Tissue macrophages act as cellular chaperones for vascular anastomosis downstream of VEGF-mediated endothelial tip cell induction.

Blood vessel networks expand in a 2-step process that begins with vessel sprouting and is followed by vessel anastomosis. Vessel sprouting is induced by chemotactic gradients of the vascular endothelial growth factor (VEGF), which stimulates tip cell protrusion. Yet it is not known which factors promote the fusion of neighboring tip cells to add new circuits to the existing vessel network. By combining the analysis of mouse mutants defective in macrophage development or VEGF signaling with live imaging in zebrafish, we now show that macrophages promote tip cell fusion downstream of VEGF-mediated tip cell induction. Macrophages therefore play a hitherto unidentified and unexpected role as vascular fusion cells. Moreover, we show that there are striking molecular similarities between the pro-angiogenic tissue macrophages essential for vascular development and those that promote the angiogenic switch in cancer, including the expression of the cell-surface proteins TIE2 and NRP1. Our findings suggest that tissue macrophages are a target for antiangiogenic therapies, but that they could equally well be exploited to stimulate tissue vascularization in ischemic disease.

[1]  T. Noda,et al.  Involvement of Flt-1 tyrosine kinase (vascular endothelial growth factor receptor-1) in pathological angiogenesis. , 2001, Cancer research.

[2]  B. Thisse,et al.  Zebrafish early macrophages colonize cephalic mesenchyme and developing brain, retina, and epidermis through a M-CSF receptor-dependent invasive process. , 2001, Developmental biology.

[3]  Christiana Ruhrberg,et al.  Neuropilin ligands in vascular and neuronal patterning. , 2009, Biochemical Society transactions.

[4]  Thomas N. Sato,et al.  Distinct roles of the receptor tyrosine kinases Tie-1 and Tie-2 in blood vessel formation , 1995, Nature.

[5]  A. Feeney,et al.  Targeted disruption of the PU.1 gene results in multiple hematopoietic abnormalities. , 1996, The EMBO journal.

[6]  B. Weinstein,et al.  Angiogenic network formation in the developing vertebrate trunk , 2003, Development.

[7]  Michael C. Ostrowski,et al.  Differentiation of the mononuclear phagocyte system during mouse embryogenesis: the role of transcription factor PU.1. , 1999, Blood.

[8]  K. Csaky,et al.  Macrophage depletion diminishes lesion size and severity in experimental choroidal neovascularization. , 2003, Investigative ophthalmology & visual science.

[9]  M Aguet,et al.  VEGF is required for growth and survival in neonatal mice. , 1999, Development.

[10]  B. Thisse,et al.  Ontogeny and behaviour of early macrophages in the zebrafish embryo. , 1999, Development.

[11]  S. Gordon,et al.  F4/80, a monoclonal antibody directed specifically against the mouse macrophage , 1981, European journal of immunology.

[12]  S. Nishikawa,et al.  The murine mutation osteopetrosis is in the coding region of the macrophage colony stimulating factor gene , 1990, Nature.

[13]  K. Alitalo,et al.  VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia , 2003, The Journal of cell biology.

[14]  Shankar Srinivas,et al.  Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus , 2001, BMC Developmental Biology.

[15]  E. Wagner,et al.  Cortical and retinal defects caused by dosage-dependent reductions in VEGF-A paracrine signaling. , 2003, Developmental biology.

[16]  U. Dirnagl,et al.  Immune surveillance of mouse brain perivascular spaces by blood‐borne macrophages , 2001, The European journal of neuroscience.

[17]  A. Sica,et al.  A distinguishing gene signature shared by tumor-infiltrating Tie2-expressing monocytes, blood "resident" monocytes, and embryonic macrophages suggests common functions and developmental relationships. , 2009, Blood.

[18]  J. Levraud,et al.  Imaging early macrophage differentiation, migration, and behaviors in live zebrafish embryos. , 2005, Methods in molecular medicine.

[19]  E. Scott,et al.  PU. 1 is not essential for early myeloid gene expression but is required for terminal myeloid differentiation. , 1995, Immunity.

[20]  M. Shibuya,et al.  M-CSF inhibition selectively targets pathological angiogenesis and lymphangiogenesis , 2009, The Journal of experimental medicine.

[21]  A. Almendros,et al.  First appearance, distribution, and origin of macrophages in the early development of the avian central nervous system , 1993, The Journal of comparative neurology.

[22]  L. Smith,et al.  Characterization of vascular development in the mouse retina. , 1988, Microvascular research.

[23]  J. Rossant,et al.  Multiple developmental roles of VEGF suggested by a LacZ-tagged allele. , 1999, Developmental biology.

[24]  J. Pollard,et al.  Osteopetrotic (op/op) mice deficient in macrophages have the ability to mount a normal T-cell-dependent immune response. , 1995, Cellular immunology.

[25]  J. Pollard,et al.  Tumor-associated macrophages press the angiogenic switch in breast cancer. , 2007, Cancer research.

[26]  A. Mantovani,et al.  Migration of human monocytes in response to vascular endothelial growth factor (VEGF) is mediated via the VEGF receptor flt-1. , 1996, Blood.

[27]  W. Wood,et al.  Mesenchymal cells engulf and clear apoptotic footplate cells in macrophageless PU.1 null mouse embryos. , 2000, Development.

[28]  Russell G Foster,et al.  Experimental validation of novel and conventional approaches to quantitative real-time PCR data analysis. , 2003, Nucleic acids research.

[29]  H. Hammes,et al.  Impaired brain angiogenesis and neuronal apoptosis induced by conditional homozygous inactivation of vascular endothelial growth factor , 2004, Thrombosis and Haemostasis.

[30]  Holger Gerhardt,et al.  Spatially restricted patterning cues provided by heparin-binding VEGF-A control blood vessel branching morphogenesis. , 2002, Genes & development.

[31]  P. Rezaie,et al.  The origin and cell lineage of microglia—New concepts , 2007, Brain Research Reviews.

[32]  J. Pollard.  Tumour-educated macrophages promote tumour progression and metastasis , 2004, Nature Reviews Cancer.

[33]  D. Hume,et al.  Origins and functions of phagocytes in the embryo. , 2000, Experimental hematology.

[34]  Y. Imai,et al.  A novel gene iba1 in the major histocompatibility complex class III region encoding an EF hand protein expressed in a monocytic lineage. , 1996, Biochemical and biophysical research communications.

[35]  A. Luttun,et al.  Revascularization of ischemic tissues by PlGF treatment, and inhibition of tumor angiogenesis, arthritis and atherosclerosis by anti-Flt1 , 2002, Nature Medicine.

[36]  I. Shiojima,et al.  Obligatory participation of macrophages in an angiopoietin 2-mediated cell death switch , 2007, Development.

[37]  M. Simon.  PU.1 and hematopoiesis: lessons learned from gene targeting experiments. , 1998, Seminars in immunology.

[38]  P. Carmeliet,et al.  VEGFR-1–Selective VEGF Homologue PlGF Is Arteriogenic: Evidence for a Monocyte-Mediated Mechanism , 2003, Circulation research.

[39]  W. Reith,et al.  Conditional gene targeting in macrophages and granulocytes using LysMcre mice , 1999, Transgenic Research.

[40]  Y. Jan,et al.  Progenitor cell maintenance requires numb and numblike during mouse neurogenesis , 2002, Nature.

[41]  M. Leduc,et al.  Potential role of microglia in retinal blood vessel formation. , 2006, Investigative ophthalmology & visual science.

[42]  R. Hoyt,et al.  Macrophage development: II. Early ontogeny of macrophage populations in brain, liver, and lungs of rat embryos as revealed by a lectin marker , 1992, The Anatomical record.

[43]  R. Zeller,et al.  Tissue remodelling through branching morphogenesis , 2009, Nature Reviews Molecular Cell Biology.

[44]  W. Wiktor-Jedrzejczak,et al.  Total absence of colony-stimulating factor 1 in the macrophage-deficient osteopetrotic (op/op) mouse. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[45]  J. Ambati,et al.  Macrophage depletion inhibits experimental choroidal neovascularization. , 2003, Investigative ophthalmology & visual science.

[46]  S. Webb,et al.  Direct observation and quantification of macrophage chemoattraction to the growth factor CSF-1. , 1996, Journal of cell science.

[47]  E. Scott,et al.  Requirement of transcription factor PU.1 in the development of multiple hematopoietic lineages. , 1994, Science.

[48]  J. Martinez-Barbera,et al.  In vivo genetic ablation by Cre‐mediated expression of diphtheria toxin fragment A , 2005, Genesis.

[49]  H. Kurz,et al.  Embryonic CNS macrophages and microglia do not stem from circulating, but from extravascular precursors , 1998, Glia.

[50]  Na Zhang,et al.  Deletion of Vascular Endothelial Growth Factor in myeloid cells accelerates tumorigenesis , 2008, Nature.

[51]  B. Weinstein,et al.  In vivo imaging of embryonic vascular development using transgenic zebrafish. , 2002, Developmental biology.

[52]  C. Nüsslein-Volhard,et al.  Live Imaging of Neuronal Degradation by Microglia Reveals a Role for v0-ATPase a1 in Phagosomal Fusion In Vivo , 2008, Cell.

[53]  Paul Martin,et al.  Dynamic analysis of filopodial interactions during the zippering phase of Drosophila dorsal closure , 2008, Development.