Analysis of the Mechanical Reaction of Human Endothelial Cells to a Single Constant or Transient Uniaxial Strain

Abstract

Many adherent cell types are continually exposed to a variety of mechanical stresses. For instance, vascular endothelial cells, alveolar cells, and cells of gastrointestinal tract experience periodic strains due to blood circulation, breathing and peristaltic activity. In order to withstand those stresses, cells have to be able to perceive them and to react accordingly through a biochemical or mechanical feedback. This ability, called mechanosensitivity, is crucial for normal cell function, proliferation, and survival. Mechanosensing is believed to be important in such processes as cancer, atherosclerosis and plaque formation. In particular, mechanical cell response is manifested in modulation of the internal stress-bearing and stress-generating structures as actin cytoskeleton and focal adhesions. The highly dynamic actin network consists of single filaments and actin bundles, connected by a variety of cross-linking proteins like α-actinin. The filaments transmit forces produced by the contracting actomyosin machinery to the cellular adhesion sites. The latter connects to transmembrane proteins anchoring to the outside of the cell, be that extracellular matrix or neighbouring cells. Thus, internally generated forces are transmitted to the environment of the cell, implying that the whole process is reciprocal. <br /> In this work the mechanical response of vascular endothelial cells was studied. These cells are known to be responsive to mechanical stimuli present in their physiological environment, where they are exposed to shear flow and pressure of the pulsating movement of blood through the vessel, and radial compression created by the smooth muscle tissue encircling the vein. Besides, endothelial cells sense the stiffness of the underlying basal membrane which is essential at counteracting in case of inflammation or atherosclerosis. Therefore, we aimed to examine the mechanical response of vein endothelial cells to an external stress. Here, cells cultivated on an elastic substratum of suitable elasticity were exposed to a uniaxial stretch in order to mimic in vivo conditions. <br /> To realize these experiments, a new setup and suitable software have been developed. The setup successfully combined live cell imaging at close to physiological conditions, traction force microscopy, and substrate stretching. Two kinds of stretch protocols were used: a constant 20% strain (also called stretch-and-hold) and a transient 20% (stretch-and-release). <br /> Cells were imaged before and after stretching for comparison. Cell traction forces were calculated by solving the Boussinesq problem for infinite layers with the help of a Fourier transform method combined with regularization. In addition, such geometrical parameters as cell area, orientation, elongation and aspect ratio were measured. The two kinds of strain protocols prompted two different cell reactions. Transient strain induced an abrupt drop of cell forces by 20% that recovered completely to the pre-stretch level within 5 min. No other visual changes of the cell behaviour were detected. Cells did not change their orientation or morphology after the stretch-release cycle. In contrast, constant strain evoked a sudden rise of contractile forces by up to 150%. These forces continued to increase for about 10 min after stretching. After that they either decreased gradually or remained at the maximal level. Surprisingly, in this strain protocol 90% of the observed cells exhibited forces that did not relax to the pre-stretch levels until the end of observation (70-100 min). At the same time, cell orientation and elongation persisted throughout measurements after stretching: cells simply followed the deformation of the substrate. <br /> The two types of experiments resulted in different kinds of mechanical response of the cell. The cell response was universal under each strain type: in practice, all cells displayed the same reaction, independently of the cell pre-stress history. The change in contractility indicated that the actomyosin activity adapted according to the applied stress. The cell orientation upon the stretch persisted in these single stretch experiments. This implies that a longer and a repetitive exposure to external loads is necessary to induce cell reorientation in either minimum stress or minimum strain direction as in cyclic stretch experiments. These observations motivate further investigations of the cell actomyosin and actin cross-linker kinetics upon single stretch or compression, as well as of gradual change of cell contractility and orientation in cyclic stretch experiments.