The asthmatic airways undergo remodeling including loss of epithelial cell integrity, mucus gland and goblet cell hypertrophy, extracellular matrix (ECM) fibrosis, angiogenesis, and increased airway smooth muscle (ASM) mass (1, 2). ASM enlargement was recognized as the main feature of airway wall thickness in asthma as early as 1922 (3). Later studies determined its pivotal role in asthmatic bronchial remodeling and hyperreactivity (4, 5). However, the source of ASM remodeling remains debated and includes epithelial to mesenchymal transition (6, 7), reduced cell apoptosis, and ASM cells hypertrophy and hyperplasia (8–12).

Tissue engineering was developed to create substitutes for injured tissues or organs (13, 14). Cells cultured on 3D substrates have a different behavior than those from 2D cultures and better mimic the in vivo environment (15–17). Therefore, protocols using 3D substrates have been adapted for studies on cellular phenotypes in disease and in response to drugs (18–20). It has been reported that compared to 2D cell culture, human umbilical artery smooth muscle cells cultured in hanging drops have an altered gene expression pattern and reduced protein levels of PCNA and increased levels of α-SMA when compared to 2D standard cell culture (21). Also, 3D co-cultures influence the phenotype of smooth muscle cells. For instance, 3D co-culturing vascular smooth muscle cells and endothelial cells, the vascular smooth muscle cells went from quiescence to contractile phenotype (22). Therefore, the composition of the bronchial scaffold and its organization could affect ASM cell development and behavior (23).

Factors triggering ASM remodeling and hyperresponsiveness remain understudied and, to the best of our knowledge, have never been explored under 3D culture conditions (24–26). In this study, we aimed to investigate the influence of bronchial extracellular matrix on the gene expression and phenotype of asthmatic ASM using a 3D cell culture. We studied an equine bronchial scaffold using decellularization and recellularization protocols previously developed (27) because of similarities reported between human and equine asthma (28, 29).

A diagram summarizing the experimental design is reported in Supplementary Material 1.

The ECM is a complex network composed of more than 300 different multi-domain proteins that releases bioactive fragments upon proteolysis (33). Through its composition and architecture, it governs the fundamental behaviors of cells and their characteristics, such as adhesion, migration, differentiation, apoptosis, and contractility (34, 35). It has been assessed that cells cultured on 3D matrices had a different behavior which is closerto in vivo conditions than on a 2D culture plates (36, 37). Thus, 3D culture has been widely used in different research studies to study the response of cells to drugs and in diseases (38, 39). Thus, we hypothesized that ASM cells cultured on bronchial scaffolds from asthmatic and healthy horses would display a distinct behavior. Our results confirmed this hypothesis, as we observed that ASM cell senescence process and gene expression varied depending on the health status of the horses from which the matrices originated. Also, there was a reduction in ASM cell number when the cells were harvested from asthmatic airways and cultured on asthmatic matrices, when compared with the control ASM cultured on control matrices. These findings indicate that both the asthmatic ECM and ASM cells may contribute to ASM remodeling in asthma.

The histomorphometry results revealed that ASM cells from healthy horses proliferated more than ASM cells from asthmatic horses whether the matrices were from asthmatic or healthy horses, suggesting that the main effector was health status of the cells. However, SEM and qualitative observations suggested that ASM cell behavior was conditioned both by the health condition of the horse providing the homing matrix and by the health status of the horse providing the cells. However, our finding of reduced number of ASM cells in asthma is in contrast with studies reporting ASM cell proliferation in both human asthma (8, 9) and equine asthma (30, 40). In horses, a dynamic equilibrium associated with increased myocyte proliferation and apoptosis was thought to be limiting ASM mass, preventing the occlusion of the airway lumen (30, 40). Both the markedly reduced cell number on histomorphometry and the presence of senescent cells on SEM we observed when recellularizing scaffolds with ASM cells from asthmatic horses suggest a shift in dynamic equilibrium that is in favor of cell death rather than cell proliferation. Asthmatic bronchial ECMs are stiffer and present altered laminin, elastin, vitronectin, collagen and fibronectin deposition and composition when compared with healthy ECMs (41–45). Considering cell-matrix “inside-out” and “outside-in” cross-talk and interdependence (23, 33), these alterations may result in modifications in the conformation of matrix integrins (23, 46), that in turn, may explain the lack of ASM cell proliferation (11, 47, 48). It is important to point out that previous studies have determined that the decellularization process we used does not alter protein composition, including that of collagen and fibronectin, nor the general architecture of the bronchi (27, 49–51).

The observation of large protein deposits on matrices from healthy horses seeded with cells from asthmatic horses and small projections on the surface of the cells was suggestive of a continued recellularization process. Indeed, it has been shown that vascular smooth muscle cells were hyperplasic on stiffened collagen fibrils (52). Increased cell deaths and senescence when ASM cells from healthy and asthmatic horses are cultured on matrices from asthmatic horses also indicate a contribution of the asthmatic ECM on ASM cell behavior.

The ECM from equine bronchi decellularized using the method implemented in this study was shown to maintain the protein composition of the native bronchi, such as that of fibronectin and collagens I and IV (27), known to promote ASM cell survival and proliferation (53). However, the lack of cell proliferation in the asthma group could be explained by the absence of structural and immune cell types in our study model, as they contribute to asthma remodeling. In equine asthma, neutrophils are the predominant granulocytes present in the airway lumen. It has been reported that ASM cell proliferation increased dose-dependently when cells are exposed to neutrophil elastase (54) or to exosomes from lipopolysaccharide-stimulated-neutrophils (26). On the other hand, neutrophil elastase along with other proteases induced ASM cell apoptosis probably resulting from ECM degradation, as fibronectin degradation products were found in the supernatant of ASM cells exposed to neutrophil elastase (55). This may lead to a dynamic equilibrium limiting ASM proliferation and ECM deposition in asthma.

Ampliseq is a sequencing technique that enables the simultaneous analysis of hundreds of genes with ultra-high multiplexed PCR. The number of reads of each gene provided by the analysis reflects its degree of expression. It is a suitable technic for exploratory studies, as it allows the identification of differentially expressed genes that could be specifically targeted in subsequent analysis. Only 4 of 70 genes related to asthmatic remodeling were differentially expressed between the control matrix/control ASM cell and asthmatic matrix/asthmatic ASM cell groups. AGC1 encodes for aggrecan, a major component of the cartilaginous extracellular matrix (56) also expressed by vascular smooth muscle cells when cultured in vitro on calcified elastin or hydroxyapatite (57). Most of the literature has linked this gene to skeleton development and disease (56, 58, 59), but it has also been associated with atopic asthma (60). MYO10 is an actin-associated molecular motor known to serve in intracellular movement (61). The in vivo role of MYO10 remains unclear, but in vitro it promotes cellular filopodia formation, and cell migration has been reported (62, 63). Therefore, MYO10 may be promoting ASM cell migration at a higher rate on matrices from healthy horses when compared with those of asthmatic horses. JAM3 is encoding for junction adhesion molecule C, a protein that regulates different cellular processes (64) and is expressed by smooth muscle cells among other cells (65–68). Using a wild-type murine model of IL-33-induced airway inflammation, downregulation of the expression of JAM3 and 60 other tight junction-coding genes was observed (69). Similarly, JAM3 expression was downregulated in the asthmatic/asthmatic group compared with control/control one, further supporting a role in asthma. TAGLN is coding for transglin-2, an actin binding protein that causes its gelation. It is highly expressed in smooth muscles (70), and its expression is upregulated in asthmatic bronchial biopsies in mild asthmatics (71). In agreement with this finding, TAGLN was also overexpressed in ASM from asthmatic horses cultured on asthmatic bronchial matrices when compared with ASM and ECM from healthy animals. Of note, other genes may have been found differently expressed with larger sample sizes.

Flow cytometry results, comparing between ASM cells and fibroblasts, confirmed the cell purity over the recellularization process, as the high co-expression of α-SMA and desmin is only displayed by the ASM cells (72, 73). Furthermore, desmin is expressed by mature muscle cells but not by fibroblasts (73). This ensured that the results obtained in this study reflect the interaction outcomes between ASM cells and bronchial extracellular matrices, as our model did not consider ASM cell interaction with other cell types.

To the best of our knowledge, this is the first attempt to evaluate the effects of asthmatic ECM on ASM cell phenotype using a biological bronchial matrix. Our results support that bronchial ECM health status determines the gene expression and behaviors of ASM cells, such as adhesion, migration, and, possibly, survival. Asthmatic ASM cells are more receptive to the influence of ECM from asthmatic origins, suggesting that the health status of both cells and matrices is the determinant of the outcome of ECM/ASM interactions in asthma. However, the model we used had limitations, namely, it did not consider the interactions of ASM cells with other structural and inflammatory cell types. Further studies including more cell types (epithelial cells, neutrophils, fibroblasts) in this 3D model would be of interest to assess the effect of ASM cell interactions with other cells implicated in the pathogenesis of asthma.

The original contributions presented in the study are publicly available. This data can be found here: https://doi.org/10.5683/SP3/4V2OBE.

The animal study was reviewed and approved by Ethical Committee of the University of Montreal.

SB participated in the design of the study, isolated ASM cells, sampled and decellularized the bronchi, cultured the ASM cells on the decellularized bronchi, sampled the 3D constructs for SEM, performed the histologic staining, conducted MOVAT and HePS staining and histomorphometry, and collected and analyzed the results of this study. MM and GS prepared and analyzed the scan microscopy images. GB did the statistical analysis of histomorphometry results and approved the statistical analysis of Amliseq and flowcytometry. J-PL participated in the design of the study, analysis of the results, and supervised the experimental protocols. All authors contributed to the article and approved the submitted version.

The Canadian Institutes of Health Research, Canada (CIHR) (Grant No: PJT-148807) and the faculty of superior and postdoctoral studies (FESP) scholarships funded this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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