As the importance of the microenvironment in governing cell responses has been demonstrated in numerous examples, three-dimensional tissue models are proving to be invaluable to studying cell and tissue physiology and pathophysiology (44
). As such, there is a growing appreciation for the importance of the biophysical environment, with appropriate mechanical cues, in both making the three-dimensional models more physiologic and also in studying the roles of such cues in pathophysiology (37
). Our results, using a physiologically relevant three-dimensional tissue model of the human airway wall, demonstrate the importance of mechanical stress in airway wall remodeling and the importance of epithelial–fibroblast crosstalk in such remodeling.
Our in vitro tissue model showed physiologically relevant cell organization and function: it has a pseudostratified epithelium with both ciliated and mucus-producing cells, and under baseline culturing conditions secreted basement membrane beneath the epithelium and type III collagen and fibronectin, among other matrix proteins, within the tissue.
SMCs were absent in our model, which is important to consider in interpreting overall remodeling response, since they can also secrete molecular regulators of airway wall remodeling and fibroblast differentiation (23
). However, their mechanical function was simulated by inducing lateral dynamic strain with a custom-made strain device. It has been reported in computational models of airway mechanics in asthma that 40% shortening of the airway smooth muscle (ASM) can result in almost total occlusion of the airway (26
), while in others, 30% shortening may translate to a > 15-fold increase in pulmonary resistance in asthmatic versus normal airways (46
). In additionally, isolated human bronchial smooth muscle cells from individuals with asthma were found to contract to a greater extent—more than 10% in length—compared with cells from normal subjects (24
). In light of these results, we chose 10% and 30% as representative strain magnitudes of ASM shortening.
In addition to strain magnitude, the duration and frequency of ASM contraction were important parameters to consider. While it is difficult to generalize the transient behavior of smooth muscle contraction, several studies have shown with isolated human bronchial ASM cells (24
) and canine muscle strips (48
) that 75% of their contractions are completed within 1.5–4 s. The time we imposed for contraction was ~ 4 s for 10% strain and ~ 12 s for 30% strain. In addition, frequency may also be an important factor in remodeling, but the duration of ASM hyperactivity can vary dramatically for individuals with asthma with variation in severity; thus it is difficult again to generalize the relevant frequencies to use for in vitro
simulations. We chose 1 and 60 cph as possible low and high frequencies of ASM contraction over a 48-h period. Therefore, with these parameters, we attempted to represent reasonably relevant values of strain that may contribute to ECM remodeling.
In our three-dimensional model, strain influenced cell–matrix and cell–cell interactions to change the architecture and organization of the ECM in the absence of inflammation. The deposition of types III and IV collagen was concentrated near the epithelium and decreased away from the epithelium, which was not observed in fibroblast-only conditions. The epithelium may play an important role as a source of profibrotic mediators such as TGF-β1
leading to matrix remodeling (8
). Furthermore, the spatial gradient in ECM protein deposition was amplified under all strain conditions compared with static controls (, data not shown for type IV collagen), suggesting that epithelial–fibroblast communication, possibly aided by convection of secreted mediators, may act to facilitate the spatial deposition of these ECM proteins.
While this study examined only a few ECM proteins, our results correlate with remodeling characteristics seen in the human airways. In individuals with asthma, the subepithelial layer of the airway wall is thickened and enriched with types III and IV collagen, as well as type V collagen, fibronectin, and tenascin (1
). Consistent with such pathologic remodeling, we observed deposition of types III and IV collagen concentrated in the subepithelial region and amplified with dynamic strain in our model. Interestingly, we found that strain decreased fibronectin deposition, indicating differential regulation of various ECM proteins in response to mechanical strain.
In addition to HBECs and HLFs, myofibroblasts may have contributed to collagen production in our system. Myofibroblasts are commonly found in the thickened subepithelial collagen layer of the asthmatic airway wall (7
), and in the lungs of patients with pulmonary fibrosis (43
). They play an important part in remodeling by matrix contraction (12
) and synthesis of types I and III collagen (52
). In our system, myofibroblasts were found in increased numbers just beneath the epithelium, in the same spatial distribution as type III collagen under conditions of mechanical strain (). These results suggest that myofibroblasts were likely an important cellular source of collagen synthesis in response to mechanical strain.
In conclusion, we have shown that mechanical strain in a tissue engineered three-dimensional co-culture model of the airway wall can be an important determinant of ECM remodeling, as indicated by deposition/secretion of ECM proteins and MMPs, and by differentiation of myofibroblasts. In our three-dimensional airway wall model, we found lateral compressive strain on airway cells resulted in upregulation of types III and IV collagen and increased levels of secreted MMP-2 and -9, but downregulation of fibronectin. In addition, type III collagen was spatially correlated with myofibroblast differentiation. Thus, our dynamic human airway wall model demonstrates the importance of mechanical strain in airway wall remodeling in a relevant three-dimensional environment, independent of inflammatory cells and their mediators.