Chemotactic Properties of Angiopoietin-1 and -2, Ligands for the Endothelial-specific Receptor Tyrosine Kinase Tie2

Angiopoietin-1 and its putative natural antagonist, angiopoietin-2, were recently isolated, and the critical role of angiopoietin-1 in embryogenic angiogenesis was demonstrated by targeted gene disruption. Specific biological effects of angiopoietin-1, however, have yet to be defined. In this study we demonstrate that angiopoietin-1, but not angiopoietin-2, is chemotactic for endothelial cells. In contrast, angiopoietin-1 as well as angiopoietin-2 exhibit no proliferative effect on endothelial cells. Excess soluble Tie2, but not Tie1 receptor, abolish the chemotactic response of endothelial cells toward angiopoietin-1. Angiopoietin-2 dose-dependently blocks directed migration toward angiopoietin-1, consistent with the role of angiopoietin-2 as a naturally occurring inhibitor of angiopoietin-1. Fibroblasts stably transfected with Tie2 receptor exhibit chemotactic responses for both angiopoietin-1 and angiopoietin-2. Fibroblasts stably expressing a transfected chimeric receptor consisting of the ectodomain of TrkC fused to the cytoplasmic domain of Tie2 also exhibit a chemotactic response to neurotrophin 3 (NT-3), a specific ligand for TrkC. Endothelial cells are shown to express angiopoietin-2 mRNA and protein, indicating the potential for autocrine activation of angiopoietin/Tie2. Finally, the demonstration that Tie2 as well as angiopoietin-1 are expressed in normal human arteries and veins suggests that the role of angiopoietin/Tie2 may extend beyond embryonic angiogenesis to maintaining integrity of the adult vasculature. Angiopoietin-1 and its putative natural antagonist, angiopoietin-2, were recently isolated, and the critical role of angiopoietin-1 in embryogenic angiogenesis was demonstrated by targeted gene disruption. Specific biological effects of angiopoietin-1, however, have yet to be defined. In this study we demonstrate that angiopoietin-1, but not angiopoietin-2, is chemotactic for endothelial cells. In contrast, angiopoietin-1 as well as angiopoietin-2 exhibit no proliferative effect on endothelial cells. Excess soluble Tie2, but not Tie1 receptor, abolish the chemotactic response of endothelial cells toward angiopoietin-1. Angiopoietin-2 dose-dependently blocks directed migration toward angiopoietin-1, consistent with the role of angiopoietin-2 as a naturally occurring inhibitor of angiopoietin-1. Fibroblasts stably transfected with Tie2 receptor exhibit chemotactic responses for both angiopoietin-1 and angiopoietin-2. Fibroblasts stably expressing a transfected chimeric receptor consisting of the ectodomain of TrkC fused to the cytoplasmic domain of Tie2 also exhibit a chemotactic response to neurotrophin 3 (NT-3), a specific ligand for TrkC. Endothelial cells are shown to express angiopoietin-2 mRNA and protein, indicating the potential for autocrine activation of angiopoietin/Tie2. Finally, the demonstration that Tie2 as well as angiopoietin-1 are expressed in normal human arteries and veins suggests that the role of angiopoietin/Tie2 may extend beyond embryonic angiogenesis to maintaining integrity of the adult vasculature. The receptor tyrosine kinase family of cell surface proteins is known to play key roles in transducing intercellular signals to the cytoplasm (1Ullrich A. Schlessinger J. Cell. 1990; 61: 203-212Abstract Full Text PDF PubMed Scopus (4619) Google Scholar). A large diversity of receptor tyrosine kinases and receptor tyrosine kinase expression patterns, which are temporally modified during development and under pathologic conditions, determines cell fate and allows tissue-specific cell responses. Ligand binding to the large extracellular domain of receptor tyrosine kinases leads to receptor dimerization and autophosphorylation of tyrosine residues on the intracellular domain of the receptor (2Schlessinger J. Ullrich A. Neuron. 1992; 9: 383-391Abstract Full Text PDF PubMed Scopus (1295) Google Scholar). A variety of Src homology 2 domain-containing proteins, which are recruited to these phosphorylation sites, have been identified (3Marschall C.J. Cell. 1995; 80: 179-185Abstract Full Text PDF PubMed Scopus (4245) Google Scholar). These proteins are regarded as initiators of different signal cascades that finally lead to specific cellular responses including proliferation, migration, differentiation, and morphologic organization in the context of surrounding tissues (4Risau W. FASEB J. 1995; 9: 926-933Crossref PubMed Scopus (528) Google Scholar). On the basis of sequence similarity and structural characteristics, it is possible to classify receptor tyrosine kinases into subfamilies (1Ullrich A. Schlessinger J. Cell. 1990; 61: 203-212Abstract Full Text PDF PubMed Scopus (4619) Google Scholar). Two receptor tyrosine kinase subfamilies are characterized by their largely endothelium-specific expression. One of them consists of the three known vascular endothelial growth factor (VEGF) 1VEGF, vascular endothelial growth factor; EC, endothelial cell; FBS, fetal bovine serum; HUVEC, human umbilical vein endothelial cell; NT-3, neurotrophin 3; PDGF, platelet-derived growth factor; EGF, epidermal growth factor; SMC, smooth muscle cells; Ang, angiopoietin; HM, human microvascular; MTS, 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium; RT, reverse transcription; PCR, polymerase chain reaction; CM, conditioned media; rTie1-Fc, Tie1 receptor-Fc. receptors Flt-1/VEGF-R1, Flk-1/KDR/VEGF-R2, and Flt-4/VEGF-R3 (5Mustonen T. Alitalo K. J. Cell Biol. 1995; 129: 895-898Crossref PubMed Scopus (477) Google Scholar, 6Joukov V. Pajusola K. Kaipainen A. Chilov D. Lahtinen I. Kukk E. Saksela O. Kalkkinen N. Alitalo K. EMBO J. 1996; 15: 290-298Crossref PubMed Scopus (1160) Google Scholar). 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Isner J.M. Am. J. Pathol. 1997; 150: 1673-1685PubMed Google these were the the of and were to of was to these in a including of the to the of the vascular these or cell as was by N. PubMed Scopus Google and was J. T. A Scholar) was by a of and for of human inhibitor and of reverse were in a and for and to with the polymerase chain S. R. H.A. PubMed Scopus Google a of and polymerase was to the of of and of the of the a was for were by of of in a and were by were The for and for human Tie2 and of for human and for human and and for human and The of was for Tie2, and and for was in using the of was in for and the were on the The of was for by with of for was as of for in human of the and Tie2 was on were and J. endothelial cells. The cells were on using FBS, and in a was and the cell was with with and as N. PubMed Scopus Google Scholar). were by on to and with a the also human and The and Tie2 were human the VEGF was the of a expression of to these cells into the was as The was with and for the cells were with of cell this was by and for the of Tie2 binding on a as S. A. V. J. C. P.C. Yancopoulos G.D. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). of binding or a signal that with cell not this Tie2 binding was protein, the was to on to and by with specific to and were expressed as of the was using for and by for A of was as cellular in response to or we with of or a known for and was shown to cell J. 1993; Scopus Google Scholar). VEGF to a in with in cell a response was and In contrast, with a to a in cell not In the of and on in was by not was as a and to a in with VEGF a in a of both to in Moreover, VEGF with or not the effect of VEGF on Similar were using of and or for The effect of with and on cell and by were in VEGF the of cells in for and not VEGF the of cells in for or effects under the of angiopoietin-1 and on cell and by were with VEGF or factor in for Cell using was and of and cells were and in a were with VEGF or factor in for Cell using was and of and cells were and a modified we the chemotactic response of to of and and it to that of which is known to be chemotactic for V. Breier G. S. L. Gertsenstein M. M. A. H. C. C. J. L. D. Risau W. A. Nature. 1996; PubMed Scopus Google Scholar). to a in directed migration of with a effect a of of and the chemotactic effect of to be that of the response and was In contrast, a not chemotactic of the effect on directed cell migration, we different and with or a to of soluble Tie2 receptor or soluble Tie1 receptor and to the of the shown in its cellular receptor to its the chemotactic effect of on In contrast, rTie1-Fc, a receptor that not was not to the that the effects are to in the and be by the of chemotactic and effects of on we a modified in which the of and the was that migration was a of and was a of In contrast, of to both of the not cell a effect of of in response to angiopoietin-1 using modified in in no factor to or no factor to or of were in the as were to for was by the of cells to the of a with were for to the of three that to a chemotactic of and is no factor to or in a of were in the as were to for was by the of cells to the of a with were for to the of three that to a chemotactic of and is the chemotactic response of and different toward and were not effect on cell however, directed migration in bovine and no effect on or human vascular was These that the chemotactic effect of is specific but not to of a have that is a naturally occurring for Tie2, we is to the chemotactic response by in of were to a of in the of a the chemotactic effect of on these cells was the of cells to of was that is to with for Tie2 receptor binding sites, but binding of to Tie2 not lead to the receptor signal to the chemotactic effects of and on cells expressing Tie2 receptors, migration using stably transfected with Tie2 receptor were and with a known chemotactic for and C. T. J. Cell. PubMed Scopus Google as a The transfected receptors were to receptor phosphorylation in to activation C. S. S. C. D. J. T. N. T. S. T. Yancopoulos G. 1997; PubMed Scopus Google Scholar). In in cell migration In contrast, stably expressing transfected Tie2 receptors demonstrated a in cell migration to for and to for the of with A of to the of the the chemotactic effect of both and on for for not toward was not by expressing Tie2 receptor demonstrated a specific chemotactic response to both and in contrast, a specific chemotactic response to Moreover, we potential in which activation of the Tie2 domain chemotactic responses. cell were stably transfected with a chimeric receptor consisting of the ectodomain of TrkC fused to the cytoplasmic domain of Tie2 D.J. Goldfarb M. Yancopoulos G.D. Cell. Full Text PDF PubMed Scopus Google Scholar). that the chimeric receptor is been shown by tyrosine phosphorylation of the of to these cells S. A. V. J. C. P.C. Yancopoulos G.D. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). these cells for migration and the responses to of cells. both as well as receptors, was as a a chemotactic response in both cell with In cells the TrkC receptor, the of not in chemotactic effect cells not In contrast, cells a chemotactic response to to indicating that activation of this chimeric receptor, to autophosphorylation of the cytoplasmic is to a signal in cellular and Tie2 mRNA expression was using in human of and shown in both as well as mRNA were in three Tie2 mRNA expression was in not express or Tie2 and as of was as for and of mRNA of for and Tie2, in with the expression of Tie2 receptor mRNA in the stably transfected was also by Tie2 mRNA was in different human endothelial in shown for as well as mRNA were not in human vascular but in as well and was on expression of as well as Tie1 and Tie2 was the mRNA in human and human In contrast, human expressed of but were for of conditioned by but no was to in that in gene Tie2 (22Sato T.N. Y. U. K. Y. M. T. H. Risau W. Qin Y. Nature. 1995; 376: PubMed Scopus Google Scholar, D.J. Gradwohl G. F. Puri M.C. Gertsenstein M. A. Breitman M.L. Dev. 1994; 8: PubMed Scopus Google Scholar) or C. S. S. P.C. S. T. Yancopoulos G.D. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar) have a role for the in embryonic are null mice (8Shalaby F. Rossant J. Yamaguchi T.P. Gertsenstein M. Wu X-F. Breitman M.L. Schuh A.C. Nature. 1995; 376: 62-66Crossref PubMed Scopus (3371) Google in Tie2 null mice are in normal and are into the vessels of Tie2 however, the of and organization for normal Similar vascular are in null mice C. S. S. P.C. S. T. Yancopoulos G.D. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google are not are with the and not with cells. These were as that the roles of the in angiogenesis into play to by the of to be of a and specific chemotactic effect of on is to the of a biological of the a and cells and expressing the Tie2 receptor, and putative basis for angiogenesis in mice Tie2 or The critical of migration, as to proliferation, in angiogenesis was demonstrated in a in the by Invest. Google Scholar). be and for 2 to of angiogenesis in this was to the of Tie2 receptor and mRNA were in normal human and Tie2 expression was recently shown by to be in adult including vascular (15Tsurumi Y. Murohara T. Krasinski K. Dongfen C. Witzenbichler B. Kearney M. Couffinhal T. Isner J.M. Nat. Med. 1997; 3: 879-886Crossref PubMed Scopus (303) Google the of Tie2 phosphorylation was as for the of Tie2 in adult consistent with that is expressed in adult tissues C. S. S. C. D. J. T. N. T. S. T. Yancopoulos G. 1997; PubMed Scopus Google Scholar). demonstration of in the adult vessel is in with this expression with the of effect of suggests possible of the in normal adult cells and maintaining endothelial integrity and of on the of the endothelial lead to of expression in surrounding cells in Tie2 receptor expression in which have with of VEGF in a effect on and of was recently demonstrated in response to (15Tsurumi Y. Murohara T. Krasinski K. Dongfen C. Witzenbichler B. Kearney M. Couffinhal T. Isner J.M. Nat. Med. 1997; 3: 879-886Crossref PubMed Scopus (303) Google Scholar). The chemotactic effect of may be involved as to VEGF in to and In the and Tie2 the of on may to the and cells as well as the of in under of as or the effect of on may be important for of as well as of toward for into may be for the of in or In this the effects of may be in a a of to as the formation may to effect of are of the of that may be of angiogenesis in The for of the which is known to be by S. J. Biol. 1995; Full Text Full Text PDF PubMed Scopus Google not to be by B. K. A. V. Y. J. L. M. B. V. U. Alitalo K. Oncogene. 1997; PubMed Scopus Google Scholar). expression of Tie2, however, been in the vascular of A. T. E. T. A. S. Alitalo K. Cancer Res. 1994; Google Scholar) and during and J. J. E. A. K. M. Alitalo K. 1992; 80: PubMed Google the that is the of receptor expression. A second ligand for the Tie2 receptor, homology in sequence with was recently identified C. S. S. C. D. J. T. N. T. S. T. Yancopoulos G. 1997; PubMed Scopus Google Scholar). Two were as to that a naturally occurring for was shown to to Tie2 expressed on transducing the receptor and was as well of receptor phosphorylation C. S. S. C. D. J. T. N. T. S. T. Yancopoulos G. 1997; PubMed Scopus Google Scholar). of to disruption of blood vessel formation in the C. S. S. C. D. J. T. N. T. S. T. Yancopoulos G. 1997; PubMed Scopus Google Scholar). in that chemotactic for be by of is consistent with these and suggests a of by binding to Tie2 autophosphorylation of the cytoplasmic tyrosine kinase Moreover, demonstration of expression in the normal vessel not been and suggests a of vessel by and The of for of cell expression in vascular of however, that expression was not to but be in as mRNA was identified by in by in human endothelial and endothelial and by in In contrast, was identified by in it was not by of human endothelial or human endothelial cells of conditioned of These to the of in endothelial expression of C. S. S. C. D. J. T. N. T. S. T. Yancopoulos G. 1997; PubMed Scopus Google Scholar). are endothelial which by were shown to express of but were for is this of expression in been shown to to cells in S. A. V. J. C. P.C. Yancopoulos G.D. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). that are to express it is to autocrine of expression the of Tie2 C. S. S. C. D. J. T. N. T. S. T. Yancopoulos G. 1997; PubMed Scopus Google Scholar) have that receptor phosphorylation in it is to for promoting Tie2 phosphorylation the receptor is expressed in demonstration of a chemotactic response of for and these to a to express a of intracellular proteins to with expressed Tie2 receptor and a signal in cell is in by that transfected with a chimeric receptor the cytoplasmic domain of Tie2 fused to TrkC extracellular binding a receptor for NT-3, be to by The effect of in endothelial cells and cells may be to a of critical in the is however, that is the of Tie2 receptor activation in both cell One potential for this is that binding of Tie2 by leads to of the receptor with as Tie1 or as yet receptor, Tie2 and phosphorylation of cytoplasmic receptor kinase including of with Tie2 and be to this The are to and of the of for and and to for