CONTENTS

ABBREVIATIONS

LIST OF FIGURES

LIST OF TABLES

1. ABSTRACT

2. INTRODUCTION

   2.1 Growth factors and their receptors

   2.2 The interaction between RTKs and their ligands

   2.3 The vascular system

      2.3.1 The development of the vascular system

2.3.1.1 The cardiovascular system

2.3.1.2 The lymphatic vascular system

2.3.1.3 Diversity of endothelial cells

      2.3.2 Regulation of angiogenesis: Stimulatory and inhibitory factors

      2.3.3 Angiogenesis in the adult organism

   2.4 The VEGF subfamily of growth factors

      2.4.1 Vascular endothelial growth factor (VEGF)

2.4.1.1 Discovery and cloning of VEGF

2.4.1.2 Genomic structure and splice variants of VEGF

2.4.1.3 Molecular characterization

2.4.1.4 VEGF receptors

2.4.1.5 VEGF expression

2.4.1.6 VEGF receptor expression

2.4.1.7 VEGF and VEGF receptor expression in neovascular processes in the adult

2.4.1.8 Biological roles of VEGF signalling

2.4.1.9 Regulation of VEGF gene expression

      2.4.2 Placenta growth factor (PlGF)

      2.4.3 VEGF-B

2.4.3.1 Cloning of VEGF-B

2.4.3.2 Genomic structure and splice variants of VEGF-B

2.4.3.3 Molecular characterization of VEGF-B

2.4.3.4 VEGF-B receptor(s)

2.4.3.5 VEGF-B expression

2.4.3.6 Biological roles of VEGF-B signalling

      2.4.4 VEGF-C

2.4.4.1 Cloning of VEGF-C

2.4.4.2 Genomic structure and splice variants of VEGF-C

2.4.4.3 Molecular characterization of VEGF-C

2.4.4.4 VEGF-C receptors

2.4.4.5 VEGF-C and VEGF-C receptor expression

2.4.4.6 Biological roles of VEGF-C signalling

3. AIMS OF THIS WORK

4. EXPERIMENTAL PROCEDURES

   4.1 Transgenic mice over-expressing VEGF-B and VEGF-C

      4.1.1 Expression vector constructs

      4.1.2 Transgenic mice

      4.1.3 Screening: PCR, Southern blotting and dot blot analysis

      4.1.4 Analysis of the mice

   4.2 Recombinant VEGF-B and VEGF-C produced with the baculovirus system

      4.2.1 Insect cell lines and culture

      4.2.2 Generation of recombinant baculoviruses by transposon-mediated insertion in E.coli

4.2.2.1 Transfer plasmids and baculovirus shuttle vector

4.2.2.2 Transposition and transfection

      4.2.3 Generation of recombinant baculovirus by allelic replacement in insect cells

4.2.3.1 Transfer plasmid and parent virus

4.2.3.2 Co-transfection

      4.2.4 Identification, plaque purification, amplification and titration of recombinant virus

      4.2.5 Harvest and characterization of the recombinant protein

4.2.5.1 Immunoprecipitation and Western blotting

4.2.5.2 Metabolic labelling

4.2.5.3 Stimulation of receptor phosphorylation by recombinant VEGF-C

      4.2.6 Large scale production and affinity purification of recombinant VEGF-C

5. RESULTS AND DISCUSSION

   5.1 Transgenic mice

      5.1.1 -MHC-VEGF-C-FL

      5.1.2 -MHC-VEGF-B167

      5.1.3 RIP-VEGF-B167

      5.1.4 RIP-VEGF-C-FL

      5.1.5 K14-VEGF-B167

      5.1.6 K14-VEGF-C-FL

5.1.6.1 Histological analysis

5.1.6.2 In situ hybridization analysis

5.1.6.3 Immunohistochemical analysis

      5.1.7 K14-NC-VEGF-C, K14-VEGF-B186

   5.2 Recombinant VEGF-B and VEGF-C

6. BIBLIOGRAPHY

7. APPENDIX

   7.1 EMBL/GenBankª accession numbers (nucleotide sequence)

   7.2 Genomic structures and mRNA splice variants of VEGF, VEGF-B and VEGF-C

   7.3 Exon/intron borders of VEGF, VEGF-B and VEGF-C genes

   7.4 Plasmid maps

   7.5 Characterization of recombinant proteins

   7.6 Pedigrees of transgenic mice

8. ACKNOWLEDGEMENTS