Search

TRPM7, a member of the melastatin subfamily of transient receptor potential channels, is suggested to be a potential candidate for a physiological Mg.sup.2+ channel. However, there is no direct evidence of Mg.sup.2+ permeation through endogenous TRPM7. To determine the physiological roles of TRPM7 in intracellular Mg.sup.2+ homeostasis, we measured the cytoplasmic free Mg.sup.2+ concentration ([Mg.sup.2+].sub.i) in TRPM7-silenced H9c2 cells. [Mg.sup.2+].sub.i was measured in a cluster of 8-10 cells using the fluorescent indicator, furaptra. TRPM7 silencing did not change [Mg.sup.2+].sub.i in Ca.sup.2+-free Tyrode's solution containing 1 mM Mg.sup.2+. Increasing the extracellular Mg.sup.2+ to 92.5 mM raised [Mg.sup.2+].sub.i in control cells (1.56 [+ or -] 0.19 mM) at 30 min, while this effect was significantly attenuated in TRPM7-silenced cells (1.12 [+ or -] 0.07 mM). The Mg.sup.2+ efflux driven by Na.sup.+ gradient was unaffected by TRPM7 silencing. These results suggest that TRPM7 regulates the rate of Mg.sup.2+ influx in H9c2 cells, although cytoplasmic Mg.sup.2+ homeostasis at basal conditions is unaffected by TRPM7 silencing. Keywords: Magnesium, TRPM7, Cardiac myoblast, H9c2, Mag-fura-2, Author(s): Michiko Tashiro[sup.1], Masato Konishi[sup.1], Ryo Kobayashi[sup.2], Hana Inoue[sup.1] and Utako Yokoyama[sup.1] Background The importance of intracellular Mg[sup.2+] has been widely recognized. Mg[sup.2+] is essential for protein synthesis, the regulation [...]

The article Epac activation inhibits IL-6-induced cardiac myocyte dysfunction., Author(s): Huiling Jin[sup.1], Takayuki Fujita[sup.1], Meihua Jin[sup.1,2], Reiko Kurotani[sup.1,3], Yuko Hidaka[sup.1], Wenqian Cai[sup.1], Kenji Suita[sup.1], Rajesh Prajapati[sup.1], Chen Liang[sup.1], Yoshiki Ohnuki[sup.4], Yasumasa Mototani[sup.4], Masanari Umemura[sup.1], Utako Yokoyama[sup.1], Motohiko Sato[sup.1,5], Satoshi [...]

PKA phosphorylates multiple molecules involved in calcium ([Ca.sup.2+]) handling in cardiac myocytes and is considered to be the predominant regulator of β-adrenergic receptor-mediated enhancement of cardiac contractility; however, recent identification of exchange protein activated by cAMP (EPAC), which is independently activated by cAMP, has challenged this paradigm. Mice lacking Epac1 (Epac1 KO) exhibited decreased cardiac contractility with reduced phospholamban (PLN) phosphorylation at serine-16, the major PKA-mediated phosphorylation site. In Epac1 KO mice, intracellular [Ca.sup.2+] storage and the magnitude of [Ca.sup.2+] movement were decreased; however, PKA expression remained unchanged, and activation of PKA with isoproterenol improved cardiac contractility. In contrast, direct activation of EPAC in cardiomyocytes led to increased PLN phosphorylation at serine-16, which was dependent on PLC and PKCe. Importantly, Epac1 deletion protected the heart from various stresses, while Epac2 deletion was not protective. Compared with WT mice, aortic banding induced a similar degree of cardiac hypertrophy in Epac1 KO; however, lack of Epac1 prevented subsequent cardiac dysfunction as a result of decreased cardiac myocyte apoptosis and fibrosis. Similarly, Epac1 KO animals showed resistance to isoproterenol- and aging-induced cardiomyopathy and attenuation of arrhythmogenic activity. These data support Epac1 as an important regulator of PKA-independent PLN phosphorylation and indicate that Epac1 regulates cardiac responsiveness to various stresses., Introduction β-Adrenergic receptor (β-AR) signaling is well established as a primary defense mechanism against acute stress or changes in hemodynamic load; however, its role in cardiac pathogenesis, although studied extensively, [...]

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.sup.-/- mice compared to those in Epac1.sup.+/+-VSMCs. The bFGF-induced phosphorylation of Akt and glycogen synthase kinase 3[beta] (GSK3[beta]), which play a role in bFGF-induced cell migration, was attenuated in Epac1.sup.-/--VSMCs. Intimal thickening induced by the insertion of a large wire was attenuated in Epac1.sup.-/- mice, and was accompanied by the decreased phosphorylation of GSK3[beta]. These data suggest that Epac1 deficiency attenuates bFGF-induced VSMC migration, possibly via Akt/GSK3[beta] pathways. Keywords: Exchange protein activated by cAMP 1, Intimal thickening, Basic fibroblast growth factor, Vascular smooth muscle cells, Migration, Author(s): Yuko Kato[sup.1,2], Utako Yokoyama[sup.1], Takayuki Fujita[sup.1], Masanari Umemura[sup.1], Tetsuo Kubota[sup.2] and Yoshihiro Ishikawa[sup.1] Introduction Intimal thickening that occurs after vascular injury resulting from percutaneous coronary intervention is a major [...]

Caveolin, a member of the membrane-anchoring protein family, accumulates various growth receptors in caveolae and inhibits their function. Upregulation of caveolin attenuates cellular proliferation and growth. However, the role of caveolin in regulating insulin signals remains controversial. Here, we demonstrate that caveolin potently enhances insulin receptor (IR) signaling when overexpressed in the liver in vivo. Adenovirus-mediated gene transfer was used to overexpress caveolin specifically in the liver of diabetic obese mice, which were generated with a high-fat diet. Expression of molecules involved in IR signaling, such as IR or Akt, remained unchanged after gene transfer. However, hepatic glycogen synthesis was markedly increased with a decrease in phosphoenolpyruvate carboxykinase protein expression. Insulin sensitivity was increased after caveolin gene transfer as determined by decreased blood glucose levels in response to insulin injection and fasting blood glucose levels. Glucose tolerant test performance was also improved. Similar improvements were obtained in [KKA.sup.y] genetically diabetic mice. Adenovirus-mediated over-expression of caveolin-3 in hepatic cells also enhanced IR signaling, as shown by increased phosphorylation of IR in response to insulin stimulation and higher glycogen synthesis at baseline. These effects were attributed mostly to increased insulin receptor activity and caveolin-mediated, direct inhibition of protein tyrosine phosphatase 1B, which was increased in obese mouse livers. In conclusion, our results suggest that caveolin is an important regulator of glucose metabolism that can enhance insulin signals. insulin receptor; diabetes mellitus doi:10.1152/ajpcell.00077.2009

Vascular remodeling after mechanoinjury largely depends on the migration of smooth muscle cells, an initial key step to wound healing. However, the role of the second messenger system, in particular, the cAMP signal, in regulating such remodeling remains controversial. Exchange protein activated by cAMP (Epac) has been identified as a new target molecule of the cAMP signal, which is independent from PKA. We thus examined whether Epac plays a distinct role from PKA in vascular remodeling. To examine the role of Epac and PKA in migration, we used primary culture smooth muscle cells from both the fetal and adult rat aorta. A cAMP analog selective to PKA, 8-(4-parachlorophenylthio)-cAMP (pCPT-cAMP), decreased cell migration, whereas an Epac-selective analog, 8-pCPT-2'-O-Me-cAMP, enhanced migration. Adenovirus-mediated gene transfer of PKA decreased cell migration, whereas that of Epac1 significantly enhanced cell migration. Striking morphological differences were observed between pCPT-cAMP-and 8-pCPT-2'-O-Me-cAMP-treated aortic smooth muscle cells. Furthermore, overexpression of Epac1 enhanced the development of neointimal formation in fetal rat aortic tissues in organ culture. When the mouse femoral artery was injured mechanically in vivo, we found that the expression of Epac1 was upregulated in vascular smooth muscle cells, whereas that of PKA was downregulated with the progress of neointimal thickening. Our findings suggest that Epac1, in opposition to PKA, increases vascular smooth muscle cell migration. Epac may thus play an important role in advancing vascular remodeling and restenosis upon vascular injury. cAMP; protein kinase A; vascular remodeling; exchange protein activated by cAMP

Preterm twins have a higher morbidity rate of patent ductus arteriosus (PDA) than do singletons. However, the effect of multiple births on maturation of the ductus arteriosus (DA) has not been reported. Because intimal thickening (IT) is required for DA anatomical closure, we examined IT development in the DA of preterm twins and singletons. Sheep DA tissues obtained from preterm fetuses were subjected to elastica van Gieson staining to evaluate IT. The total IT score in each DA was the sum of the IT scores obtained from six evenly divided parts of the DA, which was positively correlated with gestational ages in singletons. Total IT scores were smaller in preterm twins than in singletons, although no difference in gestational age, birth weight, or gender ratio was observed. These data suggest that IT development of the DA is attenuated in sheep preterm twins, which may affect the higher morbidity of PDA. Keywords: Ductus arteriosus, Intimal thickening, Preterm infant, Multiple births, Author(s): Satoko Ito[sup.1,2], Utako Yokoyama[sup.1], Junichi Saito[sup.1], Shinichi Sato[sup.2], Haruo Usuda[sup.2], Shimpei Watanabe[sup.2], Ryuta Kitanishi[sup.2], Yuichiro Miura[sup.2], Masatoshi Saito[sup.2], Takushi Hanita[sup.2], Tadashi Matsuda[sup.2] and Yoshihiro Ishikawa[sup.1] Introduction The ductus arteriosus [...]

Myocardial infarction (MI) results in left ventricular remodeling (e.g., ventricular hypertrophy, dilatation, and fibrosis). Fibrosis contributes to increased myocardial stiffening, impaired ventricular filling and function, and reduced cardiac output. Adenylyl cyclase (AC) expression and activity are reduced in animal models of heart failure. Stimulation of AC can inhibit extracellular matrix production in isolated cardiac fibroblasts; however, a role for reduced AC expression and activity in fibrosis associated with cardiac remodeling after chronic MI has never been determined. We tested the hypothesis that AC expression and activity are reduced in cardiac fihroblasts after chronic (18 wk) MI. Rats underwent coronary artery ligation or sham surgery (control), and echocardiography was used to assess left ventricular remodeling 1, 3, 5, 7, 10, 12, and 18 wk after surgery. Cardiac fibroblasts were isolated from the noninfarcted myocardium and compared for differences in AC activity and collagen synthesis. End-diastolic dimension was increased [control: 0.76 [+ or -] 0.02 cm and MI: 1.0 [+ or -] 0.02 cm (means [+ or -] SE), P < 0.001] and fractional shortening was decreased (control: 44 [+ or -] 2% and MI: 17 [+ or -] 2%, P < 0.001) in MI compared with control rats. Basal and forskolin-stimulated cAMP production were decreased by 90% and 93%, respectively, and AC5/6 expression was decreased 39% in fibroblasts isolated from MI rats compared with sham controls. Serum-stimulated collagen production was increased twofold and forskolin-mediated inhibition of collagen synthesis was reduced in fibroblasts from MI rats compared with controls. Our data demonstrate that AC expression and activity are reduced and collagen production is increased in cardiac fibroblasts of rats after MI.

Coarctation of the aorta (CoA) is defined as a congenital stenosis of the thoracic aorta and is one of the most common congenital cardiovascular diseases. Despite successful surgical treatment for CoA, arterial abnormalities, including refractory hypertension, aortic aneurysm, and proatherogenic phenotypic changes, frequently affect patients' quality of life. Emerging evidence from morphological and molecular biological investigations suggest that the area of CoA is characterized by phenotypic modulation of smooth muscle cells, intimal thickening, and impaired elastic fiber formation. These changes extend to the pre-and post-stenotic aorta and impair arterial elasticity. The aim of this review is to present current findings on the pathology and molecular mechanisms of vascular remodeling due to CoA. In particular, we will discuss the association between CoA and the ductus arteriosus since the most common site for the stenosis is in the proximity of the ductus arteriosus. Keywords: Congenital heart disease, Coarctation of the aorta, Remodeling, Ductus arteriosus, Author(s): Utako Yokoyama[sup.1], Yasuhiro Ichikawa[sup.1], Susumu Minamisawa[sup.2] and Yoshihiro Ishikawa[sup.1] Introduction Early diagnosis and appropriate management of coarctation of the aorta (CoA) have resulted in a low mortality rate associated [...]

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 Ca.sup.2+ 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. Keywords: Epac, cAMP, Catecholamine, Contractility, Cytokine, Jak-STAT, Author(s): Huiling Jin[sup.1], Takayuki Fujita[sup.1], Meihua Jin[sup.1,2], Reiko Kurotani[sup.1,3], Yuko Hidaka[sup.1], Wenqian Cai[sup.1], Kenji Suita[sup.1], Rajesh Prajapati[sup.1], Chen Liang[sup.1], Yoshiki Ohnuki[sup.4], Yasumasa Mototani[sup.4], Masanari Umemura[sup.1], Utako Yokoyama[sup.1], Motohiko Sato[sup.1,5], Satoshi [...]

Voltage-dependent [Ca.sup.2+] channels (VDCCs), which consist of multiple subtypes, regulate vascular tone in developing arterial smooth muscle, including the ductus arteriosus (DA). First, we examined the expression of VDCC subunits in the Wistar rat DA during development. Among [[alpha].sub.1]-subunits, [[alpha].sub.1C] and [[alpha].sub.1G] were the most predominant isoforms. Maternal administration of vitamin A significantly increased [[alpha].sub.1C] and [[alpha].sub.1G]-transcripts. Second, we examined the effect of VDCC subunits on proliferation of DA smooth muscle cells. We found that 1 [micro]M nitrendipine (an L-type [Ca.sup.2+] channel blocker) and kurtoxin (a T-type [Ca.sup.2+] channel blocker) significantly decreased [[sup.3]H]thymidine incorporation and that 3 [micro]M efonidipine (an L- and T-type [Ca.sup.2+] channel blocker) further decreased [[sup.3]H]thymidine incorporation, suggesting that L- and T-type [Ca.sup.2+] channels are involved in smooth muscle cell proliferation in the DA. Third, we found that a novel alternatively spliced variant of the [[alpha].sub.1C]-isoform was highly expressed in the neointimal cushion of the DA, where proliferating and migrating smooth muscle cells are abundant. The basic channel properties of the spliced variant did not differ from those of the conventional [[alpha].sub.1C]-subunit. We conclude that multiple VDCC subunits were identified in the DA, and, in particular, [[alpha].sub.1C] and [[alpha].sub.1G],-subunits were predominant in the DA. A novel spliced variant of the [[alpha].sub.1C]-subunit gene may play a distinct role in neointimal cushion formation in the DA. alternative spliced: development: gene expression: fetal circulation

Melanoma has an extremely poor prognosis due to its rapidly progressive and highly metastatic nature. Several therapeutic drugs have recently become available, but are effective only against melanoma with specific BRAF gene mutation. Thus, there is a need to identify other target molecules. We show here that Transient receptor potential, canonical 3 (TRPC3) is widely expressed in human melanoma. We found that pharmacological inhibition of TRPC3 with a pyrazole compound, Pyr3, decreased melanoma cell proliferation and migration. Similar inhibition was observed when the TRPC3 gene was silenced with short-hairpin RNA (shRNA). Pyr3 induced dephosphorylation of signal transducer and activator of transcription (STAT) 5 and Akt. Administration of Pyr3 (0.05 mg/kg) to mice implanted with human melanoma cells (C8161) significantly inhibited tumor growth. Our findings indicate that TRPC3 plays an important role in melanoma growth, and may be a novel target for treating melanoma in patients. Keywords: TRPC3, Proliferation, Migration, Melanoma, Metastasis, Pyr3, Author(s): Kayoko Oda[sup.1,2], Masanari Umemura[sup.1], Rina Nakakaji[sup.1], Ryo Tanaka[sup.1], Itaru Sato[sup.1], Akane Nagasako[sup.1], Chiaki Oyamada[sup.1], Erdene Baljinnyam[sup.3], Mayumi Katsumata[sup.1], Lai-Hua Xie[sup.3], Masatoshi Narikawa[sup.1], Yukie Yamaguchi[sup.2], Taisuke Akimoto[sup.1], Makoto Ohtake[sup.1], Takayuki [...]

Tissue‐engineered vascular grafts (TEVGs) are in urgent demand for both adult and pediatric patients. Although several approaches have utilized vascular smooth muscle cells (SMCs) and endothelial cells as cell sources for TEVGs, these cell sources have a limited proliferative capacity that results in an inability to reconstitute neotissues. Skeletal myoblasts are attractive cell sources as they possess high proliferative capacity, and they are already being tested in clinical trials for patients with ischemic cardiomyopathy. Our previous study demonstrated that periodic hydrostatic pressurization (PHP) promoted fibronectin fibrillogenesis in vascular SMCs, and that PHP‐induced extracellular matrix (ECM) arrangements enabled the fabrication of implantable arterial grafts derived from SMCs without using a scaffold material. We assessed the molecular response of human skeletal myoblasts to PHP exposure, and aimed to fabricate arterial grafts from the myoblasts by exposure to PHP. To examine the PHP‐response genes, human skeletal myoblasts were subjected to bulk RNA‐sequencing after PHP exposure. Gene‐set enrichment analysis revealed significant positive correlations between PHP exposure and vascular development‐related genes. Real‐time polymerase chain reaction (RT‐PCR) demonstrated that PHP significantly upregulated collagen and elastic fiber formation‐related gene expression, such as fibronectin, lysyl oxidase, collagen type I α1, collagen type IV α1, and tropoelastin. Based on these findings showing the potential role of PHP in vessel formation, we fabricated arterial grafts by repeated cell seeding and exposure to PHP every 24 hours. The resultant 15‐layered myoblast grafts had high collagen content, which provided a tensile rupture strength of 899 ± 104 mm Hg. Human skeletal myoblast grafts were implanted as patch grafts in the aorta of immunosuppressed rats and found to be endothelialized and completely patent until the endpoint of 60 postoperative days. Implanted human myoblasts were gradually replaced by host‐derived cells, which successfully formed vascular neotissues with layered elastic fibers. These findings suggest that human skeletal myoblasts have the potential to be a feasible cell source for scaffold‐free implantable arterial grafts under PHP culture conditions. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The article presents guidelines on working group for revision of guidelines for the use of Palivizumab in Japan. Topics include the precautions and indications on the drug's information leaflet, the appropriate use of this agent should be used on a patient by patient basis; and referring to guidelines issued by healthcare societies and other professional academic organizations.

Aims: Doxorubicin (DOX)‐induced heart failure has a poor prognosis, and effective treatments have not been established. Because DOX shows cumulative cardiotoxicity, we hypothesized that minimal cardiac remodelling occurred at the initial stage in activating cardiac fibroblasts. Our aim was to investigate the initial pathophysiology of DOX‐exposed cardiac fibroblasts and propose prophylaxis. Methods and results: An animal study was performed using a lower dose of DOX (4 mg/kg/week for 3 weeks, i.p.) than a toxic cumulative dose. Histological analysis was performed with terminal deoxynucleotidyl transferase‐mediated dUTP nick‐end labelling assay, picrosirius red staining, and immunohistochemical staining. The mechanism was analysed in vitro with a low dose of DOX, which did not induce cell apoptosis. Microarray analysis was performed. Differentially expressed genes were confirmed by enrichment analysis. Mitochondrial damage was assessed by mitochondrial membrane potential. The production of inflammatory cytokines and fibrosis markers was assessed by western blot, quantitative polymerase chain reaction, and ELISA. A phosphokinase antibody array was performed to detect related signalling pathways. Low‐dose DOX did not induced cell death, and fibrosis was localized to the perivascular area in mice. Microarray analysis suggested that DOX induced genes associated with the innate immune system and inflammatory reactions, resulting in cardiac remodelling. DOX induced mitochondrial damage and increased the expression of interleukin‐1. DOX also promoted the expression of fibrotic markers, such as alpha smooth muscle actin and galectin‐3. These responses were induced through stress‐activated protein kinase/c‐Jun NH2‐terminal kinase signalling. A peroxisome proliferator‐activated receptor (PPARγ) agonist attenuated the expression of fibrotic markers through suppressing stress‐activated protein kinase/c‐Jun NH2‐terminal kinase. Furthermore, this molecule also suppressed DOX‐induced early fibrotic responses in vivo. Conclusions: Low‐dose DOX provoked reactive fibrosis through sterile inflammation evoked by the damaged mitochondria. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

TRPM7, a member of the melastatin subfamily of transient receptor potential channels, is suggested to be a potential candidate for a physiological Mg2+ channel. However, there is no direct evidence of Mg2+ permeation through endogenous TRPM7. To determine the physiological roles of TRPM7 in intracellular Mg2+ homeostasis, we measured the cytoplasmic free Mg2+ concentration ([Mg2+]i) in TRPM7-silenced H9c2 cells. [Mg2+]i was measured in a cluster of 8–10 cells using the fluorescent indicator, furaptra. TRPM7 silencing did not change [Mg2+]i in Ca2+-free Tyrode’s solution containing 1 mM Mg2+. Increasing the extracellular Mg2+ to 92.5 mM raised [Mg2+]i in control cells (1.56 ± 0.19 mM) at 30 min, while this effect was significantly attenuated in TRPM7-silenced cells (1.12 ± 0.07 mM). The Mg2+ efflux driven by Na+ gradient was unaffected by TRPM7 silencing. These results suggest that TRPM7 regulates the rate of Mg2+ influx in H9c2 cells, although cytoplasmic Mg2+ homeostasis at basal conditions is unaffected by TRPM7 silencing. [ABSTRACT FROM AUTHOR]

Although doxorubicin (DOX)-induced cardiomyopathy causes lethal heart failure (HF), no early detection or effective treatment methods are available. The principal mechanisms of cardiotoxicity are considered to involve oxidative stress and apoptosis of cardiomyocytes. However, the effect of DOX on cardiac fibroblasts at non-lethal concentrations remains unknown. The aim of this study was to investigate the direct effect of doxorubicin on the activation of cardiac fibroblasts independent of cell death pathways. We first found that DOX induced α-SMA expression (marker of trans-differentiation) at a low concentration range, which did not inhibit cell viability. DOX also increased MMP1, IL-6, TGF-β and collagen expression in human cardiac fibroblasts (HCFs). In addition, DOX promoted Akt and Smad phosphorylation. A Smad inhibitor prevented DOX-induced α-SMA and IL-6 protein expression. An PI3K inhibitor also prevented MMP1 mRNA expression in HCFs. These findings suggest that DOX directly induces fibrotic changes in HCFs via cell death-independent pathways. Furthermore, we confirmed that these responses are organ- and species-specific for HCFs based on experiments using different types of human and murine fibroblast cell lines. These results suggest potentially new mechanisms of DOX-induced cardiotoxicity from the viewpoint of fibrotic changes in cardiac fibroblasts. [ABSTRACT FROM AUTHOR]

Although the efficacy of external stents for vein grafts in coronary artery bypass grafting has been recognized, the ideal diameter and material of the stent remain controversial. We created a new external stent made of soft polyester mesh and performed an animal experiment using canines. Bilateral saphenous vein grafts were interposed in the bilateral common carotid artery of 10 beagles. The grafts in the left carotid artery were designated as the control group, and those in the right rolled by a soft polyester mesh external stent were designated as mesh group. Two of the 10 animals were sacrificed due to severe wound infection. The other eight were observed by echography for 6 months, and then grafts were extracted and thickness of the neointima of the grafts was measured. The control group showed 146% ± 26% postoperative enlargement of the internal diameter of the vein grafts after 6 months, whereas the mesh group showed only 115% ± 15% after the same duration (P = 0.0003). The median thickness of the neointima in the mesh group (170 µm [range: 150–190]) was significantly thinner than that in the control group (260 µm [range: 220–310], P < 0.0001). Some degree of correlation between the thickness of neointima and proportion of enlargement was noted (r = 0.518, P = 0.0024). A soft polyester mesh external stent for vein grafts successfully suppressed the enlargement of the vein grafts and thickness of the neointima after 6 months. [ABSTRACT FROM AUTHOR]

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. 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 infusion of Fe(Salen) showed a greater antitumor effect than did i.v. infusion. The combination of Fe(Salen) intra‐arterial injection and AMF exposure showed a greater antitumor effect than did either Fe(Salen) or methotrexate (MTX) alone, suggesting that AMF exposure greatly enhanced the anticancer effect of Fe(Salen) by arterial injection by catheter. [ABSTRACT FROM AUTHOR]

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. The present paper aims to apply an alternating magnetic field (AMF) combined with Compound C, a cell permeable AMPK inhibitor, to a novel glioblastoma (GB) treatment. We believe that this is the first report in which an AMF is applied to treatment of GB in combination with an anti‐tumor agent, Compound C. Our results revealed that AMF further enhanced Compound C‐induced cytotoxicity through de‐phosphorylation of ERK and suppression of autophagy in GB cells. [ABSTRACT FROM AUTHOR]

Abstract: 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]