Abstract
Phenotypes of lung smooth muscle cells in health and disease are poorly characterized. This is due, in part, to a lack of methodologies that allow for the independent and direct isolation of bronchial smooth muscle cells (BSMCs) and vascular smooth muscle cells (VSMCs) from the lung. In this paper, we describe the development of a bi-fluorescent mouse that permits purification of these two cell populations by cell sorting. By subjecting this mouse to an acute allergen based-model of airway inflammation that exhibits many features of asthma, we utilized this tool to characterize the phenotype of so-called asthmatic BSMCs. First, we examined the biophysical properties of single BSMCs from allergen sensitized mice and found increases in basal tone and cell size that were sustained ex vivo. We then generated for the first time, a comprehensive characterization of the global gene expression changes in BSMCs isolated from the bi-fluorescent mice with allergic airway inflammation. Using statistical methods and pathway analysis, we identified a number of differentially expressed mRNAs in BSMCs from allergen sensitized mice that code for key candidate proteins underlying changes in matrix formation, contractility, and immune responses. Ultimately, this tool will provide direction and guidance for the logical development of new markers and approaches for studying human lung smooth muscle.
Highlights
Despite their structural and functional importance in the airway and pulmonary vasculature, defining features for bronchial smooth muscle (BSM) and vascular smooth muscle (VSM) in developing, mature, and diseased lungs have been poorly characterized
In the bi-fluorescent transgenic mouse, hrGFP is singly expressed in bronchial smooth muscle cells (BSMCs) whereas hrGFP and DsRed are doubly expressed in vascular smooth muscle cells (VSMCs) (Fig. 1A); thereby allowing the independent isolation of BSMCs and VSMC from the lung by cell sorting
Post-sort cell purity tests showed that approximately 95% of sorted populations express appropriate BSMC and VSMC markers (Figures S2, S3 in File S1)
Summary
Despite their structural and functional importance in the airway and pulmonary vasculature, defining features for bronchial smooth muscle (BSM) and vascular smooth muscle (VSM) in developing, mature, and diseased lungs have been poorly characterized. These gaps in knowledge are largely due to the lack of methodologies that allow direct and high fidelity isolation of bronchial smooth muscle cells (BSMCs) and vascular smooth muscle cells (VSMCs). The shortcomings of mouse models of airway and pulmonary vascular disease are attributable, in part, to the limited tools available to purify and analyze lung smooth muscle populations. This state-of-affairs is one of the key factors that underlie the lack of treatments aimed at reversing the pathological smooth muscle phenotypes characteristic of diseases such as asthma and pulmonary hypertension [6,7,8]
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