As the endothelial cell reorients, its tensile forces stabilize

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Abstract

When adherent cells are subjected to uniaxial sinusoidal stretch at frequencies close to physiological, their body and their contractile stress fibers realign nearly perpendicularly to the stretch axis. A common explanation for this phenomenon is that stress fibers reorient along the direction where they are unaffected by the applied cyclic stretch and thus can maintain optimal (homeostatic) tensile force. The ability of cells to achieve tensional homeostasis in response to external disturbances is important for normal physiological functions of cells and tissues and it provides protection against diseases, including cancer and atherosclerosis. However, quantitative experimental data that support the idea that stretch-induced reorientation is associated with tensional homeostasis are lacking. We observed previously that in response to uniaxial cyclic stretch of 10% strain amplitudes, traction forces of single endothelial cells reorient in the direction perpendicular to the stretch axis. Here we carried out a secondary analysis of those data to investigate whether this reorientation of traction forces is associated with tensional homeostasis. Our analysis showed that stretch-induced reorientation of traction forces was accompanied by attenuation of temporal variability of the traction field to the level that was observed in the absence of stretch. These findings represent a quantitative experimental evidence that stretch-induced reorientation of cellular traction forces is associated with the cell’s tendency to achieve tensional homeostasis.

Introduction

A ubiquitous phenomenon observed in various cell types is that in response to unidirectional cyclic stretch of the substrate, the cell body realigns globally and the cytoskeletal stress fibers realign locally. If the sinusoidal stretching frequency falls in the range of heart pulsatility, the realignment is away from the stretch axis (Hayakawa et al., 2001, Wang et al., 2000, Wang et al., 2001, Kaunas et al., 2005, Kaunas et al., 2006, Kurpinski et al., 2006). The prevailing interpretation of these observations has been that realigning contractile stress fibers away from the direction of stretch axis reduces variability of forces carried by stress fibers, which enables the cell to maintain cytoskeletal contractile stress (or tension) stable - a phenomenon known as tensional homeostasis (Brown et al., 1998). It has been argued that tensional homeostasis is essential for normal physiological functions of tissues, such as the endothelium and the epithelium, and provides protection against diseases, including atherosclerosis and cancer (cf. Chien, 2007, Paszek et al., 2005, Butcher et al., 2009, Humphrey, 2008a, Humphrey, 2008b).

Several theoretical models were advanced to explain mechanisms that govern stretch-induced reorientation of cytoskeletal stress fibers and stabilization of tensile forces carried by the fibers (cf. De et al., 2007, De and Safran, 2008, Kaunas et al., 2011, Pirentis et al., 2011). However, a direct experimental verification of tensional homeostasis associated with the reorientation is lacking. That is, the evidence that after stretch-induced reorientation is completed, the cytoskeletal stress returns to the state it had prior to stretch application has not yet been produced.

In 2012, we studied how cellular traction forces in isolated endothelial cells change in response to a slow, non-sinusoidal, cyclic, uniaxial stretch. We observed that for 10% strain amplitudes the traction field reoriented in the direction perpendicular to the stretch axis. In contrast, in the absence of stretch, the traction field did not reorient (Krishnan et al., 2012). Here we carried out a secondary analysis of those data to investigate whether the traction field reorientation is associated with the cell’s tendency to achieve tensional homeostasis. In particular, we analyzed how temporal fluctuations of the traction field around its mean value changed during reorientation. Results of our analysis indicated that those fluctuations became attenuated once the traction field reorientation was completed, suggesting that the cell achieved the state of tensional homeostasis.

Section snippets

Definition and quantification of tensional homeostasis

Tensional homeostasis in cells has often been identified with the cell’s ability to recover its baseline tension in response to applied stretch. Many experimental studies of tensional homeostasis have been centered around measurements of time-dependent changes of some scalar metric of cytoskeletal tension following static, quasi-static, or transient stretch application (Brown et al., 1998, Mizutani et al., 2004, Trepat et al., 2007, Ezra et al., 2010, Webster et al., 2014, Weng et al., 2016).

Results

Time lapses of M(t)/〈M〉 exhibited erratic fluctuations in both stretched and unstretched cases (Fig. 1). In the case of applied stretch, these fluctuations appear to be more prominent during the first hour of stretching, when the traction field reoriented, than during the second hour, when the traction field maintained its perpendicular orientation relative to the stretch axis (Fig. 1a). In the absence of stretch, no such difference was obvious (Fig. 1b).

During stretch application, the traction

Discussion

Results of our analysis demonstrated that stretch-induced reorientation of the traction field of single HUVECs was closely associated with attenuation of temporal fluctuations of the traction field. This finding supported our hypothesis that the traction field reorientation was linked to the cell’s ability to achieve and maintain tensional homeostasis. Because traction forces arise in response to cellular contraction, our results are also supportive of the notion that previously observed

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This study was supported by NSF grant CMMI-1910401 (D. Stamenović and Michael L. Smith).

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