Abstract
Asthma is associated with increased deposition and altered phenotype of airway smooth muscle (ASM) cells. However, little is known about the processes responsible for these changes. It has been suggested that alterations of the extracellular matrix (ECM) contribute to the remodeling of ASM cells in asthma. Three-dimensional matrices allow the in vitro study of complex cellular responses to different stimuli in a close-to-natural environment. Thus, we investigated the ultrastructural and genic variations of ASM cells cultured on acellular asthmatic and control bronchial matrices. We studied horses, as they spontaneously develop a human asthma-like condition (heaves) with similarities to chronic pulmonary changes observed in human asthma. Primary bronchial ASM cells from asthmatic (n = 3) and control (n = 3) horses were cultured on decellularized bronchi from control (n = 3) and asthmatic (n = 3) horses. Each cell lineage was used to recellularize six different bronchi for 41 days. Histomorphometry on HEPS-stained-recellularized matrices revealed an increased ASM cell number in the control cell/control matrix (p = 0.02) and asthmatic cell/control matrix group (p = 0.04) compared with the asthmatic cell/asthmatic matrix group. Scan electron microscopy revealed a cell invasion of the ECM. While ASM cells showed high adhesion and proliferation processes on the control ECM, the presence of senescent cells and cellular debris in the asthmatic ECM with control or asthmatic ASM cells suggested cell death. When comparing asthmatic with control cell/matrix combinations by targeted next generation sequencing, only AGC1 (p = 0.04), MYO10 (p = 0.009), JAM3 (p = 0.02), and TAGLN (p = 0.001) were differentially expressed out of a 70-gene pool previously associated with smooth muscle remodeling. To our knowledge, this is the first attempt to evaluate the effects of asthmatic ECM on an ASM cell phenotype using a biological bronchial matrix. Our results indicate that bronchial ECM health status contributes to ASM cell gene expression and, possibly, its survival.
Highlights
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]
As expected for these cell types, the co-expression of desmin and α-SMA was greater in the ASM cells than in the fibroblasts both before (p = 0.01) and during recellularization (p = 0.0001) (Figure 1)
Through its composition and architecture, it governs the fundamental behaviors of cells and their characteristics, such as adhesion, migration, differentiation, FIGURE 2 | ASM cell proliferation. (A) Hematoxylin-eosin-phloxine-safran (HEPS) staining of recellularized matrices at 200 magnification for histomorphometry. (B) Zoom on perimeter calculation on a scan section at magnification 200, the white rectangle is focused on a section containing ASM cells stained in purple with some positive nuclei identified by red arrows. (C) Point counting zoom on a scan section at magnification 200. (D) Number of nuclei per μm in the asthmatic matrix/asthmatic cell group was significantly decreased compared with the control matrix/control cell and asthmatic matrix/asthmatic cell groups
Summary
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]. The source of ASM remodeling remains debated and includes epithelial to mesenchymal transition [6, 7], reduced cell apoptosis, and ASM cells hypertrophy and hyperplasia [8,9,10,11,12]. 3D co-cultures influence the phenotype of smooth muscle cells. The composition of the bronchial scaffold and its organization could affect ASM cell development and behavior [23]
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