Updated on 2024/03/28

写真a

 
HIRASHIMA Masanori
 
Organization
Academic Assembly Institute of Medicine and Dentistry IGAKU KEIRETU Professor
Graduate School of Medical and Dental Sciences Molecular and Cellular Medicine Signal Transduction Research Professor
Title
Professor
External link

Degree

  • 博士(医学) ( 1999.3   京都大学 )

Research Interests

  • embryonic edema

  • development

  • blood vessels

  • lymphatic vessels

  • VEGF

Research Areas

  • Life Science / Pharmacology

  • Life Science / Anatomy

Research History (researchmap)

  • Niigata University   Graduate School of Medical and Dental Sciences   Professor

    2019.1

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  • Kobe University   Graduate School of Medicine   Associate Professor

    2007.9 - 2018.12

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  • Keio University   School of Medicine   Research Assistant

    2004.3 - 2007.8

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  • サミュエル・ルネンフェルド研究所   博士研究員

    1999.11 - 2004.2

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  • Kyoto University   Graduate School of Medicine

    1999.4 - 1999.10

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Research History

  • Niigata University   Graduate School of Medical and Dental Sciences Molecular and Cellular Medicine Signal Transduction Research   Professor

    2019.1

Education

  • 京都大学大学院医学研究科博士課程分子医学系専攻

    1995.4 - 1999.3

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  • Kyoto University   Faculty of Medicine   Department of Medical Science

    1989.4 - 1995.3

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Professional Memberships

  • THE JAPANESE SOCIETY ON THROMBOSIS AND HEMOSTASIS

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  • THE JAPANESE VASCULAR BIOLOGY AND MEDICINE ORGANIZATION

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  • THE JAPANESE SOCIETY OF LYMPHOLOGY

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  • THE JAPANESE PHARMACOLOGICAL SOCIETY

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Papers

  • Roles of Thromboxane Receptor Signaling in Enhancement of Lipopolysaccharide-Induced Lymphangiogenesis and Lymphatic Drainage Function in Diaphragm. Reviewed International journal

    Hiromi Matsuda, Yoshiya Ito, Kanako Hosono, Seri Tsuru, Tomoyoshi Inoue, Shuji Nakamoto, Chie Kurashige, Masanori Hirashima, Shuh Narumiya, Hirotsugu Okamoto, Masataka Majima

    Arteriosclerosis, thrombosis, and vascular biology   41 ( 4 )   1390 - 1407   2021.4

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    [Figure: see text].

    DOI: 10.1161/ATVBAHA.120.315507

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  • MAGIs regulate aPKC to enable balanced distribution of intercellular tension for epithelial sheet homeostasis. Reviewed International journal

    Kenji Matsuzawa, Hayato Ohga, Kenta Shigetomi, Tomohiro Shiiya, Masanori Hirashima, Junichi Ikenouchi

    Communications biology   4 ( 1 )   337 - 337   2021.3

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    Constriction of the apical plasma membrane is a hallmark of epithelial cells that underlies cell shape changes in tissue morphogenesis and maintenance of tissue integrity in homeostasis. Contractile force is exerted by a cortical actomyosin network that is anchored to the plasma membrane by the apical junctional complexes (AJC). In this study, we present evidence that MAGI proteins, structural components of AJC whose function remained unclear, regulate apical constriction of epithelial cells through the Par polarity proteins. We reveal that MAGIs are required to uniformly distribute Partitioning defective-3 (Par-3) at AJC of cells throughout the epithelial monolayer. MAGIs recruit ankyrin-repeat-, SH3-domain- and proline-rich-region-containing protein 2 (ASPP2) to AJC, which modulates Par-3-aPKC to antagonize ROCK-driven contractility. By coupling the adhesion machinery to the polarity proteins to regulate cellular contractility, we propose that MAGIs play essential and central roles in maintaining steady state intercellular tension throughout the epithelial cell sheet.

    DOI: 10.1038/s42003-021-01874-z

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  • Only plantar lesion of punctate palmoplantar keratoderma with a novel missense mutation in the AAGAB gene: Two Japanese familial case reports and review of reported mutations International journal

    Hasegawa, Akito, Hayashi, Ryota, Shimomura, Yutaka, Hirashima, Masanori, Abe, Riichiro

    The Journal of Dermatology   48 ( 12 )   1926 - 1930   2021

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    Punctate palmoplantar keratoderma type 1 (PPPK1) is a rare autosomal dominant disorder characterized by hyperkeratotic papules on the palms and soles. In 2012, heterozygous loss-of-function mutations in the AAGAB gene were identified as the cause of this disorder. To date, 51 AAGAB mutations have been reported in families with PPPK1, but clear genotype-phenotype correlations have not been established yet. In this report, we identified four Japanese patients with PPPK1 from two families with an identical novel heterozygous AAGAB mutation. All patients showed hyperkeratotic papules only on the soles. Direct sequencing analysis of the AAGAB gene using peripheral blood-derived genomic DNA samples revealed that all of the patients carried a heterozygous 1-bp substitution (c.844G>A, p.Glu282Lys) in exon 9, leading to a missense change. Since all patients with the same missense mutation showed a mild phenotype limited to the soles, there is thought to be a genotype-phenotype correlation regarding this mutation. The c.844G>A mutation is a known single-nucleotide polymorphism with a minor allele frequency of 0.000012. Because of its mild symptoms, individuals with this mutation can be misdiagnosed with clavus or verruca vulgaris; this suggests that there may be a high incidence of mild symptoms of skin lesions found only on the soles in patients with PPPK1. Therefore, it is necessary to consider this disease when keratotic papules are found on the soles.

    DOI: 10.1111/1346-8138.16162

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  • Blood and lymphatic systems are segregated by the FLCN tumor suppressor. Reviewed International journal

    Ikue Tai-Nagara, Yukiko Hasumi, Dai Kusumoto, Hisashi Hasumi, Keisuke Okabe, Tomofumi Ando, Fumio Matsuzaki, Fumiko Itoh, Hideyuki Saya, Chang Liu, Wenling Li, Yoh-Suke Mukouyama, W Marston Linehan, Xinyi Liu, Masanori Hirashima, Yutaka Suzuki, Shintaro Funasaki, Yorifumi Satou, Mitsuko Furuya, Masaya Baba, Yoshiaki Kubota

    Nature communications   11 ( 1 )   6314 - 6314   2020.12

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    Blood and lymphatic vessels structurally bear a strong resemblance but never share a lumen, thus maintaining their distinct functions. Although lymphatic vessels initially arise from embryonic veins, the molecular mechanism that maintains separation of these two systems has not been elucidated. Here, we show that genetic deficiency of Folliculin, a tumor suppressor, leads to misconnection of blood and lymphatic vessels in mice and humans. Absence of Folliculin results in the appearance of lymphatic-biased venous endothelial cells caused by ectopic expression of Prox1, a master transcription factor for lymphatic specification. Mechanistically, this phenotype is ascribed to nuclear translocation of the basic helix-loop-helix transcription factor Transcription Factor E3 (TFE3), binding to a regulatory element of Prox1, thereby enhancing its venous expression. Overall, these data demonstrate that Folliculin acts as a gatekeeper that maintains separation of blood and lymphatic vessels by limiting the plasticity of committed endothelial cells.

    DOI: 10.1038/s41467-020-20156-6

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  • PROX1 Is Associated with Cancer Progression and Prognosis in Gastric Cancer. Reviewed

    Ueta K, Otowa Y, Kakeji Y, Hirashima M

    Anticancer Res   38 ( 11 )   6139 - 6145   2018.11

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    DOI: 10.21873/anticanres.12966

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  • Platelets play an essential role in murine lung development through Clec-2/podoplanin interaction. Reviewed International journal

    Nagaharu Tsukiji, Osamu Inoue, Mitsuru Morimoto, Norifumi Tatsumi, Hiroaki Nagatomo, Koji Ueta, Toshiaki Shirai, Tomoyuki Sasaki, Shimon Otake, Shogo Tamura, Toshiaki Tachibana, Masataka Okabe, Masanori Hirashima, Yukio Ozaki, Katsue Suzuki-Inoue

    Blood   132 ( 11 )   1167 - 1179   2018.9

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    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

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  • VEGFR2 promotes central endothelial activation and the spread of pain in inflammatory arthritis Reviewed

    Nicholas Beazley-Long, Catherine Elizabeth Moss, William Robert Ashby, Samuel Marcus Bestall, Fatimah Almahasneh, Alexandra Margaret Durrant, Andrew Vaughan Benest, Zoe Blackley, Kurt Ballmer-Hofer, Masanori Hirashima, Richard Phillip Hulse, David Owen Bates, Lucy Frances Donaldson

    Brain, Behavior, and Immunity   74   49 - 67   2018

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    Chronic pain can develop in response to conditions such as inflammatory arthritis. The central mechanisms underlying the development and maintenance of chronic pain in humans are not well elucidated although there is evidence for a role of microglia and astrocytes. However in pre-clinical models of pain, including models of inflammatory arthritis, there is a wealth of evidence indicating roles for pathological glial reactivity within the CNS. In the spinal dorsal horn of rats with painful inflammatory arthritis we found both a significant increase in CD11b+ microglia-like cells and GFAP+ astrocytes associated with blood vessels, and the number of activated blood vessels expressing the adhesion molecule ICAM-1, indicating potential glio-vascular activation. Using pharmacological interventions targeting VEGFR2 in arthritic rats, to inhibit endothelial cell activation, the number of dorsal horn ICAM-1+ blood vessels, CD11b+ microglia and the development of secondary mechanical allodynia, an indicator of central sensitization, were all prevented. Targeting endothelial VEGFR2 by inducible Tie2-specific VEGFR2 knock-out also prevented secondary allodynia in mice and glio-vascular activation in the dorsal horn in response to inflammatory arthritis. Inhibition of VEGFR2 in vitro significantly blocked ICAM-1-dependent monocyte adhesion to brain microvascular endothelial cells, when stimulated with inflammatory mediators TNF-α and VEGF-A165a. Taken together our findings suggest that a novel VEGFR2-mediated spinal cord glio-vascular mechanism may promote peripheral CD11b+ circulating cell transmigration into the CNS parenchyma and contribute to the development of chronic pain in inflammatory arthritis. We hypothesise that preventing this glio-vascular activation and circulating cell translocation into the spinal cord could be a new therapeutic strategy for pain caused by rheumatoid arthritis.

    DOI: 10.1016/j.bbi.2018.03.012

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  • Dynamics of angiogenesis in ischemic areas of the infarcted heart Reviewed

    Koichi Kobayashi, Kengo Maeda, Mikito Takefuji, Ryosuke Kikuchi, Yoshihiro Morishita, Masanori Hirashima, Toyoaki Murohara

    SCIENTIFIC REPORTS   7 ( 1 )   7156   2017.8

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    Cardiomyocytes are susceptible to apoptosis caused by hypoxia during the acute and subacute phases of myocardial infarction (MI). Angiogenesis can reduce MI-induced damage by mitigating hypoxia. It has been speculated that the ischemic border zone is a unique area rescued by angiogenic therapy. However, the mechanism and timing for new vessel formation in the mammalian heart following hypoxia are unclear. Identifying targets that benefit from angiogenesis treatment is indispensable for the development of revolutionary therapies. Here, we describe a novel circulatory system wherein new vessels develop from the endocardium of the left ventricle to perfuse the hypoxic area and salvage damaged cardiomyocytes at 3-14 days after MI by activating vascular endothelial growth factor signaling. Moreover, enhanced angiogenesis increased cardiomyocyte survival along the endocardium in the ischemic zone and suppressed ventricular remodeling in infarcted hearts. In contrast, cardiomyocytes in the border zone's hypoxic area underwent apoptosis within 12 h of MI, and the border area that was amenable to treatment disappeared. These data indicate that the non-perfused area along the endocardium is a site of active angiogenesis and a promising target for MI treatment.

    DOI: 10.1038/s41598-017-07524-x

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  • VEGFR2 but not VEGFR3 governs integrity and remodeling of thyroid angiofollicular unit in normal state and during goitrogenesis Reviewed

    Jeon Yeob Jang, Sung Yong Choi, Intae Park, Do Young Park, Kibaek Choe, Pilhan Kim, Young Keum Kim, Byung-Joo Lee, Masanori Hirashima, Yoshiaki Kubota, Jeong-Won Park, Sheue-Yann Cheng, Andras Nagy, Young Joo Park, Kari Alitalo, Minho Shong, Gou Young Koh

    EMBO MOLECULAR MEDICINE   9 ( 6 )   750 - 769   2017.6

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    Thyroid gland vasculature has a distinguishable characteristic of endothelial fenestrae, a critical component for proper molecular transport. However, the signaling pathway that critically governs the maintenance of thyroid vascular integrity, including endothelial fenestrae, is poorly understood. Here, we found profound and distinct expression of follicular epithelial VEGF-A and vascular VEGFR2 that were precisely regulated by circulating thyrotropin, while there were no meaningful expression of angiopoietin-Tie2 system in the thyroid gland. Our genetic depletion experiments revealed that VEGFR2, but not VEGFR3, is indispensable for maintenance of thyroid vascular integrity. Notably, blockade of VEGF-A or VEGFR2 not only abrogated vascular remodeling but also inhibited follicular hypertrophy, which led to the reduction of thyroid weights during goitrogenesis. Importantly, VEGFR2 blockade alone was sufficient to cause a reduction of endothelial fenestrae with decreases in thyrotropin-responsive genes in goitrogen-fed thyroids. Collectively, these findings establish follicular VEGF-Avascular VEGFR2 axis as a main regulator for thyrotropindependent thyroid angiofollicular remodeling and goitrogenesis.

    DOI: 10.15252/emmm.201607341

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  • Sustained inflammation after pericyte depletion induces irreversible blood-retina barrier breakdown. Reviewed International journal

    Shuntaro Ogura, Kaori Kurata, Yuki Hattori, Hiroshi Takase, Toshina Ishiguro-Oonuma, Yoonha Hwang, Soyeon Ahn, Inwon Park, Wataru Ikeda, Sentaro Kusuhara, Yoko Fukushima, Hiromi Nara, Hideto Sakai, Takashi Fujiwara, Jun Matsushita, Masatsugu Ema, Masanori Hirashima, Takashi Minami, Masabumi Shibuya, Nobuyuki Takakura, Pilhan Kim, Takaki Miyata, Yuichiro Ogura, Akiyoshi Uemura

    JCI insight   2 ( 3 )   e90905   2017.2

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    In the central nervous system, endothelial cells (ECs) and pericytes (PCs) of blood vessel walls cooperatively form a physical and chemical barrier to maintain neural homeostasis. However, in diabetic retinopathy (DR), the loss of PCs from vessel walls is assumed to cause breakdown of the blood-retina barrier (BRB) and subsequent vision-threatening vascular dysfunctions. Nonetheless, the lack of adequate DR animal models has precluded disease understanding and drug discovery. Here, by using an anti-PDGFRβ antibody, we show that transient inhibition of the PC recruitment to developing retinal vessels sustained EC-PC dissociations and BRB breakdown in adult mouse retinas, reproducing characteristic features of DR such as hyperpermeability, hypoperfusion, and neoangiogenesis. Notably, PC depletion directly induced inflammatory responses in ECs and perivascular infiltration of macrophages, whereby macrophage-derived VEGF and placental growth factor (PlGF) activated VEGFR1 in macrophages and VEGFR2 in ECs. Moreover, angiopoietin-2 (Angpt2) upregulation and Tie1 downregulation activated FOXO1 in PC-free ECs locally at the leaky aneurysms. This cycle of vessel damage was shut down by simultaneously blocking VEGF, PlGF, and Angpt2, thus restoring the BRB integrity. Together, our model provides new opportunities for identifying the sequential events triggered by PC deficiency, not only in DR, but also in various neurological disorders.

    DOI: 10.1172/jci.insight.90905

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  • PKN2 is essential for mouse embryonic development and proliferation of mouse fibroblasts Reviewed

    Sally Danno, Koji Kubouchi, Mona Mehruba, Manabu Abe, Rie Natsume, Kenji Sakimura, Satoshi Eguchi, Masahiro Oka, Masanori Hirashima, Hiroki Yasuda, Hideyuki Mukai

    GENES TO CELLS   22 ( 2 )   220 - 236   2017.2

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    PKN2, a member of the protein kinase N (PKN) family, has been suggested by in vitro culture cell experiments to bind to Rho/Rac GTPases and contributes to cell-cell contact and cell migration. To unravel the in vivo physiological function of PKN2, we targeted the PKN2 gene. Constitutive disruption of the mouse PKN2 gene resulted in growth retardation and lethality before embryonic day (E) 10.5. PKN2(-/-) embryo did not undergo axial turning and showed insufficient closure of the neural tube. Mouse embryonic fibroblasts (MEFs) derived from PKN2(-/-) embryos at E9.5 failed to grow. Cre-mediated ablation of PKN2 in PKN2(flox/flox) MEFs obtained from E14.5 embryos showed impaired cell proliferation, and cell cycle analysis of these MEFs showed a decrease in S-phase population. Our results show that PKN2 is essential for mouse embryonic development and cell-autonomous proliferation of primary MEFs in culture. Comparison of the PKN2(-/-) phenotype with the phenotypes of PKN1 and PKN3 knockout strains suggests that PKN2 has distinct nonredundant functions in vivo, despite the structural similarity and evolutionary relationship among the three isoforms.

    DOI: 10.1111/gtc.12470

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  • Semaphorin 3G Provides a Repulsive Guidance Cue to Lymphatic Endothelial Cells via Neuropilin-2/PlexinD1 Reviewed

    Xinyi Liu, Akiyoshi Uemura, Yoko Fukushima, Yutaka Yoshida, Masanori Hirashima

    CELL REPORTS   17 ( 9 )   2299 - 2311   2016.11

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    The vertebrate circulatory system is composed of closely related blood and lymphatic vessels. It has been shown that lymphatic vascular patterning is regulated by blood vessels during development, but its molecular mechanisms have not been fully elucidated. Here, we show that the artery-derived ligand semaphorin 3G (Sema3G) and the endothelial cell receptor PlexinD1 play a role in lymphatic vascular patterning. In mouse embryonic back skin, genetic inactivation of Sema3G or PlexinD1 results in abnormal artery-lymph alignment and reduced lymphatic vascular branching. Conditional ablation in mice demonstrates that PlexinD1 is primarily required in lymphatic endothelial cells (LECs). In vitro analyses show that Sema3G binds to neuropilin-2 (Nrp2), which forms a receptor complex with PlexinD1. Sema3G induces cell collapse in an Nrp2/PlexinD1-dependent manner. Our findings shed light on a molecular mechanism by which LECs are distributed away from arteries and form a branching network during lymphatic vascular development.

    DOI: 10.1016/j.celrep.2016.11.008

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  • Flt1/VEGFR1 heterozygosity causes transient embryonic edema Reviewed

    Yasunori Otowa, Kazumasa Moriwaki, Keigo Sano, Masanori Shirakabe, Shigenobu Yonemura, Masabumi Shibuya, Janet Rossant, Toshio Suda, Yoshihiro Kakeji, Masanori Hirashima

    SCIENTIFIC REPORTS   6   27186   2016.6

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    Vascular endothelial growth factor-A is a major player in vascular development and a potent vascular permeability factor under physiological and pathological conditions by binding to a decoy receptor Flt1 and its primary receptor Flk1. In this study, we show that Flt1 heterozygous (Flt1(+/-)) mouse embryos grow up to adult without life-threatening abnormalities but exhibit a transient embryonic edema around the nuchal and back regions, which is reminiscent of increased nuchal translucency in human fetuses. Vascular permeability is enhanced and an intricate infolding of the plasma membrane and huge vesicle-like structures are seen in Flt1(+/-) capillary endothelial cells. Flk1 tyrosine phosphorylation is elevated in Flt1(+/-) embryos, but Flk1 heterozygosity does not suppress embryonic edema caused by Flt1 heterozygosity. When Flt1 mutants are crossed with Aspp1(-/-) mice which exhibit a transient embryonic edema with delayed formation and dysfunction of lymphatic vessels, only 5.7% of Flt1(+/-); Aspp1(-/-) mice survive, compared to expected ratio (25%). Our results demonstrate that Flt1 heterozygosity causes a transient embryonic edema and can be a risk factor for embryonic lethality in combination with other mutations causing non-lethal vascular phenotype.

    DOI: 10.1038/srep27186

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  • An Adaptor Molecule Afadin Regulates Lymphangiogenesis by Modulating RhoA Activity in the Developing Mouse Embryo Reviewed

    Takashi Majima, Keisuke Takeuchi, Keigo Sano, Masanori Hirashima, Dimitar P. Zankov, Miki Tanaka-Okamoto, Hiroyoshi Ishizaki, Jun Miyoshi, Hisakazu Ogita

    PLoS ONE   8 ( 6 )   e68134   2013.6

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    Afadin is an intracellular binding partner of nectins, cell-cell adhesion molecules, and plays important roles in the formation of cell-cell junctions. Afadin-knockout mice show early embryonic lethality, therefore little is known about the function of afadin during organ development. In this study, we generated mice lacking afadin expression in endothelial cells, and found that the majority of these mice were embryonically lethal as a result of severe subcutaneous edema. Defects in the lymphatic vessels of the skin were observed, although the morphology in the blood vessels was almost normal. Severe disruption of VE-cadherin-mediated cell-cell junctions occurred only in lymphatic endothelial cells, but not in blood endothelial cells. Knockout of afadin did not affect the differentiation and proliferation of lymphatic endothelial cells. Using in vitro assays with blood and lymphatic microvascular endothelial cells (BMVECs and LMVECs, respectively), knockdown of afadin caused elongated cell shapes and disruption of cell-cell junctions among LMVECs, but not BMVECs. In afadin-knockdown LMVECs, enhanced F-actin bundles at the cell periphery and reduced VE-cadherin immunostaining were found, and activation of RhoA was strongly increased compared with that in afadin-knockdown BMVECs. Conversely, inhibition of RhoA activation in afadin-knockdown LMVECs restored the cell morphology. These results indicate that afadin has different effects on blood and lymphatic endothelial cells by controlling the levels of RhoA activation, which may critically regulate the lymphangiogenesis of mouse embryos. © 2013 Majima et al.

    DOI: 10.1371/journal.pone.0068134

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  • EphrinB2-EphB4 Signals Regulate Formation and Maintenance of Funnel-Shaped Valves in Corneal Lymphatic Capillaries Reviewed

    Hideto Katsuta, Yoko Fukushima, Kazuichi Maruyama, Masanori Hirashima, Kohji Nishida, Shin-Ichi Nishikawa, Akiyoshi Uemura

    INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE   54 ( 6 )   4102 - 4108   2013.6

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    PURPOSE. To elucidate the role of signals mediated by EphB4 receptor tyrosine kinase and its transmembrane ephrinB2 ligand in corneal lymphatic capillaries.
    METHODS. To detect expression of ephrinB2 and EphB4 in mouse corneas, immunohistochemistry of flat-mount corneas from 6- to 10-week-old wild-type, Efnb2-lacZ, and Ephb4-lacZ mice on a C57BL/6 background was performed. To induce formation of new blood vessels and lymphatic vessels, mouse corneal epithelia were swabbed with 0.1 M sodium hydroxide. To antagonize endogenous receptor-ligand interactions in corneal lymphatic vessels, recombinant EphB4/Fc proteins were injected into the subconjunctival spaces. To visualize the corneal lymphatic flow, FITC-dextran was injected subconjunctivally.
    RESULTS. In lymphatic capillaries of adult mouse corneas, EphB4 was intensively expressed in lymphatic endothelial cells (LECs) of funnel-shaped valves, which were segregated from ephrinB2-expressing LECs. The number of corneal lymphatic valves was significantly decreased by Efnb2 haploinsufficiency, and subconjunctival EphB4/Fc injections resulted in the deformation of preexisting valves of corneal lymphatic capillaries. In alkali-burn corneas, ephrinB2 and EphB4 were highly expressed in LECs of valve-forming areas. Subconjunctival EphB4/Fc injections perturbed the morphologic maturation of new lymphatic valves, leading to reflux of FITC-dextran to peripheral lymphatic branches.
    CONCLUSIONS. The results demonstrate a pivotal role of ephrinB2-EphB4 signals in the formation and maintenance of funnel-shaped valves in corneal lymphatic capillaries, and further suggest the potential of ephrinB2-EphB4 signals as a target to therapeutically manipulate corneal lymphangiogenesis.

    DOI: 10.1167/iovs.12-11436

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  • Enhanced Angpt1/Tie2 signaling affects the differentiation and long-term repopulation ability of hematopoietic stem cells Reviewed

    Yoshiko Matsumoto Ikushima, Fumio Arai, Yuka Nakamura, Kentaro Hosokawa, Yoshiaki Kubota, Masanori Hirashima, Hirofumi Toyama, Toshio Suda

    Biochemical and Biophysical Research Communications   430 ( 1 )   20 - 25   2013.1

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    Angiopoietin-1 (Angpt1) signaling via the Tie2 receptor regulates vascular and hematopoietic systems. To investigate the role of Angpt1-Tie2 signaling in hematopoiesis, we prepared conditionally inducible transgenic (Tg) mice expressing a genetically engineered Angpt1, cartridge oligomeric matrix protein (COMP)-Angpt1. The effects of COMP-Angpt1 overexpression in osteoblasts on hematopoiesis were then investigated by crossing COMP-Angpt1 Tg mice with Col1a1-Cre Tg mice. Interestingly, peripheral blood analyses showed that 4week (wk)-old (but not 8 wk-old) Col1a1-Cre+/COMP-Angpt1+ mice had a lower percentage of circulating B cells and a higher percentage of myeloid cells than Col1a1-Cre-/COMP-Angpt1+ (control) mice. Although there were no significant differences in the immunophenotypic hematopoietic stem and progenitor cell (HSPC) populations between Col1a1-Cre+/COMP-Angpt1+ and control mice, lineage-Sca-1+c-Kit+ (LSK) cells isolated from 8 wk-old Col1a1-Cre+/COMP-Angpt1+ mice showed better long-term bone marrow reconstitution ability. These data indicate that Angpt1-Tie2 signaling affects the differentiation capacity of hematopoietic lineages during development and increases the stem cell activity of HSCs. © 2012 Elsevier Inc.

    DOI: 10.1016/j.bbrc.2012.11.002

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  • [VEGF receptors]. Reviewed

    Sano K, Hirashima M

    Nihon rinsho. Japanese journal of clinical medicine   70 Suppl 8   31 - 35   2012.11

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  • Arhgef15 Promotes Retinal Angiogenesis by Mediating VEGF-Induced Cdc42 Activation and Potentiating RhoJ Inactivation in Endothelial Cells Reviewed

    Sentaro Kusuhara, Yoko Fukushima, Shigetomo Fukuhara, Lars Martin Jakt, Mitsuhiro Okada, Yuri Shimizu, Masayuki Hata, Kohji Nishida, Akira Negi, Masanori Hirashima, Naoki Mochizuki, Shin-Ichi Nishikawa, Akiyoshi Uemura

    PLOS ONE   7 ( 9 )   e45858   2012.9

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    Background: Drugs inhibiting vascular endothelial growth factor (VEGF) signaling are globally administered to suppress deregulated angiogenesis in a variety of eye diseases. However, anti-VEGF therapy potentially affects the normal functions of retinal neurons and glias which constitutively express VEGF receptor 2. Thus, it is desirable to identify novel drug targets which are exclusively expressed in endothelial cells (ECs). Here we attempted to identify an EC-specific Rho guanine nucleotide exchange factor (GEF) and evaluate its role in retinal angiogenesis.
    Methodology/Principal Findings: By exploiting fluorescence-activated cell sorting and microarray analyses in conjunction with in silico bioinformatics analyses, we comprehensively identified endothelial genes in angiogenic retinal vessels of postnatal mice. Of 9 RhoGEFs which were highly expressed in retinal ECs, we show that Arhgef15 acted as an EC-specific GEF to mediate VEGF-induced Cdc42 activation and potentiated RhoJ inactivation, thereby promoting actin polymerization and cell motility. Disruption of the Arhgef15 gene led to delayed extension of vascular networks and subsequent reduction of total vessel areas in postnatal mouse retinas.
    Conclusions/Significance: Our study provides information useful to the development of new means of selectively manipulating angiogenesis without affecting homeostasis in un-targeted tissues; not only in eyes but also in various disease settings such as cancer.

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

    Makoto Osada, Osamu Inoue, Guo Ding, Toshiaki Shirai, Hirotake Ichise, Kazuyoshi Hirayama, Katsuhiro Takano, Yutaka Yatomi, Masanori Hirashima, Hideki Fujii, Katsue Suzuki-Inoue, Yukio Ozaki

    JOURNAL OF BIOLOGICAL CHEMISTRY   287 ( 26 )   22241 - 22252   2012.6

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    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 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-beta 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.

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  • Flt1 and Flk1 mediate regulation of intraocular pressure and their double heterozygosity causes the buphthalmia in mice Reviewed

    Keigo Sano, Osamu Katsuta, Satoshi Shirae, Yoshiaki Kubota, Masatsugu Ema, Toshio Suda, Masatsugu Nakamura, Masanori Hirashima

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   420 ( 2 )   422 - 427   2012.4

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    Flt1 and Flk1 are receptor tyrosine kinases for vascular endothelial growth factor-A which play a crucial role in physiological and pathological angiogenesis. To study genetic interaction between the Flt1 and Flk1 genes, we crossed between Flt1 and Flk1 heterozygous (Flt1(+/-) and Flk1(+/-)) mice. We found that Flt1; Flk1 double heterozygous (Flt1(+/-); Flk1(+/-)) mice showed enlarged eyes similar to the buphthalmia detected in human congenital glaucoma with elevation of intraocular pressure (IOP). Actually, IOP was elevated in Flt1(+/-); Flk1(+/-) mice and also in Flt1 or Flk1 single heterozygous mice. However, none of these mutants showed hallmarks of glaucoma such as ganglion cell death and excavation of optic disc. To clarify the pathological causes for enlarged eyes and elevated IOP, we investigate the mice from matings between Flt1(+/-) and Flk1(+/-) mice. Flt1(+/-) mice showed enlarged Schlemm's canal and disordered collagen fibers in the sclera, whereas Flk1(+/-) mice showed atrophied choriocapillaris in the choroid. These tissues are a part of the main outflow and alternative uveoscleral outflow pathway of the aqueous humor, suggesting that these pathological changes found in Flt1(+/-) and Flk1(+/-) mice are associated with the buphthalmia in Flt1(+/-); Flk1(+/-) mice. (C) 2012 Elsevier Inc. All rights reserved.

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  • The sphingosine-1-phosphate transporter Spns2 expressed on endothelial cells regulates lymphocyte trafficking in mice Reviewed

    Shigetomo Fukuhara, Szandor Simmons, Shunsuke Kawamura, Asuka Inoue, Yasuko Orba, Takeshi Tokudome, Yuji Sunden, Yuji Arai, Kazumasa Moriwaki, Junji Ishida, Akiyoshi Uemura, Hiroshi Kiyonari, Takaya Abe, Akiyoshi Fukamizu, Masanori Hirashima, Hirofumi Sawa, Junken Aoki, Masaru Ishii, Naoki Mochizuki

    JOURNAL OF CLINICAL INVESTIGATION   122 ( 4 )   1416 - 1426   2012.4

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    The bioactive lysophospholipid mediator sphingosine-l-phosphate (SIP) promotes the egress of newly formed T cells from the thymus and the release of immature B cells from the bone marrow. It has remained unclear, however, where and how SIP is released. Here, we show that in mice, the SIP transporter spinster homolog 2 (Spns2) is responsible for the egress of mature T cells and immature B cells from the thymus and bone marrow, respectively. Global Spns2-KO mice exhibited marked accumulation of mature T cells in thymi and decreased numbers of peripheral T cells in blood and secondary lymphoid organs. Mature recirculating B cells were reduced in frequency in the bone marrow as well as in blood and secondary lymphoid organs. Bone marrow reconstitution studies revealed that Spns2 was not involved in SW release from blood cells and suggested a role for Spns2 in other cells. Consistent with these data, endothelia-specific deletion of Spns2 resulted in defects of lymphocyte egress similar to those observed in the global Spns2-KO mice. These data suggest that Spns2 functions in ECs to establish the S1P gradient required for T and B cells to egress from their respective primary lymphoid organs. Furthermore, Spns2 could be a therapeutic target for a broad array of inflammatory and autoimmune diseases.

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  • Isolation and function of mouse tissue resident vascular precursors marked by myelin protein zero Reviewed

    Yoshiaki Kubota, Keiyo Takubo, Masanori Hirashima, Narihito Nagoshi, Kazuo Kishi, Yuji Okuno, Ayako Nakamura-Ishizu, Keigo Sano, Masato Murakami, Masatsugu Ema, Yoshiki Omatsu, Satoru Takahashi, Takashi Nagasawa, Masabumi Shibuya, Hideyuki Okano, Toshio Suda

    JOURNAL OF EXPERIMENTAL MEDICINE   208 ( 5 )   949 - 960   2011.5

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    Vasculogenesis describes the process of de novo vessel formation from vascular precursor cells. Although formation of the first major vessels, such as the dorsal aorta and cardinal veins, occurs during embryonic vasculogenesis, the contribution of precursor cell populations to postnatal vessel development is not well understood. Here, we identified a novel population of postnatal vascular precursor cells in mice. These cells express the Schwann cell protein myelin protein zero (Po) and exhibit a CD45(-)CD31(-)VEcad(-)c-kit(+)CXCR4(+) surface phenotype. Po(+) vascular precursors (PVPs) are recruited into the growing vasculature, and comprise a minor population of arterial endothelial cells in adult mice. Recruitment of PVPs into growing vessels is mediated by CXCL12-CXCR4 signaling, and is enhanced during vascular expansion induced by Notch inhibition. Po-specific ablation of Flk1, a receptor for VEGF, results in branching defects and insufficient arterial patterning in the retina, as well as reduced neovascularization of tumors and ischemic tissues. Thus, in postnatal mice, although growing vessels are formed primarily by angiogenesis from preexisting vessels, a minor population of arterial endothelia may be derived from tissue-resident vascular precursor cells.

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  • Sema3E-PlexinD1 signaling selectively suppresses disoriented angiogenesis in ischemic retinopathy in mice Reviewed

    Yoko Fukushima, Mitsuhiro Okada, Hiroshi Kataoka, Masanori Hirashima, Yutaka Yoshida, Fanny Mann, Fumi Gomi, Kohji Nishida, Shin-Ichi Nishikawa, Akiyoshi Uemura

    JOURNAL OF CLINICAL INVESTIGATION   121 ( 5 )   1974 - 1985   2011.5

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    During development, the retinal vasculature grows toward hypoxic areas in an organized fashion. By contrast, in ischemic retinopathies, new blood vessels grow out of the retinal surfaces without ameliorating retinal hypoxia. Restoration of proper angiogenic directionality would be of great benefit to reoxygenize the ischemic retina and resolve disease pathogenesis. Here, we show that binding of the semaphorin 3E (Sema3E) ligand to the transmembrane PlexinD1 receptor initiates a signaling pathway that normalizes angiogenic directionality in both developing retinas and ischemic retinopathy. In developing mouse retinas, inhibition of VEGF signaling resulted in downregulation of endothelial PlexinD1 expression, suggesting that astrocyte-derived VEGF normally promotes PlexinD1 expression in growing blood vessels. Neuron-derived Sema3E signaled to PlexinD1 and activated the small GTPase RhoJ in ECs, thereby counteracting VEGF-induced filopodia projections and defining the retinal vascular pathfinding. In a mouse model of ischemic retinopathy, enhanced expression of PlexinD1 and RhoJ in extraretinal vessels prevented VEGF-induced disoriented projections of the endothelial filopodia. Remarkably, intravitreal administration of Sema3E protein selectively suppressed extraretinal vascular outgrowth without affecting the desired regeneration of the retinal vasculature. Our study suggests a new paradigm for vascular regeneration therapy that guides angiogenesis precisely toward the ischemic retina.

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  • DOCK180 Is a Rac Activator That Regulates Cardiovascular Development by Acting Downstream of CXCR4 Reviewed

    Fumiyuki Sanematsu, Masanori Hirashima, Melanie Laurin, Ryosuke Takii, Akihiko Nishikimi, Keiko Kitajima, Guo Ding, Mamiko Noda, Yuzo Murata, Yoshihiko Tanaka, Sadahiko Masuko, Toshio Suda, Chikara Meno, Jean-Francois Cote, Takashi Nagasawa, Yoshinori Fukui

    CIRCULATION RESEARCH   107 ( 9 )   1102 - 1105   2010.10

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    Rationale: During embryogenesis, the CXC chemokine ligand (CXCL) 12 acts on endothelial cells to control cardiac development and angiogenesis. Although biological functions of CXCL12 are exerted in part through activation of the small GTPase Rac, the pathway leading from its receptor CXC chemokine receptor (CXCR)4 to Rac activation remains to be determined.
    Objective: DOCK180 (dedicator of cytokinesis), an atypical Rac activator, has been implicated in various cellular functions. Here, we examined the role of DOCK180 in cardiovascular development.
    Methods and Results: DOCK180 associates with ELMO (engulfment and cell motility) through the N-terminal region containing a Src homology 3 domain. We found that targeted deletion of the Src homology 3 domain of DOCK180 in mice leads to embryonic lethality with marked reduction of DOCK180 expression at the protein level. These mutant mice, as well as DOCK180-deficient mice, exhibited multiple cardiovascular abnormalities resembling those seen in CXCR4-deficient mice. In DOCK180 knocked down endothelial cells, CXCL12-induced Rac activation was impaired, resulting in a marked reduction of cell motility.
    Conclusions: These results suggest that DOCK180 links CXCR4 signaling to Rac activation to control endothelial cell migration during cardiovascular development. (Circ Res. 2010;107:1102-1105.)

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  • Glomerular Structure and Function Require Paracrine, Not Autocrine, VEGF-VEGFR-2 Signaling Reviewed

    Karen Sison, Vera Eremina, Hans Baelde, Wang Min, Masanori Hirashima, I. George Fantus, Susan E. Quaggin

    JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY   21 ( 10 )   1691 - 1701   2010.10

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    VEGF is a potent vascular growth factor produced by podocytes in the developing and mature glomerulus. Specific deletion of VEGF from podocytes causes glomerular abnormalities including profound endothelial cell injury, suggesting that paracrine signaling is critical for maintaining the glomerular filtration barrier (GFB). However, it is not clear whether normal GFB function also requires autocrine VEGF signaling in podocytes. In this study, we sought to determine whether an autocrine VEGF-VEGFR-2 loop in podocytes contributes to the maintenance of the GFB in vivo. We found that induced, whole-body deletion of VEGFR-2 caused marked abnormalities in the kidney and also other tissues, including the heart and liver. By contrast, podocyte-specific deletion of the VEGFR-2 receptor had no effect on glomerular development or function even up to 6 months old. Unlike cell culture models, enhanced expression of VEGF by podocytes in vivo caused foot process fusion and alterations in slit diaphragm-associated proteins; however, inhibition of VEGFR-2 could not rescue this defect. Although VEGFR-2 was dispensable in the podocyte, glomerular endothelial cells depended on VEGFR-2 expression: postnatal deletion of the receptor resulted in global defects in the glomerular microvasculature. Taken together, our results provide strong evidence for dominant actions of a paracrine VEGF-VEGFR-2 signaling loop both in the developing and in the filtering glomerulus. VEGF produced by the podocyte regulates the structure and function of the adjacent endothelial cell.

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  • Essential in Vivo Roles of the C-type Lectin Receptor CLEC-2: Embryonic/neonatal lethality of CLEC-2-deficient mice by blood/lymphatic misconnections and impaired thrombus formation of CLEC-2-deficient platelets Reviewed

    Katsue Suzuki-Inoue, Osamu Inoue, Guo Ding, Satoshi Nishimura, Kazuya Hokamura, Koji Eto, Hirokazu Kashiwagi, Yoshiaki Tomiyama, Yutaka Yatomi, Kazuo Umemura, Yonchol Shin, Masanori Hirashima, Yukio Ozaki

    JOURNAL OF BIOLOGICAL CHEMISTRY   285 ( 32 )   24494 - 24507   2010.8

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    CLEC-2 has been described recently as playing crucial roles in thrombosis/hemostasis, tumor metastasis, and lymphangiogenesis. The snake venom rhodocytin is known as a strong platelet activator, and we have shown that this effect is mediated by CLEC-2 (Suzuki-Inoue, K., Fuller, G. L., Garc a, 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). Podoplanin, which is expressed on the surface of tumor cells, is an endogenous ligand for CLEC-2 and facilitates tumor metastasis by inducing platelet aggregation. Mice deficient in podoplanin, which is also expressed on the surface of lymphatic endothelial cells, show abnormal patterns of lymphatic vessel formation. In this study, we report on the generation and phenotype of CLEC-2-deficient mice. These mice are lethal at the embryonic/neonatal stages associated with disorganized and blood-filled lymphatic vessels and severe edema. Moreover, by transplantation of fetal liver cells from Clec-2(-/-) or Clec-2(+/+) embryos, we were able to demonstrate that CLEC-2 is involved in thrombus stabilization in vitro and in vivo, possibly through homophilic interactions without apparent increase in bleeding tendency. We propose that CLEC-2 could be an ideal novel target protein for an anti-platelet drug, which inhibits pathological thrombus formation but not physiological hemostasis.

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  • Identification of targets of Prox1 during in vitro vascular differentiation from embryonic stem cells: functional roles of HoxD8 in lymphangiogenesis Reviewed

    Kaori Harada, Tomoko Yamazaki, Caname Iwata, Yasuhiro Yoshimatsu, Hitoshi Sase, Koichi Mishima, Yasuyuki Morishita, Masanori Hirashima, Yuichi Oike, Toshio Suda, Naoyuki Miura, Tetsuro Watabe, Kohei Miyazono

    JOURNAL OF CELL SCIENCE   122 ( 21 )   3923 - 3930   2009.11

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    During lymphatic development, Prox1 plays central roles in the differentiation of blood vascular endothelial cells (BECs) into lymphatic endothelial cells (LECs), and subsequently in the maturation and maintenance of lymphatic vessels. However, the molecular mechanisms by which Prox1 elicits these functions remain to be elucidated. Here, we identified FoxC2 and angiopoietin-2 (Ang2), which play important roles in the maturation of lymphatic vessels, as novel targets of Prox1 in mouse embryonic-stem-cell-derived endothelial cells (MESECs). Furthermore, we found that expression of HoxD8 was significantly induced by Prox1 in MESECs, a finding confirmed in human umbilical vein endothelial cells (HUVECs) and human dermal LECs (HDLECs). In mouse embryos, HoxD8 expression was significantly higher in LECs than in BECs. In a model of inflammatory lymphangiogenesis, diameters of lymphatic vessels of the diaphragm were increased by adenovirally transduced HoxD8. We also found that HoxD8 induces Ang2 expression in HDLECs and HUVECs. Moreover, we found that HoxD8 induces Prox1 expression in HUVECs and that knockdown of HoxD8 reduces this expression in HDLECs, suggesting that Prox1 expression in LECs is maintained by HoxD8. These findings indicate that transcriptional networks of Prox1 and HoxD8 play important roles in the maturation and maintenance of lymphatic vessels.

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  • Impaired vascular development in the yolk sac and allantois in mice lacking RA-GEF-1 Reviewed

    Hoshimi Kanemura, Takaya Satoh, Shymaa E. Bilasy, Shuji Ueda, Masanori Hirashima, Tohru Kataoka

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   387 ( 4 )   754 - 759   2009.10

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    RA-GEF-1 is a guanine nucleotide exchange factor for the small GTPase Rap1. RA-GEF-1 knockout mice show defects in vascular development starting around 7.5 days post coitum and die by 9.5 days post coitum. Here, we employed in vitro culture systems for allantois explants and endothelial cells to gain insights into the mechanism for RA-GEF-1-mediated regulation of embryonic vascular network formation. The development of the vascular plexus and the accumulation of VE-cadherin at cell-cell junctions were significantly impaired in the RA-GEF-1 knockout allantois and yolk sac. Rap1 activation as visualized by an activation-specific probe was also diminished by RA-GEF-1 knockout. Reduced accumulation of VE-cadherin at cell-cell junctions and defects in blood vessel formation in vitro due to the lack of RA-GEF-1 were suppressed by ectopic expression of constitutively activated Rap1. Overall, these results suggest the involvement of Rap1 downstream of RA-GEF-1 in the regulation of vascular network formation in mouse embryos. (C) 2009 Elsevier Inc. All rights reserved.

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  • Alternatively spliced vascular endothelial growth factor receptor-2 is an essential endogenous inhibitor of lymphatic vessel growth Reviewed

    Romulo J. C. Albuquerque, Takahiko Hayashi, Won Gil Cho, Mark E. Kleinman, Sami Dridi, Atsunobu Takeda, Judit Z. Baffi, Kiyoshi Yamada, Hiroki Kaneko, Martha G. Green, Joe Chappell, Joerg Wilting, Herbert A. Weich, Satoru Yamagami, Shiro Amano, Nobuhisa Mizuki, Jonathan S. Alexander, Martha L. Peterson, Rolf A. Brekken, Masanori Hirashima, Seema Capoor, Tomohiko Usui, Balamurali K. Ambati, Jayakrishna Ambati

    NATURE MEDICINE   15 ( 9 )   1023 - U74   2009.9

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    Disruption of the precise balance of positive and negative molecular regulators of blood and lymphatic vessel growth can lead to myriad diseases. Although dozens of natural inhibitors of hemangiogenesis have been identified, an endogenous selective inhibitor of lymphatic vessel growth has not to our knowledge been previously described. We report the existence of a splice variant of the gene encoding vascular endothelial growth factor receptor-2 (Vegfr-2) that encodes a secreted form of the protein, designated soluble Vegfr-2 (sVegfr-2), that inhibits developmental and reparative lymphangiogenesis by blocking Vegf-c function. Tissue-specific loss of sVegfr-2 in mice induced, at birth, spontaneous lymphatic invasion of the normally alymphatic cornea and hyperplasia of skin lymphatics without affecting blood vasculature. Administration of sVegfr-2 inhibited lymphangiogenesis but not hemangiogenesis induced by corneal suture injury or transplantation, enhanced corneal allograft survival and suppressed lymphangioma cellular proliferation. Naturally occurring sVegfr-2 thus acts as a molecular uncoupler of blood and lymphatic vessels; modulation of sVegfr-2 might have therapeutic effects in treating lymphatic vascular malformations, transplantation rejection and, potentially, tumor lymphangiogenesis and lymphedema.

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  • Loss of Notch signalling induced by Dll4 causes arterial calibre reduction by increasing endothelial cell response to angiogenic stimuli Reviewed

    Rui Benedito, Alexandre Trindade, Masanori Hirashima, Domingos Henrique, Luis Lopes da Costa, Janet Rossant, Parkash S. Gill, Antonio Duarte

    BMC DEVELOPMENTAL BIOLOGY   8   117   2008.12

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    Background: In the vascular system, Notch receptors and ligands are expressed mainly on arteries, with Delta-like 4 (Dll4) being the only ligand known to be expressed early during the development of arterial endothelial cells and capillaries. Dll4 null embryos die very early in development with severely reduced arterial calibre and lumen and loss of arterial cell identity.
    Results: The current detailed analysis of these mutants shows that the arterial defect precedes the initiation of blood flow and that the arterial Dll4(-/-) endothelial cells proliferate and migrate more actively. Dll4(-/-) mutants reveal a defective basement membrane around the forming aorta and increased endothelial cell migration from the dorsal aorta to peripheral regions, which constitute the main causes of arterial lumen reduction in these embryos. The increased proliferation and migration of Dll4(-/-) endothelial cells was found to coincide with increased expression of the receptors VEGFR-2 and Robo4 and with downregulation of the TGF-beta accessory receptor Endoglin.
    Conclusion: Together, these results strongly suggest that Notch signalling can increase arterial stability and calibre by decreasing the response of arterial endothelial cells to local gradients of proangiogenic factors like VEGF.

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  • Leukemia inhibitory factor regulates microvessel density by modulating oxygen-dependent VEGF expression in mice Reviewed

    Yoshiaki Kubota, Masanori Hirashima, Kazuo Kishi, Colin L. Stewart, Toshio Suda

    JOURNAL OF CLINICAL INVESTIGATION   118 ( 7 )   2393 - 2403   2008.7

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    To meet tissue requirements for oxygen, capillaries must be properly distributed without excess or shortage. In this process, tissue oxygen concentration is well known to determine capillary density via the hypoxia-induced cascade, in which HIFs and VEGF play key roles. However, some additional mechanisms modulating this cascade are suggested to be involved in precise capillary network formation. Here, we showed that leukemia inhibitory factor (LIF) was predominantly expressed in developing endothelium, while its receptor was expressed in surrounding cells such as retinal astrocytes. The retinas of Lif(-/-) mice displayed increased microvessel density accompanied by sustained tip cell activity, due to increased VEGF expression by astrocytes in the vascularized area. Lif(-/-) mice resisted hyperoxygen insult in the oxygen-induced retinopathy model, whereas they paradoxically had increased numbers of neovascular tufts. In an in vitro study, LIF inhibited hypoxia-induced VEGF expression and proliferation in cultured astrocytes. Lif(-/-) mice also exhibited similarly increased microvessel density and upregulated VEGF in various tissues outside the retina. Together, these findings suggest that tissues and advancing vasculature communicate to ensure adequate vascularization using LIF as well as oxygen, which suggests a new strategy for antiangiogenic therapy in human diseases such as diabetic retinopathy and cancer.

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  • Lymphatic vessel assembly is impaired in Aspp1-deficient mouse embryos Reviewed

    Masanori Hirashima, Keigo Sano, Toru Morisada, Kyoko Murakami, Janet Rossant, Toshio Suda

    DEVELOPMENTAL BIOLOGY   316 ( 1 )   149 - 159   2008.4

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    We previously identified apoptosis stimulating protein of p53 (Aspp1) as an endothelial-specific gene functioning in mouse embryogenesis. To investigate the in vivo role of Asppl, we generated Asppl knockout mice by targeted disruption. Aspp1(-/-) embryos showed subcutaneous edema and disorganized lymphatic vasculature. Morphological changes in lymphatic endothelial cells and isolated lymphatic islands were detected in Aspp1(-/-) embryos. Lymphangiography by injecting dye subcutaneously into the embryonic forelimb showed defective lymphatic drainage function and obstruction in collecting lymphatic vessels of Aspp1(-/-) embryos. Interestingly, Aspp1(-/-) adult mice resolved these lymphatic functional defects seen during embryogenesis, but lymphangiography in Aspp1(-/-) adult mice revealed abnormal patterns in collecting lymphatic vessels. Since Aspp proteins reportedly enhance apoptotic activity of p53, we asked whether p53 deficiency also affected lymphatic vessel development. Analysis of p53 knockout or Asppl; p53 double knockout mice showed that p53 loss did not affect lymphatic vessels. These results indicate that Asppl plays a crucial role in the initial assembly and function of lymphatic vessels during mouse development in a p53-independent manner. Here we report novel lymphatic vascular phenotypes in Aspp1(-/-) mice; subcutaneous edema detected only during embryogenesis, delayed lymphatic vessel formation, and mispatterned collecting lymphatic vessels. (C) 2008 Elsevier Inc. All rights reserved.

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  • Ras signaling directs endothelial specification of VEGFR2(+) vascular progenitor cells Reviewed

    Kyoko Kawasaki, Tetsuro Watabe, Hitoshi Sase, Masanori Hirashima, Hiroshi Koide, Yasuyuki Morishita, Keiko Yuki, Toshikuni Sasaoka, Toshio Suda, Motoya Katsuki, Kohei Miyazono, Keiji Miyazawa

    JOURNAL OF CELL BIOLOGY   181 ( 1 )   131 - 141   2008.4

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    Vascular endothelial growth factor receptor 2 (VEGFR2) transmits signals of crucial importance to vasculogenesis, including proliferation, migration, and differentiation of vascular progenitor cells. Embryonic stem cell-derived VEGFR2(+) mesodermal cells differentiate into mural lineage in the presence of platelet derived growth factor (PDGF)-BB or serum but into endothelial lineage in response to VEGF-A. We found that inhibition of H-Ras function by a farnesyltransferase inhibitor or a knockdown technique results in selective suppression of VEGF-A-induced endothelial specification. Experiments with ex vivo whole-embryo culture as well as analysis of H-ras(-/-) mice also supported this conclusion. Furthermore, expression of a constitutively active H-Ras[G12V] in VEGFR2(+) progenitor cells resulted in endothelial differentiation through the extracellular signal-related kinase (Erk) pathway. Both VEGF-A and PDGF-BB activated Ras in VEGFR2(+) progenitor cells 5 min after treatment. However, VEGF-A, but not PDGF-BB, activated Ras 6-9 h after treatment, preceding the induction of endothelial markers. VEGF-A thus activates temporally distinct Ras-Erk signaling to direct endothelial specification of VEGFR2(+) vascular progenitor cells.

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  • VEGFR1 tyrosine kinase signaling promotes lymphangiogenesis as well as angiogenesis indirectly via macrophage recruitment Reviewed

    Masato Murakami, Yujuan Zheng, Masanori Hirashima, Toshio Suda, Yohei Morita, Jun Ooehara, Hideo Ema, Guo-Hua Fong, Masabumi Shibuya

    ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY   28 ( 4 )   658 - 664   2008.4

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    Objective - Angiogenesis and lymphangiogenesis are complex phenomena that involve the interplay of several growth factors and receptors. Recently, we have demonstrated that in Keratin-14 (K14) promoter-driven Vegf-A transgenic (Tg) mice, not only angiogenesis but also lymphangiogenesis is stimulated. However, the mechanism by which VEGFR1 is involved in lymphangiogenesis remains unclear.
    Methods and Results - To examine how important the tyrosine kinase (TK) of VEGFR1 is in lymphangiogenesis in K14 Vegf-A Tg mice, we crossed the K14 Vegf-A Tg mice with Vegfr1-tk- deficient mice to generate double mutant K14 Vegf-A Tg Vegfr1 tk(-/-) mice. K14 Vegf-A Tg Vegfr1 tk(-/-) mice exhibit a remarkable decrease in lymphangiogensis as well as angiogenesis in subcutaneous tissues. To address the mechanism underlying the decrease in lymphangiogensis, we investigated the recruitment of monocyte-macrophage-lineage cells into the skin. The recruitment of VEGFR1-expressing macrophages driven by VEGF-A was reduced in K14 Vegf-A Tg Vegfr1 tk(-/-) mice. Vegf-A Tg mice that received Vegfr1-tk- deficient bone marrow showed a reduction of macrophage recruitment, lymphangiogenesis and angiogenesis compared with those in K14 Vegf-A Tg mice.
    Conclusions - VEGFR1 signaling promotes lymphangiogenesis as well as angiogenesis mainly by increasing bone marrow - derived macrophage recruitment.

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  • Prox1 induces lymphatic endothelial differentiation via integrin alpha 9 and other signaling cascades Reviewed

    Koichi Mishima, Tetsuro Watabe, Akira Saito, Yasuhiro Yoshimatsu, Natsuko Imaizumi, Shinji Masui, Masanori Hirashima, Tohru Morisada, Yuichi Oike, Makoto Araie, Hitoshi Niwa, Hajime Kubo, Toshio Suda, Kohei Miyazono

    MOLECULAR BIOLOGY OF THE CELL   18 ( 4 )   1421 - 1429   2007.4

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    During embryonic lymphatic development, a homeobox transcription factor Prox1 plays important roles in sprouting and migration of a subpopulation of blood vessel endothelial cells (BECs) toward VEGF-C-expressing cells. However, effects of Prox1 on endothelial cellular behavior remain to be elucidated. Here, we show that Prox1, via induction of integrin alpha 9 expression, inhibits sheet formation and stimulates motility of endothelial cells. Prox1-expressing BECs preferentially migrated toward VEGF-C via up-regulation of the expression of integrin a9 and VEGF receptor 3 (VEGFR3). In mouse embryos, expression of VEGFR3 and integrin a9 is increased in Prox1-expressing lymphatic endothelial cells (LECs) compared with BECs. Knockdown of Prox1 expression in human LECs led to decrease in the expression of integrin a9 and VEGFR3, resulting in the decreased chemotaxes toward VEGF-C. These findings suggest that Prox1 plays important roles in conferring and maintaining the characteristics of LECs by modulating multiple signaling cascades and that integrin a9 may function as a key regulator of lymphangiogenesis acting downstream of Prox1.

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  • Dosage-sensitive requirement for mouse Dll4 in artery development Reviewed

    A Duarte, M Hirashima, R Benedito, A Trindade, P Diniz, E Bekman, L Costa, D Henrique, J Rossant

    GENES & DEVELOPMENT   18 ( 20 )   2474 - 2478   2004.10

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    Involvement of the Notch signaling pathway in vascular development has been demonstrated by both gain- and loss-of-function mutations in humans, mice, and zebrafish. In zebrafish, Notch signaling is required for arterial identity by suppressing the venous fate in developing artery cells. In mice, the Notch4 receptor and the Delta-like 4 (Dll4) ligand are specifically expressed in arterial endothelial cells, suggesting a similar role. Here we show that the Dll4 ligand alone is required in a dosage-sensitive manner for normal arterial patterning in development. This implicates Dll4 as the specific mammalian endothelial ligand for autocrine endothelial Notch signaling, and suggests that Dll4 may be a suitable target for intervention in arterial angiogenesis.

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  • Gene-trap expression screening to identify endothelial-specific genes Reviewed

    M Hirashima, A Bernstein, WL Stanford, J Rossant

    BLOOD   104 ( 3 )   711 - 718   2004.8

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    The endothelial cell is a key cellular component for blood vessel formation. Many signaling receptors expressed in endothelial cells play critical roles in vascular development during embryogenesis. However, downstream response genes required for vascular differentiation are still not clearly identified. Here we describe the development of a protocol for gene-trap expression screening in embryonic stem (ES) cells for endothelial-specific genes. ES cells were differentiated into endothelial cells on an OP9 feeder cell layer in 96-well plates. in a pilot screen, 5 gene-trapped ES cell lines showed an up-regulated expression of the gene trap lacZ reporter out of 864 ES clones screened. One of the trapped genes was endoglin, an endothelial-specific transforming growth factor-beta type III receptor, and another was ASPP1, a p53-binding protein. In vivo expression analysis of the lacZ reporter confirmed that both genes are specifically expressed in endothelial cells during early mouse embryogenesis. Gene-trap expression screening can thus be used to identify early endothelial-specific genes and analyze their function in mice.

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  • Targeted mutations of the juxtamembrane tyrosines in the Kit receptor tyrosine kinase selectively affect multiple cell lineages Reviewed

    Y Kimura, N Jones, M Kluppel, M Hirashima, K Tachibana, JB Cohn, JL Wrana, T Pawson, A Bernstein

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   101 ( 16 )   6015 - 6020   2004.4

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    Loss-of-function mutations in the murine dominant white spotting /c-kit locus affect a diverse array of biological processes and cell lineages and cause a range of phenotypes, including severe anemia, defective pigmentation, sterility, mast cell deficits, a lack of interstitial cells of Cajal, spatial learning memory deficits, and defects in peripheral nerve regeneration. Here we show that tyrosine residues 567 and 569 in the juxtamembrane (Jx) domain of the murine Kit receptor tyrosine kinase are crucial for the function of Kit in melanogenesis and mast cell development, but are dispensable for the normal development of erythroid, interstitial cells of Cajal and germ cells. Furthermore, adult mice lacking both tyrosines exhibit splenomegaly, dysregulation of B-cell and megakaryocyte development, and enlarged stomachs. Analysis of signal transduction events induced by the mutant receptors after ligand stimulation indicates that Jx tyrosine mutations diminish receptor autophosphorylation and selectively attenuate activation of extracellular signal-regulated kinase/mitogen-activated protein kinases. Together, these observations demonstrate that the Jx domain of Kit plays a cell-type specific regulatory role in vivo and illustrate how engineered mutations in Kit can be used to understand the complex biological and molecular events that result from activating a receptor tyrosine kinase.

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  • Trophoblast expression of fms-like tyrosine kinase 1 is not required for the establishment of the maternal-fetal interface in the mouse placenta Reviewed

    M Hirashima, Y Lu, L Byers, J Rossant

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   100 ( 26 )   15637 - 15642   2003.12

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    Fms-like tyrosine kinase 1 (Flt1)/vascular endothelial growth factor (VEGF) receptor 1, a receptor for VEGF-A and placental growth factor, is expressed in the spongiotrophoblast layer that segregates the maternal and fetal vasculature in the mouse placenta. A soluble form of Flt1 (sFlt1) produced in the mouse and human placenta can also be detected in the maternal blood. Levels of maternal sFlt1 are elevated in preeclampsia, suggesting that placental sFlt1 plays roles in regulating the maternal vasculature during pregnancy. However, it remains to be determined whether placental Flt1/sFlt1 serves as a regulator of VEGF-A activity in the placenta per se. Here, we investigated the placental development in Flt1-deficient mice. Flt1 is expressed in a subpopulation of ectoplacental cone cells and later marks the spongiotrophoblast cells, peri/endovascular trophoblast cells, and trophoblast glycogen cells. The labyrinth of Flt1(lacZ/lacZ) placentae lacked the fetal capillary network because of a defect in allantoic mesoderm invasion. To address whether the absence of Flt1 in the trophoblast alone affects placental development, we investigated chimeric placentae comprised of Flt1(lacZ/lacZ) trophoblast and Flt1(+/+) mesoderm, generated by tetraploid aggregation. Fetal growth was supported normally, and no defect in the formation of placental circulation into the maternal spiral artery or invasion of peri/endovascular trophoblast was detected. These findings indicate that trophoblast-derived Flt1/sFlt1 is dispensable for the initial establishment of the maternal-fetal interface in the mouse placenta. Targeting maternal sFlt1 levels for treatment of preeclampsia may thus be possible without affecting the proper formation of the placenta.

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  • A chemically defined culture of VEGFR2(+) cells derived from embryonic stem cells reveals the role of VEGFR1 in tuning the threshold for VEGF in developing endothelial cells Reviewed

    M Hirashima, M Ogawa, S Nishikawa, K Matsumura, K Kawasaki, M Shibuya, SI Nishikawa

    BLOOD   101 ( 6 )   2261 - 2267   2003.3

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    Vascular endothelial growth factor (VEGF) is a major growth factor for developing endothelial cells (ECs). Embryonic lethality due to haploinsufficiency of VEGF in the mouse highlighted the strict dose dependency of VEGF on embryonic vascular development. Here we investigated the dose-dependent effects of VEGF on the differentiation of ES cell-derived fetal liver kinase 1 (FIk-1)/VEGF receptor 2(+) (VEGFR2(+)) mesodermal cells into ECs on type IV collagen under a chemically defined serum-free condition. These cells could grow even in the absence of VEGF, but differentiated mostly into mural cells positive for alpha-smooth muscle actin. VEGF supported in a dose-dependent manner the differentiation into ECs defined by the expression of VE-cadherin, platelet-endothelial cell adhesion molecule 1 (PECAM-1)/ CD31, CD34, and TIE2/TEK. VEGF requirement was greater at late than at early phase of culture during EC development, whereas response of VEGFR2(+) cells to VEGF-E, which is a virus-derived ligand for VEGFR2 but not for Flt-1/VEGFR1, was not dose sensitive even at late phase of culture. Delayed expression of VEGFR1 correlated with increased dose dependency of VEGF. These results suggested that greater requirement of VEGF in the maintenance than induction of ECs was due to the activity of VEGFR1 sequestering VEGF from VEGFR2 signal. The chemically defined serum-free culture system described here provides a new tool for assessing different factors for the proliferation and differentiation of VEGFR2(+) mesodermal cells.

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  • Combinatorial effects of Flk1 and Tal1 on vascular and hematopoietic development in the mouse Reviewed

    M Ema, P Faloon, WL Zhang, M Hirashima, T Reid, WL Stanford, S Orkin, K Choi, J Rossant

    GENES & DEVELOPMENT   17 ( 3 )   380 - 393   2003.2

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    Mouse embryos mutant for the VEGF receptor, VEGFR2, Flk-1, or Kdr, fail to form both endothelial and hematopoietic cells, suggesting a possible role in a common progenitor to both lineages. The transcription factor Tall (Scl), is not expressed in Flk1(-/-) embryos, consistent with a downstream role in the Flk1 pathway. We tested whether expression of Tall under the Flk1 promoter was sufficient to rescue the loss of endothelial and hematopoietic cells in Flk1 mutants. Only partial rescue of hematopoiesis and endothelial development was observed in vivo. However, Flk1(-/Tal1) embryonic stem (ES) cells were capable of blast colony formation in vitro at levels equivalent to Flk1(+/-) heterozygotes. Ectopic expression of Tall under the Flk1 promoter in Flk1(+/-) mouse embryos or ES cells caused no obvious pathology but increased the number of blast colony forming cells (BL-CFCs) and enhanced their hematopoietic potential. These single-cell-derived BL-CFCs also produced smooth muscle cells in vitro. Increased Tall expression inhibited smooth muscle differentiation in this assay, whereas loss of Tall promoted smooth muscle formation. We propose a model in which the combinatorial effects of Flk1 and Tall act to regulate cell fate choice in early development into hematopoietic, endothelial, and smooth muscle lineages.

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  • Modulation of VEGFR-2-mediated endothelial-cell activity by VEGF-C/VEGFR-3 Reviewed

    K Matsumura, M Hirashima, M Ogawa, H Kubo, H Hisatsune, N Kondo, S Nishikawa, T Chiba, SI Nishikawa

    BLOOD   101 ( 4 )   1367 - 1374   2003.2

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    Vascular endothelial growth factor (VEGF) receptor 3 (VEGFR-3), a receptor for VEGF-C, was shown to be essential for angiogenesis as well as for lymphangiogenesis. Targeted disruption of the VEGFR-3 gene in mice and our previous study using an antagonistic monoclonal antibody (MoAb) for VEGFR-3 suggested that VEGF-C/VEGFR-3 signals might be involved in the maintenance of vascular integrity. In this study we used an in vitro embryonic stem (ES) cell culture system to maintain the VEGFR-3(+) endothelial cell (EC) and investigated the role of VEGFR-3 signals at the cellular level. In this system packed clusters of ECs were formed. Whereas addition of exogenous VEGF-A induced EC dispersion, VEGF-C, which can also stimulate VEGFR-2, promoted EC growth without disturbing the EC clusters. Moreover, addition of AFL4, an antagonistic MoAb for VEGFR-3, resulted in EC dispersion. Cytological analysis showed that VEGF-A- and AFL4-treated ECs were indistinguishable in many aspects but were distinct from the cytological profile induced by antagonistic MoAb for VIE-cadherin (VECD-1). As AFL4-induced EC dispersion requires VEGF-A stimulation, it is likely that VEGFR-3 signals negatively modulate VEGFR-2. This result provides new insights into the involvement of VEGFR-3 signals in the maintenance of vascular integrity through modulation of VEGFR-2 signals. Moreover, our findings suggest that the mechanisms underlying AFL4-induced EC dispersion are distinct from those underlying VECD-1-induced dispersion for maintenance of EC integrity. (C) 2003 by The American Society of Hematology.

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  • Recombinant angiopoietin-1 restores higher-order architecture of growing blood vessels in mice in the absence of mural cells Reviewed

    A Uemura, M Ogawa, M Hirashima, T Fujiwara, S Koyama, H Takagi, Y Honda, SJ Wiegand, GD Yancopoulos, SI Nishikawa

    JOURNAL OF CLINICAL INVESTIGATION   110 ( 11 )   1619 - 1628   2002.12

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    Interactions between endothelial cells (ECs) and perivascular mural cells (MCs) via signaling molecules or physical contacts are implicated both in vascular remodeling and maintenance of vascular integrity. However, it remains unclear how MCs regulate the morphogenic activity of ECs to form an organized vascular architecture, comprising distinct artery, vein, and capillary, from a simple mesh-like network. A clear elucidation of this question requires an experimental model system in which ECs are separated from MCs and yet form vascular structures. Here we report that injection of an antagonistic mAb against PDGFR-P into murine neonates provides such an experimental system in the retina by completely blocking MC recruitment to developing vessels. While a vascular network was formed even in the absence of MCs, it was poorly remodeled and leaky. Using this vascular system ideal for direct assessment of the activities of MC-derived molecules, we show that addition of recombinant modified angiopoietin-1 restored a hierarchical vasculature, and also rescued retinal edema and hemorrhage in the complete absence of MCs. These observations demonstrate the potential of Ang1 as a new therapeutic modality for MC dropout in diseases such as diabetic retinopathies.

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  • Zinc finger protein, Hzf, is required for megakaryocyte development and hemostasis Reviewed

    Y Kimura, A Hart, M Hirashima, C Wang, D Holmyard, J Pittman, XL Pang, CW Jackson, A Bernstein

    JOURNAL OF EXPERIMENTAL MEDICINE   195 ( 7 )   941 - 952   2002.4

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    Using an expression gene trapping strategy, we recently identified a novel gene, hematopoletic zinc finger (Hzf) which encodes a protein containing three C2H2-type zinc fingers that is predominantly expressed in megakaryocytes. Here, we have examined the in vivo function of Hzf by gene targeting and demonstrated that Hzf is essential for megakaryopoiesis and hemostasis in vivo. Hzf-deficient mice exhibited a pronounced tendency to rebleed and had reduced alpha-granule substances in both megakaryocytes and platelets. These in-ice also had large, faintly stained platelets, whereas the numbers of both megakaryocytes and platelets were normal. These results indicate that Hzf plays important roles in regulating the synthesis of a-granule Substances and/or their packing into alpha-granules during the process of megakaryopoiesis.

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  • Flk1-positive cells derived from embryonic stem cells serve as vascular progenitors Reviewed

    J Yamashita, H Itoh, M Hirashima, M Ogawa, S Nishikawa, T Yurugi, M Naito, K Nakao, S Nishikawa

    NATURE   408 ( 6808 )   92 - 96   2000.11

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    Interaction between endothelial cells and mural cells (pericytes and vascular smooth muscle) is essential for vascular development and maintenance(1-4). Endothelial cells arise from Flk1-expressing (Flk1(+)) mesoderm cells(5), whereas mural cells are believed to derive from mesoderm, neural crest or epicardial cells and migrate to form the vessel wall(6-8). Difficulty in preparing pure populations of these lineages has hampered dissection of the mechanisms underlying vascular formation. Here we show that Flk1(+) cells derived from embryonic stem cells can differentiate into both endothelial and mural cells and can reproduce the vascular organization process. Vascular endothelial growth factor promotes endothelial cell differentiation, whereas mural cells are induced by platelet-derived growth factor-BB. Vascular cells derived from Flk1(+) cells can organize into vessel-like structures consisting of endothelial tubes supported by mural cells in three-dimensional culture. Injection of Flk1(+) cells into chick embryos showed that they can incorporate as endothelial and mural cells and contribute to the developing vasculature in vivo. Our findings indicate that Flk1(+) cells can act as `vascular progenitor cells' to form mature vessels and thus offer potential for tissue engineering of the vascular system.

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  • Maturation of embryonic stem cells into endothelial cells in an in vitro model of vasculogenesis Reviewed

    M Hirashima, H Kataoka, S Nishikawa, N Matsuyoshi, SI Nishikawa

    BLOOD   93 ( 4 )   1253 - 1263   1999.2

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    A primitive vascular plexus is formed through coordinated regulation of differentiation, proliferation, migration, and cell-cell adhesion of endothelial cell (EC) progenitors. In this study, a culture system was devised to investigate the behavior of purified EC progenitors in vitro. Because Flk-1(+) cells derived from ES cells did not initially express other EC markers, they were sorted and used as EC progenitors. Their in vitro differentiation into ECs, via vascular endothelial-cadherin (VE-cadherin)(+) platelet-endothelial cell adhesion molecule-1 (PECAM-1)(+) CD34(-) to VE-cadherin(+) PECAM-1(+) CD34(+) stage, occurred without exogenous factors, whereas their proliferation, particularly at low cell density, required OP9 feeder cells. On OP9 feeder layer, EC progenitors gave rise to sheet-like clusters of Flk-1(+) cells, with VE-cadherin concentrated at the cell-cell junction. The growth was suppressed by Flt-1-IgG1 chimeric protein and dependent on vascular endothelial growth factor (VEGF) but not placenta growth factor (PIGF). Further addition of VEGF resulted in cell dispersion, indicating the role of VEGF in the migration of ECs as well as their proliferation. Cell-cell adhesion of ECs in this culture system was mediated by VE-cadherin. Thus, the culture system described here is useful in dissecting the cellular events of EC progenitors that occur during vasculogenesis and in investigating the molecular mechanisms underlying these processes. (C) 1999 by The American Society of Hematology.

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  • Progressive lineage analysis by cell sorting and culture identifies FLK1(+)VE-cadherin(+) cells at a diverging point of endothelial and hemopoietic lineages Reviewed

    S Nishikawa, S Nishikawa, M Hirashima, N Matsuyoshi, H Kodama

    DEVELOPMENT   125 ( 9 )   1747 - 1757   1998.5

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    Totipotent murine ES cells have an enormous potential for the study of cell specification. Here we demonstrate that ES cells can differentiate to hemopoietic cells through the proximal lateral mesoderm, merely upon culturing in type TV collagen-coated dishes. Separation of the Flk1(+) mesoderm from other cell lineages was critical for hemopoietic cell differentiation, whereas formation of the embryoid body was not. Since the two-dimensionally spreading cells can be monitored easily in real time, this culture system will greatly facilitate the study of the mechanisms involved in the cell specification to mesoderm, endothelial, and hemopoietic cells. In the culture of ES cells, however, lineages and stages of differentiating cells can only be defined by their own characteristics. We showed that a combination of monoclonal antibodies against E-cadherin, Flk1/KDR, PDGF receptor alpha, VE-cadherin, CD45 and Ter119 was sufficient to define most intermediate stages during differentiation of ES cells to blood cells. Using this culture system and surface markers,we determined the following order for blood cell differentiation: ES cell (E-cadherin(+)Flk1(-)PDGFR alpha(-)), proximal lateral mesoderm (E-cadherin(-)Flk1(+)VE-cadherin(-)), progenitor with hemoangiogenic potential (Flk1(+)VE-cadherin(+)CD45(-)), hemopoietic progenitor (CD45(+)c-Kit(+)) and mature blood cells (c-Kit(-)CD45(+) or Ter119(+)), though direct differentiation of blood cells from the Flk1(+)VE-cadherin(-) stage cannot be ruled out. Not only the VE-cadherin(+)CD45(-) population generated from ES cells but also those directly sorted from the yolk sac of 9.5 dpc embryos have a potential to give rise to hemopoietic cells. Progenitors with hemoangiogenic potential were identified in both the Flk1(+)VE-cadherin(-) and Flk1(+)VE-cadherin(+) populations by the single cell deposition experiment. This line of evidence implicates Flk1(+)VE-cadherin(+) cells as a diverging point of hemopoietic and endothelial cell lineages.

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  • Variation of hepatitis B virus precore/core gene sequence in acute and fulminant hepatitis B Reviewed

    TT Aye, T Uchida, SO Becker, M Hirashima, T Shikata, F Komine, M Moriyama, Y Arakawa, S Mima, M Mizokami, JYN Lau

    DIGESTIVE DISEASES AND SCIENCES   39 ( 6 )   1281 - 1287   1994.6

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    Variations of the hepatitis B virus (HBV) precore/core sequence has been shown to play a role in the development of active liver disease in chronic hepatitis B. Whether this is also an important viral factor in the pathogenesis of acute and fulminant hepatitis B is unknown. To determine the precore/core gene sequence in patients with acute and fulminant hepatitis B, II patients with fulminant hepatitis B and seven patients with acute hepatitis B were studied. The sequences of precore/core gene were determined by direct sequencing of the polymerase chain reaction amplicons generated from the HBV isolated from patients' serum. For the II patients with fulminant hepatitis B, the precore/core regions were successfully amplified in 10 patients. Eight patients exhibited precore stop codon mutations. In addition, nine of the 10 fulminant hepatitis B patients had frequent nucleotide substitutions with corresponding changes in the predicted amino acid sequences in the mid-core and the 5' terminus region of the core gene. In contrast, precore stop codon mutants were not detected, and variations of the HBV core gene were minimal inpatients with acute hepatitis B. The association of HBV precore mutants and HBV core gene variations with fulminant hepatitis B and not acute hepatitis B suggested that these variations may be important in modulating the clinical course of HBV infection.

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  • Detection of precore/core-mutant hepatitis B virus (HBV) genome in patients with acute or fulminant hepatitis without serological markers for recent HBV infection Reviewed

    T Uchida, TT Aye, SO Becker, M Hirashima, T Shikata, F Komine, M Moriyama, Y Arakawa, S Takase, S Mima

    JOURNAL OF HEPATOLOGY   18 ( 3 )   369 - 372   1993.7

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    To confirm the possibility that some hepatitis B virus (HBV) variants do not induce HB s antigen (HBsAg), anti-HB core antibody (anti-HBc) and anti-HBc IgM in a transient infection, polymerase chain reaction (PCR) was performed in 20 patients with acute hepatitis and 7 patients with fulminant hepatitis. Patients were diagnosed with non-A, non-B hepatitis by serological markers at admission. PCR successfully amplified the precore/core gene in 5 (25%) of the patients with acute hepatitis and 2 (29%) of the patients with fulminant hepatitis. Subsequent sequencing revealed frequent mutations including precore-defects in the precore/core gene.

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MISC

  • Regulation of endothelial cell differentiation and arterial specification by VEGF and Notch signaling Invited

    Masanori Hirashima

    ANATOMICAL SCIENCE INTERNATIONAL   84 ( 3 )   95 - 101   2009.9

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    Analysis of molecular and cellular mechanisms underlying vascular development in vertebrates indicates that initially vasculogenesis occurs when a primary capillary plexus forms de novo from endothelial cell precursors derived from nascent mesodermal cells. Transplantation experiments in avian embryos demonstrate that embryonic endothelial cells originate from two different mesodermal lineages: splanchnic mesoderm and somites. Genetic analysis of mouse and zebrafish reveals that vascular endothelial growth factor (VEGF)/Flk1 and Notch signaling play crucial roles throughout embryonic vascular development. VEGFA plays a major role in endothelial cell proliferation, migration, survival, and regulation of vascular permeability. Flk1, the primary VEGFA receptor, is the earliest marker of the developing endothelial lineage and is essential for endothelial differentiation during vasculogenesis. Notch signaling has been demonstrated to directly induce arterial endothelial differentiation. Recent studies suggest that Notch signaling is activated downstream of VEGF signaling and negatively regulates VEGF-induced angiogenesis and suppresses aberrant vascular branching morphogenesis. In addition to altering endothelial cell fate through Notch activation, VEGFA directly guides endothelial cell migration in an isoform-dependent manner, modifying vascular patterns. Interestingly, genetic studies in mice show that many molecules involved in VEGF or Notch signaling must be tightly regulated for proper vascular formation. Taken together, VEGF and Notch signaling apparently coordinate vascular patterning by regulating each other.

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  • Differentiation of arterial and venous endothelial cells and vascular morphogenesis Invited

    M Hirashima, T Suda

    ENDOTHELIUM-JOURNAL OF ENDOTHELIAL CELL RESEARCH   13 ( 2 )   137 - 145   2006.3

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    The vascular system is comprised of an organized hierarchical structure of arteries, veins, and capillaries. Recent studies in zebrafish, chick, and mouse reveal that the identity of artery and vein is governed by genetic factors as well as blood flow. The ephrin/Eph system establishes arterial and venous endothelial cell identity, and is important for structural segregation between arteries and veins. Analyses using loss- or gain-of-function mutations in zebrafish and mice show that Su(H)/RBP-J-dependent Delta/Notch signaling is a key mediator of arterial endothelial cell fate decision and vascular patterning. Vascular endothelial growth factor has also been shown to work upstream of Notch and is a key player in arteriogenesis. On the other hand, an orphan nuclear receptor, COUP-TFII, induces venous endothelial cell differentiation by suppressing the Notch signaling. Arteriovenous malformations are frequently induced by a loss of arterial and venous cell specification. These insights indicate that the balance of these genetic factors and modification by epigenetic factors such as hemodynamics and oxygen tension are important for proper endothelial cell identities in vascular morphogenesis.

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  • Vascular development and patterning: making the right choices Invited

    J Rossant, M Hirashima

    CURRENT OPINION IN GENETICS & DEVELOPMENT   13 ( 4 )   408 - 412   2003.8

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    The developing vascular system is regulated by a series of intrinsic and extrinsic signaling interactions that result in the formation and specialization of vessels that circulate blood and lymph around the body. Signaling molecules, such as vascular epithelial growth factors (VEGFs) and angiopoietins, that establish the cellular identity of the endothelial cells in the early embryo also play roles in establishing whether developing vessels will be arterial, venous or lymphatic. Genetic studies in zebrafish and mice have suggested a key role for Notch signaling, downstream of VEGF-A, in specifying arterial versus venous fate, and for VEGF-C and -D, along with the transcription factor, Prox1, in specifying lymphatic fate.

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  • Cell biology of vascular endothelial cells. Invited

    Nishikawa SI, Hirashima M, Nishikawa S, Ogawa M

    Ann N Y Acad Sci   947   35 - 40   2001.12

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  • Hematopoietic tissues, as a playground of receptor tyrosine kinases of the PDGF-receptor family Invited

    H Yoshida, N Takakura, M Hirashima, H Kataoka, K Tsuchida, S Nishikawa, S Nishikawa

    DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY   22 ( 3 )   321 - 332   1998.5

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    Three receptor tyrosine kinases of the PDGF receptor family (RTKP) that clustered within 1000 Kb of the mouse chromosome 5 constitute an interesting unit that are expressed in three distinct cell lineages essential for constructing hematopoietic tissues. Namely, the c-kit gene that is expressed in hematopoietic stem cells is flanked by pdgfr alpha and flk genes expressed respectively in stromal cells and vascular endothelial cells. In this article, we review our results on their expression in the embryonic hematopoietic tissues. We found that co-expression of Flk1 and c-Kit was frequently detected either in vascular endothelial cells or hematopoietic cells in the early hematopoietic tissues. On the other hand, the three RTKPs are expressed in different cell lineages in the fetal liver. On the basis of this finding, we propose two modes of embryonic hematopoiesis; hematogenic angiopoiesis and hematopoiesis. (C) 1998 Elsevier Science Ltd. All rights reserved.

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Awards

  • 岡本研究奨励賞(第24回)

    2009.9   成人血管病研究振興財団   内皮細胞分化と血管・リンパ管形成に関する研究

    Hirashima Masanori

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  • Best Young Investigator Award

    2006.12   第14回日本血管生物医学会学術集会  

    平島 正則

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Research Projects

  • 血小板とリンパ管内皮細胞の相互作用を標的にした新たなリンパ浮腫治療の開発

    Grant number:22K09854

    2022.4 - 2025.3

    System name:科学研究費助成事業

    Research category:基盤研究(C)

    Awarding organization:日本学術振興会

    植木 春香, 田中 宏明, 平島 正則, 松田 健, 椎谷 友博

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    Grant amount:\4030000 ( Direct Cost: \3100000 、 Indirect Cost:\930000 )

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  • リンパ管・血管分離を維持する血小板放出因子の作用機序

    Grant number:22K06788

    2022.4 - 2025.3

    System name:科学研究費助成事業

    Research category:基盤研究(C)

    Awarding organization:日本学術振興会

    椎谷 友博, 平島 正則

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    Grant amount:\4160000 ( Direct Cost: \3200000 、 Indirect Cost:\960000 )

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  • リンパ節におけるリンパ洞フィルターの分子・細胞基盤と免疫応答制御

    Grant number:21H02653

    2021.4 - 2024.3

    System name:科学研究費助成事業

    Research category:基盤研究(B)

    Awarding organization:日本学術振興会

    片貝 智哉, 奥田 修二郎, 平島 正則

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    Grant amount:\17420000 ( Direct Cost: \13400000 、 Indirect Cost:\4020000 )

    リンパ節において、リンパ管を介して到達した抗原や液性因子に対する素早い応答は、リンパ洞の独特な組織構造と濾過機能に依存し、免疫・生理学的にも極めて重要な過程であると考えられる。しかし、このリンパ洞フィルターの構造や機能の詳細については未だ不明な点が多い。本研究は、リンパ節の特定領域に局在する特殊なリンパ管内皮細胞、間質ストローマ細胞、組織マクロファージにより構成されるリンパ洞フィルターの組織・細胞構造の分子基盤と構築原理、および免疫・生理学的意義の究明を目的とする。
    初年度は、野生型マウスや各種遺伝子発現レポーターマウスを用いて、リンパ節の洞内皮細胞、ストローマ細胞、マクロファージを含む免疫細胞の各種マーカーについて、共焦点および多光子励起レーザー顕微鏡による高解像度顕微観察を行い、リンパ洞フィルターの分子細胞構成・微細構造の詳細を明らかにした。また、蛍光標識デキストラン(分子量可変)、ポリスチレンビーズ(粒径可変)、黄色ブドウ球菌、酵母菌、LPS、卵白アルブミン(OVA)をマウス皮下に投与し、浸出リンパ節のリンパ洞フィルターによる捕捉・移送の経時的観察を行うとともに、フローサイトメトリーによりマクロファージサブセットへの取り込みを定量的に評価した。さらに、クロドロン酸内包リポソーム投与によるマクロファージ除去の影響を検討した。これらの解析により、髄洞の一部が網目状のリンパ管内皮細胞ネットワークと髄質マクロファージにより特殊な物理フィルター構造を形成し、リンパ節におけるリンパ液フィルターの本体であることを明確にした。我々は、この領域を辺縁髄洞接続帯(SMB)領域と名付けた。

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  • 血小板のリンパ組織発生における役割:生理活性物質の運び手としての血小板

    Grant number:20H03709

    2020.4 - 2023.3

    System name:科学研究費助成事業

    Research category:基盤研究(B)

    Awarding organization:日本学術振興会

    井上 克枝, 築地 長治, 平島 正則, 佐々木 知幸, 白井 俊光

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    Grant amount:\8840000 ( Direct Cost: \6800000 、 Indirect Cost:\2040000 )

    私達は、肺とリンパ管の発生にはリンパ管内皮の膜蛋白ポドプラニン(PDPN)と血小板活性化受容体CLEC-2の結合により活性化された血小板から放出されるTGF-βが必須であることを報告した。CLEC-2 nullマウスでは全身のリンパ節が欠損し、肺、肝臓などにリンパ球の集簇した異所性リンパ組織(ELS)が認められた。以上より私達は「胎生期に生理活性物質を満載した血小板が、CLEC-2を介して様々な細胞のPDPNに触れ、特異的な箇所で活性物質を放出して器官発生が進行する」という全身に共通の血小板による発生制御機構が存在すると考えた。本課題ではリンパ節発生をモデルとし、血小板がいつ、どこで、どのようにしてリンパ節発生を制御するかと、血小板が運ぶ生理活性物質の実態を明らかにする。
    令和2年度までに、CLEC-2下流のシグナル分子であるSyk null マウス、PDPN null マウス、リンパ管特異的 PDPN 欠損マウスには鼠経リンパ節が欠如していることを見出し、リンパ節の形成には血小板活性化とリンパ管内皮 PDPN が必要であることが示された。しかし、令和3年度にリンパ管特異的 PDPN 欠損マウスのリンパ節をさらに調べたところ、小型のリンパ節が確認される個体も存在した。文献的には、Prox1欠損マウスなど、リンパ管が欠損したマウスでも、リンパ節は認められるが、通常よりも小型と報告されている。また、リンパ節を形成するLTi, LTo細胞でのPDPNを検討するため、免疫染色を行ったところ、CD3陽性のLTi細胞の周囲を取り囲むように、PDPN陽性のLTo様細胞が認められた。以上より、リンパ管内皮PDPNは、リンパ節原基の形成には不要だが、その成熟には必要であると考えた。リンパ節の成熟には、LTo細胞のPDPNが関与するとの仮説を立てた。

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  • ヒト胎児疾患モデルマウスの新規スクリーニング法の確立

    2017.4 - 2019.3

    System name:学術研究助成基金助成金/挑戦的研究(萌芽)

    平島 正則

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    Authorship:Principal investigator  Grant type:Competitive

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  • 生理的リンパ管・静脈吻合の形成機構と機能的意義

    2014.4 - 2017.3

    System name:学術研究助成基金助成金/基盤研究(C)

    平島 正則

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    Authorship:Principal investigator  Grant type:Competitive

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  • A-STEP「高眼圧の早期診断と創薬に向けたスクリーニング系の開発」

    2013

    System name:研究成果最適展開支援プログラム フィージビリティスタディステージ 探索タイプ

    平島 正則

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    Authorship:Principal investigator  Grant type:Competitive

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  • リンパ管腔形成と維持におけるAspp1の役割と分子機構

    2012.4 - 2014.3

    System name:科学研究費補助金/新学術領域研究

    平島 正則

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    Authorship:Principal investigator  Grant type:Competitive

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  • リンパ管内皮細胞の分子生物学的解析~プロスタノイドレセプターを巡って~

    2010

    System name:科学研究費補助金/基盤研究(C)

    野村 正

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    Grant type:Competitive

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  • 胎生期浮腫を示す内皮細胞遺伝子変異マウスの病態と分子機構

    2010

    System name:科学研究費補助金/基盤研究(C)

    平島 正則

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    Authorship:Principal investigator  Grant type:Competitive

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  • Analysis of the function of Rap1-activating factors which mediate the cross-talks between different species of small G proteins

    Grant number:20390080

    2008 - 2010

    System name:Grants-in-Aid for Scientific Research

    Research category:Grant-in-Aid for Scientific Research (B)

    Awarding organization:Japan Society for the Promotion of Science

    KATAOKA Tohru, SATOH Takaya, SHIMA FUMI, EDAMATSU Hironori, HIRASHIMA Masanori

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    Grant amount:\19630000 ( Direct Cost: \15100000 、 Indirect Cost:\4530000 )

    RA-GEF-1, RA-GEF-2, and phospholipase Cε? (PLCε) are specific activators (guanine nucleotide exchange factors) for Rap1, which mediate cross-talks between different species of small G proteins. We have elucidated crucial functions of RA-GEF-1 in fetal blood vessel formation and neuronal migration, of RA-GEF-2 in sperm formation, and of PLCε in inflammation and carcinogenesis through generation and phenotypic characterization of whole body and conditional knockout mice of the respective genes.

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  • 血管内皮細胞の動・静脈アイデンティティーを決定する細胞外環境

    2007

    System name:科学研究費補助金/特定領域研究

    平島 正則

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    Authorship:Principal investigator  Grant type:Competitive

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  • マウス胚における動・静脈形成

    2007

    System name:科学研究費補助金/若手研究(B)

    平島 正則

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    Authorship:Principal investigator  Grant type:Competitive

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Teaching Experience (researchmap)

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Teaching Experience

  • 医学論文を読む(ジャーナルクラブ)A

    2023
    Institution name:新潟大学

  • 先端医科学研究概説

    2022
    Institution name:新潟大学

  • 人体の構造と機能II(生理学)

    2021
    Institution name:新潟大学

  • 基礎薬理

    2019
    Institution name:新潟大学

  • 統合臨床医学

    2019
    -
    2020
    Institution name:新潟大学

  • 医学研究実習

    2019
    -
    2020
    Institution name:新潟大学

  • 先端医科学研究概説

    2019
    -
    2020
    Institution name:新潟大学

  • 病態薬理Ⅱ

    2019
    Institution name:新潟大学

  • 病態薬理Ⅰ

    2019
    Institution name:新潟大学

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