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

Blood vessels are continuously exposed to mechanical forces that lead to ad aptive remodeling and atherosclerosis. Although there have been many studie s characterizing the responses of vascular cells to mechanical stimuli, the precise mechanical characteristics of the forces applied to cells to elici t these responses are not clear. We designed a magnetic exposure system cap able of producing a defined normal force on ferromagnetic beads that are sp ecifically bound to cultured cells coated with extracellular matrix protein s or integrin-specific antibodies. Rat aortic smooth muscle cells were incu bated with engineered fibronectin-coated ferromagnetic beads and then expos ed to a magnetic field. With activation of extracellular signal-regulated m itogen-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 t o2.5 pN per cell. The peak response occurred after 5 minutes of exposure an d slowly decreased to baseline after 30 minutes. A cyclic, rather than stat ic, force was required for this activation, and the frequency-response curv e increased approximate to2-fold between 0.5 and 2.0 Hz. Vitronectin- and b eta (3) antibody-coated beads showed a response nearly identical to those c oated with engineered fibronectin, whereas forces applied to beads coated w ith 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 sm ooth muscle cells occurs through specific integrin receptors and requires a cyclic force with a magnitude estimated to be in the piconewton range.