Es [7,8], we have shown that the antifibrotic effect of transplanted MSCs was associated and probably mediated by their down-modulation of hyperoxia-induced pulmonary inflammatory responses. Significant attenuation of hyperoxiainduced increase in both fibrogenic cytokines such as TGF-b and TIMP-1 [34] along with other inflammatory cytokines, and collagen levels in both HT3 and HT3+10, but not in HT10 support the assumption that the early timing of MSCs Mirin transplantation is critical for their best anti-inflammatory and the ensuing anti-fibrotic effects. In the present study, hyperoxia-induced decrease in growth factors such as VEGF and HGF was significantly up-regulated in both HT3 and HT3+10, but not in HT10 despite higher donor cell localization in the lung tissue at P21 in HT10 than in HT3. These findings suggest that the protective effects of MSCs transplantation such as promotion of angiogenesis, anti-apoptotic effects and reduced inflammation are strongly associated orTiming of MSCs Injection for Hyperoxic Lung InjuryFigure 7. Histograms of inflammatory cytokines and chemokines in the hyperoxic lung injury after MSCs transplantation. TNF-a, IL1a, IL-1b, IL-6, and TGF-b levels measured by ELISA (A) and TIMP-1, CXCL7, RANTES, L-selectin and sICAM-1 levels measured by protein array at P21 in the rat lung tissue (B,C). NC, Normoxia control group; HC, Salmon calcitonin web hyperoxia control group; HT3, hyperoxia with stem cell transplantation group at P3; HT10, hyperoxia with stem cell treatment group at 1379592 P10; HT3+10, hyperoxia with stem cell treatment group at P3 and P10. Data; mean6SEM. *P,0.05 compared to NC, # P,0.05 compared to HC,{ P,0.05 compared to HT3, { P,0.05 compared to HT10. doi:10.1371/journal.pone.0052419.gTiming of MSCs Injection for Hyperoxic Lung InjuryFigure 8. Inflammation with fibrosis in the hyperoxic lung injury after MSCs transplantation. ED1 positive cells indicative of alveolar macrophage were labeled with FITC (green) and the nuclei were labeled with DAPI (blue) (Scale bar; 25 mm) (at top) and number of observed ED1 positive cells per high power field (below) (A), myeloperoxidase activity (B), and collagen levels (C) of the rat P21 lung tissues. NC, Normoxia control group; HC, hyperoxia control group; HT3, hyperoxia with stem cell transplantation group at P3; HT10, hyperoxia with stem cell treatment group at P10; HT3+10, hyperoxia with stem cell treatment group at P3 and P10. Data; mean6SEM. *P,0.05 compared to NC, # P,0.05 compared to HC,{ P,0.05 compared to HT3, { P,0.05 compared to HT10. doi:10.1371/journal.pone.0052419.gprobably mediated by enhanced secretion of these growth factors [35]. Moreover, as the full blown host inflammatory milieu might hinder the secretion of these growth factors by MSCs [36] the timing of MSCs transplantation early in the inflammation isessential for enhanced expression of these growth factors. Further studies will be necessary to clarify this. In summary, intratracheal transplantation of human UCB derived MSCs time-dependently attenuated hyperoxia-induced lung pathology such as decreased alveolarization and increasedFigure 9. VEGF and HGF in the hyperoxic lung injury after MSCs transplantation. Representative RT-PCR blots (at top) and densitometric histograms (below) for HGF (A) and VEGF measured with ELISA (B) in the P21 rat lungs. NC, 18325633 Normoxia control group; HC, hyperoxia control group; HT3, hyperoxia with stem cell transplantation group at P3; HT10, hyperoxia with stem cell treatment group at P10;.Es [7,8], we have shown that the antifibrotic effect of transplanted MSCs was associated and probably mediated by their down-modulation of hyperoxia-induced pulmonary inflammatory responses. Significant attenuation of hyperoxiainduced increase in both fibrogenic cytokines such as TGF-b and TIMP-1 [34] along with other inflammatory cytokines, and collagen levels in both HT3 and HT3+10, but not in HT10 support the assumption that the early timing of MSCs transplantation is critical for their best anti-inflammatory and the ensuing anti-fibrotic effects. In the present study, hyperoxia-induced decrease in growth factors such as VEGF and HGF was significantly up-regulated in both HT3 and HT3+10, but not in HT10 despite higher donor cell localization in the lung tissue at P21 in HT10 than in HT3. These findings suggest that the protective effects of MSCs transplantation such as promotion of angiogenesis, anti-apoptotic effects and reduced inflammation are strongly associated orTiming of MSCs Injection for Hyperoxic Lung InjuryFigure 7. Histograms of inflammatory cytokines and chemokines in the hyperoxic lung injury after MSCs transplantation. TNF-a, IL1a, IL-1b, IL-6, and TGF-b levels measured by ELISA (A) and TIMP-1, CXCL7, RANTES, L-selectin and sICAM-1 levels measured by protein array at P21 in the rat lung tissue (B,C). NC, Normoxia control group; HC, hyperoxia control group; HT3, hyperoxia with stem cell transplantation group at P3; HT10, hyperoxia with stem cell treatment group at 1379592 P10; HT3+10, hyperoxia with stem cell treatment group at P3 and P10. Data; mean6SEM. *P,0.05 compared to NC, # P,0.05 compared to HC,{ P,0.05 compared to HT3, { P,0.05 compared to HT10. doi:10.1371/journal.pone.0052419.gTiming of MSCs Injection for Hyperoxic Lung InjuryFigure 8. Inflammation with fibrosis in the hyperoxic lung injury after MSCs transplantation. ED1 positive cells indicative of alveolar macrophage were labeled with FITC (green) and the nuclei were labeled with DAPI (blue) (Scale bar; 25 mm) (at top) and number of observed ED1 positive cells per high power field (below) (A), myeloperoxidase activity (B), and collagen levels (C) of the rat P21 lung tissues. NC, Normoxia control group; HC, hyperoxia control group; HT3, hyperoxia with stem cell transplantation group at P3; HT10, hyperoxia with stem cell treatment group at P10; HT3+10, hyperoxia with stem cell treatment group at P3 and P10. Data; mean6SEM. *P,0.05 compared to NC, # P,0.05 compared to HC,{ P,0.05 compared to HT3, { P,0.05 compared to HT10. doi:10.1371/journal.pone.0052419.gprobably mediated by enhanced secretion of these growth factors [35]. Moreover, as the full blown host inflammatory milieu might hinder the secretion of these growth factors by MSCs [36] the timing of MSCs transplantation early in the inflammation isessential for enhanced expression of these growth factors. Further studies will be necessary to clarify this. In summary, intratracheal transplantation of human UCB derived MSCs time-dependently attenuated hyperoxia-induced lung pathology such as decreased alveolarization and increasedFigure 9. VEGF and HGF in the hyperoxic lung injury after MSCs transplantation. Representative RT-PCR blots (at top) and densitometric histograms (below) for HGF (A) and VEGF measured with ELISA (B) in the P21 rat lungs. NC, 18325633 Normoxia control group; HC, hyperoxia control group; HT3, hyperoxia with stem cell transplantation group at P3; HT10, hyperoxia with stem cell treatment group at P10;.
