Biomedical subjects

Masanori Hirashima

Publications and source records attributed to Masanori Hirashima.

11 recordsLinked to original sources

PubMed2004

Dosage-sensitive requirement for mouse Dll4 in artery development.

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.

Animals↗

PubMed2004

Gene-trap expression screening to identify endothelial-specific genes.

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.

Animals↗

PubMed2004

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

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.

Animals↗

PubMed2003

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

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 Flt1lacZ/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 Flt1lacZ/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.

Animals↗

PubMed2003

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

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 Tal1 (Scl), is not expressed in Flk1(-/-) embryos, consistent with a downstream role in the Flk1 pathway. We tested whether expression of Tal1 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 Tal1 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 Tal1 expression inhibited smooth muscle differentiation in this assay, whereas loss of Tal1 promoted smooth muscle formation. We propose a model in which the combinatorial effects of Flk1 and Tal1 act to regulate cell fate choice in early development into hematopoietic, endothelial, and smooth muscle lineages.

Animals↗

PubMed2003

Vascular development and patterning: making the right choices.

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.

Angiopoietins↗

PubMed2002

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.

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 (Flk-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.

Animals↗

PubMed2002

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

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 VE-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.

Animals↗

PubMed2002

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

Using an expression gene trapping strategy, we recently identified a novel gene, hematopoietic zinc finger (Hzf), which encodes a protein containing three C(2)H(2)-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 mice 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 alpha-granule substances and/or their packing into alpha-granules during the process of megakaryopoiesis.

Animals↗

PubMed2002

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

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-beta 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.

Angiogenesis Inducing Agents↗

PubMedDate not supplied

Differentiation of arterial and venous endothelial cells and vascular morphogenesis.

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.

Animals↗