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Hypoxia-preconditioned mesenchymal stem cells attenuate bleomycin-induced pulmonary fibrosis.

Lan YW, Choo KB, Chen CM, Hung TH, Chen YB, Hsieh CH, Kuo HP, Chong KY - Stem Cell Res Ther (2015)

Bottom Line: The pulmonary respiratory functions significantly improved for up to 18 days of hypoxia-preconditioned MSC treatment.Histopathologic examination observed a significant amelioration of the lung fibrosis.Our data further demonstrated that upregulation of hepatocyte growth factor possibly played an important role in mediating the therapeutic effects of transplanted hypoxia-preconditioned MSCs.

View Article: PubMed Central - PubMed

Affiliation: Division of Biotechnology, Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan, Republic of China. bublelanwilliam@gmail.com.

ABSTRACT

Introduction: Idiopathic pulmonary fibrosis is a progressive diffuse parenchymal lung disorder of unknown etiology. Mesenchymal stem cell (MSC)-based therapy is a novel approach with great therapeutic potential for the treatment of lung diseases. Despite demonstration of MSC grafting, the populations of engrafted MSCs have been shown to decrease dramatically 24 hours post-transplantation due to exposure to harsh microenvironments. Hypoxia is known to induce expression of cytoprotective genes and also secretion of anti-inflammatory, anti-apoptotic and anti-fibrotic factors. Hypoxic preconditioning is thought to enhance the therapeutic potency and duration of survival of engrafted MSCs. In this work, we aimed to prolong the duration of survival of engrafted MSCs and to enhance the effectiveness of idiopathic pulmonary fibrosis transplantation therapy by the use of hypoxia-preconditioned MSCs.

Methods: Hypoxic preconditioning was achieved in MSCs under an optimal hypoxic environment. The expression levels of cytoprotective factors and their biological effects on damaged alveolar epithelial cells or transforming growth factor-beta 1-treated fibroblast cells were studied in co-culture experiments in vitro. Furthermore, hypoxia-preconditioned MSCs (HP-MSCs) were intratracheally instilled into bleomycin-induced pulmonary fibrosis mice at day 3, and lung functions, cellular, molecular and pathological changes were assessed at 7 and 21 days after bleomycin administration.

Results: The expression of genes for pro-survival, anti-apoptotic, anti-oxidant and growth factors was upregulated in MSCs under hypoxic conditions. In transforming growth factor-beta 1-treated MRC-5 fibroblast cells, hypoxia-preconditioned MSCs attenuated extracellular matrix production through paracrine effects. The pulmonary respiratory functions significantly improved for up to 18 days of hypoxia-preconditioned MSC treatment. Expression of inflammatory factors and fibrotic factor were all downregulated in the lung tissues of the hypoxia-preconditioned MSC-treated mice. Histopathologic examination observed a significant amelioration of the lung fibrosis. Several LacZ-labeled MSCs were observed within the lungs in the hypoxia-preconditioned MSC treatment groups at day 21, but no signals were detected in the normoxic MSC group. Our data further demonstrated that upregulation of hepatocyte growth factor possibly played an important role in mediating the therapeutic effects of transplanted hypoxia-preconditioned MSCs.

Conclusion: Transplantation of hypoxia-preconditioned MSCs exerted better therapeutic effects in bleomycin-induced pulmonary fibrotic mice and enhanced the survival rate of engrafted MSCs, partially due to the upregulation of hepatocyte growth factor.

No MeSH data available.


Related in: MedlinePlus

Hypoxia-preconditioned mesenchymal stem cell transplantation downregulates expression of inflammatory factors in the bleomycin-induced pulmonary fibrosis mouse model. Quantitative real-time RT-PCR was performed to analyze the expression of inflammatory factors (A) interleukin (IL)-6 and (B) pro-IL-1β in the lung tissues of animals that received normoxia-preconditioned mesenchymal stem cells (NP-MSCs), hypoxia-preconditioned mesenchymal stem cells (HP-MSCs) or phosphate-buffered saline (PBS) for 18 days following bleomycin (BLM) administration on day 3. Values are normalized to the GAPDH values and expressed relative to the PBS group. *P < 0.05, **P < 0.01. Each dot represents an individual mouse with the mean shown for n > 5 per group. (C) Western blot analysis of pro-IL-1β and IL-1β in whole lung tissues from each group. Ctrl, control.
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Fig5: Hypoxia-preconditioned mesenchymal stem cell transplantation downregulates expression of inflammatory factors in the bleomycin-induced pulmonary fibrosis mouse model. Quantitative real-time RT-PCR was performed to analyze the expression of inflammatory factors (A) interleukin (IL)-6 and (B) pro-IL-1β in the lung tissues of animals that received normoxia-preconditioned mesenchymal stem cells (NP-MSCs), hypoxia-preconditioned mesenchymal stem cells (HP-MSCs) or phosphate-buffered saline (PBS) for 18 days following bleomycin (BLM) administration on day 3. Values are normalized to the GAPDH values and expressed relative to the PBS group. *P < 0.05, **P < 0.01. Each dot represents an individual mouse with the mean shown for n > 5 per group. (C) Western blot analysis of pro-IL-1β and IL-1β in whole lung tissues from each group. Ctrl, control.

Mentions: To determine the effects of HP-MSCs on inflammation and fibrosis in the BLM-induced pulmonary fibrosis model, expression levels of possible mediators that may be involved, including pro-inflammatory cytokine and fibrotic factors, were determined. The mRNA level of the inflammation-mediating IL-6 was significantly upregulated 21 days after BLM treatment compared with the PBS control group (0.91 ± 0.32 versus 5.81 ± 4.01, P < 0.01; Figure 5A). On the other hand, pro-IL-1β showed no significant upregulation at the mRNA level, but the protein levels of the precursor and mature IL-1β were markedly overexpressed (Figure 5B,C). However, there was a significant reduction in the expression of these inflammatory mediators in the HP-MSC transplantation group (IL-6 mRNA level: 0.69 ± 0.44 in BLM HP-MSC group versus 4.65 ± 4.04 (BLM NP-MSC) and 5.81 ± 4.01 (BLM control); pro-IL-1β mRNA level: 0.41 ± 0.20 in BLM HP-MSC group versus 0.86 ± 0.30 (BLM NP-MSCs) and 1.02 ± 0.30 (BLM control); Figure 5A-C). Interestingly, there was no discernible difference in the levels of inflammation between the BLM NP-MSCs and BLM control groups (Figure 5A-C). Furthermore, expression of two factors mediating fibrosis, namely collagen type III and connective tissue growth factor (CTGF), was significantly upregulated at day 21 post-BLM treatment, but expression of collagen type III and CTGF was clearly reduced on HP-MSC transplantation (collagen type III mRNA level: 1.04 ± 0.72 in BLM HP-MSC group versus 3.62 ± 2.24 (BLM NP-MSC) and 5.40 ± 1.90 (BLM control); CTGF mRNA level: 0.66 ± 0.58 in BLM HP-MSC group versus 3.86 ± 3.70 (BLM NP-MSC) and 8.58 ± 7.91 (BLM control); Figure 6A,B). Moreover, there was clearly collagen deposition after BLM treatment, but only low collagen content could be detected in the HP-MSC transplanted mice which was comparable to the normal level (8.87 ± 7.20 μg/mg wet lung tissue in BLM HP-MSC group versus 20.17 ± 15.36 (BLM NP-MSCs) and 28.48 ± 3.06 (BLM control) and 8.02 ± 4.26 (PBS control); Figure 6C). Noting the downregulation of inflammatory and fibrotic mediators, other possible cytoprotective mediators were further investigated by examining the expression levels of HGF in whole-lung tissues by western blot analysis. HGF showed decreased levels in the BLM only and MSC-treated mice, but was overexpressed in the HP-MSC-transplanted mice (Figure 6D). Taken together, transplantation of HP-MSCs exerted better inhibitory effects on upregulating pro-inflammatory and fibrotic factors and on inducing the accumulation of collagen than in the BLM NP-MSC group (Figures 5 and 6).Figure 5


Hypoxia-preconditioned mesenchymal stem cells attenuate bleomycin-induced pulmonary fibrosis.

Lan YW, Choo KB, Chen CM, Hung TH, Chen YB, Hsieh CH, Kuo HP, Chong KY - Stem Cell Res Ther (2015)

Hypoxia-preconditioned mesenchymal stem cell transplantation downregulates expression of inflammatory factors in the bleomycin-induced pulmonary fibrosis mouse model. Quantitative real-time RT-PCR was performed to analyze the expression of inflammatory factors (A) interleukin (IL)-6 and (B) pro-IL-1β in the lung tissues of animals that received normoxia-preconditioned mesenchymal stem cells (NP-MSCs), hypoxia-preconditioned mesenchymal stem cells (HP-MSCs) or phosphate-buffered saline (PBS) for 18 days following bleomycin (BLM) administration on day 3. Values are normalized to the GAPDH values and expressed relative to the PBS group. *P < 0.05, **P < 0.01. Each dot represents an individual mouse with the mean shown for n > 5 per group. (C) Western blot analysis of pro-IL-1β and IL-1β in whole lung tissues from each group. Ctrl, control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig5: Hypoxia-preconditioned mesenchymal stem cell transplantation downregulates expression of inflammatory factors in the bleomycin-induced pulmonary fibrosis mouse model. Quantitative real-time RT-PCR was performed to analyze the expression of inflammatory factors (A) interleukin (IL)-6 and (B) pro-IL-1β in the lung tissues of animals that received normoxia-preconditioned mesenchymal stem cells (NP-MSCs), hypoxia-preconditioned mesenchymal stem cells (HP-MSCs) or phosphate-buffered saline (PBS) for 18 days following bleomycin (BLM) administration on day 3. Values are normalized to the GAPDH values and expressed relative to the PBS group. *P < 0.05, **P < 0.01. Each dot represents an individual mouse with the mean shown for n > 5 per group. (C) Western blot analysis of pro-IL-1β and IL-1β in whole lung tissues from each group. Ctrl, control.
Mentions: To determine the effects of HP-MSCs on inflammation and fibrosis in the BLM-induced pulmonary fibrosis model, expression levels of possible mediators that may be involved, including pro-inflammatory cytokine and fibrotic factors, were determined. The mRNA level of the inflammation-mediating IL-6 was significantly upregulated 21 days after BLM treatment compared with the PBS control group (0.91 ± 0.32 versus 5.81 ± 4.01, P < 0.01; Figure 5A). On the other hand, pro-IL-1β showed no significant upregulation at the mRNA level, but the protein levels of the precursor and mature IL-1β were markedly overexpressed (Figure 5B,C). However, there was a significant reduction in the expression of these inflammatory mediators in the HP-MSC transplantation group (IL-6 mRNA level: 0.69 ± 0.44 in BLM HP-MSC group versus 4.65 ± 4.04 (BLM NP-MSC) and 5.81 ± 4.01 (BLM control); pro-IL-1β mRNA level: 0.41 ± 0.20 in BLM HP-MSC group versus 0.86 ± 0.30 (BLM NP-MSCs) and 1.02 ± 0.30 (BLM control); Figure 5A-C). Interestingly, there was no discernible difference in the levels of inflammation between the BLM NP-MSCs and BLM control groups (Figure 5A-C). Furthermore, expression of two factors mediating fibrosis, namely collagen type III and connective tissue growth factor (CTGF), was significantly upregulated at day 21 post-BLM treatment, but expression of collagen type III and CTGF was clearly reduced on HP-MSC transplantation (collagen type III mRNA level: 1.04 ± 0.72 in BLM HP-MSC group versus 3.62 ± 2.24 (BLM NP-MSC) and 5.40 ± 1.90 (BLM control); CTGF mRNA level: 0.66 ± 0.58 in BLM HP-MSC group versus 3.86 ± 3.70 (BLM NP-MSC) and 8.58 ± 7.91 (BLM control); Figure 6A,B). Moreover, there was clearly collagen deposition after BLM treatment, but only low collagen content could be detected in the HP-MSC transplanted mice which was comparable to the normal level (8.87 ± 7.20 μg/mg wet lung tissue in BLM HP-MSC group versus 20.17 ± 15.36 (BLM NP-MSCs) and 28.48 ± 3.06 (BLM control) and 8.02 ± 4.26 (PBS control); Figure 6C). Noting the downregulation of inflammatory and fibrotic mediators, other possible cytoprotective mediators were further investigated by examining the expression levels of HGF in whole-lung tissues by western blot analysis. HGF showed decreased levels in the BLM only and MSC-treated mice, but was overexpressed in the HP-MSC-transplanted mice (Figure 6D). Taken together, transplantation of HP-MSCs exerted better inhibitory effects on upregulating pro-inflammatory and fibrotic factors and on inducing the accumulation of collagen than in the BLM NP-MSC group (Figures 5 and 6).Figure 5

Bottom Line: The pulmonary respiratory functions significantly improved for up to 18 days of hypoxia-preconditioned MSC treatment.Histopathologic examination observed a significant amelioration of the lung fibrosis.Our data further demonstrated that upregulation of hepatocyte growth factor possibly played an important role in mediating the therapeutic effects of transplanted hypoxia-preconditioned MSCs.

View Article: PubMed Central - PubMed

Affiliation: Division of Biotechnology, Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan, Republic of China. bublelanwilliam@gmail.com.

ABSTRACT

Introduction: Idiopathic pulmonary fibrosis is a progressive diffuse parenchymal lung disorder of unknown etiology. Mesenchymal stem cell (MSC)-based therapy is a novel approach with great therapeutic potential for the treatment of lung diseases. Despite demonstration of MSC grafting, the populations of engrafted MSCs have been shown to decrease dramatically 24 hours post-transplantation due to exposure to harsh microenvironments. Hypoxia is known to induce expression of cytoprotective genes and also secretion of anti-inflammatory, anti-apoptotic and anti-fibrotic factors. Hypoxic preconditioning is thought to enhance the therapeutic potency and duration of survival of engrafted MSCs. In this work, we aimed to prolong the duration of survival of engrafted MSCs and to enhance the effectiveness of idiopathic pulmonary fibrosis transplantation therapy by the use of hypoxia-preconditioned MSCs.

Methods: Hypoxic preconditioning was achieved in MSCs under an optimal hypoxic environment. The expression levels of cytoprotective factors and their biological effects on damaged alveolar epithelial cells or transforming growth factor-beta 1-treated fibroblast cells were studied in co-culture experiments in vitro. Furthermore, hypoxia-preconditioned MSCs (HP-MSCs) were intratracheally instilled into bleomycin-induced pulmonary fibrosis mice at day 3, and lung functions, cellular, molecular and pathological changes were assessed at 7 and 21 days after bleomycin administration.

Results: The expression of genes for pro-survival, anti-apoptotic, anti-oxidant and growth factors was upregulated in MSCs under hypoxic conditions. In transforming growth factor-beta 1-treated MRC-5 fibroblast cells, hypoxia-preconditioned MSCs attenuated extracellular matrix production through paracrine effects. The pulmonary respiratory functions significantly improved for up to 18 days of hypoxia-preconditioned MSC treatment. Expression of inflammatory factors and fibrotic factor were all downregulated in the lung tissues of the hypoxia-preconditioned MSC-treated mice. Histopathologic examination observed a significant amelioration of the lung fibrosis. Several LacZ-labeled MSCs were observed within the lungs in the hypoxia-preconditioned MSC treatment groups at day 21, but no signals were detected in the normoxic MSC group. Our data further demonstrated that upregulation of hepatocyte growth factor possibly played an important role in mediating the therapeutic effects of transplanted hypoxia-preconditioned MSCs.

Conclusion: Transplantation of hypoxia-preconditioned MSCs exerted better therapeutic effects in bleomycin-induced pulmonary fibrotic mice and enhanced the survival rate of engrafted MSCs, partially due to the upregulation of hepatocyte growth factor.

No MeSH data available.


Related in: MedlinePlus