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ABSTRACT: Phosphatidylinositol (PtdIns) 3-kinase (PI3K) family regulates diverse cellular processes including cell proliferation, migration and vesicular trafficking through catalyzing 3-phosphorylation of phosphoinositides. In contrast to class I PI3Ks including p110α and p110β, functional roles of class II PI3Ks comprising PI3K-C2α, PI3K-C2β and PI3K-C2γ are little understood. The lysophospholipid mediator sphingosine-1-phosphate (S1P) plays the important roles in regulating vascular functions including vascular formation and barrier integrity via the G-protein coupled receptors S1P1-3. We studied roles of PI3K-C2α in S1P-induced endothelial cell (EC) migration and tube formation. S1P stimulated cell migration and activation of Akt, ERK and Rac1, the latter of which acts as a signaling molecule essential for cell migration and tube formation, via S1P1 in ECs. Knockdown of either PI3K-C2α or class I p110β markedly inhibited S1P-induced migration, lamellipodium formation and tube formation whereas that of p110α or Vps34 did not. Only p110β was necessary for S1P-iduced Akt activation, but both PI3K-C2α and p110β were required for Rac1 activation. Fluorescent resonance energy transfer (FRET) imaging showed that S1P induced Rac1 activation in both the plasma membrane and PtdIns-3-phosphate (PtdIns(3)P)-enriched endosomes. Knockdown of PI3K-C2α but not p110β markedly reduced PtdIns(3)P-enriched endosomes and suppressed endosomal Rac1 activation. Also, knockdown of PI3K-C2α but not p110β suppressed S1P-induced S1P1 internalization into PtdIns(3)P-enriched endosomes. Finally, pharmacological inhibition of endocytosis suppressed S1P-induced S1P1 internalization, Rac1 activation, migration and tube formation. These observations indicate that PI3K-C2α plays the crucial role in S1P1 internalization into intracellular vesicular compartment, Rac1 activation on endosomes, and thereby migration through regulating vesicular trafficking in EC.
Journal of Biological Chemistry 11/2012; · 4.77 Impact Factor
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Kazuaki Yoshioka,
Kotaro Yoshida,
Hong Cui,
Tomohiko Wakayama,
Noriko Takuwa,
Yasuo Okamoto,
Wa Du,
Xun Qi,
Ken Asanuma,
Kazushi Sugihara, [......],
Masaya Ueno,
Shoichi Iseki,
Robert J Schwartz,
Hiroshi Okamoto,
Takehiko Sasaki,
Osamu Matsui,
Masahide Asano,
Ralf H Adams,
Nobuyuki Takakura, Yoh Takuwa
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ABSTRACT: The class II α-isoform of phosphatidylinositol 3-kinase (PI3K-C2α) is localized in endosomes, the trans-Golgi network and clathrin-coated vesicles; however, its functional role is not well understood. Global or endothelial-cell-specific deficiency of PI3K-C2α resulted in embryonic lethality caused by defects in sprouting angiogenesis and vascular maturation. PI3K-C2α knockdown in endothelial cells resulted in a decrease in the number of PI3-phosphate-enriched endosomes, impaired endosomal trafficking, defective delivery of VE-cadherin to endothelial cell junctions and defective junction assembly. PI3K-C2α knockdown also impaired endothelial cell signaling, including vascular endothelial growth factor receptor internalization and endosomal RhoA activation. Together, the effects of PI3K-C2α knockdown led to defective endothelial cell migration, proliferation, tube formation and barrier integrity. Endothelial PI3K-C2α deficiency in vivo suppressed postischemic and tumor angiogenesis and diminished vascular barrier function with a greatly augmented susceptibility to anaphylaxis and a higher incidence of dissecting aortic aneurysm formation in response to angiotensin II infusion. Thus, PI3K-C2α has a crucial role in vascular formation and barrier integrity and represents a new therapeutic target for vascular disease.
Nature medicine 09/2012; 18(10):1560-9. · 27.14 Impact Factor
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ABSTRACT: Fibrosis is a pathological process characterized by massive deposition of extracellular matrix (ECM) such as type I/III collagens and fibronectin that are secreted by an expanded pool of myofibroblasts, which are phenotypically altered fibroblasts with more contractile, proliferative, migratory and secretory activities. Fibrosis occurs in various organs including the lung, heart, liver and kidney, resulting in loss of normal tissue architecture and functions. Myofibroblasts could originate from multiple sources including tissue-resident fibroblasts, epithelial and endothelial cells through mechanisms of epithelial/endothelial-mesenchymal transition (EMT/EndMT), and bone marrow-derived circulating progenitors called fibrocytes. Emerging evidence in recent years shows that sphingosine-1-phosphate (S1P) acts on several types of target cells and is engaged in pro-fibrotic inflammatory process and fibrogenic process through multiple mechanisms, which include vascular permeability change, leukocyte infiltration, and migration, proliferation and myofibroblast differentiation of fibroblasts. Many of these S1P actions are receptor subtype-specific. In these actions, S1P has multiple cross-talks with other cytokines, particularly transforming growth factor-β (TGFβ), which plays a major role in fibrosis. The cross-talks include the regulation of S1P production through altered expression and activity of sphingosine kinases in fibrotic lesions, altered expression of S1P receptors, and S1P receptor-mediated transactivation of TGFβ signaling pathway. These cross-talks may give rise to a feed-forward, amplifying loop between S1P and TGFβ, and possibly with other cytokines in stimulating fibrogenesis. Another lysophospholipid mediator lysophosphatidic acid has also been recently implicated in fibrosis. The lysophospholipid signaling pathways represent novel, promising therapeutic targets for treating refractory fibrotic diseases. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.
Biochimica et Biophysica Acta 06/2012; · 4.66 Impact Factor
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ABSTRACT: Sphingosine-1-phosphate (S1P), which acts as both the extracellular and intracellular messenger, exerts pleiotropic biological activities including regulation of formation of the vasculature, vascular barrier integrity, and lymphocyte trafficking. Many of these S1P actions are mediated by five members of the G protein-coupled S1P receptors (S1P(1) -S1P(5) ) with overlapping but distinct coupling to heterotrimeric G proteins. The biological activities of S1P are based largely on the cellular actions of S1P on migration, adhesion, and proliferation. Notably, S1P often exhibits receptor subtype-specific, bimodal effects in these cellular actions. For example, S1P(1) mediates cell migration toward S1P, that is, chemotaxis, via G(i) /Rac pathway whereas S1P(2) mediates inhibition of migration toward a chemoattractant, that is, chemorepulsion, via G(12/13) /Rho pathway, which induces Rac inhibition. In addition, S1P(1) mediates stimulation of cell proliferation through the G(i) -mediated signaling pathways including phosphatidylinositol 3-kinase (PI3K)/Akt and ERK whereas S1P(2) mediates inhibition of cell proliferation through mechanisms involving G(12/13) /Rho/Rho kinase/PTEN-dependent Akt inhibition. These differential effects of S1P receptor subtypes on migration and proliferation lead to bimodal regulation of various biological responses. An observed biological response is likely determined by an integrated outcome of the counteracting signals input by S1P receptor subtypes. More recent studies identified the new intracellular targets of S1P including the inflammatory signaling molecule TRAF2 and histone deacetylases HDAC1 and HDAC2. These interactions of S1P regulate NF-κB activity and gene expression, respectively. Development of S1P receptor agonists and antagonists with improved receptor subtype-selectivity, inhibitors, or modulators of sphingolipid-metabolizing enzymes, and their optimal drug delivery system provide novel therapeutic tactics. © 2012 International Union of Biochemistry and Molecular Biology, Inc.
BioFactors 06/2012; 38(5):329-37. · 4.93 Impact Factor
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Yuko Kageyama,
Hitoshi Ikeda,
Naoko Watanabe,
Masakazu Nagamine,
Yoshika Kusumoto,
Mitsuru Yashiro,
Yumiko Satoh,
Tatsuo Shimosawa,
Koji Shinozaki,
Tomoaki Tomiya, [......],
Takako Nishikawa,
Natsuko Ohtomo,
Yasushi Tanoue,
Hiromitsu Yokota,
Takatoshi Koyama,
Kazuhiro Ishimaru,
Yasuo Okamoto, Yoh Takuwa,
Kazuhiko Koike,
Yutaka Yatomi
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ABSTRACT: Sinusoidal vasoconstriction, in which hepatic stellate cells operate as contractile machinery, has been suggested to play a pivotal role in the pathophysiology of portal hypertension. We investigated whether sphingosine 1-phosphate (S1P) stimulates contractility of those cells and enhances portal vein pressure in isolated perfused rat livers with Rho activation by way of S1P receptor 2 (S1P(2) ). Rho and its effector, Rho kinase, reportedly contribute to the pathophysiology of portal hypertension. Thus, a potential effect of S1P(2) antagonism on portal hypertension was examined. Intravenous infusion of the S1P(2) antagonist, JTE-013, at 1 mg/kg body weight reduced portal vein pressure by 24% without affecting mean arterial pressure in cirrhotic rats induced by bile duct ligation at 4 weeks after the operation, whereas the same amount of S1P(2) antagonist did not alter portal vein pressure and mean arterial pressure in control sham-operated rats. Rho kinase activity in the livers was enhanced in bile duct-ligated rats compared to sham-operated rats, and this enhanced Rho kinase activity in bile duct-ligated livers was reduced after infusion of the S1P(2) antagonist. S1P(2) messenger RNA (mRNA) expression, but not S1P(1) or S1P(3) , was increased in bile duct-ligated livers of rats and mice and also in culture-activated rat hepatic stellate cells. S1P(2) expression, determined in S1P 2LacZ/+ mice, was highly increased in hepatic stellate cells of bile duct-ligated livers. Furthermore, the increase of Rho kinase activity in bile duct-ligated livers was observed as early as 7 days after the operation in wildtype mice, but was less in S1P 2-/- mice. Conclusion: S1P may play an important role in the pathophysiology of portal hypertension with Rho kinase activation by way of S1P(2) . The S1P(2) antagonist merits consideration as a novel therapeutic agent for portal hypertension. (HEPATOLOGY 2012).
Hepatology 04/2012; 56(4):1427-38. · 11.66 Impact Factor
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ABSTRACT: Thrombin induces vascular responses including the promotion of tissue factor (TF) and plasminogen activator inhibitor-1 (PAI-1) protein expression, which is modulated by small GTPases RhoA and Rac1, Ca(2+) signaling and reactive oxygen species (ROS). Recent studies have shown that membrane type 1 matrix metalloproteinase (MT1-MMP) functions not only as a protease but also as a signaling molecule. In this study, we hypothesized that MT1-MMP may mediate RhoA and Rac1 activation and their downstream events in thrombin-stimulated endothelial cells.
We used cultured human aortic endothelial cells (HAECs). MT1-MMP was silenced by small interfering RNA (siRNA). RhoA was inhibited by C3 exoenzyme, whereas adenovirus-mediated gene transfection of dominant negative RhoA and Rac1 was used for the inhibition of RhoA and Rac1. RhoA and Rac1 activation was determined by pull-down assays. Intracellular Ca(2+) concentrations ([Ca(2+)](i)) were fluorescently measured by fura-2 assay. NADPH oxidase activity was determined by lucigenin-enhanced chemiluminescence.
Inhibition of RhoA attenuated thrombin-triggered [Ca(2+)](i) increase and TF and PAI-1 expression in HAECs, whereas thrombin-triggered ROS generation and TF and PAI-1 expression were blocked by inhibition of Rac1. Silencing of MT1-MMP attenuated thrombin-triggered RhoA and Rac1 activation, resulting in the attenuation of downstream events including Ca(2+) signaling, NADPH oxidase activity, ROS generation, and TF and PAI-1 expression.
The present study shows that MT1-MMP mediates the RhoA/Ca(2+) and Rac1/NADPH oxidase-dependent signaling pathways in thrombin-induced vascular responses.
Journal of atherosclerosis and thrombosis 06/2011; 18(9):762-73. · 2.69 Impact Factor
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Seikagaku. The Journal of Japanese Biochemical Society 06/2011; 83(6):536-44. · 0.04 Impact Factor
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ABSTRACT: Radial arteries are increasingly used as conduits for coronary artery bypass grafts. However, vasospasm continues to be a major concern in radial artery grafts. Rho kinase plays a critical role in vascular contraction through phosphorylation of the regulatory subunit myosin phosphatase targeting subunit 1 (MYPT1) of the myosin light chain phosphatase to inhibit myosin light chain phosphatase in vascular smooth muscle. The purpose of this study was to evaluate the inhibitory effects of fasudil, a clinically used Rho kinase inhibitor, on Rho kinase activity, myosin light chain phosphorylation, in vitro contraction, and in situ vasospasm in radial arteries of patients undergoing coronary artery bypass grafting surgery.
The inhibitory efficacy of fasudil on vasoconstrictor-induced contraction and phosphorylation of MYPT1 was examined in radial artery rings. In situ phosphorylation of MYPT1 was evaluated in nonspastic and spastic radial arteries, and the effects of intraluminal administration of fasudil and verapamil-glyceryl trinitrate (VG) on in situ free blood flow and phosphorylation of MYPT1 and myosin light chain were compared in spastic radial arteries.
Both fasudil and VG nearly fully inhibited noradrenaline- and serotonin-induced contraction of radial artery rings. However, fasudil but not VG abolished MYPT1 phosphorylation. In spastic radial arteries phosphorylation of MYPT1 and myosin light chain was increased compared with that seen in nonspastic arteries. Intraradial administration of fasudil induced a much larger increase in in situ free blood flow compared with VG treatment. This antispastic effect of fasudil was accompanied by marked decreases in phosphorylation of MYPT1 and myosin light chain.
Fasudil is a very effective Rho kinase inhibitor that deinhibits myosin light chain phosphatase and powerfully relieves vasospasm in situ in radial arteries.
The Journal of thoracic and cardiovascular surgery 03/2011; 142(2):e59-65. · 3.41 Impact Factor
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ABSTRACT: Sphingosine-1-phosphate (S1P) is a plasma lipid mediator with multiple roles in mammalian development, physiology and pathophysiology. It is constitutively produced mostly by erythrocytes by the action of sphingosine kinase 1 (SphK1), resulting in high (∼0.5 micromolar) steady-state plasma S1P content and steep S1P concentration gradient imposed between plasma/lymph/tissue interstitial fluid. S1P is also locally produced by activated platelets and tumor cells, in the latter case SphK1 is a downstream target of activated Ras mutant and hypoxia, and is frequently upregulated especially in advanced stages of tumors. Most if not all of the S1P actions in vertebrates are mediated through evolutionarily conserved G protein-coupled S1P receptor family. Ubiquitously expressed mammalian subtypes S1PR1, S1PR2 and S1PR3 mediate pleiotropic actions of S1P in diverse cell types, through coupling to distinctive repertoire of heterotrimeric G proteins. S1PR1 and S1PR3 mediate directed cell migration toward S1P through coupling to G(i) and activating Rac, a Rho family small G protein essential for cell migration. Indeed, S1PR1 expressed in lymphocytes directs their egress from lymph nodes into lymph and recirculation, serving as the target for downregulation by the immunosuppressant FTY720 (fingolimod). S1PR1 in endothelial cells plays an essential role in vascular maturation in embryonic stage, and mediates angiogenic and vascular protective roles of S1P which include eNOS activation and maintenance of barrier integrity. It is likely that S1PR1 and SphK1 expressed in host endothelial cells and tumor cells act in concert in a paracrine loop to contribute to tumor angiogenesis, tumor invasion and progression. In sharp contrast, S1PR2 mediates S1P inhibition of Rac at the site downstream of G(12/13)-mediated Rho activation, thus identified as the first G protein-coupled receptor that negatively regulates Rac and cell migration. S1PR2 could also mediate inhibition of Akt and cell proliferation/survival signaling via Rho-ROCK-PTEN pathway. S1PR2 expressed in tumor cells mediates inhibition of cell migration and invasion in vitro and metastasis in vivo. Moreover, S1PR2 expressed in host endothelial cells and tumor-infiltrating myeloid cells in concert mediates potent inhibition of tumor angiogenesis and tumor growth in vivo, with inhibition of VEGF expression and MMP9 activity. These recent findings provide further basis for S1P receptor subtype-specific, novel therapeutic tactics for individualized treatment of patients with cancer.
American journal of cancer research. 01/2011; 1(4):460-81.
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Hiroshi Ohkawara,
Toshiyuki Ishibashi,
Shu-ichi Saitoh,
Nobutaka Inoue,
Koichi Sugimoto,
Masashi Kamioka,
Hironori Uekita,
Takashi Kaneshiro,
Katsuya Ando, Yoh Takuwa,
Yukio Maruyama,
Yasuchika Takeishi
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ABSTRACT: Small GTPases RhoA and Rac1 play crucial roles in endothelial dysfunction and reactive oxygen species (ROS) generation. We reported evidence that in thrombin-stimulated endothelial cells, rapid geranylgeranylation is an essential process for full activation of unprocessed RhoA, which is blocked by statin. In this study, we examined the effects of intravenous administration of pravastatin on thrombin-triggered vascular responses in vivo, as well as on the lipid modification of unprocessed forms of RhoA and Rac1 and their activation induced by thrombin.
Thrombin (50 U/kg) was intravenously injected with or without 0.3 mg/kg pravastatin into Wistar and spontaneously hypertensive rats. Coadministration of pravastatin prevented thrombin-induced impaired endothelium-dependent coronary vasodilation and down-regulated Akt/endothelial nitric oxide synthase (eNOS) phosphorylation within 1 h, as well as the down-regulation of eNOS protein expression within 4 h. In addition, thrombin increased Rac1/p47(phox)-dependent NAD(P)H oxidase activities of rat aortas within 1 h, resulting in ROS generation, which was prevented by the coadministration of pravastatin. Furthermore, the coadministration of pravastatin prevented thrombin-induced conversion of unprocessed RhoA and Rac1 into the geranylgeranylated forms as well as GTP-loading and membrane translocation within 1 h.
Intravenous injection of pravastatin prevents impaired NO-dependent vasodilation and Rac1/NAD(P)H oxidase-mediated-ROS generation by blocking the down-regulation of Akt/eNOS pathways and the full activation of unprocessed RhoA and Rac1 in vivo.
Cardiovascular research 12/2010; 88(3):492-501. · 5.80 Impact Factor
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ABSTRACT: Sphingosine-1-phosphate (S1P) is a blood-borne lipid mediator with pleiotropic biological activities. S1P acts via the specific cell surface G-protein-coupled receptors, S1P(1-5). S1P(1) and S1P(2) were originally identified from vascular endothelial cells (ECs) and smooth muscle cells, respectively. Emerging evidence shows that S1P plays crucial roles in the regulation of vascular functions, including vascular formation, barrier protection and vascular tone via S1P(1), S1P(2) and S1P(3). In particular, S1P regulates vascular formation through multiple mechanisms; S1P exerts both positive and negative effects on angiogenesis and vascular maturation. The positive and negative effects of S1P are mediated by S1P(1) and S1P(2), respectively. These effects of S1P(1) and S1P(2) are probably mediated by the S1P receptors expressed in multiple cell types including ECs and bone-marrow-derived cells. The receptor-subtype-specific, distinct effects of S1P favor the development of novel therapeutic tactics for antitumor angiogenesis in cancer and therapeutic angiogenesis in ischemic diseases.
World journal of biological chemistry. 10/2010; 1(10):298-306.
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ABSTRACT: Rho-mediated inhibition of myosin light chain (MLC) phosphatase (MLCP), together with Ca(2+)-dependent MLC kinase activation, constitutes the major signaling mechanisms for vascular smooth muscle contraction. We recently unveiled the involvement of Ca(2+)-induced, phosphoinositide 3-kinase (PI3K) class IIα isoform (PI3K-C2α)-dependent Rho activation and resultant Rho kinase-dependent MLCP suppression in membrane depolarization- and receptor agonist-induced contraction. It is unknown whether Ca(2+)- and PI3K-C2α-dependent regulation of MLCP is altered in vascular smooth muscle of hypertensive animals and is involved in hypertension. Therefore, we studied the role of the Ca(2+)-PI3K-C2α-Rho-MLCP pathway in spontaneously hypertensive rats (SHRs). PI3K-C2α was readily detected in various vascular beds of Wistar-Kyoto rats and activated by high KCl. High KCl also stimulated vascular Rho activity and phosphorylation of the MLCP regulatory subunit MYPT1 at Thr(853) in a PI3K inhibitor wortmannin-sensitive manner. In mesenteric and other vessels of SHRs at the hypertensive but not the prehypertensive stage, the activity of PI3K-C2α but not class I PI3K p110α was elevated with concomitant rises of Rho activity and Thr(853)-phosphorylation of MYPT1, as compared with normotensive controls. Infusion of the Ca(2+) channel antagonist nicardipine reduced blood pressure with suppression of vascular activity of PI3K-C2α-Rho and phosphorylation of MYPT1 in hypertensive SHRs. Infusion of wortmannin lowered blood pressure with inhibition of PI3K-C2α-Rho activities and MYPT1 phosphorylation in hypertensive SHRs. These observations suggest that an increased activity of the Ca(2+)-PI3K-C2α-Rho signaling pathway with resultant augmented MLCP suppression contributes to hypertension in SHRs. The Ca(2+)- and PI3K-C2α-dependent Rho stimulation in vascular smooth muscle may be a novel, promising target for treating hypertension.
Hypertension 10/2010; 56(5):934-41. · 6.21 Impact Factor
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Masashi Kamioka,
Toshiyuki Ishibashi,
Hiroshi Ohkawara,
Ryoji Nagai,
Koichi Sugimoto,
Hironori Uekita,
Takanori Matsui,
Sho-Ichi Yamagishi,
Katsuya Ando,
Takayuki Sakamoto,
Nobuo Sakamoto, Yoh Takuwa,
Ikuo Wada,
Masashi Shiomi,
Yukio Maruyama,
Yasuchika Takeishi
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ABSTRACT: An advanced glycation end products (AGE)/a receptor for AGE (RAGE) axis plays a key role in diabetic vascular complications. Membrane type 1-matrix metalloproteinase (MT1-MMP) has been shown to function not only as a proteolytic enzyme but also as a signaling molecule. In this study, we investigated the role of MT1-MMP in the AGE/RAGE-triggered signaling pathways in cultured rabbit smooth muscle cells (SMCs) and the molecular interaction between RAGE and MT1-MMP in vitro and in vivo. In SMCs, AGE-activated Rac1 and p47(phox) within 1 min, NADPH oxidase activity and reactive oxygen species (ROS) generation within 5 min, and NF-κB phosphorylation within 15 min, thereby inducing redox-sensitive molecular expression. Silencing of RAGE by small-interfering RNA (siRNA) blocked the AGE-induced signaling pathways. AGE-induced geranylgeranyl transferase I (GGTase I) activity, Rac1·p47(phox) activation, NADPH oxidase activity, ROS generation, and molecular expression were also markedly attenuated by silencing of MT1-MMP. An inhibitor of GGTase I mimicked the effects of MT1-MMP-specific siRNA. Fluorescent immunohistochemistry revealed that MT1-MMP was partially co-localized with RAGE in SMCs, and RAGE was found to form a complex with MT1-MMP in both cultured SMCs and the aortae of diabetic rats by immunoprecipitation. Furthermore, MT1-MMP and RAGE formed a complex in the aortic atherosclerotic lesions of hyperlipidemic rabbits. We show that MT1-MMP plays a crucial role in RAGE-activated NADPH oxidase-dependent signaling pathways and forms a complex with RAGE in the vasculature, thus suggesting that MT1-MMP may be a novel therapeutic target for diabetic vascular complications.
Journal of Cellular Physiology 10/2010; 226(6):1554-63. · 3.87 Impact Factor
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Fei Wang,
Yasuo Okamoto,
Isao Inoki,
Kazuaki Yoshioka,
Wa Du,
Xun Qi,
Noriko Takuwa,
Koichi Gonda,
Yasuhiko Yamamoto,
Ryunosuke Ohkawa,
Takumi Nishiuchi,
Naotoshi Sugimoto,
Yutaka Yatomi,
Kunitoshi Mitsumori,
Masahide Asano,
Makoto Kinoshita, Yoh Takuwa
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ABSTRACT: Sphingosine-1-phosphate (S1P) is a biologically active sphingolipid that has pleiotropic effects in a variety of cell types including ECs, SMCs, and macrophages, all of which are central to the development of atherosclerosis. It may therefore exert stimulatory and inhibitory effects on atherosclerosis. Here, we investigated the role of the S1P receptor S1PR2 in atherosclerosis by analyzing S1pr2-/- mice with an Apoe-/- background. S1PR2 was expressed in macrophages, ECs, and SMCs in atherosclerotic aortas. In S1pr2-/-Apoe-/- mice fed a high-cholesterol diet for 4 months, the area of the atherosclerotic plaque was markedly decreased, with reduced macrophage density, increased SMC density, increased eNOS phosphorylation, and downregulation of proinflammatory cytokines compared with S1pr2+/+Apoe-/- mice. Bone marrow chimera experiments indicated a major role for macrophage S1PR2 in atherogenesis. S1pr2-/-Apoe-/- macrophages showed diminished Rho/Rho kinase/NF-κB (ROCK/NF-κB) activity. Consequently, they also displayed reduced cytokine expression, reduced oxidized LDL uptake, and stimulated cholesterol efflux associated with decreased scavenger receptor expression and increased cholesterol efflux transporter expression. S1pr2-/-Apoe-/- ECs also showed reduced ROCK and NF-κB activities, with decreased MCP-1 expression and elevated eNOS phosphorylation. Pharmacologic S1PR2 blockade in S1pr2+/+Apoe-/- mice diminished the atherosclerotic plaque area in aortas and modified LDL accumulation in macrophages. We conclude therefore that S1PR2 plays a critical role in atherogenesis and may serve as a novel therapeutic target for atherosclerosis.
The Journal of clinical investigation 10/2010; 120(11):3979-95. · 15.39 Impact Factor
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ABSTRACT: Gomisin A (GA) is an active ingredient of the fruits of Schisandra chinensis which has been widely used as a tonic in traditional Korean medicine. GA induces not only endothelium-dependent but also endothelium-independent relaxation in an isolated rat's thoracic aorta. This study was aimed to investigate the molecular mechanism by which GA induces endothelium-independent vasorelaxation. Rat aortic rings were denuded of endothelium, mounted in organ baths, and subjected to contraction or relaxation. We measured the amount of GTP RhoA as well as the phosphorylation level of 20 kDa myosin light chains (MLC₂₀), myosin phosphatase-targeting subunit 1 (MYPT1) and protein kinase C-potentiated inhibitory protein for heterotrimeric myosin light-chain phosphatase of 17 kDa (CPI17). Pretreatment with GA dose-dependently inhibited the concentration-response curves in response to sodium fluoride (NaF) or thromboxane A(2) agonist U46619, but not to phorbol 12, 13-dibutyrate (PDBu). GA decreased the activation of RhoA as well as the phosphorylation level of MLC₂₀, MYPT1(Thr₈₅₅), and CPI17 induced by 8.0 mM NaF or 30 nM U46619. However, K+ channel blockers such as glibenclamide, apamin, or charybdotoxin did not affect the vascular relaxation induced by GA. Furthermore, GA did not affect the level of phosphorylation of CPI17 induced by PDBu. GA reduces vascular contraction through inhibition of RhoA/Rho-kinase pathway in endothelium-denuded rat aorta.
Archiv für Experimentelle Pathologie und Pharmakologie 10/2010; 383(1):45-56. · 2.65 Impact Factor
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Xun Qi,
Yasuo Okamoto,
Tomomi Murakawa,
Fei Wang,
Osamu Oyama,
Ryunosuke Ohkawa,
Kazuaki Yoshioka,
Wa Du,
Naotoshi Sugimoto,
Yutaka Yatomi,
Noriko Takuwa, Yoh Takuwa
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ABSTRACT: Therapeutic angiogenesis is a promising strategy for treating ischemia. The lysophospholipid mediator sphingosine-1-phosphate (S1P) acts on vascular endothelial cells to stimulate migration and tube formation, and plays the critical role in developmental angiogenesis. We developed poly(lactic-co-glycolic-acid) (PLGA)-based S1P-containing microparticles (PLGA-S1P), which are biodegradable and continuously release S1P, and studied the effects of PLGA-S1P on neovascularization in murine ischemic hindlimbs. Intramuscular injections of PLGA-S1P stimulated blood flow in C57BL/6 mice dose-dependently, with repeated administrations at a 3-day interval, rather than a single bolus or 6-day interval, over 28 days conferring the optimal stimulating effect. In Balb/c mice that exhibit limb necrosis and dysfunction due to retarded blood flow recovery, injections of PLGA-S1P stimulated blood flow with alleviation of limb necrosis and dysfunction. PLGA-S1P alone did not induce edema in ischemic limbs, and rather blocked vascular endothelial growth factor-induced edema. PLGA-S1P not only increased the microvessel densities in ischemic muscle, but promoted coverage of vessels with smooth muscle cells and pericytes, thus stabilizing vessels. PLGA-S1P stimulated Akt and ERK with increased phosphorylation of endothelial nitric oxide synthase in ischemic muscle. The effects of the nitric oxide synthase inhibitor, Nomega-nitro-L-arginine methylester, showed that PLGA-S1P-induced blood flow stimulation was partially dependent on nitric oxide. Injections of PLGA-S1P also increased the expression of angiogenic factors and the recruitment of CD45-, CD11b- and Gr-1-positive myeloid cells, which are implicated in post-ischemic angiogenesis, into ischemic muscle. These results indicate that PLGA-based, sustained local delivery of S1P is a potentially useful therapeutic modality for stimulating post-ischemic angiogenesis.
European journal of pharmacology 03/2010; 634(1-3):121-31. · 2.59 Impact Factor
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ABSTRACT: Sphingosine-1-phosphate (S1P) has been implicated in tumor angiogenesis by acting through the G(i)-coupled chemotactic receptor S1P(1). Here, we report that the distinct receptor S1P(2) is responsible for mediating the G(12/13)/Rho-dependent inhibitory effects of S1P on Akt, Rac, and cell migration, thereby negatively regulating tumor angiogenesis and tumor growth. By using S1P(2)(LacZ/+) mice, we found that S1P(2) was expressed in both tumor and normal blood vessels in many organs, in both endothelial cells (EC) and vascular smooth muscle cells, as well as in tumor-associated, CD11b-positive bone marrow-derived cells (BMDC). Lewis lung carcinoma or B16 melanoma cells implanted in S1P(2)-deficient (S1P(2)(-/-)) mice displayed accelerated tumor growth and angiogenesis with enhanced association of vascular smooth muscle cells and pericytes. S1P(2)(-/-) ECs exhibited enhanced Rac activity, Akt phosphorylation, cell migration, proliferation, and tube formation in vitro. Coinjection of S1P(2)(-/-) ECs and tumor cells into wild-type mice also produced a relative enhancement of tumor growth and angiogenesis in vivo. S1P(2)(-/-) mice were also more efficient at recruiting CD11b-positive BMDCs into tumors compared with wild-type siblings. Bone marrow chimera experiments revealed that S1P(2) acted in BMDCs to promote tumor growth and angiogenesis. Our results indicate that, in contrast to endothelial S1P(1), which stimulates tumor angiogenesis, S1P(2) on ECs and BMDCs mediates a potent inhibition of tumor angiogenesis, suggesting a novel therapeutic tactic for anticancer treatment.
Cancer Research 01/2010; 70(2):772-81. · 7.86 Impact Factor
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Yuya Yoshida,
Mitsutoshi Nakada,
Naotoshi Sugimoto,
Tomoya Harada,
Yasuhiko Hayashi,
Daisuke Kita,
Naoyuki Uchiyama,
Yutaka Hayashi,
Akihiro Yachie, Yoh Takuwa,
Jun-ichiro Hamada
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ABSTRACT: Sphingosine-1-phosphate (S1P) is a bioactive lipid that signals through a family of G protein-coupled receptors consisting of 5 members termed S1P1–5, and it regulates cellular proliferation, migration and survival. We investigated the expression and role of S1P receptors in glioma. Human glioma expressed S1P1, S1P2, S1P3, and S1P5 by quantitative real-time PCR analysis. Expression of the S1P1 was significantly lower in glioblastoma than in the normal brain (p < 0.01) and diffuse astrocytoma (p < 0.05). Immunoblotting showed that normal brain expressed more S1P1 protein than did glioblastoma. Immunohistochemistry showed that S1P1 was localized predominantly in the astrocytes in the normal brain, but no staining was observed in glioblastoma. Downregulation of S1P1 expression correlated with poor survival of patients with glioblastoma (p < 0.05). S1P1 small interfering RNA promoted cell proliferation in high-expressor glioma cell lines (T98G, G112). Cell proliferation was promoted by the pertussis toxin, which deactivates Gi/o type of G proteins; the S1P1 is exclusively coupled to these proteins. Forced expression of the S1P1 in low-expressor cell lines (U87, U251) resulted in decreased cell growth and led to suppressed tumor growth in transplanted gliomas in vivo. Furthermore, we found a significant association between the S1P1 expression and early growth response-1, a transcriptional factor that exhibits tumor suppression in glioblastoma cells (p < 0.05). These data indicate that the downregulation of S1P1 expression enhances the malignancy of glioblastoma by increasing cell proliferation and correlates with the shorter survival of patients with glioblastoma.
International Journal of Cancer 10/2009; 126(10):2341 - 2352. · 5.44 Impact Factor
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Yuya Yoshida,
Mitsutoshi Nakada,
Naotoshi Sugimoto,
Tomoya Harada,
Yasuhiko Hayashi,
Daisuke Kita,
Naoyuki Uchiyama,
Yutaka Hayashi,
Akihiro Yachie, Yoh Takuwa,
Jun-Ichiro Hamada
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ABSTRACT: Sphingosine-1-phosphate (S1P) is a bioactive lipid that signals through a family of G protein-coupled receptors consisting of 5 members termed S1P(1-5), and it regulates cellular proliferation, migration and survival. We investigated the expression and role of S1P receptors in glioma. Human glioma expressed S1P(1), S1P(2), S1P(3), and S1P(5) by quantitative real-time PCR analysis. Expression of the S1P(1) was significantly lower in glioblastoma than in the normal brain (p < 0.01) and diffuse astrocytoma (p < 0.05). Immunoblotting showed that normal brain expressed more S1P(1) protein than did glioblastoma. Immunohistochemistry showed that S1P(1) was localized predominantly in the astrocytes in the normal brain, but no staining was observed in glioblastoma. Downregulation of S1P(1) expression correlated with poor survival of patients with glioblastoma (p < 0.05). S1P(1) small interfering RNA promoted cell proliferation in high-expressor glioma cell lines (T98G, G112). Cell proliferation was promoted by the pertussis toxin, which deactivates G(i/o) type of G proteins; the S1P(1) is exclusively coupled to these proteins. Forced expression of the S1P(1) in low-expressor cell lines (U87, U251) resulted in decreased cell growth and led to suppressed tumor growth in transplanted gliomas in vivo. Furthermore, we found a significant association between the S1P(1) expression and early growth response-1, a transcriptional factor that exhibits tumor suppression in glioblastoma cells (p < 0.05). These data indicate that the downregulation of S1P(1) expression enhances the malignancy of glioblastoma by increasing cell proliferation and correlates with the shorter survival of patients with glioblastoma.
International Journal of Cancer 10/2009; 126(10):2341-52. · 5.44 Impact Factor
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Noriko Takuwa,
Sei-Ichiro Ohkura,
Shin-Ichiro Takashima,
Keisuke Ohtani,
Yasuo Okamoto,
Tamotsu Tanaka,
Kaoru Hirano,
Soichiro Usui,
Fei Wang,
Wa Du, [......],
Kiyomi Mizugishi,
Yasuni Nakanuma,
Isao Ishii,
Masayuki Takamura,
Shuichi Kaneko,
Shosuke Kojo,
Kiyoshi Satouchi,
Kunitoshi Mitumori,
Jerold Chun, Yoh Takuwa
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ABSTRACT: Sphingosine kinase 1 (SPHK1), its product sphingosine-1-phosphate (S1P), and S1P receptor subtypes have been suggested to play protective roles for cardiomyocytes in animal models of ischaemic preconditioning and cardiac ischaemia/reperfusion injury. To get more insight into roles for SPHK1 in vivo, we have generated SPHK1-transgenic (TG) mice and analysed the cardiac phenotype.
SPHK1-TG mice overexpressed SPHK1 in diverse tissues, with a nearly 20-fold increase in enzymatic activity. The TG mice grew normally with normal blood chemistry, cell counts, heart rate, and blood pressure. Unexpectedly, TG mice with high but not low expression levels of SPHK1 developed progressive myocardial degeneration and fibrosis, with upregulation of embryonic genes, elevated RhoA and Rac1 activity, stimulation of Smad3 phosphorylation, and increased levels of oxidative stress markers. Treatment of juvenile TG mice with pitavastatin, an established inhibitor of the Rho family G proteins, or deletion of S1P3, a major myocardial S1P receptor subtype that couples to Rho GTPases and transactivates Smad signalling, both inhibited cardiac fibrosis with concomitant inhibition of SPHK1-dependent Smad-3 phosphorylation. In addition, the anti-oxidant N-2-mercaptopropyonylglycine, which reduces reactive oxygen species (ROS), also inhibited cardiac fibrosis. In in vivo ischaemia/reperfusion injury, the size of myocardial infarct was 30% decreased in SPHK1-TG mice compared with wild-type mice.
These results suggest that chronic activation of SPHK1-S1P signalling results in both pathological cardiac remodelling through ROS mediated by S1P3 and favourable cardioprotective effects.
Cardiovascular research 09/2009; 85(3):484-93. · 5.80 Impact Factor
