| In the asthmatic lung, airway smooth muscle (ASM) constantly experiences characteristically-elevated internal contractile stress as well as external mechanical stress (MS) due to breathing, which may be elevated by symptoms. The ASM cytoskeleton is highly adaptable, responding dynamically to external MS and inflammatory mediators, leading to altered structure and function that may enhance airway narrowing in asthma. In this thesis, we developed methodology and software to apply a technique known as optical magnetic twisting cytometry to investigate the roles of external MS; internal MS, or tone; and inflammation - alone and in combination - on the functional response of the cytoskeleton.;We applied external MS to cultured human bronchial ASM cells acutely (90 min.) by sinusoidal rotation of integrin-bound microbeads (4.5 mum diameter) on cells that had been incubated with proinflammatory cytokine, interleukin-1beta (IL-1beta, 20 ng/mL), for 20 hr. As previously established, IL-1beta incubation increased contractility and decreased relaxation induced by beta-agonists, promoting hyperresponsiveness. We found this occurred via cytoskeletal stiffening of 34.2% associated with Rho activation, actin polymerization, and focal adhesion growth, all mediated by cyclooxygenase (COX)-2-dependent pathways. MS similarly induced cytoskeletal stiffening of 29.8% and focal adhesion growth, but this was independent of COX-2 and without increased contractility. Airway inflammation thus increased contractile function via stress-independent cytoskeletal remodeling.;In another study, chronic (5 days) external and predominantly-uniaxial MS was applied to the basal surface of ASM cell cultures (5%, 0.3 Hz), during which tone was either decreased by repeated addition of forskolin or increased by repeated addition of either carbachol or histamine, relative to sham. Cells aligned and baseline stiffness increased with strain, but decreased tone inhibited both effects (p<0.05). Importantly, strain reversed previously-reported inhibition of myosin light-chain kinase (MLCK) content by tone in unstrained cells. Strain and tone together increased both MLCK and myosin light-chain phosphorylation, leading to an increase in contractility of 176%. Along with increased contractile protein content and activity, observed strain-dependent changes in cytoskeletal organization contributed to the increase in contractility.;Taken together, the presented data suggest that treatment with corticosteroids and bronchodilators may lead to improved airway function by a novel mechanism involving the reduction of cytoskeletal remodeling-induced increases in ASM contractility. |
