Cells sense and respond to the physical attributes of their local environment, a concept embodied by the terms mechanosensing and mechanotransduction. Advances in surface chemistry and nanotechnology have provided unique insights into the ability of cells to adjust their shape and motility to minute changes in the chemical and physical features of their immediate surroundings (1). A remarkable discovery is that cells can sense nanometer-scale variations in the average spacing of randomly organized integrin ligands (2,3). Fibroblasts adhere, migrate, and proliferate on surfaces with average spacings of the tripeptide arginine-glycine-aspartic acid (RGD) of <70 nm, whereas they adhere poorly and migrate erratically when integrin ligands are spaced farther apart (2). Importantly, the 10–200 nm scale of average ligand spacing is physiologically relevant because the nanoscaled and periodic spacing is similar to that found in fibronectin and collagen fibers (4,5,6).
The concept of mechanotransduction appears to be particularly relevant for endothelial cells. Interactions between endothelial cells and the extracellular matrix (ECM) control many vascular processes (7), including permeability (8), sensitivity to growth factors (e.g., responsiveness to vascular endothelial growth factor (VEGF) stimulation (9)), and transformation into a proliferative and invasive phenotype that is characteristic of angiogenesis (10). However, although the importance of cell-matrix interactions for the functioning of the endothelium is recognized, little is known about how fundamental physical features of the matrix, such as the average spacing of integrin ligands, affects the behavior of endothelial cells.
Endothelial adhesion to the ECM is facilitated by integrins (11). Engaged integrins cluster together with cytoskeletal and signaling proteins to form focal adhesions (FAs) and complexes (11). These complexes control a range of cell activation responses, including cell polarization and migration, membrane trafficking, cell cycle progression, gene expression, and oncogenic transformation (7,12,13,14). Signaling at FAs also includes VEGF-induced intracellular calcium fluxes, activation of phosphatydylinositol-3 (PI3) kinase and mitogen-activated protein (MAP) kinases, and, further downstream, activation of endothelial nitric oxide synthase (eNOS) (15). Curiously, although integrins have no intrinsic enzymatic activity (14), in many cases they enable growth factor signals, that is, growth factor signaling does not occur unless integrins are occupied (9,10). Hence, VEGF and integrin form a functional partnership in endothelial cells; however, how integrin spacing and FA organization influence VEGF signaling is currently not known.
The exposure of endothelial cells to RGD peptides, which are found in fibronectin and recognized by the integrins αvβ3 and α5β1, sensitizes endothelial cells to angiogenic transformation (10,16). In this study, we sought to determine how endothelial cells respond to the average spacing of randomly distributed RGD ligands by assessing FAs, integrin activation, and VEGF-induced signaling and migration. To that end, we functionalized silicon surfaces with average RGD spacing from nano- to micrometers. We found that nanoscaled variations in integrin ligand spacing govern membrane order within FAs, which in turn determines signaling efficiency and cell migration. Taken together, our results suggest that the spatial arrangement of the local cellular environment may significantly contribute to the proangiogenic behavior of endothelial cells.
Guillaume Le Saux†, ‡, Astrid Magenau†, Krishanthi Gunaratnam†, Kristopher A. Kilian‡, ¶, Till Böcking‡, §, J. Justin Gooding‡, , and Katharina Gaus†,
