The Effect Of Epithelium To Airway Smooth Muscle Under Pressure In Two Co-culture Models

Asthma has become an increasingly serious threat to human health, but its pathogenesis has not yet completely understood. The hallmarks of asthma include airway inflammation, hyperresponsiveness, and remodeling, involving many factors such as cell fibrosis, mucosal shedding and the precipitation of basement membrane, alteration of smooth muscle. All of these changes will ultimately lead to enhanced airway contractility, and reduced or diminished airway diastolic capacity, resulting in airway narrowing and respiratory congestion.When the airway narrows, not only its area and diameter will change, but also the airway wall will fold at the same time, thus leading to the extrusion of epithelial layer. Increasing evidence indicates that the pressure on the epithelium due to this extrussion causes responses in the epithelium by releasing inflammatory cytokines that may play important roles in mediating airway dysfunction. Because smooth muscle layer and the epithelium are neighboring in the airway wall, it may be possible that the pressure exerted on the epithelium may indirectly influence the airway smooth muscle. However, there is little study with respect to this possibility.Therefore, we examined murine airway smooth muscle cells (mASMC) in coculture with epithelial cells (AEC cell line CCC000149) that were either or not subjected to externally applied pressure. We designed two different co-culture models, namely 1) epithelial cells and airway smooth muscle cells in cell culture were separately inoculated in the culture dish bottom of embedded device (Transwell) and the bottom of the culture chamber, knowned as indirect co-culture; 2) epithelial cells and airway smooth muscle cells were separately seeded in the inner and outer surfaces of the bottom of the culture chamber, known as direct co-culture. Then, we used an in-house built pressure system to exert pressure on the cultured epithelial cells. The pressure was loaded at 0, 2, 4, or 6 kPa for 8, 12, 24, 36, or 48 h, respectively. Under these different experimental conditions, we examined the mASMC in terms of proliferation by MTT, cell morphology inverted optical microscopy; and cell stiffness by atomic force microscopy.Our resutls show that, as compared to in single culture, mASMC in both co-culture models grew increasingly faster, became wider and more flat, also stiffened increasingly as the extermal pressure increased. Furthermore, these changes in mASMC due to applied pressure appeared to be more pronounced in the direct co-culture model than in the indirect co-culture model. These results indicate that the pressure applied on the epithelial cells indeed influenced the nearby airway smooth muscle cells.This study has, to certain extent, simulated in vivo airway narrowing when the extruded epithelium and the airway smooth muscle coexist and interact in response to the pressure applied on the epithelium. The findings from this study are not only important to correct undertanding of airway pathobiology in asthma, but may also provide useful information in regards tissue engineering.