The Effects Of Three-Dimensional Matrix Stiffening On Vascular Structure And Integrity In Angiogenesis

The extracellular environment is an essential mediator of blood vessel health and provides both chemical and mechanical stimuli to influence endothelial cell behavior. While historically there has been significant emphasis placed on the chemical regulators of angiogenesis, the role of the mechanical environment is less well known. Interestingly, the mechanical properties of tissues are altered in many disease states, leading to impaired vascular function. Herein, we tune the mechanical properties of collagen-based scaffolds using nonenzymatic glycation to show that angiogenesis is differentially regulated by matrix stiffness. Importantly, our methods de-couple matrix stiffness from matrix density and fiber structure in collagen gels. Endothelial cell spreading increases with matrix stiffness, as do the number and length of angiogenic sprouts. Increased stiffness also promotes increased branching in sprouts that form from spheroids, and it disrupts endothelial barrier function. In the first steps towards translating these findings in vivo, we used a murine tumor model of stiffening to show that vascular density and the localization of mural cells are altered within murine mammary tumors where collagen cross-linking has been disrupted. Additionally, we studied breast tumors isolated from patients and found that a specific splice variant of fibronectin, a protein known to be required for neovessel formation which is typically associated with angiogenic blood vessels, is present within the vasculature of human breast tumors but not in patient-matched normal tissue. Together, these data show that the tumor vasculature is inherently different than that of normal tissue and suggest that matrix stiffness may play a role in these alterations. To study the interplay and balance between chemical factors and matrix stiffness, we developed a versatile microfluidic platform to expose cells cultured on substrates of well-characterized, tunable stiffness to well-defined, stable chemical gradients. The utility of this platform was validated by imposing a chemical gradient onto vascular smooth muscle cells to show that podosomes preferentially form upstream in a gradient. We anticipate that this device, with modifications, can be adapted for the study of angiogenesis in response to simultaneous chemical cues and matrix stiffness. Taken together, these data demonstrate that matrix stiffness regulates the formation and function of angiogenic vasculature. The data suggest that therapeutically targeting stiffness or endothelial cell response to stiffening may help maintain and restore vessel structure and function to minimize metastasis and aid in drug delivery