Heaves is a debilitating and incurable noninfectious lung disease of horses associated with alteration of the airway epithelium1 and increases in amounts of ASM2,3 and extracellular matrix deposition.4 These changes are only partially reversible even after a year of inhalational corticosteroid treatment and strict environmental control.5 A need therefore exists for the development of medications capable of preventing or even reversing the airway remodeling that is characteristic of this common condition.
Three-dimensional tissue-engineered bronchial constructs have been successfully used to study cell-cell interactions and cell-matrix remodeling6–8 and for large throughput assessment of novel therapeutic targets for airway remodeling associated with heaves.9 These bronchi models lack ASM, which is important to include because the contractile properties of ASM contribute to airway hyperresponsiveness.10 Airway smooth muscle also has the ability to synthesize and release bioactive inflammatory mediators, including cytokines, chemokines, and growth factors, possibly contributing to the airway wall remodeling.11
Mature contracting smooth muscle cells are unique in their ability to dedifferentiate into a proliferative phenotype and even to transdifferentiate into a panel of myocyte subpopulations.12–14 These phenotypic changes depend on numerous factors in the local microenvironment of the cells, including the composition of extracellular matrix, the presence of cytokines and growth factors, and the mechanical stress to which the tissue is exposed.15–19
The proliferative phenotype observed in culture is associated with decreases in expression of contractile proteins, including α-SMA, SMMHC, and desmin.11,20–22 Studies21,23,24 have revealed a rapid reduction in these contractile proteins when tracheal smooth muscle cells are cultured at a low cell density and in the presence of fetal bovine serum. Cells reassume their contractile state (ie, they express contractile proteins), but only when they reach confluency and are deprived of growth factors.21
Whether ASM cells isolated from endobronchial biopsy specimens maintain their initial phenotype (ie, their contractile phenotype) in long-term cell culture has not yet been reported, to the authors’ knowledge. This information would be important, given that the number of cells generally required for tissue engineering applications necessitates cell expansion in culture, and variations during the several passages required to achieve that aim could result in inconsistent or even erroneous conclusions.
Members of our research group were the first to produce 3-D human healthy and asthmatic bronchi in culture by use of the tissue engineering approach as well as to isolate epithelial cells and fibroblasts from human endobronchial biopsy specimens.7,25 Repetitive sampling of ASM cells from the same horses during both the clinical and subclinical stages of heaves would allow the creation of an equine bronchial model containing ASM. Therefore, we hypothesized that the initial phenotype of ASM cells isolated from endobronchial biopsy specimens would be maintained in long-term cell culture.
The objective of the study reported here was to develop a sampling protocol for isolating ASM cells from freshly harvested equine lungs and from endobronchial biopsy specimens from healthy and heaves-affected horses in clinical remission from the disease and to determine whether these cells would maintain a contractile phenotype in long-term cell culture. The overall intention was to obtain cells that would meet the prerequisites for the production of tissue-engineered 3-D equine bronchi in vitro, incorporating an ASM layer.
Supported by the Canadian Institutes of Health Research (grant No. MOF102751).
Presented in part as an abstract at the American Thoracic Society International Conference, San Diego, May 2014.
The authors thank Guy Beauchamp for performing the statistical analyses and Michela Bullone for providing the photomicrographs.
α-Smooth muscle actin
Airway smooth muscle
Dulbecco Modified Eagle Medium
Mean fluorescence intensity
Smooth muscle myosin heavy chain
Standard fenestrated and smooth, 2.3-m biopsy forceps, Olympus Medical Systems Corp, Center Valley, Pa.
Roche Diagnostics, Laval, QC, Canada.
Cederlane Labs, Burlington, ON, Canada.
Sigma-Aldrich, Oakville, ON, Canada.
Bristol-Myers Squibb, New York, NY.
DMEM nutrient and F-12 mixture, Thermo Fisher Scientific, Burlington, ON, Canada.
Thermo Fisher Scientific, Burlington, ON, Canada.
Nunc Lab-Tek II, Fisher Scientific Co, Ottawa, ON, Canada.
Vector Laboratories, Burlington, ON, Canada.
Abcam, Toronto, ON, Canada.
Biomed Technologies, Mount Arlington, NJ.
Axio Imager M1 equipped with an AxioCam MRm, Zeiss Canada, North York, ON, Canada.
Cytofix/Cytoperm, BD Biosciences, Mississauga, ON, Canada.
BD Biosciences, Mississauga, ON, Canada.
FACSCalibur, BD Biosciences, Mississauga, ON, Canada.
BCA protein assay kit, Fisher Scientific Co, Ottawa, ON, Canada.
Mini-PROTEAN TGX stain-free precast gel, Bio-Rad Laboratories, St-Laurent, QC, Canada.
Millipore Canada Ltd, Etobicoke, ON, Canada.
Cell Signaling Technology, Whitby, ON, Canada.
BM chemiluminescence western blotting substrate, Thermo Fisher Scientific, Burlington, ON, Canada.
Fusion FX7 system, Montreal Biotech Inc, Dorval, QC, Canada.
Quantity One 4.5.0 image, Bio-Rad Laboratories Inc, Saint-Laurent, QC, Canada.
DMC-SZ7 10X optical zoom, Panasonic Canada Inc, Calgary, Canada.
Image J software, Research Services Branch, National Institutes of Health, Bethesda, Md.
SAS, version 9.3, SAS Institute Inc, Cary, NC.
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