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New prospects in the roles of the C-terminal domains of VEGF-A and their cooperation for ligand binding,cellular signaling and vessels formation.

Delcombel R, Janssen L, Vassy R, Gammons M, Haddad O, Richard B, Letourneur D, Bates D, Hendricks C, Waltenberger J, Starzec A, Sounni NE, Noël A,Deroanne C, Lambert C, Colige A.

Angiogenesis. 2013 Apr;16(2):353-71.

 

Laboratory of Connective Tissues Biology, GIGA-R, University of Liege, Avenue de l’Hopital 3, 4000, Liege, Belgium, [email protected].

 

Abstract

VEGF-A is a crucial growth factor for blood vessel homeostasis and pathological angiogenesis. Due to alternative splicing of its pre-mRNA, VEGF-A is produced under several isoforms characterized by the combination of their C-terminal domains, which determines their respective structure, availability and affinity for co-receptors. As controversies still exist about the specific roles of these exon-encoded domains, we systematically compared the properties of eight natural and artificial variants containing the domains encoded by exons 1-4 and various combinations of the domains encoded by exons 5, 7 and 8a or 8b. All the variants (VEGFa, VEGFb, VEGFa, VEGFb, VEGFa, VEGFb, VEGFa, VEGFb) have a similar affinity for VEGF-R2, as determined by Surface plasmon resonance analyses. They strongly differ however in terms of binding to neuropilin-1 and heparin/heparan sulfate proteoglycans. Data indicate that the 6 amino acids encoded by exon 8a must be present and cooperate with those of exons 5 or 7 for efficientbinding, which was confirmed in cell culture models. We further showed that VEGFb has inhibitory effects in vitro, as previously reported, but that the shortest VEGF variant possessing also the 6 amino acids encoded by exon 8b (VEGFb) is remarkably proangiogenic, demonstrating the critical importance of domain interactions for defining the VEGF properties. The number, size and localization of newly formed blood vessels in a model of tumour angiogenesis strongly depend also on the C-terminal domain composition, suggesting that association of several VEGF isoforms may be more efficient for treating ischemic diseases than the use of any single variant.

 

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Additional Information

 

VEGF-A, a major regulator of angiogenesis, is involved in physio-pathological processes such as development, menstrual cycle, wound healing, cancer and several eyes diseases. Its expression is regulated at the transcriptional level by activation of its promoter but also by post-translational mechanisms, mainly in hypoxic conditions. Alternative splicing of the pre-mRNA is also a potent regulator of VEGF-A activity as it induces the production of several VEGF isoforms possessing specific properties. While the role of the constant amino-terminal portion encoded by exons 1 to 4 (E1-E4) is currently well defined, the functions of the alternative carboxy-terminal domains (E5-E8a/E8b) have to be deeply characterized in order to better understand why the different VEGF variants have very specific properties regarding the regulation of angiogenesis.

 

Our work, aimed at better defining the roles and potential cooperation of the domains encoded by the E5 to E8a/E8b terminal exons. First, we produced and purified relevant VEGF variants: VEGF111a (E1-E4, E8a), VEGF111b (E1-E4, E8b), VEGF121a (E1-E4, E5, E8a), VEGF121b (E1-E4, E5, E8b), VEGF155a (E1-E4, E7, E8a), VEGF155b (E1-E4, E7, E8b), VEGF165a (E1-E4, E5, E7, E8a) and VEGF165b (E1-E4, E5, E7, E8b).

 

We then performed a characterization at a biochemical level by determining their affinity to VEGF (co-)receptors. All variants have a similar affinity for VEGF-R2, as determined by Surface Plasmon Resonance analyses. They strongly differ however in terms of binding to neuropilin-1 and heparin/heparin sulfate proteoglycans.  Data indicate that the 6 amino acids encoded by E8a must be present and cooperate with those of E5, newly identified binding site in this work, or E7 for efficient binding. Studying their resistance to proteolysis, we highlighted new cleavage sites for plasmin hosted in E7 encoded domain.

 

Finally we evaluated of their role on angiogenesis by measuring their effects on various endothelial cell types in vitro. These data confirmed effect observed for (co-)receptors binding. We further showed that VEGF165b has inhibitory effects in vitro, as previously reported, but that the shortest VEGF variant possessing also the 6 amino acids encoded by E8b (VEGF111b) is remarkably pro-angiogenic, demonstrating the critical importance of domain interactions for defining the VEGF properties. On tumoural angiogenesis in vivo model, the number, size and localization of newly formed blood vessels strongly depend also on the C-terminal domain composition, suggesting that association of several VEGF isoforms may be more efficient for treating ischemic diseases than the use of any single variant.

 

New prospects in the roles of the C-terminal domains of VEGF-A