Therapeutic angiogenesis is likely to require the administration of factors that complement each other. Activation of the receptor tyrosine kinase (RTK) Flk1 by vascular endothelial growth factor (VEGF) is crucial, but molecular interactions of other factors with VEGF and Flk1 have been studied to a limited extent. Here we report that placental growth factor (PGF, also known as PlGF) regulates inter- and intramolecular cross talk between the VEGF RTKs Flt1 and Flk1. Activation of Flt1 by PGF resulted in intermolecular transphosphorylation of Flk1, thereby amplifying VEGF-driven angiogenesis through Flk1. Even though VEGF and PGF both bind Flt1, PGF uniquely stimulated the phosphorylation of specific Flt1 tyrosine residues and the expression of distinct downstream target genes. Furthermore, the VEGF/PGF heterodimer activated intramolecular VEGF receptor cross talk through formation of Flk1/Flt1 heterodimers. The inter- and intramolecular VEGF receptor cross talk is likely to have therapeutic implications, as treatment with VEGF/PGF heterodimer or a combination of VEGF plus PGF increased ischemic myocardial angiogenesis in a mouse model that was refractory to VEGF alone.
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Rivard, A. et al. Age-dependent impairment of angiogenesis. Circulation 99, 111–120 (1999).
Couffinhal, T. et al. Impaired collateral vessel development associated with reduced expression of vascular endothelial growth factor in ApoE−/− mice. Circulation 99, 3188–3198 (1999).
Waltenberger, J. Impaired collateral vessel development in diabetes: potential cellular mechanisms and therapeutic implications. Cardiovasc. Res. 49, 554–560 (2001).
De Falco, S., Gigante, B. & Persico, M. Structure and function of placental growth factor. Trends Cardiovasc. Med. 12, 241–246 (2002).
Schlessinger, J. Cell signaling by receptor tyrosine kinases. Cell 103, 211–225 (2000).
Ferrara, N. Role of vascular endothelial growth factor in regulation of physiological angiogenesis. Am. J. Physiol. Cell. Physiol. 280, C1358–C1366 (2001).
Park, J.E., Chen, H.H., Winer, J., Houck, K.A. & Ferrara, N. Placenta growth factor. Potentiation of vascular endothelial growth factor bioactivity, in vitro and in vivo, and high affinity binding to Flt-1 but not to Flk-1/KDR. J. Biol. Chem. 269, 25646–25654 (1994).
Hiratsuka, S., Minowa, O., Kuno, J., Noda, T. & Shibuya, M. Flt-1 lacking the tyrosine kinase domain is sufficient for normal development and angiogenesis in mice. Proc. Natl. Acad. Sci. USA 95, 9349–9354 (1998).
Hiratsuka, S. et al. Involvement of Flt-1 tyrosine kinase (vascular endothelial growth factor receptor-1) in pathological angiogenesis. Cancer Res. 61, 1207–1213 (2001).
Luttun, A. et al. Revascularization of ischemic tissues by PlGF treatment, and inhibition of tumor angiogenesis, arthritis and atherosclerosis by anti-Flt1. Nat. Med. 8, 831–840 (2002).
Zeng, H., Dvorak, H.F. & Mukhopadhyay, D. VPF/VEGF receptor-1 down-modulates VPF/VEGF receptor-2 mediated endothelial cell proliferation, but not migration, through phosphatidylinositol 3-kinase dependent pathways. J. Biol. Chem. 276, 26969–26979 (2001).
Zeng, H., Zhao, D. & Mukhopadhyay, D. Flt-1-mediated down-regulation of endothelial cell proliferation through pertussis toxin-sensitive G proteins, beta gamma subunits, small GTPase CDC42, and partly by Rac-1. J. Biol. Chem. 277, 4003–4009 (2002).
Gille, H. et al. A repressor sequence in the juxtamembrane domain of Flt-1 (VEGFR-1) constitutively inhibits vascular endothelial growth factor-dependent phosphatidylinositol 3′-kinase activation and endothelial cell migration. EMBO J. 19, 4064–4073 (2000).
Yang, S. et al. Vascular endothelial growth factor-induced genes in human umbilical vein endothelial cells. Relative roles of KDR and Flt-1 receptors. Arterioscler. Thromb. Vasc. Biol. 22, 1797–1803 (2002).
Rahimi, N., Dayanir, V. & Lashkari, K. Receptor chimeras indicate that the VEGFR-1 modulates mitogenic activity of VEGFR-2 in endothelial cells. J. Biol. Chem. 275, 16986–16992 (2000).
Kanno, S. et al. Roles of two VEGF receptors, Flt-1 and KDR, in the signal transduction of VEGF effects in human vascular endothelial cells. Oncogene 19, 2138–2146 (2000).
Carmeliet, P. et al. Synergism between vascular endothelial growth factor and placental growth factor contributes to angiogenesis and plasma extravasation in pathological conditions. Nat. Med. 7, 575–583 (2001).
Adini, A., Kornaga, T., Firoozbakht, F. & Benjamin, L.E. Placental growth factor is a survival factor for tumor endothelial cells and macrophages. Cancer Res. 62, 2749–2752 (2002).
Odorisio, T. et al. Mice overexpressing placenta growth factor exhibit increased vascularization and vessel permeability. J. Cell Sci. 115, 2559–2567 (2002).
Hattori, K. et al. Placental growth factor reconstitutes hematopoiesis by recruiting VEGFR1(+) stem cells from bone-marrow microenvironment. Nat. Med. 8, 841–849 (2002).
Castellon, R. et al. Effects of angiogenic growth factor combinations on retinal endothelial cells. Exp. Eye Res. 74, 523–535 (2002).
Kendall, R.L., Wang, G. & Thomas, K.A. Identification of a natural soluble form of the vascular endothelial growth factor receptor, FLT-1, and its heterodimerization with KDR. Biochem. Biophys. Res. Commun. 226, 324–328 (1996).
Huang, K., Andersson, C., Roomans, G.M., Ito, N. & Claesson-Welsh, L. Signaling properties of VEGF receptor-1 and -2 homo- and heterodimers. Int. J. Biochem. Cell Biol. 33, 315–324 (2001).
Cao, Y., Linden, P., Shima, D., Browne, F. & Folkman, J. In vivo angiogenic activity and hypoxia induction of heterodimers of placenta growth factor/vascular endothelial growth factor. J. Clin. Invest. 98, 2507–2511 (1996).
DiSalvo, J. et al. Purification and characterization of a naturally occurring vascular endothelial growth factor.placenta growth factor heterodimer. J. Biol. Chem. 270, 7717–7723 (1995).
Eriksson, A. et al. Placenta growth factor-1 antagonizes VEGF-induced angiogenesis and tumor growth by the formation of functionally inactive PGF-1/VEGF heterodimers. Cancer Cell 1, 99–108 (2002).
Takahashi, T., Yamaguchi, S., Chida, K. & Shibuya, M. A single autophosphorylation site on KDR/Flk-1 is essential for VEGF-A- dependent activation of PLC-gamma and DNA synthesis in vascular endothelial cells. EMBO J. 20, 2768–2778 (2001).
Li, B. et al. Receptor-selective variants of human vascular endothelial growth factor. Generation and characterization. J. Biol. Chem. 275, 29823–29828 (2000).
Yu, Y. et al. Direct identification of a major autophosphorylation site on vascular endothelial growth factor receptor Flt-1 that mediates phosphatidylinositol 3′-kinase binding. Biochem. J. 358, 465–472 (2001).
Igarashi, K. et al. Tyrosine 1213 of Flt-1 is a major binding site of Nck and SHP-2. Biochem. Biophys. Res. Commun. 246, 95–99 (1998).
Ito, N., Wernstedt, C., Engstrom, U. & Claesson-Welsh, L. Identification of vascular endothelial growth factor receptor-1 tyrosine phosphorylation sites and binding of SH2 domain-containing molecules. J. Biol. Chem. 273, 23410–23418 (1998).
Ito, N., Huang, K. & Claesson-Welsh, L. Signal transduction by VEGF receptor-1 wild type and mutant proteins. Cell Signal. 13, 849–854 (2001).
Heymans, S. et al. Inhibition of plasminogen activators or matrix metalloproteinases prevents cardiac rupture but impairs therapeutic angiogenesis and causes cardiac failure. Nat. Med. 5, 1135–1142 (1999).
Thuringer, D., Maulon, L. & Frelin, C. Rapid transactivation of the vascular endothelial growth factor receptor KDR/Flk-1 by the bradykinin B2 receptor contributes to endothelial nitric-oxide synthase activation in cardiac capillary endothelial cells. J. Biol. Chem. 277, 2028–2032 (2002).
Lowes, V.L., Ip, N.Y. & Wong, Y.H. Integration of signals from receptor tyrosine kinases and G protein- coupled receptors. Neurosignals 11, 5–19 (2002).
Wang, Y. et al. Interplay between integrins and FLK-1 in shear stress-induced signaling. Am. J. Physiol. Cell. Physiol. 283, C1540–C1547 (2002).
Gingras, D., Lamy, S. & Beliveau, R. Tyrosine phosphorylation of the vascular endothelial-growth-factor receptor-2 (VEGFR-2) is modulated by Rho proteins. Biochem. J. 348, 273–280 (2000).
Carmeliet, P. et al. Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature 380, 435–439 (1996).
Davis-Smyth, T., Presta, L.G. & Ferrara, N. Mapping the charged residues in the second immunoglobulin-like domain of the vascular endothelial growth factor/placenta growth factor receptor Flt-1 required for binding and structural stability. J. Biol. Chem. 273, 3216–3222 (1998).
Cunningham, S.A., Arrate, M.P., Brock, T.A. & Waxham, M.N. Interactions of FLT-1 and KDR with phospholipase C gamma: identification of the phosphotyrosine binding sites. Biochem. Biophys. Res. Commun. 240, 635–639 (1997).
Songyang, Z. et al. SH2 domains recognize specific phosphopeptide sequences. Cell 72, 767–778 (1993).
Wiesmann, C. et al. Crystal structure at 1.7 A resolution of VEGF in complex with domain 2 of the Flt-1 receptor. Cell 91, 695–704 (1997).
Iyer, S. et al. The crystal structure of human placenta growth factor-1 (PGF-1), an angiogenic protein, at 2.0 A resolution. J. Biol. Chem. 276, 12153–12161 (2001).
Landgren, E., Schiller, P., Cao, Y. & Claesson-Welsh, L. Placenta growth factor stimulates MAP kinase and mitogenicity but not phospholipase C-gamma and migration of endothelial cells expressing Flt 1. Oncogene 16, 359–367 (1998).
Shevchenko, A., Wilm, M., Vorm, O. & Mann, M. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal. Chem. 68, 850–858 (1996).
Puskas, L.G., Zvara, A., Hackler, L., Jr. & Van Hummelen, P. RNA amplification results in reproducible microarray data with slight ratio bias. Biotechniques 32, 1330–1334 (2002).
The authors thank G. Breier for VEGF receptor constructs; S. Suarez (Switzerland), P. Schaeffer (Synthélabo), N. Meyer (Ulm), M. Tucci (Geymonat), M. Röttgen (Basel), and K. Bijnens, A. Bouché, K. Clijsters, S. De Cat, K. De Roover, E. Gils, B. Hermans, S. Jansen, L. Kieckens, Y.W. Man, A. Manderveld, K. Maris, T. Vancoetsem, A. Vandenhoeck, A. Van den Boomen, P. Van Wesemael and S. Wyns (Leuven) for assistance; and B. Nürnberg for discussion. This work was supported in part by the European Union (Biomed BMH4-CT98-3380), Actie Levenslijn (#7.0019.98), European Union (Fifth Framework QLRT-2001-01955), Fonds voor Wetenschappelijk Onderzoek (#G012500), Associazone Italiana per la Ricerca sul Cancro, Istituto Superiore di Sanità (Programma Nazionale AIDS 1998), Deutsche Forschungsgemeinschaft (SFB451/B1, SFB497/C1, SPP1069/Do688/1-1 and Wa734/6-2), Hauptabteilung fur die Sicherheit der Kernanlagen der Schweiz (#65680), Ulm University Medical Center (#P.570) and the European Molecular Biology Organization (fellowship to M.A). David Communi and Didier Communi are Research Associates at the Fonds National pour la Recherche Scientifique.
The authors declare no competing financial interests.
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Autiero, M., Waltenberger, J., Communi, D. et al. Role of PlGF in the intra- and intermolecular cross talk between the VEGF receptors Flt1 and Flk1. Nat Med 9, 936–943 (2003). https://doi.org/10.1038/nm884
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