Progressive accumulation of α-synuclein (α-syn) has been associated with Parkinson's Disease (PD) and Dementia with Lewy Body (DLB). The mechanisms through which α-syn leads to neurodegeneration are not completely clear; however, formation of various oligomeric species have been proposed to play a role. Antibody therapy has shown effectiveness at reducing α-syn accumulation in the CNS. Both active and passive immunization strategies have been utilized in animal models and human patients to alleviate the effects of toxic protein aggregation associated with various neurodegenerative diseases. Initial immunotherapeutic studies showed that vaccination with full-length human α-syn protein in α-syn tg mouse models of DLB decreased accumulation of aggregated α-syn and reduced neurodegeneration. Likewise, passive immunization with antibodies against α-syn reduced memory and neurodegenerative deficits by promoting clearance of α-syn via autophagy or microglia-dependent degradation. Furthermore, immunization to certain epitopes of α-syn also reduce α-syn propagation to glial and neuronal cells. We and others have also investigated the use of conformation specific antibodies that recognize only oligomeric versus monomeric or fibrilar α-syn. To date, immunotherapeutic approaches suggest a viable therapeutic approach for the treatment of neurodegenerative diseases that progress with the accumulation and prion-like propagation of toxic protein aggregates.

A commonality between numerous neurodegenerative disorders is the generation of different self-aggregating proteins/peptides whose presence and accumulation both define the disease and drive neurotoxic pathological cascades that result in disease progression. These proteins include amyloid-β peptide (Aβ) generated from amyloid precursor protein (APP) in Alzheimer's disease (AD), α-synuclein (α-syn) in Parkinson's disease (PD), Tau in tauopathies and prions in transmissible spongiform encephalopathies. The oligomerization/self-aggregation and resulting neurotoxicity of such proteins is generally concentration dependent, and agents that selectively reduce their rate of synthesis in a well-tolerated manner possess therapeutic potential. Posiphen ((+)-N-phenylcarbamoyleseroline tartrate) was selected from a screen of 144 analogues generated to lower APP mRNA translation at the level of its 5'-untranslated region. In neuronal cellular studies, the brain of wild type and AD transgenic (APP+PS1) mice, and in CSF of humans with mild cognitive impairment (MCI), Posiphen lowers APP as well as Aβ levels without adverse actions, and is hence being evaluated as a new treatment strategy for AD and MCI. The molecular machinery underpinning Posiphen's repression of APP mRNA translation appears to be relevant to α-syn (Friedlich et al., Mol Psychiatry 12:222, 2007) and, quite possibly, other proteins. In neuronal cellular and in vivo studies, Posiphen lowers α-syn levels and provides neurotrophic actions – augmenting neurogenesis. Posiphen hence possesses potential therapeutic actions across neurodegenerative disorders, including AD, PD and ischemic stroke.

α-Synuclein (αS) is a small, cytosolic protein containing 140 amino acids. It is abundantly expressed in the brain, where it is located in presynaptic nerve terminals. The identification of a missense mutation in the SNCA gene in pedigrees of Parkinson's disease (PD) sheds light on the nature of Lewy bodies, and subsequent immunohistochemical work with the antibodies has revealed that αS is the major component of Lewy bodies (LBs) in PD and dementia with Lewy bodies, as well as glial cytoplasmic inclusions in multiple system atrophy (MSA). The distribution and spreading of these pathologies are closely correlated with disease symptoms and progression. Recent studies have demonstrated that the abnormal forms of αS (synthetic αS fibrils or pathological αS from brains of patients) have prion-like properties which can convert normal αS to an abnormal form in vitro and in vivo. Intracerebral injection of these pathological αS into transgenic or wild-type mouse brains induced prion-like propagation of abnormal αS pathology. Furthermore, injection of the αS fibrils into striatum of wild-type marmoset brains also resulted in spreading of abundant αS pathologies, including robust Lewy body-like inclusions in TH-positive neurons and a significant decrease in the numbers, suggesting the retrograde spreading of abnormal αS and the toxicities. These results strongly suggest that cell to cell transmission of pathological &alphaS play a key role in the progression of α-synucleinopathies. The in vitro and in vivo seeded αS aggregation models may contribute to pharmacology for evaluation and development of disease-modifying therapies for PD and relating disorders.

Stressful events represent a major risk factor for neuropsychiatric disorders. Whereas the effects of chronic stress have been widely investigated in animal models, the long-term consequences of acute stressors have been little studied, despite it is known that even a single trauma may induce a disorder in humans (e.g., PTSD).

Recently, we have dissected the destabilizing effects of acute stress on the excitatory glutamate system. Acute inescapable footshock stress induced a rapid and sustained (at least up to 24 h) enhancement of depolarization-evoked glutamate release/transmission in the prefrontal cortex (PFC). Acute stress also dramatically increased the total number of excitatory synapses in PFC, while at the same time caused a significant atrophy of apical dendrites, measured already 24 h after stress exposur,e and sustained for at least 14 days. Intriguingly, both prior chronic treatment with traditional antidepressants (2 weeks), and single administration of ketamine (KET; 10 mg/kg) 24 h before stress blocked the enhancement of glutamate release. To dissect adaptive and maladaptive mechanisms underlying acute stress response, we are now using sucrose intake (SI) test, a standard behavioral test for anhedonia, allowing to identify animals resilient/vulnerable towards acute stress.

We are also using the Chronic Mild Stress (CMS) model of depression to look at the effects of chronic stress and of the antidepressant mechanism of KET. Rats were subjected to CMS for 5 weeks. Sucrose Preference Test (SPT) was used to distinguish stress-resilient (CMS-R) from vulnerable (CMS-V) rats. KET was acutely administered to CMS-V 24 hours before sacrifice. A decrease in basal and depolarization-evoked glutamate release was measured in synaptosomes from the hippocampus of CMS-V. In situ hybridization showed reduced dendritic trafficking of BDNF mRNA in CA1 and CA3 of CMS-V. Morphological analysis of CA3 pyramidal neurons showed a reduction in total length and branching of apical dendrites. KET reversed most of these CMS- induced changes in CMS-V.

Overall, our results showed that both acute and chronic stress induce a functional and structural remodeling of excitatory synapses, while acute ketamine restores most of the maladaptive changes induced by stress.

Current pharmacological research in psychiatry encourages sex-specific therapeutic intervention. This reflects the rate of successful treatment for environmental- and genetic-related psychiatric diseases in men and women. Males and females use distinct brain circuits to cope with similar challenges. The hippocampus is a crucial target of stress and sex hormones, because of its neuroanatomical connectivity and expression of steroid hormone receptors. We explored sex and genotype differences on hippocampal transcriptome and behavior in response to stress and estradiol in heterozygous BDNF Val66Met (HET-Met) mice, a model of genetic sensitivity to stress. RNA sequencing of ribosome-bound mRNA from hippocampal CA3 neurons demonstrated a sexually dimorphic transcriptome, with HET-Met female mice displaying greater gene expression change than HET-Met males, when compared to their respective wild-type mice. Interestingly, genes common to both sexes were regulated by genotype in opposite direction. HET-Met mice of both sexes showed a pre-stressed translational phenotype, in which the same genes that were induced as a function of genotype, without applied stress, were also induced in wild-type mice by an acute stressor. Pre-stressed gene networks included epigenetic modifiers and glucocorticoid-binding genes. Thus, the translational response to stress in CA3 neurons was sex- and genotype-specific. Behaviorally, males of either genotype did not exhibit cognitive deficits. In females, spatial memory was affected only in HET-Met mice, regardless of stress exposure. This effect was not observed in ovariectomized HET-Met females, suggesting that circulating ovarian hormones induce cognitive impairment in Met carriers. Ovariectomized HET-Met mice also displayed anxiety- and depressive-like behavior when treated with estradiol. This was associated with changes in unique gene networks in the ventral hippocampus of estradiol-treated Met carriers. We found that estradiol add-back in ovariectomized HET-Met mice affected the Extra Sex Combs and Enhancer of Zeste complex, ESC/E(Z), a gene-silencing epigenetic cluster altered in women suffering from premenstrual dysphoric disorder (PMDD). Tissues isolated from HET-Met mice and women with PMDD were compared using RNA-seq data, revealing gene expression similarities that transcended species and cell types. These findings shed light on sex-specific epigenetic targets for the treatment of mental illnesses.

Transient focal cerebral ischemia is the most common type of stroke caused by occlusion of a cerebral artery. It causes both acute and chronic dysfunctions in brain, lowers the quality of life in patients for a long period of lifetime. Neurons in the ischemic core including cerebral cortex and some parts of striatum are immediately damaged after ischemia-reperfusion, sometimes followed by the delayed neuronal death in the areas distant from ischemic core. Hippocampus is a vulnerable region known to show the delayed neuronal death after transient cerebral ischemia, leading cognitive and other dysfunctions in chronic stage. In this symposium, I introduce a possible rat model of post-ischemic depression associated with the neurodegeneration in hippocampal dentate gyrus (DG), which is sensitive to antidepressant.

　In the rat model of cerebral ischemia operated with transient right middle cerebral artery occlusion (MCAO, 90min), cognitive functions assessed by Y-maze and novel object recognition tests were impaired. Moreover, significant aggravation of anhedonia assessed by sucrose preference test was observed after 20 weeks of MCAO. As many previous studies reported, neuronal death of the ipsilateral pyramidal neurons in the hippocampal CA1 area started after a few days of reperfusion, reached to the maximal loss of neurons within one week. Further immunohistochemical analysis also showed apoptotic neuronal death of the granular cells (GC) in ipsilateral DG started from two weeks after MCAO, lead to the significant loss of GC at 20 weeks after MCAO. Proliferation and differentiation of the neural stem cells both in ipsi- and contralateral subgranular zone (SGZ) of DG were increased transiently after reperfusion, however, were almost ceased after 6 weeks of MCAO. Chronic administration of an antidepressant imipramine or fluvoxamine started after one week of reperfusion prevented MCAO rats both from aggravation of anhedonia and from neuronal loss of GC in ipsilateral DG with increasing the survival signal characterized by Bcl-2, and partially recovered the neurogenesis in SGZ.

　Pathogenesis of post-stroke depression (PSD) is considered to be multifactorial with neurodegenerative, psychogenic, and genetic mechanisms. This rat MCAO-based ischemia model could explain PSD by hippocampal neurodegeneration treatable with antidepressant.

MicroRNAs have emerged as key factors in development, neurogenesis and synaptic functions in the central nervous system. In the present study, we investigated a pathophysiological significance of microRNA-188-5p (miR-188-5p) in Alzheimer's disease (AD). We found that oligomeric Aβ _1-42 treatment diminished miR-188-5p expression in primary hippocampal neuron cultures and that miR-188-5p rescued the Aβ _1-42-mediated synapse elimination and synaptic dysfunctions. Moreover, the impairments in cognitive function and synaptic transmission observed in 7-month-old five familial AD (5XFAD) transgenic mice, were ameliorated via viral-mediated expression of miR-188-5p. miR-188-5p expression was down-regulated in the brain tissues from AD patients and 5XFAD mice. The addition of miR-188-5p rescued the reduction in dendritic spine density in the primary hippocampal neurons treated with oligomeric Aβ _1-42 and cultured from 5XFAD mice. The reduction in the frequency of mEPSCs was also restored by addition of miR-188-5p. The impairments in basal fEPSPs and cognition observed in 7-month-old 5XFAD mice were ameliorated via the viral-mediated expression of miR-188-5p in the hippocampus. Furthermore, we found that miR-188 expression is CREB-dependent. Taken together, our results suggest that dysregulation of miR-188-5p expression contributes to the pathogenesis of AD by inducing synaptic dysfunction and cognitive deficits associated with Aβ-mediated pathophysiology in the disease.

Abstract not yet available.

Inflammation also as well as oxidative stress independently or concomitantly lead to atherosclerosis through progression of endothelial dysfunction, resulting in cardiovascular complications. In a clinical setting, subjects with helicobacter pylori (HP), Kawasaki disease and periodontitis and patients with Buerger's disease (TAO) and atherosclerotic peripheral arterial disease (ASO) are ideal models for determining how endothelium-dependent vasodilation is affected by inflammation. Flow-mediated vasodilation (FMD) as an index of endothelial function was significantly lower and the levels of hs-CRP was significantly higher in subjects with seropositive antibodies to HP than in subjects with seronegative antibodies to HP. FMD was significantly lower in Kawasaki disease without classical cardiovascular risk factors than in controls. FMD in children with aneurysm was significantly impaired compared to that in those without. Vascular response to acetylcholine was significantly less in patients with periodontitis than in healthy control subjects. After endothelial nitric oxide synthase (eNOS) inhibitor L-NMMA infusion, there was no significant difference between vascular response to acetylcholine in the two groups. Vascular response to acetylcholine was increased significantly by treatment, whereas there was no significant difference between vascular response to acetylcholine in the untreated group before and after treatment. L-NMMA completely abolished the periodontal therapy-induced augmentation of vascular response to acetylcholine. Serum concentrations of IL-6 and hs-CRP were significantly higher in patients with periodontitis than in healthy subjects. Treatment of periodontitis significantly decreased serum concentrations of IL-6 and hs-CRP. There was no significant difference in FMD between the TAO group and ASO group. The number of circulating progenitor cells (CPCs) and migration of circulating CPCs were similar in the TAO group and control group, whereas the number of circulating CPCs were significantly lower in the ASO group than in other groups. There was a significant correlation between CRP and FMD and migration of CPCs. Both inflammation and endothelial dysfunction independently or concomitantly lead to atherosclerosis, resulting in cardiovascular complications. From a clinical perspective, it is important to select an appropriate intervention that is effective in improving endothelial dysfunction. Therapy for inflammation should improve endothelial function through an increase in NO bioavailability.

Plasma estrogen level is closely related to cardiovascular health. Estrogen has complex effects that are both dependent and independent on transcriptional activities and are mediated by at least three estrogen receptors. The novel G protein-coupled estrogen receptor 1 (GPER) regulates many cardiovascular functions such as the control of fat metabolism, insulin resistance and glucose tolerance, and vascular tone and cardiac diastolic functions. However, effects of its activation and mechanisms of action are not well understood. Both Ca2+ entry and Ca2+ efflux are essential in shaping intracellular Ca2+ signals important for cardiovascular functions. Calmodulin, the ubiquitous transducer of Ca2+ signals, is required for the activities of up to 300 intracellular proteins, yet is insufficiently expressed for all its binding sites. This renders an intracellular shortage and dynamic competition for calmodulin across cardiovascular tissues. Factors that control Ca2+ influx, Ca2+ efflux and calmodulin-dependent activities thus plays important roles in the regulation of cardiovascular activities. Key data will be presented on the effects and mechanisms of GPER in the control of Ca2+ entry and efflux, the role of GPER in a feed-forward loop that regulates functional linkage in the network of calmodulin-dependent proteins and its direct involvement in cardiac pressor response. A mechanistic model for the role of GPER as a moderator of cardiovascular functions will be presented. The data and summative model encourage exploitation of GPER activities in the management of cardiovascular morbidity.

Hypertension is common and in the majority of cases its cause remains unknown. Recent interest has focused on the role of macrophages (M Φ) in blood pressure (BP) regulation. Endothelin-1 (ET-1) is the most potent endogenous vasoconstrictor mediating its effects through two receptors - the endothelin-A receptor (ET_A) and endothelin-B (ET_B) receptor. The ET _B receptor has a specific role in ET-1 clearance. We investigated the role of the M Φ ET_B receptor in a model of angiotensin II (Ang II)-mediated end-organ damage.

MΦ ET_B receptor deficient mice (LysMET_B^-/-) and controls were exposed to Ang II infusion for 6 weeks under a high salt diet. We assessed BP via telemetry, cardiac structure and function and endothelial function by Doppler ultrasound, end-organ injury and plasma and urine ET-1.

At baseline, components of BP did not differ between groups and increased similarly with Ang II. Whereas after 6 weeks of Ang II LysMET_B^-/- and controls had similar left ventricular hypertrophy and cardiac insufficiency, endothelial function was better in LysMET_B^-/- at both baseline and after Ang II (% dilation of basilar artery in response to CO₂, LysMET_B^-/- vs. controls: baseline: 20 vs.11%, p<0.01; at 6 weeks: 11 vs.0%, p<0.01). Baseline renal function and proteinuria did not differ between groups. After Ang II, LysMET_B^-/-showed similar renal function compared to controls but less proteinuria (urine albumin:creat, mg/mmol: 208 ± 10 vs. 530 ± 25, p<0.01), glomerulosclerosis (34 ± 2 vs. 61 ± 4%, p<0.001), and fewer renal MΦ compared to controls (F4/80 staining per high power field, LysMET_B^-/- vs. controls: 1.1 ± 0.7 vs.3.2 ± 0.5%, p=0.02), although similar levels of CD3⁺ T cells. Plasma ET-1 was no different at baseline but increased more in LysMET_B^-/- with Ang II (LysMET_B^-/- vs. controls after 6 weeks Ang II: 3.7 ± 0.7 vs.1.4 ± 0.2 pg/ml, p=0.03). Urine ET-1 was similar baseline and 6 weeks.

Deletion of the MΦ ET_BR is associated with a blunting of the effects of systemic Ang II infusion as reflected by less endothelial dysfunction, reduced inflammation and end-organ damage. The mechanisms for these effects are the focus of ongoing research.

An increase in cytosolic free Ca2+ concentration ([Ca2+]cyt) in pulmonary artery smooth muscle cells (PASMCs) triggers pulmonary vasoconstriction and stimulates PASMC proliferation and migration leading to pulmonary artery wall thickening. In this study, we report that STIM2, a Ca2+ sensor in the sarcoplasmic reticulum (SR) membrane, is required for raising the resting [Ca2+]cyt in PASMCs from patients with pulmonary arterial hypertension (PAH) and activating downstream signaling cascades that stimulate PASMC proliferation and inhibit PASMC apoptosis. Downregulation of STIM2 in PAH-PASMCs by CRISPR/Cas9 reduces the resting [Ca2+]cyt, while overexpression of STIM2 in normal PASMCs increases the resting [Ca2+]cyt. The increased resting [Ca2+]cyt in PAH-PASMCs is associated with enhanced phosphorylation (p) of CREB, STAT3 and AKT, increased NFAT nuclear translocation, and elevated level of Ki67 (a marker of cell proliferation). Furthermore, the STIM2-associated increase in the resting [Ca2+]cyt also upregulates the anti-apoptotic protein Bcl-2 in PAH-PASMCs. Downregulation of STIM2 in PAH-PASMCs with siRNA a) decreases the level of pCREB, pSTAT3 and pAKT and inhibits NFAT nuclear translocation, thereby attenuating proliferation in PAH-PASMC, and b) decreases Bcl-2, which leads to an increase of apoptosis. In summary, these data indicate that upregulated STIM2 in PAH-PASMCs, by raising the resting [Ca2+]cyt, contributes to enhancing PASMC proliferation by activating the CREB, STAT3, AKT and NFAT signaling pathways and promoting quiescent PASMCs to enter the cell cycle and go through the mitosis. The STIM2-associated increase in the resting [Ca2+]cyt is also involved in upregulating Bcl-2 that makes PAH-PASMCs resistant to apoptosis, and thus plays an important role in sustained pulmonary vasoconstriction and excessive pulmonary vascular remodeling in patients with PAH.

Recent studies highlighted that coronary microvascular disease is one of the leading causes of mortality and morbidity in diabetes. However, the molecular mechanisms in which the microvascular in the heart becomes more contractile and the number of capillary density gets less in diabetes are not well understood. In this study, we investigated the role of HuR (ELAV-like protein 1) in the development of coronary microvascular disease in diabetes. HuR is an RNA binding protein that regulates the stability of many RNAs. The protein levels of HuR was significantly decreased in coronary endothelial cells (CECs) isolated from type 2 diabetic mice and in human CECs from type 2 diabetic patients compared to the control CECs. Endothelium-specific HuR knockout (HuRKO) mice exhibited reduced coronary flow velocity reserve (CFVR, an indicator of coronary microvascular function), decreased capillary density in the left ventricle, and attenuated endothelium-derived hyperpolarization (EDH)-dependent relaxation in coronary arteries. A PCR-array using CECs isolated from Wt and HuRKO mice identified 20 genes as a target of HuR. We have extensive research experience in one of 20 genes, connexin 40 (Cx40), and Cx40 mRNA level was significantly decreased in CECs isolated from HuRKO mice. Therefore, we examined HuR-Cx40 interaction and the role of Cx40 in endothelial dysfunction in diabetes. Cx40 predominantly expresses in vascular ECs and regulates EDH-dependent vascular relaxation and angiogenesis. RNA immunoprecipitation confirmed the binding between HuR and Cx40mRNA and it was significantly decreased in diabetes. Cx40 protein expression was significantly lower in CECs in diabetic mice and diabetic patients than the control CECs. Both Cx40 KO mice and diabetic mice exhibited decreased CFVR and capillary density and attenuated EDH-dependent relaxation compared to their controls. Cx40 overexpression increased tube formation in high-glucose treated CECs toward the level seen in control CECs. EC-specific Cx40 overexpression significantly increased CFVR in diabetic mice. These data suggest that decreased HuR expression leads to downregulation of Cx40, decreases EDH-dependent relaxation and capillary density, and subsequently reduces CFVR in diabetic mice. Overexpression of HuR and/or Cx40 would be a potential treatment for coronary microvascular disease in diabetes.

Background: Arterial remodeling and endothelial dysfuntions are defining features of age-dependent hypertension and the molecular key determinant of this phenomenon involves oxidative stress induced-dysregulation of protective signaling pathways. Purinergic receptors (P2YRs), activated by adenine, uridine nucleotides and nucleotide sugars, play pivotal roles in cardiovascular homeostasis. P2Y6R, an inflammation inducible G protein-coupled receptor is ubiquitously expressed in cardiovascular system and was shown to play significant role in vascular tone regulation. Study in mice shows that P2Y6R-G12/13 signaling activation triggers pressure overload-induced cardiac fibrosis, an event speculated to be the upstream mediator of Ang II Type 1 receptor (AT1R) signaling. Here, we inverstigate the role of P2Y6R in Ang II-induced pathophysiology during aging in mice model.

Methods: P2Y6R knockout mice and littermate control were infused with Ang II for 4 weeks. Blood pressure was monitored weekly. At the end of experimental period, abdominal aorta were isolated and tested for vascular remodeling and endothelial function. in vitro studies on smooth muscle cells were carriend out to investigate the interaction between P2Y6R and AT1R.

Results: We found that deletion of P2Y6R attenuated Ang II-induced increase in blood pressure, vascular remodeling, oxidative stress and endothelial dysfunctions in mice. P2Y6R promoted Ang II-induced hypertension and vascular remodeling in mice ina an age-dependent manner. P2Y6R abundance increased with age in vascular smooth muscle cells. AT1R and P2Y6R formed stable heterodimers, which enhanced G protein-dependent vascular hypertrophy but reduces beta arrestin-dependent AT1R internalization. Moreover, the age-related formation of heterodimer between AT1R and P2Y6R was disrupted by MRS2578, a P2Y6R-selective inhibitor.

Conclusion: These results suggest that increased formation of AT1R-P2Y6R heterodimers with age may increase the likelihood of hypertension induced by Ang II. Targeting P2Y6R oligomerization could be a new strategy to reduce the risk of age associated cardiovacular diseases.

Portal hypertension is a major complication in patients with chronic liver diseases and cirrhosis, but its pathogenic mechanism remains unclear. Vascular tone of portal vein smooth muscles is regulated by the activities of several ion channels including Ca²⁺-activated Cl^- (Cl_Ca) channel. TMEM16A is mainly responsible for Cl_Ca channel conductance in vascular smooth muscle cells including portal vein smooth muscle cells (PVSMCs). Here, the functional expression of TMEM16A channels in portal hypertension was analyzed using two experimental animal models, bile duct ligation (BDL) mice with cirrhotic portal hypertension and partial portal vein ligation (PPVL) mice with idiopathic non-cirrhotic portal hypertension. Expression analyses revealed that TMEM16A was downregulated in BDL-PVSMCs by approximately 50% but not in PPVL-PVSMCs. Whole-cell Cl_Ca currents were significantly reduced in BDL-PVSMCs compared to sham- and PPVL-PVSMCs. Portal vein smooth muscles from sham and PPVL mice showed spontaneous contractions which were sensitive to a specific inhibitor of TMEM16A, T16A_inh-A01. On the other hand, portal vein smooth muscles from BDL mice represented similar spontaneous contractions, however, the TMEM16A-mediated component was clearly attenuated. This TMEM16A downregulation was mimicked by the exposure to angiotensin II in normal PVSMCs. The angiotensin II-induced downregulation of TMEM16A was reversed by the treatment with an angiotensin II receptor blocker, candesartan. These results suggest that the reduced Cl_Ca channel activity due to TMEM16A downregulation causes a lower membrane excitability of portal vein smooth muscles and results in preventing from development of portal hypertension.

Individual variation in drug response is a major issue in the area of pharmacotherapy and of a drug development. The pharmacological response variation may cause therapeutic failure or adverse drug reactions in patients who have unusual genotype. It has been well known that the inter-individual variation of the pharmacological responses is related to the heterogeneity of the disease and other clinical variables such as age, gender, diet, drug-drug interactions, and hepatic and renal dysfunction etc. In addition to these environmental factors, it is clear that genetic factors also contribute to the individual variation of drug responses of therapeutic agents, in both pharmacokinetics and pharmacodynamics.

The pharmacogenetic variation of drug disposition causes wide variation of drug concentrations in plasma and probably in the action site, which is usually related to the genetic difference of drug metabolizing enzymes and/or drug transporters. The drug metabolizing enzymes, in particular cytochrome P450 enzymes play a pivotal role in the elimination of many therapeutic drugs. The genetic variation of drug metabolizing enzymes may cause extensive individual variation and/or ethnic difference of plasma drug concentrations, which is related to the variable therapeutic effect and toxic side effect among patients who took same dose of therapeutic drugs. Therefore, many of drug metabolizing enzymes showing genetic polymorphism are already implemented on the clinical practice for the personalized precision pharmacotherapy, including CYP2D6 for eliglustat, CYP2C9 for warfarin, CYP2C19 for clopidogrel etc. In addition to the genotype guided dose regiment selection, therapeutic drug monitoring and/or model based dose prediction will be also good tools to be implemented to the optimum personalized precision pharmacotherapy. The presentation will cover the issues on the current status and perspectives on the pharmacogenomics of drug metabolism and the clinical implementation for the personalized precision medicine/ dosing in the clinical practice.

Membrane transporters are critical determinants of drug action and disposition. In this presentation, I will describe genetic variation in membrane transporters in ethnically diverse populations, focusing in particular on polymorphisms in transporters that play a role in clinical drug response. The use of genomewide methods to discover endogenous substrates and biomarkers for transporters in the solute carrier superfamily (SLC) will be described. In addition, I will present recent studies in our laboratory focused on genetic variants in the organic anion transporter, OATP2B1, the organic cation transporter, OCT1 and the glucose transporter, GLUT2, which has been associated with response to the anti-diabetic drug, metformin, in large ethnically diverse populations. Overall, polymorphisms in transporters are increasingly being recognized for their role in drug disposition and therapeutic and adverse drug response.

MicroRNAs (miRNAs), short (18-24 nucleotides in length) non-coding RNAs, play key roles in the post-transcriptional regulation of gene expression. They are first transcribed from genomic DNA as long primary transcripts called pri-miRNAs. Each pri-miRNA is cleaved to a precursor miRNA (pre-miRNA) having a hairpin stem-loop structure and the pre-miRNA is then processed into a single-stranded mature miRNA. The mature miRNA forming RNA-induced silencing complex (RISC) with some proteins binds to target mRNAs to cause gene silencing through translational repression or mRNA degradation. To date, more than 2,500 miRNAs have been identified in human. miRNAs regulate every biological processes such as differentiation, cell proliferation and apoptosis. Evidence is accumulating that miRNAs regulate the expression of genes related with drug response and toxicity.

　Single nucleotide polymorphisms (SNPs) are present not only genes encoding mRNA but also genes encoding miRNA. SNPs in mature miRNA, pri-miRNA, and pre-miRNA could modify various biological processes by influencing target selection or miRNA processing. When a SNP exists in the seed sequence of mature miRNAs, it can affect miRNA-mRNA interactions. When a SNP exists in the pri-miRNA or pre-miRNAs sequence, it may affect the expression level of the corresponding mature miRNA and lead to modifications in target gene expression levels. Recently, some miRNA-SNPs have been reported to be linked with altered drug response and toxicity.

　We performed Next-Generation Sequencing, targeting ～1,900 kinds of pre-miRNA genes, to characterize SNPs or variants on miRNA genes in Japanese. Approximately 600 kinds of miRNA-SNPs were found, and each subject had ～170 miRNA-SNPs on average. Being consistent with a previous report, SNPs on the seed sequence in the mature miRNAs are rare. By the comparison of the allele frequency of miRNA-SNPs between healthy subjects and cancer patients, SNPs that appear to be linked with cancer risk or drug response and toxicity could be identified. The underlying mechanisms for the association of the miRNA-SNPs with the altered drug response will be discussed. miRNA-SNPs can be novel biomarkers for drug response and toxicity, and may be utilized to optimize individualized medicine.

Since transporters and metabolic enzymes are expressed in various human tissues, variations in ADME genes significantly contribute to individual differences in PK/PD profiles of drugs. Epigenetic mechanisms, such as DNA methylation and microRNA, alter the expression of genes without changing DNA sequences, leading to above-mentioned variations. MATE1, which mediates the excretion of organic cations into bile and urine, has an approximately 20-fold interindividual variability in its hepatic expression. Hepatic mRNA expression levels negatively correlated with methylation levels of the CpG island in the 27 kb upstream of the TSS. Some evidences including the luciferase reporter assay indicated that the 5' CpG island of SLC47A1 acts as an enhancer for the expression, and the DNA methylation level is expected as a biomarker for MATE1 function. Recently, we found that hepatic and intestinal CYP3A4 expressions are also regulated by DNA methylation at the regions on the chromosome 7q21. BCRP, another important drug transporter, has functionally significant 421C>A mutation in the ABCG2 gene, which is a useful biomarker for describing large interindividual differences in the PK profile of sulfasalazine (SASP). However, large inter-genotypic variability still exists in spite of the incorporation of this mutation into the PK of SASP. Since miR-328 negatively regulates BCRP expressions (both mRNA and protein levels) in human tissues, we hypothesized that exosomal miR-328 in plasma, which leaks from the intestine, is a possible biomarker for estimation BCRP activity in the human intestine. We established an immunoprecipitation-based quantitative method for circulating intestine-derived miR-328 in plasma using an anti-glycoprotein A33 antibody. Interestingly, intestine-derived exosomal miR-328 levels positively correlated (p < 0.05) with SASP AUC, suggesting that subjects with high miR-328 levels have low intestinal BCRP activity, resulting in the high AUC of SASP. This is the first study to show circulating tissue-specific exosomal microRNA in plasma has potential as a possible biomarker for estimating transporter function in an organ.

Understanding the molecular basis for the anti-inflammatory efficacy of glucocorticoids has revealed that the majority of therapeutic (and detrimental) actions are mediated by genomic actions. That is, by the ligand-bound glucocorticoid receptor (GR) recruiting epigenetic modifying cofactors which alter the accessibility of the transcriptional apparatus to genomic loci. The development of dissociated GR ligands, which are able to repress inflammatory gene activation, without stimulating the metabolic genes thought to be associated with adverse effects, looked initially promising in vitro, but failed to live up to expectations in vivo. Recent advances in understanding both GR pharmacology and the regulation of gene expression suggest that a more nuanced understanding is required. The pharmacological effects of GR activation are much more complex than classical transactivation versus transrepression, nor can one GR action neatly account for adverse effects, and another for therapeutic effects. Given this complexity, how then are we to improve the safety and efficacy profile of glucocorticoid drugs?

In this talk I will discuss recent evidence that biased signalling at the GR can be rationally harnessed to only modulate the expression of certain genes using quantitative analytical pharmacology approaches. Furthermore, I will discuss how advances in genomics give us a better understanding of the mechanism of action of glucocorticoids. Indeed, the majority of GR binding sites are in intergenic or intronic regions distal to transcription start sites, suggesting they work as enhancer elements, rather than the classical idea of response elements in the proximal promoter. Finally, I will discuss how we may even be able to modulate the inflammatory response by targeting the epigenetic machinery itself.

Steroid receptors play key roles in multiple pathologies, and as such, are common targets for therapeutic intervention. This is exemplified in breast and prostate cancer treatments, where drugs that target estrogen receptor (ER) and androgen receptor (AR) signaling pathways have significantly improved patient outcomes. Though the acquisition of therapy resistance remains a common clinical challenge, the persistent expression of these receptors in recurrent tumors and the success of combined therapies that target distinct regulatory pathways lends hope to development of new complementary therapies that take advantage of insight gained from mechanistic studies of how these receptor pathways can be modulated. Ubiquitylation is an emerging arena for targeted therapies. Ubiquitin, a small protein that covalently modifies substrates, can modulate receptors at multiple mechanistic levels. Most prominent is the role of ubiquitin modification in the turnover and stability of receptor proteins. The regulation of receptors by the ubiquitin-proteasome system (UPS), has given rise to a class of degrader compounds, SERDS and SARDs, many of which are now in clinical trial. New areas that are evolving in the field exploit the broader activities of inhibitors of the ubiquitin-proteasome system that also affect receptor synthesis and transcriptional signaling. Chemical biology and small molecule inhibitors are also making strides toward targeting components of the ubiquitin machinery. Evidence highlighting mechanisms by which ubiquitin and UPS inhibitors regulate ER and AR biology in cancer will be presented.

The androgen receptor (AR), a nuclear receptor superfamily transcription factor, plays a key role in prostate cancer. AR signaling is the principal target for prostate cancer treatment, but current androgen-deprivation therapies (ADT) cannot completely abolish AR signaling owing to the heterogeneity of prostate cancers. Therefore, unraveling the mechanism of AR reactivation in androgen-depleted conditions can identify effective prostate cancer therapeutic targets. Increasing evidence indicates that AR activity is mediated by the interplay of modifying/demodifying enzymatic co-regulators. To better understand the mechanism of AR transcriptional activity regulation, we used antibodies against AR for affinity purification and identified the deubiquitinating enzyme ubiquitin-specific protease 7 (USP7) as a novel AR co-regulator in prostate cancer cells. We showed that USP7 associates with AR in an androgen-dependent manner and mediates AR deubiquitination. Sequential ChIP assay indicated that USP7 forms a complex with AR on androgen-responsive elements (AREs) of target genes upon stimulation with the androgen 5-alpha-dihydrotestosterone (DHT). Further investigation indicated that USP7 is necessary to facilitate and maintain androgen-activated AR binding to chromatin. Transcriptome profile analysis of USP7-knockdown LNCaP cells also revealed the essential role of USP7 in the expression of a subset of androgen-responsive genes. Hence, inhibition of USP7 represents a compelling therapeutic strategy for the treatment of prostate cancer.

Background: The histone deacetylases (HDAC) play an important role in the transcriptional regulation of eukaryotic gene expression by modifying the acetylation state of histones and other important proteins. The HDAC enzymes are composed of 18 family members classified in four classes depending on sequence identity and domain organization. The 11 so-called classical HDAC enzymes of class I, II, and IV are Zn2+ dependent. The remaining seven class III HDAC enzymes are referred to as sirtuin (SIRT) enzymes and require NAD+ as an essential cofactor. Aberrant HDAC enzyme function has been implicated in many diseases including various forms of cancer, asthma and allergic diseases, and inflammatory and CNS disorders.

Previously we showed evidence that inhibition of histone deacetylases (HDAC) attenuate development of hypertension in DOCA-induced hypertensive rats and spontaneously hypertensive rats.

Objective: We hypothesized that HDAC inhibition attenuates development of hypertension and hyperglycemia induced by Cushing's syndrome.

Methods and Results: Expression of target genes was measured by quantitative real-time PCR (qPCR). Recruitment of hormone receptor on promoters of target genes was analyzed by chromatin immunoprecipitation (ChIP) assay. Interaction between hormone receptor and HDACs was analyzed by co-immunoprecipitation (CoIP). Animal model of Cushing's syndrome was established by subcutaneous implantation of osmotic mini-pump containing dexamethasone (Dex, 10 ug/day) or adrenocorticotrophic hormone (ACTH, 40 ng/day) for 4 weeks. Blood pressure was determined by tail-cuff method. Blood glucose level was analyzed by Accu-Check Performa. Dex and ACTH infusion induced hypertension and hyperglycemia which were attenuated by administration of valproic acid (VPA), a class I and IIa HDAC inhibitor. Expression of hormone target genes related with sodium reabsorption and gluconeogenesis was elevated by Dex and ACTH infusion, which was repressed by VPA administration in the kidney. In addition, enrichment of hormone receptor and RNA polymerase II on the promoters of target genes was elevated by Dex and ACTH infusion, which was attenuated by VPA administration.

Conclusion: HDAC inhibition attenuates development of hypertension and hyperglycemia induced by Cushing's syndrome.

Prostaglandins (PGs) play roles in various types of inflammatory diseases by exerting their pro-inflammatory actions. Particularly, PGE₂ has been reported to work as one of the pro-inflammatory mediators during the pathological processes in various peripheral tissues. The actions of PGE₂ are mediated by four PGE receptor subtypes, EP1, EP2, EP3, and EP4: EP1 receptor is coupled to intracellular Ca²⁺ mobilization, EP2 and EP4 are coupled to stimulation of adenylate cyclase, and EP3 is coupled mainly to inhibition of adenylate cyclase. Both pharmacological and genetic analyses have clarified which EP subtype are involved in each of PGE₂ actions (Narumiya S, Sugimoto Y, et al. Physiol. Rev. 79: 1193, 1999; Sugimoto Y, and Narumiya S. J. Biol. Chem. 282: 11613, 2007). For instance, EP3 receptor is involved in inflammation-associated fever generation, and EP1 is involved in thermal hyperalgesia. However, until recently, it was unknown which EP subtypes mediates PGE₂-induced inflammatory response, such as enhancement of vasopermeability, edema formation and leukocyte infiltration. In order to clarify these points, we employed arachidonate-induced and PGE₂-elicited dermatitis models and examined the effect of each EP gene disruption on this model. Finally, we uncovered that PGE₂-EP3 signaling triggers acute inflammatory responses by mast cell activation in the skin (Morimoto K, Shirata N, et al. J. Immunol. 192: 1130, 2014). In addition to the roles of PGE₂-EP3 signaling in mast cell activation, we would like to discuss on potential roles of PGI₂ in the modulation of inflammatory mast cell responses.

Although epidemiologic and experimental observations support the hypothesis that chronic inflammation and diet are risk factors for colorectal cancer, the precise mechanisms by which these factors contribute to the development of cancer are poorly understood. Evidence for the link between inflammation and cancer comes from epidemiologic and clinical studies showing that use of nonsteroidal anti-inflammatory drugs (NSAIDs) reduce the relative risk for developing colorectal cancer (CRC) by 40-50%. NSAIDs exert some of their anti-inflammatory and anti-tumor effects by targeting cyclooxygenase enzymes (COX-1 and COX-2). Metabolism of arachidonic acid, a major ingredient in animal fats, by cyclooxygenase enzymes provides one mechanism for the contribution of dietary fats and chronic inflammation to carcinogenesis. COX-2-derived prostaglandin E₂ (PGE₂) is a pro-inflammatory mediator that promotes tumor progression, metastatic spread, cell motility and modulates the immune cells within the tumor microenvironment.

Considering the importance of PGE₂ signaling in inflammation and colorectal carcinogenesis, the aim of our work is to determine how PGE₂ modulates the tumor microenvironment in order to promote tumor progression. We have found that PGE₂ regulates a number of genes involved in many of the basic hallmarks of cancer some of which are context specific. Via its effect on cancer stem cells (CSCs), it can increase the numbers of CSCs and their metastatic spread to the liver. Via its effects on CXCL1 and other CXCR2 ligands it modulates the number of myeloid derived suppressor cells in the tumor microenvironment. PGE₂ can also modulate the "pre-metastatic" niche prior to cancer cell metastasis. All of these issues will be discussed along with unpublished data that has been recently acquired by the laboratory.

Injury of the aortic endothelium occurs frequently due to disturbed flow-induced abnormal shear stress, metabolic abnormalities and hypertension. However, chronic endothelial injury contributes to atherosclerosis and aneurysm. Hemodynamic shear stress response of the endothelium is one the key stimuli. Pulsatile laminar shear forces promote normal homeostasis whereas disturbed shear forces which are common in geometrically challenged areas of the vasculature (branch points, lesser curvature) lead to endothelial injury and dysfunction. We recently showed that sphingosine 1-phosphate (S1P) receptor-1 GFP signaling mice shows marked activation of β-arrestin-coupled GPCR signaling (Galvani et al. (2015) Science Signaling) at sites of endothelial injury. Given the fact that S1pr1 is an inhibitor of vascular injury and atherosclerosis as shown in the aforementioned study, we sorted GFP+ aortic endothelial cells (injured aortic endothelial cells; IAEC) and compared with GFP- aortic endothelial cells (healthy aortic endothelial cells; HAEC). IAEC and HAEC from normal S1pr1 GFP signaling mice (N=4-5; age ～ 8-10 weeks) were used for systems level chromatin profiling (ATACseq) and RNA expression (RNAseq). From this dataset, we found enrichment of signal transduction pathways influencing immune cell infiltration, inflammation, endothelial mesenchymal transition (EndMT), lipid uptake and cell proliferation. Expression of unique transcription factors were modulated and their binding sites were enriched in the open chromatin of IAEC and HAEC. Open chromatin sites were associated with genes that regulate immune cell infiltration, inflammation, EndMT, lipid uptake and cell proliferation. These data suggest that either S1pr1 β-arrestin coupling which is associated with GPCR endocytosis and biased signaling regulates transcriptional events involved in endothelial injury. Alternatively only some of these genes are regulated by S1pr1 signaling whereas the others are regulated directly by disturbed flow-induced biomechanical signaling. We will present our recent data in this area of investigation. These studies are expected to further increase our understanding of endothelial injury at the molecular level and may ultimately lead to the development of novel approaches to treat endothelial injury which occurs in many diseases including chronic inflammatory, autoimmune, cardiovascular, neurological and metabolic conditions.

Cyclooxygenase 2 (COX-2) and microsomal prostaglandin E synthase-1 (mPGES-1)-derived Prostaglandin E2 (PGE2) induces angiogenesis especially in tumor microenvironment via PGE2 receptor (EP). But the long terms clinical use of COX-2 inhibitor increased risk of ischemic cardiovascular diseases. Immune cells, especially regulatory T cells (Treg) related to cancer growth and angiogenesis in myocardium following acute infarction. It was reported that PGE2 modulated function and differentiation of Treg. Based on these previous reports, we hypothesized COX-2 and mPGES-1-derived PGE2 and EP signaling contribute to recovery from ischemia by accumulation of Treg. Ischemic hind limb model was made by femoral artery ligation. Blood flow recovery was significantly suppressed in Aspirin- and the COX-2 inhibitor-treated mice compared with vehicle-treated mice. The mRNA expressions of COX-2, and mPGES-1 together with those of FOXP3, a specific transcription factor for Treg, in the ischemic muscle was significantly suppressed in Aspirin- and COX-2 inhibitor-treated mice. Compared with wild type (WT) mice, mPGES-1 deficient mice (mPGES-1KO) showed significantly delayed blood flow recovery. Immunohistochemical analysis against FOXP3 showed that the number of accumulated FOXP3+ cells in the ischemic tissues was decreased in Aspirin-, COX-2 inhibitor-treated mice and mPGES-1KO compared to vehicle-treated mice. The number of accumulated FOXP3+ cells and blood flow recovery was significantly suppressed by injecting folate receptor 4 (FR4) antibody in WT (P<0.05) but not in mPGES-1KO. PGE2 binds to EP receptor, EP1-EP4. In order to estimate which EP receptor is predominantly involved in recovery from ischemia, we analyzed the expression of EP receptors in the ischemic tissues and thymus.Compared to other EP receptors, the expression of EP4 in the ischemic tissues was significantly enhanced compared to naive tissues. In vitro study showed that the expression of EP4 in Treg from thymus under CD3 antibody stimulation was suppressed in mPGES-1KO compared to WT (P<0.05). Moreover, the blood flow recovery was significantly suppressed in EP4KO compared to EP4WT (P<0.05). These results suggested that COX-2 derived PGE2 induces blood flow recovery from ischemia by accumulating Treg via EP4. Highly selective EP4 agonist might be useful for treating peripheral artery disease.

Biosimilar is biological product which is highly similar to the reference product not withstanding minor differences in clinical inactive components and there are no clinically meaningful differences between the biological product and the reference product in terms of the safety, purity, and potency of the product. Generic drug is a copy of a small-molecule (chemical) drug, which can be fully defined structurally and reproduced with an identical chemical structure. However biosimilars can never be identical to reference product due to complex manufacturing process of biologic agents. For the biosimilar, a greater emphasis is on nonclinical (physicochemical, biologic, and animal) development compared with originator emphasis on clinical trials. A comparative clinical study for a biosimilar development program should be designed to investigate whether there are clinically meaningful differences between the proposed product and the reference product. Extrapolation of clinical trial results between disease states, such as rheumatic disease and gastrointestinal diseases should proceed with caution, since safety and immunogenicity issues may arise.

To date, safety profiles of the biosimilars have been consistent with those of the originator biologics. But anti-drug antibody response to biosimilars may lead to a number of deleterious consequences, such as loss of efficacy, altered pharmacokinetics, cross-reactivity to endogenous protein and adverse events. Because of high cost biologics, biosimilar therapy could reduce the pressure on healthcare budgets. It increases patients' access to biologics and further to new regimens and new drugs. It reduces cost of expensive biologic medicinal products and contribute to reduce the price of reference biologics.

Therefore regulatory bodies need to ensure that appropriate analytical, preclinical and clinical studies are done to ensure comparable safety and efficacy between biosimilar and bio-originator. Switching between a bio-originator and a biosimilar appears not to compromise efficacy and safety. However multiple switching, between biosimilar and biosimilar, remains an unknown. More studies, and more real-world data will be needed to ensure that safety is maintained.

For the approved biosimilars for infliximab, etanercept, adalimumab and rituximab, the similarity in efficacy, safety and immunogenicity was demonstrated. But in some points, no clinically significant differences were noted (eg., injection site reaction and immunogenicity).

Interleukin (IL)-6 has a wide range of effects on immune cells and contributes to the pathogenesis of autoimmune diseases. In the synovial tissue of rheumatoid arthritis (RA), fibroblast-like synoviocytes mainly expressed IL-6, which induces production of inflammatory mediators, such as chemokines and adhesion molecules, enhancement of angiogenesis and osteoclastogenesis, and differentiation into Th17 and plasma cell. Therefore, IL-6 signaling was thought to be a therapeutic target for RA. IL-6 is engaged by IL-6 receptor and IL-6 signal transducer/gp130, which leads to the formation of the dimer as a signaling-competent hexamer. Tocilizumab, humanized anti-IL-6 receptor monoclonal antibody (mAb), was launched on 2008 in Japan for RA. It has been shown that tocilizumab is effective for the arthritis and also inhibits bone destruction. Tocilizumab monotherapy, without methotrexate, is also effective. Tocilizumab is now world widely used for RA. Tocilizumab was also used for juvenile idiopathic arthritis and Castleman disease. Moreover, recently tocilizumab has been approved for Takayasu arteritis and giant cell arteritis. Tocilizumab may also be effective for polymyalgia rheumatica. However, treatment of tocilizumab increases frequency of serious infection and sometimes induces neutrocytopenia, thrombocytopenia, and dyslipidemia. Sarilumab, human mAb against anti-IL-6 receptor, was just approved for RA in this year. Sarilumab as well as tocilizumab binds domain 2 of IL-6 receptor. Sarilumab showed similar effects and similar adverse events for RA as tocilizumab. Sirukumab, human anti-IL-6 mAb, also showed efficacy for RA. However, US Food and Drug Administration did not approve it for RA because of slight increase of numbers of dead patients and cardiovascular events. Blockade of IL-6 signaling is effective for RA. It is expected that more IL-6 blocking drugs will be developed.

Treatment for rheumatoid arthritis (RA) has been revolutionized by the use of biologics, although some limitation still exists. Kinase inhibitors are orally available and numbers of compounds have shown efficacy in animal models but not in human with favorable benefit-to-risk ratio until the Janus kinase (JAK) inhibitors. Tofacitinib ansbaricitinib are the JAK inhibitors approved in Japan for treatment of RA showing similar efficacy with biologics and a couple of JAK inhibitors with different specificity are on phase III clinical trials.

JAK is a tyrosine kinase in the cytoplasm constituting a family with JAK1, JAK2, JAK3 and TYK2. JAKs are expressed universally except for JAK3 which is expressed only on hematopoietic cells and constitutively binds to the specific receptors. Specific combination of JAKs are activate depending on the ligand. Multiple cytokines and hormones activate JAKs and some of them are deeply involved in the pathology of RA. Hence JAK inhibitors are able to inhibit multiple cytokines and hormones in multiple cells which is in contrast to biologics. Interestingly, other kinases such as Burton's tyrosine kinase are also targeted for treatment of RA.

In order to elucidate the mechanism of JAK inhibitors we evaluated lymphocytes from patients treated with tofacitinib. As a result, decreased proliferation of CD4+ T cells correlated with clinical efficacy and low CD8+ T cells before tofacitinib treatment was a risk for infection. In order to investigate the effect on synovitis, synovium and cartilage from RA patients undergoing joint replacement was implanted to an immunodeficient mouse. Tofacitinib significantly decrease human IL-6, IL-8 and matrix metalloproteinase-3 in mouse serum and synovium invasion into the cartilage. Effect on dendritic cells was also analyzed and revealed that co-stimulatory molecule expression was suppressed followed with suppressed proliferation of co-cultured allogenic CD4+ T cells. In conclusion, JAK inhibitors suppress the immune system in multiple methods which results in anti-rheumatic effect similar to biologics with some specific side-effects.

In the last two decades, a variety of targeted biologic and synthetic molecules have been developed to treat rheumatoid arthritis. Rheumatologists and patients now have therapeutic options with varying mechanisms of action and targets. Remarkably, the safety profile of these agents are fairly similar, although differences do exist particularly with certain types of infections according to drug class. This lecture will explore the unique safety aspects of various targeted therapies and synthetic DMARDs. The emergence of JAK inhibitors provides unique challenges from a safety standpoint, particularly with regard to infection and potentially venous thromboembolism. Advances in the safety of both biologics and synthetic DMARDs include the ability of these agents to be steroid sparing. Advances in vaccination and screening for certain types of infections have also become more reliable, and these will be reviewed during this lecture.

The last two decades have witnessed the role of dipeptidyl peptidase-4 inhibitors (DPP-4i) in translation of the incretin-research science into clinically meaningful outcomes in management of type 2 diabetes (T2DM). The conceptual change shifted emphasis from a gluco-centric approach to holistic exploration of the underlying pathophysiological processes. This was conceivable as the discovery of glucagon like peptide-1 (GLP-1) in 1987 was followed by the understanding how DPP-4i's could prevent GLP-1 inactivation. The discovery of the world's first DPP-4i, DPP-728, and its pharmacologically improved version with optimised binding kinetics, vildagliptin, in 1998, lead to proof-of-concept studies establishing the prospect of DPP-4 inhibition in glycaemic control.

During the last 20 years, a vast array of preclinical and clinical studies has demonstrated the detailed mechanism of action of vildagliptin and its selectivity in blocking of inactivation of GLP-1 and glucose-dependent insulinotropic polypeptide (GIP). Its clinical efficacy and safety in monotherapy and as an add-on therapy highlighted also the importance of acknowledging hypoglycaemia and weight gain as barriers to optimised care in T2DM rather than signs of progression of the underlying condition.

During the development of DPP-4i's the regulatory requirements for new anti-diabetic drugs underwent considerable changes, with increased emphasis on safety. This led to the systematic collection of adjudicated cardiovascular (CV) safety data for meta-analysis or initiation of large, standardised CV safety studies. Furthermore, the growing awareness and the rapidly changing demographics of T2DM worldwide and its precursor, obesity, demanded a more diverse approach to recruitment, conduction and reporting in randomised and real-world clinical trials; novel clinically relevant questions were yet to set new standards for foundation for clinical evidence for optimised management of T2DM. Finally, the global financial crisis of the last decade forced a new awareness on the health economics- obliging conversion of the clinical findings beyond patient outcomes to be assessed from the perspective cost-effectiveness. These unique developments in the global landscape, and the role the DPP-4i's, specifically vildagliptin, have played a significant role in research advancement and how via translation clinical pharmacology findings into clinical practise optimisation of diabetes care could improve in all diverse populations with T2DM worldwide.

Abstract not yet available.

Adiponectin (Ad) is an antidiabetic adipokine, which binds to its receptors AdipoR1/R2 (Nature, 2003), leading to activation of AMPK and PPAR pathways, respectively (Nature med. 2007, Nature 2010). Recently, small-molecule AdipoR agonist was shown to ameliorate diabetes and increase exercise endurance, and at the same time prolong shortened lifespan in obesity (Nature 2013). Although AdipoRs are predicted to contain seven-transmembrane (7TM) domains, with an internal N-terminus and an external C-terminus, which is opposite to GPCRs, crystal structures of AdipoRs remained to be determined.

In this study, we optimized properties of AdipoRs by mutations, and then used Fv fragment of an anti-AdipoR monoclonal antibody and lipidic cubic phase for crystallization. Then, we successfully determined crystal structures of human AdipoR1/R2, and found that overall structures of AdipoRs are indeed distinct from those of GPCRs (Nature 2015). Moreover, as for common functions of AdipoRs such as Ad binding, mutational analyses of conserved residues between AdipoR1/R2 shown by crystal structures revealed that Ad may broadly interact with extracellular surface of AdipoRs, in a different manner from previously anticipated. In contrast, as for specific functions of AdipoRs such as specific intracellular signal transduction pathways, mutational analyses of specific residues shown by crystal structures gave important insight into understanding each AdipoR specific intracellular signaling mechanisms.

The seven-transmembrane domain of both AdipoR1 and AdipoR2 was shown to have a cavity with a zinc-binding site, which contains unidentified extra electron densities. It was thus suggested that these electron densities may represent potential substrates for AdipoR hydrolytic activity or their products. For development of best-in-class AdipoR agonists, optimization of AdipoRon based on 3D structure of AdipoRon-AdipoR complex should be most important.

In conclusion, mutational analyses based on crystal structures revealed novel structural and functional properties of AdipoRs, including 7TM architecture and a putative Ad-binding surface, which are completely distinct from those of GPCRs, thus highlighting uniqueness of AdipoRs. This study should open new avenues toward elucidation of an unprecedented paradigm of signal transduction and development and optimization of AdipoR agonists.

Activins, members of transforming growth factor  (TGF superfamily proteins, are known to play pivotal roles in the reproductive and developmental processes and their variety of functions have recently been explored in many cell types, while the role in glucose metabolism is poorly understood. Here we show that administration or overexpression of Activin B, which is endogenously produced in liver in the fasted state, markedly reduces blood glucose levels in both obese diabetic mice and insulin deficient diabetic mice. Activin B exerts glucose lowering effects via induction of FGF21 through the canonical pathway, suppression of gluconeogenesis and increased insulin secretion through the non-canonical pathway. Although expression of Activin B is not altered by obesity, expression of FSTL3, known as an inhibitory molecule for TGF superfamily proteins, in adipocytes is increased by obesity and strongly correlates with BMI and insulin resistance in mice and humans. Indeed, metabolically beneficial effects of Activin B is completely canceled by co-administration of FSTL3, while suppression of FSTL3 improves glucose homeostasis in obese mice. Thus, increasing expression or action of Activin B may be a novel therapeutic strategy to cure diabetes.

Metabolic syndrome (MetS) is a highly prevalent complex trait despite recent advances in pathophysiology and pharmacological treatment. MetS can begin in early life by so-called the developmental origins of health and disease (DOHaD). The DOHaD concept also offers reprogramming strategies aiming at shifting therapeutic interventions from adulthood to early life, even before clinical symptoms are evident.

Based on those two concepts, this talk will present the evidence for the existence of, and the programming mechanisms in, oxidative stress that may lead to MetS. Despite the diversity in early-life insults, emerging evidence from animal studies indicates the existence of several common pathways that may contribute to the pathogenesis of MetS of developmental origin. One of the possible mechanisms of developmental origins of MetS is induction of oxidative stress, characterized by an imbalance between free radical production and antioxidant defenses, mainly related to dysregulation of reactive oxygen species (ROS) and nitric oxide (NO). The embryo is developed in a relatively low-oxygen environment and is highly vulnerable to oxidant injury. A number of recent studies support the importance of oxidative stress in relation to MetS of developmental origin. These models, including preeclampsia, caloric restriction, maternal diabetes, prenatal dexamethasone exposure, high fructose intake, maternal smoking, and low-protein diet, have been studied.

This will be followed by potential pharmacological interventions targeting on oxidative stress that may serve as a reprogramming strategy to counter the rising epidemic of MetS. We point out that before patients could benefit from this strategy, the most pressing issue is for the growing body of evidence from animal studies in support of pharmacological intervention as a reprogramming strategy to long-term protect against MetS of developmental origins to be validated clinically and the critical window, drug dose, dosing regimen, and therapeutic duration identified.

Epidemiological studies have shown that age is the dominant risk factor for lifestyle-related diseases. The incidence and the prevalence of diabetes, heart failure, coronary heart disease and hypertension increase with advancing age. However, the molecular mechanisms underlying the increased risk of such diseases that is conferred by aging remain unclear. Cellular senescence is originally described as the finite replicative lifespan of human somatic cells in culture. Cellular senescence is accompanied by a specific set of phenotypic changes in morphology and gene expression including negative regulators of the cell cycle such as p53. Primary cultured cells from patients with premature aging syndromes are known to have a shorter lifespan than cells from age-matched healthy persons. It is also reported that the number of senescent cells increases in various tissues with advancing age. Interestingly, such accumulation of senescent cells in aged animals is attenuated by caloric restriction that regulates the lifespan regulatory system and delays age-associate phenotypes. I therefore hypothesize that cellular senescence in vivo contributes to the pathogenesis of age-associated disease. An important feature shared by several types of senescent cells is persistent up-regulation of inflammatory molecules and accumulating evidence has suggested a critical role of senescence-induced inflammation in metabolic and cardiovascular disease. Here I will present our recent data on the role of cellular senescence in age-related pathologies and will discuss the potential of anti-senescence as a novel therapeutic strategy for age-associated diseases.

The origins of susceptibility for cardiometabolic diseases in adult could be traced back to early life, a theory referred to as developmental origins of adult health and disease. It is now recognized that maternal diet impacts significantly the susceptibility to metabolic syndrome in adult offspring; however, how the maternal nutritional insults alter organogenesis and physiological adaptations resulting in the manifestation of cardiometabolic diseases and organ-specific programming in adulthood are largely unknown. Employing a rodent model of developmental programming of metabolic syndrome in response to maternal high fructose diet (HFD), this synopsis summarizes our recent work in the identification of mitochondrial dysfunction and tissue oxidative stress at the brain in the programming of metabolic syndrome in adult offspring. Maternal exposure to HFD (60%) during gestation and lactation induces metabolic traits of hyperinsulinemia, hypertriglyceridemia, insulin resistance, and the increase in blood pressure in young offspring at the age of 12 weeks old. This maternal HFD-programmed cardiometabolic disorder is associated with an increase in the reactive oxygen species production, a reduction in nitric oxide (NO) generation, as well as the decreases in mitochondrial DNA (mtDNA) copy number and the expressions of peroxisome-proliferator-activated receptor γ coactivator-1α (PGC-1α), the nuclear DNA-encoded transcription factors for mitochondrial biogenesis, in the brain stem. Our next-generation RNA sequencing (NGS) findings further indicate that maternal HFD induces long-term transcriptional changes in the brain stem of adult offspring, promoting neural programming. Oral treatment with resveratrol or metformin to the HFD-fed mother significantly preserves mitochondrial biogenesis and bioenergetics. The same treatments also attenuate the enhanced redox-sensitive signaling, leading to protection against tissue oxidative stress, preservation of NO signaling in the brain, and delay in the programming of metabolic syndrome and the development of programmed hypertension in adult offspring. Together, our data suggest pivotal roles of brain mitochondrial dysfunction and tissue oxidative stress in the programming of adult cardiometabolic disorder, and that these signals could be targets for the development of therapeutic interventions to de-program adult metabolic syndrome of developmental origin.

Asthma affects more than 10% of the population. Severe steroid-dependent asthma occurs in ～5% of patients but is estimated to account for 50% of the cost. The only mechanistically new therapies for asthma in the past 30 years, target so-called T2 asthma, which is driven by T helper 2 -type cytokines, with antibodies targeting interleukin-5 or its receptor. These biological therapies do not meet the needs of the majority of the severe steroid dependent asthma subgroup. Our efforts have been directed to identifying pathways which underlie the remodelling response, which is prominent in severe asthma. This focus on structural cell types within the airway wall has identified that the mechanical microenvironment impacts on the pharmacology of the airway, a phenomenon more generally referred to as mechanopharmacology (Krishnan et al, 2016). Recent work has identified casein kinase 1delta /epsilon (CK1 delta/epsilon) as a downstream effector of the remodelling and steroid resistance inducing effects of transforming growth factor (Xia et al 2017). This CK1 delta/epsilon pathway is also implicated in regulating the circadian clock "signalling circuit" via phosphorylation of CLOCK repressors, period and cryptochrome. The connection of CK1delta /epsilon to glucocorticoid activity holds the potential to explain the well-known connection between circadian rhythm and severity of allergy and asthma symptoms, that are more pronounced in the early morning. In addition, there is evidence of anti-allergic effects of CK1 delta/epsilon inhibitors that occur independently of actions on glucocorticoids. We are now examining the clinical development potential of the CK1 delta/epsilon inhibitors in treatment of severe asthma and data on the prototypical inhibitor, PF670462 and on novel agents will be presented.

Krishnan, R., Park, J-A., Seow, C.Y., Lee, P.V.S., Stewart, A.G. (2016). Cellular biomechanics in drug screening and evaluation: Mechanopharmacology. Trends in Pharmacological Sciences 37:87-100.

Xia, Y.C., Radwan, A., Keenan, C.R., Langenbach, S.Y. Li, M Radojicic, D., Londrigan, S.L, Gualano, R.C., Stewart, A.G. (2017). Glucocorticoid insensitivity in virally infected airway epithelial cells is dependent on transforming growth factor-beta activity. PLOS Pathogens http://dx.doi.org/10.1371/journal.ppat.1006138

Fibrosis is pathologically defined in all organs as the terminal mode of the organ. Today, with the introduction of HRCT examination, the UIP pattern is also widely used in routine practice as a form of the poorest prognosis in images. Moreover, while image inspection is inferior to judgment of pathological level, it carries superiority which can reproduce the change over time. In this field, however, the evaluation of the prognostic index based on remarkable development of molecular genetics and biomarkers was delayed.

　On the other hand, in IPF, the change amount of respiratory function (FVC has the most reproducibility) is the most accurate prognostic index. Many clinical trials have shown scientific evidence in recent years.

　Antifibrotic drugs, pirfenidone, resulted in Japan developing its own clinical trial, and the % VC drops by about 50% annually. Prior to the European countries and the United States, it was approved as the IPF treatment drug only in Japan in 2008. Currently permits and licenses are spreading all over the world.

On the other hand, from the results of PANCER trial, treatment of corticosteroid for the suppression of inflammation has been proved to deteriorate the life prognosis of IPF, and it is recommended not to use it in GL. Therefore, instead of "anti-inflammation", "inhibition of fibrosis" has been shown to inhibit the progression of IPF and provably improve the prognosis of life. The second Nintedanib is three growth factor inhibitors developed for the same fibrosis inhibition purpose. It was distributed to the market from 2014.

ILD other than IPF, even if inflammation destroys the existing structure of the lung, fibrosis progresses with aging. There is a limit with anti-inflammatory medicine alone.

Due to the transition of treatment concept, the disease concept also changes. FVC decline is due to the progress of fibrosis. Biomarker research corresponding to disease behavior is underway.

Asthma and COPD remain respiratory diseases with significant unmet needs. Current treatment relies mainly on the use of inhaled bronchodilators for treatment of symptoms and inhaled glucocovrticosteroids as anti-inflammatory drugs. Phosphodiesterase 3 (PDE3) is now recognised as the predominant PDE in airway smooth muscle and PDE4 as the predominant PDE in the majority of inflammatory cells implicated in the pathogenesis of asthma and COPD. Inhibition of PDE3 induces airway smooth muscle relaxation and inhibition of PDE4 inhibits the activation of most inflammatory cells. We have developed a number of drugs exhibiting both PDE3 and PDE4 inhibition in the same molecule that have both bronchodilator and anti-inflammatory activity. An exemplar of this drug class showing "bifunctional" activity is RPL 554 which has now been demonstrated to cause significant bronchodilator activity in patients with asthma or COPD (1). In addition at bronchodilator doses nebuliser RPL 554 inhibits LPS-induced recruitment of inflammatory cells into the lungs of healthy volunteers confirming its anti-inflammatory effects. To date these "bifunctional" actions have not been associated with any significant gastrointestinal or cardiovascular side effects and therefore this class of drug shows promise as a future treatment strategy for patients with asthma or COPD.

(1) Franciosi et al, Lancet Respiratory Medicine, 1: 714-727 (2013)

Inhaled corticosteroid is the first-line controller for asthma and COPD. However, about 10% of the asthmatics (severe/refractory asthma) and most of the COPD patients are resistant to the beneficial effects of corticosteroids. There is a pressing unmet medical need to develop novel therapeutic agents to restore corticosteroid efficacy in affected patients. There have been reports showing the promise of theophylline and rapamycin in reversing steroid resistance in COPD. Our laboratory has demonstrated that andrographolide, a bioactive diterpenoid lactone isolated from the plant Andrographis paniculata, is an effective anti-inflammatory and anti-oxidative compound in both asthma and COPD experimental models. In a severe asthma mouse model using combined IFN-γ/LPS exposure, production of IL-27 and methacholine-induced airway hyperresponsiveness (AHR) were found to be corticosteroid-resistant. Andrographolide was found to restore the anti-inflammatory effect of dexamethasone in LPS/IFN-γ;-induced IL-27 levels in bronchoalveolar lavage (BAL) fluid and AHR in mice. LPS/IFN-γ markedly reduced the nuclear level of histone deacetylase-2 (HDAC2), an essential epigenetic enzyme that mediates corticosteroid anti-inflammatory actions. Andrographolide significantly restored nuclear HDAC2 levels and diminished total HAT/HDAC activity ratio in mouse lungs exposed to LPS/IFN-γ, probably via suppression of PI3K/Akt/HDAC2 phosphorylation and up-regulation of the antioxidant transcription factor Nrf2 level. In a cigarette smoke (CS)-induced COPD model, andrographolide markedly restored dexamethasone actions in inhibiting CS-induced lung neutrophilia. In addition, andrographolide facilitated dexamethasone actions to suppress BAL fluid IL-6, IL-1β, KC and IL-17 levels. In lung lysates, andrographolide markedly restored total nuclear HDAC activity. The complete steroid re-sensitization mechanism of andrographolide remains to be unraveled. Nevertheless, our existing data strongly implicate a potentially novel steroid re-sensitizing activity of andrographolide in both severe asthma and COPD models.

This work was supported by a CREATE Grant R-184-000-269-592 by the National Research Foundation (NRF) of Singapore to W.S.F.W.

The functions of the lower urinary tract, to store and periodically release urine, are dependent on the activity of smooth and striated muscles in the urinary bladder, urethra, and external urethral sphincter. This activity is unusual in its pattern and organized by neural control mechanisms in the brain, spinal cord, and peripheral ganglia. Many of the neural circuits have switch-like or phasic patterns of activity, unlike the tonic patterns characteristic of other autonomic pathways such as those to cardiovascular organs. In addition, micturition is under voluntary control and depends on learned behavior that develops during maturation of the nervous system, whereas many other visceral functions are regulated involuntarily.

Various neurotransmitters, including acetylcholine, norepinephrine, dopamine, serotonin, excitatory and inhibitory amino acids, adenosine triphosphate, nitric oxide, and neuropeptides, both in the periphery including bladder urothelium and in the central nervous system have been implicated in the neural regulation of the lower urinary tract. Due to these complex controlling mechanisms of micturition, injuries or diseases of the nervous system, as well as drugs and disorders of the peripheral organs, can produce voiding dysfunctions such as urinary frequency, urgency, pain and incontinence or inefficient voiding and urinary retention.

For the treatment of overactive bladder (OAB) targeting the peripheral nervous system, muscarinic receptor antagonists have classically been used; however, β3-adrenergic receptor agonists have recently been developed and proven effective for OAB. For the treatment of lower urinary tract symptoms (LUTS) with benign prostatic hyperplasia (BPH), which includes both storage and voiding symptoms, α1-adrenergic receptor antagonists and, more recently, phosphodiesterase inhibitors have been prescribed. Although they are still in preclinical stages, pharmacological modulation of urothelial-afferent interactions targeting purinergic, muscarinic/nicotinic cholinergic and neurokinin receptors has been investigated. Also, pharmacological targets for LUTS treatments have been found in the central nervous system (spinal cord and brain) including those in serotonergic, noradrenergic, dopaminergic/adenosinergic and GABA/glycinergic systems. Thus, in the first portion of this symposium, I will overview and discuss the recent advancements of neuropharmacology of lower urinary tract function and dysfunction.

Originally thought to act only as a barrier, the functions performed by the urothelium are now known to include a primary role in bladder sensation and powerful influences over detrusor contraction. During bladder filling, stretch of urothelial cells induces the release of chemical mediators including ATP, acetylcholine, prostaglandins and nitric oxide. The activation of sensory nerve fibres by ATP and PGE2 is well established, while our understanding of the role of other mediators is less complete.

The urothelium and lamina propria also develop tonic contractions which can be induced by neurotransmitters and drugs acting via a number of receptors including muscarinic, alpha- and beta-adrenergic, tachykinin and serotonergic receptors. These are the same receptors that regulate contractions of the detrusor muscle and thus drugs used clinically to treat overactive bladder will also influence these urothelial/lamina propria responses resulting in altered bladder function. In addition to tonic contractions to drugs, the bladder urothelium/lamina propria also has pacemakers which generate spontaneous contractile activity. This is enhanced during bladder stretch by a mechanism involving acetylcholine release from the urothelium. This finding is particularly interesting when one considers that muscarinic antagonists in the clinical setting act on bladder function during the filling stage of the micturition cycle (ie. during bladder stretch).

These mechanisms involving urothelial mediators and urothelium/lamina propria contractile activity may be necessary for maintaining continence, and changes in function have been observed in a number of conditions including enhanced release of ATP in interstitial cystitis, overactive bladder and ageing. Changes in urothelial function are also found following intravesical drug treatments with cytotoxic drugs or systemic treatment with cyclophosphamide where urinary metabolites cause bladder inflammation and urothelial damage. These urothelial changes may be responsible for the severe urological side effects observed following treatment with cytotoxic drugs.

In conclusion, urothelial functions are regulated by a number of receptors that will be affected during treatment for urinary tract conditions, including anti-cholinergics, alpha-blockers and beta-agonists, whilst cytotoxic drugs directly or indirectly damage the urothelium to cause bladder dysfunction.

The storage and periodic elimination of urine depends on coordinated function of the lower urinary tract (LUT), which consists of the urinary bladder, bladder neck, urethra and urethral sphincter. This coordinated function of the LUT is controlled by complex neural circuits, including the central nervous system, and it involves pathways at the brain and spinal cord mediated by multiple neurotransmitters.

Recent studies in animals and humans have revealed new insights of micturition reflex into sensory and motor mechanisms in the central nervous system. During storage phase, the bladder is distended with a low level of afferent firing, which in turn stimulates sympathetic outflow to the bladder outlet and somatic outflow to the urethral sphincter for continence. Afferent nerves from the bladder project to region of the spinal cord, and ascending afferents from the spinal cord synapse on the midbrain periaqueductal gray (PAG). Further, afferent fibers are relayed via various areas in the brain, including the prefrontal cortex (PFC). The PFC inhibits the PAG and pontine micturition center (PMC). At starting elimination of urine, the PFC relaxes inhibition of the PAG, which excites the PMC. During voiding phase, this stimulates the parasympathetic out flow to the bladder as well as inhibits sympathetic and somatic outflow to the urethral outlet. Regarding neurotransmitters, many studies revealed that multiple neurotransmitters in the spinal cord and brain were associated with micturition reflex. Our group also revealed that some neurotransmitters involved regulation of micturition reflex using microdialysis in the PAG and PFC.

Thus, since LUT function is regulated through complex neural circuits, changes in these neural pathways occur and LUT function is disrupted in various injuries and diseases of the central nervous system. Therefore, further studies of the central nervous system can provide tips of the new treatments in neurogenic LUT dysfunction. We would like to review results of recent development including our studies in the central nervous system regarding LUT function, and introduce promising treatments in LUT dysfunction.

Bladder outlet obstruction (BOO) due to benign prostatic hyperplasia (BPH) is one of the main causes of lower urinary tract symptoms (LUTS) in ageing men. However, many clinical studies have reported the presence of LUTS in unobstructed patients, and bladder dysfunction has been implicated as a pathophysiology of LUTS in unobstructed patients. Lower urinary tract dysfunction (LUTD) is induced by multiple factors including BOO, detrusor overactivity or detrusor underactivity (DUA), chronic inflammation and ageing. For the past few decades, some researchers have been focusing on the bladder ischemia or vascular endothelial function relating to the emergence of LUTD. Bladder ischemia could occur independently of BOO in aging people and the patients with vascular risk factors such as hypertension, diabetes, hyperlipidemia and smoking. However, it's still challenging to clinically demonstrate bladder ischemia due to the lack of established methods for measuring bladder blood flow in a reliable and non-invasive way. Only a few clinical studies using the Doppler ultrasonography showed that the surgical or medical treatment of BPH could improve the bladder blood flow along with the improvement of LUTS. Animal studies using chronic bladder ischemia model without BOO showed that the severity or long duration of bladder ischemia could induce neuronal degeneration, significant decrease in detrusor contractility, and bladder fibrotic change, leading to DUA. Drugs that might have any effects on the blood flow, oxidative stress, inflammation or endothelial function could improve the bladder ischemic changes or LUTD in animal models of BOO and chronic bladder ischemia. Therapeutic agents used for patients with BPH include alpha-adrenergic antagonist, 5-alpha reductase inhibitor and phosphodiesterase-5 inhibitor, and all these drugs have been reported to have an action mechanism on blood flow and endothelial function.

　Thus, a lot of clinical and basic studies indicate a significant role of bladder blood flow in the development of LUTD. Targeting the bladder blood flow and vascular endothelial function could be a promising strategy for treatment of LUTD in aging population in the future.

The calcium signal has a vital role in many processes important in breast cancer progression, including cellular proliferation and migration. An increasing number of calcium permeable ion channels including TRPV6 and TRPV4 have been linked to breast cancers of the basal molecular subtype, which have a significant overlap with triple negative breast cancers; a subtype where the need for new therapeutic approaches has been highlighted. Pharmacological inhibition of calcium permeable ion channels which are a feature of breast cancers of the basal molecular subtype represent new opportunities to attenuate breast cancer cell proliferation and/or migration. Calcium signalling also plays a critical role in the initiation or modulation of cell death pathways, however, the possible opportunity of targeting calcium permeable ion channels in triple negative breast cancer cells has been less extensively investigated. Our studies demonstrated that pharmacological activation of TRPV4 in MDA-MB-468 triple negative basal-like breast cancer cells can induce oncosis and apoptosis. Genetically encoded calcium indicators when combined with automated epifluorescence microscopy is now providing new insights into calcium signalling in triple negative breast cancer cells during apoptotic and necrotic stimuli.

Normal cell progression to their malignant derivatives is associated with remodeling of the proteins controlling such major cellular functions as apoptosis, proliferation and migration. Here, we show that prostate cancer cells use TRP, ORAI and STIM protein redistribution as an oncogenic switch mechanism resulting from genomic and microenvironment perturbations that disrupt the equilibrium of channels and favors the formation of novel channel complexes. This remodeling of Ca²⁺ signaling in turn induces cell progression to a more aggressive phenotype. We also present new mechanistical aspects explaining the major role of ORAI 1 and STIM1 in cancer stem cells.

Our study specifically positions calcium channels and their regulatory proteins at the center of molecular machinery linking deregulated metabolism, calcium homeostasis, and oncogenesis.

The type 2 ryanodine receptor (RyR2) is a Ca²⁺ release channel on the endoplasmic reticulum (ER) and plays a pivotal role in E-C coupling in the heart. Mutations in RyR2 have been linked to various types of cardiac arrhythmias such as catecholaminergic polymorphic ventricular tachycardia (CPVT), idiopathic ventricular fibrillation (IVF), long QT syndrome (LQTS), and so on. These mutations are supposed to develop arrhythmia via abnormal Ca²⁺ signaling in cardiac cells. However, the underlying cellular mechanisms are largely unclear, and hence the effective therapies for them remain to be established. To address these issues, we have developed an efficient approach for functional evaluation of mutant RyR2s by measurements of cytoplasmic and ER [Ca²⁺] and Ca²⁺ dependent Ca²⁺ release activity by [3H]ryanodine binding assay using HEK293 expression system (Murayama et al, Human Mut., 2016, Uehara et al. J. Gen. Physiol., 2017, Fujii et al. Heart Rhythm, 2017). We have found that all CPVT-related mutations exhibit gain-of-function (GOF) phenotypes whereas other IVF and LQTS-related mutations include both GOF and loss-of-function (LOF) phenotypes. In this presentation, I will summarize functional properties of RyR2 mutants related to a variety of cardiac arrhythmias, and show impact of them on Ca²⁺ signaling in cultured cardiomyocytes expressing mutant RyRs. Furthermore, as an attempt to develop therapies for RyR2-related arrhythmias, I will show the effects of RyR2-specific inhibitors, which were recently discovered by our high-throughput screening approach, on Ca²⁺ homeostasis in cardiac and non-cardiac cells expressing mutant RyR2s.

Venoms are widely used by spiders, scorpions, reptiles, cnidarians, fish, as well as some mammals and marsupials to deter predators and incapacitate prey. Many contain hundreds to thousands of individual components with distinct pharmacological activity. Defensive envenomations often cause notoriously intense pain resulting from activation of sensory neurons by specific venom components. This process commonly involves the activation of various ion channels/receptors on the neuronal cell membrane, and invariably leads to an increase in intracellular calcium concentration.

We used a high-throughput, plate reader-based imaging assay and a high-content, microscope-based calcium imaging assay to isolate toxins with specific action on sensory neurons. Pharmacological characterisation of spitting cobra, spider and scorpion venom components revealed the presence of both non-specific, pore-forming toxin components that non-selectively activate sensory neurons, as well as compounds selectively activating calcium-permeable transient receptor potential channels and voltage-gated sodium channels.

The mechanism of action of these venom peptides can provide considerable insight into the pathophysiology of pain and may lead to the identification of novel compounds with analgesic activity.