Control of VEGF‐Endothelial Response by ECM Stiffness

Abstract

Vascular endothelial growth factor (VEGF) drives both endothelial cell maintenance and normal/pathological angiogenesis. Recent research has suggested that mechanical signaling due to the stiffness of the cell contact surface may also have profound effects on endothelial cell behavior. To study VEGF signaling as a function of substrate stiffness, endothelial cells were cultured on fibronectin linked polyacrylamide gels and treated with VEGF. To analyze the response to VEGF we developed a custom MATLAB software script to analyze calcium flux movies. The script is able to individually identify cells, normalize each calcium signal value to the cell's individual background, and analyze the data. A variety of outputs are produced such as the magnitude of response, number of local maximums each cell experiences and clusters of coordinated cells. Utilizing this tool, we observed that VEGF‐stimulated intracellular calcium signaling varies in both magnitude and signaling kinetics depending on the stiffness of the substrate. At low VEGF concentrations we found maximal response (2–3.5 fold increases) at moderate stiffness whereas at high concentrations of VEGF the softest substrates showed the most intense response, but the response dissipated the fastest. We show that extracellular matrix stiffness can differentially drive endothelial response to VEGF. Supported by HL088572 and AHAF M2012014.