Age is the most significant risk factor for atherosclerosis; however, the link between age and atherosclerosis is poorly understood. During both aging and atherosclerosis progression, the blood vessel wall stiffens owing to alterations in the extracellular matrix. Using in vitro and ex vivo models of vessel-wall stiffness and aging, we show that stiffening of extracellular matrix within the intima promotes endothelial cell permeability—a hallmark of atherogenesis. When cultured on hydrogels fabricated to match the elasticity of young and aging intima, endothelial monolayers exhibit increased permeability and disrupted cell-cell junctions on stiffer matrices. In parallel experiments, we showed a corresponding increase in cell-cell junction width with age in ex vivo aortas from young (10 weeks) and old (21 to 25 months) healthy mice. To investigate the mechanism by which matrix stiffening alters monolayer integrity, we found that cell contractility increases with increased matrix stiffness, mechanically destabilizing cell-cell junctions. This increase in endothelial permeability results in increased leukocyte extravasation, which is a critical step in atherosclerotic plaque formation. Mild inhibition of Rho-dependent cell contractility using Y-27632, an inhibitor of Rho-associated kinase, or siRNA restored monolayer integrity in vitro and in vivo. Our results suggest that extracellular matrix stiffening alone, which occurs during aging, can lead to endothelial monolayer disruption and atherosclerosis pathogenesis. Because previous therapeutics designed to decrease vascular stiffness have been met with limited success, our findings could be the basis for the design of therapeutics that target the Rho-dependent cellular contractile response to matrix stiffening, rather than stiffness itself, to more effectively prevent atherosclerosis progression.

Cancer cells exist in a mechanically and chemically heterogeneous microenvironment which undergoes dynamic changes throughout neoplastic progression. During metastasis, cells from a primary tumor acquire characteristics that enable them to escape from the primary tumor and migrate through the heterogeneous stromal environment to establish secondary tumors. Despite being linked to poor prognosis, there are no direct clinical tests available to diagnose the likelihood of metastasis. Moreover, the physical mechanisms employed by metastatic cancer cells to migrate are poorly understood. Because metastasis of most solid tumors requires cells to exert force to reorganize and navigate through dense stroma, we investigated differences in cellular force generation between metastatic and non-metastatic cells. Using traction force microscopy, we found that in human metastatic breast, prostate and lung cancer cell lines, traction stresses were significantly increased compared to non-metastatic counterparts. This trend was recapitulated in the isogenic MCF10AT series of breast cancer cells. Our data also indicate that increased matrix stiffness and collagen density promote increased traction forces, and that metastatic cells generate higher forces than non-metastatic cells across all matrix properties studied. Additionally, we found that cell spreading for these cell lines has a direct relationship with collagen density, but a biphasic relationship with substrate stiffness, indicating that cell area alone does not dictate the magnitude of traction stress generation. Together, these data suggest that cellular contractile force may play an important role in metastasis, and that the physical properties of the stromal environment may regulate cellular force generation. These findings are critical for understanding the physical mechanisms of metastasis and the role of the extracellular microenvironment in metastatic progression.

Tumor microvasculature tends to be malformed, more permeable, and more tortuous than vessels in healthy tissue, effects that have been largely attributed to up-regulated VEGF expression. However, tumor tissue tends to stiffen during solid tumor progression, and tissue stiffness is known to alter cell behaviors including proliferation, migration, and cell-cell adhesion, which are all requisite for angiogenesis. Using in vitro, in vivo, and ex ovo models, we investigated the effects of matrix stiffness on vessel growth and integrity during angiogenesis. Our data indicate that angiogenic outgrowth, invasion, and neovessel branching increase with matrix cross-linking. These effects are caused by increased matrix stiffness independent of matrix density, because increased matrix density results in decreased angiogenesis. Notably, matrix stiffness up-regulates matrix metalloproteinase (MMP) activity, and inhibiting MMPs significantly reduces angiogenic outgrowth in stiffer crosslinked gels. To investigate the functional significance of altered endothelial cell behavior in response to matrix stiffness, we measured endothelial cell barrier function on substrates mimicking the stiffness of healthy and tumor tissue. Our data indicate that barrier function is impaired and the localization of vascular endothelial cadherin is altered as function of matrix stiffness. These results demonstrate that matrix stiffness, separately from matrix density, can alter vascular growth and integrity, mimicking the changes that exist in tumor vasculature. These data suggest that therapeutically targeting tumor stiffness or the endothelial cell response to tumor stiffening may help restore vessel structure, minimize metastasis, and aid in drug delivery.tumor stiffness | endothelial cells | vascular permeability | glycation | extracellular matrix T he ingrowth of newly sprouted blood vessels is necessary for solid tumor growth, and tumor vasculature is typically malformed, leakier, and more tortuous than the vasculature of normal tissues (1-3). Generally, aberrant tumor vasculature is considered to be caused by up-regulated VEGF expression resulting in chaotic vascular growth and failure to establish mature, well-regulated networks (4, 5). Here, we propose a different hypothesis, namely that extracellular matrix (ECM) mechanical properties also contribute to the aberrant vascular phenotype seen in tumors.Solid tumor tissue is typically stiffer than native, healthy tissue (1, 6). Increased ECM stiffness within tumors is caused primarily by both increased collagen deposition and increased cross-linking within the tumor stroma (7). Increased ECM density and crosslinking are associated with poor prognosis in a number of cancers (8, 9). Many studies have investigated the role of matrix density on angiogenesis and, in both collagen and fibrin matrices, have shown that angiogenesis decreases with increasing matrix concentration (10-13). Increased matrix density appears to act as a physical barrier that restricts cell migration, and cells rely on matr...

Cells generate traction stresses against their substrate during adhesion and migration, and traction stresses are used in part by the cell to sense the substrate. While it is clear that traction stresses, substrate stiffness, and cell area are related, it is unclear whether or how area and substrate stiffness affect force generation in cells. Moreover, multiple studies have investigated traction stresses of single cells, but few have focused on forces exerted by cells in contact, which more closely mimics the in vivo environment. Here, cellular traction forces were measured where cell area was modulated by ligand density or substrate stiffness. We coupled these measurements with a multilinear regression model to show that both projected cell area and underlying substrate stiffness are significant predictors of traction forces in endothelial cells, and interestingly, substrate ligand density is not. We further explored the effect of cell-cell contact on the interplay between cell area, substrate stiffness, and force generation and found that again both area and stiffness play a significant role in cell force generation. These data indicate that cellular traction force cannot be determined by cell area alone and that underlying substrate stiffness is a significant contributor to traction force generation.

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.