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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

Hypoxic preconditioning promotes cell proliferation and decreases hydrogen peroxide- or belomycin-induced cell death in mesenchymal stem cells. (A) Mesenchymal stem cells (MSCs) were stained with 1 μM JC-10 for 30 minutes, and were then analyzed by flow cytometry. Owing to the dual wavelength emission of JC-10, most stained cells distributed at the double-positive region (top right quadrant). Electronic compensation was made to correct the bleed of the green (monomer) and red (aggregate) fluorescence signals into the FL2 and FL1 channels. Right column shows compensated data. The gated region represented the higher mitochondrial membrane potential cell population. The histogram on the right shows the quantification of three independent normoxia-preconditioned MSCs (NP-MSCs) and hypoxia-preconditioned MSCs (HP-MSCs) samples of JC-10 staining. (B) Cell number of NP-MSCs and HP-MSCs was determined by an automated cell counter. (C) Cell viability of NP-MSCs and HP-MSCs treated with the indicated H2O2 concentration for 1 hour as assessed by flow cytometric analysis with Annexin V/propidium iodide (PI) staining. Percentages of Annexin V/PI-double positive cells (dead cells population) are shown. (D) MTT assays of cell viability of bleomycin-treated mouse lung epithelial cells (MLE-12) in the presence of NP-MSC- or HP-MSC-conditioned medium for 48 hours. *P < 0.05, **P < 0.01, differences between HP-MSCs and the NP-MSC controls.
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Fig2: Hypoxic preconditioning promotes cell proliferation and decreases hydrogen peroxide- or belomycin-induced cell death in mesenchymal stem cells. (A) Mesenchymal stem cells (MSCs) were stained with 1 μM JC-10 for 30 minutes, and were then analyzed by flow cytometry. Owing to the dual wavelength emission of JC-10, most stained cells distributed at the double-positive region (top right quadrant). Electronic compensation was made to correct the bleed of the green (monomer) and red (aggregate) fluorescence signals into the FL2 and FL1 channels. Right column shows compensated data. The gated region represented the higher mitochondrial membrane potential cell population. The histogram on the right shows the quantification of three independent normoxia-preconditioned MSCs (NP-MSCs) and hypoxia-preconditioned MSCs (HP-MSCs) samples of JC-10 staining. (B) Cell number of NP-MSCs and HP-MSCs was determined by an automated cell counter. (C) Cell viability of NP-MSCs and HP-MSCs treated with the indicated H2O2 concentration for 1 hour as assessed by flow cytometric analysis with Annexin V/propidium iodide (PI) staining. Percentages of Annexin V/PI-double positive cells (dead cells population) are shown. (D) MTT assays of cell viability of bleomycin-treated mouse lung epithelial cells (MLE-12) in the presence of NP-MSC- or HP-MSC-conditioned medium for 48 hours. *P < 0.05, **P < 0.01, differences between HP-MSCs and the NP-MSC controls.

Mentions: Mitochondria are critical oxygen sensors linked to various protective effects, such as enhancement of antioxidant defense, cell survival and anti-apoptosis [39,40]. To determine the mitochondrial functions in HP-MSCs in relation to NP-MSCs, the mitochondrial membrane potential in MSCs was assessed using membrane-permeant dual-emission potential-sensitive JC-10 dye and flow cytometry analysis. Dot plot revealed that all JC-10-stained cells distributed into the double-positive region (Figure 2A; left panel). Due to the dual wavelength emission of JC-10, electronic compensation was used to correct for spillage of the green (monomer) and red (aggregate) fluorescence signals into the FL2 and FL1 channels, respectively [41]. The compensated results showed a 14.8% higher red fluorescence signal in HP-MSCs when compared with that in NP-MSCs (approximately 5.9% higher red fluorescence signal), representing a 2.5-fold increment (Figure 2A; right panel).Figure 2


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)

Hypoxic preconditioning promotes cell proliferation and decreases hydrogen peroxide- or belomycin-induced cell death in mesenchymal stem cells. (A) Mesenchymal stem cells (MSCs) were stained with 1 μM JC-10 for 30 minutes, and were then analyzed by flow cytometry. Owing to the dual wavelength emission of JC-10, most stained cells distributed at the double-positive region (top right quadrant). Electronic compensation was made to correct the bleed of the green (monomer) and red (aggregate) fluorescence signals into the FL2 and FL1 channels. Right column shows compensated data. The gated region represented the higher mitochondrial membrane potential cell population. The histogram on the right shows the quantification of three independent normoxia-preconditioned MSCs (NP-MSCs) and hypoxia-preconditioned MSCs (HP-MSCs) samples of JC-10 staining. (B) Cell number of NP-MSCs and HP-MSCs was determined by an automated cell counter. (C) Cell viability of NP-MSCs and HP-MSCs treated with the indicated H2O2 concentration for 1 hour as assessed by flow cytometric analysis with Annexin V/propidium iodide (PI) staining. Percentages of Annexin V/PI-double positive cells (dead cells population) are shown. (D) MTT assays of cell viability of bleomycin-treated mouse lung epithelial cells (MLE-12) in the presence of NP-MSC- or HP-MSC-conditioned medium for 48 hours. *P < 0.05, **P < 0.01, differences between HP-MSCs and the NP-MSC controls.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig2: Hypoxic preconditioning promotes cell proliferation and decreases hydrogen peroxide- or belomycin-induced cell death in mesenchymal stem cells. (A) Mesenchymal stem cells (MSCs) were stained with 1 μM JC-10 for 30 minutes, and were then analyzed by flow cytometry. Owing to the dual wavelength emission of JC-10, most stained cells distributed at the double-positive region (top right quadrant). Electronic compensation was made to correct the bleed of the green (monomer) and red (aggregate) fluorescence signals into the FL2 and FL1 channels. Right column shows compensated data. The gated region represented the higher mitochondrial membrane potential cell population. The histogram on the right shows the quantification of three independent normoxia-preconditioned MSCs (NP-MSCs) and hypoxia-preconditioned MSCs (HP-MSCs) samples of JC-10 staining. (B) Cell number of NP-MSCs and HP-MSCs was determined by an automated cell counter. (C) Cell viability of NP-MSCs and HP-MSCs treated with the indicated H2O2 concentration for 1 hour as assessed by flow cytometric analysis with Annexin V/propidium iodide (PI) staining. Percentages of Annexin V/PI-double positive cells (dead cells population) are shown. (D) MTT assays of cell viability of bleomycin-treated mouse lung epithelial cells (MLE-12) in the presence of NP-MSC- or HP-MSC-conditioned medium for 48 hours. *P < 0.05, **P < 0.01, differences between HP-MSCs and the NP-MSC controls.
Mentions: Mitochondria are critical oxygen sensors linked to various protective effects, such as enhancement of antioxidant defense, cell survival and anti-apoptosis [39,40]. To determine the mitochondrial functions in HP-MSCs in relation to NP-MSCs, the mitochondrial membrane potential in MSCs was assessed using membrane-permeant dual-emission potential-sensitive JC-10 dye and flow cytometry analysis. Dot plot revealed that all JC-10-stained cells distributed into the double-positive region (Figure 2A; left panel). Due to the dual wavelength emission of JC-10, electronic compensation was used to correct for spillage of the green (monomer) and red (aggregate) fluorescence signals into the FL2 and FL1 channels, respectively [41]. The compensated results showed a 14.8% higher red fluorescence signal in HP-MSCs when compared with that in NP-MSCs (approximately 5.9% higher red fluorescence signal), representing a 2.5-fold increment (Figure 2A; right panel).Figure 2

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