Endothelialization of blood-contacting devices requires biochemical and biomechanical cues to support stable endothelial monolayers. During transanastomotic endothelialization, endothelial cells migrate from compliant native vessels onto stiffer device surfaces, experiencing changing mechanical environments. The present study examined how substrate stiffness and prior mechanical exposure influence temporal remodeling of collagen-binding integrins and downstream endothelialization processes on bioactive hydrogels.
Human coronary artery endothelial cells (HCECs) were cultured on bioactive hydrogels engineered to span physiologically relevant stiffness ranges. The design aimed to model the mechanical transition cells undergo when moving from compliant native tissues to stiffer blood-contacting materials. The investigators measured expression of collagen-binding integrins, focal adhesion maturation via vinculin association, and cellular behaviors important for endothelialization: proliferation, migration, progression to confluence, and transition to quiescence.
A principal finding was that increasing substrate stiffness led to a time-dependent upregulation of a2b1 integrin expression in HCECs. In contrast, expression of a1b1 integrin did not change with substrate stiffness. The differential regulation indicates that distinct collagen-binding integrins respond differently to mechanical cues, with a2b1 showing particular sensitivity to the rigidity of the substrate.
Upregulation of a2b1 on stiffer hydrogels was accompanied by enhanced focal adhesion maturation, as evidenced by increased vinculin-associated adhesion structures. These changes at the adhesion complex level correlated with accelerated endothelialization behavior: cells on stiffer substrates exhibited increased proliferation and migration, progressed to confluence more rapidly, and reached a quiescent state after one week. Taken together, the data indicate that a stiffer extracellular mechanical environment promotes both integrin-mediated adhesion maturation and the cellular behaviors necessary to establish a continuous endothelial layer.
To better model transanastomotic migration and probe the influence of prior mechanical exposure, the authors performed transfer experiments in which cells initially expanded on compliant hydrogels were moved onto stiff substrates. These transferred cells showed transient reductions in a2b1 expression at early timepoints when compared with cells expanded on tissue culture polystyrene prior to seeding on experimental substrates. Importantly, these differences in integrin expression were not persistent: transferred cells ultimately showed no sustained differences in focal adhesion maturation, proliferation, migration, confluence, or timing of quiescence relative to controls on stiff substrates.
This outcome indicates that while mechanical history can transiently modulate integrin expression, the current mechanical environment exerts the dominant influence on adhesion complex maturation and the functional endothelialization processes required to form a stable monolayer.
The study demonstrates that substrate stiffness is a primary regulator of early endothelialization processes on bioactive hydrogels. Although mechanical history (prior expansion on compliant substrates) produced short-lived effects on a2b1 levels, these effects were superseded by the stiffness of the new substrate and did not alter longer-term adhesion maturation or functional outcomes. Based on these observations, the authors propose that targeting a2b1-mediated mechanotransduction could be a rational design strategy for blood-contacting biomaterials to promote rapid endothelial coverage while permitting eventual endothelial quiescence.
This report is presented as a preprint and has not been certified by peer review. Specific experimental details such as exact stiffness values, time-resolved quantitative metrics, and methodological parameters are contained in the full manuscript but are not reproduced in this summary of the abstract. The work lists funding from the National Institutes of Health (R01 HL180615). The authors declared no competing interests.
Collectively, the findings clarify how current substrate mechanics shape integrin-dependent adhesion and endothelialization and identify a2b1 as a key mechanosensitive integrin relevant to the design of blood-contacting hydrogels and devices.