Orientation of apical and basal actin stress fibers in isolated and subconfluent endothelial cells as an early response to cyclic stretching

Abstract

We investigated the response of apical and basal actin stress fibers (SFs) and its dependency on cell confluency for endothelial cells subjected to cyclic stretching. Porcine aortic endothelial cells from the 2nd and 5th passages were transferred to a fibronectin-coated silicone chamber with 5000-8000 cells/cm2 (isolated condition), positioning the cells apart, or with 25,000-27,000 cells/cm2 (subconfluent condition), allowing cell-to-cell contact. The substrate was stretched cyclically by 0.5 Hz for 2 h with a peak strain on the substrate that was 15% in the stretch direction and -4% in the transverse direction. The actin filaments (AFs) were stained with rhodamine phalloidin and their orientations were examined under a confocal laser scanning microscope. In the basal region, SFs formed in all of the cells under both the isolated and subconfluent conditions. We observed an average of 5 and 9 SFs per cell under the isolated and subconfluent conditions, respectively, in the fluorescent images of the apical region. We also observed cells that were bush-like without apical AFs or apical SFs. On average, the SFs in the subconfluent cells oriented in the direction of minimal strain, while the SFs in the isolated cells oriented in the direction of a 2% compressive strain. These results suggest that such differential response may be due to differences in the transmission of mechanical stretching to the central and apical regions of the cell through the SFs. We also speculate that cell-to-cell contact might change the strength, orientation, and anchorage of apical AFs and play a critical role in mechanical signal transduction.