Epithelial to Mesenchymal Transition in Murine Tracheal Allotransplantation: An Immunohistochemical Observation

Abstract

Background: Aberrant epithelial repair is a crucial event in the airway remodeling which characterizes obliterative bronchiolitis (OB) in the transplanted lungs. Epithelial to mesenchymal transition (EMT) has been implicated in the pathogenesis of OB after lung transplantation. During EMT, cells lose epithelial properties and gain properties of mesenchymal cells. Recent data from epithelial cell lines and human airway tissue from recipients of lung transplantations suggest that EMT may have important role in OB. However, few studies have proved the occurrence of EMT in animal models. The aim of this study was to clarify in animal transplant model whether EMT was involved in airway remodeling. Methods: We performed orthotopic tracheal transplantation from BALB/c to C57BL/6 mice. As a control group, syngeneic tracheas were implanted into BALB/c mice. After transplantation, five allogeneic and two syngeneic recipients were sacrificed at predetermined days from day 2 to 12, day 14, and 21. Histology was evaluated on hematoxylin-eosin (H&E) staining. Expression of the epithelial marker (E-cadherin), the mesenchymal marker (α-SMA and S100A4), and EMT-related transcription factor (ZEB1) were studied by immunohistochemistry. Results: In allogeneic grafts, following remarkable pseudostratified respiratory epithelium and subepithelial inflammatory cell infiltration (by day 8), denuded and flattened epithelium and subepithelial fibrosis (from day 8) was observed on serial H&E stains. Epithelial changes were dynamic and earlier than subepithelial fibrosis. At day 14, epithelium was flattened. At day 21, subepithelial spindle-shaped cells like fibroblasts were numerous (Figure 1–). On serial immunohistochemical evaluation of these rejecting tracheas, α-SMA positive epithelial cells emerged by day 6 and are most prominent at day 7 (Figure 1–B). α-SMA positive epithelial cells were frequently lining just above basement membrane. After day 9 few intraepithelial α-SMA positive cells were observed correspond with epithelial denuded stage. S100A4 was also expressed in epithelial cells. The expression of E-Cadherin attenuated in α-SMA positive epithelial cells (Figure 1–C). Before a few days prior to intraepithelial expression of α-SMA, transcription factor ZEB1 emerged in the nucleus of epithelial cells. The expression was greatest from day 2 to 4 (Figure 1–D).[Figure 1]Conclusions: After alloimmune injury, remarkable phenotypic change was observed in the epithelial cells in the allografts: remarkable expression of EMT-related transcription factor ZEB1, subsequent emergence of mesenchymal marker α-SMA and S100A4, and attenuation of E-cadherin expression in α-SMA positive epithelial cells. These findings were observed several days prior to subepithealial prominent fibrous formation, and may suggest that epithelial cells play an important role in airway remodeling through epithelial to mesenchymal transition.