Multiscale Stress Analysis of Sheared and Focally-Adhered Endothelial Cells: Role of Subcellular Matrix Moduli in Focal Adhesion-Mediated Signaling

Abstract

Focal adhesions (FAs) and their associated integrins are thought to act as mechanosensors and transducers of shear stress into intracellular biochemical signals. However, to date there exists no quantification of the magnitude of forces generated at integrin molecules in response to apically-applied fluid shear stress. Thus, we used finite element analysis of fluid dynamics and cellular stresses to compute FA stresses from solid models of focally-adhered endothelial cells. These models were developed from quantitative 3-D microscopy and total internal reflection fluorescence (TIRF) microscopy of calcein-stained endothelial cells. Extrusion coupling variables mapped stresses from the macroscale cell model to individual microscale 3-D models of FAs determined from quantitative TIRF. Included in the microscale FA model were moduli for subcellular matrix (SCM) (e.g. hyaluronan, hyaluronaic acid and other glycocalyx constituents) and extracellular matrix (ECM) (e.g. collagen, fibronectin). Integrin forces were estimated from assumed bonds densities and computed FA stresses. Maximal bond tension obtained from the simulation for a single integrin-extracellular matrix (ECM) bond was .1pN. Thus, it is unlikely that integrin-ECM bonds or chemical activities are appreciably affected by shear stress. The computational model, however, supports an alternative model of activation in and reorganization of FAs in which shear stress-induced forces cause the membrane to bend toward and away from the ECM immediately upstream and downstream of the FA, respectively. The simulation also suggests that the elasticity of the SCM plays an important role in modulating shear-induced FA reorganization. These results support a new model of endothelial cell activation by shear stress in which integrins and FAs participate in the directional biasing of force-induce signaling but do not initiate it.