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

摘要 哮喘目前用靶向炎症的药物（例如皮质类固醇）和随后的免疫抑制剂来治疗。 支气管收缩（β2肾上腺素能受体激动剂），导致气道狭窄。虽然有各种各样的 在抑制细胞力产生和收缩的机制中，短效和长效支气管扩张剂起作用 通过一个单一的行动机制，这具有负面影响，因为适应长效β 激动剂导致短效β激动剂“救援吸入器”的功效降低。需要新的药物 其通过与β激动剂正交的途径靶向气道平滑肌收缩。但 目前还没有进行靶向细胞力产生的高通量筛选的方法。我们已经开发 一种基于微技术的高通量筛选方法来表征细胞在 单细胞水平。我们假设，干扰气道平滑肌细胞收缩的新药， 通过不同的途径发挥作用，并为哮喘患者带来新的治疗选择。目标1 我们将进行高通量筛选，以确定放松气道平滑肌收缩的化合物， 细胞我们将在组织模型中验证命中化合物-精确切割肺切片。我们还预计， 气道平滑肌收缩的抑制剂可以通过针对其它收缩剂的反筛选来开发。 细胞我们的平台允许联合测量免疫荧光，钙水平和收缩 单细胞的表型。在目标2中，我们将使用这种能力来解决钙动员是否 增加并足以通过收缩激动剂引起HASM细胞缩短。分子输入调节 平滑肌肌动球蛋白跨桥循环和收缩的强度仍然不太清楚， 控制平滑肌张力的输入的种类更多。此外，我们将使用这个平台来识别新的表面 与高反应性收缩表型相关的标记物，突出潜在的关键ASM亚群 与疾病有关。这种表面标记物也将有助于设计细胞靶向抗收缩药物， 哮喘在未来

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.