A Balance of Substrate Mechanics and Matrix Chemistry Regulates Endothelial Cell Network Assembly

Abstract

Driven by specific extracellular matrix cues, endothelial cells can spontaneously assemble into networks. Cell network assembly is, in part, dictated by both substrate stiffness and extracellular matrix chemistry; however, the balance between substrate mechanics and matrix chemistry in promoting cell network assembly is not well understood. Because both mechanics and chemistry can alter cell-substrate and cell-cell adhesion, we hypothesized that cell network assembly can be promoted on substrates that minimize cell-substrate adhesivity while promoting cell-cell connections. To investigate these hypotheses, bovine aortic endothelial cells (BAEC) were seeded on variably compliant polyacrylamide (PA) substrates derivatized with type I collagen and observed over time. Our results indicate that cell network assembly can be induced on substrates that are sufficiently compliant (Young's modulus, E = 200 Pa) and present significant amounts of substrate-bound ligand, and on substrates that are stiffer (E = 10,000 Pa) but which present less adhesive ligand. In both of these cases, cellsubstrate adhesivity is decreased, which may enhance cellcell adhesivity. Moreover, our data indicate that fibronectin polymerization stabilizes cell-cell contacts and is necessary for network formation to occur regardless of substrate compliance or the density of substrate-bound ligand. These data demonstrate the balance between substrate mechanics and chemistry in directing cell network assembly.

Regulation of Endothelial Cell Adherence and Elastic Modulus by Substrate Stiffness

“…In accordance to this, we observed rounded cell morphologies with diffuse actin fibers on the soft substrate while cells cultured on the hard substrate exhibited more spread resting shapes with organized actin fibers. This is in agreement with published data on the increase in spread area of endothelial cells in response to increasing substrate stiffness (Reinhart-King et al 2005;Califano & Reinhart-King 2008).…”

Vascular Morphogenesis of Human Umbilical Vein Endothelial Cells on Cell-Derived Macromolecular Matrix Microenvironment

“…ECs growing on compliant substrates prefer cell-cell connections for network formation, whereas on stiffer substrates, cells prefer to remain well-dispersed, without the formation of significant cell-cell connections. 24 We postulated that PDM and FDM might have the stiffness similar to that of BM, and thus, support the vascular morphogenesis of ECs. Interestingly enough, present measurement of average matrix elasticity of PDM (10.5 kPa) matched well with that of BM ECM that surrounds striated muscle fibers (*10 kPa).…”

Review of Collagen I Hydrogels for Bioengineered Tissue Microenvironments: Characterization of Mechanics, Structure, and Transport

“…58,86,87 ECM stiffness has also been linked with endothelial integrity and consequently regulation of angiogenesis. 13,77,88,89 The link between tissue stiffness and cell response has two major implications for tissue engineering using collagen hydrogels. For in vitro hydrogels meant to mimic specific tissues, hydrogel mechanical properties must be matched to the corresponding in vivo tissue properties in order to obtain physiologic behavior of cells in the hydrogel.…”