Radiation-induced pneumonitis and subsequent fibrosis are major dose-limiting complications for patients receiving thoracic RT. Various mouse strains have been used to study radiation-induced normal tissue injury; their response is strain-dependent. In the current study, we demonstrated for the first time, i] the sensitivity of young adult female FVB/N mice to thoracic irradiation and ii] the involvement of EMT in radiation-induced lung injury.
In our study, irradiating the thorax of young adult female FVB/N mice with single doses of 12 and 13 Gy led to 50% mortality at 14 and 11 weeks postirradiation, respectively (). In contrast, Steel et al
reported that the median survival time of C57BL mice after doses of 12–20 Gy is between 200–300 days 
. Yang et al
reported that ~50% of the C57BL mice died within 32 weeks after a single 14.5 Gy dose of WTI 
. O'Brien et al. reported that the mean survival time of C57BL mice was 21.8±1.5 weeks after a single 18 Gy dose of WTI 
. Histological analysis of lung tissues from FVB/N mice revealed an extensive alveolar inflammation and regional fibrosis 14 weeks after a single 12 Gy dose of WTI (). In contrast, fibrotic lesions were observed 24 and 26 weeks postirradiation in fibrosis-prone C57/B6 mice after single 12 Gy and 12.5 Gy doses of WTI, respectively 
. These results indicate that the lungs of young adult female FVB/N mice are radiosensitive, making them an appropriate model for studying both radiation-induced pneumonitis and fibrosis, particularly since many transgenic mice are created on a FVB/N background.
One feature of radiation pneumonitis is excessive leakage of fluid from the vessels into the alveolar space, which could impede alveolar gas exchange. Aquaporin-5 has been described as playing a critical role in maintaining water permeability across the cell membrane and also in removal of pulmonary edema fluid from the alveolar space 
. Aquaporin-5 knock-out mice exhibit a 90% decrease in airspace-capillary water permeability, suggesting an important role for aquaporin-5 in maintaining normal lung physiological function 
. Decreased expression of aquaporin 5 has been reported in pathological conditions such as acute lung injury 
and lung fibrosis 
. Moreover, a significant reduction in aquaporin-5 mRNA and protein level has been associated with pulmonary inflammation and edema resulting from adenoviral infection 
. Gabazza et al. noted decreased expression of aquaporin-5 in alveolar type I cells in a mouse model of bleomycin-induced lung fibrosis; aquaporin-5 knock-out mice revealed a fibrotic phenotype 
. Taken together, these data indicate that down-regulation of aquaporin-5 results in abnormal lung fluid metabolism in many diseases. In our study, a decrease in the aquaporin-5 protein level was measured in lungs of mice after a single 12 Gy dose or a fractionated 30 Gy dose of WTI (); a time-dependent decrease in the aquaporin-5 protein level was measure after a fractionated 24 Gy dose of WTI (). These novel findings suggest that aquaporin-5 may be an important biomarker of radiation-induced lung injury.
The lung alveolar epithelium is in direct contact with air. AE1 cells are terminally differentiated, highly susceptible to injury, and incapable of self-renewal. In contrast, AE2 cells are highly resistant to insults, able to self-renew, and serve as stem cells for producing AE1cells; AE2 cells are known as the defenders of the alveoli 
. When the alveolar epithelium is damaged, AE2 cells start proliferating and transdifferentiate into AE1 cells to re-establish a functional alveolar epithelium. Alveolar epithelial injury followed by abnormal epithelial repair appears to be a key pathological feature of lung fibrosis. Increased proliferation/hyperplasia of AE2 cells has been frequently observed in injured and irradiated lungs 
. Pro-SP-c, a surfactant protein, is expressed only by AE2 cells; thus, it has been used as a marker of type II cell differentiation in the mammalian lung 
. The increased number of pro-SP-c+
cells () and the increased pro-SP-c protein levels () measured after both single and fractionated doses of WTI likely reflects proliferation of AE2 cells in an attempt to repair the radiation-induced lung damage.
Myofibroblasts, α-SMA expressing fibroblasts, are a prominent source of type I collagen and fibrogenic/inflammatory cytokines in fibrotic lesions. Several origins for myofibroblasts have been proposed; resident fibroblasts seem to be the most common one. Bone marrow-derived circulating cells have also been suggested as an alternative source of myofibroblasts. Epperly et al. demonstrated that marrow-derived cells constitute 20 to 50% of the cells in radiation-induced fibrotic areas using GFP–positive bone marrow cells 
. Consistent with these results, collagen-producing lung fibroblasts derived from bone marrow progenitor cells have been detected in fibrotic tissues in bleomycin-induced fibrosis 
. However, these marrow-derived fibroblasts did not express α-SMA and were resistant to the fibroblast to myofibroblast conversion by TGF-ß1 
. Recent studies indicate that myofibroblasts can also arise from resident epithelial cells 
that undergo EMT. Liu et al. 
reported expression of α-SMA and vimentin in kidney tubular epithelial cells in a rat model of radiation nephropathy, supporting the hypothesis that radiation leads to the transition of tubular epithelial cells to myofibroblasts. EMT and increased cell motility has also been reported in the irradiated human lung A549 cells 
. Using an in vitro
model, we have demonstrated that irradiation of AE2 cells resulted a transition of epithelial to a myofibroblast-like phenotype, which was mediated by the ROS/ERK/GSK-3ß/Snail pathway 
. However, EMT in the irradiated lung has not been previously reported. In this study, a significant decrease in the protein levels of the epithelial cell markers, E-cadherin () and aquaporin-5 (), was measured after both single and fractionated doses of WTI. This was accompanied by a concomitant increase in vimentin (), a mesenchymal marker. Moreover, double immunoflorescence staining showed co-localization of the pro-SP-c and α-SMA proteins in the alveoli of irradiated lungs (), implying that AE2 cells had gained a mesenchymal-like phenotype. Our data suggest that AE2 to mesenchymal transition occurs in the irradiated lungs of FVB/N mice. However, it is not known if other epithelial cells in the lung can differentiate into myofibroblasts. The relative contributions of epithelial cells in the irradiated lung to the overall increase in the myofibroblast population and the pathogenic role that EMT plays in radiation-induced lung fibrosis remain to be investigated.
TGF-β1 plays an important role in the development of lung fibrosis 
. Increased TGF-β1 expression leads to recruitment of monocytes and macrophages to the inflammatory site, enhances the maturation and activation of fibroblasts, and stimulates EMT 
. Chronic radiation-induced lung injury has been reported to increase expression and activation of TGF-β1, which leads to parenchymal cell depletion and excess fibrosis 
. TGF-β1 expression in the plasma of patients immediately after RT has been used as an important marker to predict the risk for radiation-induced lung injury 
. In our study, both single and fractionated doses of WTI increased the TGF-β1 protein levels in the lung (). The sustained increase in the TGF-ß1 protein level in the irradiated lungs of FVB/N mice further demonstrates the important role of TGF-ß1 in the pathogenesis of radiation-induced lung injury.
Recently, chromatin remodeling via posttranslational histone modification was found to function in a genome-wide manner and contributes to an extensive range of biological functions 
. Histone deacetylases (HDACs) are known as modulators of gene transcription, which is important for cell function, proliferation and differentiation. HDAC inhibitors have been reported to induce protein hyperacetylation, chromatin remodeling, transcriptional activation and repression, cell-cycle arrest, and cell death 
. Preclinical studies indicate that HDAC inhibitors can effectively block cardiac, skin, liver and renal fibrosis 
. In vitro
and in vivo
investigations indicate that HDAC inhibitors modulate fibrosis by suppressing ECM production, inhibiting myofibroblast activation, blocking EMT, and/or reducing pro-inflammatory cytokine production 
. Furthermore, recent studies suggest that topical treatment of rat skin with HDAC inhibitor 4-Phenyl butyrate has been shown to promote wound healing, reduce skin fibrosis, and decrease tumorigenesis after irradiation 
. In light of these findings, effect of on HDAC inhibitors on radiation-induced lung damage in FVB/N mice is underway in our lab.
In summary, the current findings indicate that the lungs of female FVB/N mice are radiosensitive and represent an appropriate model for investigating radiation-induced lung inflammation and fibrosis. The marked changes in the levels of the alveolar epithelial proteins, E-cadherin, aquaporin-5, and pro-SP-c, appears to be associated with the development of radiation-induced lung injury in FVB/N mice, suggesting that these proteins may serve as sensitive indicators of radiation-induced lung damage. WTI resulted in the loss of epithelial markers and a subsequent increase in the levels of mesenchymal proteins, indicating that EMT occurs in irradiated lung tissue. Although the in vivo significance of EMT is unclear, radiation-induced alterations in the alveolar epithelium phenotype implicate impairment of alveolar epithelial function in producing fibrosis. Future research to identify and quantify the specific mechanisms involved in producing radiation-induced fibrosis should provide targets for the development of interventions that prevent/ameliorate this devastating complication of WTI.