Tissue assembly is a fundamental biological process that arises from complex cell-cell and cell-extracellular matrix interactions. Angiogenesis is the process of capillary formation that enables normal physiological responses like wound healing and mediates disease states like tumorigenesis. During angiogenesis, capillary endothelial cells degrade the basement membrane, proliferate, migrate, and assemble a new vascular network. While there is much focus on growth factor signaling cascades that enable angiogenesis, less attention has been paid to the role of mechanics in capillary formation. Notably, capillary network assembly has been demonstrated on compliant, but not stiff, substrates suggesting that the mechanical microenvironment also mediates angiogenesis. However, it is unknown whether, or how, substrate stiffness regulates capillary network assembly.;Herein, we demonstrate that substrate stiffness regulates capillary network assembly and mediates endothelial cell behaviors that enable assembly. Compliant (E1 kPa), substrates promote the self-assembly of endothelial cell networks that result from a balance of cell-cell and cell-matrix adhesion. Substrate stiffness alters the localization of VE-cadherin and focal adhesions, mediators of endothelial cell-cell and cell-matrix adhesion, respectively. Endothelial network assembly also requires polymerization of the matrix protein fibronectin that stabilizes cell-cell interactions. Analogously, we demonstrate that mammary cell network assembly is also sensitive to substrate stiffness and requires the deposition of laminin. Our findings indicate that compliant substrates foster network assembly by promoting cell-cell adhesion, cell-matrix interactions, and reducing cell-matrix adhesion.;We further investigate the role of substrate stiffness in mediating changes in cell shape and contractility. We determine that substrate stiffness and ligand density alter cell area, and that both stiffness and cell area are significant predictors of traction force generation in endothelial cells during cell-cell contact. In addition, we demonstrate that substrate stiffness alters the synthesis and deposition of fibronectin and extra domain B-fibronectin, an isoform preferentially localized to neovasculature, by modulating cell shape and the directionality of traction forces in endothelial cells.;Taken together, these data demonstrate that substrate stiffness regulates capillary network assembly by altering endothelial cell behaviors that facilitate assembly. These findings contribute to the understanding of how the mechanical microenvironment regulates capillary network assembly and enable approaches to control angiogenesis for therapeutic use.
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