Matnix-bound sixth Ig-like domain of cell adhesion molecule L1 acts as an angiogenic factor by ligating αvβ3-integrin and activating VEGF-R2

Angiogenic signals can be matrix attached or freely diffusible. Here, the sixth Ig-like domain of L1 (L1Ig6), a ligand for alphavbeta3-integrin, was investigated. This domain was expressed as a fusion protein having a substrate sequence for factor XIII to enable covalent binding into three-dimensional fibrin matrices. Matrix-bound L1Ig6 induced endothelial cell (EQ process extension in vitro, which was associated with ligation and phosphorylation of alphavbeta3-integrin. VEGF-R2 and alphavbeta3 were observed to co-associate after stimulation with either L1Ig6 or VEGF-A(165), whereas no co-association with bFGF-R was observed. Furthermore, VEGF-R2 was tyrosine phosphorylated after stimulation with L1Ig6, even in the absence of exogenous VEGF-A165, indicating close cooperation between VEGF-R2 and (alphavbeta3. Angiogenesis was investigated in vivo by stimulating chicken chorioallantoic membranes (CAMs) with L1Ig6-modified matrices with or without co-incorporation of VEGF-A165 or bFGF. Matrix-immobilized L1Ig6 induced angiogenesis to a similar degree as VEGF-A165; co-stimulation with bFGF increased vascular branching, whereas VEGF-A(165) did not. Matrix-immobilized L1Ig6 induced up-regulation of alphav in CAMs by a similar degree as stimulation with VEGF-A 165, and this up-regulation was increased further by co-stimulation with matrix-bound L1Ig6 and VEGF-A(165), alpha5 and beta1 levels were not increased. The similarity of action of matrix-bound L1Ig6 and soluble VEGF-A(165) indicate a close link between specific ligation of (alphavbeta3-integrin and VEGF-R2 and suggest the possible use of matrix-bound L1Ig6 in local therapeutic angiogenesis. (C) 2004 Elsevier Inc. All fights reserved.