Characterization of Biomechanical Properties of Primary Endothelial Cells Exposed to Shear Stress

Abstract

Mechanotransduction, the process where cells convert biophysical signals into chemical signals, directs the behavior and function of endothelial cells (ECs) because of their unique position on the luminal surface of blood vessels. ECs are in direct contact with blood and can sense changes in shear stress caused by alterations in flow rate. To investigate the biophysical implications of this response we recapitulated physiological levels of shear stress in vitro using two systems, imaged and measured the consequent cytoskeletal changes with immunofluorescence, and quantified cortical stiffness distributions. We developed a sampling method for Atomic Force Microscopy (AFM) stiffness measurements by which force-indentation data is collected in a uniform grid (force map) over multiple cells within a monolayer. In this way, the sampling bias resulting from the selection of force-indentation measurements was reduced. Hertzian contact modeling for AFM was used to specifically isolate the cortical stiffness of the cell using a pyramidal indenter and small indentation depths (100 nm). Nonparametric statistics were used to compare the distributions of moduli from a given experiment, as the data distribution was not Gaussian. Actin filament alignment was quantified using Hough Transforms on immunofluorescence images and increased when cells were exposed to shear stress. The medians of stiffness distributions within sheared EC monolayers increased in magnitude when compared to static monolayers, yielding a method-dependent increase in cortical stiffness of up to 60%. This demonstrates that physiological levels of shear stress dramatically affect ECs morphology in a way that cannot be simulated under static conditions. We concluded that the biophysical milieu of a cell's physiological microenvironment must be recapitulated in order to represent characteristic cellular mechanical properties in vitro.