Tsukasa Ohmori

Sphingosine 1-phosphate: synthesis and release.

Sphingosine 1-phosphate (Sph-1-P) is a bioactive sphingolipid, acting both as an intracellular second messenger and extracellular mediator, in mammalian cells. In cell types where Sph-1-P acts as an intracellular messenger, stimulation-dependent synthesis of Sph-1-P, possibly resulting from sphingosine (Sph) kinase activation, is essential. Since this important kinase has recently been cloned, precise regulation of intracellular Sph-1-P synthesis will be clarified in the near future. As an intercellular mediator, elucidation of sources for extracellular Sph-1-P is important, in addition to identification of the cell surface receptors for this phospholipid. Blood platelets are very unique in that they store Sph-1-P abundantly (possibly due to the existence of highly active Sph kinase and a lack of Sph-1-P lyase) and release this bioactive lipid extracellularly upon stimulation. It is likely that platelets are an important source for extracellular Sph-1-P, especially for plasma and serum Sph-1-P. Platelet-derived Sph-1-P seems to play an important role in vascular biology.

Essential roles of sphingosine 1-phosphate/S1P1 receptor axis in the migration of neural stem cells toward a site of spinal cord injury

Neural stem/progenitor cells (NSPCs) migrate toward a damaged area of the central nervous system (CNS) for the purpose of limiting and/or repairing the damage. Although this migratory property of NSPCs could theoretically be exploited for cell‐based therapeutics of CNS diseases, little is known of the mechanisms responsible for migratory responses of NSPCs. Here, we found that sphingosine 1‐phosphate (Sph‐1‐P), a physiological lysophospholipid mediator, had a potent chemoattractant activity for NSPCs, in which, of Sph‐1‐P receptors, S1P1 was abundantly expressed. Sph‐1‐P‐induced NSPC migration was inhibited by the pretreatment with pertussis toxin, Y‐27632 (a Rho kinase inhibitor), and VPC23019 (a competitive inhibitor of S1P1 and S1P3). Sph‐1‐P does not act as intracellular mediator or in an autocrine manner, because [3H]sphingosine, incorporated into NSPCs, was mainly converted to ceramide and sphingomyeline intracellularly, and the stimulation‐dependent formation and extracellular release of Sph‐1‐P were not observed. Further, Sph‐1‐P concentration in the spinal cord was significantly increased at 7 days after a contusion injury, due to accumulation of microglia and reactive astrocytes in the injured area. This locally increased Sph‐1‐P concentration contributed to the migration of in vivo transplanted NSPCs through its receptor S1P1, given that lentiviral transduction of NSPCs with a short hairpin RNA interference for S1P1 abolished in vivo NSPC migration toward the injured area. This is the first report to identify a physiological role for a lipid mediator in NSPC migration toward a pathological area of the CNS and further indicates that the Sph‐1‐P/S1P1 pathway may have therapeutic potential for CNS injuries.

Aspirin resistance detected with aggregometry cannot be explained by cyclooxygenase activity : involvement of other signaling pathway(s) in cardiovascular events of aspirin-treated patients

Summary.  Objectives: Although the concept of aspirin resistance is extensively reported in medical literature, its precise mechanisms and clinical outcomes are largely unknown. In this study, we examined individual thromboxane biosynthesis and platelet aggregation in aspirin‐treated patients, and whether the results of a platelet aggregation test influenced clinical outcomes. Results: Subjects taking 81 mg of aspirin (n = 50) and controls (n = 38) were evaluated for platelet aggregation and platelet cyclooxygenase‐1 (COX‐1) activity by measuring collagen‐induced thromboxane B2 production. For aggregometry, both light transmission (LT) and laser‐light scattering methods were employed to quantitatively evaluate aggregate sizes and numbers. Aspirin treatment resulted in the inhibition of collagen‐induced platelet aggregation, particularly the transition from small to large platelet aggregates. Although platelet COX‐1 activity seemed to be uniformly inhibited in all patients, platelet aggregation studies showed great inter‐individual differences; variation in platelet COX‐1 activity only accounted for 6–20% of the individual aggregations. Factor analysis revealed the existence of a common factor (other than platelet COX‐1) that explained 48.4% of the variations in platelet aggregation induced by collagen, adenosine diphosphate (ADP), and collagen‐related peptide. We then prospectively enrolled 136 aspirin‐treated patients in our study, and we found that being in the upper quartile level of LT, or with large aggregate formation induced by collagen, was an independent risk factor for developing cardiovascular events within 12 months [hazard ratio (HR) = 7.98, P = 0.008 for LT; HR = 7.76, P = 0.007 for large aggregates]. On the other hand, the existence of diabetes mellitus was an independent risk factor for overall outcomes (HR 1.30–11.9, P = 0.015–0.033). Conclusions: Aspirin resistance expressed as unsuppressed platelet COX‐1 activity is a rare condition in an out‐patient population. Other factor(s) affecting collagen‐induced platelet aggregation may influence early outcomes in aspirin‐treated patients.

Plasminogen Activator Inhibitor 1 Promotes a Poor Prognosis in Sepsis-Induced Disseminated Intravascular Coagulation

Sepsis-induced disseminated intravascular coagulation (DIC) is a serious condition because it is closely linked to the development of multiple organ dysfunctions.We compared molecular fibrinolysis markers for 117 patients with sepsis-induced DIC and 1627 patients with nonseptic DIC. Levels of fibrinogen and fibrin degradation products and D-dimer were significantly lower in sepsis-induced DIC cases than in nonseptic DIC cases. In septic DIC cases, plasma plasminogen activator inhibitor 1 (PAI-1) levels were significantly higher than in nonseptic DIC cases. D-dimer levels were negatively correlated with plasma PAI-1 levels in septic DIC cases. Multiple Organ Dysfunction Scores were significantly higher in septic DIC patients with PAI-1 levels >90 ng/mL than in the group with PAI-1 levels <30 ng/mL. The Kaplan-Meier survival functions until 28 days after DIC diagnosis were significantly lower in the group with PAI-1 levels >90 ng/mL than in the other groups. In a multivariate analysis, plasma PAI-1 levels at DIC diagnosis were an independent risk factor for mortality in sepsis-induced DIC (hazard ratio, 1.012; P = .008). These data suggest that plasma PAI-1 plays an important role in sustaining DIC in septic DIC cases and contributes to multiple organ failure and decreased survival in such patients.

Gi-mediated Cas Tyrosine Phosphorylation in Vascular Endothelial Cells Stimulated with Sphingosine 1-Phosphate POSSIBLE INVOLVEMENT IN CELL MOTILITY ENHANCEMENT IN COOPERATION WITH Rho-MEDIATED PATHWAYS

Since blood platelets release sphingosine 1-phosphate (Sph-1-P) upon activation, it is important to examine the effects of this bioactive lipid on vascular endothelial cell functions from the viewpoint of platelet-endothelial cell interactions. In the present study, we examined Sph-1-P-stimulated signaling pathways related to human umbilical vein endothelial cell (HUVEC) motility, with a special emphasis on the cytoskeletal docking protein Crk-associated substrate (Cas). Sph-1-P stimulated tyrosine phosphorylation of Cas, which was inhibited by the Giinactivator pertussis toxin but not by the Rho inactivator C3 exoenzyme or the Rho kinase inhibitor Y-27632. Fyn constitutively associated with and phosphorylated Cas, suggesting that Cas tyrosine phosphorylation may be catalyzed by Fyn. Furthermore, upon HUVEC stimulation with Sph-1-P, Crk, through its SH2 domain, interacted with tyrosine-phosphorylated Cas, and the Cas-Crk complex translocated to the cell periphery (membrane ruffles), through mediation of Gi (Fyn) but not Rho. In contrast, tyrosine phosphorylation of focal adhesion kinase, and formation of stress fibers and focal adhesion were mediated by Rho but not Gi(Fyn). Finally, Sph-1-P-enhanced HUVEC motility, assessed by a phagokinetic assay using gold sol-coated plates and a Boydens chamber assay, was markedly inhibited not only by pertussis toxin (or the Fyn kinase inhibitor PP2) but also by C3 exoenzyme (or Y-27632). In HUVECs stimulated with Sph-1-P, these data suggest the following: (i) cytoskeletal signalings may be separable into Gi-mediated signaling pathways (involving Cas) and Rho-mediated ones (involving FAK), and (ii) coordinated signalings from both pathways are required for Sph-1-P-enhanced HUVEC motility. Since HUVECs reportedly express the Sph-1-P receptors EDG-1 (coupled with Gi) and EDG-3 (coupled with G13 and Gq) and the EDG-3 antagonist suramin was found to block specifically Rho-mediated responses, it is likely that Cas-related responses following Gi activation originate from EDG-1, whereas Rho-related responses originate from EDG-3.

Antagonism of Sphingosine 1-Phosphate Receptor-2 Enhances Migration of Neural Progenitor Cells Toward an Area of Brain Infarction

Background and Purpose— We have previously shown that the sphingosine 1-phosphate (S1P)/S1P receptor-1 (S1P1R) axis contributes to the migration of transplanted neural progenitor cells (NPCs) toward areas of spinal cord injury. In the current study, we examined a strategy to increase endogenous NPC migration toward the injured central nervous system to modify S1PR. Methods— S1P concentration in the ischemic brain was measured in a mouse thrombosis model of the middle cerebral artery. NPC migration in vitro was assessed by a Boyden chamber assay. Endogenous NPC migration toward the insult was evaluated after ventricular administration of the S1P2R antagonist JTE-013. Results— The concentration of S1P in the brain was increased after ischemia and was maximal 14 days after the insult. The increase in S1P in the infarcted brain was primarily caused by accumulation of microglia at the insult. Mouse NPCs mainly expressed S1P1R and S1P2R as S1PRs, and S1P significantly induced the migration of NPCs in vitro through activation of S1P1R. However, an S1P1R agonist failed to have any synergistic effect on S1P-mediated NPC migration, whereas pharmacologic or genetic inhibition of S1P2R by JTE-013 or short hairpin RNA expression enhanced S1P-mediated NPC migration but did not affect proliferation and differentiation. Interestingly, administration of JTE-013 into a brain ventricle significantly enhanced endogenous NPC migration toward the area of ischemia. Conclusions— Our findings suggest that S1P is a chemoattractant for NPCs released from an infarcted area and regulation of S1P2R function further enhances the migration of NPCs toward a brain infarction.

IL-1α induces thrombopoiesis through megakaryocyte rupture in response to acute platelet needs

An alternative pathway triggering enhanced platelet release from bone marrow megakaryocytes via a rupture-based mechanism is regulated by IL-1α in response to acute platelet requirements.

Efficient expression of a transgene in platelets using simian immunodeficiency virus-based vector harboring glycoprotein Ibα promoter: in vivo model for platelet-targeting gene therapy

Platelets release several mediators that modify vascular integrity and hemostasis. In the present study, we developed a technique for efficient transgene expression in platelets in vivo and examined whether this targeted‐gene‐product delivery system using a platelet release reaction could be exploited for clinical applications. Analysis of luciferase reporter gene constructs driven by platelet‐specific promoters (the GPIIb, GPIbα, and GPVI) revealed that the GPIbα promoter was the most potent in the megakaryoblastic cell line UT‐7/TPO and human CD34+‐derived megakaryocytes. Transduction of UT‐7/TPO;CD34+‐derived megakaryocytes; and c‐Kit+, ScaI+, and Lineage− (KSL) murine hematopoietic stem cells with a simian immunodeficiency virus (SIV)‐based lentiviral vector carrying eGFP resulted in efficient, dose‐dependent expression of eGFP, and the GPIbα promoter seemed to bestow megakaryocytic‐specific expression. Transplantation of KSL cells transduced with SIV vector containing eGFP into mice showed that there was preferable expression of eGFP in platelets driven by the GPIbα promoter [7–11% for the cytomeglovirus (CMV) promoter, 16–27% for the GPIbα promoter]. Furthermore, transplantation of ex vivo‐transduced KSL cells by SIV vector carrying human factorVIII (hFVIII) driven by the GPIbα promoter induced the production of detectable transcripts of the hFVIII gene and the hFVIII antigen in bone marrow and spleen for at least 90 days and partially corrected the hemophilia A phenotype. Platelet‐targeting gene therapy using SIV vectors appears to be promising for gene therapy approaches toward not only inherited platelet diseases but also other hemorrhagic disorders such as hemophilia A.—Ohmori, T., Mimuro, J., Takano, K., Madoiwa, S., Kashiwakura, Y., Ishiwata, A., Niimura, M., Mitomo, K., Tabata, T., Hasegawa, M., Ozawa, K., Sakata, Y. Efficient expression of a transgene in platelets using simian immunodeficiency virus‐based vector harboring glycoprotein Ibα promoter: in vivo model for platelet‐targeting gene therapy. FASEB J. 20, E769–E779 (2006)

Sphingosine 1-Phosphate Stimulates Gi- and Rho-Mediated Vascular Endothelial Cell Spreading and Migration

Sphingosine 1-phosphate (Sph-1-P) is a bioactive lipid released from activated platelets, which may be involved in angiogenesis. We, hence, investigated Sph-1-P effects on human umbilical vein endothelial cells (HUVECs) from a viewpoint of angiogenesis. Sph-1-P facilitated HUVEC spreading on the basement membrane component Matrigel, at concentrations ranging from 10 to 250 nM. This stimulatory response induced by Sph-1-P was blocked by pertussis toxin and C3 transferase (from Clostridium botulinum), which inactivate G(i)-type heterotrimeric G protein and Rho, respectively. Furthermore, Sph-1-P, in the modified Boydens chamber assay, stimulated HUVEC migration in a concentration-dependent manner, up to 250 nM. Checkerboard analysis revealed that Sph-1-P markedly induces directional migration (chemotaxis), but a random motility (chemokinesis) was also enhanced. The stimulatory effect of Sph-1-P on HUVEC migration was much stronger than that of other bioactive lipids, and again inhibited by pertussis toxin and by C3 transferase. Our present results that Sph-1-P induces endothelial spreading and migration through G(i)-coupled cell surface receptor(s) and Rho are consistent with a recent report on the role of this platelet-derived sphingolipid as a novel regulator of angiogenesis.

ENPP2 contributes to adipose tissue expansion and insulin resistance in diet-induced obesity.

Body weight is tightly regulated by food intake and energy dissipation, and obesity is related to decreased energy expenditure (EE). Herein, we show that nucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2, autotaxin) is an adipose-derived, secreted enzyme that controls adipose expansion, brown adipose tissue (BAT) function, and EE. In mice, Enpp2 was highly expressed in visceral white adipose tissue and BAT and is downregulated in hypertrophied adipocytes/adipose tissue. Enpp2+/− mice and adipocyte-specific Enpp2 knockout mice fed a high-fat diet showed smaller body weight gains and less insulin resistance than control mice fed the same diet. BAT was functionally more active and EE was increased in Enpp2-deficient mice. In humans, ENPP2 expression in subcutaneous fat and ENPP2 levels in serum were reduced in obese subjects. Taken together, our results establish ENPP2 as an adipose-derived, secreted enzyme that regulates adipose obesity and systemic metabolism. They also suggest ENPP2 could be a useful therapeutic target for the treatment of metabolic disease.