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1.
J Immunol ; 198(8): 3318-3325, 2017 04 15.
Article in English | MEDLINE | ID: mdl-28258201

ABSTRACT

IL-33, required for viral clearance by cytotoxic T cells, is generally expressed in vascular endothelial cells in healthy human tissues. We discovered that endothelial IL-33 expression was stimulated as a response to adenoviral transduction. This response was dependent on MRE11, a sensor of DNA damage that can also be activated by adenoviral DNA, and on IRF1, a transcriptional regulator of cellular responses to viral invasion and DNA damage. Accordingly, we observed that endothelial cells responded to adenoviral DNA by phosphorylation of ATM and CHK2 and that depletion or inhibition of MRE11, but not depletion of ATM, abrogated IL-33 stimulation. In conclusion, we show that adenoviral transduction stimulates IL-33 expression in endothelial cells in a manner that is dependent on the DNA-binding protein MRE11 and the antiviral factor IRF1 but not on downstream DNA damage response signaling.


Subject(s)
Adenoviridae Infections/immunology , DNA Damage/immunology , Human Umbilical Vein Endothelial Cells/immunology , Interleukin-33/immunology , Adenoviridae , Adenoviridae Infections/metabolism , DNA-Binding Proteins/immunology , DNA-Binding Proteins/metabolism , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Immunoblotting , Interferon Regulatory Factor-1/immunology , Interferon Regulatory Factor-1/metabolism , Interleukin-33/biosynthesis , MRE11 Homologue Protein , Polymerase Chain Reaction , Transfection
2.
Sci Rep ; 5: 9261, 2015 Mar 20.
Article in English | MEDLINE | ID: mdl-25791723

ABSTRACT

Vascular endothelial cells present luminal chemokines that arrest rolling leukocytes by activating integrins. It appears that several chemokines must form higher-order oligomers to elicit proper in vivo effects, as mutants restricted to forming dimers have lost the ability to recruit leukocytes to sites of inflammation. Here, we show for the first time that the chemokine RANTES/CCL5 binds to the surface of human endothelial cells in a regular filamentous pattern. Furthermore, the filaments bound to the surface in a heparan sulfate-dependent manner. By electron microscopy we observed labeling for RANTES on membrane projections as well as on the remaining plasma membrane. Mutant constructs of RANTES restricted either in binding to heparin, or in forming dimers or tetramers, appeared either in a granular, non-filamentous pattern or were not detectable on the cell surface. The RANTES filaments were also present after exposure to flow, suggesting that they can be present in vivo. Taken together with the lacking in vivo or in vitro effects of RANTES mutants, we suggest that the filamentous structures of RANTES may be of physiological importance in leukocyte recruitment.


Subject(s)
Biopolymers/metabolism , Chemokine CCL5/metabolism , Endothelium, Vascular/metabolism , Endothelium, Vascular/cytology , Endothelium, Vascular/drug effects , Heparitin Sulfate/pharmacology , Human Umbilical Vein Endothelial Cells , Humans , Interferon-gamma/pharmacology , Tumor Necrosis Factor-alpha/pharmacology
3.
PLoS One ; 8(5): e62994, 2013.
Article in English | MEDLINE | ID: mdl-23671648

ABSTRACT

Lesions of hyaline cartilage do not heal spontaneously, and represent a therapeutic challenge. In vitro engineering of articular cartilage using cells and biomaterials may prove to be the best solution. Patients with osteoarthritis (OA) may require tissue engineered cartilage therapy. Chondrocytes obtained from OA joints are thought to be involved in the disease process, and thus to be of insufficient quality to be used for repair strategies. Bone marrow (BM) derived mesenchymal stem cells (MSCs) from healthy donors may represent an alternative cell source. We have isolated chondrocytes from OA joints, performed cell culture expansion and tissue engineering of cartilage using a disc-shaped alginate scaffold and chondrogenic differentiation medium. We performed real-time reverse transcriptase quantitative PCR and fluorescence immunohistochemistry to evaluate mRNA and protein expression for a range of molecules involved in chondrogenesis and OA pathogenesis. Results were compared with those obtained by using BM-MSCs in an identical tissue engineering strategy. Finally the two populations were compared using genome-wide mRNA arrays. At three weeks of chondrogenic differentiation we found high and similar levels of hyaline cartilage-specific type II collagen and fibrocartilage-specific type I collagen mRNA and protein in discs containing OA and BM-MSC derived chondrocytes. Aggrecan, the dominant proteoglycan in hyaline cartilage, was more abundantly distributed in the OA chondrocyte extracellular matrix. OA chondrocytes expressed higher mRNA levels also of other hyaline extracellular matrix components. Surprisingly BM-MSC derived chondrocytes expressed higher mRNA levels of OA markers such as COL10A1, SSP1 (osteopontin), ALPL, BMP2, VEGFA, PTGES, IHH, and WNT genes, but lower levels of MMP3 and S100A4. Based on the results presented here, OA chondrocytes may be suitable for tissue engineering of articular cartilage.


Subject(s)
Cartilage, Articular/cytology , Chondrocytes/cytology , Mesenchymal Stem Cells/cytology , Osteoarthritis/pathology , Tissue Engineering/methods , Aged , Aggrecans/genetics , Aggrecans/metabolism , Alginates/chemistry , Bone Morphogenetic Protein 2/genetics , Bone Morphogenetic Protein 2/metabolism , Cartilage, Articular/metabolism , Cell Differentiation/genetics , Cells, Cultured , Chondrocytes/metabolism , Chondrogenesis/genetics , Collagen Type I/genetics , Collagen Type I/metabolism , Collagen Type II/genetics , Collagen Type II/metabolism , Gene Expression , Glucuronic Acid/chemistry , Hedgehog Proteins/genetics , Hedgehog Proteins/metabolism , Hexuronic Acids/chemistry , Humans , Immunohistochemistry , Mesenchymal Stem Cells/metabolism , Middle Aged , Osteoarthritis/genetics , Osteoarthritis/metabolism , Osteopontin/genetics , Osteopontin/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Time Factors , Tissue Scaffolds/chemistry , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/metabolism , Wnt Proteins/genetics , Wnt Proteins/metabolism
4.
Arterioscler Thromb Vasc Biol ; 33(2): e47-55, 2013 Feb.
Article in English | MEDLINE | ID: mdl-23162017

ABSTRACT

OBJECTIVE: Interleukin (IL)-33 is a nuclear protein that is released from stressed or damaged cells to act as an alarmin. We investigated the effects of IL-33 on endothelial cells, using the prototype IL-1 family member, IL-1ß, as a reference. METHODS AND RESULTS: Human umbilical vein endothelial cells were stimulated with IL-33 or IL-1ß, showing highly similar phosphorylation of signaling molecules, induction of adhesion molecules, and transcription profiles. However, intradermally injected IL-33 elicited significantly less proinflammatory endothelial activation when compared with IL-1ß and led us to observe that quiescent endothelial cells (ppRb(low)p27(high)) were strikingly resistant to IL-33. Accordingly, the IL-33 receptor was preferentially expressed in nonquiescent cells of low-density cultures, corresponding to selective induction of adhesion molecules and chemokines. Multiparameter phosphoflow cytometry confirmed that signaling driven by IL-33 was stronger in nonquiescent cells. Manipulation of nuclear IL-33 expression by siRNA or adenoviral transduction revealed no functional link between nuclear, endogenous IL-33, and exogenous IL-33 responsiveness. CONCLUSIONS: In contrast to other inflammatory cytokines, IL-33 selectively targets nonquiescent endothelial cells. By this novel concept, quiescent cells may remain nonresponsive to a proinflammatory stimulus that concomitantly triggers a powerful response in cells that have been released from contact inhibition.


Subject(s)
Cell Proliferation , Dermatitis/immunology , Endothelial Cells/immunology , Inflammation Mediators/metabolism , Interleukins/metabolism , Skin/blood supply , Adenoviridae/genetics , Animals , Biopsy , Cells, Cultured , Cellular Senescence , Cyclin-Dependent Kinase Inhibitor p27/metabolism , Dermatitis/pathology , E-Selectin/metabolism , Endothelial Cells/pathology , Female , Flow Cytometry , Genetic Vectors , Human Umbilical Vein Endothelial Cells/immunology , Humans , Interleukin-1beta/metabolism , Interleukin-33 , Interleukins/genetics , JNK Mitogen-Activated Protein Kinases/metabolism , Mice , Mice, Inbred C57BL , NF-kappa B/metabolism , Neovascularization, Physiologic , Phosphorylation , RNA Interference , Receptors, Interleukin/metabolism , Retinoblastoma Protein/metabolism , Signal Transduction , Transcription, Genetic , Transduction, Genetic , Transfection , p38 Mitogen-Activated Protein Kinases/metabolism
5.
Am J Pathol ; 181(3): 1099-111, 2012 Sep.
Article in English | MEDLINE | ID: mdl-22809957

ABSTRACT

The molecular mechanisms that drive expression of the alarmin interleukin-33 (IL-33) in endothelial cells are unknown. Because nuclear IL-33 is a marker of endothelial cell quiescence (corroborated in this study by coexpression of cyclin-dependent kinase inhibitor p27(Kip1)), we hypothesized that Notch signaling might be involved in regulating IL-33 expression. Activation of Notch1 by immobilized Notch ligands was sufficient to induce nuclear IL-33 expression in cultured endothelial cells. Conversely, IL-33 expression was inhibited by the γ-secretase inhibitor DAPT or by inhibiting the function of Dll4, Jagged1, Notch1, or the canonical Notch transcription factor RBP-Jκ. Insensitivity to cycloheximide indicated that IL-33 was a direct target of Notch signaling, well in line with the identification of several conserved RBP-Jκ binding sites in the IL33 gene. The in vivo expression of Dll4 but not of Jagged1 was well correlated with expression of IL-33 in quiescent vessels, and subcutaneous injection of DAPT in healthy skin reduced IL-33 expression, indicating that Notch signaling was involved. On the other hand, loss of IL-33 during angiogenesis occurred despite sustained Dll4 and Notch1 expression, suggesting that other signals may override the IL-33-driving signal in this context. Taken together, our data demonstrate that endothelial nuclear IL-33 is induced by Notch and that Dll4 may be the dominant ligand responsible for this signaling in vivo.


Subject(s)
Endothelial Cells/cytology , Endothelial Cells/metabolism , Interleukins/metabolism , Receptor, Notch1/metabolism , Adaptor Proteins, Signal Transducing , Amyloid Precursor Protein Secretases/antagonists & inhibitors , Amyloid Precursor Protein Secretases/metabolism , Animals , Binding Sites , Biomarkers/metabolism , Calcium-Binding Proteins/metabolism , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Dipeptides/pharmacology , Down-Regulation/drug effects , Down-Regulation/genetics , Endothelial Cells/drug effects , Endothelial Cells/enzymology , Female , Genetic Loci/genetics , Genome, Human/genetics , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Immunoglobulin J Recombination Signal Sequence-Binding Protein/metabolism , Intercellular Signaling Peptides and Proteins/metabolism , Interleukin-33 , Interleukins/genetics , Jagged-1 Protein , Male , Membrane Proteins/metabolism , Neovascularization, Physiologic/drug effects , Neovascularization, Physiologic/genetics , Protein Binding/drug effects , Rats , Rats, Wistar , Receptor, Notch1/antagonists & inhibitors , Serrate-Jagged Proteins , Signal Transduction/drug effects , Signal Transduction/genetics , Wound Healing/drug effects
6.
Tissue Eng Part A ; 17(7-8): 1003-13, 2011 Apr.
Article in English | MEDLINE | ID: mdl-21087199

ABSTRACT

We have used a disc-shaped self-gelling alginate hydrogel as a scaffold for in vitro chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells. The comparison of monolayer cells and alginate embedded cells with or without differentiation medium allowed us to perform a detailed kinetic study of the expression of a range of genes and proteins known to be involved in chondrogenesis, using real-time polymerase chain reaction, fluorescence immunohistochemistry, and glycosaminoglycan measurement in the supernatant. mRNA encoding type II collagen (COL2), COL10, aggrecan, and SOX5, 6, and 9 were greatly elevated already at day 7, whereas COL1 and versican mRNA were gradually reduced. COL2 and aggrecan were dispersed throughout the extracellular matrix at day 21, whereas COL10 distribution was mainly intra/pericellular. COL1 seemed to be produced by only some of the cells. SOX proteins were predominantly localized in the nuclei. Then, using microarray analysis, we identified a signature cluster of extracellular matrix and transcription factor genes upregulated during chondrogenesis similar to COL2A1, and clusters of genes involved in immune responses, blood vessel development, and cell adhesion downregulated similar to the chemokine CXCL12. Analysis of the signature chondrogenic clusters, including novel potential marker genes identified here, may provide a better understanding of how the stem cell fate could be directed to produce perfect hyaline cartilage implants.


Subject(s)
Alginates/chemistry , Bone Marrow Cells/cytology , Chondrogenesis/physiology , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/metabolism , Tissue Scaffolds/chemistry , Adult , Aggrecans/genetics , Cells, Cultured , Chondrogenesis/genetics , Collagen Type II/genetics , Collagen Type X/genetics , Computational Biology , Glucuronic Acid/chemistry , Hexuronic Acids/chemistry , Humans , Microscopy, Fluorescence , Middle Aged , Multigene Family/genetics , Multigene Family/physiology , Oligonucleotide Array Sequence Analysis , Reverse Transcriptase Polymerase Chain Reaction , SOX9 Transcription Factor/genetics , SOXD Transcription Factors/genetics
7.
Am J Pathol ; 177(6): 2804-15, 2010 Dec.
Article in English | MEDLINE | ID: mdl-21037074

ABSTRACT

Interleukin-33 (IL-33) is a novel member of the interleukin-1 family that induces mucosal pathology in vivo and may drive fibrosis development and angiogenesis. To address its potential role in inflammatory bowel disease, we explored its tissue expression in biopsy specimens from untreated ulcerative colitis patients, observing a 2.6-fold up-regulation of IL-33 mRNA levels, compared to controls. Immunohistochemical analyses of surgical specimens showed that a prominent source of IL-33 in ulcerative colitis lesions were ulceration-associated myofibroblasts that co-expressed the fibroblast marker heat shock protein 47, platelet-derived growth factor receptor (PDGFR)ß, and, in part, the myofibroblast marker α-smooth muscle actin (SMA). In contrast, IL-33-positive myofibroblasts were almost absent near the deep fissures seen in Crohn's disease. A screen of known and putative activators of IL-33 in cultured fibroblasts revealed that the Toll-like receptor-3 agonist poly (I:C) was among the strongest inducers of IL-33 and that it synergized with transforming growth factor-ß, a combination also known to boost myofibroblast differentiation. Experimental wound healing in rat skin revealed that the de novo induction of IL-33 in pericytes and the possible activation of scattered, tissue-resident IL-33(+)PDGFRß(+)αSMA(-) fibroblast-like cells were early events that preceded the later appearance of IL-33(+)PDGFRß(+)αSMA(+) cells. In conclusion, our data point to a novel role for IL-33 in mucosal healing and wound repair and to an interesting difference between ulcerative colitis and Crohn's disease.


Subject(s)
Colitis, Ulcerative/genetics , Inflammatory Bowel Diseases/genetics , Interleukins/genetics , Myofibroblasts/metabolism , Animals , Biopsy , Case-Control Studies , Cells, Cultured , Colitis, Ulcerative/metabolism , Colitis, Ulcerative/pathology , Colon/drug effects , Colon/metabolism , Colon/pathology , Crohn Disease/genetics , Crohn Disease/metabolism , Crohn Disease/pathology , Gene Expression/drug effects , Humans , Inflammatory Bowel Diseases/metabolism , Inflammatory Bowel Diseases/pathology , Interleukin-33 , Interleukins/metabolism , Myofibroblasts/drug effects , Myofibroblasts/pathology , Poly I-C/pharmacology , Rats , Toll-Like Receptor 3/agonists , Transforming Growth Factor beta/pharmacology , Wound Healing/genetics , Wound Healing/physiology
8.
Arch Oral Biol ; 54(11): 1002-7, 2009 Nov.
Article in English | MEDLINE | ID: mdl-19740458

ABSTRACT

OBJECTIVE: In search for possible novel genes that may be involved in tooth development, we analysed the genome-wide transcriptome of developing mandibular tooth germs of mouse during embryonic and early life and selected family-with-sequence-similarity-46, member A (Fam46a) gene for further expression analysis. METHODS: We applied microarray, quantitative real time polymerase chain reaction and in situ hybridisation methods for the expression study of the mouse Fam46a gene. RESULTS: We found the family-with-sequence-similarity-46, member A (Fam46a) gene to be highly expressed and further verify its temporo-spatial expression in the mouse tooth. CONCLUSION: We have shown that Fam46a is expressed in ameloblasts' nuclei of tooth germs and hypothesise that it might act together with morphogenetic factors important for the formation of enamel in mouse tooth.


Subject(s)
Amelogenesis/genetics , Proteins/genetics , Tooth Germ/embryology , Ameloblasts/metabolism , Animals , Gene Expression , Gene Expression Regulation, Developmental , In Situ Hybridization , Mice , Mice, Inbred Strains , Oligonucleotide Array Sequence Analysis , Polymerase Chain Reaction , Tooth Germ/growth & development
9.
J Biol Chem ; 284(35): 23532-9, 2009 Aug 28.
Article in English | MEDLINE | ID: mdl-19578117

ABSTRACT

Sorting of proteins to Weibel-Palade bodies (WPB) of endothelial cells allows rapid regulated secretion of leukocyte-recruiting P-selectin and chemokines as well as procoagulant von Willebrand factor (VWF). Here we show by domain swap studies that the exposed aspartic acid in loop 2 (Ser(44)-Asp(45)-Gly(46)) of the CXC chemokine interleukin (IL)-8 is crucial for targeting to WPB. Loop 2 also governs sorting of chemokines to alpha-granules of platelets, but the fingerprint of the loop 2 of these chemokines differs from that of IL-8. On the other hand, loop 2 of IL-8 closely resembles a surface-exposed sequence of the VWF propeptide, the region of VWF that directs sorting of the protein to WPB. We conclude that loop 2 of IL-8 constitutes a critical signal for sorting to WPB and propose a general role for this loop in the sorting of chemokines to compartments of regulated secretion.


Subject(s)
Endothelial Cells/metabolism , Interleukin-8/chemistry , Interleukin-8/metabolism , Weibel-Palade Bodies/metabolism , Amino Acid Sequence , Cells, Cultured , Endothelial Cells/chemistry , Female , Humans , Interleukin-8/genetics , Molecular Sequence Data , Protein Structure, Tertiary , Protein Transport , Sequence Alignment , Weibel-Palade Bodies/chemistry , Weibel-Palade Bodies/genetics
10.
Trends Immunol ; 30(5): 227-33, 2009 May.
Article in English | MEDLINE | ID: mdl-19359217

ABSTRACT

Cytokines are thought to exert biological effects through their specific cell surface membrane receptors but increasing evidence suggests that some also function within the nucleus. Here, we review current knowledge of such cytokines, including the novel interleukin (IL)-1 family member IL-33. Its extracellular function has attracted much recent attention as a ligand for the Th2-associated ST2 receptor, but the discoveries of its nuclear functions and modes of secretion are only just beginning to surface. We review the currently available data on IL-33 regulation, nuclear function and release and discuss them in the context of other intranuclear cytokines and the prototype alarmin HMGB1, considering to what extent IL-33 can be seen as a novel member of the alarmin family.


Subject(s)
Interleukins/physiology , Animals , Cytokines/physiology , HMGB1 Protein/physiology , Humans , Interleukin-1/physiology , Interleukin-33 , Models, Biological , Nuclear Proteins/physiology
11.
Am J Pathol ; 173(4): 1229-42, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18787100

ABSTRACT

Interleukin (IL)-33 is a novel member of the IL-1 family of cytokines that promotes Th2 responses in lymphocytes as well as the activation of both mast cells and eosinophils via the ST2 receptor. Additionally, IL-33 has been proposed to act as a chromatin-associated transcriptional regulator in both endothelial cells of high endothelial venules and chronically inflamed vessels. Here we show that nuclear IL-33 is expressed in blood vessels of healthy tissues but down-regulated at the earliest onset of angiogenesis during wound healing; in addition, it is almost undetectable in human tumor vessels. Accordingly, IL-33 is induced when cultured endothelial cells reach confluence and stop proliferating but is lost when these cells begin to migrate. However, IL-33 expression was not induced by inhibiting cell cycle progression in subconfluent cultures and was not prevented by antibody-mediated inhibition of VE-cadherin. Conversely, IL-33 knockdown did not induce detectable changes in either expression levels or the cellular distribution of either VE-cadherin or CD31. However, activation of endothelial cell cultures with either tumor necrosis factor-alpha or vascular endothelial growth factor and subcutaneous injection of these cytokines led to a down-regulation of vascular IL-33, a response consistent with both its rapid down-regulation in wound healing and loss in tumor endothelium. In conclusion, we speculate that the proposed transcriptional repressor function of IL-33 may be involved in the control of endothelial cell activation.


Subject(s)
Cell Nucleus/metabolism , Endothelium/metabolism , Inflammation/metabolism , Interleukins/metabolism , Neovascularization, Pathologic/metabolism , Animals , Blood Vessels/drug effects , Blood Vessels/metabolism , Cell Count , Cell Movement/drug effects , Cell Nucleus/drug effects , Cells, Cultured , Cytokines/pharmacology , Down-Regulation/drug effects , Endothelium/drug effects , Endothelium/pathology , Female , Health , Humans , Interleukin-33 , Neoplasms/metabolism , Neoplasms/pathology , Rats , Tumor Necrosis Factor-alpha/pharmacology , Vascular Endothelial Growth Factor A/pharmacology , Wound Healing/drug effects
12.
BMC Genomics ; 8: 11, 2007 Jan 09.
Article in English | MEDLINE | ID: mdl-17212827

ABSTRACT

BACKGROUND: Large-scale mutagenesis screens in the zebrafish employing the mutagen ENU have isolated several hundred mutant loci that represent putative developmental control genes. In order to realize the potential of such screens, systematic genetic mapping of the mutations is necessary. Here we report on a large-scale effort to map the mutations generated in mutagenesis screening at the Max Planck Institute for Developmental Biology by genome scanning with microsatellite markers. RESULTS: We have selected a set of microsatellite markers and developed methods and scoring criteria suitable for efficient, high-throughput genome scanning. We have used these methods to successfully obtain a rough map position for 319 mutant loci from the Tübingen I mutagenesis screen and subsequent screening of the mutant collection. For 277 of these the corresponding gene is not yet identified. Mapping was successful for 80 % of the tested loci. By comparing 21 mutation and gene positions of cloned mutations we have validated the correctness of our linkage group assignments and estimated the standard error of our map positions to be approximately 6 cM. CONCLUSION: By obtaining rough map positions for over 300 zebrafish loci with developmental phenotypes, we have generated a dataset that will be useful not only for cloning of the affected genes, but also to suggest allelism of mutations with similar phenotypes that will be identified in future screens. Furthermore this work validates the usefulness of our methodology for rapid, systematic and inexpensive microsatellite mapping of zebrafish mutations.


Subject(s)
Chromosome Mapping , Microsatellite Repeats , Mutation , Zebrafish/embryology , Zebrafish/genetics , Animals , Female , Genome , Male , Mutagenesis , Phenotype
13.
Methods Mol Biol ; 360: 253-68, 2007.
Article in English | MEDLINE | ID: mdl-17172733

ABSTRACT

The future ability to manipulate the growth of new blood vessels (angiogenesis) holds great promise for treating ischemic disease and cancer. Several models of human in vivo angiogenesis have been described, but they seem to depend on transgenic support and have not been validated in a therapeutic context. Here, we describe an in vivo assay that mimics human angiogenesis in which native human umbilical vein-derived endothelial cells are suspended in a liquid laminin/collagen gel (Matrigel), injected into immunodeficient mice, and develop into mature, functional vessels that vascularize the Matrigel plug in the course of 30 d. Moreover, we demonstrate how to target this process therapeutically by sustained delivery of the angiogenesis inhibitor endostatin from subcutaneously implanted microosmotic pumps.


Subject(s)
Collagen , Drug Evaluation, Preclinical/methods , Endothelium, Vascular/cytology , Laminin , Neovascularization, Physiologic/drug effects , Proteoglycans , Angiogenesis Inducing Agents/pharmacology , Animals , Cells, Cultured , DNA-Binding Proteins/genetics , Drug Combinations , Endostatins/pharmacology , Endothelium, Vascular/drug effects , Humans , Immunologic Deficiency Syndromes/genetics , Injections, Subcutaneous , Mice , Mice, Knockout , Platelet Endothelial Cell Adhesion Molecule-1/analysis , Umbilical Veins/cytology
14.
Curr Biol ; 16(12): 1244-8, 2006 Jun 20.
Article in English | MEDLINE | ID: mdl-16782017

ABSTRACT

Lymphangiogenesis results in the formation of a vascular network distinct from arteries and veins that serves to drain interstitial fluid from surrounding tissues and plays a pivotal role in the immune defense of vertebrates as well as in the progression of cancer and other diseases . In mammals, lymph vessels are lined by endothelial cells possibly sprouting from embryonic veins, and their development appears to be critically dependent on the function of PROX1 and VEGFC signaling . The existence of a lymphatic system in teleosts has been a matter of debate for decades. Here we show on the morphological, molecular, and functional levels that zebrafish embryos develop a lymphatic vasculature that serves to retrieve components of the interstitium to the lymph system. We demonstrate the existence of vessels that are molecularly and functionally distinct from blood vessels and show that the development of these vessels depends on Vegfc and VEGFR-3/Flt4 signaling. These findings imply that the molecular components controlling lymphangiogenesis in zebrafish and mammals are conserved and that the zebrafish lymphatic system develops early enough to allow in vivo observations, lineage tracing, and genetic as well as pharmacological screens.


Subject(s)
Lymphatic Vessels/embryology , Signal Transduction , Vascular Endothelial Growth Factor C/metabolism , Zebrafish Proteins/metabolism , Zebrafish/embryology , Angiopoietin-2/metabolism , Animals , Animals, Genetically Modified/anatomy & histology , Animals, Genetically Modified/metabolism , Biological Transport , Embryo, Nonmammalian/cytology , Embryo, Nonmammalian/metabolism , Endothelium, Lymphatic/cytology , Endothelium, Lymphatic/metabolism , Extracellular Fluid/metabolism , Green Fluorescent Proteins/analysis , Lymphatic Vessels/metabolism , Vascular Endothelial Growth Factor Receptor-3/metabolism , Zebrafish/genetics , Zebrafish/metabolism
15.
Development ; 129(12): 2851-65, 2002 Jun.
Article in English | MEDLINE | ID: mdl-12050134

ABSTRACT

A number of studies have suggested that retinoic acid (RA) is an important signal for patterning the hindbrain, the branchial arches and the limb bud. Retinoic acid is thought to act on the posterior hindbrain and the limb buds at somitogenesis stages in chick and mouse embryos. Here we report a much earlier requirement for RA signalling during pre-segmentation stages for proper development of these structures in zebrafish. We present evidence that a RA signal is necessary during pre-segmentation stages for proper expression of the spinal cord markers hoxb5a and hoxb6b, suggesting an influence of RA on anteroposterior patterning of the neural plate posterior to the hindbrain. We report the identification and expression pattern of the zebrafish retinaldehyde dehydrogenase2 (raldh2/aldh1a2) gene. Raldh2 synthesises retinoic acid (RA) from its immediate precursor retinal. It is expressed in a highly ordered spatial and temporal fashion during gastrulation in the involuting mesoderm and during later embryogenesis in paraxial mesoderm, branchial arches, eyes and fin buds, suggesting the involvement of RA at different times of development in different functional contexts. Mapping of the raldh2 gene reveals close linkage to no-fin (nof), a newly discovered mutant lacking pectoral fins and cartilaginous gill arches. Cloning and functional tests of the wild-type and nof alleles of raldh2 reveal that nof is a raldh2 mutant. By treating nof mutants with RA during different time windows and by making use of a retinoic acid receptor antagonist, we show that RA signalling during pre-segmentation stages is necessary for anteroposterior patterning in the CNS and for fin induction to occur.


Subject(s)
Aldehyde Oxidoreductases/genetics , Aldehyde Oxidoreductases/metabolism , Central Nervous System/embryology , Signal Transduction , Tretinoin/metabolism , Zebrafish/embryology , Amino Acid Sequence , Animals , Body Patterning , Chromosome Mapping , Cloning, Molecular , Embryo, Nonmammalian , Female , Gene Expression Regulation, Developmental , Genetic Markers , Limb Buds/metabolism , Male , Molecular Sequence Data , Mutation , Retinal Dehydrogenase , Rhombencephalon/embryology , Sequence Homology, Amino Acid , Spinal Cord/embryology
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