Substrate stiffening promotes endothelial monolayer disruption through enhanced physical forces

Area of Science:

• Biophysics
• Cell Biology
• Vascular Biology

Background:

• Vascular leakage is a hallmark of inflammatory diseases, driven by endothelial cell (EC) monolayer integrity.
• EC monolayer integrity depends on cell-cell/substrate contacts, soluble mediators, and biomechanical forces.
• The role of substrate stiffness in mediating these interactions remains largely uninvestigated.

Purpose of the Study:

• To investigate how substrate stiffness influences endothelial monolayer disruption.
• To determine the role of cell-cell/substrate contacts, soluble mediators, and physical forces in stiffness-dependent disruption.

Main Methods:

• Utilized traction force microscopy to measure forces between endothelial cells and between cells and the substrate.
• Applied a hyperpermeability stimulus (thrombin) to assess force modulation and gap formation on substrates of varying stiffness.
• Investigated the involvement of Rho kinase signaling pathway.

Main Results:

• Greater cell-cell and cell-substrate forces were observed on stiffer substrates.
• Thrombin significantly enhanced these forces and induced inter-endothelial cell gaps on stiffer substrates, but not on softer ones.
• Increased Rho kinase activity correlated with stiffness-dependent force enhancement; its inhibition reduced baseline forces and thrombin-induced gap formation.

Conclusions:

• Substrate stiffness plays a critical role in endothelial monolayer integrity by modulating physical forces.
• Stiffness-dependent enhancement of physical forces contributes to vascular leakage.
• This study highlights a novel mechanism where the physical microenvironment, specifically substrate stiffness, influences endothelial barrier function.