VEGFR-3VEGFR-3 LigandsLigands andand LymphangiogenesisLymphangiogenesis HelsinkiHelsinki 20022002 Michael Michael JeltschJeltsch VEGFR-3 Ligands and Lymphangiogenesis Michael Jeltsch Molecular/Cancer Biology Laboratory Biomedicum Helsinki and Haartman Institute Department of Biosciences, Division of Biochemistry Faculty of Science University of Helsinki Finland Academic dissertation To be publicly discussed with the permission of the Faculty of Science of the University of Helsinki in the lecture hall 3, Biomedicum Helsinki, Haartmaninkatu 8, Helsinki, on November 29th, 2002, at 12:00. Helsinki 2002 Supervised by Dr. Kari Alitalo Molecular/Cancer Biology Laboratory University of Helsinki Reviewed by Dr. Jussi Taipale School of Medicine, Department of Molecular Biology and Genetics The Johns Hopkins University and Dr. Lena Claesson-Welsh Institute for Genetics and Pathology University of Uppsala Thesis opponent Dr. Carl-Henrik Heldin Ludwig Institute for Cancer Research, Uppsala Branch ISBN 952-91-5016-4 (printed version) ISBN 952-10-0651-X (PDF) ISBN 952-10-0652-8 (HTML) http://ethesis.helsinki.fi Yliopistopaino Helsinki 2002 Contents List of Figures........................................................................................................................... 4 Abbreviations ........................................................................................................................... 5 List of Original Publications................................................................................................... 6 Abstract..................................................................................................................................... 7 Review of the Literature.......................................................................................................... 8 1 The Two Vascular Systems............................................................................................... 8 2 The Cardiovascular System.............................................................................................. 8 3 Form and Function of the Lymphatic System .................................................................. 9 3.1 Drainage ................................................................................................................................... 11 3.2 Immune Defence ...................................................................................................................... 11 3.3 Intestinal Absorption ................................................................................................................ 11 3.4 Lymph Formation ..................................................................................................................... 12 3.5 Lymph Propulsion .................................................................................................................... 12 3.6 Pre-lymphatic Channels ........................................................................................................... 12 4 Development of the Lymphatic System ......................................................................... 13 4.1 Centrifugal Sprouting ............................................................................................................... 14 4.2 Centripetal Sprouting................................................................................................................ 14 5 Lymphatics in Different Vertebrates............................................................................... 14 5.1 Fish ........................................................................................................................................... 14 5.2 Amphibians and Reptiles.......................................................................................................... 15 5.3 Birds and Mammals.................................................................................................................. 15 6 Pathology........................................................................................................................ 16 6.1 Blood Vessels and Lymphatic Vessels in Disease .................................................................... 16 6.2 Lymphedema ............................................................................................................................ 16 6.2.1 Hereditary Lymphedema Type I and II................................................................................ 17 6.2.2 Lymphedema-Distichiasis Syndrome.................................................................................. 17 6.2.3 Lymphangiectasia ................................................................................................................ 17 6.3 Lymphatic Neoplasms .............................................................................................................. 17 6.4 Tumor Lymphangiogenesis ...................................................................................................... 18 6.5 Therapeutic Lymphangiogenesis.............................................................................................. 18 7 VEGFs............................................................................................................................ 18 7.1 VEGF - Master Effector of Angiogenesis ................................................................................ 19 7.2 PlGF, VEGF-B and Other Problem Children ........................................................................... 24 7.3 The Lymphangiogenic Factors: VEGF-C and VEGF-D .......................................................... 25 7.3.1 Molecular Features of VEGF-C and VEGF-D .................................................................... 25 7.3.2 Structure of VEGFR-3 and Its Ligands ............................................................................... 26 7.3.3 VEGF-C Expression and Its Regulation.............................................................................. 27 7.3.4 VEGF-C Signaling .............................................................................................................. 27 Outline of the Study............................................................................................................... 29 Materials and Methods.......................................................................................................... 30 Results and Discussion........................................................................................................... 32 1 VEGF-C is Lymphangiogenic........................................................................................ 32 2 Mature VEGF-C is Lymphangiogenic in the Chick CAM............................................. 32 3 VEGF-D is a Ligand for VEGFR-2 and VEGFR-3 ....................................................... 33 4 Uncoupling of Receptor Binding from Specificity Allows to Create a Super-VEGF.... 34 Acknowledgements ................................................................................................................ 36 References............................................................................................................................... 37 List of Figures Abbreviations List of Figures Figure 1. Schematic view of the lymphatic system 10 Figure 2. VEGF family members and their receptors 19 Figure 3. Alignment of selected VEGFs 21 Figure 4. Structure of VEGFs: Uncoupling receptor binding and specificity 23 4 5 List of Figures Abbreviations Abbreviations Akt Oncogene from AKR thymoma mouse Bcl-2 B-cell lymphomal leukemia oncoprotein BR3P Balbiani ring 3 protein CAM Chorioallantoic membrane E Embryonic day EpoR Erythropoietin receptor FGF Fibroblast growth factor FOXC2 Winged helix/forkhead transcription factor subclass C member 2 Grb2 Growth factor receptor-bound protein 2 kDa Kilodalton MAPK Mitogen-activated protein kinase OMIM Online Mendelian inheritance in men PDGF Platelet-derived growth factor PDGFR Platelet-derived growth factor receptor PI3-K Phosphatidyl inositol-3-kinase PKC Protein kinase C PLCγ Phospholipase C-gamma PlGF Placenta growth factor Ras p21 oncoprotein from rat sarcoma Shc Src homology and collagen domain STAT Signal transducer and activators of transcription VEGF Vascular endothelial growth factor VEGFR Vascular endothelial growth factor receptor VHD VEGF homology domain 4 5 List of Original Publications Abstract List of Original Publications This thesis is based on the following articles, which will be referred to by their Roman numer- als. I. Jeltsch, M.*, Kaipainen, A.*, Joukov, V., Meng, X., Lakso, M., Rauvala, H., Swartz, M., Fukumura, D., Jain, R. K. and Alitalo, K. (1997): Hyperplasia of lymphatic ves- sels in VEGF-C transgenic mice [published erratum appeared 1997 in Science 277, 463]. Science 276, 1423-1425. II. Oh, S. J., Jeltsch, M. M., Birkenhager, R., McCarthy, J. E., Weich, H. A., Christ, B., Alitalo, K. and Wilting, J. (1997): VEGF and VEGF-C: specific induction of angiogenesis and lymphangiogenesis in the differentiated avian chorioallantoic membrane. Developmental Biology 188, 96-109. III. Achen, M. G., Jeltsch, M., Kukk, E., Makinen, T., Vitali, A., Wilks, A. F., Alitalo, K. and Stacker, S. A. (1998): Vascular endothelial growth factor D (VEGF-D) is a ligand for the tyrosine kinases VEGF receptor 2 (Flk1) and VEGF receptor 3 (Flt4). Proceedings of the National Academy of Science of the USA 95, 548-553. IV. Jeltsch, M., Karpanen, T. and Alitalo, K. (manuscript submitted, 2002): Identification