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

摘要 目前哮喘的治疗方法是针对炎症的药物(如皮质类固醇)和随后的 导致呼吸道狭窄的支气管收缩(β-2肾上腺素能受体激动剂)。尽管有各种各样的 在抑制细胞力量产生和收缩的机制中，短效和长效的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，因为适应了长效的Beta 激动剂会导致短效β-激动剂“救援吸入剂”的疗效降低。对新药的需求 通过与β受体激动剂的正交通路靶向气道平滑肌的收缩。然而，在那里 目前还没有进行针对细胞力量产生的高通量筛选的方法。我们已经开发出 一种基于微技术的高通量筛选方法来表征细胞力在 单细胞水平。我们假设，干扰呼吸道平滑肌细胞收缩的新药可以 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。在目标1中 我们将进行高通量筛选，以确定能松弛呼吸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--精确切割的肺切片。我们还预计，有选择性的 通过对其他收缩物质的反筛选，可以开发出抑制呼吸道平滑肌收缩的药物 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，我们将使用这一能力来解决钙动员是否 收缩激动剂增加并足以引起HASM细胞缩短。调节的分子输入 肌动球蛋白跨桥循环和收缩强度仍然知之甚少。 控制平滑肌张力的更大种类的输入。此外，我们还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突出潜在的关键ASM亚群 与疾病有关的。这种表面标志物也将有助于设计细胞靶向的抗收缩药物 未来的哮喘。

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.