antibody, low molecular weight compound and their combination treatment groups. Anti-PD-L1 antibody was administered intraperitoneally twice a week for two weeks. The low molecular weight compound was administered orally once a day for 14 days. The tumor diameters were measured to calculate the tumor volumes. Observation and measurement were performed for 14 days after the initiation of administration. The tumor was excised and dispersed to analyze tumor-infiltrating lymphocytes（TILs）. The dispersed cells were stained with fluorescent-labeled antibodies and analyzed using the flow cytometer.

As a result, a proportion of TILs subsets including regulatory T cells (Treg), CD8⁺ T cells, tumor-associated macrophages (TAM) and myeloid-derived suppressor cells (MDSC) were altered by administration of drugs. It suggested that the evaluation system described above is useful for combined efficacy study of anti-PD-L1 antibody with low molecular weight compound in the tumor-bearing mouse model.

Antitumor activities of L-MTP-PE (Liposome-entrapped myuramyl tripeptide phosphatidylethanolamine) in the combination treatment with chemo- or immune-therapeutic antitumor agents against various syngeneic tumors were tested. Against liver metastasis model of M5076 carcinoma, L-MTP-PE showed a tendency of elongation of survival days by its single treatment, however, elongation with statistical significance was observed in the combination treatment with 5-FU. Against Meth A fibrosarcoma system, L-MTP-PE showed a significant elongation of survival days in spite of its non-effect on tumor growth, when combined with 5-FU. L-MTP-PE also enhanced antitumor effect of OK-432 (picibanil), a bacterial immunotherapeutic agent against MM46 mammary carcinoma. In parallel with enhanced antitumor activity, TNF production induced by OK-432 was potentiated when primed with L-MTP-PE. These data suggest that L-MTP-PE seems to elongate the survival days of solid tumor bearing mice due to its saving effect on chemotherapeutic drug-induced immunosuppression and that L-MTP-PE also may potentiate the antitumor effect of immunotherapeutic agent OK-432 by the enhanced production of TNF.

Renal cell carcinoma (RCC) accounts for 2–3% of all malignant tumors. Even with oncologic removal, about 40% of patients will develop metastases after surgical resection. The five-year survival probability of patients with metastatic renal cell carcinoma is less than 10% because of the cancer's resistance to chemotherapy and radiotherapy. Thus, there is an urgent need to establish novel therapeutic approaches for metastatic RCC treatment. The metastatic cascade has been reported to be modulated by an endogenous carcinostatic 15-deoxy-Δ^{12, 14}-prostaglandin J₂ (15d-PGJ₂). A nuclear receptor of 15d-PGJ₂ is peroxisome-proliferator activated receptor γ (PPARγ), and its membrane receptor is chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2). 15d-PGJ₂ has also been reported to reduce cell migration, stimulate focal adhesion disaggregation, and induce filamentous actin realignment. In the present study, we evaluated the effects of 15d-PGJ₂ on the migration of Caki-2 RCC cells. Although treatment with low concentrations of 15d-PGJ₂ did not cause apoptosis, it did decrease the migration of Caki-2 cells. PPARγ and CRTH2 did not mediate the inhibitory effect of 15d-PGJ₂ on the migration of Caki-2 cells. Our present study proposes the therapeutic potential of 15d-PGJ₂ for prevention of RCC metastasis.

We reported that differentiation-inducing factor-1 (DIF-1) inhibited the proliferation of various cancer cells including malignant melanoma and that DIF-1 prevented lung colony formation in a mouse model of metastatic melanoma. However, the mechanisms of this action remain to be elucidated. In the present study, we investigated the anti-metastatic effects of DIF-1 in human malignant melanoma A2058 cells. Activities of cell migration and invasion were measured by the wound healing assay and cell invasion assay, respectively. Activities of cell adhesion to extracellular matrix (ECM) and vascular endothelial cells were measured by using ECM-coated plate and human umbilical vein endothelial cells (HUVECs). Expression levels of signaling molecules were measured by Western blotting. DIF-1 suppressed the phosphorylation levels of signal transducer and activator of transcription 3 (STAT3) and subsequently reduced a variety of genes related to cell migration and invasion such as matrix metalloproteinase-2, vimentin, N-cadherin and twist, resulting in the inhibition of cell migration and invasion. Further, DIF-1 inhibited the melanoma cell adhesion to ECM and HUVECs. These results suggested that DIF-1 suppresses the detachment of cancer cells from the primary tumor by inhibiting cell migration and invasion, and also prevents circulating cancer cells from adhering to vascular endothelial cells. Therefore, DIF-1 may have potential to be a lead chemical compound for developing a novel anti-metastatic agent against malignant melanoma.

Probenecid is a well-known uricosuric agent used for gout treatment through blockage of urate transporter in renal tubules. On the other hand, probenecid can also inhibit multiple channels and transporters including multidrug resistance protein-1 (MRP-1). Therefore, probenecid might support the effect of anti-cancer drugs via inhibition of efflux of these drugs from tumor cells.

Conventionally, cancer cell lines have been cultured as two-dimensional (2D) monolayer. However, such condition does not reflect the real tumor situation in vivo. Recently, 3D culture techniques have developed to examine the cancer cell lines with more natural tumor property. In this study, we evaluated the effect of probenecid on prostate cancer cell lines which cultured as multicellular tumor spheroids by culturing in ultra-low attachment plate.

Prostate cancer cell line 22Rv1 cultured as spheroid showed lower sensitivity against anti-cancer drug doxorubicin, compared to cells cultured as monolayer. Probenecid treated-spheroid was more sensitive against doxorubicin. Interestingly, we found that probenecid itself has anti-tumor activity in concentration dependent manner. Probenecid was more effective to 3D cultured spheroid than 2D cultured monolayer. In this presentation, we also show the result of other prostate cancer cell lines and discuss the anti-cancer mechanism of probenecid.

Choline is an organic cation that plays a critical role in the structure and function of biological membranes. Intracellular choline accumulation through choline transporters is the rate-limiting step in phospholipid metabolism, and it is a prerequisite for cell proliferation. In this study, we examined the functional characterization of choline transporters in U251MG glioma cells. Furthermore, we searched for compounds that inhibit choline uptake as well as cell proliferation in a plant-derived natural organic compound library. Choline transporter-like protein 1 (CTL1) and CTL2 mRNA are highly expressed. CTL1 and CTL2 were located in the cell membrane and intracellular compartment, respectively. [³H]Choline uptake was mediated by a single Na⁺-independent, intermediate-affinity transport system. We found two hit compounds that inhibit choline uptake and cell proliferation from 500 plant-derived natural organic compounds. These hit compounds reduced cell survival and enhanced caspase-3/7 activity. One of the compounds inhibited tumor growth in U251MG cell xenograft model mice. These results suggest that CTL1 is functionally expressed in glioma cells and are also involved in abnormal proliferation. Identification of this CTL1-mediated choline transport system provides a potential new target for glioma therapy.

Primary effusion lymphoma (PEL) is defined as a rare subtype of non-Hodgkin's B-cell lymphoma which is caused by Kaposi's sarcoma-associated herpesvirus (KSHV) in immunosuppressed patients. PEL is frequently resistant to conventional chemotherapies such as CHOP. Therefore, novel therapeutic options for PEL have been expected. We had reported that a pyrrolidinium fullerene derivative induces apoptosis via Akt suppression in PEL.

Here, we have synthesized eight pyridinium-type cationic fullerene derivatives and evaluated cytotoxic effects of them against PEL. The pyridinium fullerenes decreased the cell viability of PEL compared with KSHV-uninfected B-lymphomas. The most potent derivative suppressed Wnt signaling by β-catenin downregulation in PEL cells, whereas it did not affect MAPKs, NF-kB and Akt signaling. The fullerene derivative decreased not β-catenin mRNA, but β-catenin protein in PEL cells. NF-kB, MAPK, and Wnt pathways are constitutively activated in PEL, and these activations are thought to be necessary for cell survival and growth of PEL. We consider that the pyridinium fullerene exerts an anti-PEL activity by disrupting Wnt signaling. Now, we are attempting to elucidate the mechanism of β-catenin downregulation by the fullerene derivative.

Cancer cachexia is the metabolic wasting syndrome that the cancer will release a lot of cytokines and result in metabolic abnormalities and anorexia. The rate of the cancer death has been revealed almost 30% in the cachexia patients. Cancer cachexia can vary according to tumor type, site, mass, and host genotype. Clinical studies showed that more than 60% of head and neck cancer (HNC) patients might develop cancer cachexia. Several animal models have been established to elucidate the importance of pro-cachectic cytokines, such as TNF-α and interleukin 6 (IL-6) in the pathogenesis of cancer cachexia. Unfortunately, the pathogenesis of HNC cachexia is still unknown. Our preliminary results demonstrated that IFIT2, an interferon-induced protein with tetratricopeptide repeat 2 (IFIT2) depletion enhances expression of TNF-α, a well-known cancer-cachexia related cytokine in HNC cells. Thus, this study aims to explore the effect of IFIT2 depletion on HNC cachexia. To the end, a murine model was established by injecting the IFIT2 depleted HNC cells. Moreover, the body weight and survival rate were significantly decreased in IFIT2-depleted cells bearing mice as compared to control mice. The quadriceps had a 28.6% reduction in cachectic mice. Similarly, the gastrocnemius had a 33.3% reduction in cachectic mice. These results suggest that IFIT2-depleted HNC cells bearing mice may act as a model for studies on HNC cachexia.

Introduction: Chromone ring constitutes basic skeleton of secondary metabolites in various plants. We have previously investigated 16 groups of chromone derivatives (239 compounds) for their tumor-specificity against human oral squamous cell carcinoma (OSCC) cell lines. Since 3-styrylchromone derivative showed prominent tumor-specificity, we performed here QSAR analysis with 14 newly synthesized 3-styrylchromones.  Method: Tumor-specificity (TS) was calculated by dividing the mean 50% cytotoxic concentration (CC₅₀) for three human oral normal cells (gingival fibroblast, periodontal ligament fibroblast, pulp cell) (A) by that for four OSCC cells (Ca9-22, HSC-2, HSC-3, and HSC-4) (B) (T =A/B).  PSE value that ​​reflects both tumor-specificity and cytotoxicity against cancer cells were calculated as follow: PSE= TS×100/B. Induction of apoptosis was evaluated by cell sorter. QSAR analysis was performed to determine the correlation between cytotoxicity and tumor-specificity of test compounds with 3,167 chemical descriptors, calculated from the most stabilized structure of 3-styrylchromone derivatives. Results and Discussion: Two compounds [7, 14] showed higher tumor-specificity (TS = 301, 182; PSE = 49842, 27898) than doxorubicin (TS = 55, PSE = 24954) and 5-FU (TS = 16; PSE = 26). When the 6 and 7th positions of chromone ring was H and OCH3 group, respectively, higher tumor-specificity was observed. Tumor-specificity was not increased, by introduction of OH, OCH3, Cl, or F into the 3, 4, 5 positions of the benzene ring. Treatment of HSC-2 cells with [7,14] induced the accumulation of HSC-2 cells in the subG1 and G2/M phases, suggesting the induction of apoptosis. The tumor-specificity of 3-styrylchromone derivatives were most correlated with descriptors for molecule shape and electronic charge. The present study suggests the applicability of 3-styrylchromone derivatives as seed compounds for exploring new anticancer drugs.

Advanced glycation end products (AGEs), produced by non-enzymatic glycation between sugar (or its metabolites) and the amino residues on biomolecules, exert the inflammatory response via the stimulation of some pattern recognition receptors including RAGE. AGEs are known to increase in various age-related diseases, suggesting their involvement in chronic inflammation and tissue remodeling. Therefore, it is likely that the regulation of AGEs signaling will be the potent therapeutic target for prevention and treatment of age-related diseases. Previously, we purified the AGEs-binding factor using affinity gel with AGEs as specific ligand. In this study, we analyzed its properties, and tried to identify the peptides antagonizing the AGEs-RAGE binding. By in vitro AGEs-RAGE binding assay, AGEs-affinity chromatography and MALDI-TOF mass analysis, AGEs-binding factor with molecular mass of 70 kD was isolated. This factor inhibited AGEs-RAGE binding in concentration-dependent manner, and it was revealed that the inhibitory region existed near the N-terminus by the analysis using overlapping-peptides. Additionally, two minimal peptides exhibiting the inhibitory activity were identified. These findings suggested that AGEs-binding factor and its derived inhibitory peptides will have the potential usefulness for regulating chronic inflammation and tissue remodeling.

Prostaglandin E₂ (PGE₂) are known to be involved in inflammation and cancer. There are four subtypes of E-type prostanoid (EP) receptors, EP1 to EP4, for PGE₂. Among them, EP2 receptor and EP4 receptor are frequently confused because they are both coupled with Gs-protein. Although, we have previously shown that EP4 receptor is additionally coupled with Gi-protein. PGE₂ is metabolized to 15-keto-PGE₂ by the action of 15-hydroxy prostaglandin dehydrogenase. 15-keto-PGE₂ has been considered as an inactive form of PGE₂. However, we thought 15-keto-PGE₂ may activate EP receptor subtypes as biased agonist, since the only difference between PGE₂ and 15-keto-PGE₂ is a hydroxyl or a carbonyl functional group at position 15. Here we found that 15-keto-PGE₂ acts as a full agonist for EP2 receptor, while acting as a partial agonist for EP4 receptor. In addition, when compared to the affinity and efficacy, it was found that PGE₂ is tend to activate EP4 receptor, but when it is metabolized to 15-keto-PGE₂, it prefers to activate EP2 receptor. Thus, 15-keto-PGE₂ may not be just an inactive form of PGE₂, but may involve in the biological and physiological roles that need to be elucidated.

The structure of piceatannol (PIC) is similar to that of a polyphenol resveratrol (RSV), an activator of an NAD⁺-dependent protein deacetylase SIRT1. However, whether the PIC exhibit cytoprotective effect through the SIRT1 activation remains unclear. Here we compared to the cytoprotective effect through SIRT1 activation of PIC and RSV.

[Results and Methods] We used the C2C12 myoblasts in this experiment. Treatment with antimycin A, an inhibitor of complex III that induces reactive oxygen species (ROS), fluorescence of MitoSOX Red, an indicator of ROS, was increased 12-fold compared with control, but treatment with PIC or RSV suppressed AA-induced ROS reduced by 72% and 26%, respectively.

Treatment with AA significantly increased apoptosis and necrosis, but treatment with PIC or RSV significantly decreased AA-induced apoptosis and necrosis. In SIRT1 knockdown cells with siRNA, the anti-apoptotic effect of RSV was completely inhibited, whereas, the anti-apoptotic effect of PIC was partially retained.

RSV and PIC similarly decreased acetyl-histone H3 levels, suggesting SIRT1 activation.

RSV increased the expression of antioxidative enzymes such as SOD2 and catalase. On the other hand, PIC elevated catalase, but not SOD2. In the presence of deacetylase inhibitors, neither RSV nor PIC changed the acetyl-histone H3 level and antioxidant expression levels.

[Conclusion]

These results suggest that PIC has unknown cytoprotective mechanisms via SIRT1 activation independent pathway, unlike RSV.

The tumor microenvironment is a complex tissue which is described as the accumulation of various stromal cells, sustaining angiogenesis and redox imbalance. Especially, tumor-associated macrophages (TAMs) are one of the major components of tumor tissues, and they play pivotal roles in prompting the various tumor growths by producing growth factors. Previously, we have reported that Transient receptor potential melastatin 2 (TRPM2), a ROS-sensitive Ca²⁺ channel, is abundantly expressed in macrophages and regulate immune responses by tuning various gene expressions. Here, we found that deletion of TRPM2 gene inhibited tumor growth, and the tumors developing in these conditions were characterized by a high density network of immature vessels, severe haemorrhage and increased hypoxia due to non-productive angiogenesis. In addition, TAMs isolated from TRPM2 knock out mice showed strong expression of proangiogenic factor VEGF according to the enhanced activity of transcription factor Stat3. Importantly, the intratumoral injection of angiostatic soluble VEGFR-1 in tumor-bearing TRPM2 knockout mice led to a rescue of tumor growth. We also found that the activation of TRPM2 channel induced by H₂O₂ suppress the activity of Stat3. TRPM2 protein showed physical interaction with Stat3 protein, and their complex was degraded gradually in the presence of H₂O₂. Together, our results suggest that TRPM2-Stat3 complex promotes functional blood vessel formation by controlling the VEGF levels depending on the environmental oxygen/redox conditions.

Effects of CA metabolites on β-AR-mediated relaxation were investigated in mouse (β₁)/GP (β₂) trachea and rat TA (β₃). Among tested seven CA metabolites, metadrenaline (MA) relaxed GP trachea even in the presence of clorgiline (CLG, MAO_A inhibitor). In mouse trachea, only in the presence of CLG, normetadrenaline (NMA) and MA significantly inhibited isoprenaline (ISO)-induced relaxation, which was also inhibited by 3,4-dihydroxyphenylglycol (DHPG) in the presence of 3,5-dinitrocatechol (3,5-DNC, COMT inhibitor). In GP trachea, NMA, MA, 3,4-dihydroxymandelic acid (DOMA), and DHPG significantly augmented ISO-induced relaxation, which was inhibited by NMA, and MA in the presence of 3,5-DNC or CLG plus 3,5-DNC, and by DHPG in the presence of 3,5-DNC. In rat TA, DHPG significantly inhibited the relaxation to CGP-12177A (β₃-AR partial agonist) in the presence of 3,5-DNC. Our findings indicate that 1) MA may have β₂-AR agonistic action; 2) NMA/MA have β₁-/β₂-AR antagonistic action although they enhance β₂-AR-mediated tracheal relaxation in the absence of CA metabolic inhibitors; 3) DHPG shows β₁-/β₂-/β₃-AR antagonistic action, and this is particularly remarkable for β₃-AR. Our observations may partly explain some of the pathologies associated with pheochromocytoma, which is characterized by elevated CA metabolites levels.

The TRPA1 channel is Ca²⁺-permeable non-selective cation channel that shows exquisite sensitivity to reactive oxygen species (ROS). While accumulating evidence has indicated that TRPA1 mediates various physiological functions, such as pain sensing, in mice, the function of TRPA1 is not fully understood in human. Here, we identified and characterized novel splice variants of TRPA1 from human brain cDNA library, one of which excludes the 5′ part of exon 2. Our electrophysiological and intercellular Ca²⁺ measurements revealed that this TRPA1 variant has higher redox sensitivity than intact TRPA1 despite no difference in the sensitivity to the TRPA1 agonist, allyl isothiocyanate. Interestingly, overexpression of the variant increases mitochondrial ROS levels. Altogether, these results suggest that the novel human TRPA1 splice variant exhibits hyper sensitivity to ROS by changing the cellular redox status.

Human muscarinic receptor, M₂ is one of the five subtypes of muscarinic receptors belonging to the family of G protein-coupled receptors. Muscarinic receptors are targets for multiple neurodegenerative diseases. The challenge has been designing subtype selective ligands against one of the five muscarinic receptors. We report high resolution structures of a thermostabilized mutant M₂ receptor bound to a subtype selective antagonist AF-DX 384 and a non-selective antagonist NMS. The thermostabilizing mutation S110R in M₂ was predicted using a theoretical strategy previously developed in our group. Comparison of the crystal structures and pharmacological properties of the M₂ receptor shows that the Arg in the S110R mutant mimics the stabilizing role of the sodium cation, that is known to allosterically stabilize inactive state(s) of class A GPCRs. Molecular Dynamics simulations reveal that tightening of the ligand-residue contacts in M₂ receptor compared to M₃ receptor leads to subtype selectivity of AF-DX 384.

The bioactive lipid sphingosine 1-phosphate (S1P) binds to five known G protein-coupled receptors, S1P_1–5, and acts as a second messenger during cell signaling. Among them FTY720 targeting S1P₁ is used for immunosuppressive agent for the treatment of autoimmune disease. However, FTY720 acts through multiple S1P receptors, the mechanism of action through one or more of these receptors may account for its side effects. In 2011, the X-ray crystal structure of antagonist-bound inactive state S1P₁ was solved, but FTY720 is an agonist. Solving the structure of agonist-bound active state S1P₁ is expected not only to elucidate the mechanism of S1P_1, but to design of a more selective and effective drug.

First, we attempted purification of S1P₁R and G protein complex for structural analysis. However, the expression level of wild-type S1P₁ is very low. To improve this problem, co-expressing dominant negative Gi and Gβγ with S1P₁ increased the yield and enhance the stability of S1P₁-G protein heterotrimer complexes. Negative stain electron microscope (EM) and 2D class averages revealed uniformity and stable complex particles suitable for cryo-EM.

Endothelin receptors (ETRs) is one of G protein coupled receptors, and consist of ET type A receptor (ET_AR) and ET type B receptor (ET_BR). The overexpression of endothelin (ET)-1 or ETRs is related to malignancy of human cancer, although ET-1 was originally identified as an endothelium-derived vasocontractile peptide. In cancer cells, ET-1 activates various signaling pathways, including mitogen-activated protein kinase, phosphatidylinositol 3-kinase, protein kinase C through ETRs, although the mechanisms by which ET-1 activates these signaling pathways remain uncertain. Here, we found that ETRs interacted with annexin A2, which is overexpressed in various cancers. Annexin A2 bound to ET_AR and ET_BR. Upon ET-1 stimulation, serine phosphorylation of annexin A2 increased, while there is no change in tyrosine phosphorylation of annexin A2. Furthermore, we found that annexin A2 silencing suppressed activation of ERK upon ET-1 stimulation. These results suggested that interaction of ETRs and annexin A2 may play important roles in activation of extracellular signal-regulated kinase upon ET-1 stimulation.

Aquaporin 3 (AQP3) is a water and hydrogen peroxide (H₂O₂)-transporter expressed in various epithelial cells and in macrophages. Here, we developed an anti-AQP3 monoclonal antibody (mAb) that inhibited AQP3-facilitated H₂O₂ transport and prevented liver injury in an experimental animal model. Using AQP3 knockout (AQP3^−/−) mice in a CCl₄-induced model of liver injury and fibrosis, we found that AQP3-facilitated H₂O₂ uptake into macrophages was responsible for nuclear factor-κB (NF-κB) cell signaling and macrophage activation during acute liver inflammation. The hepatic inflammation, oxidative stress, and stellate cell activation caused by activated macrophages was dependent on macrophage AQP3 expression. Administration of an anti-AQP3 mAb, which targeted an extracellular epitope on AQP3, prevented liver injury by a mechanism involving inhibition of AQP3-mediated H₂O₂ transport and macrophage activation. These findings implicate the involvement of macrophage AQP3 in liver injury, and provide evidence for mAb inhibition of AQP3-mediated H₂O₂ transport as therapy for macrophage-dependent liver injury.

Glycine is a co-agonist at NMDA receptors and an agonist at glycine receptors.　Glycine transporters reuptake glycine from the synaptic cleft and regulate glycine concentration. Glycine transporter subtype 1 (GlyT1) is abundant in astrocytes and GlyT1 inhibitors have analgesic effect. We found that GlyT1 mRNA expression in C6 glioma cells was increased during amino acid starvation. To investigate the mechanisms underlying GlyT1 mRNA upregulation, we focused on ATF4 (activating transcription factor 4) that is activated during amino acid starvation and mTOR (mammalian target of rapamycin) that is inactivated during amino acid starvation. Tunicamycin, an ER stress inducer that upregulates ATF4 expression, led to increase of GlyT1. Next, we examined the involvement of mTOR in GlyT1 expression. The mTOR inhibitor rapamycin increased GlyT1 mRNA expression in the culture medium with amino acids. Moreover, ATF4 mRNA expression was also increased by rapamycin. These results indicate that ATF4 increases GlyT1 and mTOR downregulates GlyT1 gene expression.