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

强制气道平滑肌细胞表型分析以开发新型哮喘疗法

基本信息

批准号：
9452964
负责人：
Dino Di Carlo
金额：
$ 23.48万
依托单位：
UNIVERSITY OF CALIFORNIA LOS ANGELES
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-04-01 至 2019-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9452964
关键词：
Actomyosin Acute Address Adrenal Cortex Hormones Adrenergic Agonists Affect Agonist Animal Model Asthma Biological Assay Bronchoconstriction Bronchodilator Agents Calcium Cardiac Myocytes Cells Chemicals Clinical Cyclic AMP Data Development Disease Drug Screening Drug Targeting Effectiveness Emergency Situation Epigenetic Process Extracellular Matrix FDA approved Fibroblasts Future Generations Genomics Human Immunofluorescence Immunologic Inflammation Inflammation Mediators Inhalation Inhalators Lead Legal patent Libraries Link Lung Measurement Measures Methods Molecular Monitor Muscle Contraction Muscle Tonus Muscle relaxation phase Myosin Light Chain Kinase Natural Products Opsonin Pathway interactions Patients Pattern Pharmaceutical Preparations Pharmacology Phenotype Play Potassium Channel Relaxation Respiratory physiology Role Second Messenger Systems Signal Transduction Slice Smooth Muscle Smooth Muscle Myocytes Specificity Speed Stains Surface System Techniques Testing Time Tissue Model Tissues Vascular Smooth Muscle airway hyperresponsiveness asthma inhaler asthmatic patient base beta-2 Adrenergic Receptors cell type constriction design drug discovery experimental study high throughput screening inhibitor/antagonist macrophage mechanical behavior molecular phenotype mortality new therapeutic target novel novel therapeutics prevent release of sequestered calcium ion into cytoplasm respiratory smooth muscle response screening tool

项目摘要

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.

抽象的目前用靶向注射（例如皮质类固醇）和随后的药物治疗哮喘支气管收缩（β2肾上腺素能受体激动剂），导致气道变窄。虽然有多种多样抑制细胞力产生和收缩，短效和长效支气管扩张剂操作员的机制通过一种具有负面后果的作用机理，因为适应了长效beta 激动剂会导致短暂作用的β激动剂“救援遗传”的效率降低。需要新药该靶向气道通过正交途径到达β激动剂的平滑肌收缩力。但是，那里目前没有执行针对细胞力产生的高通量屏幕的方法。我们已经发展了一种基于微技术的高通量筛选方法，以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的新药可以可以发现通过单独的途径起作用，并为哮喘患者提供新的治疗选择。在目标1中我们将进行高通量屏幕，以识别在气道平滑肌中放松收缩的化合物细胞。我们将验证组织模型中的命中化合物 - 精确切割的肺切片。我们还期望有选择性气道平滑肌收缩的抑制剂可以通过对其他收缩的反筛查来开发细胞。我们的平台允许对免疫荧光，钙水平和收缩的合并测量单细胞的表型。在AIM 2中，我们将使用此能力来解决钙动员是否为收缩激动剂的增加且足以唤起HASM细胞的缩短。调节的分子输入平滑肌肌动菌素跨桥骑自行车和收缩强度的理解程度较低控制平滑肌音调的较大种类的输入。另外，我们将使用此平台来识别新的表面与超响应性收缩表型相关的标记突出了潜在的关键ASM亚群参与疾病。这样的表面标记还将有助于设计针对细胞的抗收缩药物的未来的哮喘。