Makoto Osada

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.

Platelet Activation Receptor CLEC-2 Regulates Blood/Lymphatic Vessel Separation by Inhibiting Proliferation, Migration, and Tube Formation of Lymphatic Endothelial Cells

Background: Mice deficient in the platelet receptor CLEC-2 for podoplanin showed impaired blood/lymphatic vessel separation. Results: Functions of lymphatic endothelial cells are inhibited by platelet releasates and BMP-9, which we identified as a novel releasate. Conclusion: Granule contents including BMP-9 released upon platelet activation by CLEC-2-podoplanin interaction may contribute to the separation in vivo. Significance: We proposed a novel mechanism of platelet-mediated blood/lymphatic vessel separation. The platelet activation receptor CLEC-2 plays crucial roles in thrombosis/hemostasis, tumor metastasis, and lymphangiogenesis, although its role in thrombosis/hemostasis remains controversial. An endogenous ligand for CLEC-2, podoplanin, is expressed in lymphatic endothelial cells (LECs). We and others have reported that CLEC-2-deficiency is lethal at mouse embryonic/neonatal stages associated with blood-filled lymphatics, indicating that CLEC-2 is essential for blood/lymphatic vessel separation. However, its mechanism, and whether CLEC-2 in platelets is necessary for this separation, remains unknown. We found that specific deletion of CLEC-2 from platelets leads to the misconnection of blood/lymphatic vessels. CLEC-2+/+ platelets, but not by CLEC-2−/− platelets, inhibited LEC migration, proliferation, and tube formation but had no effect on human umbilical vein endothelial cells. Additionally, supernatants from activated platelets significantly inhibited these three functions in LECs, suggesting that released granule contents regulate blood/lymphatic vessel separation. Bone morphologic protein-9 (BMP-9), which we found to be present in platelets and released upon activation, appears to play a key role in regulating LEC functions. Only BMP-9 inhibited tube formation, although other releasates including transforming growth factor-β and platelet factor 4 inhibited proliferation and/or migration. We propose that platelets regulate blood/lymphatic vessel separation by inhibiting the proliferation, migration, and tube formation of LECs, mainly because of the release of BMP-9 upon activation by CLEC-2/podoplanin interaction.

Expression of the LIM Proteins Paxillin and Hic-5 in Human Tissues

The LIM domain is a protein-protein interaction motif critically involved in a variety of fundamental biological processes, including cytoskeletal organization, cell lineage specification, and organ development. In this study we examined the expression of the LIM proteins paxillin and Hic-5 in adult human tissues by immunohistochemistry and immunoblotting. Paxillin expression was widespread and observed both in non-muscle and muscle tissues. Of the latter, paxillin was mainly expressed in multinuclear striated muscle. In contrast, Hic-5 showed restricted expression and was expressed in muscle tissues, mainly in mononuclear smooth muscle. Taken together with previous findings, it appears likely that the counterbalance between paxillin and Hic-5 may be deeply involved in muscle differentiation.

C‐type lectin‐like receptor 2 promotes hematogenous tumor metastasis and prothrombotic state in tumor‐bearing mice

Essentials The role of C‐type lectin‐like receptor‐2 (CLEC‐2) in cancer progression is unclear. CLEC‐2‐depleted mouse model is generated by using a rat anti‐mouse CLEC‐2 monoclonal antibody. CLEC‐2 depletion inhibits hematogenous tumor metastasis of podoplanin‐expressing B16F10 cells. CLEC‐2 depletion prolongs cancer survival by suppressing thrombosis and inflammation.

Podoplanin-positive periarteriolar stromal cells promote megakaryocyte growth and proplatelet formation in mice by CLEC-2

Megakaryopoiesis is the hierarchical differentiation of hematopoietic stem cells into megakaryocytes. Differentiating megakaryocytes undergo maturation characterized by endomitosis and produce numerous platelets through proplatelet formation. C-type lectin-like receptor 2 (CLEC-2) is a podoplanin (PDPN) receptor mainly expressed on platelets and megakaryocytes. Deletion of platelet/megakaryocyte CLEC-2 causes thrombocytopenia in mice; however, its contribution to megakaryopoiesis remains unknown. Here, we show that megakaryopoiesis is promoted through the CLEC-2/PDPN interaction in the vicinity of arterioles in the bone marrow (BM). We have also identified PDPN-expressing BM arteriolar stromal cells, tentatively termed as BM fibroblastic reticular cell (FRC)-like cells. Platelet/megakaryocyte-specific CLEC-2 conditional knockout (cKO) mice showed a decrease in the number of immature megakaryocytes. CLEC-2 wild-type megakaryocyte expansion was augmented in vitro by the addition of recombinant PDPN, but not cKO megakaryocytes. Moreover, megakaryocyte colonies were colocalized with periarteriolar BM FRC-like cells in the BM. Coculture of megakaryocytes with BM FRC-like cells augmented megakaryocyte expansion, which was dependent upon the CLEC-2/PDPN interaction. Furthermore, we found that the CLEC-2/PDPN interaction induces BM FRC-like cells to secrete chemokine (C-C motif) ligand 5 (CCL5) to facilitate proplatelet formation. These observations indicate that a reciprocal interaction between CLEC-2 on megakaryocytes and PDPN on BM FRC-like cells contributes to the periarteriolar megakaryopoietic microenvironment in mouse BM.

Physiologic and pathophysiologic roles of interaction between C-type lectin-like receptor 2 and podoplanin: partners from in utero to adulthood

A platelet activation receptor, C‐type lectin‐like receptor 2 (CLEC‐2), has been identified as a receptor for a platelet‐activating snake venom, rhodocytin. CLEC‐2 protein is highly expressed in platelets/megakaryocytes, and at lower levels in liver Kupffer cells. Recently, podoplanin has been revealed as an endogenous ligand for CLEC‐2. Podoplanin is expressed in certain types of tumor cells, fibroblastic reticular cells (FRCs) in lymph nodes, kidney podocytes, and lymphatic endothelial cells, but not in vascular endothelial cells. CLEC‐2 in platelets cannot have access to podoplanin under normal conditions, but they interact with each other under pathologic conditions or during developmental stages, and play various pathophysiologic roles. CLEC‐2 facilitates hematogenous metastasis of podoplanin‐expressing tumors. During development, the interaction between CLEC‐2 and podoplanin in lymphatic endothelial cells or neuroepithelial cells facilitates blood–lymphatic vessel separation and cerebrovascular patterning and integrity, respectively. In adulthood, platelet CLEC‐2 binding to FRCs is crucial for maintenance of the integrity of high endothelial venules in lymph nodes. Podoplanin‐expressing FRC‐like cells have recently been identified in the bone marrow, and facilitate megakaryocyte proliferation and proplatelet formation by binding to megakaryocyte CLEC‐2. Podoplanin is inducibly expressed in liver monocytes and keratinocytes during Salmonella infection and wound healing, and regulates thrombus formation in the liver and controlled wound healing, respectively. By binding to unknown ligands, platelet CLEC‐2 regulates the maintenance of vascular integrity during inflammation, thrombus stability under flow, and maintenance of quiescence of hematopoietic stem cells. Podoplanin is expressed in various cells, and additional roles of the CLEC‐2–podoplanin interaction will be revealed in the future.

Vascular Smooth Muscle Cells Stimulate Platelets and Facilitate Thrombus Formation through Platelet CLEC-2: Implications in Atherothrombosis.

The platelet receptor CLEC-2 is involved in thrombosis/hemostasis, but its ligand, podoplanin, is expressed only in advanced atherosclerotic lesions. We investigated CLEC-2 ligands in vessel walls. Recombinant CLEC-2 bound to early atherosclerotic lesions and normal arterial walls, co-localizing with vascular smooth muscle cells (VSMCs). Flow cytometry and immunocytochemistry showed that recombinant CLEC-2, but not an anti-podoplanin antibody, bound to VSMCs, suggesting that CLEC-2 ligands other than podoplanin are present in VSMCs. VSMCs stimulated platelet granule release and supported thrombus formation under flow, dependent on CLEC-2. The time to occlusion in a FeCl3-induced animal thrombosis model was significantly prolonged in the absence of CLEC-2. Because the internal elastic lamina was lacerated in our FeCl3-induced model, we assume that the interaction between CLEC-2 and its ligands in VSMCs induces thrombus formation. Protein arrays and Biacore analysis were used to identify S100A13 as a CLEC-2 ligand in VSMCs. However, S100A13 is not responsible for the above-described VSMC-induced platelet activation, because S100A13 is not expressed on the surface of normal VSMCs. S100A13 was released upon oxidative stress and expressed in the luminal area of atherosclerotic lesions. Suspended S100A13 did not activate platelets, but immobilized S100A13 significantly increased thrombus formation on collagen-coated surfaces. Taken together, we proposed that VSMCs stimulate platelets through CLEC-2, possibly leading to thrombus formation after plaque erosion and stent implantation, where VSMCs are exposed to blood flow. Furthermore, we identified S100A13 as one of the ligands on VSMCs.

Independence of tumor necrosis factor-alpha-induced adhesion molecule expression from sphingosine 1-phosphate signaling in vascular endothelial cells.

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Causes of Thrombocytopenia in Chronic Hepatitis C Viral Infection

We retrospectively studied 89 patients with chronic hepatitis C virus (HCV) infection, including 50 chronic hepatitis (CH) cases, 18 liver cirrhosis (LC) cases, and 21 LC with hepatocellular carcinoma (LC + HCC) cases, with regard to various factors related with thrombocytopenia. The platelet count decreased with the stage advancement of liver diseases. Multiple regression analysis revealed that splenomegaly and von Willebrand factor (vWF) were explanatory variables that correlated with thrombocytopenia. Splenomegaly appears to be the most responsible factor, although there are a considerable number of thrombocytopenic cases without splenomegaly, suggesting other factors may also be responsible. The vWF level is inversely correlated with the platelet count. Soluble thrombomodulin, a marker of endothelial dysfunction, increases with the advancement of liver fibrosis. It is positively correlated with vWF and inversely with the platelet count. Our present results imply that vascular endothelial dysfunction is also involved in thrombocytopenia during chronic HCV infection.