Yutaka Yatomi

Autotaxin Stabilizes Blood Vessels and Is Required for Embryonic Vasculature by Producing Lysophosphatidic Acid

Autotaxin (ATX) is a cancer-associated motogen that has multiple biological activities in vitro through the production of bioactive small lipids, lysophosphatidic acid (LPA). ATX and LPA are abundantly present in circulating blood. However, their roles in circulation remain to be solved. To uncover the physiological role of ATX we analyzed ATX knock-out mice. In ATX-null embryos, early blood vessels appeared to form properly, but they failed to develop into mature vessels. As a result ATX-null mice are lethal around embryonic day 10.5. The phenotype is much more severe than those of LPA receptor knock-out mice reported so far. In cultured allantois explants, neither ATX nor LPA was angiogenic. However, both of them helped to maintain preformed vessels by preventing disassembly of the vessels that was not antagonized by Ki16425, an LPA receptor antagonist. In serum from heterozygous mice both lysophospholipase D activity and LPA level were about half of those from wild-type mice, showing that ATX is responsible for the bulk of LPA production in serum. The present study revealed a previously unassigned role of ATX in stabilizing vessels through novel LPA signaling pathways.

Involvement of the Snake Toxin Receptor CLEC-2, in Podoplanin-mediated Platelet Activation, by Cancer Cells

Podoplanin (aggrus), a transmembrane sialoglycoprotein, is involved in tumor cell-induced platelet aggregation, tumor metastasis, and lymphatic vessel formation. However, the mechanism by which podoplanin induces these cellular processes including its receptor has not been elucidated to date. Podoplanin induced platelet aggregation with a long lag phase, which is dependent upon Src and phospholipase Cγ2 activation. However, it does not bind to glycoprotein VI. This mode of platelet activation was reminiscent of the snake toxin rhodocytin, the receptor of which has been identified by us as a novel platelet activation receptor, C-type lectin-like receptor 2 (CLEC-2) (Suzuki-Inoue, K., Fuller, G. L., Garcia, A., Eble, J. A., Pohlmann, S., Inoue, O., Gartner, T. K., Hughan, S. C., Pearce, A. C., Laing, G. D., Theakston, R. D., Schweighoffer, E., Zitzmann, N., Morita, T., Tybulewicz, V. L., Ozaki, Y., and Watson, S. P. (2006) Blood 107, 542–549). Therefore, we sought to evaluate whether CLEC-2 serves as a physiological counterpart for podoplanin. Association between CLEC-2 and podoplanin was confirmed by flow cytometry. Furthermore, their association was dependent on sialic acid on O-glycans of podoplanin. Recombinant CLEC-2 inhibited platelet aggregation induced by podoplanin-expressing tumor cells or lymphatic endothelial cells, suggesting that CLEC-2 is responsible for platelet aggregation induced by endogenously expressed podoplanin on the cell surfaces. These findings suggest that CLEC-2 is a physiological target protein of podoplanin and imply that it is involved in podoplanin-induced platelet aggregation, tumor metastasis, and other cellular responses related to podoplanin.

Sphingosine 1-phosphate induces contraction of coronary artery smooth muscle cells via S1P2

OBJECTIVES Sphingosine 1-phosphate (Sph-1-P), a bioactive lipid derived from activated platelets, may play an important role in coronary artery spasm and hence the pathogenesis of ischemic heart diseases, since we reported that a decrease in coronary blood flow was induced by this lysophospholipid in an in vivo canine heart model [Cardiovasc. Res. 46 (2000) 119]. In this study, metabolism related to and cellular responses elicited by Sph-1-P were examined in human coronary artery smooth muscle cells (CASMCs). METHODS AND RESULTS [3H]Sphingosine (Sph), incorporated into CASMCs, was converted to [3H]Sph-1-P intracellularly, but its stimulation-dependent formation and extracellular release were not observed. Furthermore, the cell surface Sph-1-P receptors of S1P family (previously called EDG) were found to be expressed in CASMCs. Accordingly, Sph-1-P seems to act as an extracellular mediator in CASMCs. Consistent with Sph-1-P-elicited coronary vasoconstriction in vivo, Sph-1-P strongly induced CASMC contraction, which was inhibited by JTE-013, a newly-developed specific antagonist of S1P(2) (EDG-5). Furthermore, C3 exoenzyme or Y-27632 inhibited the CASMC contraction induced by Sph-1-P, indicating Rho involvement. Finally, exogenously-added [3H]Sph-1-P underwent a rapid degradation. Since lipid phosphate phosphatases, ectoenzymes capable of dephosphorylating Sph-1-P, were expressed in CASMCs, Sph-1-P may be dephosphorylated by the ectophosphatases. CONCLUSIONS Sph-1-P, derived from platelets and dephosphorylated on the cell surface, may induce the contraction of coronary artery smooth muscle cells through the S1P(2)/Rho signaling.

Enhancement of sphingosine 1-phosphate-induced migration of vascular endothelial cells and smooth muscle cells by an EDG-5 antagonist

Sphingosine 1-phosphate (Sph-1-P), a bioactive lysophospholipid capable of inducing a wide spectrum of biological responses, acts as an intercellular mediator, through interaction with the endothelial differentiation gene (EDG)/S1P family of G protein-coupled receptors. In this study, the effects of JTE-013, a specific antagonist of the migration-inhibitory receptor EDG-5, on Sph-1-P-elicited responses were examined in human umbilical vein endothelial cells (HUVECs) and vascular smooth muscle cells (SMCs), which expressed EDG-5 protein weakly and abundantly, respectively. This pyrazolopyridine compound reversed the inhibitory effect of Sph-1-P on SMC migration and further enhanced Sph-1-P-stimulated HUVEC migration. In contrast, its effect on Sph-1-P-induced intracellular Ca(2+) mobilization was marginal. Our results indicate that specific regulation of Sph-1-P-modulated migration responses in vascular cells can be achieved by EDG-5 antagonists and that manipulation of Sph-1-P biological activities by each EDG antagonist may lead to a therapeutical application to control vascular diseases.

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.

Both plasma lysophosphatidic acid and serum autotaxin levels are increased in chronic hepatitis C.

Objectives Recent accumulating evidence indicates that lysophosphatidic acid (LPA) is a lipid mediator, abundantly present in blood, with a wide range of biologic actions including the regulation of proliferation and contraction in liver cells. Although it is speculated that LPA might play a role in pathophysiologic processes in vivo, not only its role but also even a possible alteration in its blood concentration under specific diseases is essentially unknown. Autotaxin (ATX), originally purified as an autocrine motility factor for melanoma cells, was revealed to be a key enzyme in LPA synthesis. We determined LPA and ATX levels in the blood of patients with liver disease. Methods ATX activity was measured by determining choline with the substrate of lysophosphatidylcholine, and the LPA level by an enzymatic cycling method in 41 patients with chronic hepatitis C. Results The serum ATX activity and plasma LPA level were significantly increased in patients, and were correlated positively with serum hyaluronic acid, and negatively with platelets, albumin, and prothrombin time. The plasma LPA level was strongly correlated with serum ATX activity. There were significant correlations between the histologic stage of fibrosis and both the serum ATX activity and plasma LPA level. Conclusions The serum ATX activity and plasma LPA level are increased in chronic hepatitis C in association with liver fibrosis. Our study may provide the first evidence showing a significant increase of both ATX and LPA in the blood under a specific disease.

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.

Sphingosine 1-Phosphate in Vascular Biology: Possible Therapeutic Strategies to Control Vascular Diseases

Blood platelets are very unique in that they store sphingosine 1-phosphate (Sph-1-P) abundantly (possibly due to the existence of highly active sphingosine kinase and a lack of Sph-1-P lyase) and release this bioactive lipid extracellularly upon stimulation. Vascular endothelial cells (ECs) and smooth muscle cells (SMCs) respond dramatically to this platelet-derived bioactive lipid mainly through a family of G protein-coupled Sph-1-P receptors named S1P1, 2, 3, 4, and 5, originally referred to as EDG-1, 5, 3, 6, and 8, respectively. In fact, the importance of Sph-1-P in platelet-vascular cell interactions has been revealed in a number of recent reports. Through interaction with ECs, Sph-1-P can mediate physiological wound healing processes such as vascular repair, although this important bioactive lipid can become atherogenic and thrombogenic, and cause or aggravate cardiovascular diseases especially under certain pathological conditions. On the other hand, Sph-1-P induces vasoconstriction through interaction with SMCs. It is likely that regulation of Sph-1-P biological activities is important for the therapeutical purpose to control vascular disorders. Particularly, the development of specific S1P receptor agonists or antagonists seems a reasonable strategy to selectively regulate the bioactivity of Sph-1-P, considering that a great diversity of Sph-1-P actions has been reported and that this diversity depends mainly on the S1P receptor subtype involved. In this review, I will summarize recent findings on possible roles of Sph-1-P in vascular biology and its therapeutical implications.

Serum autotaxin measurement in haematological malignancies: a promising marker for follicular lymphoma

Autotaxin (ATX) is a tumour cell motility‐stimulating factor originally isolated from melanoma cell supernatants. ATX is identical to lysophospholipase D, which produces a bioactive lipid mediator, lysophosphatidic acid (LPA), from lysophosphatidylcholine. ATX is overexpressed in various malignancies, including Hodgkin lymphoma, and ATX may stimulate tumour progression via LPA production. The present study measured the serum ATX antigen levels in patients with haematological malignancies using a recently developed automated enzyme immunoassay. The serum ATX antigen levels in patients with B‐cell neoplasms, especially follicular lymphoma (FL), were higher than those in healthy subjects. Serum ATX antigen levels in FL patients were associated with tumour burden and changed in parallel with the patients’ clinical courses. The serum ATX antigen levels were little affected by inflammation, unlike the soluble interleukin‐2 receptor and β2‐microglobulin levels. As expected, the plasma LPA levels in FL patients were correlated with the serum ATX antigen levels. Given that leukaemic tumour cells from FL patients expressed ATX, the shedding of ATX from lymphoma cells probably leads to the elevation of serum ATX antigen levels. Our results suggest that the serum ATX antigen level may be a promising and novel marker for FL.

Hyperfibrinogenemia is associated with lymphatic as well as hematogenous metastasis and worse clinical outcome in T2 gastric cancer

BackgroundAbnormal hemostasis in cancer patients has previously been described, however the correlation between the plasma fibrinogen level and cancer metastasis and prognosis has not been reported in a large-scale clinical study.MethodsPreoperative plasma fibrinogen levels were retrospectively examined in 405 patients who underwent surgery for advanced gastric cancer. The association of fibrinogen levels with clinical/pathological findings and clinical outcome was evaluated.ResultsThere was a positive correlation between plasma fibrinogen levels and the depth of invasion (p < 0.05). Hyperfibrinogenemia (>310 mg/dl) was independently associated with lymph node (Odds Ratio; 2.342, P = 0.0032) and liver (Odds Ratio; 2.933, P = 0.0147) metastasis, not with peritoneal metastasis in this series. Patients with hyperfibrinogenemia showed worse clinical outcome in T2 gastric cancer, however, there was no correlation of plasma fibrinogen level with prognosis in T3/T4 gastric cancer.ConclusionOur results might support the idea that hyperfibrinogenemia can augment lymphatic and hematogeneous metastasis of advanced gastric cancer, which is major determinant of the prognosis in T2 gastric cancer. Therefore, in the situation without peritoneal involvement, hyperfibrinogenemia is a useful biomarker to predict the possible metastasis and worse clinical outcome in T2 gastric cancer.