Substrate stiffness regulates arterial-venous differentiation of endothelial progenitor cells via the Ras/Mek pathway

Cells sense and respond to the biophysical properties of their surrounding environment by interacting with the extracellular matrix (ECM). Therefore, the optimization of these cell-matrix interactions is critical in tissue engineering. The vascular system is adapted to specific functions in diverse tissues and organs. Appropriate arterial-venous differentiation is vital for the establishment of functional vasculature in angiogenesis. Here, we have developed a polydimethylsiloxane (PDMS)-based substrate capable of simulating the physiologically relevant stiffness of both venous (7kPa) and arterial (128kPa) tissues. This substrate was utilized to investigate the effects of changes in substrate stiffness on the differentiation of endothelial progenitor cells (EPCs). As EPCs derived from mouse bone marrow were cultured on substrates of increasing stiffness, the mRNA and protein levels of the specific arterial endothelial cell marker ephrinB2 were found to increase, while the expression of the venous marker EphB4 decreased. Further experiments were performed to identify the mechanotransduction pathway involved in this process. The results indicated that substrate stiffness regulates the arterial and venous differentiation of EPCs via the Ras/Mek pathway. This work shows that modification of substrate stiffness may represent a method for regulating arterial-venous differentiation for the fulfilment of diverse functions of the vasculature.