Regional atherosclerotic plaque properties in ApoE-/- mice quantified by atomic force, immunofluorescence, and light microscopy.

Elucidating regional material properties of arterial tissue is fundamental to predicting transmural stresses and understanding how tissue stiffness influences cellular responses and vice versa. Atomic force microscopy (AFM) was used to measure point-wise the axial compressive stiffness of healthy aortas and atherosclerotic plaques at micron level separation distances. Cross sections of plaques were obtained from a widely used animal model of atherosclerosis (ApoE-/- mice). Median point-wise values of material stiffness were 18.7 and 1.5 kPa for the unloaded healthy wall (n = 25 specimens) and plaque (n = 18), respectively. When the healthy wall was distended uniformly during AFM testing, two mechanically distinct populations emerged from comparisons of normal cumulative distributions, with median values of 9.8 and 76.7 kPa (n = 16). The higher values of stiffness may have been due to extended elastin, which was not present in the plaques. Rather, most plaques were identified via standard and immunofluorescent histology to be largely lipid laden, and they exhibited a nearly homogeneous linear elastic behavior over the small AFM indentations. Understanding the mechanics and mechanobiological factors involved in lesion development and remodeling could lead to better treatments for those lesions that are vulnerable to rupture.

Time-dependent changes in smooth muscle cell stiffness and focal adhesion area in response to cyclic equibiaxial stretch.

Observations from diverse studies on cell biomechanics and mechanobiology reveal that altered mechanical stimuli can induce significant changes in cytoskeletal organization, focal adhesion complexes, and overall mechanical properties. To investigate effects of short-term equibiaxial stretching on the transverse stiffness of and remodeling of focal adhesions in vascular smooth muscle cells, we developed a cell-stretching device that can be combined with both atomic force and confocal microscopy. Results demonstrate that cyclic 10%, but not 5%, equibiaxial stretching at 0.25 Hz significantly and rapidly alters both cell stiffness and focal adhesion associated paxillin and vinculin. Moreover, measured changes in stiffness and focal adhesion area from baseline values tend to correlate well over the durations of stretching studied. It is suggested that remodeling of focal adhesions plays a critical role in regulating cell stiffness by recruiting and anchoring actin filaments, and that cells rapidly remodel in an attempt to maintain a homeostatic biomechanical state when perturbed above a threshold value.

Measurements of the cross-bridge attachment/detachment process within intact sarcomeres by surface plasmon resonance.

We have developed a surface plasmon resonance (SPR) system to monitor the cross-bridge attachment/detachment process within intact sarcomeres from mouse heart muscle. SPR occurs when laser light energy is transferred to surface plasmons that are resonantly excited in a metal (gold) film. This resonance manifests itself as a minimum in the reflection of the incident laser light and occurs at a characteristic angle. The angle of the SPR occurrence depends on the dielectric permittivity of the sample medium adjacent to the gold film. Purified sarcomeric preparations are immobilized onto the gold film in the presence of a relaxing solution. Replacement of the relaxing solution with increasing Ca(2+) concentration solution activates the cross-bridge interaction and produces an increase in the SPR angle. These results imply that the interaction of myosin heads with actin within an intact sarcomere changes the dielectric permittivity of the sarcomeric structure. In addition, we further verify that SPR measurements can detect the changes in the population of the attached cross-bridges with altered concentrations of phosphate, 2,3-butanedione monoxime, or adenosine triphosphate at a fixed calcium concentration, which have been shown to reduce the force and increase the cross-bridge population in attached state. Thus, our data provide the first evidence that the SPR technique allows the monitoring of the cross-bridge attachment/detachment process within intact sarcomeres.