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1.
Blood ; 125(24): 3769-77, 2015 Jun 11.
Article in English | MEDLINE | ID: mdl-25908104

ABSTRACT

Mice with a constitutive or platelet-specific deletion of the C-type-lectin-like receptor (CLEC-2) exhibit hemorrhaging in the brain at mid-gestation. We sought to investigate the basis of this defect, hypothesizing that it is mediated by the loss of CLEC-2 activation by its endogenous ligand, podoplanin, which is expressed on the developing neural tube. To induce deletion of podoplanin at the 2-cell stage, we generated a podoplanin(fl/fl) mouse crossed to a PGK-Cre mouse. Using 3-dimensional light-sheet microscopy, we observed cerebral vessels were tortuous and aberrantly patterned at embryonic (E) day 10.5 in podoplanin- and CLEC-2-deficient mice, preceding the formation of large hemorrhages throughout the fore-, mid-, and hindbrain by E11.5. Immunofluorescence and electron microscopy revealed defective pericyte recruitment and misconnections between the endothelium of developing blood vessels and surrounding pericytes and neuro-epithelial cells. Nestin-Cre-driven deletion of podoplanin on neural progenitors also caused widespread cerebral hemorrhaging. Hemorrhaging was also seen in the ventricles of embryos deficient in the platelet integrin subunit glycoprotein IIb or in embryos in which platelet α-granule and dense granule secretion is abolished. We propose a novel role for podoplanin on the neuro-epithelium, which interacts with CLEC-2 on platelets, mediating platelet adhesion, aggregation, and secretion to guide the maturation and integrity of the developing vasculature and prevent hemorrhage.


Subject(s)
Brain/blood supply , Brain/embryology , Cerebrovascular Circulation , Lectins, C-Type/genetics , Membrane Glycoproteins/genetics , Animals , Blood Platelets/metabolism , Body Patterning , Brain/metabolism , Gene Deletion , Gene Expression Regulation, Developmental , Intracranial Hemorrhages/genetics , Intracranial Hemorrhages/metabolism , Lectins, C-Type/metabolism , Membrane Glycoproteins/metabolism , Mice, Inbred C57BL , Platelet Activation , Platelet Aggregation , Platelet Membrane Glycoprotein IIb/genetics , Platelet Membrane Glycoprotein IIb/metabolism
2.
Blood ; 125(1): 144-54, 2015 Jan 01.
Article in English | MEDLINE | ID: mdl-25352128

ABSTRACT

We have used a novel knockin mouse to investigate the effect of disruption of phosphotyrosine binding of the N-terminal SH2 domain of Syk on platelet activation by GPVI, CLEC-2, and integrin αIIbß3. The Syk(R41Afl/fl) mouse was crossed to a PF4-Cre(+) mouse to induce expression of the Syk mutant in the megakaryocyte/platelet lineage. Syk(R41Afl/fl;PF4-Cre) mice are born at approximately 50% of the expected frequency and have a similar phenotype to Syk(fl/fl;PF4-Cre) mice, including blood-lymphatic mixing and chyloascites. Anastomosis of the venous and lymphatic vasculatures can be seen in the mesenteric circulation accounting for rapid and continuous mixing of the 2 vasculatures. Platelet activation by CLEC-2 and GPVI is abolished in Syk(R41Afl/fl;PF4-Cre) platelets. Syk phosphorylation on Tyr519/20 is blocked in CLEC-2-stimulated platelets, suggesting a model in which binding of Syk via its N-terminal SH2 domain regulates autophosphorylation. In contrast, outside-in signaling by integrin αIIbß3 is not altered, but it is inhibited in the presence of inhibitors of Src and Syk tyrosine kinases. These results demonstrate that αIIbß3 regulates Syk through an ITAM-independent pathway in mice and provide novel insight into the course of events underlying Syk activation and hemITAM phosphorylation by CLEC-2.


Subject(s)
Blood Platelets/metabolism , Intracellular Signaling Peptides and Proteins/genetics , Lectins, C-Type/metabolism , Phosphoproteins/metabolism , Platelet Glycoprotein GPIIb-IIIa Complex/metabolism , Platelet Membrane Glycoproteins/metabolism , Protein-Tyrosine Kinases/genetics , Amino Acid Motifs , Amino Acid Sequence , Animals , Intracellular Signaling Peptides and Proteins/metabolism , Mice , Mice, Transgenic , Phosphorylation , Phosphotyrosine/metabolism , Platelet Activation , Platelet Aggregation , Protein Structure, Tertiary , Protein-Tyrosine Kinases/metabolism , Purpura, Thrombocytopenic, Idiopathic/metabolism , Signal Transduction/drug effects , Syk Kinase , src Homology Domains
3.
Blood ; 123(20): 3200-7, 2014 May 15.
Article in English | MEDLINE | ID: mdl-24532804

ABSTRACT

The importance of CLEC-2, a natural ligand/receptor for Gp38/Podoplanin, in the formation of the lymphatic vasculature has recently been demonstrated. As the development and maintenance of lymph nodes (LNs) is dependent on the formation of the lymphatic vasculature and the differentiation of Gp38/Podoplanin(+) stromal cells, we investigated the role of CLEC-2 in lymphoneogenesis and LN homeostasis. Using constitutive Clec1b(-/-) mice, we showed that while CLEC-2 was not necessary for initiation of the LN anlage, it was required at late stages of development. Constitutive deletion of CLEC-2 induced a profound defect in lymphatic endothelial cell proliferation, resulting in lack of LNs at birth. In contrast, conditional deletion of CLEC-2 in the megakaryocyte/platelet lineage in Clec1b(fl/fl)PF4-Cre mice led to the development of blood-filled LNs and fibrosis, in absence of a proliferative defect of the lymphatic endothelial compartment. This phenotype was also observed in chimeric mice reconstituted with Clec1b(fl/fl)PF4-Cre bone marrow, indicating that CLEC-2 expression in platelets was required for LN integrity. We demonstrated that LNs of Clec1b(fl/fl)PF4-Cre mice are able to sustain primary immune responses but show a defect in immune cell recirculation after repeated immunizations, thus suggesting CLEC-2 as target in chronic immune response.


Subject(s)
Lectins, C-Type/genetics , Lectins, C-Type/metabolism , Lymph Nodes/growth & development , Animals , Blood Platelets/metabolism , Cell Proliferation , Cells, Cultured , Endothelium, Lymphatic/cytology , Endothelium, Lymphatic/metabolism , Gene Deletion , Lymph Nodes/cytology , Lymph Nodes/metabolism , Lymphangiogenesis , Megakaryocytes/metabolism , Mice , Mice, Inbred C57BL
4.
Adv Anat Embryol Cell Biol ; 214: 93-105, 2014.
Article in English | MEDLINE | ID: mdl-24276889

ABSTRACT

Blood platelets have recently been proposed to play a critical role in the development and repair of the lymphatic system. The platelet C-type lectin receptor CLEC-2 and its ligand, the transmembrane protein Podoplanin, which is expressed at high levels on lymphatic endothelial cells (LECs), are required to prevent mixing of the blood and lymphatic vasculatures during mid-gestation. A similar defect is seen in mice deficient in the tyrosine kinase Syk, which plays a vital role in mediating platelet activation by CLEC-2. Furthermore, blood-lymphatic mixing is also present in mice with platelet-/megakaryocyte-specific deletions of CLEC-2 and Syk, suggesting that the phenotype is platelet in origin. The molecular basis of this effect is not known, but it is independent of the major platelet receptors that support hemostasis, including integrin αIIbß3 (GPIIb-IIIa). Radiation chimeric mice reconstituted with CLEC-2-deficient or Syk-deficient bone marrow exhibit blood-lymphatic mixing in the intestines, illustrating a role for platelets in repair and growth of the lymphatic system. In this review, we describe the events that led to the identification of this novel role of platelets and discuss possible molecular mechanisms and the physiological and pathophysiological significance.


Subject(s)
Blood Platelets/physiology , Lymphangiogenesis , Lymphatic Vessels/embryology , Animals , Humans , Lectins, C-Type/metabolism , Lymphatic Vessels/physiology , Membrane Glycoproteins/metabolism
5.
PLoS One ; 8(11): e80294, 2013.
Article in English | MEDLINE | ID: mdl-24282533

ABSTRACT

Airway branching morphogenesis in utero is essential for optimal postnatal lung function. In the fetus, branching morphogenesis occurs during the pseudoglandular stage (weeks 9-17 of human gestation, embryonic days (E)11.5-16.5 in mouse) in a hypercalcaemic environment (~1.7 in the fetus vs. ~1.1-1.3 mM for an adult). Previously we have shown that fetal hypercalcemia exerts an inhibitory brake on branching morphogenesis via the calcium-sensing receptor. In addition, earlier studies have shown that nifedipine, a selective blocker of L-type voltage-gated Ca(2+) channels (VGCC), inhibits fetal lung growth, suggesting a role for VGCC in lung development. The aim of this work was to investigate the expression of VGCC in the pseudoglandular human and mouse lung, and their role in branching morphogenesis. Expression of L-type (CaV1.2 and CaV1.3), P/Q type (CaV2.1), N-type (CaV2.2), R-type (CaV2.3), and T-type (CaV3.2 and CaV3.3) VGCC was investigated in paraffin sections from week 9 human fetal lungs and E12.5 mouse embryos. Here we show, for the first time, that Cav1.2 and Cav1.3 are expressed in both the smooth muscle and epithelium of the developing human and mouse lung. Additionally, Cav2.3 was expressed in the lung epithelium of both species. Incubating E12.5 mouse lung rudiments in the presence of nifedipine doubled the amount of branching, an effect which was partly mimicked by the Cav2.3 inhibitor, SNX-482. Direct measurements of changes in epithelial cell membrane potential, using the voltage-sensitive fluorescent dye DiSBAC2(3), demonstrated that cyclic depolarisations occur within the developing epithelium and coincide with rhythmic occlusions of the lumen, driven by the naturally occurring airway peristalsis. We conclude that VGCC are expressed and functional in the fetal human and mouse lung, where they play a role in branching morphogenesis. Furthermore, rhythmic epithelial depolarisations evoked by airway peristalsis would allow for branching to match growth and distension within the developing lung.


Subject(s)
Calcium Channels/physiology , Calcium/blood , Lung/embryology , Morphogenesis , Animals , Epithelium/metabolism , Humans , Mice , Muscle, Smooth/metabolism
6.
Arterioscler Thromb Vasc Biol ; 33(5): 926-34, 2013 May.
Article in English | MEDLINE | ID: mdl-23448972

ABSTRACT

OBJECTIVE: Platelet inhibition is a major strategy to prevent acute ischemic cardiovascular and cerebrovascular events, which may, however, be associated with an increased bleeding risk. The (hem)immunoreceptor tyrosine activation motif-bearing platelet receptors, glycoprotein VI (GPVI) and C-type lectin-like receptor 2 (CLEC-2), might be promising antithrombotic targets because they can be depleted from circulating platelets by antibody treatment, leading to sustained antithrombotic protection, but only moderately increased bleeding times in mice. APPROACH AND RESULTS: We investigated whether both (hem)immunoreceptor tyrosine activation motif-bearing receptors can be targeted simultaneously and what the in vivo consequences of such a combined therapeutic GPVI/CLEC-2 deficiency are. We demonstrate that isolated targeting of either GPVI or CLEC-2 in vivo does not affect expression or function of the respective other receptor. Moreover, simultaneous treatment with both antibodies resulted in the sustained loss of both GPVI and CLEC-2, while leaving other activation pathways intact. However, GPVI/CLEC-2-depleted mice displayed a dramatic hemostatic defect and profound impairment of arterial thrombus formation. Furthermore, a strongly diminished hemostatic response could also be reproduced in mice genetically lacking GPVI and CLEC-2. CONCLUSIONS: These results demonstrate that GPVI and CLEC-2 can be simultaneously downregulated in platelets in vivo and reveal an unexpected functional redundancy of the 2 receptors in hemostasis and thrombosis. These findings may have important implications of the potential use of anti-GPVI and anti-CLEC-2-based agents in the prevention of thrombotic diseases.


Subject(s)
Hemostasis , Lectins, C-Type/physiology , Platelet Membrane Glycoproteins/physiology , Thrombosis/prevention & control , Animals , Lectins, C-Type/antagonists & inhibitors , Male , Mice , Mice, Inbred C57BL , Platelet Membrane Glycoproteins/antagonists & inhibitors
7.
Blood ; 119(7): 1747-56, 2012 Feb 16.
Article in English | MEDLINE | ID: mdl-22186994

ABSTRACT

The C-type lectin receptor CLEC-2 signals through a pathway that is critically dependent on the tyrosine kinase Syk. We show that homozygous loss of either protein results in defects in brain vascular and lymphatic development, lung inflation, and perinatal lethality. Furthermore, we find that conditional deletion of Syk in the hematopoietic lineage, or conditional deletion of CLEC-2 or Syk in the megakaryocyte/platelet lineage, also causes defects in brain vascular and lymphatic development, although the mice are viable. In contrast, conditional deletion of Syk in other hematopoietic lineages had no effect on viability or brain vasculature and lymphatic development. We show that platelets, but not platelet releasate, modulate the migration and intercellular adhesion of lymphatic endothelial cells through a pathway that depends on CLEC-2 and Syk. These studies found that megakaryocyte/platelet expression of CLEC-2 and Syk is required for normal brain vasculature and lymphatic development and that platelet CLEC-2 and Syk directly modulate lymphatic endothelial cell behavior in vitro.


Subject(s)
Blood Platelets/metabolism , Cell Lineage/genetics , Growth and Development/genetics , Intracellular Signaling Peptides and Proteins/physiology , Lectins, C-Type/physiology , Megakaryocytes/metabolism , Protein-Tyrosine Kinases/physiology , Animals , Animals, Newborn , Blood Platelets/physiology , Cell Differentiation/genetics , Cell Differentiation/physiology , Cell Lineage/physiology , Cells, Cultured , Embryo, Mammalian , Female , Gene Expression Regulation, Developmental , Growth and Development/immunology , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Lectins, C-Type/genetics , Lectins, C-Type/metabolism , Megakaryocytes/physiology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Pregnancy , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/metabolism , Syk Kinase , Thrombopoiesis/genetics , Thrombopoiesis/physiology
9.
Pflugers Arch ; 458(6): 1007-22, 2009 Oct.
Article in English | MEDLINE | ID: mdl-19484257

ABSTRACT

The capacity to sense and adapt to changes in environmental cues is of paramount importance for every living organism. From yeast to man, cells must be able to match cellular activities to growth environment and nutrient availability. Key to this process is the development of membrane-bound systems that can detect modifications in the extracellular environment and to translate these into biological responses. Evidence gathered over the last 15 years has demonstrated that many of these cell surface "sensors" belong to the G protein-coupled receptor superfamily. Crucial to our understanding of nutrient sensing in mammalian species has been the identification of the extracellular Ca(2+)/cation-sensing receptor, CaR. CaR was the first ion-sensing molecule identified in man and genetic studies in humans have revealed the importance of the CaR in mineral ion metabolism. Latter, it has become apparent that the CaR also plays an important role outside the Ca(2+) homeostatic system, as an integrator of multiple environmental signals for the regulation of many vital cellular processes, from cell-to-cell communication to secretion and cell survival/cell death. Recently, novel aspects of receptor function reveal an unexpected role for the CaR in the regulation of growth and development in utero.


Subject(s)
Receptors, Calcium-Sensing/genetics , Animals , Bone and Bones/metabolism , Calcium Signaling/physiology , Cardiovascular System/metabolism , Gastrointestinal Tract/metabolism , Gene Expression Regulation , Gene Expression Regulation, Developmental , Homeostasis/physiology , Humans , Kidney/physiology , Kidney Tubules/metabolism , Lung/embryology , Neoplasms/physiopathology , Nervous System/embryology , Skin/metabolism
10.
J Physiol ; 586(24): 6007-19, 2008 Dec 15.
Article in English | MEDLINE | ID: mdl-18955379

ABSTRACT

Postnatal lung function is critically dependent upon optimal embryonic lung development. As the free ionized plasma calcium concentration ([Ca(2+)](o)) of the fetus is higher than that of the adult, the process of lung development occurs in a hypercalcaemic environment. In the adult, [Ca(2+)](o) is monitored by the G-protein coupled, extracellular calcium-sensing receptor (CaR), but neither its ontogeny nor its potential role in lung development are known. Here, we demonstrate that CaR is expressed in the mouse lung epithelium, and that its expression is developmentally regulated, with a peak of expression at embryonic day 12.5 (E12.5) and a subsequent decrease by E18, after which the receptor is absent. Experiments carried out using the lung explant culture model in vitro show that lung branching morphogenesis is sensitive to [Ca(2+)](o), being maximal at physiological adult [Ca(2+)](o) (i.e. 1.0-1.3 mM) and lowest at the higher, fetal (i.e. 1.7 mM) [Ca(2+)](o). Administration of the specific CaR positive allosteric modulator, the calcimimetic R-568, mimics the suppressive effects of high [Ca(2+)](o) on branching morphogenesis while both phospholipase C and PI3 kinase inhibition reverse these effects. CaR activation suppresses cell proliferation while it enhances intracellular calcium signalling, lung distension and fluid secretion. Conditions which are restrictive either to branching or to secretion can be rescued by manipulating [Ca(2+)](o) in the culture medium. In conclusion, fetal Ca(2+)(o), acting through a developmentally regulated CaR, is an important extrinsic factor that modulates the intrinsic lung developmental programme. Our observations support a novel role for the CaR in preventing hyperplastic lung disease in utero.


Subject(s)
Calcium/metabolism , Embryo, Mammalian/embryology , Lung/embryology , Receptors, Calcium-Sensing/physiology , Aniline Compounds/pharmacology , Animals , Animals, Newborn , Calcium/pharmacology , Calcium Signaling/drug effects , Calcium Signaling/physiology , Carbachol/pharmacology , Cell Proliferation/drug effects , Chromones/pharmacology , Embryo, Mammalian/metabolism , Enzyme Inhibitors/pharmacology , Estrenes/pharmacology , Female , Gene Expression Regulation, Developmental/drug effects , Lung/cytology , Lung/metabolism , Male , Mesoderm/embryology , Mesoderm/metabolism , Mice , Mice, Inbred C57BL , Morphogenesis/drug effects , Morpholines/pharmacology , Phenethylamines , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors , Pregnancy , Propylamines , Pyrrolidinones/pharmacology , Receptors, Calcium-Sensing/genetics , Reverse Transcriptase Polymerase Chain Reaction , Tissue Culture Techniques , Type C Phospholipases/antagonists & inhibitors , Type C Phospholipases/metabolism
11.
Neuron Glia Biol ; 3(3): 233-44, 2007 Aug.
Article in English | MEDLINE | ID: mdl-18634614

ABSTRACT

Expression of the human epidermal growth factor receptor (EGFR) in murine Schwann cells results in loss of axon-Schwann cell interactions and collagen deposition, modeling peripheral nerve response to injury and tumorigenesis. Mast cells infiltrate nerves in all three situations. We show that mast cells are present in normal mouse peripheral nerve beginning at 4 weeks of age, and that the number of mast-cells in EGFR(+) nerves increases abruptly at 5-6 weeks of age as axons and Schwann cells dissociate. The increase in mast cell number is preceded and accompanied by elevated levels of mRNAs encoding the mast-cell chemoattractants Rantes, SCF and VEGF. Genetic ablation of mast cells and bone marrow reconstitution in W(41) x EGFR(+) mice indicate a role for mast cells in loss of axon-Schwann cell interactions and collagen deposition. Pharmacological stabilization of mast cells by disodium cromoglycate administration to EGFR(+) mice also diminished loss of axon-Schwann cell interaction. Together these three lines of evidence support the hypothesis that mast cells can contribute to alterations in peripheral nerves.

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