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* From the Department of Pathology, Nippon Medical School, Tokyo, Department of 1st Internal Medicine, Kumamoto University, School of Medicine, Kumamoto, Japan.
Correspondence to: Yasuhiro Terasaki, MD, Second Department of Pathology, Kumamoto University, 2-2-1 Honjou, Kumamoto City, 860-0811, Kumamoto, Japan; e-mail:terayasu{at}kaiju.medic.kumamoto-u.ac.jp
Epimorphin was identified as a mesenchymal cell surface-associated protein that is essential for epithelial morphogenesis. In the embryonic mouse skin and lung, epimorphin is localized strongly to the border of mesenchymal and epithelial cells by using the epimorphin antibody.1 Using primary cultured rat hepatocytes, it also has been shown that epimorphin induces the formation of hepatocyte spheroids with a bile canaliculi-like structure, which maintained albumin production even in the absence of growth factors.2 Pulmonary fibrosis is thought to be a result of the process of wound healing, and the process takes place mainly within the intra-alveolar space, which in part mimics the process of fetal lung development.3 We investigated the temporal and spatial changes in the distribution of epimorphin protein and the expression of its messenger RNA in bleomycin-induced pulmonary fibrosis in mice.
Materials and Methods
Pulmonary fibrosis was induced in 8-week-old male ICR mice by a single intratracheal instillation of bleomycin. On selected days after injection (days 0, 3, 7, 14, 21, 28, 35, 42, and 56), we harvested the lungs and investigated. We have performed immunohistochemical analysis for epimorphin using light microscopy and confocal microscopy and also have performed the analysis by immunoelectron microscopy and in situ hybridization. We have added quantitative analysis for epimorphin by Northern blotting.
Results
Epimorphin immunoreactivity is seen in the bronchiolar, vascular, and alveolar walls of healthy lungs. On day 7 after bleomycin, epimorphin-positive immunoreactivity also is seen in the early stage of intra-alveolar fibrosis. Fourteen days after treatment, intra-alveolar fibrosis is clearly detectable, and epimorphin immunoreactivity is detected in the alveolar walls and in the areas of intra-alveolar fibrosis. Keratin-positive epithelial cells are observed in the healthy alveolar walls but are not yet present in the intra-alveolar fibrosis. This finding is confirmed by double-labeling of confocal images showing the strong epimorphin-positive intra-alveolar fibrosis area without overlaying keratin-positive regenerating epithelial cells. Twenty-one days after treatment, the triple-labeling of confocal images clearly confirmed that at this stage, epimorphin-positive intra-alveolar fibrotic lesions are being reepithelialized by regenerating type II pneumocytes double positive (dark red color) for keratin (blue color) and surfactant protein C (SP-C; red color). Confocal images also clearly confirmed that epimorphin is localized in vimentin-positive stromal cells and in the surrounding extracellular matrix. By day 35, strong epimorphin immunoreactivity was observed in the fibrotic areas, and confocal images clearly confirm that the epimorphin-positive fibrotic lesions (green color) are now almost completely overlaid with regenerating epithelial cells with SP-C (dark red color) and without SP-C (blue color); (Fig 1 , left, a). By day 56, some of the mildly fibrotic area was healed, but some of the severely fibrotic area remained scarred. There was less epimorphin immunoreactivity in the fibrotic areas than in the previous stages. Other paired serial sections showed the presence of vimentin-positive cells within areas of dense fibrosis where decreased expression of epimorphin also was demonstrated.
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Discussion
The observed distribution of epimorphin protein and the expression of its messenger RNA in healthy mouse lungs were consistent with previous reports4 and suggest that epimorphin is active in healthy adult tissue, although its specific function is not yet known. It is known, however, that under physiologic conditions, alveolar epithelial cell turnover time in mouse lung ranges from 28 to 35 days, and the daily endothelial cell turnover is estimated to be about 1%.5 Thus, in the healthy adult lung, epimorphin may serve as an epithelial and endothelial cell morphogen, maintaining the cell turnover necessary for normal structure and function.
Our findings clearly demonstrate the expression of epimorphin in mesenchymal cells situated within active fibrotic lesions and the presence of epimorphin in the extracellular matrix in the vicinity of those cells. Those findings were consistent with previous reports demonstrating the expression of epimorphin in mesenchymal cells in the rat fetal and postnatal intestine by in situ hybridization6 and the high affinity of epimorphin for the basement membrane component in Matrigel (Collaborative Research; Lexington, MA).1 Furthermore, the expression of epimorphin was found to be elevated before and during the period of active reepithelialization of fibrotic lesions, after which epimorphin expression declined. The time course of the expression of epimorphin in our model was similar to that seen in fetal rat intestine, where epimorphin messenger RNA was strongly expressed during lumen formation and villus morphogenesis, then decreased gradually but continued at a lower level even in the postnatal period.6
It has been suggested that the function of epimorphin is to induce epithelial cell differentiation. However, in the early stages of lung fibrosis in our study, epimorphin expression was strongly detected in areas of active fibrotic lesions in which reepithelialization had not yet occurred. This high levels of expression continued until day 28, by which time reepithelialization was virtually complete. The extracellular matrix present in early fibrotic lesions is known to contain fibronectin and other adhesion molecules as ligands for regenerating alveolar epithelial cells for successful repair.7 Therefore, we suggest that a key role for the epimorphin present in the early fibrotic lesions is to participate in the adhesion of the regenerating epithelial cells as well as to the differentiation of the regenerating epithelial cells from SP-C–positive type II pneumocytes to SP-C–negative type I pneumocytes. In conclusion, epimorphin may have important roles as a morphogen not only in embryonic lungs but also in adult lungs during the process of wound healing and may contribute to the remodeling of pulmonary fibrosis via epithelial-mesenchymal interactions.
Footnotes
Abbreviation: SP-C = surfactant protein C
References
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