Krilleke, D.; (2006) Structural and functional analysis of the heparin-binding domain of VEGF164. Doctoral thesis , University of London.

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Abstract

Several of the multitude of functions attributed to Vascular Endothelial Growth Factor-A (VEGF-A) are coordinated by its various isoforms, which are generated as a result of alternative splicing from a single gene. Despite the fact that the VEGF isoforms exhibit distinct biochemical properties, little has been done to clarify their functions and their contributions to physiological and pathological processes. In this thesis I describe the biochemical and biological characterization of the heparin-binding domain of mouse VEGF 164 through structure-function analysis. To investigate the functional significance of heparin binding, mutations were introduced into exon 7 of VEGF 164 to identify essential residues for heparin binding. Three key amino acids involved in this interaction were identified. Mutants with alterations in these amino acids were unable to bind heparin and were compromised in their ability to bind to cell-surface heparan sulfate. These mutants, however, retained wild-type like potency in inducing tissue factor expression in vitro and microvessel growth ex vivo, and maintained the capacity to bind to the receptors neuropilin-1, VEGFR-1, and VEGFR-2. A second goal of this work was to better define the role of VEGF 164 in mediating inflammatory processes during pathological vascularization of the retina. Analysis of VEGF 164-deficient (VEGF1ZU/ iao) mice subjected to neovascularization-inducing conditions and rats injected intravitreally with recombinant VEGF variants demonstrated that endogenous and exogenous VEGF 164 increases leukocyte adhesiveness to retinal vessels compared to the non-heparin-binding isoform, VEGF 120. Interestingly, the three basic residues that confer heparin binding of VEGF 164, appear to be critical for its pro inflammatory activity, but not for its angiogenic activity. In addition, both mutants (and VEGF 120) exhibited a reduced affinity for VEGFR-1, a leukocyte-expressed receptor that mediates VEGF-induced migration. Results of in vivo experiments using P1GF, VEGF-E, as well as a VEGFR-1 neutralizing antibody, further demonstrate a role for VEGFR-1 in retinal leukostasis.

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