Force phenotyping of airway smooth muscle cells to develop novel asthma therapies

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ABSTRACT Asthma is currently treated with drugs that target inflammation (e.g. corticosteroids) and the subsequent bronchoconstriction (β2 adrenergic receptor agonists) that leads to airway narrowing. Although there are a variety of mechanisms to inhibit cell force generation and contraction, short- and long-acting bronchodilators operate through a single mechanism of action, which has negative consequences, since adaptation to a long-acting beta agonist leads to reduced efficacy of short-acting beta agonist “rescue inhalers.” There is a need for new drugs that target airway smooth muscle contractility through orthogonal pathways to the beta agonists. However, there are no current methods to perform high-throughput screens targeting cell force generation. We have developed a microtechnology-based high-throughput screening approach to characterize cellular force generation at the single-cell level. We hypothesize that new drugs that interfere with airway smooth muscle cell contractility can be found that act through separate pathways and lead to new treatment options for asthma patients. In Aim 1 we will conduct a high-throughput screen to identify compounds that relax contraction in airway smooth muscle cells. We will validate hit compounds in a tissue model - precision cut lung slices. We also anticipate that selective inhibitors of airway smooth muscle contraction can be developed by counter-screening against other contractile cells. Our platform allows for combined measurement of immunofluorescence, calcium levels, and contractile phenotypes for single cells. In Aim 2 we will use this capability to address whether calcium mobilization is increasing and sufficient to evoke HASM cell shortening by contractile agonists. Molecular inputs that modulate smooth muscle actomyosin cross-bridge cycling and the strength of contraction remain less understood given the larger variety of inputs that control smooth muscle tone. Also, we will use this platform to identify new surface markers associated with hyper-responsive contractile phenotypes highlighting potential key ASM subpopulations involved in disease. Such surface markers would also assist in designing cell-targeted anti-contractility drugs for asthma in the future.