Integrin-mediated mechanotransduction in vascular smooth muscle cells - Frequency and force response characteristics

Blood vessels are continuously exposed to mechanical forces that lead to adaptive remodeling and atherosclerosis. Although there have been many studies characterizing the responses of vascular cells to mechanical stimuli, the precise mechanical characteristics of the forces applied to cells to elicit these responses are not clear. We designed a magnetic exposure system capable of producing a defined normal force on ferromagnetic beads that are specifically bound to cultured cells coated with extracellular matrix proteins or integrin-specific antibodies. Rat aortic smooth muscle cells were incubated with engineered fibronectin-coated ferromagnetic beads and then exposed to a magnetic field. With activation of extracellular signal-regulated mitogen-activated protein kinase 1/2 (ERK 1/2(MAPK)) used as a prototypical marker for cell responsiveness to mechanical forces, Western blot analysis demonstrated an increase in phosphorylated ERK 1/2MAPK expression reaching a maximal response of a 3.5-fold increase at a total force of approximate to2.5 pN per cell. The peak response occurred after 5 minutes of exposure and slowly decreased to baseline after 30 minutes. A cyclic, rather than static, force was required for this activation, and the frequency-response curve increased approximate to2-fold between 0.5 and 2.0 Hz. Vitronectin- and beta (3) antibody-coated beads showed a response nearly identical to those coated with engineered fibronectin, whereas forces applied to beads coated with alpha (2) and beta (1) antibodies did not significantly activate ERK 1/2(MAPK). Mechanical activation of the ERK 1/2(MAPK) system in rat aortic smooth muscle cells occurs through specific integrin receptors and requires a cyclic force with a magnitude estimated to be in the piconewton range.