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Abstract: Intestinal subepithelial myofibroblasts (ISMFs) are crucial for barrier formation against inflammatory stimuli. Physical injury induces cyclooxygenase-2 (COX-2) expression, which accelerates wound healing by ISMFs. However, the mechanism of COX-2 induction remains unclear. Physically damaged cells release ATP. Here, we investigate the role of ATP-purinergic signaling in wound-induced COX-2 induction in ISMFs. By 24h post-injury, bovine ISMFs had migrated to and closed the wounded area. A COX inhibitor, indomethacin or a purinergic P2 receptor antagonist, suramin, inhibited wound healing. However, additional treatment with indomethacin did not influence wound healing in suramin-treated ISMFs. RT-PCR showed an increase in COX-2 mRNA expression 2h post-injury, which was inhibited by suramin. These results suggest that ATP mediates wound-induced COX-2 elevation. We next assessed the contribution of various purinergic receptors in COX-2 induction. An ATP analog, ATPγS and a purinergic P2Y1, 11–13 receptors agonist, ADP, were among the agents tested which increased COX-2 expression. ATPγS-induced COX-2 mRNA expression was suppressed by suramin or a purinergic P2Xs, P2Y1, 4, 6, and 13 receptors antagonist, PPADS. These data suggest the involvement of Gq-coupled purinergic P2Y1 receptor or Gi-coupled purinergic P2Y13 receptor in COX-2 induction. U73122, an inhibitor of phospholipase C, which is a downstream signal of Gq protein, showed suppression of COX-2 mRNA expression. However, pertussis toxin, a Gi inhibitor, did not show suppression. We also revealed that inhibitors of p38 MAPK and PKC inhibited ATPγS-induced COX-2 mRNA expression. Collectively, purinergic P2Y1 receptor signaling mediates wound-induced COX-2 expression through p38 MAPK and PKC pathways in ISMFs. [Copyright &y& Elsevier]

Abstract: Prostaglandin E2 (PGE2) is one major prostanoid produced under inflammatory situation. Although PGE2 is known to induce vascular contraction, its detailed mechanism remains unknown. In the present study, we investigated the signaling pathway underlying PGE2-induced smooth muscle contraction in rat mesenteric artery. PGE2 (0.3–30μM) concentration-dependently caused contraction in endothelium-denuded artery. RT-PCR showed that this artery expresses mRNAs for all four prostanoid EP receptors (prostanoid EP1–4). Among selective agonists for PGE2 receptors, only a prostanoid EP3 receptor agonist, ONO-AE-248 (0.3–30μM) induced contraction. Consistently, pretreatment with a prostanoid EP3 antagonist (L-798106, 1μM) significantly but not completely inhibited the PGE2-induced contraction. Interestingly, pretreatment with a prostanoid FP antagonist (AL8810, 1μM) or a TP antagonist (SQ29548, 10nM) also partially inhibited the PGE2-induced contraction. Since ONO-AE-248 (10μM) did not influence intracellular Ca2+ concentration in mesenteric artery, we next examined the involvement of Ca2+-independent contractile pathway including PKCs and ROCK in prostanoid EP3-mediated contraction. Pretreatment with bisindolyl-maleimide I (a general PKC inhibitor, 1μM), Ro-31-8425 (a conventional PKC and PKCε inhibitor, 1μM), rottlerin (a selective PKCδ inhibitor, 1μM) and Y-27632 (a ROCK inhibitor, 1μM) but not Go 6976 (a conventional PKC inhibitor, 1μM) attenuated 10μM ONO-AE-248-induced vascular contraction. In western blot analysis, we confirmed that the treatment with ONO-AE-248 (10μM, 30min) phosphorylated PKCδ (Thr505) and PKCε (Ser729). These results suggest that PGE2 induces vascular smooth muscle contraction via prostanoid EP3, FP and TP receptors in rat mesenteric artery. Prostanoid EP3-mediated contraction is ascribed to Ca2+-independent contractile pathway including PKCδ, ε and ROCK. [Copyright &y& Elsevier]

Abstract: Halichlorine, isolated from a marine sponge Halichondria okadai Kadota, has a unique structure and its physiological activity is virtually unknown. In the present study, we investigated the direct effect of halichlorine on vascular contractility. In endothelium-denuded rat aorta, while the treatment of halichlorine (0.01–10μM) did not induce vascular contraction, halichlorine (0.01–10μM) dose-dependently inhibited both the steady-state precontractions induced by high K+ (65.4mM) and phenylephrine (1μM). The vasodilator effect of halichlorine (10μM) on high K+ (65.4mM)-induced contraction was more potent than that on phenylephrine (1μM)-induced contraction (65.4mM high K+: 72.7±3.4%; 1μM phenylephrine: 34.7±2.3%). To investigate the mechanism underlying the suppressive effect of halichlorine on vascular contractility, we examined the effect of halichlorine on intracellular Ca2+ concentration in vascular smooth muscle with a fluorescent Ca2+ indicator, fura-2. Treatment of halichlorine (10μM) significantly inhibited the sustained [Ca2+]i elevation induced by high K+ (65.4mM) (45.3±5.5%). Furthermore, current measurements by whole-cell mode patch-clamp recording in rat aortic smooth muscle cells (A7r5 cells) demonstrated that halichlorine (10μM) decreased the current density of the L-type Ca2+ channel (peak Ca2+-channel current densities: −2.09±0.27pA/pF for control; −0.58±0.07pA/pF for halichlorine). These results suggest that halichlorine inhibits L-type Ca2+ channels in vascular smooth muscle cells, which inhibit intracellular Ca2+ influx, and then reduce vascular contractions. [Copyright &y& Elsevier]

Abstract: Increased intrahepatic resistance is one of the major characteristics of cirrhotic liver, in which extravascular cells including liver myofibroblasts (MFs) abnormally contract. Although several studies provided evidence that various prostaglandins (PG) are involved in liver cirrhosis, the role of PGD2 remains unknown. In this study, we investigated the effect of PGD2 on the contractile properties of liver MFs. Cultured rat liver MFs were used at passages 4–7. A collagen gel contraction assay was used for the evaluation of the MFs contraction. mRNA expression was assessed by semi-quantitative RT-PCR. Intracellular Ca2+ concentrations ([Ca2+]i) were measured by monitoring the fluorescence intensity of fura-2. PGD2 (1–10 μM) induced liver MF contraction in a dose-dependent manner with [Ca2+]i elevation. Pretreatment with 300 nM LaCl3, a nonselective Ca2+ channel blocker abolished the 10 μM PGD2-induced MFs contraction. RT-PCR revealed that three distinct PGD2 responsive receptors, prostanoid DP receptor, chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) and thromboxane A2 receptor (prostanoid TP receptor), were expressed in liver MFs. While prostanoid DP receptor agonist and CRTH2 agonist didn''t induce contraction, 0.01–1 μM U46619 (11α, 9α-epoxymethano-PGH2, prostanoid TP receptor agonist) caused robust contraction with [Ca2+]i elevation. Furthermore, pretreatment with prostanoid TP receptor antagonists ramatroban (1 μM) or SQ29548 ([1S-[1α, 2α(Z), 3α, 4α]]-7-[3-[[2-[(phenyl amino)carbonyl]hydrazino]methyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid, 1 μM) completely suppressed PGD2-induced contraction and [Ca2+]i elevation. Additionally, we observed that BW245C (1–10 μM) decreased basal MF contraction. These results suggest that PGD2 induces rat liver MF contraction with an increase in [Ca2+]i through prostanoid TP receptor. [Copyright &y& Elsevier]

Summary: The Akt signaling pathway controls several cellular functions in the cardiovascular system; however, its role in atherogenesis is unknown. Here, we show that the genetic ablation of Akt1 on an apolipoprotein E knockout background (ApoE−/−Akt1−/− ) increases aortic lesion expansion and promotes coronary atherosclerosis. Mechanistically, lesion formation is due to the enhanced expression of proinflammatory genes and endothelial cell and macrophage apoptosis. Bone marrow transfer experiments showing that macrophages from ApoE−/−Akt1−/− donors were not sufficient to worsen atherogenesis when transferred to ApoE−/− recipients suggest that lesion expansion in the ApoE−/−Akt1−/− strain might be of vascular origin. In the vessel wall, the loss of Akt1 increases inflammatory mediators and reduces eNOS phosphorylation, suggesting that Akt1 exerts vascular protection against atherogenesis. The presence of coronary lesions in ApoE−/−Akt1−/− mice provides a new model for studying the mechanisms of acute coronary syndrome in humans. [Copyright &y& Elsevier]

Various cellular signals initiate calcium entry into cells, and there is evidence that lipid rafts and caveolae may concentrate proteins that regulate transmembrane calcium fluxes. Here, using mice deficient in caveolin-1 (Cav-1) and Cav-1 knock-out reconstituted with endothelium-specific Cav-1, we show that Cav-1 is essential for calcium entry in endothelial cells and governs the localization and protein-protein interactions between transient receptor channels C4 and C1. Thus, Cav-1 is required for calcium entry in vascular endothelial cells and perhaps other specialized cell types containing caveolae. [ABSTRACT FROM AUTHOR]

Abstract: In the present study, we assessed the effects of dexamethasone on fetal bovine serum-induced dysfunction of mesenteric endothelial cells using an organ culture procedure. In rabbit mesenteric arteries cultured in the presence of 10% fetal bovine serum for 7 days, the endothelium-dependent, nitric oxide (NO)-mediated relaxations caused by substance P and ionomycin were decreased as compared to those in non-treated arteries. Dexamethasone (3 μM) inhibited the proliferative stimuli-induced endothelial dysfunction without affecting the contractility or NO susceptibility of smooth muscle cells. Cross-sectioned hematoxylin–eosin staining and whole-mount CD31 staining indicated that chronic proliferative stimulation induced detachment of endothelial cells from the tunica intima in some regions, and also caused thickening of the arterial wall and shortening of the internal diameter. Endothelial NO synthesis (eNOS) mRNA expression was also decreased by the treatment with fetal bovine serum. The dexamethasone treatment did not inhibit the smooth muscle hypertrophy, but it inhibited the peeling of endothelial cells and recovered the eNOS mRNA expression. These results suggest that DEX ameliorate the impairments of arterial relaxation induced by proliferative stimuli and that these beneficial effects may be mediated by maintaining the adhesion of endothelial cells to the vascular wall and/or by recovering eNOS mRNA expression. [Copyright &y& Elsevier]

Abstract: Intestinal subepithelial myofibroblasts (IMFs) exist just under the epithelial membrane directly facing the mucosal microvascular capillary surface distributed in the lamina propria. In the gastrointestinal tract, ATP is released from epithelial and endothelial cells in response to mechanical stimuli. Although it has been reported that mechanical stimuli evoke synchronized Ca2+ waves in cultured IMFs, the contractile responses by ATP stimulation have not been examined. The aim of this study was to clarify the mechanism of the contraction of IMFs in response to ATP. ATP (1–30μM) induced contraction in a concentration-dependent manner. These contractions were inhibited by LaCl3 (100–300μM) and by Ca2+-free solution (0.5mM EGTA). Fura-2/Ca2+ signals indicated that ATP (1–10μM) elicited transient increases in intracellular Ca2+ concentration ([Ca2+]i). In addition, αβ-methylene-ATP (10, 30 and 300μM), a broad spectrum P2X agonist at a concentration higher than 100μM, induced neither contraction nor [Ca2+]i rise. UTP (1–30μM), a selective P2Y2 and P2Y4 agonist in rodent, induced concentration-dependent contractions and [Ca2+]i increases, whereas ADP and UDP (10μM) did not induce contractions. Pretreatment with suramin (30–100μM), a relatively selective P2Y2 antagonist, strongly inhibited ATP- and UTP-induced contractions and [Ca2+]i increases. However, pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonate (PPADS: 10–30μM), a receptor antagonist for several P2X and P2Y but less effective to P2Y2 receptor, failed to inhibit ATP- and UTP-induced contractions and [Ca2+]i increases. By RT-PCR, mRNA expressions of the P2Y1 and P2Y2 receptors, but not P2Y4 or P2Y6, were detected in IMFs. These results suggest that ATP induces [Ca2+]i-dependent contraction in IMFs, which is mediated through the P2Y2 receptor. [Copyright &y& Elsevier]

Abstract: Objective: In this study, we examined the synergistic effects of the two potent pathogenic factors, platelet-derived growth factor-BB (PDGF-BB) and endothelin-1 (ET-1) to induce vascular hyperplasia using ex vivo organ-culture system. Methods and results: In organ-cultured rat tail arteries, concomitant treatment with 100ng/ml PDGF-BB and 300nM ET-1 for 4 days induced medial hyperplasia with increased smooth muscle cell proliferation. Concomitant treatment with PDGF-BB (10–300nM) and ET-1 (30nM–1μM) dose-dependently suppressed contractile responses to high K+ and norepinephrine. This dyscontractility was accompanied by decreased α-actin protein expression. In all series of experiments, concomitant treatment with PDGF-BB and ET-1 exhibited stronger effects than sole treatment with PDGF-BB (100ng/ml) or ET-1 (300nM). Western blot analysis revealed that concomitant treatment with PDGF-BB and ET-1 synergistically phosphorylated extracellular signal-regulated kinase 1 and 2 (ERK1/2), Akt, and a downstream target of mammalian target of rapamycin (mTOR), p70 ribosomal S6 kinase in cultured artery. Consistently, a MAPK/ERK kinase (MEK) inhibitor, PD98059 (30μM), a phosphoinositide 3-kinase (PI3K) inhibitor, LY294002, and an mTOR inhibitor, rapamycin (30nM), partially restored PDGF-BB and ET-1-induced hyperplastic changes. Conclusions: We evidenced for the first time at tissue level that PDGF-BB and ET-1 synergistically accelerate vascular smooth muscle hyperplastic changes and lose its contractility, at least partially through ERK1/2, Akt, and mTOR activation. [Copyright &y& Elsevier]

Abstract: We evaluated the pharmacological effect of statins (3-hydroxy-3-methylglutaryl-CoA reductase inhibitors) on mast cell degranulation in RBL-2H3 cells. A hydrophilic statin (pravastatin) did not inhibit degranulation induced by dinitrophenol-human serum albumin (DNP-HSA); in contrast, lipophilic statins (simvastatin, fluvastatin and atorvastatin) inhibited DNP-HSA-induced degranulation in that order. The inhibitory effects were completely attenuated by simultaneous treatment with 100–1000 µM mevalonic acid for 4 h. We used fluvastatin to clarify the mechanism of the statin-mediated inhibitory action of mast cell degranulation. Fluvastatin (3 µM) had no effect on Ca2+ release from the endoplasmic reticulum or Ca2+ influx in the DNP-HSA- or thapsigargin-stimulated cells. Fluvastatin treatment also had no effect on the total granule content of the cell or sensitivity to DNP-HSA and IgE. Fluvastatin (3 µM, 24 h treatment) also failed to affect the morphology, proliferation, and viability of RBL-2H3 cells. Geranylgeranyl transferase inhibitor, GGTI-286 (20 µM), but not farnesyl transferase inhibitor, FPTIII (20 µM), inhibited the DNP-HSA-induced degranulation. The GGTI-286-induced inhibitory action was not associated with a decrease in the cytoplasmic Ca2+ level. In conclusion, fluvastatin at a lower concentration range inhibited DNP-HSA-induced degranulation without affecting the cytoplasmic Ca2+ response and also without changing the amount of granule content and proliferation of the mast cells. The statin-induced inhibitory action may be mediated by the suppression of geranylgeranyl transferase via the depletion of intracellular mevalonic acid. [Copyright &y& Elsevier]

Abstract: Because rapid and uniform laser heating can fix the neck-drawing point in continuous drawing of PTT fiber, we have successfully analyzed the fiber structure development in the continuous drawing process by in-situ measurement with a time resolution of less than 1ms. In this study, we investigated fiber structure development for PTT around the neck point controlled with a CO2 laser-heated apparatus during continuous drawing, through on-line measurements of WAXD, SAXS, and fiber temperature. Fiber temperature attained by laser radiation initiated a rise around −3mm in relation to the neck point at 0mm, and increased to about 90°C, which is past the 45°C T g for PTT. The instantaneous increase in fiber temperature continued with a vertical ascent, with plastic deformation around the neck point. The crystalline diffraction pattern was revealed initially at the elapsed time of 0.415ms immediately after necking, and remained fairly constant with elapsed time. The ultimate crystalline diffraction pattern for a completely drawn fiber showed little difference from that at the initial stage. In PET a two-dimensionally ordered structure in the form of a mesophase was detected immediately after the necking, whereas in PTT the phenomenon was not observed. With elapsed time, the d spacing of (002) plane decreased gradually due to transformation of the initial all-trans conformation into trans-gauche-gauche-trans conformation, and ultimately the PTT molecular chain could favorably adopt the trans-gauche-gauche-trans conformation. SAXS pattern immediately after the necking revealed an X-shape; the scattering intensity concentrated on meridian directions due to individual crystal development, and at 2ms two-pointed scattering started to appear. Past 8ms, the typical two-pointed scattering pattern was prominent and its intensity increased with elapsed time. Long period decreased with increasing elapsed time, but the crystallite size of meridian (002) plane hardly changed. The decrease in long period might be caused by chain relaxation in the amorphous region. [Copyright &y& Elsevier]

Myoferlin and dysferlin are members of the ferlin family of membrane proteins. Recent studies have shown that mutation or genetic disruption of myoferlin or dysferlin promotes muscular dystrophy-related phenotypes in mice, which are the result of impaired plasma membrane integrity. However, no biological functions have been ascribed to myoferlin in non-muscle tissues. Herein, using a proteomic analysis of endothelial cell (EC) caveolae/lipid raft microdomains we identified myoferlin in these domains and show that myoferlin is highly expressed in ECs and vascular tissues. The loss of myoferlin results in lack of proliferation, migration, and nitric oxide (NO) release in response to vascular endothelial growth factor (VEGF). Western blotting and surface biotinylation experiments show that loss of myoferlin reduces the expression level and autophosphorylation of VEGF receptor-2 (VEGFR-2) in native ECs. In a reconstituted cell system, transfection of myoferlin increases VEGFR-2 membrane expression and autophosphorylation in response to VEGF. In vivo, VEGFR-2 levels and VEGF-induced permeability are impaired in myoferlin-deficient mice. Mechanistically, myoferlin forms a complex with dynamin-2 and VEGFR-2, which prevents CBL-dependent VEGFR-2 polyubiquitination and proteasomal degradation. These data are the first to report novel biological activities for myoferlin and reveal the role of membrane integrity to VEGF signaling. [ABSTRACT FROM AUTHOR]

Zinc was discovered to be a novel second messenger in immunoreactive cells. We synthesized a novel free zinc chelator, IPZ-010. Here, we investigated the effects of IPZ-010 in a mouse postoperative ileus model and determined the effects of zinc signal inhibition as a new therapeutic strategy against postoperative ileus. Zinc waves were measured in bone marrow-derived mast cells (BMMCs) loaded with a zinc indicator, Newport green. Degranulation and cytokine expression were measured in BMMCs and bone marrow-derived macrophages (BMDMs). Postoperative ileus model mice were established with intestinal manipulation. Mice were treated with IPZ-010 (30 mg/kg, s.c. or p.o.) 1 h before and 2 h and 4 h after intestinal manipulation. Gastrointestinal transit, inflammatory cell infiltration, and expression of inflammatory mediators were measured. Free zinc waves occurred following antigen stimulation in BMMCs and were blocked by IPZ-010. IPZ-010 inhibited interleukin-6 secretion and degranulation in BMMCs. IPZ-010 inhibited tumor necrosis factor-α mRNA expression in BMMCs stimulated with lipopolysaccharide or adenosine triphosphate, whereas IPZ-010 had no effects on tumor necrosis factor-α mRNA expression in BMDMs stimulated with lipopolysaccharide or adenosine triphosphate. In postoperative ileus model mice, IPZ-010 inhibited leukocyte infiltration and cytokine expression, which ameliorated gastrointestinal transit. Furthermore, ketotifen (1 mg/kg) induced similar effects as IPZ-010. These effects were not amplified by co-administration of IPZ-010 and ketotifen. IPZ-010 inhibited zinc waves, resulting in inhibition of inflammatory responses in activated BMMCs in vitro. Targeting zinc waves in inflammatory cells may be a novel therapeutic strategy for treating postoperative ileus. [ABSTRACT FROM AUTHOR]