Extracellular Matrix Remodeling by Dynamic Strain in a Three-Dimensional Tissue-Engineered Human Airway Wall Model

Abstract

Airway wall remodeling is a hallmark of asthma, characterized by subepithelial thickening and extracellular matrix (ECM) remodeling. Mechanical stress due to hyperresponsive smooth muscle cells may contribute to this remodeling, but its relevance in a threedimensional environment (where the ECM plays an important role in modulating stresses felt by cells) is unclear. To characterize the effects of dynamic compression in ECM remodeling in a physiologically relevant three-dimensional environment, a tissue-engineered human airway wall model with differentiated bronchial epithelial cells atop a collagen gel containing lung fibroblasts was used. Lateral compressive strain of 10 or 30% at 1 or 60 cycles per hour was applied using a novel straining device. ECM remodeling was assessed by immunohistochemistry and zymography. Dynamic strain, particularly at the lower magnitude, induced airway wall remodeling, as indicated by increased deposition of types III and IV collagen and increased secretion of matrix metalloproteinase-2 and -9. These changes paralleled increased myofibroblast differentiation and were fibroblast-dependent. Furthermore, the spatial pattern of type III collagen deposition correlated with that of myofibroblasts; both were concentrated near the epithelium and decreased diffusely away from the surface, indicating some epithelial control of the remodeling response. Thus, in a physiologically relevant three-dimensional model of the bronchial wall, dynamic compressive strain induced tissue remodeling that mimics many features of remodeling seen in asthma, in the absence of inflammation and dependent on epithelial-fibroblast signaling.Keywords: asthma; collagen; fibrosis; in vitro; myofibroblast Remodeling of the airway wall in asthma is characterized by thickening of the subepithelial lamina reticularis, increase in smooth muscle mass, fibroblast hyperplasia, mucus hypersecretion, edema, and angiogenesis (1-6). The subepithelial fibrosis that is often part of this remodeling is further characterized by the differentiation of fibroblasts to myofibroblasts, which are more contractile than fibroblasts and are likely to contribute to the alignment and stiffening of the airway matrix by secretion of types I, III, and V collagen (7-14). In addition, matrix metalloproteinases (MMPs) and their inhibitors (tissue inhibitors of metalloproteinases [TIMPs]), particularly MMP-2 and -9 and TIMP-1, are also increased in the airways in asthma, as they are key players in extracellular matrix (ECM) remodeling (15, 16). Evidence from clinical studies supports the concept that airway remodeling contributes to irreversible airflow obstruction and loss of pulmonary function over time in patients with chronic asthma (17, 18).The complex interplay of factors contributing to airway wall remodeling, which includes inflammatory and mechanical factors, is not well understood. Immune cells recruited to the inflamed airways release cytokines such as RANTES (CCL5), IL-13, TGF-␤ 1 , and cysteinyl leukotrienes C4, D4, and E4 (19-21) that c...

Effects of dynamic compression on lentiviral transduction in an in vitro airway wall model

“…We first sought to optimize culture conditions for epithelial ciliogenesis. Compared with our previous work (11,12), we found that the presence of calcium, ethanolamine, and phospho-ethanolamine as well as higher concentrations of retinoic acid (30 ng/ml) and bovine pituitary extract (200 g/ml) improved the extent of ciliogenesis and cilia length (Fig. 3).…”

“…It is likely that this increased susceptibility is at least partly caused by epithelial activation and damage and by factors including cytokines secreted by mesenchymal and inflammatory cells, which in turn cause loss of barrier function. However, asthmatic airways also experience compressive strain due to frequent brochocon- striction, and it has been demonstrated in vitro that mechanical compressive stress itself, at physiologically relevant levels, can activate the epithelium in asthmatic-like ways and induce airway wall remodeling events in the absence of inflammatory mediators (11,12,52,60,67,68).…”

“…The utility of previously developed in vitro models for studying transepithelial transport-related phenomena is limited by the level of bronchial epithelial cell differentiation and ciliary function attainable (10 -12, 50, 51, 55). Culture conditions that were previously developed to promote epithelial differentiation towards a pseudostratified state include coculture with fibroblasts within a 3D matrix (1,11,50,51,63,64,75), the addition of specific factors to a serum-free media (15,27,55), and culture in ALI after epithelial cells have reached confluence (3,15,18,49,65). We further optimized the medium composition for ciliogenesis and cultured for 21 days at ALI.…”

“…The dynamic strain culture device (Fig. 1B) has been previously described (11). HLFs were suspended at 500,000 cells/ml in 2.5 mg/ml type I collagen (BD Biosciences, Basel, Switzerland) and allowed to polymerize at 37°C, 5% CO2 within the culture device.…”

“…In particular, several different in vitro models of the bronchial airway wall have been used to demonstrate that mechanical stress, at levels relevant to bronchoconstriction, can itself induce changes in gene and protein expression in epithelial cells and fibroblasts that are consistent with airway wall remodeling events, as well as matrix remodeling and smooth muscle cell (SMC) migration and proliferation (11,12,31,42,60,66,73).…”