The Role of Endothelial Mechanosensing in Capillary Development and Organization.

Abstract

Ischemic injury is a leading cause of morbidity and mortality with the most common causes being heart attack, stroke, and peripheral artery disease. Therapies attempt to improve healing, in part, by promoting angiogenesis in these ischemic sites. Angiogenic invasion and maturation into a new capillary network may be affected by the altered microstructure and the mechanical properties of the ischemic tissue, in particular, the extracellular matrix (ECM). It is known that endothelial cells (EC) are mechanosensitive and reorient in response to both shear and normal stresses in vessels. Further, they generate traction forces and displacements in 2D culture to coordinate motion. However, the question of whether EC use cell-generated ECM forces to communicate in 3D culture to direct capillary organization and anastomosis is currently unresolved. Hydrogels formed from natural extracellular matrix (ECM) proteins readily support the formation of vasculature in vitro. The ECM is a highly ordered meshwork of various macromolecules. This anisotropic microstructure produces non-linear viscoelastic mechanical properties which confound attempts towards modeling the mechanical environment around cells. To overcome these issues, we developed a biosynthetic hydrogel consisting of polyethylene glycol diacrylamide conjugated to macromolecular type-I collagen. Through acrylamide-based cross-links, these materials allow for independent control of physical properties and bulk ligand concentration. Photoencapsulation of EC and fibroblasts within this hydrogel material and their subsequent co-culture led to the formation of capillary vessel-like networks with well-defined hollow lumens. Patterned hydrogel constructs were produced to assess angiogenic invasion independently of other stages of EC organization. ECM displacements were observed over time and mechanical modeling was used to compute cell-generated stresses and strains. We found that regions of strain exceeding 9% and stress exceeding 1,500 pico-Newtons per square micron co-localized with regions of capillary invasion (r=0.44). Thus, capillaries were found to generate stresses which propagated though the ECM. Through these studies, we developed an engineered ECM which enabled the magnitudes of cell-generated stresses during a complex 3D morphogenetic process to be quantified for the first time. These findings could yield a better understanding of the physical principles guiding capillary morphogenesis and provide new strategies for treating ischemic disease.