Abstract
OBJECTIVE To develop a method to maintain the initial phenotype of airway smooth muscle (ASM) cells isolated from equine endobronchial biopsy specimens in long-term cell culture.
SAMPLE Endobronchial tissue specimens (8 to 10/horse) collected from the lungs of previously healthy horses at necropsy (n = 12) and endobronchial biopsy specimens collected from standing, sedated, heaves-affected horses in clinical remission of the disease (5) and control horses (4).
PROCEDURES A sampling protocol was developed to recover and maintain a contractile phenotype in ASM cells from endobronchial specimens from freshly harvested equine lungs and from healthy and heaves-affected horses. Immunologic techniques were used to evaluate the contractile phenotype of ASM cells in culture.
RESULTS Characteristic ASM cells were successfully cultured from endobronchial tissue or biopsy specimens from both healthy and heaves-affected horses, and their contractile phenotype was maintained for up to 7 passages. Moreover, the capacity of cells at the seventh passage to contract in a collagen gel in response to methacholine was maintained.
CONCLUSIONS AND CLINICAL RELEVANCE ASM cells isolated from equine endobronchial tissue and biopsy specimens were able to maintain a contractile phenotype in long-term cell cultures, suggesting they could be used for tissue engineering and in vitro studies of equine ASM cells.
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.
Materials and Methods
Ethics statement
All experimental procedures were performed in accordance with the Canadian Council for Animal Care guidelines and were approved by the Animal Care Committee for the Faculty of Veterinary Medicine of the Université de Montréal (protocol No. Rech-1324).
Animals
Tissue specimens were collected from the lungs of 12 healthy horses that had been slaughtered at an abattoir and biopsy specimens from 5 standing, sedated heaves-affected horses in clinical remission from the disease and 4 control horses without heaves. All horses from which lungs were obtained had been declared as healthy by the state veterinarian or an inspector under the supervision of the veterinarian within 24 hours prior to death. A routine postmortem examination and inspection had also been performed, including examination of the thoracic viscera. Lungs were obtained from those horses within 30 minutes after death and kept on ice until endobronchial tissue specimens were collected (from similar sites as for endobronchial biopsy specimens collected from live horses) ≤ 90 minutes later.
The other 9 horses had been part of a larger study26 involving evaluation of markers of systemic inflammation in horses with heaves. These horses, the experimental protocol, and physiologic, inflammatory, and selected airway remodelling features are described elsewhere.26 Briefly, heaves-affected horses had a documented 3- to 10-year history of reversible airway obstruction (abnormal lung function) and inflammation when exposed to hay (airway neutrophilia > 25% as indicated by results of cytologic evaluation of bronchoalveolar lavage fluid). Control horses had been deemed healthy on the basis of results of physical examination, cytologic evaluation, and hematologic analyses.
Endobronchial specimen collection
Endobronchial biopsy specimens (8 to 10/horse) were obtained from sedated horses with videoendoscopic guidance from the third- to fifth-generation bronchial carinas by use of disposable forcepsa as described elsewhere.27 Endobronchial tissue specimens (8 to 10/horse) were also collected from freshly harvested lungs of the 12 horse cadavers at the same anatomic sites. Specimens (ranging from 2 to 3 mm) were washed 3 times in sterile PBS solution (pH, 7.4) and then placed in a Petri dish. A binocular dissecting microscope (14× magnification) and light-emitting diode were then used to facilitate careful removal of the epithelium, which appeared as a fine yellowish superficial layer, and any cartilage from the surrounding tissues.
ASM cell isolation
Isolation of ASM cells from endobronchial biopsy and tissue specimens was performed by enzymatic digestion with collagenase Hb (0.125 U/mL) and elastasec (1 U/mL), with trypsin inhibitord (1 mg/mL) and a combination of penicillin-streptomycin (10,000 U/mL) and antifungal,e under rotational movement for 3 hours in a humidified incubator at 37°C with 5% CO2. The digested cells were then seeded at a density of 300,000 cells/cm3 in DMEM-nutrient mixturef (3:1) supplemented with sodium pyruvateg (100 μM), adenined (2.4 mg/L), 10% fetal bovine serum, penicillin-streptomycin, and antifungal. The medium was changed every 48 hours, and cells were passaged at a ratio of 1:3 with trypsin every 7 to 10 days.
Fibroblasts
To exclude the possibility of contamination by fibroblasts and to ensure that our isolation technique was highly enriched for ASM cells, primary dermal fibroblasts were also evaluated. These cells were isolated from skin biopsy specimens from 3 healthy horses. Briefly, biopsy specimens were rapidly washed in PBS solution in a Petri dish, cut into small fragments, and placed on thermolysin solutiond (500 μg/mL) for 5 hours at 37°C. The sample was then recovered and digested with collagenase H (0.125 U/mL) under rotational movement for 3 hours28 in a humidified incubator at 37°C with 5% CO2.27 Cells were then cultured in DMEM-nutrient mixturef (3:1) containing 10% fetal bovine serum. After 24 hours, few cells had attached to the plates and the nonadherent cells were removed by medium change. After 7 to 8 days, adherent cells reached 80% to 90% confluency with spindle-shaped morphology.
Immunofluorescence evaluation
Immunofluorescence analysis was performed of contractile protein isolated from endobronchial specimens (8 to 10/horse) from 4 live horses and the lungs of 12 horse cadavers and of the cultured primary dermal fibroblasts. Freshly isolated or trypsinized ASM and fibroblast cells were washed twice in PBS solution, cytocentrifuged on glass microscope slides at 200 × g for 5 minutes, and fixed in 100% acetone at −20°C for 20 minutes. Alternatively, cultured cells were labeled directly on chamber slidesh for immunostaining. Cells were then washed twice in PBS solution and kept at 4°C until examined and stained with anti-α-SMA, anti-desmin, and anti-SMMHC antibodies.
Briefly, for the staining process, cells were blocked with 2% goat serumi diluted in PBS solution, then incubated with anti-α-SMAd (dilution, 1/250) antibody for 1 hour at room temperature (approx 22°C) and with anti-desminj (dilution, 1/200) or anti-SMMHC IgGk (dilution, 1/300) antibody for 1 and 2 hours, respectively, at room temperature. After several washes, cells were incubated with fluorescent dye-conjugated goat anti-rabbit IgG antibody (dilution, 1/1,000) and fluorescent dye-conjugated goat anti-mouse IgG antibodyg (dilution, 1/1,000) for 1 hour at room temperature in the dark. Specificity of the staining was confirmed with the appropriate isotype antibody (mouse IgG2ad as a negative control substance for detection of anti-α-SMA antibody and rabbit IgGi as a negative control substance for detection of anti-desmin and anti-SMMHC antibodies). Diamidino-2-phenylindole dyeg (0.5μg/mL) was applied as a nuclear counterstain.
Slides were washed 3 times with PBS solution, then coverslips were added with a solution containing 30% glycerolg and 0.4% gelatin (wt:vol). Images were acquired with an imaging microscope.l Percentages of cells expressing α-SMA, desmin, and SMMHC were determined by examination of 400 cells in randomly selected fields (2 to 5/cytopreparation) at high (400×) magnification.
Flow cytometry
Flow cytometry analysis was performed of contractile protein isolated from endobronchial tissue specimens (8 to 10/horse) from the lungs of 8 horses at necropsy. Isolated ASM cells (106 cells) were washed twice in PBS solution, counted, and fixed for 20 minutes in a cell fixation-permeabilization solution.m After 3 washes in wash buffer,n cells were stained for intracellular markers with anti-α-SMA, anti-desmin, and anti-SMMHC antibodies. Briefly, ASM cells (106 cells/100 μL) were stained for 1 hour with anti-α-SMAd (dilution, 1/250), anti-desminj (dilution, 1/200), and anti-SMMHCj (dilution, 1/300) antibodies. All incubation steps were performed at 4°C. Cells were then washed 3 times in wash buffer and incubated for 30 minutes in the dark with fluorescent dye-conjugated anti-IgG antibodyg (dilution, 1/1,000). Cells were washed twice and suspended in 400 μL of PBS solution.
Flow cytometry was performed with acquisition of 10,000 events by use of the software provided with the flow cytometry instrument.o Isotype-matched control antibodies (mouse IgG2a and rabbit IgG) were used as negative control substances. All signals greater than those of the isotype-matched control antibodies were considered positive, and degree of staining was evaluated as the mean fluorescence intensity and mean percentage of positive cells.
Western blot analysis
Western blot analysis was performed of contractile protein isolated from endobronchial tissue specimens (8 to 10/horse) from the lungs of 4 horse cadavers. Cellular extracts (20 μg) were collected after cell trypsinization at each passage, and protein concentrations were measured by use of a protein assay kitp in accordance with the manufacturer's instructions. Electrophoresis was performed by use of a polyacrylamide gel,q after which a polyvinylidene difluoride membraner was applied. Membranes were blocked with 5% milk and incubated with anti-α-SMA (dilution, 1/500), anti-desmin (dilution, 1/300), and anti-SMMHC (dilution, 1/300) antibodies. After several washes, membranes were further incubated with horseradish peroxidase-conjugated goat anti-rabbit and mouse antibodiess (dilution, 1/10,000), and signals were detected by use of chemiluminescence blotting substrate.t Membranes were scanned with a chemiluminescence imaging system,u and band intensity was measured with an analysis software.v
Collagen gel contraction assay
The contractile capacity of ASM cells isolated from endobronchial tissue specimens obtained from the lungs of 3 horse cadavers was evaluated by assessing their ability to contract collagen (type I) gel in response to methacholine. Briefly, a collagen type I solutiond (2 mg/mL) containing the ASM cells from the seventh passage was loaded in a 24-well culture plate and allowed to polymerize at 37°C and equilibrate overnight. The resulting gels were carefully detached from each culture well by use of a sterile clamp and transferred into a 6-well plate containing 3 mL of DMEMf with or without methacholined (100μM). Photographs were taken at various points (0, 5, 10, 15, 20, and 30 minutes and 2, 24, 48, and 72 hours) of incubation with or without methacholine by use of a camera.w The brightness of each image was adjusted prior to analysis with imaging software.x The surface area of each gel was then measured, and gel contraction was measured at each photograph point. The experiment was repeated 3 times; each time, a different culture at the seventh passage was used.
Statistical analyses
Differences in contractile proteins among passages were evaluated by means of a repeated-measures linear model.y When differences were significant, values of each passage were compared with those of the first passage by means of the Dunnett post hoc test. Differences between groups of horses (with vs without heaves) were evaluated by use of the unpaired t test. Values of P ≤ 0.05 were considered significant.
Results
Animals
The 5 heaves-affected horses (3 females and 2 males) had a mean age of 21 years (range, 19 to 23 years), and the 4 healthy control horses (4 females) had a mean age of 15 years (range, 11 to 20 years; P = 0.06). Mean body weight of heaves-affected horses (472 kg; range, 442 to 530 kg) was not significantly (P = 0.21) different from that of control horses (484 kg; range, 477 to 490 kg).
Endobronchial biopsy and tissue specimens
The epithelium of endobronchial biopsy and tissue specimens was visible as a fine yellow layer on the edge of the specimens under the binocular dissecting microscope and as purple-stained regions on the histologic sections (Figure 1). This epithelium was easily removed in most instances. The ASM layer of the biopsy specimens was indistinguishable from the surrounding extracellular matrix.
Representative photomicrographs of histologic preparations of endobronchial tissue specimens from lungs freshly harvested from the cadavers of previously healthy horses (A, C, and D) and illustration of smooth muscle and epithelial layers observed in a specimen (B). A—Smooth muscle cells are visible in pink, the epithelium layer in purple, and connective tissues in yellow. Hematoxylin phloxine saffron stain; bar = 1 mm. B—Dissection involved removal of the epithelium, which was identified as a fine yellow layer under a binocular dissecting microscopic (14× magnification). C—After the epithelial layer had been dissected away from the specimen, primarily ASM cells remained. Hematoxylin phloxine saffron stain; bar = I mm. D—An epithelial layer is shown after dissection. Hematoxylin phloxine saffron stain; bar = 100 μm.
Citation: American Journal of Veterinary Research 78, 3; 10.2460/ajvr.78.3.359
Cell isolation
After enzymatic digestion of biopsy specimens, 2 distinct cell types were identified in the first days of cell culture. The first type had a spindle-shaped morphology with tight elongations, establishing connection with adjacent cells (Figure 2). The second cell type was observed in small numbers (≤ 15% of all cells) and resembled smooth muscle cells with what has been described as a proliferative phenotype29 or fibroblasts.30 After a few days of culture, cells appeared as a homogenous population as a result of the faster growth of the second cell type. At confluency, cells grew in overlapping layers, as has been reported for tracheal ASM cells,30,31 and had a characteristic hill-and-valley appearance. Eighty percent confluence was observed in a mean of 7 days.
Representative phase-contrast microscopic images of cells with typical smooth muscle morphology that were isolated from endobronchial tissue specimens from the freshly harvested lungs of horse cadavers. Cells were cultured for up to 7 days until they reached confluency, and selected images represent 8 independent experiments. A—Most cells were spindle shaped. Bar = 50 μm. B—Large, rounded cells with numerous organelles were occasionally identified. Bar = 50 μm. C—Cells in monolayers were most often grouped and growing in a concentric pattern. Confluent cells had the characteristic hill-and-valley appearance of cultured ASM cells. Bar = 100 μm.
Citation: American Journal of Veterinary Research 78, 3; 10.2460/ajvr.78.3.359
Detection of contractile proteins
In preliminary experiments, the presence of α-SMA, desmin, and SMMHC in mesenchymal cell cultures pertaining to 6 horses was evaluated up to the third passage by use of immunolabeling techniques. These mesenchymal cells had uniform staining for α-SMA throughout the 3 passages, with α-SMA being more densely distributed in stress fibers that aligned radially at the cell periphery (Figure 3). The myosin staining was concentrated in the outer cytoplasm, with sparsely distributed myosin spots appearing in extended leading lamellae. Myosin aggregates aligned into small bars and appeared as well-defined striations alternating with α-SMA from the leading edge toward the inner cytoplasm. Desmin was distributed evenly within the cell. These characteristics were observed in both the cytopreparations and the chamber slides. Results confirmed the presence of a contractile cell phenotype in the mesenchymal cell cultures.
Fluorescent microscopic images of equine bronchial mesenchymal cells (A to F) and skin fibroblasts (G and H) that were cultured for 3 passages and stained with fluorescent anti-α-SMA (green; all images), anti-desmin (red; A, D, and G), or anti-SMMHC (red; B, C, E, and H) antibody stains and 4′,6-diamidino-2-phenylindole (blue nuclei) or with fluorescent isotype control antibodies (F). Cells were immunolabeled in monolayer on chamber slides (A to C) or directly on cytopreparations (D to H). The myosin staining was concentrated in the outer cytoplasm (B, C, and E), with sparsely distributed myosin spots (arrowheads) appearing in extended leading lamellae (C). Myosin aggregates aligned into small bars and appeared as well-defined striations alternating with α-SMA from the leading edge toward the inner cytoplasm. Desmin was distributed evenly within cells (A and D). These results confirmed the presence of a contractile cell phenotype in the mesenchymal cultures. Bar = 50 μm in all images.
Citation: American Journal of Veterinary Research 78, 3; 10.2460/ajvr.78.3.359
Expression of the contractile proteins remained stable from the first to third passages; from 61% to 51% of cells having positive staining for α-SMA, respectively; and from 15% to 17% of cells expressing desmin, whereas the proportion of SMMHC-positive cells increased slightly from 14% to 21%. In comparison, immunofluorescence staining of fibroblasts revealed that α-SMA was expressed by only 2.5% of the cells, whereas no cells expressed desmin or SMMHC (Figure 4). No staining of ASM cells was observed when isotype-matched control antibodies were used.
Mean percentage of equine bronchial mesenchymal cells (from 10 biopsy specimens collected from each of 6 healthy horses) with positive staining for the proteins described in Figure 3 after the first (P1), second (P2), and third (P3) passage of cell culture. Error bars represent SD. No significant differences were identified among passages for each protein.
Citation: American Journal of Veterinary Research 78, 3; 10.2460/ajvr.78.3.359
Characterization and quantification of contractile proteins over time
Plots of forward light scatter versus side light scatter data obtained via flow cytometry revealed a homogenous cell distribution in endobronchial tissue specimens from the freshly harvested lungs of 5 horse cadavers (Figure 5). At first passage, 87%, 72%, and 78% of ASM cells expressed α-SMA, desmin, and SMMHC, respectively, whereas 29%, 14%, and 15% had positive staining for α-SMA, desmin, and SMMHC, respectively.
Scatterplots of side light scatter (SSC-H) versus forward light scatter (FSC-H; A) and percentages of cells with positive staining for α-SMA, desmin, SMMHC, and their isotype control (CTL) substances (B) in flow cytometry analysis of ASM cells isolated from endobronchial biopsy specimens from freshly harvested lungs of 3 horse cadavers and primary dermal fibroblasts isolated from skin biopsy specimens from 3 healthy horses. Data are representative of 3 independent experiments. B—Data were obtained at the first passage of cell culture. CTL = Control. FL1-H = Intensity (height) in the FL1 channel (green fluorescence). FL2-H = Intensity (height) in the FL2 channel (orange fluorescence). M1 = Histogram marker 1.
Citation: American Journal of Veterinary Research 78, 3; 10.2460/ajvr.78.3.359
The percentage of ASM cells that expressed α-SMA remained stable (approx 84%) up to the seventh passage, with no significant differences observed between the first and subsequent passages (Figure 6). Approximately 55% and 66% of the cells expressed desmin and SMMHC, respectively. By contrast, MFI (reflecting the quantity of protein in cells) varied significantly among the 7 passages for α-SMA (P = 0.03), desmin (P = 0.01), and SMMHC (P = 0.005). Specifically, MFI differed significantly between first and third passages for α-SMA (P = 0.04), and between first and second passages (P = 0.01) and first and third passages (P = 0.02) for SMMHC. Patterns in MFI over time were similar for the 3 contractile proteins, with a transient decrease in expression observed at second and third passages. From the fourth passage, MFI of these proteins remained stable and was similar to the intensity measured after the first passage.
Mean percentages of ASM cells isolated from endobronchial tissue specimens from the freshly harvested lungs of 5 horse cadavers with positive staining for α-SMA (A), desmin (C), and SMMHC (E) and mean MFI values (arbitrary units) for staining for α-SMA (B), desmin (D), and SMMHC (F). Error bars represent SD. *Values indicated by the ends of the horizontal bars differ significantly (P ≤ 0.05).
Citation: American Journal of Veterinary Research 78, 3; 10.2460/ajvr.78.3.359
Results of western blot analyses were similar to those of flow cytometry (Figure 7). Quantification of the various band intensities revealed no significant difference among the intensity of expression of the 3 contractile proteins between the first and subsequent passages, although there was a significant (P = 0.04) difference among passages for α-SMA.
Photographs of results of western blot analysis of α-SMA, desmin, and SMMHC in ASM cells isolated from endobronchial tissue specimens from the freshly harvested lungs of 4 horse cadavers and cultured over 7 passages (P1 to P7; A) and graphs of mean band intensities for α-SMA (B), desmin (C), and SMMHC (D). In panel A, molecular weights for each detected protein are indicated on the side of the blots. In panels B, C, and D, error bars represent SD.
Citation: American Journal of Veterinary Research 78, 3; 10.2460/ajvr.78.3.359
Functional contractility of ASM cells
The degree of contraction of a collagen gel by ASM cells in response to methacholine was significant, starting 5 minutes after exposure to methacholine (Figure 8). This cell behavior was followed by time-dependent spontaneous contractions and related to cytoskeletal reorganization.32
Representative photographs showing degrees of gel contraction in response to ASM cells being exposed to vehicle (DMEM) or 100μM methacholine (A) and overall contraction curves (B) for ASM cells isolated from endobronchial tissue specimens from the lungs of 3 horse cadavers. Results of 1 representative experiment (of 3 total experiments) are shown.
Citation: American Journal of Veterinary Research 78, 3; 10.2460/ajvr.78.3.359
Contractile phenotype in ASMs from live healthy horses and horses with heaves
Cells isolated from endobronchial biopsy specimens from 5 heaves-affected horses in clinical remission from the disease and 4 healthy control horses (10 specimens/horse) and cultured to 7 passages similarly expressed α-SMA and desmin. No significant difference was identified between heaves-affected and control horses in the percentage of cells expressing α-SMA (P = 0.89) or desmin (P = 0.51; Figure 9). However, a significantly greater proportion of ASM cells from specimens collected from live horses (n = 4) had expression of α-SMA (P = 0.04) and desmin (P = 0.005) than did cells from specimens collected from horses at necropsy (6).
Mean percentages of ASM cells isolated from endobronchial biopsy specimens (n = 90) collected from live healthy horses (n = 5) and heaves-affected (4) horses in clinical remission with positive immunofluorescence results for α-SMA and desmin at first passage of cell culture (A) and of ASM cells isolated from similar specimens (100) collected from live healthy horses (4) and healthy horses at necropsy (6; B). See Figure 6 for remainder of key.
Citation: American Journal of Veterinary Research 78, 3; 10.2460/ajvr.78.3.359
Discussion
Airway smooth muscle cells are involved in many lung diseases of humans and other animals, and intrinsic differences in the behavior of these cells in culture suggest a possible contribution to inflammatory and remodeling processes affecting the airways. In the study reported here, a method was developed for isolation and culture of ASM cells from endobronchial biopsy specimens obtained from horses at necropsy, and our findings were confirmed by use of similar specimens obtained from live heaves-affected horses in clinical remission from the disease and healthy control horses. Expression of the contractile proteins α-SMA, desmin, and SMMHC transiently decreased in these cells during the first 3 passages in culture and were then restored after the fourth passage, with approximately 80% of cells having positive results for α-SMA and 60% having positive results for desmin and SMMHC, suggesting transcriptional regulation of these contractile proteins after few passages.
The ASM cells used in the present study were isolated from 8 to 10 endobronchial biopsy specimens from each horse, as is commonly performed in humans.33 This amount of specimens was generally sufficient for establishing primary ASM cell cultures and evaluating the function of contractile proteins by flow cytometry or immunofluorescence. Expression of contractile proteins was evaluated by immunofluorescence cell staining on cytopreparations and chamber slides, by western blot analysis, and by flow cytometry. Flow cytometry provided a more objective, reproducible, and sensitive assessment than the other methods did. Isolated ASM cells achieved confluency and a characteristic hill-and-valley pattern within 7 days of culture, as has been reported for human trachealis muscle cells.23 Contrary to findings for canine tracheal muscle,12 however, flow cytometry analysis of ASM cells in the present study did not reveal any distinct subpopulations and ASM cells appeared homogenous throughout all passages.
The reason for the decrease in the degree of contractile protein expression observed in the second and third passages was unclear but could have been a consequence of digestion or time necessary to reach the balance between the different cell populations. As has been reported,34 2 distinct cell types were identified via microscopy during the first passage. The less common cell type was faster growing, possibly representing proliferative ASM, whereas the more common type was slower growing, appearing to be ASM in a contractile state. We speculated that the proliferative cell type, initially less abundant in the first passage, was overrepresented in the second and third passages to finally decrease in subsequent passages. This initial loss of contractile proteins should be taken into account when studying the kinetics of phenotypic changes in bronchial tissues.
Despite heterogeneity in the cell populations initially recovered, cells expressing proteins associated with a contractile phenotype were present up to the seventh passage. Using immunofluorescence cell staining, we found that the cell localization within individual ASM cells of the 3 contractile proteins evaluated was similar to that in previous reports35–38 regarding human cells. Although our findings indicated that ASM cells were successfully maintained in culture, α-SMA and desmin can also be found in other cell types. Even SMMHC,39 which is considered specific for smooth muscle cells,13 may also be expressed by myofibroblasts in certain conditions.40,41 The presence of myofibroblasts, and possibly fibroblasts, mixed with the contractile smooth muscle cells may therefore have contributed to the observed partial expression of contractile proteins.
Cells with positive staining for α-SMA but not for desmin or SMMHC may have been fibroblasts, possibly evolving into myofibroblasts.40,42 However, in the culture conditions used, fibroblasts expressed these contractile proteins only weakly, if at all. Furthermore, a wide spectrum of possible ASM cell phenotypes may also coexist in culture,24,43 with mixed or intermediary phenotypes expressing various combinations of contractile proteins. The presence of other cell types such as epithelial cells was excluded on the basis of visible morphology and the selective medium used.
The collagen gel contraction assay used in the present study is a physiologic in vitro model used to investigate the mechanism of cytoskeletal reorganization or stress fiber formation leading to smooth muscle contraction. The ASM cells at the seventh passage were hyperresponsive (within a few minutes) to methacholine, a bronchoconstrictor agent commonly used to evaluate the behavior of ASM cells in vitro and in vivo,32,44 demonstrating that contractile mechanisms of the ASM cells isolated from endobronchial biopsy specimens were maintained. Although no significant difference was identified between live healthy and heaves-affected horses (in clinical remission) in the expression of contractile proteins, a difference was found between ASM cells isolated from specimens obtained from cadavers of previously healthy horses and those from live healthy horses. These results suggested that a delay as brief as 2 hours between sample collection and processing may be sufficient to alter the phenotype of the cells.
In the study reported here, a protocol was established to isolate equine ASM cells from endobronchial tissue and biopsy specimens from horses and the temporal pattern of change in these cells and the stability of their phenotype was elucidated. Unlike in previous studies involving human smooth muscle cells cultured from endobronchial biopsy specimens, the epithelial layer and cartilage were first dissected and the enzymatic dispersion technique45 was subsequently used to isolate smooth muscle cells because this technique reportedly allows maintenance of muscle cells in a morphologically well-differentiated and contractile state.30,46 The ASM cells were maintained in an enriched culture medium at a specific ratio (3:1), offering optimal growth with the highest condition for maintenance of the contractile phenotype. We therefore concluded that, as reported for cells obtained from lungs harvested at autopsy or open lung biopsy specimens from humans,14,34,47 ASM cells cultured from equine endobronchial biopsy specimens had phenotypic heterogeneity in vitro but maintained a contractile state through 7 passages and could serve for 3-D bronchi engineering.
Acknowledgments
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.
ABBREVIATIONS
| α-SMA | α-Smooth muscle actin |
| ASM | Airway smooth muscle |
| DMEM | Dulbecco Modified Eagle Medium |
| MFI | Mean fluorescence intensity |
| SMMHC | Smooth muscle myosin heavy chain |
Footnotes
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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