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Introduction: EP4 is one of the prostaglandin E2 (PGE2) receptors. We previously reported that EP4 promoted cell migration via Ca2+ signaling, however the underlying mechanism has remained unclear. In Ca2+ signaling, Store-operated Ca2+entry (SOCE) is a major mechanism of Ca2+ import from the extracellular to the intracellular space. Orai1, one of the selective Ca2+ channels, plays an important role in SOCE. We focused on the correlation with EP4 and Orai1. Additionally, we found that EP4 promoted the phosphorylation of calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) and AMP-activated protein kinase (AMPK) located downstream of CaMKK2, i.e., cell energy regulators. AMPK activation can control positively or negatively the cellular adenosine triphosphate (ATP) supply. Therefore, we investigated how EP4 regulated the mitochondrial function via Ca2+ and promoted the cell migration in oral cancer cells. Materials and Methods: HSC-3, human tongue squamous cell carcinoma cell line was used. ONO-AE1-437 was used as EP4 agonist. HSC-3 cells were transduced with Orai1 shRNA, and control shRNA using lentivirus. The migration ability was evaluated by scratch assay. Measurement of intracellular Ca2+ concentration was measured using Fura-2. Immunoprecipitation was performed to evaluate the interaction between EP4 and Orai1. XF Cell Mito Stress Test and XF Real-Time ATP Rate Assay were performed to evaluate the mitochondrial aspiration and the ATP production using Extracellular Flux Analyzer. Results: Scratch assay showed EP4 promoted cell migration in oral cancer cells (n=4, p Conclusion: We concluded that EP4 regulated the mitochondrial function via Ca2+ through Orai1 and promoted the cell migration in oral cancer cells. Citation Format: Rina Nakakaji, Masanari Umemura, Kohei Osawa, Soichiro Ishikawa, Kenji Mitsudo, Yoshihiro Ishikawa. EP4 increases the mitochondrial respiration and promotes cell migration in oral cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4835.

Introduction: Lymph node metastasis caused by migration of oral cancer cells is an important prognostic factor. We previously reported that EP4, a prostaglandin E2 (PGE2) receptor, regulates the cell migration in oral cancer cells via Ca2+ signaling. However, how Ca2+ signaling regulates cell migration remains unclear. The intracellular Ca2+ regulates a variety of signaling pathways, either directly or by forming complexes with proteins such as calmodulin (CaM), and then phosphorylates calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2). Furthermore, Ca2+ signaling is directly or indirectly involved in cancer cell proliferation and migration. Therefore, we hypothesized that EP4 regulates the cell migration via CaMKK2 and its downstream signaling in oral cancer. Materials and Methods: Human gingival fibroblasts, HGnF and Human tongue squamous cell carcinoma cell lines, HSC-3 were used. EP4 agonist (ONO-AE1-437) and CaMKK2 inhibitor (STO-609) were used. HSC-3 cells were transduced with CaMKK2 shRNA, and scramble control shRNA using lentivirus. The protein expressions were evaluated by western blotting. The intracellular calcium concentrations were measured using Fura-2, the Fluorescent Ca2+ Indicator. The cell migration ability was evaluated by scratch assay. Immunocytochemistry was performed to evaluate the lamellipodium. The mRNA transcriptions of the mitochondrial associated genes were evaluated by real-time qPCR. The intracellular adenosine triphosphate (ATP) production and the reactive oxygen species (ROS) production were also evaluated by luciferase activity assay and DCFH-DA staining. Results: EP4 protein expression of HSC-3 cells was higher than that of HGnF cells (n=4, p Conclusion: EP4/CaMKK2/AMPK pathway is a novel signal transduction in oral cancer and may be a new target for oral cancer therapy. Citation Format: Soichiro Ishikawa, Masanari Umemura, Rina Nakakaji, Akane Nagasako, Kagemichi Nagao, Yuto Mizuno, Fumina Suzuki, Kohei Osawa, Mitomu Kioi, Kenji Mitsudo, Yoshihiro Ishikawa. EP4 promoted cell migration via mitochondrial biogenesis in oral cancer cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3619.

Doxorubicin (DOX)-induced cardiomyopathy has a poor prognosis. No early detection or effective treatment methods are available in clinical. The mechanisms of cardiotoxicity were considered as oxidative stress and apoptosis in cardiomyocytes. However, the effect of DOX on cardiac fibroblasts remains to be developed. We investigated the direct effect of DOX on the function of human cardiac fibroblasts (HCFs) independently of cell death pathway. Animal study showed that lower dose of DOX (4 mg/kg/week for 3 weeks, i.p.) than a toxic cumulate dose, induced perivascular fibrosis without cell death in hear of mice. DOX increased the protein expression of α-SMA (a marker of trans-differentiation) in HCFs culture cells, indicating that DOX promoted the trans-differentiation of HCFs into myofibroblast. DOX also increased the mRNA and protein expression of matrix metalloproteinase (MMP)-1 in less than 0.1 μM which did not induce cell apoptosis of HCFs cells via PI3K/Akt pathway in HCFs. DOX increased Interleukin-6 (IL-6) via transforming growth factor (TGF)-β/Smad pathway. In addition, DOX induced the mitochondrial damage and increased the expression of Interleukin-1 (IL-1) via stress-activated protein kinases (SAPK)/ c-Jun NH-2termial kinase (JNK). A peroxisome proliferator-activated receptor gamma (PPARγ) agonist, pioglitazone hydrochloride attenuated the expression of fibrotic marker such as α-SMA and galectin-3 and collagen1 via SAPK/JNK signaling. Pioglitazone also suppressed DOX-induced early fibrotic response in vivo. In conclusion, these findings suggested that low dose DOX induced reactive fibrotic change of cardiac fibroblasts via cell death-independent pathway. There may be potentially new mechanisms of DOX induced cardiotoxicity in clinical usage.

Supplemental Digital Content is available in the text., Objective: The ductus arteriosus (DA) is a fetal artery connecting the aorta and pulmonary arteries. Progressive matrix remodeling, that is, intimal thickening (IT), occurs in the subendothelial region of DA to bring anatomic DA closure. IT is comprised of multiple ECMs (extracellular matrices) and migrated smooth muscle cells (SMCs). Because glycoprotein fibulin-1 binds to multiple ECMs and regulates morphogenesis during development, we investigated the role of fibulin-1 in DA closure. Approach and Results: Fibulin-1–deficient (Fbln1−/−) mice exhibited patent DA with hypoplastic IT. An unbiased transcriptome analysis revealed that EP4 (prostaglandin E receptor 4) stimulation markedly increased fibulin-1 in DA-SMCs via phospholipase C-NFκB (nuclear factor κB) signaling pathways. Fluorescence-activated cell sorting (FACS) analysis demonstrated that fibulin-1 binding protein versican was derived from DA-endothelial cells (ECs). We examined the effect of fibulin-1 on directional migration toward ECs in association with versican by using cocultured DA-SMCs and ECs. EP4 stimulation promoted directional DA-SMC migration toward ECs, which was attenuated by either silencing fibulin-1 or versican. Immunofluorescence demonstrated that fibulin-1 and versican V0/V1 were coexpressed at the IT of wild-type DA, whereas 30% of versican-deleted mice lacking a hyaluronan binding site displayed patent DA. Fibulin-1 expression was attenuated in the EP4-deficient mouse (Ptger4−/−) DA, which exhibits patent DA with hypoplastic IT, and fibulin-1 protein administration restored IT formation. In human DA, fibulin-1 and versican were abundantly expressed in SMCs and ECs, respectively. Conclusions: Fibulin-1 contributes to DA closure by forming an environment favoring directional SMC migration toward the subendothelial region, at least, in part, in combination with EC-derived versican and its binding partner hyaluronan.

The EP4 prostanoid receptors are one of four receptor subtypes for prostaglandin E2 (PGE2). Therefore, EP4 may play an important role in cancer progression. However, little information is available regarding their function per se, including migration and the cellular signaling pathway of EP4 in oral cancer. First, we found that mRNA and protein expression of EP4 was abundantly expressed in human‐derived tongue squamous cell carcinoma cell lines HSC‐3 and OSC‐19. The EP4 agonist (ONO‐AE1‐437) significantly promoted cell migration in HSC‐3 cells. In contrast, knockdown of EP4 reduced cell migration. Furthermore, we confirmed that knockdown of EP4 suppressed metastasis of oral cancer cells in the lungs of mice in vivo. Therefore, we focused on the mechanism of migration/metastasis in EP4 signaling. Interestingly, EP4 agonist significantly induced intracellular Ca2+ elevation not in only oral cancer cells but also in other cells, including normal cells. Furthermore, we found that EP4 activated PI3K and induced Ca2+ influx through Orai1 without activation of store depletion and stromal interaction molecule 1 (STIM1). Immunoprecipitation showed that EP4 formed complexes with Orai1 and TRPC1, but not with STIM. Moreover, the EP4 agonist ONO‐AE1‐437 phosphorylated ERK and activated MMP‐2 and MMP‐9. Knockdown of Orai1 negated EP4 agonist‐induced ERK phosphorylation. Taken together, our data suggested that EP4 activated PI3K and then induced Ca2+ influx from the extracellular space through Orai1, resulting in ERK phosphorylation and promoting cell migration. Migration is regulated by EP4/PI3K/Orai1 signaling in oral cancer., EP4 activated PI3K and then induced Ca2+ influx from the extracellular space through Orai1, resulting in ERK phosphorylation and promoting cell migration.

Prevention of cardiomyocyte death is an important therapeutic strategy for heart failure. In this study, we focused on translationally controlled tumor protein (TCTP), a highly conserved protein that is expressed ubiquitously in mammalian tissues, including heart. TCTP plays pivotal roles in survival of certain cell types, but its function in cardiomyocytes has not been examined. We aimed to clarify the role of TCTP in cardiomyocyte survival and the underlying mechanism. Here, we demonstrated that downregulation of TCTP with siRNA induced cell death of cardiomyocytes with apoptotic and autophagic features, accompanied with mitochondrial permeability transition pore (mPTP) opening. TCTP loss did not induce cell death of cardiac fibroblasts. Bcl-2/adenovirus E1B 19-kDa interacting protein 3 (Bnip3) was found to mediate the TCTP-loss-induced cardiomyocyte death. In exploring the clinical significance of the TCTP expression in the heart, we found that DOX treatment markedly downregulated the protein expression of TCTP in cultured cardiomyocytes and in mouse heart tissue. Exogenous rescue of TCTP expression attenuated DOX-induced cardiomyocyte death. In mice, cardiomyocyte-specific overexpression of TCTP resulted in decreased susceptibility to DOX-induced cardiac dysfunction, accompanied with attenuated induction of Bnip3. Dihydroartemisinin, a pharmacological TCTP inhibitor, induced development of heart failure and cardiomyocyte death in control mice, but not in mice with cardiomyocyte-specific TCTP overexpression. Our findings revealed TCTP has a pivotal role in cardiomyocyte survival, at least in part through a Bnip3-dependent mechanism. TCTP could be considered as a candidate therapeutic target to prevent DOX-induced heart failure.

We previously identified a novel nanomagnetic particle, N,N'-bis(salicylidene)ethylenediamine iron [Fe(Salen)]. Fe(Salen) not only shows antitumor effects but also magnetic properties. We found that Fe(Salen) can be used for magnet-guided drug delivery and visualization of accumulated drug by magnetic resonance imaging (MRI) because of its magnetism. In addition, Fe(Salen) can generate heat by itself when exposed to an alternating current magnetic field (AMF), resulting in a hyperthermia effect. Herein, we partly elucidated the antitumor mechanism of Fe(Salen) and carried out an i.v. repeated dose toxicity study to decide the therapeutic amount. Furthermore, we evaluated the antitumor effect of selective intra-arterial injection or i.v. injection of Fe(Salen) by catheter and the hyperthermia effect of Fe(Salen) when exposed to AMF in vivo. We used a rabbit model grafted with VX2 cells (rabbit squamous cell carcinoma) on the right leg. Intra-arterial injection of Fe(Salen) showed a greater antitumor effect than did i.v. injection. The combination of Fe(Salen) intra-arterial injection and AMF exposure showed a greater antitumor effect than did either Fe(Salen) or methotrexate (MTX) without AMF exposure, suggesting that AMF exposure greatly enhanced the antitumor effect of Fe(Salen) by arterial injection by catheter. This is the first report that the effectiveness of Fe(Salen) was evaluated in the point of administration route; that is, selective intra-arterial injection by catheter. Taken together, these results indicate a new administration route; that is, selective arterial injection of Fe(Salen) by catheter, and the development of a new strategy of simultaneous hyperthermia-chemotherapy in the future.

Ubiquitination is a representative, reversible biological process of the post-translational modification of various proteins with multiple catalytic reaction sequences, including ubiquitin itself, in addition to E1 ubiquitin activating enzymes, E2 ubiquitin conjugating enzymes, E3 ubiquitin ligase, deubiquitinating enzymes, and proteasome degradation. The ubiquitin–proteasome system is known to play a pivotal role in various molecular life phenomena, including the cell cycle, protein quality, and cell surface expressions of ion-transporters. As such, the failure of this system can lead to cancer, neurodegenerative diseases, cardiovascular diseases, and hypertension. This review article discusses Nedd4-2/NEDD4L, an E3-ubiquitin ligase involved in salt-sensitive hypertension, drawing from detailed genetic dissection analysis and the development of genetically engineered mice model. Based on our analyses, targeting therapeutic regulations of ubiquitination in the fields of cardio-vascular medicine might be a promising strategy in future. Although the clinical applications of this strategy are limited, compared to those of kinase systems, many compounds with a high pharmacological activity were identified at the basic research level. Therefore, future development could be expected.

We previously reported the efficacy of anti‐cancer therapy with hyperthermia using an alternating magnetic field (AMF) and a magnetic compound. In the course of the study, unexpectedly, we found that an AMF enhances the cytotoxicity of Compound C, an activated protein kinase (AMPK) inhibitor, although this compound is not magnetic. Therefore, we examined the cellular mechanism of AMF‐induced cytotoxicity of Compound C in cultured human glioblastoma (GB) cells. An AMF (280 kHz, 250 Arms) for 30 minutes significantly enhanced the cytotoxicity of Compound C and promoted apoptosis towards several human GB cell lines in vitro. The AMF also increased Compound C‐induced cell‐cycle arrest of GB cells at the G2 phase and, thus, inhibited cell proliferation. The AMF increased Compound C‐induced reactive oxygen species production. Furthermore, the AMF decreased ERK phosphorylation in the presence of Compound C and suppressed the protective autophagy induced by this compound. The application of an AMF in cancer chemotherapy may be a simple and promising method, which might reduce the doses of drugs used in future cancer treatment and, therefore, the associated side effects.

// Rina Nakakaji 1, 2, * , Masanari Umemura 1, * , Kenji Mitsudo 2 , Jeong-Hwan Kim 1 , Yujiro Hoshino 3 , Itaru Sato 1, 2 , Takatsugu Masuda 4 , Masahiro Yamamoto 5 , Mitomu Kioi 2 , Toshiyuki Koizumi 2 , Takayuki Fujita 1 , Utako Yokoyama 1 , Masaki Iida 2 , Motohiko Sato 6 , Hiroshi Sato 7 , Shoko Murofushi 7 , Sayaka Shibata 8 , Ichio Aoki 8 , Haruki Eguchi 7 , Iwai Tohnai 2 and Yoshihiro Ishikawa 1 1 Cardiovascular Research Institute, Yokohama City University Graduate School of Medicine, Yokohama, Japan 2 Department of Oral and Maxillofacial Surgery, Yokohama City University Graduate School of Medicine, Yokohama, Japan 3 Department of Environment and Natural Sciences, Yokohama National University Graduate School of Environment and Information Sciences, Yokohama, Japan 4 Tokyo Neutron Science Laboratory, Tokyo University Institute for Solid State Physics, Kashiwa, Japan 5 Department of Chemistry of Functional Molecules, Konan University Faculty of Science and Engineering, Kobe, Japan 6 Department of Physiology, Aichi Medical University, Nagakute, Japan 7 Advanced Applied Science Department, Research Laboratory, IHI Corporation, Yokohama, Japan 8 Molecular Imaging Center, National Institute of Radiological Sciences, Chiba, Japan * These authors contributed equally to this work Correspondence to: Masanari Umemura, email: umemurma@yokohama-cu.ac.jp Yoshihiro Ishikawa, email: yishikaw@med.yokohama-cu.ac.jp Keywords: iron-salen; taxol; oral cancer; paclitaxel; magnetism Received: July 14, 2017 Accepted: February 20, 2018 Epub: February 26, 2018 Published: March 20, 2018 ABSTRACT N,N’-Bis(salicylidene)ethylenediamine iron (Fe(Salen)) is an anti-cancer agent with intrinsic magnetic property. Here, we covalently linked Fe(Salen) to paclitaxel (PTX), a widely used anti-cancer drug, to obtain a magnetized paclitaxel conjugate (M-PTX), which exhibited magnetic characteristics for magnet-guided drug delivery and MRI visualization. M-PTX increased apoptosis and G2/M arrest of cultured human oral cancer cell lines in the same manner as PTX. Furthermore, marked contrast intensity was obtained in magnetic resonance imaging (MRI) of M-PTX. In a mouse oral cancer model, a permanent magnet placed on the body surface adjacent to the tumor resulted in distinct accumulation of M-PTX, and the anti-cancer effect was greater than that of M-PTX without the magnet. We believe that this strategy may improve future cancer chemotherapy by providing conventional anti-cancer drugs with novel functionalities such as magnet-guided drug delivery or MRI-based visualization/quantitation of drug distribution.

Sympathetic activation causes clinically important arrhythmias including atrial fibrillation (AF) and ventricular tachyarrhythmia. Although the usefulness of β-adrenergic receptor blockade therapy is widely accepted, its multiple critical side effects often prevent its initiation or continuation. The aim of this study is to determine the advantages of vidarabine, an adenylyl cyclase (AC)-targeted anti-sympathetic agent, as an alternative treatment for arrhythmia. We found that vidarabine, which we identified as a cardiac AC inhibitor, consistently shortens AF duration and reduces the incidence of sympathetic activation-induced ventricular arrhythmias. In atrial and ventricular myocytes, vidarabine inhibits adrenergic receptor stimulation-induced RyR2 phosphorylation, sarcoplasmic reticulum (SR) Ca2+ leakage, and spontaneous Ca2+ release from SR, the last of which has been considered as a potential arrhythmogenic trigger. Moreover, vidarabine also inhibits sympathetic activation-induced reactive oxygen species (ROS) production in cardiac myocytes. The pivotal role of vidarabine's inhibitory effect on ROS production with regard to its anti-arrhythmic property has also been implied in animal studies. In addition, as expected, vidarabine exerts an inhibitory effect on AC function, which is more potent in the heart than elsewhere. Indexes of cardiac function including ejection fraction and heart rate were not affected by a dosage of vidarabine sufficient to exert an anti-arrhythmic effect. These findings suggest that vidarabine inhibits catecholamine-induced AF or ventricular arrhythmia without deteriorating cardiac function in mice.

Pro-inflammatory cytokines are released in septic shock and impair cardiac function via the Jak-STAT pathway. It is well known that sympathetic stimulation leads to coupling of the β-adrenergic receptor/Gs/adenylyl cyclase, a membrane-bound enzyme that catalyzes the conversion of ATP to cAMP, thereby stimulating protein kinase A (PKA) and ultimately compensating for cardiac dysfunction. The mechanism of such compensation by catecholamine has been traditionally understood as PKA-mediated enforcement of cardiac contractility. We hypothesized that exchange protein activated by cyclic AMP (Epac), a new target of cAMP signaling that functions independently of protein kinase A, also plays a key role in protection against acute stresses or changes in hemodynamic overload. Lipopolysaccharide injection induced cytokine release and severe cardiac dysfunction in mouse. In mouse overexpressing Epac1 in the heart, however, the magnitude of such dysfunction was significantly smaller. Epac1 overexpression inhibited the Jak-STAT pathway, as indicated by decreased phosphorylation of STAT3 and increased SOCS3 expression, with subsequent inhibition of iNOS expression. In cultured cardiomyocytes treated with isoproterenol or forskolin, the increase of SOCS3 expression was blunted when Epac1 or PKCα was silenced with siRNA. Activation of the cAMP/Epac/PKCα pathway protected the heart against cytokine-induced cardiac dysfunction, suggesting a new role of catecholamine signaling in compensating for cardiac dysfunction in heart failure. Epac1 and its downstream pathways may be novel targets for treating cardiac dysfunction in endotoxemia.

Pro-inflammatory cytokines are released in septic shock and impair cardiac function via the Jak-STAT pathway. It is well known that sympathetic and thus catecholamine signaling is activated thereafter to compensate for cardiac dysfunction. The mechanism of such compensation by catecholamine signaling has been traditionally understood to be cyclic AMP-dependent protein kinase (PKA)-mediated enforcement of cardiac contractility. We hypothesized that the exchange protein activated by cAMP (Epac), a newly identified target of cAMP signaling that functions independently of PKA, also plays a key role in this mechanism. In cultured cardiac myocytes, activation of Epac attenuated the inhibitory effect of interleukin-6 on the increase of intracellular Ca2+ concentration and contractility in response to isoproterenol, most likely through inhibition of the Jak-STAT pathway via SOCS3, with subsequent changes in inducible nitric oxide synthase expression. These findings suggest a new role of catecholamine signaling in compensating for cardiac dysfunction in heart failure. Epac and its downstream pathway may be a novel target for treating cardiac dysfunction in endotoxemia.

Background It has been suggested that protein directly activated by cAMP (Epac), one of the downstream signaling molecules of β-adrenergic receptor (β-AR), may be an effective target for the treatment of arrhythmia. However, there have been no reports on the anti-arrhythmic effects or cardiac side-effects of Epac1 inhibitors in vivo. Methods and Results: In this study, the roles of Epac1 in the development of atrial and ventricular arrhythmias are examined. In addition, we examined the usefulness of CE3F4, an Epac1-selective inhibitor, in the treatment of the arrhythmias in mice. In Epac1 knockout (Epac1-KO) mice, the duration of atrial fibrillation (AF) was shorter than in wild-type mice. In calsequestrin2 knockout mice, Epac1 deficiency resulted in a reduction of ventricular arrhythmia. In both atrial and ventricular myocytes, sarcoplasmic reticulum (SR) Ca2+ leak, a major trigger of arrhythmias, and spontaneous SR Ca2+ release (SCR) were attenuated in Epac1-KO mice. Consistently, CE3F4 treatment significantly prevented AF and ventricular arrhythmia in mice. In addition, the SR Ca2+ leak and SCR were significantly inhibited by CE3F4 treatment in both atrial and ventricular myocytes. Importantly, cardiac function was not significantly affected by a dosage of CE3F4 sufficient to exert anti-arrhythmic effects. Conclusions These findings indicated that Epac1 is involved in the development of atrial and ventricular arrhythmias. CE3F4, an Epac1-selective inhibitor, prevented atrial and ventricular arrhythmias in mice.

Colorectal anastomotic leakage is one of the most feared and fatal complications of colorectal surgery. To date, no external coating material that can prevent anastomotic leakage has been developed. As myoblasts possess anti-inflammatory capacity and improve wound healing, we developed a multilayered human skeletal muscle myoblast (HSMM) sheet by periodic exposure to supraphysiological hydrostatic pressure during repeated cell seeding. We assessed whether the application of an HSMM sheet can promote the healing process after colonic anastomosis. Partial colectomy and insufficient suturing were employed to create a high-risk colo-colonic anastomosis model in 60 nude rats. Rats were divided into a control group ( n = 30) and an HSMM sheet group ( n = 30). Macroscopic findings, anastomotic bursting pressure, and histology at the colonic anastomotic site were evaluated on postoperative day (POD) 3, 5, 7, 14, and 28. The application of an HSMM sheet significantly suppressed abscess formation at the anastomotic site compared to the control group on POD3 and 5. The anastomotic bursting pressure in the HSMM sheet group was higher than that in the control group on POD3 and 5. Inflammatory cell infiltration in the HSMM sheet group was significantly suppressed compared to that in the control group throughout the time course. Collagen deposition in the HSMM sheet group on POD3 was significantly abundant compared to that in the control group. Regeneration of the mucosa at the colonic anastomotic site was promoted in the HSMM sheet group compared to that in the control group on POD14 and 28. Immunohistochemical analysis demonstrated that surviving cells in the HSMM sheet gradually decreased with postoperative time and none were detected on POD14. These results suggest that the application of a multilayered HSMM sheet may prevent postoperative colonic anastomotic leakage.

Type 5 adenylyl cyclase (AC5) plays an important role in the development of chronic catecholamine stress-induced heart failure and arrhythmia in mice. Epac (exchange protein activated by cAMP), which is directly activated by cAMP independent of protein kinase A, has been recently identified as a novel mediator of cAMP signaling in the heart. However, the role of Epac in AC5-mediated cardiac dysfunction and arrhythmias remains poorly understood. We therefore generated AC5 transgenic mice (AC5TG) with selective disruption of the Epac1 gene (AC5TG-Epac1KO), and compared their phenotypes with those of AC5TG after chronic isoproterenol (ISO) infusion. Decreased cardiac function as well as increased susceptibility to pacing-induced atrial fibrillation (AF) in response to ISO were significantly attenuated in AC5TG-Epac1KO mice, compared to AC5TG mice. Increased cardiac apoptosis and cardiac fibrosis were also concomitantly attenuated in AC5TG-Epac1KO mice compared to AC5TG mice. These findings indicate that Epac1 plays an important role in AC5-mediated cardiac dysfunction and AF susceptibility.

BACKGROUND Extremely preterm infants frequently have patent ductus arteriosus (PDA). Recent recommendations include immediately beginning amino acid supplementation in extremely preterm infants. However, the effect of amino acids on closure of the ductus arteriosus (DA) remains unknown.Methods and Results:Aminogram results in human neonates at day 2 revealed that the plasma glutamate concentration was significantly lower in extremely preterm infants (

Abdominal aortic aneurysm (AAA) is a progressive disease that has an increasing prevalence with aging, but no effective pharmacological therapy to attenuate AAA progression is currently available. We reported that the prostaglandin E receptor EP4 plays roles in AAA progression. Here, we show the effect of CJ-42794, a selective EP4 antagonist, on AAA using two mouse models (angiotensin II- and CaCl2 -induced AAAs) and human aortic smooth muscle cells isolated from AAA tissue. Oral administration of CJ-42794 (0.2 mg/kg per day) for 4 weeks significantly decreased AAA formation in ApoE-/- mice infused with angiotensin II (1 μg/kg per min), in which elastic fiber degradation and activations of matrix metalloproteinase (MMP)-2 and MMP-9 were attenuated. Interleukin-6 (IL-6) proteins were highly expressed in the medial layer of angiotensin II-induced mouse AAA tissues, whereas this expression was significantly decreased in mice treated with CJ-42794. AAA formation induced by periaortic CaCl2 application in wild-type mice was also reduced by oral administration of CJ-42794 for 4 weeks. After oral administration of CJ-42794 beginning 2 weeks after periaortic CaCl2 application and continuing for an additional 4 weeks, the aortic diameter and elastic fiber degradation grade were significantly smaller in CJ-42794-treated mice than in untreated mice. Additionally, in smooth muscle cells isolated from human AAA tissues, stimulation of CJ-42794 inhibited PGE2 -induced IL-6 secretion in a dose-dependent manner and decreased PGE2 -induced MMP-2 activity. These data suggest that inhibition of EP4 has the potential to be a pharmacological strategy for attenuation of AAA progression.

Vascular smooth muscle cell (VSMC) migration and the subsequent intimal thickening play roles in vascular restenosis. We previously reported that an exchange protein activated by cAMP 1 (Epac1) promotes platelet-derived growth factor (PDGF)-induced VSMC migration and intimal thickening. Because basic fibroblast growth factor (bFGF) also plays a pivotal role in restenosis, we examined whether Epac1 was involved in bFGF-mediated VSMC migration. bFGF-induced lamellipodia formation and migration were significantly decreased in VSMCs obtained from Epac1−/− mice compared to those in Epac1+/+-VSMCs. The bFGF-induced phosphorylation of Akt and glycogen synthase kinase 3β (GSK3β), which play a role in bFGF-induced cell migration, was attenuated in Epac1−/−-VSMCs. Intimal thickening induced by the insertion of a large wire was attenuated in Epac1−/− mice, and was accompanied by the decreased phosphorylation of GSK3β. These data suggest that Epac1 deficiency attenuates bFGF-induced VSMC migration, possibly via Akt/GSK3β pathways.

Transforming growth factor-β1 (TGF-β1) induces phenotypic changes in fibroblasts to become myofibroblasts with increased production of extracellular matrix (ECM) components and cytokines. It is also known that excessive activation of myofibroblasts accelerates cardiac fibrosis, remodeling, and thus cardiac dysfunction. However, no effective therapy has been established to prevent this process although recent clinical studies have demonstrated the effectiveness of hyperthermia in cardiac dysfunction. The aim of this study was to examine the molecular mechanism of hyperthermia on TGF-β1-mediated phenotypic changes in cardiac fibroblasts. TGF-β1 increased the expression of IL-6, α-smooth muscle actin (α-SMA), and collagen in human cardiac fibroblasts (HCFs). Hyperthermia (42 °C) significantly prevented these changes, i.e., increases in IL-6, α-SMA, and collagen, as induced by TGF-β1 in a time-dependent manner. Immunoblotting showed that hyperthermia decreased Akt/S6K signaling, but did not affect Smad2 and Smad3 signaling. Pharmacological inhibition of Akt signaling mimicked these effects of hyperthermia. Furthermore, hyperthermia treatment prevented cardiac fibrosis in Ang II infusion mice model. Putting together, our findings suggest that hyperthermia directly inhibits TGF-β-mediated activation of HCFs via suppressing Akt/S6K signaling.

Aims The ductus arteriosus (DA) closes after birth to adapt to the robust changes in hemodynamics, which require intimal thickening (IT) to occur. The smooth muscle cells of the DA have been reported to play important roles in IT formation. However, the roles of the endothelial cells (ECs) have not been fully investigated. We herein focused on tissue-type plasminogen activator (t-PA), which is a DA EC dominant gene, and investigated its contribution to IT formation in the DA. Methods and results ECs from the DA and aorta were isolated from fetal rats using fluorescence-activated cell sorting. RT-PCR showed that the t-PA mRNA expression level was 2.7-fold higher in DA ECs than in aortic ECs from full-term rat fetuses (gestational day 21). A strong immunoreaction for t-PA was detected in pre-term and full-term rat DA ECs. t-PA-mediated plasminogen-plasmin conversion activates gelatinase matrix metalloproteinases (MMPs). Gelatin zymography revealed that plasminogen supplementation significantly promoted activation of the elastolytic enzyme MMP-2 in rat DA ECs. In situ zymography demonstrated that marked gelatinase activity was observed at the site of disruption in the internal elastic laminae (IEL) in full-term rat DA. In a three-dimensional vascular model, EC-mediated plasminogen-plasmin conversion augmented the IEL disruption. In vivo administration of plasminogen to pre-term rat fetuses (gestational day 19), in which IT is poorly formed, promoted IEL disruption accompanied by gelatinase activation and enhanced IT formation in the DA. Additionally, experiments using five human DA tissues demonstrated that the t-PA expression level was 3.7-fold higher in the IT area than in the tunica media. t-PA protein expression and gelatinase activity were also detected in the IT area of the human DAs. Conclusion t-PA expressed in ECs may help to form IT of the DA via activation of MMP-2 and disruption of IEL.

Myocardial β-adrenergic receptor (β-AR) β1- and β2-subtypes are highly homologous, but play opposite roles in cardiac apoptosis and heart failure, as do cardiac adenylyl cyclase (AC) subtypes 5 (AC5) and 6 (AC6): β1-AR and AC5 promote cardiac remodeling, while β2-AR and AC6 activate cell survival pathways. However, the mechanisms involved remain poorly understood. We hypothesized that AC5 is coupled preferentially to β1-AR rather than β2-AR, and we examined this idea by means of pharmacological and genetic approaches. We found that selective inhibition of AC5 with 2'5'-dideoxyadenosine significantly suppressed cAMP accumulation and cardiac apoptosis induced by selective β1-AR stimulation, but had no effect on cAMP accumulation and cardiac apoptosis in response to selective β2-AR stimulation. The results of selective stimulation of β1-AR and β2-AR in neonatal cardiac myocytes prepared from wild-type and AC5-knockout mice were also consistent with the idea that β1-AR selectively couples with AC5. We believe these results are helpful for understanding the mechanisms underlying the different roles of AR subtypes in healthy and diseased hearts.

Cyclic adenosine monophosphate (cAMP) and Ca(2+) levels may oscillate in harmony within excitable cells; a mathematical oscillation loop model, the Cooper model, of these oscillations was developed two decades ago. However, in that model all adenylyl cyclase (AC) isoforms were assumed to be inhibited by Ca(2+), and it is now known that the heart expresses multiple AC isoforms, among which the type 5/6 isoforms are Ca(2+)-inhibitable whereas the other five (AC2, 3, 4, 7, and 9) are not. We used a computational systems biology approach with CellDesigner simulation software to develop a comprehensive graphical map and oscillation loop model for cAMP and Ca(2+). This model indicated that Ca(2+)-mediated inhibition of AC is essential to create oscillations of Ca(2+) and cAMP, and the oscillations were not altered by incorporation of phosphodiesterase-mediated cAMP hydrolysis or PKA-mediated inhibition of AC into the model. More importantly, they were created but faded out immediately in the co-presence of Ca(2+)-noninhibitable AC isoforms. Because the subcellular locations of AC isoforms are different, spontaneous cAMP and Ca(2+) oscillations may occur within microdomains containing only Ca(2+)-inhibitable isoforms in cardiac myocytes, which might be necessary for fine tuning of excitation-contraction coupling.

Shortage of autologous blood vessel sources and disadvantages of synthetic grafts have increased interest in the development of tissue-engineered vascular grafts. However, tunica media, which comprises layered elastic laminae, largely determines arterial elasticity, and is difficult to synthesize. Here, we describe a method for fabrication of arterial grafts with elastic layer structure from cultured human vascular SMCs by periodic exposure to extremely high hydrostatic pressure (HP) during repeated cell seeding. Repeated slow cycles (0.002 Hz) between 110 and 180 kPa increased stress-fiber polymerization and fibronectin fibrillogenesis on SMCs, which is required for elastic fiber formation. To fabricate arterial grafts, seeding of rat vascular SMCs and exposure to the periodic HP were repeated alternatively ten times. The obtained medial grafts were highly elastic and tensile rupture strength was 1451 ± 159 mmHg, in which elastic fibers were abundantly formed. The patch medial grafts were sutured at the rat aorta and found to be completely patent and endothelialized after 2.5 months, although tubular medial constructs implanted in rats as interpositional aortic grafts withstood arterial blood pressure only in early acute phase. This novel organized self-assembly method would enable mass production of scaffold-free arterial grafts in vitro and have potential therapeutic applications for cardiovascular diseases.

Aims At birth, dynamic changes occur in serum components and haemodynamics, such as closure of the ductus arteriosus (DA). A previous study demonstrated that, in full-term human neonates, serum osmolality decreased transiently after birth, but recovered over the next few days. However, the significance of this transient decrease in osmolality has never been addressed. The objective of the present study was to examine the role of changes in serum osmolality after birth in DA closure. Methods and results We found that rats exhibited a similar transient hypoosmolality after birth. Hypotonic stimulation induced constriction of DA rings and increased Ca2+ transient in DA smooth muscle cells, but not in the aorta. The hypoosmotic sensor transient receptor potential melastatin 3 (TRPM3) was highly expressed in the rat DA, and TRPM3 silencing abolished the Ca2+ response to hypoosmolality. Pregnenolone sulfate stimulation of TRPM3 induced rat DA constriction ex vivo and in vivo. Furthermore, hyper tonic fluid injection impaired rat DA closure. In humans, neonatal serum hypoosmolality was observed in relatively mature preterm infants (≥28 weeks). In extremely preterm infants (

Fibroblast growth factor (FGF2) regulates endothelial and melanoma cell migration. The binding of FGF2 to its receptor requires N-sulfated heparan sulfate (HS) glycosamine. We have previously reported that Epac1, an exchange protein activated by cAMP, increases N-sulfation of HS in melanoma. Therefore, we examined whether Epac1 regulates FGF2-mediated cell-cell communication. Conditioned medium (CM) of melanoma cells with abundant expression of Epac1 increased migration of human umbilical endothelial cells (HUVEC) and melanoma cells with poor expression of Epac1. CM-induced increase in migration was inhibited by antagonizing FGF2, by the removal of HS and by the knockdown of Epac1. In addition, knockdown of Epac1 suppressed the binding of FGF2 to FGF receptor in HUVEC, and in vivo angiogenesis in melanoma. Furthermore, knockdown of Epac1 reduced N-sulfation of HS chains attached to perlecan, a major secreted type of HS proteoglycan that mediates the binding of FGF2 to FGF receptor. These data suggested that Epac1 in melanoma cells regulates melanoma progression via the HS-FGF2-mediated cell-cell communication.