A New Approach for the Study of Lung Smooth Muscle Phenotypes and Its Application in a Murine Model of Allergic Airway Inflammation

<div><p>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 <i>ex vivo</i>. 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.</p></div

Separation of BSMCs and VSMCs from the lung using the bi-fluorescent <i>αSMA-hrGFP;NG2-DsRed</i> mouse.

<p>(<b>A</b>) Expression pattern of hrGFP and DsRed in BSMCs and VSMCs in the lung of an <i>αSMA-hrGFP;NG2-DsRed</i> mouse. Both BSMCs and VSMCs are hrGFP⁺. BSMCs are DsRed⁻ whereas VSMCs are DsRed⁺. Asterisk (*) indicates the blood vessel in the lung the airway. Arrows (>) indicate the airway. Scale bar: 20 µm. (<b>B</b>) Algorithm for bronchial and vascular smooth muscle cell identification by flow cytometry. CD31⁺ endothelial cells and CD45⁺ immune cells were separated from dissociated lung preparation (left panel) followed by evaluation of hrGFP and DsRed distribution within CD31⁻CD45⁻ population (middle panel). CD31⁻CD45⁻hrGFP⁺DsRed⁻ population corresponds to BSMCs (pointed by an arrow). CD45⁻CD31⁻hrGFP⁺DsRed⁺ population is enriched in vascular smooth muscle cells (VSMCs). (<b>C</b>) Relative <i>Notch3</i> mRNA expression in isolated singly hrGFP⁺ cells and doubly hrGFP⁺DsRed⁺ cells from lung cell preparation as determined by qPCR followed by normalization to 18S. Data show mean ± SD and are representative of three independent experiments. ***p<0.001.</p

Measurement of changes in physical properties of individual BSMCs from OVA sensitized mice.

<p>BSMCs isolated from PBS-sensitized control and OVA sensitized <i>αSMA-hrGFP;NG2-DsRed</i> mice were allowed to attach to collagen I gel in culture for 72 hrs before assays. (<b>A</b>) Images of hrGFP⁺ BSMCs from PBS control and OVA sensitized mice (insert) and their traction maps on collagen I gels. (<b>B</b>) Contractile moment measurement of individual BSMCs from control and sensitized mice based on their traction maps. The line represents the mean of individual measurements of control (n = 20) and acute asthmatic (n = 22) cells. (<b>C</b>) Measurement of cell size of BSMCs from PBS sensitized control and OVA sensitized mice. The line represents the mean of individual measurements. **p<0.008.</p

Gene sets misregulated in BSMCs from OVA sensitized mice as compared to PBS sensitized control.

<p>The five most significantly down- and up-regulated gene sets among the MSigDB canonical pathways, Gene Ontology categories, and motif gene sets are shown, ordered in each subsection by CAMERA <i>p</i>-value. No motif gene set was up-regulated with p<0.05, so these are excluded. The size column gives the number of genes in the gene set after restricting to members with mouse orthologs represented by probe sets on the arrays.</p