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

摘要 目前治疗哮喘的方法是针对炎症的药物（例如皮质类固醇）和随后的药物 支气管收缩（β2 肾上腺素能受体激动剂），导致气道变窄。虽然有各种各样的 抑制细胞力产生和收缩的机制，短效和长效支气管扩张剂起作用 通过单一的作用机制，这会产生负面后果，因为适应了长效贝塔 激动剂会导致短效β激动剂“救援吸入器”的功效降低。需要新药 通过与β受体激动剂的正交途径来靶向气道平滑肌收缩力。然而，有 目前尚无针对细胞力生成进行高通量筛选的方法。我们开发了 一种基于微技术的高通量筛选方法，用于表征细胞力的产生 单细胞水平。我们假设干扰气道平滑肌细胞收缩性的新药可以 被发现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，以确定能够放松气道平滑肌收缩的化合物 细胞。我们将在组织模型（精密切割的肺切片）中验证命中化合物。我们还预计，选择性 气道平滑肌收缩抑制剂可以通过对抗其他收缩性药物的反筛选来开发。 细胞。我们的平台可以联合测量免疫荧光、钙水平和收缩力 单细胞的表型。在目标 2 中，我们将使用此功能来解决钙动员是否 增加并足以通过收缩激动剂引起 HASM 细胞缩短。调节分子输入 鉴于平滑肌肌动球蛋白跨桥循环和收缩强度的了解仍然较少 控制平滑肌张力的输入种类更多。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了潜在的关键 ASM 亚群 涉及疾病。这种表面标记物还有助于设计细胞靶向抗收缩药物 将来会出现哮喘。

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.