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Substrate stiffness heterogeneities disrupt endothelial barrier integrity in a micropillar model of heterogeneous vascular stiffening.

ABSTRACT: Intimal stiffening has been linked with increased vascular permeability and leukocyte transmigration, hallmarks of atherosclerosis. However, recent evidence indicates age-related intimal stiffening is not uniform but rather characterized by increased point-to-point heterogeneity in subendothelial matrix stiffness, the impact of which is much less understood. To investigate the impact of spatially heterogeneous matrix rigidity on endothelial monolayer integrity, we develop a micropillar model to introduce closely-spaced, step-changes in substrate rigidity and compare endothelial monolayer phenotype to rigidity-matched, uniformly stiff and compliant substrates. We found equivalent disruption of adherens junctions within monolayers on step-rigidity and uniformly stiff substrates relative to uniformly compliant substrates. Similarly, monolayers cultured on step-rigidity substrates exhibited equivalent percentages of leukocyte transmigration to monolayers on rigidity-matched, uniformly stiff substrates. Adherens junction tension and focal adhesion density, but not size, increased within monolayers on step-rigidity and uniformly stiff substrates compared to more compliant substrates suggesting that elevated tension is disrupting adherens junction integrity. Leukocyte transmigration frequency and time, focal adhesion size, and focal adhesion density did not differ between stiff and compliant sub-regions of step-rigidity substrates. Overall, our results suggest that endothelial monolayers exposed to mechanically heterogeneous substrates adopt the phenotype associated with the stiffer matrix, indicating that spatial heterogeneities in intimal stiffness observed with age could disrupt endothelial barrier integrity and contribute to atherogenesis.

SUBMITTER: VanderBurgh JA

PROVIDER: S-EPMC6301132 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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Substrate stiffness heterogeneities disrupt endothelial barrier integrity in a micropillar model of heterogeneous vascular stiffening.

VanderBurgh Jacob A JA Hotchkiss Halie H Potharazu Archit A Taufalele Paul V PV Reinhart-King Cynthia A CA

Integrative biology : quantitative biosciences from nano to macro 20181201 12

Intimal stiffening has been linked with increased vascular permeability and leukocyte transmigration, hallmarks of atherosclerosis. However, recent evidence indicates age-related intimal stiffening is not uniform but rather characterized by increased point-to-point heterogeneity in subendothelial matrix stiffness, the impact of which is much less understood. To investigate the impact of spatially heterogeneous matrix rigidity on endothelial monolayer integrity, we develop a micropillar model to ...[more]

PMID: 30382278

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Project description:Fibrosis is associated with aging and many cardiac pathologies. It is characterized both by myofibroblast differentiation and by excessive accumulation of extracellular matrix proteins. Fibrosis-related tissue remodeling results in significant changes in tissue structure and function, including passive mechanical properties. This research area has gained significant momentum with the recent development of new tools and approaches to better characterize and understand the ability of cells to sense and respond to their biophysical environment. We use a novel hydrogel, termed CyPhyGel, to provide an advanced in vitro model of remodeling-related changes in tissue stiffness. Based on light-controlled dimerization of a Cyanobacterial Phytochrome, it enables contactless and reversible tuning of hydrogel mechanical properties with high spatial and temporal resolution. Human primary atrial fibroblasts were cultured on CyPhyGels. After 4 days of culturing on stiff (~4.6 kPa) or soft (~2.7 kPa) CyPhyGels, we analyzed fibroblast cell area and stiffness. Cells grown on the softer substrate were smaller and softer, compared to cells grown on the stiffer substrate. This difference was absent when both soft and stiff growth substrates were combined in a single CyPhyGel, with the resulting cell areas being similar to those on homogeneously stiff gels and cell stiffnesses being similar to those on homogeneously soft substrates. Using CyPhyGels to mimic tissue stiffness heterogeneities in vitro, our results confirm the ability of cardiac fibroblasts to adapt to their mechanical environment, and suggest the presence of a paracrine mechanism that tunes fibroblast structural and functional properties associated with mechanically induced phenotype conversion toward myofibroblasts. In the context of regionally increased tissue stiffness, such as upon scarring or in diffuse fibrosis, such a mechanism could help to prevent abrupt changes in cell properties at the border zone between normal and diseased tissue. The light-tunable mechanical properties of CyPhyGels and their suitability for studying human primary cardiac cells make them an attractive model system for cardiac mechanobiology research. Further investigations will explore the interactions between biophysical and soluble factors in the response of cardiac fibroblasts to spatially and temporally heterogeneous mechanical cues.

Dataset Information

Substrate stiffness heterogeneities disrupt endothelial barrier integrity in a micropillar model of heterogeneous vascular stiffening.

Publications

Substrate stiffness heterogeneities disrupt endothelial barrier integrity in a micropillar model of heterogeneous vascular stiffening.

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