A new approach for the study of lung smooth muscle phenotypes and its application in a murine model of allergic airway inflammation

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.

Figure 1. Separation of BSMCs and VSMCs from the lung using the bi-fluorescent αSMA-hrGFP;NG2-DsRed mouse.

(A) Expression pattern of hrGFP and DsRed in BSMCs and VSMCs in the lung of an αSMA-hrGFP;NG2-DsRed 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) 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). (C) Relative Notch3 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.

Figure 2. Characterization of an allergen sensitized mouse model of asthma using αSMA-hrGFP;NG2-DsRed mouse.

(A) A protocol to induce acute allergic airway inflammation using OVA sensitization and challenge. Mice sensitized with saline were control. (B) Airway reactivity in response to increasing doses of Mch in control (•) and OVA-sensitized (▴) mice by Flexivent. The measurements represented Rn values. (C) Serum levels of OVA-specific IgE in control (▪) and OVA-sensitized (•) mice, as measured by ELISA. (D) PAS staining for mucin production in the lung epithelium of control and OVA-sensitized mice. (E) The expression of hrGFP and DsRed in the lungs of OVA-sensitized mice. Scale bar, 20 µm. *P<0.05.

Figure 3. Measurement of changes in physical properties of individual BSMCs from OVA sensitized mice.

BSMCs isolated from PBS-sensitized control and OVA sensitized αSMA-hrGFP;NG2-DsRed mice were allowed to attach to collagen I gel in culture for 72 hrs before assays. (A) Images of hrGFP⁺ BSMCs from PBS control and OVA sensitized mice (insert) and their traction maps on collagen I gels. (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. (C) 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.

Figure 4. Validation of selected deregulated genes in BSMCs from OVA sensitized mice identified by the array.

(A, B) Real time PCR validation of selected up-regulated genes (A) and down-regulated genes (B) genes in BSMCs from OVA sensitized mice. Results were normalized to 18S. Data show mean ± SD and are representative of three independent experiments. *p<0.05. (C) Immuno-histochemistry for collagen 6 alpha 5 (COL6A5) and the adrenergic β2-receptor (ADRB2) in lungs of PBS sensitized control and OVA sensitized mice. Lung sections of control and sensitized mice were stained side-by-side under the same condition. Black arrows indicate BSMC layer. Scale bar: 20 µm. (D) Flow cytometry for the ADRB2 receptor in non-permeabilized BSMCs from PBS sensitized control (red) and OVA sensitized (blue) mice. Data is representative of two independent experiments.