Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

Lymphatic vessels form a systemic network that maintains interstitial fluid homeostasis and regulates immune responses and is strictly separated from the circulatory system. During embryonic development, lymphatic endothelial cells originate from blood vascular endothelial cells in the cardinal veins and form lymph sacs. Platelets are critical for separating lymph sacs from the cardinal veins through interactions between CLEC-2 (C-type lectin-like receptor-2) and PDPN (podoplanin) in lymphatic endothelial cells. Therefore, deficiencies of these genes cause blood-filled lymphatic vessels, leading to abnormal lymphatic vessel maturation. The junction between the thoracic duct and the subclavian vein has valves and forms physiological thrombi dependent on CLEC-2/PDPN signaling to prevent blood backflow into the thoracic duct. In addition, platelets regulate lymphangiogenesis and maintain blood/lymphatic separation in pathological conditions, such as wound healing and inflammatory diseases. More recently, it was reported that the entire hemostatic system is involved in lymphangiogenesis. Thus, the hemostatic system plays a crucial role in the establishment, maintenance, and rearrangement of lymphatic networks and contributes to body fluid homeostasis, which suggests that the hemostatic system is a potential target for treating lymphatic disorders. This review comprehensively summarizes the role of the hemostatic system in lymphangiogenesis and lymphatic vessel function and discusses challenges and future perspectives.

DOI： 10.1161/ATVBAHA.123.318824

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1080/09537104.2023.2244594.

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.jtha.2023.07.005.

Language：English   Publishing type：Research paper (scientific journal)  

Bone marrow development and endochondral bone formation occur simultaneously. During endochondral ossification, periosteal vasculatures and stromal progenitors invade the primary avascular cartilaginous anlage, which induces primitive marrow development. We previously determined that bone marrow podoplanin (PDPN)-expressing stromal cells exist in the perivascular microenvironment and promote megakaryopoiesis and erythropoiesis. In this study, we aimed to examine the involvement of PDPN-expressing stromal cells in postnatal bone marrow generation. Using histological analysis, we observed that periosteum-derived PDPN-expressing stromal cells infiltrated the cartilaginous anlage of the postnatal epiphysis and populated on the primitive vasculature of secondary ossification center. Furthermore, immunophenotyping and cellular characteristic analyses indicated that the PDPN-expressing stromal cells constituted a subpopulation of the skeletal stem cell lineage. In vitro xenovascular model cocultured with human umbilical vein endothelial cells and PDPN-expressing skeletal stem cell progenies showed that PDPN-expressing stromal cells maintained vascular integrity via the release of angiogenic factors and vascular basement membrane-related extracellular matrices. We show that in this process, Notch signal activation committed the PDPN-expressing stromal cells into a dominant state with basement membrane-related extracellular matrices, especially type IV collagens. Our findings suggest that the PDPN-expressing stromal cells regulate the integrity of the primitive vasculatures in the epiphyseal nascent marrow. To the best of our knowledge, this is the first study to comprehensively examine how PDPN-expressing stromal cells contribute to marrow development and homeostasis.

DOI： 10.1016/j.jbc.2022.101833

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

BACKGROUND: Erythropoiesis is a complex multistep process by which erythrocytes are produced. C-type lectin-like receptor 2 (CLEC-2) is a podoplanin (PDPN) receptor almost exclusively expressed on the surface of platelets and megakaryocytes. Deletion of megakaryocyte/platelet CLEC-2 was reported to cause anemia along with thrombocytopenia in mice. PDPN-expressing stromal cells in the bone marrow (BM) were also reported to facilitate megakaryocyte expansion and maturation depending on the CLEC-2/PDPN interaction. OBJECTIVES: We investigated how specific deletion of CLEC-2 in megakaryocytes/platelets leads to anemia. METHODS: We used flow-cytometry to analyze maturation of erythroblasts, apoptotic cell death, and cell cycle distribution. CLEC-2 stimulated PDPN-expressing stromal cell conditioned medium was analyzed by cytokine array and ELISA, and co-cultured with immature erythroblasts. Cytokine levels in serum and BM extracellular fluid were quantified by ELISA. RESULTS: We observed increased apoptosis of BM erythroblasts in megakaryocyte/platelet-specific CLEC-2 conditional knockout (Clec1bΔPLT ) mice. Moreover, PDPN-expressing stromal cells in the BM secreted insulin-like growth factor 1 (IGF-1) depending on the CLEC-2/PDPN interaction. Pretreatment with IGF-1 receptor inhibitor increased apoptosis rate and decreased the proliferation of erythroblasts in vitro. Furthermore, in Clec1bΔPLT mice, IGF-1 concentrations in serum and BM extracellular fluid were decreased, and IGF-1 replacement in Clec1bΔPLT mice attenuated anemia. CONCLUSIONS: Our findings suggest that IGF-1 secretion from PDPN-expressing stromal cells by CLEC-2 stimulation positively regulates erythroblasts. This novel mechanism of erythropoiesis regulation indicates that a microenvironment consisting of megakaryocytes and PDPN-expressing stromal cells supports erythropoiesis.

DOI： 10.1111/jth.15317

PubMed

Authorship：Lead author,　Corresponding author   Language：Japanese   Publishing type：(MISC) Summary of the papers read (national conference and other science council)   Publisher：(一社)日本血栓止血学会  

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Society of Hematology  

<title>Abstract</title>
There is increasing evidence that platelets participate in multiple pathophysiological processes other than thrombosis and hemostasis, such as immunity, inflammation, embryonic development, and cancer progression. A recent study revealed that heme (hemin)-activated platelets induce macrophage extracellular traps (METs) and exacerbate rhabdomyolysis-induced acute kidney injury (RAKI); however, how hemin activates platelets remains unclear. Here, we report that both C-type lectin-like receptor-2 (CLEC-2) and glycoprotein VI (GPVI) are platelet hemin receptors and are involved in the exacerbation of RAKI. We investigated hemin-induced platelet aggregation in humans and mice, binding of hemin to CLEC-2 and GPVI, the RAKI-associated phenotype in a mouse model, and in vitro MET formation. Using western blotting and surface plasmon resonance, we showed that hemin activates human platelets by stimulating the phosphorylation of SYK and PLCγ2 and directly binding to both CLEC-2 and GPVI. Furthermore, hemin-induced murine platelet aggregation was partially reduced in CLEC-2–depleted and FcRγ-deficient (equivalent to GPVI-deficient) platelets and almost completely inhibited in CLEC-2–depleted FcRγ-deficient (double-knockout) platelets. In addition, hemin-induced murine platelet aggregation was inhibited by the CLEC-2 inhibitor cobalt hematoporphyrin or GPVI antibody (JAQ-1). Renal dysfunction, tubular injury, and MET formation were attenuated in double-knockout RAKI mice. Furthermore, in vitro MET formation assay showed that the downstream signaling pathway of CLEC-2 and GPVI is involved in MET formation. We propose that both CLEC-2 and GPVI in platelets play an important role in RAKI development.

DOI： 10.1182/bloodadvances.2020001698

PubMed

Language：Japanese   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

DOI： 10.1002/jbmr.4045

PubMed

Authorship：Lead author,　Corresponding author   Language：Japanese   Publishing type：(MISC) Summary of the papers read (national conference and other science council)   Publisher：(一社)日本血栓止血学会  

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley Online Library  

DOI： 10.1002/rth2.12338

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

DOI： 10.1111/jth.14726

PubMed

Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：一般社団法人 日本血液学会  

DOI： 10.11406/rinketsu.60.1283

PubMed

Authorship：Lead author,　Last author,　Corresponding author   Language：Japanese   Publishing type：(MISC) Introduction and explanation (scientific journal)  

Authorship：Lead author,　Corresponding author   Language：Japanese   Publishing type：(MISC) Introduction and explanation (scientific journal)  

Authorship：Lead author   Language：Japanese   Publishing type：(MISC) Introduction and explanation (scientific journal)   Publisher：日本リンパ学会  

Authorship：Lead author,　Corresponding author   Language：Japanese   Publishing type：(MISC) Introduction and explanation (scientific journal)   Publisher：日本リンパ学会  

Authorship：Lead author   Language：Japanese   Publishing type：(MISC) Introduction and explanation (commerce magazine)   Publisher：科学評論社  

Authorship：Lead author,　Corresponding author   Language：Japanese   Publishing type：(MISC) Introduction and explanation (commerce magazine)   Publisher：科学評論社  

Language：English   Publishing type：Research paper (scientific journal)   Publisher：The Japanese Society of Hematology  

Soluble forms of platelet membrane proteins are released upon platelet activation. We previously reported that soluble C-type lectin-like receptor 2 (sCLEC-2) is released as a shed fragment (Shed CLEC-2) or as a whole molecule associated with platelet microparticles (MP-CLEC-2). In contrast, soluble glycoprotein VI (sGPVI) is released as a shed fragment (Shed GPVI), but not as a microparticle-associated form (MP-GPVI). However, mechanism of sCLEC-2 generation or plasma sCLEC-2 has not been fully elucidated. Experiments using metalloproteinase inhibitors/stimulators revealed that ADAM10/17 induce GPVI shedding, but not CLEC-2 shedding, and that shed CLEC-2 was partially generated by MMP-2. Although MP-GPVI was not generated, it was generated in the presence of the ADAM10 inhibitor. Moreover, antibodies against the cytoplasmic or extracellular domain of GPVI revealed the presence of the GPVI cytoplasmic domain, but not the extracellular domain, in the microparticles. These findings suggest that most of the GPVI on microparticles are induced to shed by ADAM10; MP-GPVI is thus undetected. Plasma sCLEC-2 level was 1/32 of plasma sGPVI level in normal subjects, but both soluble proteins significantly increased in plasma of patients with acute coronary syndrome. Thus, sCLEC-2 and sGPVI are released by different mechanisms and released in vivo upon platelet activation.

DOI： 10.1007/s12185-019-02680-4

PubMed

Language：Japanese   Publishing type：(MISC) Introduction and explanation (scientific journal)  

Authorship：Lead author,　Corresponding author   Language：Japanese   Publishing type：(MISC) Introduction and explanation (scientific journal)   Publisher：日本検査血液学会  

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Society of Hematology  

Platelets participate in not only thrombosis and hemostasis but also other pathophysiological processes, including tumor metastasis and inflammation. However, the putative role of platelets in the development of solid organs has not yet been described. Here, we report that platelets regulate lung development through the interaction between the platelet-activation receptor, C-type lectin-like receptor-2 (Clec-2; encoded by Clec1b), and its ligand, podoplanin, a membrane protein. Clec-2 deletion in mouse platelets led to lung malformation, which caused respiratory failure and neonatal lethality. In these embryos, α-smooth muscle actin-positive alveolar duct myofibroblasts (adMYFs) were almost absent in the primary alveolar septa, which resulted in loss of alveolar elastic fibers and lung malformation. Our data suggest that the lack of adMYFs is caused by abnormal differentiation of lung mesothelial cells (luMCs), the major progenitor of adMYFs. In the developing lung, podoplanin expression is detected in alveolar epithelial cells (AECs), luMCs, and lymphatic endothelial cells (LECs). LEC-specific podoplanin knockout mice showed neonatal lethality and Clec1b-/--like lung developmental abnormalities. Notably, these Clec1b-/--like lung abnormalities were also observed after thrombocytopenia or transforming growth factor-β depletion in fetuses. We propose that the interaction between Clec-2 on platelets and podoplanin on LECs stimulates adMYF differentiation of luMCs through transforming growth factor-β signaling, thus regulating normal lung development.

DOI： 10.1182/blood-2017-12-823369

PubMed

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1182/bloodadvances.2018016261.

Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

The platelet activation receptor C-type lectin-like receptor 2 (CLEC-2) interacts with podoplanin on the surface of certain types of tumor cells, and this interaction facilitates tumor metastasis. CLEC-2 is also involved in thrombus formation and its stabilization. Because CLEC-2-depleted mice are protected from experimental lung metastasis and thrombus formation and do not show increased bleeding time, CLEC-2 may serve as a good target for antimetastatic or antithrombotic drugs. We screened 6770 compounds for their capability to inhibit CLEC-2-podoplanin binding using an enzyme-linked immunosorbent assay. In the first screening round, 63 compounds were identified and further evaluated by flow cytometry using CLEC-2-expressing cells. We identified protoporphyrin IX (H2-PP) as the most potent inhibitor and modified its hematoporphyrin moiety to be complexed with cobalt (cobalt hematoporphyrin [Co-HP]), which resulted in an inhibitory potency much stronger than that of H2-PP. Surface plasmon resonance analysis and molecular docking study showed that Co-HP binds directly to CLEC-2 at N120, N210, and K211, previously unknown podoplanin-binding sites; this binding was confirmed by analysis of CLEC-2 mutants with alterations in N120 and/or K211. Co-HP at a concentration of 1.53 μM inhibited platelet aggregation mediated through CLEC-2, but not that mediated through other receptors. IV administration of Co-HP to mice significantly inhibited hematogenous metastasis of podoplanin-expressing B16F10 cells to the lung as well as in vivo arterial and venous thrombosis, without a significant increase in tail-bleeding time. Thus, Co-HP may be a promising molecule for antimetastatic and antiplatelet treatment that does not cause bleeding tendency.

DOI： 10.1182/bloodadvances.2018016261

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：International Society on Thrombosis Haemostasis  

Essentials We generated recombinant rhodocytin that could aggregate platelets via CLEC-2. Recombinant wild-type rhodocytin formed heterooctamer with four α- and β-subunits. Asp 4 in α-subunit of rhodocytin was required for binding to CLEC-2. Inhibitory mutant of rhodocytin blocked podoplanin-dependent hematogenous metastasis. Summary: Background Rhodocytin, a disulfide-linked heterodimeric C-type lectin from Calloselasma rhodostoma consisting of α-subunits and β-subunits, induces platelet aggregation through C-type lectin-like receptor 2 (CLEC-2). CLEC-2 is a physiological binding partner of podoplanin (PDPN), which is expressed on some tumor cell types, and is involved in tumor cell-induced platelet aggregation and tumor metastasis. Thus, modified rhodocytin may be a possible source of anti-CLEC-2 drugs for both antiplatelet and antimetastasis therapy. However, its molecular function has not been well characterized, because of the lack of recombinant rhodocytin that induces platelet aggregation. Objective To produce recombinant rhodocytin, in order to verify its function with mutagenesis, and to develop an anti-CLEC-2 drug based on the findings. Methods We used Chinese hamster ovary cells to express recombinant rhodocytin (wild-type [WT] and mutant), which was analyzed for induction/inhibition of platelet aggregation with light transmission aggregometry, the formation of multimers with blue native PAGE, and binding to CLEC-2 with flow cytometry. Finally, we investigated whether mutant rhodocytin could suppress PDPN-induced metastasis in an experimental lung metastasis mouse model. Results Functional WT] rhodocytin (αWTβWT) was obtained by coexpression of both subunits. Asp4 in α-subunits of rhodocytin was required for CLEC-2 binding. αWTβWT formed a heterooctamer similarly to native rhodocytin. Moreover, an inhibitory mutant of rhodocytin (αWTβK53A/R56A), forming a heterotetramer, bound to CLEC-2 without inducing platelet aggregation, and blocked CLEC-2–PDPN interaction-dependent platelet aggregation and experimental lung metastasis. Conclusion These findings provide molecular characterization information on rhodocytin, and suggest that mutant rhodocytin could be used as a therapeutic agent to target CLEC-2.

DOI： 10.1111/jth.13987

Scopus

Language：English   Publishing type：(MISC) Introduction and explanation (scientific journal)  

Authorship：Lead author,　Corresponding author   Language：English   Publishing type：(MISC) Introduction and explanation (scientific journal)  

C-type lectin-like receptor 2 (CLEC-2) has been identified on the surface of platelets as a receptor for a platelet activating snake venom, rhodocytin/aggretin. CLEC-2 belongs to a C-type lectin superfamily and binds to a sialoglycoprotein, podoplanin, in vivo. Platelets play a crucial role in hemostasis and thrombosis, but recent studies have uncovered multiple roles of platelets beyond hemostasis in physiology and pathology. The interaction between platelet CLEC-2 and podoplanin is the key to several roles of platelets beyond hemostasis. The spatial and temporal expression patterns of podoplanin regulate vascular/lymphatic development, maintenance of vascular integrity, tissue regeneration, and some pathological processes including tumor metastasis and thromboinflammation. CLEC-2 facilitates blood/lymphatic vessel separation during embryonic development by binding to podoplanin on lymphatic endothelial cells. The leakage of platelets from hyperpermeable vessels for maintaining vascular integrity during inflammation depends on CLEC-2. During wound healing, the expression of podoplanin in keratinocytes is upregulated, which helps in the process. Podoplanin is expressed on the surface of tumor cells and facilitates hematogenous metastasis by inducing platelet aggregation through CLEC-2. During thrombotic processes, such as development of deep vein thrombosis, podoplanin is upregulated on unknown cells in the vessel wall in the area of inflammation, facilitates thrombus formation, and promotes further inflammation by binding to CLEC-2. In this article, the roles of platelets beyond hemostasis are comprehensively reviewed.

DOI： 10.1055/s-0037-1604090

PubMed

Authorship：Lead author   Language：Japanese   Publishing type：(MISC) Introduction and explanation (scientific journal)  

Authorship：Lead author,　Corresponding author   Language：Japanese   Publishing type：(MISC) Introduction and explanation (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY  

Background and aim: The aim of the present study was to investigate the role of C-type lectin receptor (CLEC)-2 in liver regeneration following partial liver resection in mice. Materials and methods: Irradiated chimeric mice transplanted with fetal liver cells from wildtype (WT) mice, CLEC-2-deleted (KO) mice or mice with CLEC-2 deleted specifically from platelets (flKO) were generated. Mice underwent 70% partial hepatectomy (PH). Immunohistochemical staining was performed to investigate the expression of the endogenous ligand for CLEC-2, podoplanin. The accumulation of platelets in the liver was also quantified. The hepatic expression of the IL-6/gp130 and STAT3, Akt and ERK1/2 was also examined. Results: The liver/body weight ratio and expression of all cell proliferation markers were significantly lower in the flKO group than in the WT group. The expression of phosphorylated (p) Akt and pERK1/2 was similar in the WT and flKO groups. On the other hand, the expression of pSTAT3 and IL-6 was significantly stronger in the WT group than in the flKO group. The expression of podoplanin was detected in the hepatic sinusoids of both groups. However, the extent to which platelets accumulated in hepatic sinusoids was significantly less in the flKO group than in the WT group. Conclusion: CLEC-2 was involved in hepatic regeneration after liver resection and CLEC-2-related liver regeneration was attributed to the interaction between platelets and sinusoidal endothelial cells.

DOI： 10.1111/jth.13672

Web of Science

Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY  

Background C-type lectin-like receptor 2 (CLEC-2) is a platelet activation receptor of sialoglycoprotein podoplanin, which is expressed on the surface of certain types of tumor cells. CLEC-2-podoplanin interactions facilitate hematogenous tumor metastasis. However, direct evidence of the role of CLEC-2 in hematogenous metastasis and cancer progression is lacking.
Objective and methods We generated immunological CLEC-2-depleted mice by using anti-mouse CLEC-2 monoclonal antibody 2A2B10 and investigated whether CLEC-2 promoted hematogenous tumor metastasis and tumor growth and exacerbated the prognosis of mice bearing podoplanin-expressing B16F10 melanoma cells.
Results Our results showed that hematogenous metastasis was significantly inhibited in CLEC-2-depleted mice. B16F10 cells co-cultured with wild-type platelets, but not with CLEC-2-deficient platelets, showed increased proliferation. However, B16F10 cell proliferation was not inhibited in CLEC-2-depleted mice. Histological analysis showed that thrombus formation in tumor vessels was significantly inhibited and functional vessel density was significantly increased in CLEC-2-depleted mice. These data suggest that CLEC-2 deficiency may inhibit thrombus formation in tumor vessels and increase the density of functional vessels, thus improving oxygen and nutrient supply to tumors, indirectly promoting tumor proliferation. Furthermore, the overall survival of CLEC-2-depleted mice was significantly prolonged, which may be due to the suppression of thrombus formation in the lungs and subsequent inhibition of systemic inflammation and cachexia.
Conclusions These data provide a rationale for the targeted inhibition of CLEC-2 as a new strategy for preventing hematogenous tumor metastasis and for inhibiting cancer-related thromboembolism.

DOI： 10.1111/jth.13604

Web of Science

Authorship：Lead author   Language：Japanese   Publishing type：(MISC) Introduction and explanation (scientific journal)  

Authorship：Lead author,　Corresponding author   Language：Japanese   Publishing type：(MISC) Introduction and explanation (scientific journal)  

Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Society of Hematology  

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. Coculturc of megakaryocytes with BM FRC-like cells augmt nted 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 mcgakaryopoiotic microenvironment in mouse BM.

DOI： 10.1182/blood-2015-08-663708

Scopus

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

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.

DOI： 10.1371/journal.pone.0139357

Web of Science

Language：English   Publishing type：Research paper (scientific journal)  

Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS INC  

Detection of platelet activation in vivo is useful to identify patients at risk of thrombotic diseases. Platelet factor 4 (PF4) and beta-thromboglobulin (beta-TG) are used for this purpose; however, they are easily released upon the minimal platelet activation that occurs during sampling. Soluble forms of several platelet membrane proteins are released upon platelet activation; however, the soluble form of C-type lectin-like receptor 2 (sCLEC-2) has not yet been fully investigated. Western blotting with an anti-CLEC-2 antibody showed that sCLEC-2 was released from washed human platelets stimulated with collagen mimetics. To detect sCLEC-2 in plasma, we established a sandwich enzyme-linked immunosorbent assay (ELISA) using F(ab')2 anti-CLEC-2 monoclonal antibodies. Although plasma mixed with citrate, adenosine, theophylline and adenosine (CTAD) is needed for the PF4 and beta-TG assays, effects of anti-coagulants (EDTA, citrate and CTAD) on the sCLEC-2 ELISA were negligible. Moreover, while special techniques are required for blood sampling and sample preparation for PF4 and beta-TG assay, the standard blood collections procedures used in daily clinical laboratory tests have shown to suffice for sCLEC-2 analysis. In this study, we found that two forms of sCLEC-2 are released after platelet activation: a shed fragment and a microparticle-bound full-length protein, both of which are detected by the sCLEC-2 ELISA. The average concentration of sCLEC-2 in the plasma of 10 healthy individuals was 97 +/- 55 pg/ml, whereas that in the plasma of 25 patients with diabetes mellitus (DM) was 149 +/- 260 pg/ml. A trend towards an increase in sCLEC-2 concentration in the DM patients may reflect in vivo platelet activation in the patients, suggesting that sCLEC-2 may have clinical significance as a biomarker of in vivo platelet activation.

DOI： 10.3109/09537104.2015.1021319

Web of Science

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Hedgehog (Hh) signaling plays pivotal roles in morphogenesis of several embryonic tissues, including the primitive gut. In the mouse embryo, Sonic hedgehog (Shh) is expressed in endodermal epithelia from the oral cavity to the intestine, and contributes to cell proliferation in the underlying mesenchyme and subsequent differentiation into the gastrointestinal smooth muscle. Three evolutionary conserved non-coding sequences in the region upstream of the Shh coding sequence contain endoderm-specific enhancers for Shh expression. Although Shh expression in the endodermal epithelial lining is mostly attributed to these three enhancers, none of them regulates gene expression in the gastroesophageal epithelium. Here, we found that a 1.7 Kb fragment located 100 Kb upstream of the Shh coding sequence contains a functional element for Shh expression in endodermal organs, including the esophagus and stomach. Compared with the three known endodermal enhancers, this novel enhancer shows less evolutionary conservation, even among rodents. In mouse embryonic endodermal tissues, the seamless expression of Shh is achieved by a patchwork of multiple enhancers with different rates of evolution. (C) 2014 Published by Elsevier Ireland Ltd.

DOI： 10.1016/j.mod.2013.09.003

Web of Science

Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

The retinal pigment epithelium (RPE) shares its developmental origin with the neural retina (NR). When RPE development is disrupted, cells in the presumptive RPE region abnormally differentiate into NR-like cells. Therefore, the prevention of NR differentiation in the presumptive RPE area seems to be essential for regionalizing the RPE during eye development. However, its molecular mechanisms are not fully understood. In this study, we conducted a functional inhibition of a transcription factor Otx2, which is required for RPE development, using early chick embryos. The functional inhibition of Otx2 in chick eyes, using a recombinant gene encoding a dominant negative form of Otx2, caused the outer layer of the optic cup ( the region forming the RPE, when embryos normally develop) to abnormally form an ectopic NR. In that ectopic NR, the characteristics of the RPE did not appear and NR markers were ectopically expressed. Intriguingly, the repression of Otx2 function also caused the ectopic expression of Fgf8 and Sox2 in the outer layer of the optic cup ( the presumptive RPE region of normally developing eyes). These two factors are known to be capable of inducing NR cell differentiation in the presumptive RPE region, and are not expressed in the normally developing RPE region. Here, we suggest that Otx2 prevents the presumptive RPE region from forming the NR by repressing the expression of both Fgf8 and Sox2 which induce the NR cell fate. Citation: Nishihara D, Yajima I, Tabata H, Nakai M, Tsukiji N, et al. (2012) Otx2 Is Involved in the Regional Specification of the Developing Retinal Pigment Epithelium by Preventing the Expression of Sox2 and Fgf8, Factors That Induce Neural Retina Differentiation. PLoS ONE 7(11): e48879. doi:10.1371/journal.pone.0048879

DOI： 10.1371/journal.pone.0048879

Web of Science

Authorship：Lead author,　Corresponding author   Language：Japanese   Publishing type：(MISC) Summary of the papers read (national conference and other science council)  

J-GLOBAL

Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.ydbio.2008.11.029

Authorship：Lead author,　Corresponding author   Language：Japanese   Publishing type：(MISC) Summary of the papers read (national conference and other science council)  

J-GLOBAL

Authorship：Lead author,　Corresponding author   Language：English   Publishing type：(MISC) Summary of the papers read (international conference)   Publisher：BLACKWELL PUBLISHING  

Web of Science

Authorship：Lead author,　Corresponding author   Language：Japanese   Publishing type：(MISC) Summary of the papers read (national conference and other science council)  

J-GLOBAL

Authorship：Lead author,　Corresponding author   Language：English   Publishing type：(MISC) Summary of the papers read (national conference and other science council)   Publisher：ZOOLOGICAL SOC JAPAN  

Web of Science

Authorship：Lead author,　Corresponding author   Language：Japanese   Publishing type：(MISC) Summary of the papers read (national conference and other science council)  

J-GLOBAL

Event date： 2022.7

Language：English   Presentation type：Poster presentation  

Venue：London  

Language：English   Presentation type：Poster presentation  

Venue：London  

Event date： 2022.6

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：仙台国際センター　+Web  

Event date： 2022.6

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：仙台国際センター　+Web  

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：仙台国際センター +Web  

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：仙台国際センター +Web  

Event date： 2021.11

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：WEB開催  

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：WEB開催  

Event date： 2021.9

Language：Japanese   Presentation type：Oral presentation(general)  

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：Web開催  

Event date： 2021.5

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：オンライン開催  

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：オンライン開催  

Event date： 2020.7

Language：English   Presentation type：Oral presentation(general)  

Venue：Virtual  

Language：English   Presentation type：Oral presentation(general)  

Venue：Virtual  

Event date： 2020.6

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：大阪（web開催）  

Event date： 2020.6

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：大阪（web開催）  

Event date： 2020.6

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：大阪（web開催）　  

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：大阪（web開催）  

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：大阪（web開催）  

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：大阪（web開催）  

Event date： 2019.7

Language：English   Presentation type：Oral presentation(general)  

OC 79.2

Language：English   Presentation type：Oral presentation(general)  

Venue：オーストラリア・メルボルン  

OC 79.2

Event date： 2019.7

Language：English   Presentation type：Poster presentation  

PB0988

Language：English   Presentation type：Poster presentation  

Venue：オーストラリア・メルボルン  

PB0988

Event date： 2019.7

Language：Japanese   Presentation type：Public discourse, seminar, tutorial, course, lecture and others  

Language：Japanese   Presentation type：Public discourse, seminar, tutorial, course, lecture and others  

Venue：東京都品川区  

Event date： 2019.6

Language：Japanese   Presentation type：Poster presentation  

Venue：三重県津市  

P-053

Language：Japanese   Presentation type：Poster presentation  

Venue：三重県津市  

P-053

Event date： 2019.6

Language：Japanese   Presentation type：Oral presentation(invited, special)  

Venue：三重県津市  

学術奨励賞受賞講演「基礎」

Event date： 2019.6

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：三重県津市  

O-017

Language：Japanese   Presentation type：Oral presentation(invited, special)  

Venue：三重県津市  

学術奨励賞受賞講演「基礎」

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：三重県津市  

O-017

Event date： 2019.4

Language：Japanese   Presentation type：Symposium workshop panel(nominated)  

Venue：山梨県小淵沢  

Language：Japanese   Presentation type：Symposium workshop panel(nominated)  

Venue：山梨県小淵沢  

Event date： 2019.3

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：山梨県甲府市  

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：山梨県甲府市  

Event date： 2018.10

Language：English   Presentation type：Oral presentation(general)  

Language：English   Presentation type：Oral presentation(general)  

Venue：大阪・大阪国際会議場  

Event date： 2018.9

Language：Japanese   Presentation type：Poster presentation  

Venue：京都・国立京都国際会館  

Language：Japanese   Presentation type：Poster presentation  

Venue：京都・国立京都国際会館  

Event date： 2018.6

Language：English   Presentation type：Symposium workshop panel(nominated)  

Event date： 2018.6

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：北海道・札幌  

Event date： 2018.6

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：北海道・札幌  

Event date： 2018.6

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：北海道・札幌  

Language：English   Presentation type：Symposium workshop panel(nominated)  

Venue：北海道・札幌  

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：北海道・札幌  

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：北海道・札幌  

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：北海道・札幌  

Event date： 2018.6

Language：Japanese   Presentation type：Oral presentation(invited, special)  

Venue：青森県弘前市  

Language：Japanese   Presentation type：Oral presentation(invited, special)  

Venue：青森県弘前市  

Event date： 2017.7

Language：English   Presentation type：Oral presentation(general)  

Venue：Berlin, Germany  

Event date： 2017.7

Language：English   Presentation type：Poster presentation  

Venue：Berlin, Germany  

Event date： 2017.7

Language：English   Presentation type：Poster presentation  

Venue：Berlin, Germany  

Language：English   Presentation type：Oral presentation(general)  

Venue：Berlin, Germany  

Language：English   Presentation type：Poster presentation  

Venue：Berlin, Germany  

Language：English   Presentation type：Poster presentation  

Venue：Berlin, Germany  

Event date： 2017.6

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：名古屋国際会議場  

Event date： 2017.6

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：名古屋国際会議場  

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：名古屋国際会議場  

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：名古屋国際会議場  

Event date： 2016.6

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：奈良  

Event date： 2016.6

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：奈良  

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：奈良  

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：奈良  

Event date： 2016.6

Language：Japanese   Presentation type：Other  

Venue：長浜バイオ大学  

Language：Japanese   Presentation type：Other  

Venue：長浜バイオ大学  

Event date： 2016.6

Language：English   Presentation type：Poster presentation  

Language：English   Presentation type：Poster presentation  

Venue：東京大学  

Event date： 2015.6

Language：English   Presentation type：Oral presentation(general)  

Language：English   Presentation type：Oral presentation(general)  

Venue：トロント、カナダ  

Event date： 2015.6

Language：English   Presentation type：Oral presentation(general)  

Event date： 2015.6

Language：English   Presentation type：Oral presentation(general)  

Language：English   Presentation type：Oral presentation(general)  

Venue：第25回国際血栓止血学会（カナダトロント、2015.6.20-25）  

Language：English   Presentation type：Oral presentation(general)  

Venue：トロント、カナダ  

Event date： 2015.6

Language：English   Presentation type：Oral presentation(general)  

Event date： 2015.6

Language：English   Presentation type：Oral presentation(general)  

Language：English   Presentation type：Oral presentation(general)  

Venue：トロント、カナダ  

Language：English   Presentation type：Oral presentation(general)  

Venue：トロント、カナダ  

Event date： 2015.6

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：名古屋  

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：名古屋  

Event date： 2015.6

Language：English   Presentation type：Poster presentation  

Language：English   Presentation type：Poster presentation  

Venue：第48回日本発生生物学会（茨城県つくば市、2014.6.2-5）  

Event date： 2015.5

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：山梨・甲府  

Event date： 2015.5

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：山梨・甲府  

Event date： 2015.5

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：山梨・甲府  

Event date： 2015.5

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：山梨・甲府  

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：山梨・甲府  

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：山梨・甲府  

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：山梨・甲府  

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：山梨・甲府  

Event date： 2015.5

Language：English   Presentation type：Oral presentation(general)  

Language：English   Presentation type：Oral presentation(general)  

Venue：山梨・甲府  

Event date： 2015.5

Language：English   Presentation type：Poster presentation  

Language：English   Presentation type：Poster presentation  

Venue：第９回Aso International Meeting（熊本県南阿蘇郡、2015.5.14-16）  

Event date： 2015.4

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：日本呼吸器学会細胞・分子生物学学術部会サテライトミーティング（東京都千代田区、2015.4.16）  

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：日本呼吸器学会細胞・分子生物学学術部会サテライトミーティング（東京都千代田区、2015.4.16）  

Event date： 2015.1

Language：English   Presentation type：Poster presentation  

Language：English   Presentation type：Poster presentation  

Venue：TGF-βファミリーシグナル国際共同研究拠点 第４回国際シンポジウム（茨城県つくば市、2015.1.12, 13）  

Event date： 2014.5

Language：Japanese   Presentation type：Oral presentation(general)  

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：第47回日本発生生物学会（愛知県名古屋市、2014.5.27-30）  

Award type：Award from Japanese society, conference, symposium, etc. 

Award type：Award from Japanese society, conference, symposium, etc. 

Award type：Award from Japanese society, conference, symposium, etc.  Country：Japan

全てのポスター発表の中から、特に優秀な5つの演題が選出され、表彰されました。

Award type：Award from Japanese society, conference, symposium, etc.  Country：Japan

全てのポスター発表の中から、特に優秀な5つの演題が選出され、表彰されました。

学会口頭発表
タイトル：UNEXPECTED ROLE OF PLATELETS IN LUNG DEVELOPMENT DEPENDING ON A PLATELET ACTIVATION RECEPTOR, CLEC-2