Therapeutic benefits of young, but not old, adipose-derived mesenchymal stem cells in a chronic mouse model of bleomycin-induced pulmonary fibrosis

Abstract

The observation that pulmonary inflammatory lesions and bleomycin (BLM)-induced pulmonary fibrosis spontaneously resolve in young mice, whereas remaining irreversible in aged mice suggests that impairment of pulmonary regeneration and repair is associated with aging. Because mesenchymal stem cells (MSCs) may promote repair after injury, we postulated that differences in MSCs from aged mice may underlie postinjury fibrosis in aging. The potential for young-donor MSCs to inhibit BLM-induced pulmonary fibrosis in aged male mice (>22 months) has not been studied. Adipose-derived MSCs (ASCs) from young (4 months) and old (22 months) male mice were infused 1 day after intratracheal BLM administration. At 21-day sacrifice, aged BLM mice demonstrated lung fibrosis by Ashcroft score, collagen content, and α(v)-integrin messenger RNA (mRNA) expression. Lung tissue from aged BLM mice receiving young ASCs exhibited decreased fibrosis, matrix metalloproteinase (MMP)-2 activity, oxidative stress, and markers of apoptosis vs BLM controls. Lung mRNA expression of tumor necrosis factor-alpha was also decreased in aged BLM mice receiving young-donor ASCs vs BLM controls. In contrast, old-donor ASC treatment in aged BLM mice did not reduce fibrosis and related markers. On examination of the cells, young-donor ASCs had decreased mRNA expression of MMP-2, insulin-like growth factor (IGF) receptor, and protein kinase B (AKT) activation compared with old-donor ASCs. These results show that the BLM-induced pulmonary fibrosis in aged mice could be blocked by young-donor ASCs and that the mechanisms involve changes in collagen turnover and markers of inflammation.

Copyright © 2015 Elsevier Inc. All rights reserved.

Figures

Figure 1

Schematic diagram of treatment Day 0 (D0)=time of bleomycin instillation. Day 1 (D1)=infusion of either saline or ASCs. Day 21(D21)=sacrifice.

Figure 2. Adipose-derived mesenchymal stem cells (ASCs) isolated from 4 month and 22 month old male C57BL/6 mice demonstrate adipogenic and osteogenic differentiation potential

The mouse mesenchymal stem cell functional identification kit (R&D Systems, Minneapolis, MN) was used to stimulate differentiation of ASCs into adipocytes (left) and osteoblasts (right), per the manufacturer’s instructions. Briefly, adipocytes were stained using goat anti-mouse FABP4 primary antibody, and osteocytes were stained using goat anti-mouse osteopontin primary antibody. Subsequently, both were exposed to donkey anti-goat IgG affinity purified PAb NorthernLights 557 fluorochrome-labeled secondary antibody (R&D Systems). Immunocytochemical staining of differentiated cells are presented with DAPI (blue) and NorthernLights (red) fluorescence imaging at magnification 40x.

Figure 3. Bleomycin (BLM) pulmonary fibrosis in 22 month old male C57BL/6 mice is inhibited by young (yASC) but not old ASCs (oASC) at 21 day sacrifice

Histologic examination of Masson’s-Trichrome stained lung tissue was performed as described in Methods. Representative photomicrographs of the lungs of 22 month male C57BL/6 mice infused with saline (n=8), BLM (n=8), BLM+yASCs (n=7), or BLM+oASCs (n=9). Trichrome stained images at 2× (upper), 20× (left), and 40× (right) magnification.

Figure 4. Bleomycin (BLM)-induced increase in markers of pulmonary fibrosis in the lungs of old male C57BL/6 are only inhibited by young adipose derived mesenchymal stem cells (yASC) not old ASCs (oASC)

A) Ashcroft scores were used to evaluate the degree of fibrosis. Data are graphed as the mean score of 32 fields/section of lung. Saline (n=8), BLM (n=8), BLM+yASCs (n=5), BLM+oASCs (n=7). B) Collagen content was estimated by hydroxyproline assay as described in methods. Data are graphed as μg/mg of lung tissue. Saline (n=8), BLM (n=8), BLM+yASCs (n=7), BLM+oASCs (n=8). C) αv-integrin mRNA expression was determined by RT-PCR as a marker of fibrosis. Data are graphed normalized for 18S content. Saline (n=7), BLM (n=12), BLM+yASCs (n=5), BLM+oASCs (n=5), *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 5. Bleomycin (BLM)-induced MMP-2 activity and AKT phosphorylation are inhibited in the lungs of aged male C57BL/6 mice by infusion of young adipose derived mesenchymal stem cells (yASC) but not old ASCs (oASC)

A) Zymography was performed on lung tissue protein extracts from aged male C57BL/6 mice receiving saline, BLM, BLM+yASC, or BLM+oASC to measure MMP-2 activity. A representative zymogram is shown of two individual mice/group. Data are graphed as the mean ± SEM of n=5–6/group. B) Western blots were performed on lung tissue protein extracts from aged male C57BL/6 mice receiving saline, BLM, BLM+yASC, or BLM+oASC to measure AKT phosphorylation. A representative Western blot is shown of two individual mice/group. Data are graphed as the mean ± SEM of n=5–9/group.* p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 6. Markers of apoptosis are increased in lung tissue of aged male C57BL/6 after bleomycin (BLM) fibrosis, and inhibited by infusion of young adipose derived mesenchymal stem cells (yASC) but not old ASCs (oASC)

A) ApopTag assay was performed on lung tissue sections from aged male C57BL/6 mice receiving saline, bleomycin (BLM), BLM+yASC, or BLM+oASC. Panels are stained with FITC (nuclear TUNEL labeling) and DAPI (nuclear staining) as indicated in the Methods section. B) Western blots were performed on lung tissue protein extracts from aged male C57BL/6 mice receiving saline, BLM, BLM+yASC, or BLM+oASC to measure the ratio of cleaved caspase-9 relative to total caspase-9. A representative Western blot is shown of two individual mice/group. Data are graphed as the mean ± SEM of n=4–10/group.* p<0.05, ***p<0.001, ****p<0.0001.

Figure 7. Old adipose derived mesenchymal stem cells (oASC) prevent or slow resolution of Bleomycin (BLM)-induced pulmonary fibrosis in the lungs of young male C57BL/6 mice

A) Collagen content was estimated by hydroxyproline assay as described in methods. Data are graphed as μg/mg of lung tissue. B) αv-integrin mRNA expression was determined by RT-PCR as a marker of fibrosis. Data are graphed normalized for 18S content. *p<0.05. n=5 individual mice for BLM, n=7 individual mice for BLM+oASC.