Endothelial cells are known to change their shapes in alignment with the direction perpendicular to that of cyclic stretch. However, the mechanisms are still not clear. In this study, we developed a new cell-stretching device which allows continuous observation of cyclically stretched/relaxed cells. Using this apparatus, morphological changes of single cells in response to cyclic stretch (1 Hz, 10%, 2 hours) were visualized and analyzed. The data showed that there were two major patterns of morphological changes of ECs, depending on their initial shapes. While ECs with round shapes at the initial state changed their shapes to become more elongated in a direction perpendicular to that of stretch, cells oriented and elongated in the stretch direction at the initial state changed their shapes to first become rounder and then elongated to align perpendicular to the stretch direction at the second step. These results suggest that cell retraction as well as elongation is important in the stretch-induced endothelial morphological changes, and that the elucidation of the mechanisms of the morphological changes requires studies of cell retraction, elongation, and their coordination at the single-cell level.

Cells under cyclic stretch sense their environments and induce responses such as actin stress fiber (SF) reorientation and morphological changes. These physiological responses are thought to occur when cells sense incompatibility between SF orientation and stretching direction. This hypothesis requires existence of SFs. However, such existence of SFs in cells under cyclic stretch remains unclear since few studies attempted to track the existence of SFs throughout cyclic stretch. In order to track the existence of SFs throughout cyclic stretch, high time resolution time-lapse imaging was improved in two points. First, SFs were clearly imaged with coexpression of DsRed-zyxin and GFP-actin. Second, time resolution was improved so that fluorescence images were obtained every 28 sec. With the improved high time resolution time-lapse imaging, it was revealed, for the first time, that SFs could exist continuously throughout cyclic stretch. Moreover, physiological responses including morphological change as well as SF reorientation occurred during the time when SFs formed incompatibility between SF orientation and stretching direction. These results demonstrated that SFs continuously existed in cells under cyclic stretch and in turn suggested that continuous presence of incompatibility between orientation of long-lasting SFs and the stretching direction might be important for mechanosensing which induces physiological responses.

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