Spacing of Integrin Ligands Influences Signal Transduction in Endothelial Cells

The physical attributes of the extracellular matrix play a key role in endothelium function by modulating the morphology and phenotype of endothelial cells. Despite the recognized importance of matrix-cell interactions, it is currently not known how the arrangement of adhesive ligands affects the morphology, signal transduction processes, and migration of endothelial cells. We aimed to study how endothelial cells respond to the average spatial arrangement of integrin ligands. We designed functionalized silicon surfaces with average spacing ranging from nanometers to micrometers of the peptide arginine-glycine-aspartic acid (RGD). We found that endothelial cells adhered to and spread on surfaces independently of RGD-to-RGD spacing. In contrast, organization within focal adhesions (FAs) was extremely sensitive to ligand spacing, requiring a nanoscaled average RGD spacing of 44 nm to form lipid raft domains at FAs. The localized membrane organization strongly correlated with the signaling efficiencies of integrin activation and regulated vascular endothelial growth factor (VEGF)-induced signaling events. Importantly, this modulation in signal transduction directly affected the migratory ability of endothelial cells. We conclude that endothelial cells sense nanoscaled variations in the spacing of integrin ligands, which in turn influences signal transduction processes. Average RGD spacing similar to that found in fibronectin leads to lipid raft accumulation at FAs, enhances sensitivity to VEGF stimulation, and controls migration in endothelial cells.