Investigating the integrative role of substrate stiffness, strain, and tone on the functional and phenotypic response of airway smooth muscle in asthmatic airways

Abstract

In recent years, studies have shown that substrate stiffness is a potent modulator of cellular function, gene expression, and morphology in many cell types. These observations may be of important relevance to asthma pathology as remodeling of the airway is thought to create a stiffer microenvironment for ASM cells. Indeed, ASM cells demonstrate a more contractile phenotype on stiffer substrates in vitro. However, the mechanotransduction process by which cells sense the external mechanical environment is an active system that relies on dynamic changes in cell tone, structure, and signaling pathways. Therefore, the ability of the cell to sense and respond to the stiffness of the external environment may be upset by other stimuli that affect external or internal mechanics. In the airway, ASM cells are constantly exposed to oscillatory strain due to breathing and in asthma, these cells also maintain higher tone. To better model the mechanical environment of the asthmatic airway, I attempted to integrate either chronic tone modulation or strain into the well-characterised polyacrylamide substrate stiffness model. The increase in contractile phenotype on stiff substrates was confirmed, and, for the first time, was shown to correlate with transcriptional regulation of smMHC. Interestingly, these effects were time-dependent and only observable after 7 days of cell culture. Strain was applied to the model by successfully binding PA gels to Flexcell silicone membranes. However, the inability to fully polymerise PA gels soft enough to accurately mimic the healthy airway environment, likely due to the use of oxygen plasma, prevented further use of this model. Chronic relaxation of ASM cells on PA gels had little effect on substrate stiffness-dependent changes, yet independently regulated transcription of SMA and Sm22. These results provide evidence for independent mechanotransduction pathways in ASM cells, implicating both airway remodeling and cell tone as necessary therapeutic targets in asthma.