Hypoxia-preconditioned mesenchymal stem cells attenuate bleomycin-induced pulmonary fibrosis
© Lan et al. 2015
Received: 9 January 2015
Accepted: 21 April 2015
Published: 20 May 2015
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.
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.
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.
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.
Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive form of interstitial lung disease characterized by an intricate cytokine network and abnormal deposition of mesenchymal cells [1,2]. To date, there are no effective and approved treatments for IPF , making IPF the most life-threatening idiopathic disease with a median survival of about 3 years from initial diagnosis.
Mesenchymal stem cell (MSC)-based cell therapy is a potential therapeutic approach for the treatment of various lung diseases [4,5]. Several studies have demonstrated that MSCs home to sites of lung injury, an event also associated with reduced tissue damage, inhibited production of proinflammatory mediators, decreased extracellular matrix collagen deposition, and generally contributed to tissue repair [6,7]. In addition, MSCs secrete paracrine factors with anti-inflammatory, anti-apoptotic and anti-fibrotic functions [8,9]. Despite reported success, it was demonstrated that the amount of engrafted MSCs decreased dramatically after 24 hours of transplantation due to exposure to toxic and oxidative microenvironments . Recent studies involving overexpressing anti-oxidant [11,12] or anti-apoptotic genes or growth factors in engrafted stem cells have shown improved survival following transplantation . In addition, cells, tissues or whole animals may be preconditioned by sub-lethal exposure to selected stresses to induce prior expression of cytoprotective genes before subsequent lethal challenges . Cellular preconditioning may include exposure of cells to physiological stimuli such as hypoxia, heat shock, small-molecule pharmacological agents, cytokines, growth factors or biophysical stimuli. Alternatively, cellular preconditioning may be achieved by genetic manipulation by overexpression of anti-apoptotic proteins, chemokine receptors, growth factors or pro-survival genes prior to transplantation [15-18].
Adopting appropriate preconditioning strategies may provide a simple yet effective way of promoting survival and regenerative properties, and also the tissue repair capability of transplanted cells in stem cell-based therapy . Recent studies have also shown that short-term exposure of MSCs to sub-lethal hypoxia before transplantation enhances cell survival and downregulation of apoptosis-related pathways [19,20]. Furthermore, hypoxic preconditioning also induces the expression of pro-survival markers , chemoattractants  and growth factors involved in cell proliferation, anti-oxidation, anti-apoptosis and angiogenesis  in MSCs. Hypoxic preconditioning and stem cell transplantation have been extensively studied in a number of organs and tissues in relation to diseases to enhance therapeutic effects; the studies have included ischemic diseases such as stroke and myocardial infarction [19,24-26], traumatic brain injuries , diabetes mellitus , inflammatory bowel disease , and acute kidney [22,30] and liver injuries . However, there have been no published studies on using hypoxia-preconditioned MSCs (HP-MSCs) in the treatment of pulmonary fibrosis.
In this work, we investigated if hypoxic preconditioning of MSCs would lead to comprehensive regulation that promotes cell survival, enhances cytoprotective and growth factor action, and attenuates extracellular matrix (ECM) production. These effects were anticipated to result in inhibited inflammatory response, fibrotic factor production, and significant improvements in lung functions in a lung fibrosis animal model.
Material and methods
Bleomycin sulfate from Streptomyces verticillus was obtained from Sigma-Aldrich (St Louis, MO, USA). The c-Met inhibitor PHA-665752 was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).
MSCs were isolated from the bone marrow of C57BL/6 female mice. MSCs were purchased from Life Technologies (GIBCO mouse C57BL/6 MSCs; Carlsbad, CA, USA) following the guidelines of Good Manufacturing Practices for medical derivatives, Code of Federal Regulations Title 21 (21 CFR), Part 820 of the US Food and Drug Administration regulation. The female murine alveolar epithelial cell line (MLE-12; CRL-2110) was purchased from American Type Culture Collection (Manassas, VA, USA). Both cell lines were maintained in Dulbecco’s modified Eagle’s medium/Ham’s Nutrient Mixture F-12 (Life Technologies) supplemented with 10% fetal bovine serum (Life Technologies), 2 mM L-glutamine (Life Technologies) and 1% penicillin/streptomycin (Life Technologies). Human 14-week male embryonal lung cell line (MRC-5; BCRC-60023) was purchased from Bioresource Collection and Research Center (Hsinchu, Taiwan). MRC-5 cells were maintained in Eagle’s minimal essential medium (Life Technologies) supplemented with 10% fetal bovine serum (Life Technologies) and 1% penicillin/streptomycin (Life Technologies), and were incubated at 37°C in a 5% CO2 incubator.
Viral production and viral transduction
Virus stocks were prepared by co-transfecting the pLenti6/v5-GW/lacZ plasmid (Life Technologies) with three packaging plasmids, pMDLg/pRRE, CMV-VSVG and RSV-Rev, into 293 T cells following the method of Chen and colleagues . The viral supernatants were harvested 36 to 48 hours later, filtered and centrifuged at 20,000 × g for 90 minutes. The viral titer was determined by the method of end-point dilution through counting the number of infected red cells at ×100 magnification under a fluorescence microscope 96 hours after infection to 293 T cells. Titer in transducing units was computed as follows: (TU)/mL = (the numbers of red fluorescent cells) × (dilution factor)/(volume of virus solution). Titers of the viral particles were quantified by HIV-quantification enzyme-linked immunosorbent assay kit. MSCs were seeded in 12-well plates and the cells were transduced with an equal ratio of viral particles of pLenti6/v5-GW/lacZ virus particle and the stably transduced cells were designated as β-Gal-MSCs.
MSCs were grown to confluency and were changed to fresh complete medium before hypoxia treatment using a finely-controlled ProOx-C-chamber system (Biospherix, Redfield, NY, USA) for 24 hours. The oxygen concentration in the chamber was maintained at 1.5% with a residual gas mixture composed of 5% carbon dioxide and balanced nitrogen. Normoxia-treated MSCs used as a control were cultured in 95% atmospheric air and 5% CO2 for 24 hours. Conditioned medium was collected from MSCs cultured in normoxic or hypoxic conditions.
Measurement of mitochondrial membrane potential
Mitochondrial membrane potential was assessed using a sensitive fluorescent probe JC-10 (Enzo Life Sciences Inc., Farmingdale, NY, USA). MSCs were incubated with JC-10 (1 μM) at 37°C for 30 minutes. JC-10 is capable of selectively entering into mitochondria, and reversibly changes its color from green (JC-10 monomeric form) to orange (JC-10 aggregate form) as membrane potentials increase. Both colors can be detected using flow cytometers (FACSCalibur; BD Biosciences, San Jose, CA, USA).
Mesenchymal stem cells and MRC-5 co-culture assay
MSCs were plated at a density of 1 × 105 cells/well and MRC-5 cells were plated at a density of 2 × 105 cells/well in transwells (BD Biosciences) and 6-well culture plates (BD Biosciences), respectively, and the cells were cultured overnight. MSCs were then treated with the indicated oxygen concentrations in hypoxic treatment for 24 hours. MRC-5 cells were treated with or without 2.5 ng/mL transforming growth factor (TGF)-β1 (Sino Biological Inc., Beijing, China) for 24 hours. After removing the medium, hypoxia-pretreated MSCs in transwells were co-cultured with the MRC-5 cells for 24 hours. The MRC-5 cells were harvested for detection of fibronectin mRNA expression level by quantitative real-time RT-PCR.
MRC-5 cells were plated at a density of 2 × 105 cells/well in 6-well culture plates (BD Biosciences) and cells were treated with or without indicated concentrations of PHA665752 (Sigma-Aldrich) and 2.5 ng/mL TGF-β1 (Sino Biological) for 24 hours. After removing the medium, hypoxia-pretreated MSCs in transwells were co-cultured with the PHA665752-treated MRC-5 cells for 24 hours. The MRC-5 cells were harvested for detection of fibronectin mRNA expression level by quantitative real-time RT-PCR.
Cell proliferation and viability test
MSCs were plated at a density of 5 × 104 cells/well in a 12-well culture plate (BD Biosciences) and incubated overnight to allow adhesion. The cells were then treated with the indicated concentrations of oxygen for 24 hours. The cells were detached by trypsinization and were counted in triplicate using a Countess™ automated cell counter (Life Technologies). Cell viability was measured using 0.4% (w/v) trypan-blue (Sigma-Aldrich) exclusion and the Countess™ software.
Cell viability MTT assay
MLE-12 cells were plated at a density of 1 × 105 cells/well in a 24-well culture plate (BD Biosciences) and were cultured to confluency. The cells were treated with bleomycin (BLM; 13.5 μM) in the presence of normoxia-preconditioned MSC (NP-MSC)- or HP-MSC-conditioned medium for 48 hours. The cell viability was then detected using the MTT method at a wavelength of 540 nm by VERSAMax spectrophotometry (Molecular Devices, Sunnyvale, CA, USA) as previous described .
Short hairpin RNA and transient transfection
The hepatocyte growth factor (HGF) short hairpin RNA (shRNA) clone (TRCN0000336131; GGTAAAGGAGGCAGCTATAAA) targeted at the mouse HGF transcript was purchased from the National RNAi Core Facility (Academia Sinica, Taipei, Taiwan). MSCs were plated at a density of 1 × 105 cells/well in a 24-well plate and were transfected with 2.5 μg HGF shRNA plasmid premixed with 2.5 μl Lipofactamine™ LTX (Life Technologies) for 24 hours. At the end of the incubation, MSCs were exposed to hypoxia for 24 hours. Following hypoxic preconditioning, cells were used in co-culture assays.
Annexin V/propidium iodide double-staining assay
At 1 hour post-H2O2 (Sigma-Aldrich) treatment (0.5, 1, and 2 μM), cell viability of NP-MSCs and HP-MSCs were detected by Annexin V/propidium iodide (PI) staining (Strong Biotech Co., Taipei, Taiwan). Briefly, 1 × 105 cells were labeled with 1 μL Annexin V and PI in a binding buffer for 15 minutes. The cells were analyzed using a flow cytometer (FACSCalibur; BD Biosciences). The percentage of cell numbers in each quadrant was calculated using CELLQUEST software (BD Biosciences).
RNA isolation and quantitative real-time RT-PCR
Forward: AGAGACGGCC GCATCTTCTT
Western blot analysis
Total cellular proteins were isolated from cell lines by the PRO-PREP™ Protein Extraction Solution (Intron Biotechnology, Kyonggi-do, Korea). Western blot analysis was performed as described previously . Briefly, 25 or 50 μg total protein from cell lysates or conditioned media was loaded onto each lane and the proteins were separated in SDS-PAGE (Bio-Rad Laboratories, Hercules, CA, USA). After electrophoresis, the resolved proteins were transferred to PVDF membrane (Millipore, Billerica, MA, USA). The membranes were blocked with 5% skimmed milk powder (Anchor, Kowloon, Hong Kong) in phosphate-buffered saline (PBS)-Tween (PBS containing 0.1% Tween-20 (both Sigma-Aldrich)) for 1 hour and probed overnight with the following antisera at appropriate dilutions: anti-heme oxygenase 1 (HO-1; MBL International, Woburn, MA, USA; 1:1,000), anti-fibronectin, anti-HGF, anti-vascular endothelial growth factor (VEGF) and anti-interleukin (IL)-1β (Santa Cruz Biotechnology; 1:1,000), primary antibodies detecting pan-AKT or phospho-AKT S473 (Cell Signaling Technology, Beverly, MA, USA; 1:2,000), and anti-β-actin (Millipore; 1:10,000) antisera in PBS-Tween. Identification of each protein was achieved with the Western Lighting Plus Reagent (Perkin Elmer, Waltham, MA, USA) using an appropriate horseradish peroxidase-conjugated secondary antibody (Jackson Immuno Research Laboratories, West Grove, PA, USA). Protein levels in the western blot analysis were detected and quantified by the LAS-3000 chemiluminescence detection device (Fujifilm, Valhalla, NY, USA). To adjust for loading differences, the optical density of each protein was normalized to that of the β-actin band.
Animal model of bleomycin-induced lung fibrosis
Eight-week old male C57BL/6JNarl mice were purchased from the National Laboratory Animal Center (Taipei, Taiwan). The mice were maintained in an air-conditioned animal facility under constant temperature and humidity conditions with a 12:12 light-dark cycle and were allowed ad libitum diet and drinking water. Mice were randomly picked to different groups and there were at least six or more mice in each group. All experimental procedures were approved by the Institutional Animal Care and Use Committee of the Chang Gung University. Bleomycin sulfate (Sigma-Aldrich) stock was prepared by dissolving in sterile PBS at 10 mg/mL and storing in small aliquots at 4°C. Mice were anesthetized by isoflurane (Abbott Laboratories, Abbott Park, IL, USA) inhalation and BLM was instilled intratracheally at 1.5 mg/kg body weight in 50 μL sterile PBS. All animals received intratracheal instillation of either BLM or PBS on day 0. On day 3, mice were randomly selected for intratracheal injection (5 × 105 cells in 50 μl PBS) of normoxia- or hypoxia-pretreated MSCs, or just PBS. On day 4 and day 18 after stem cell transplantation, animals were placed in the whole-body plethysmograph tanks for analysis of pulmonary functions. At each time point, the mice were sacrificed by an overdose of 2.5% avertin (Sigma-Aldrich).
Noninvasive measurement of pulmonary functions by whole-body plethysmography
The baseline enhanced respiratory pause (Penh) in unrestrained conscious mice was measured by whole-body plethysmography (Buxco Electronic Inc., Wilmington, NC, USA). Mice were placed in the chamber and allowed to equilibrate for approximately 10 minutes. According to the manufacturer’s instructions, Penh is calculated by the formula Penh = (Te/Rt-1) × (PEF/PIF), where Te is expiratory time (seconds); Rt is relaxation time (seconds), defined as the time of “volume” decay to 35% of the total expiratory pressure signal (area under the box pressure signal at expiration); PEF is peak expiratory flow (ml/second); and PIF is peak inspiratory flow (ml/second). Penh reflects changes in the waveform of the box pressure signal during both inspiration and expiration, and combines these changes with the timing comparison of early and late expiration during the animal’s spontaneous breathing. Penh was measured for 3 minutes for each experiment.
Left lungs were fixed with 10% formalin (Macron Fine Chemicals™, Avantor Performance Materials, Center Valley, PA, USA) and were embedded in paraffin sections before staining with hematoxylin and eosin (Sigma-Aldrich) according to standard protocols. Briefly, 4-μm serial step sections were taken along the longitudinal axis of the lobe. The fixed distance between the sections was calculated to allow systematic sampling of 10 sections across the whole lobe. The severity of lung fibrosis was assessed by measuring the Masson’s trichrome staining . The Ashcroft score was used for quantitative histological analysis of fibrotic changes induced by BLM. Five fields within each lung section were observed, and the score of fibrosis ranged from 0 (normal lung) to 8 (total fibrous obliteration of the field) .
Immunohistochemical staining of β-gal
Immunohistochemistry was performed to evaluate engraftment of transplanted β-Gal-MSCs in vivo. Paraffin-fixed sections were microwave-heated (750 W, three 5-minute cycles) in 0.01 M citrate (pH 6.0) or 1-mM EDTA (pH 8.0) solution. Immunostaining was performed with a 1:1,000 dilution of the anti-β-Galactosidase antibody (ab9361; Abcam Inc, Cambridge, MA, USA) using the Dako-REAL, Alkaline-Phosphatase/RED detection system (Dako, Glostrup, Denmark). Hematoxylin was used for counterstaining according to the manufacturer’s protocol. The stained sections were scanned by the HistoFAXS (TissueFAX plus; TissueGnostics, Vienna, Austria).
The Sircol collagen assay (Biocolor Ltd., Belfast, UK), a dye-binding method designed for the analysis of acid and pepsin-soluble collagens, was used to detect the amounts of collagenous protein in lung tissues. The stained tissue homogenate released Sircol red dyes by an alkali reagent (1 N NaOH, Sigma-Aldrich) and measured by VERSAMax spectrophotometry (Molecular Devices). The amounts (μg) of collagenous proteins in each tissue were determined by OD at 550 nm.
Data are either shown as individual data points in a vertical scatter dot plot, with a line to indicate the mean, or as bar graphs showing mean ± standard deviation (SD). Comparisons between two groups were analyzed using the two-tailed Student’s t-test. For multiple comparisons, one-way analysis of variance analysis was used, followed by the Tukey’s post hoc test for analyzing parametric data. All statistical analyses were performed using Graph Pad Prism (GraphPad Software, Inc., San Diego, CA, USA). *P < 0.05, **P < 0.01 and ***P < 0.001 are considered statistically significant.
Hypoxic preconditioning upregulates expression of cytoprotective and regenerative factors in mesenchymal stem cells
Hypoxic preconditioning stabilizes the mitochondrial membrane potential in mesenchymal stem cells
Hypoxic preconditioning promotes cell proliferation, reduces hydrogen peroxide-induced cytotoxicity in mesenchymal stem cells and attenuates bleomycin-induced alveolar epithelial cell death
The effects of hypoxic culture on MSC proliferation and expansion efficiency are dependent on the sources of MSCs, oxygen concentration, seeding density and the culturing duration of the MSCs . MSCs cultured in hypoxic conditions increased by about 40% in cell number when compared to normoxic cells (cell number (×105) of NP-MSCs = 4.50 ± 0.62, versus HP-MSCs = 6.27 ± 0.58; P < 0.01; Figure 2B). The hypoxic conditions used showed no adverse effects on cell viability (data not shown). To study the effects of hypoxic preconditioning on H2O2-induced cytotoxicity, HP-MSCs were cultured for 24 hours, followed by exposure to H2O2 for 1 hour. Results showed that H2O2 induced cell death in a dose-dependent manner, as measured by annexin V/PI staining (1 μM H2O2 treatment with NP-MSCs (39.67 ± 3.49) versus HP-MSCs (26.83 ± 2.62); 2 μM H2O2 treatment with NP-MSCs (62.70 ± 1.82) versus HP-MSCs (51.95 ± 3.29); Figure 2C). As anticipated, there were significant degrees of protection against H2O2-induced cell death in HP-MSCs compared with NP-MSCs (Figure 2C). Mouse alveolar epithelial cells (MLE-12) were further used to examine the cytoprotective effects of paracrine factors secreted by MSCs in attenuating BLM-induced cell death. MLE-12 cells were cultured in the presence of conditioned medium (CM) from NP-MSCs or HP-MSCs while under BLM treatment, followed by MTT assays. CM from HP-MSC cultures was able to improve lung epithelial cell survival compared to the control and NP-MSC CM during BLM treatment (58.70 ± 2.73 versus 69.27 ± 0.63, P < 0.01; Figure 2D).
Hypoxia-preconditioned mesenchymal stem cell-conditioned medium attenuates extracellular matrix production through transforming growth factor-β1-mediated Akt signaling
Hence, we used two approaches to examine whether HP-MSCs attenuated ECM production through HGF. Firstly, genetic knock-down of HGF with short hairpins (shRNAs) in MSCs was used, followed by the use of an in vitro co-culture system to assess the anti-fibrotic effects. After 24 hours of co-culture, MSCs transfected with shRNA showed about a 50% decrease in the HGF mRNA level compared to cells transfected with the control scramble shRNA plasmid. Under hypoxic conditions, expression levels of HGF were significantly decreased in MSCs transfected with shRNA compared to the control group (data not shown). On the other hand, knockdown of HGF also abrogated the suppressive effects of HP-MSCs on TGF-β1-induced overexpression of fibronectin (2.64 ± 0.07 after co-culture with control media versus 1.68 ± 0.06 (HP-MSCs) and 2.29 ± 0.22 (HP-shHGF), expressed as relative gene expression changes; Figure 3C). Secondly, pharmacological inhibition of c-Met with its specific inhibitor, PHA-665752, to suppress HGF signaling in MRC-5 cells was also tested. Results showed that ECM production significantly increased in the MRC-5 cells treated with TGF-β1 plus PHA-665752. Although treatment with HP-MSC CM obviously suppressed expression of fibronectin, co-treatment with HP-MSC CM and PHA-665752 significantly inhibited the suppressive effects (3.55 ± 0.04 after treatment with HP-MSC CM versus 4.61 ± 0.05 (control media) and 4.88 ± 0.07 (HP-MSC CM plus PHA-665752), expressed as relative gene expression changes; Figure 3D). The results indicated that paracrine factors, such as HGF, derived from CM of HP-MSCs play a crucial role for attenuation of ECM production.
Improvement of pulmonary respiratory functions of hypoxia-preconditioned mesenchymal stem cell treatment in the bleomycin-induced pulmonary fibrosis mouse model
Downregulated expression of inflammatory and fibrotic factors in hypoxia-preconditioned mesenchymal stem cell-transplanted bleomycin-induced pulmonary fibrosis mouse model
Hypoxia-preconditioned mesenchymal stem cell transplantation alleviates histological changes in the lungs of the bleomycin-induced pulmonary fibrosis mouse model
Our data indicated that hypoxic preconditioning enhanced the survival rate of engrafted MSCs, exerted better therapeutic effects and improved lung functions in BLM-induced pulmonary fibrotic mice.
Accumulating evidence suggests that stem cell-based therapies may be used for lung regeneration and modulation of inflammatory and fibrotic processes . Ortiz and colleagues  first reported that systemically engrafted MSCs possessed the ability to ameliorate fibrotic effects in mice challenged with BLM. Other reports have shown that multiple mechanisms are probably involved in the engrafted MSCs leading to improved outcome in animal models with pulmonary fibrosis [7,46]. In this work, we are the first to demonstrate that engraftment of HP-MSCs rendered better therapeutic effects than untreated MSCs in a BLM-induced pulmonary fibrosis animal model. Hypoxic preconditioning upregulated cytoprotective and regenerative genes, stabilized mitochondrial membrane potentials, promoted cell proliferation and acted against H2O2-induced cell death in the treated MSCs. Moreover, HP-MSCs attenuated BLM-induced cell apoptosis and ECM production through TGF-β1-mediated Akt signaling via paracrine effects. HP-MSC engraftment improved lung functions, reduced lung edema and the levels of pro-inflammatory and fibrotic factors.
Although recent studies indicated that both local (intratracheal) and systemic (intravenous or intraperitoneal) infusion may be effective in attenuating lung injury, the optimal route of delivery remains unclear . On the other hand, systemic administration of MSCs showed some difficult conditions for application to the clinical setting, such as the recipient requiring total body irradiation to promote stem cell engraftment . Alternatively, considering the advantages of local administration to target the lung tissue, several recent studies reported that a lower dosage of stem cells are needed [48,49] through the intratracheal route and showed more effective airway regeneration [50,51] compared with the intravenous route.
Although MSC-based cell therapy has shown great potential in cell repair, the therapeutic effects of MSC-based approaches have met a bottleneck following transplantation. Leblond and colleagues  reported that a large number of the engrafted cells were trapped in the damaged lungs during short-term treatment. Less than 5% of implanted cells remained detectable in the injured lungs 7 days after transplantation . Our results also showed that the number of engrafted MSCs dramatically decreased with time after transplantation (data not shown). To resolve poor therapeutic efficacy of long-term MSC transplantation, recent studies have proposed various strategies aiming to precondition implanted MSCs by sublethal physical and chemical stresses or genetic and pharmacological stimuli to increase tolerance of the transplanted cells to withstand the severe microenvironment of the transplanted sites [11-13,53].
Murry and colleagues  first described the phenomenon termed “ischemic preconditioning” which attenuated myocardium damage induced by ischemia. Our data suggest that hypoxic preconditioning may be a better solution for the transplanted cells to adapt to low oxygen tension microenvironment, or in organs with lesions, through systematically activating and altering a series of cellular responses [55-57]. Many of these alterations, including improved growth kinetics, homing ability, survival, regenerative, and genetic stability of the implanted cells are characterized by upregulation of cytoprotective genes HIF-1α, erythropoietin receptor, B-cell lymphoma 2, HGF and VEGF [21,40,58-60]. In addition, these cytoprotective factors exert both autocrine and paracrine effects . In agreement with previous studies, our in vitro studies showed that HP-MSCs upregulated expression of cytoprotective genes (Figure 1). The reason why 1.5% O2 concentration was chosen in this study was that 1.5% mimics the low oxygen concentration (1 to 2%) naturally encountered by MSCs in their bone marrow niche [62-64]. It has been reported that short-term (24 hour) exposure of MSCs to hypoxia at 1.5% O2 stabilizes the HIF-1 protein, and clearly promotes proliferation, cell cycle progression and migration . The viability of MSCs was not adversely affected by 24 hours of hypoxia at 1.5% O2 (data not shown). The mRNA expression level of HIF-1α reached the peak level at 12 hours of hypoxic exposure (Figure 1A), whereas the HIF-1α protein level peaked after 24 hours of exposure (Figure 1E). On the other hand, western blot analyses showed that hypoxic conditions caused a time-dependent increase in the levels of the cytoprotective proteins (Figure 1), reaching maximum at 24 hours after hypoxic preconditioning.
The strategies of HP-MSC transplantation have shown potential benefits in several experimental disease models in acute kidney injury  and ischemic diseases [25,27,60,66]. Studies showed that therapeutic effects were improved on hypoxic treatment due to activation of HIF-1α followed by upregulation of growth factors and downregulation of pro-inflammatory cytokines and chemokines in hypoxic MSCs . Such events positively support HP-MSCs to overcome limitations and circumvent hindrances in cell-based therapy . Here, we found that intratracheal instillation of HP-MSCs provided better therapeutic effects than untreated MSCs after BLM exposure, and protected lung tissues from injuries by improving the pulmonary functions (Figure 4) and reducing the extent of inflammation (Figure 5) and fibrosis (Figure 6).
Mounting evidence has shown that BLM as a chemotherapeutic agent has undesirable side-effects in inducing lung injuries and triggering apoptosis of alveolar epithelial cells . In addition, either BLM or downstream TGF-β1 may induce ECM synthesis, and ECM deposition is mediated through Akt signaling in fibroblasts . Hence, dysregulation of intercellular cross-talk between alveolar epithelia and fibroblasts is a critical event in the fibrosis process. The net balance is disturbed when alveolar epithelial cells die and then factors such as TGF-β1 shift the balance toward enhancement of fibroblast proliferation and ECM production . Our study showed that HP-MSCs possessed better cytoprotective abilities to resist stimuli-induced alveolar epithelial cell death (Figure 2B,C) and to attenuate Akt-mediated ECM production in fibroblast cells through paracrine effects (Figure 3). Consistent with other studies, transplantated MSCs site-specifically migrate into damaged alveolar epithelial cells, and reduced alveolar epithelial cell apoptosis [45,69], resulting in the differentiation of MSCs into alveolar epithelial cells  through paracrine mechanisms . Hence, transplantation of HP-MSCs inhibited production of pro-inflammatory mediators (Figure 5), attenuated ECM deposition (Figure 6C) and reduced alveolar wall thickness (Figure 7D) in the fibrosis lung model.
Many reports have shown some benefits from using MSCs to attenuate inflammatory response and fibrosis. However, the therapeutic efficacy may be affected by the transplanted cell number, cell ages, timing of transplantation, and treatment period . Most studies assessed the therapeutic effects at day 7 or day 14 after BLM treatment, but only a few studies showed the longer period of treatment effects. Aguilar and colleagues noted that delivery of MSCs at the time of BLM injury and 3 days later, the end point of their experiments found that MSCs showed no effect in attenuating the ECM deposition and histological damage . Moodley and colleagues noted that there was no change in aberrant lung collagen deposition in the MSC treatment group at day 21 after BLM injury . Moreover, Cargnoni and colleagues noted that transplantation of MSCs slightly modulated lung inflammation during long-term treatment . Similar results were shown in our study; NP-MSC treatment showed slight suppression of ECM deposition and no effect on inflammatory response at day 21 after BLM treatment. Therefore, HP-MSCs were more effective at reducing inflammatory and ECM production (Figures 4, 5 and 6).
Recent studies have demonstrated that HGF plays a key role in preventing fibrosis or scar formation after injuries [76,77]. Moreover, HGF exerts multiple protective effects on injured tissues via mitogenic, motogenic, anti-apoptosis, and anti-inflammatory and anti-fibrogenic signals [78-82]. Reports also showed that TGF-β1-induced fibrosis could be prevented by treatment with HGF through inhibiting myofibroblast differentiation [83,84] and ECM production [85-87]. In agreement with this reported evidence, the regenerative factor HGF was significantly upregulated in the HP-MSC-treated fibrotic lungs in the present study (Figure 6D).
In summary, we report here that transplantation of HP-MSCs improves pulmonary functions and reduces inflammatory and fibrotic mediators in a BLM-induced pulmonary fibrosis model. The upregulation of HGF might play a key role in mediating the therapeutic effects of transplanted HP-MSCs.
connective tissue growth factor
hepatocyte growth factor
heme oxygenase 1
hypoxia-preconditioned mesenchymal stem cell
idiopathic pulmonary fibrosis
mesenchymal stem cell
normoxia-preconditioned mesenchymal stem cell
enhanced respiratory pause
short hairpin RNA
transforming growth factor
vascular endothelial growth factor
This work was supported in part by the Ministry of Science and Technology (Taiwan), NSC 95-2320-B-182-028-MY3, NSC 99-2632-B-182-001-MY3 and MOST 103-2320-B-182-021, the Chang Gung Memorial Hospital Grant (CMRPD 34012, CMRPD 180133, CMRPD 1B0471, CMRPD 1B0472, and CMRPD 1B0473) and Taiwan Ministry of Education (EMRPD 1C0121). The authors thank Molecular Imaging Center, Chang Gung Memorial Hospital, Linkou Medical Center for tissue slide imaging by HistoFAXS, and Professor Mei-Ling Kuo, Department of Microbiology and Immunology, College of Medicine, Chang Gung University, for technical advice of noninvasive measurement of pulmonary function by WBP.
- Ley B, Collard HR, King Jr TE. Clinical course and prediction of survival in idiopathic pulmonary fibrosis. Am J Respir Crit Care Med. 2011;183:431–40.PubMedView ArticleGoogle Scholar
- Rafii R, Juarez MM, Albertson TE, Chan AL. A review of current and novel therapies for idiopathic pulmonary fibrosis. J Thorac Dis. 2013;5:48–73.PubMed CentralPubMedGoogle Scholar
- Maher TM, Wells AU, Laurent GJ. Idiopathic pulmonary fibrosis: multiple causes and multiple mechanisms? Eur Respir J. 2007;30:835–9.PubMedView ArticleGoogle Scholar
- Bonfield TL, Caplan AI. Adult mesenchymal stem cells: an innovative therapeutic for lung diseases. Discov Med. 2010;9:337–45.PubMedGoogle Scholar
- Inamdar AC, Inamdar AA. Mesenchymal stem cell therapy in lung disorders: pathogenesis of lung diseases and mechanism of action of mesenchymal stem cell. Exp Lung Res. 2013;39:315–27.PubMedView ArticleGoogle Scholar
- Katsha AM, Ohkouchi S, Xin H, Kanehira M, Sun R, Nukiwa T, et al. Paracrine factors of multipotent stromal cells ameliorate lung injury in an elastase-induced emphysema model. Mol Ther. 2011;19:196–203.PubMed CentralPubMedView ArticleGoogle Scholar
- Toonkel RL, Hare JM, Matthay MA, Glassberg MK. Mesenchymal stem cells and idiopathic pulmonary fibrosis. Potential for clinical testing. Am J Respir Crit Care Med. 2013;188:133–40.PubMedView ArticleGoogle Scholar
- Akram KM, Samad S, Spiteri MA, Forsyth NR. Mesenchymal stem cells promote alveolar epithelial cell wound repair in vitro through distinct migratory and paracrine mechanisms. Respir Res. 2013;14:9.PubMed CentralPubMedView ArticleGoogle Scholar
- Meirelles Lda S, Fontes AM, Covas DT, Caplan AI. Mechanisms involved in the therapeutic properties of mesenchymal stem cells. Cytokine Growth Factor Rev. 2009;20:419–27.PubMedView ArticleGoogle Scholar
- Leblond AL, Naud P, Forest V, Gourden C, Sagan C, Romefort B, et al. Developing cell therapy techniques for respiratory disease: intratracheal delivery of genetically engineered stem cells in a murine model of airway injury. Hum Gene Ther. 2009;20:1329–43.PubMed CentralPubMedView ArticleGoogle Scholar
- Greenberger JS. Gene therapy approaches for stem cell protection. Gene Ther. 2008;15:100–8.PubMedView ArticleGoogle Scholar
- Sakata H, Niizuma K, Wakai T, Narasimhan P, Maier CM, Chan PH. Neural stem cells genetically modified to overexpress Cu/Zn-superoxide dismutase enhance amelioration of ischemic stroke in mice. Stroke. 2012;43:2423–9.PubMed CentralPubMedView ArticleGoogle Scholar
- Trivedi P, Tray N, Nguyen T, Nigam N, Gallicano GI. Mesenchymal stem cell therapy for treatment of cardiovascular disease: helping people sooner or later. Stem Cells Dev. 2010;19:1109–20.PubMedView ArticleGoogle Scholar
- Ogle ME, Yu SP, Wei L. Primed for lethal battle: a step forward to enhance the efficacy and efficiency of stem cell transplantation therapy. J Thorac Cardiovasc Surg. 2009;138:527.PubMed CentralPubMedView ArticleGoogle Scholar
- Baraniak PR, McDevitt TC. Stem cell paracrine actions and tissue regeneration. Regen Med. 2010;5:121–43.PubMed CentralPubMedView ArticleGoogle Scholar
- Haider KH, Ashraf M. Preconditioning approach in stem cell therapy for the treatment of infarcted heart. Prog Mol Biol Transl Sci. 2012;111:323–56.PubMedView ArticleGoogle Scholar
- Noort WA, Feye D, Van Den Akker F, Stecher D, Chamuleau SA, Sluijter JP, et al. Mesenchymal stromal cells to treat cardiovascular disease: strategies to improve survival and therapeutic results. Panminerva Med. 2010;52:27–40.PubMedGoogle Scholar
- Ranganath SH, Levy O, Inamdar MS, Karp JM. Harnessing the mesenchymal stem cell secretome for the treatment of cardiovascular disease. Cell Stem Cell. 2012;10:244–58.PubMed CentralPubMedView ArticleGoogle Scholar
- Theus MH, Wei L, Cui L, Francis K, Hu X, Keogh C, et al. In vitro hypoxic preconditioning of embryonic stem cells as a strategy of promoting cell survival and functional benefits after transplantation into the ischemic rat brain. Exp Neurol. 2008;210:656–70.PubMedView ArticleGoogle Scholar
- Tsai CC, Yew TL, Yang DC, Huang WH, Hung SC. Benefits of hypoxic culture on bone marrow multipotent stromal cells. Am J Blood Res. 2012;2:148–59.PubMed CentralPubMedGoogle Scholar
- Chacko SM, Ahmed S, Selvendiran K, Kuppusamy ML, Khan M, Kuppusamy P. Hypoxic preconditioning induces the expression of prosurvival and proangiogenic markers in mesenchymal stem cells. Am J Physiol Cell Physiol. 2010;299:C1562–70.PubMed CentralPubMedView ArticleGoogle Scholar
- Liu H, Liu S, Li Y, Wang X, Xue W, Ge G, et al. The role of SDF-1-CXCR4/CXCR7 axis in the therapeutic effects of hypoxia-preconditioned mesenchymal stem cells for renal ischemia/reperfusion injury. PLoS One. 2012;7, e34608.PubMed CentralPubMedView ArticleGoogle Scholar
- Ohnishi S, Yasuda T, Kitamura S, Nagaya N. Effect of hypoxia on gene expression of bone marrow-derived mesenchymal stem cells and mononuclear cells. Stem Cells. 2007;25:1166–77.PubMedView ArticleGoogle Scholar
- Francis KR, Wei L. Human embryonic stem cell neural differentiation and enhanced cell survival promoted by hypoxic preconditioning. Cell Death Dis. 2010;1, e22.PubMed CentralPubMedView ArticleGoogle Scholar
- Jaussaud J, Biais M, Calderon J, Chevaleyre J, Duchez P, Ivanovic Z, et al. Hypoxia-preconditioned mesenchymal stromal cells improve cardiac function in a swine model of chronic myocardial ischaemia. Eur J Cardiothorac Surg. 2013;43:1050–7.PubMedView ArticleGoogle Scholar
- Muscari C, Giordano E, Bonafe F, Govoni M, Pasini A, Guarnieri C. Priming adult stem cells by hypoxic pretreatments for applications in regenerative medicine. J Biomed Sci. 2013;20:63.PubMed CentralPubMedView ArticleGoogle Scholar
- Chang CP, Chio CC, Cheong CU, Chao CM, Cheng BC, Lin MT. Hypoxic preconditioning enhances the therapeutic potential of the secretome from cultured human mesenchymal stem cells in experimental traumatic brain injury. Clin Sci (Lond). 2013;124:165–76.View ArticleGoogle Scholar
- Mottaghi S, Larijani B, Sharifi AM. Apelin 13: a novel approach to enhance efficacy of hypoxic preconditioned mesenchymal stem cells for cell therapy of diabetes. Med Hypotheses. 2012;79:717–8.PubMedView ArticleGoogle Scholar
- Watanabe S, Arimura Y, Nagaishi K, Isshiki H, Onodera K, Nasuno M, et al. Conditioned mesenchymal stem cells produce pleiotropic gut trophic factors. J Gastroenterol. 2014;49:27082.Google Scholar
- Yu X, Lu C, Liu H, Rao S, Cai J, Liu S, et al. Hypoxic preconditioning with cobalt of bone marrow mesenchymal stem cells improves cell migration and enhances therapy for treatment of ischemic acute kidney injury. PLoS One. 2013;8, e62703.PubMed CentralPubMedView ArticleGoogle Scholar
- Yu J, Yin S, Zhang W, Gao F, Liu Y, Chen Z, et al. Hypoxia preconditioned bone marrow mesenchymal stem cells promote liver regeneration in a rat massive hepatectomy model. Stem Cell Res Ther. 2013;4:83.PubMed CentralPubMedView ArticleGoogle Scholar
- Chen YB, Lan YW, Hung TH, Chen LG, Choo KB, Cheng WTK, et al. Mesenchymal stem cell-based HSP70 promoter-driven VEGFA induction by resveratrol alleviates elastase-induced emphysema in a mouse model. Cell Stress Chaperones. 2015;20:643–52. doi:10.1007/s12192-015-0588-x.
- Hung TH, Hsu SC, Cheng CY, Choo KB, Tseng CP, Chen TC, et al. Wnt5A regulates ABCB1 expression in multidrug-resistant cancer cells through activation of the non-canonical PKA/β-catenin pathway. Oncotarget. 2014;5:12273–90.PubMed CentralPubMedGoogle Scholar
- Chong KY, Lai CC, Su CY. Inducible and constitutive HSP70s confer synergistic resistance against metabolic challenges. Biochem Biophys Res Commun. 2013;430:774–9.PubMedView ArticleGoogle Scholar
- Hung TH, Chen CM, Tseng CP, Shen CJ, Wang HL, Choo KB, et al. FZD1 activates protein kinase C delta-mediated drug-resistance in multidrug-resistant MES-SA/ Dx5 cancer cells. Int J Biochem Cell Biol. 2014;53:55–65.PubMedView ArticleGoogle Scholar
- Wen ST, Chen W, Chen HL, Lai CW, Yen CC, Lee KH, et al. Amniotic fluid stem cells from EGFP transgenic mice attenuate hyperoxia-induced acute lung injury. PLoS One. 2013;8, e75383.PubMed CentralPubMedView ArticleGoogle Scholar
- Hubner RH, Gitter W, El Mokhtari NE, Mathiak M, Both M, Bolte H, et al. Standardized quantification of pulmonary fibrosis in histological samples. Biotechniques. 2008;44:507–11.PubMedView ArticleGoogle Scholar
- Wenger RH. Cellular adaptation to hypoxia: O2-sensing protein hydroxylases, hypoxia-inducible transcription factors, and O2-regulated gene expression. FASEB J. 2002;16:1151–62.PubMedView ArticleGoogle Scholar
- Skarka L, Ostadal B. Mitochondrial membrane potential in cardiac myocytes. Physiol Res. 2002;51:425–34.PubMedGoogle Scholar
- Wang JA, Chen TL, Jiang J, Shi H, Gui C, Luo RH, et al. Hypoxic preconditioning attenuates hypoxia/reoxygenation-induced apoptosis in mesenchymal stem cells. Acta Pharmacol Sin. 2008;29:74–82.PubMedView ArticleGoogle Scholar
- Mathur A, Hong Y, Kemp BK, Barrientos AA, Erusalimsky JD. Evaluation of fluorescent dyes for the detection of mitochondrial membrane potential changes in cultured cardiomyocytes. Cardiovasc Res. 2000;46:126–38.PubMedView ArticleGoogle Scholar
- Fuentes-Calvo I, Blazquez-Medela AM, Eleno N, Santos E, Lopez-Novoa JM, Martinez-Salgado C. H-Ras isoform modulates extracellular matrix synthesis, proliferation, and migration in fibroblasts. Am J Physiol Cell Physiol. 2012;302:C686–97.PubMedView ArticleGoogle Scholar
- Goc A, Choudhary M, Byzova TV, Somanath PR. TGFbeta- and bleomycin-induced extracellular matrix synthesis is mediated through Akt and mammalian target of rapamycin (mTOR). J Cell Physiol. 2011;226:3004–13.PubMed CentralPubMedView ArticleGoogle Scholar
- Oury TD, Thakker K, Menache M, Chang LY, Crapo JD, Day BJ. Attenuation of bleomycin-induced pulmonary fibrosis by a catalytic antioxidant metalloporphyrin. Am J Respir Cell Mol Biol. 2001;25:164–9.PubMedView ArticleGoogle Scholar
- Ortiz LA, Gambelli F, McBride C, Gaupp D, Baddoo M, Kaminski N, et al. Mesenchymal stem cell engraftment in lung is enhanced in response to bleomycin exposure and ameliorates its fibrotic effects. Proc Natl Acad Sci U S A. 2003;100:8407–11.PubMed CentralPubMedView ArticleGoogle Scholar
- Tzouvelekis A, Ntolios P, Bouros D. Stem cell treatment for chronic lung diseases. Respiration. 2013;85:179–92.PubMedView ArticleGoogle Scholar
- Chimenti L, Luque T, Bonsignore MR, Ramirez J, Navajas D, Farre R. Pre-treatment with mesenchymal stem cells reduces ventilator-induced lung injury. Eur Respir J. 2012;40:939–48.PubMedView ArticleGoogle Scholar
- Chang YS, Choi SJ, Sung DK, Kim SY, Oh W, Yang YS, et al. Intratracheal transplantation of human umbilical cord blood-derived mesenchymal stem cells dose-dependently attenuates hyperoxia-induced lung injury in neonatal rats. Cell Transplant. 2011;20:1843–54.PubMedView ArticleGoogle Scholar
- Chang YS, Oh W, Choi SJ, Sung DK, Kim SY, Choi EY, et al. Human umbilical cord blood-derived mesenchymal stem cells attenuate hyperoxia-induced lung injury in neonatal rats. Cell Transplant. 2009;18:869–86.PubMedView ArticleGoogle Scholar
- Wong AP, Dutly AE, Sacher A, Lee H, Hwang DM, Liu M, et al. Targeted cell replacement with bone marrow cells for airway epithelial regeneration. Am J Physiol Lung Cell Mol Physiol. 2007;293:L740–52.PubMedView ArticleGoogle Scholar
- Wong AP, Keating A, Lu WY, Duchesneau P, Wang X, Sacher A, et al. Identification of a bone marrow-derived epithelial-like population capable of repopulating injured mouse airway epithelium. J Clin Invest. 2009;119:336–48.PubMed CentralPubMedGoogle Scholar
- Chistiakov DA. Endogenous and exogenous stem cells: a role in lung repair and use in airway tissue engineering and transplantation. J Biomed Sci. 2010;17:92.PubMed CentralPubMedView ArticleGoogle Scholar
- Haider H, Ashraf M. Preconditioning and stem cell survival. J Cardiovasc Transl Res. 2010;3:89–102.PubMedView ArticleGoogle Scholar
- Murry CE, Jennings RB, Reimer KA. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation. 1986;74:1124–36.PubMedView ArticleGoogle Scholar
- Haque N, Rahman MT, Abu Kasim NH, Alabsi AM. Hypoxic culture conditions as a solution for mesenchymal stem cell based regenerative therapy. Sci World J. 2013;2013:632972.View ArticleGoogle Scholar
- Yan F, Yao Y, Chen L, Li Y, Sheng Z, Ma G. Hypoxic preconditioning improves survival of cardiac progenitor cells: role of stromal cell derived factor-1alpha-CXCR4 axis. PLoS One. 2012;7, e37948.PubMed CentralPubMedView ArticleGoogle Scholar
- Yu SP, Wei Z, Wei L. Preconditioning strategy in stem cell transplantation therapy. Transl Stroke Res. 2013;4:76–88.PubMed CentralPubMedView ArticleGoogle Scholar
- Hu X, Yu SP, Fraser JL, Lu Z, Ogle ME, Wang JA, et al. Transplantation of hypoxia-preconditioned mesenchymal stem cells improves infarcted heart function via enhanced survival of implanted cells and angiogenesis. J Thorac Cardiovasc Surg. 2008;135:799–808.PubMedView ArticleGoogle Scholar
- Rosova I, Dao M, Capoccia B, Link D, Nolta JA. Hypoxic preconditioning results in increased motility and improved therapeutic potential of human mesenchymal stem cells. Stem Cells. 2008;26:2173–82.PubMed CentralPubMedView ArticleGoogle Scholar
- Wei L, Fraser JL, Lu ZY, Hu X, Yu SP. Transplantation of hypoxia preconditioned bone marrow mesenchymal stem cells enhances angiogenesis and neurogenesis after cerebral ischemia in rats. Neurobiol Dis. 2012;46:635–45.PubMed CentralPubMedView ArticleGoogle Scholar
- Gnecchi M, Zhang Z, Ni A, Dzau VJ. Paracrine mechanisms in adult stem cell signaling and therapy. Circ Res. 2008;103:1204–19.PubMed CentralPubMedView ArticleGoogle Scholar
- Chow DC, Wenning LA, Miller WM, Papoutsakis ET. Modeling pO(2) distributions in the bone marrow hematopoietic compartment. II Modified Kroghian models Biophys J. 2001;81:685–96.Google Scholar
- Cipolleschi MG, Dello Sbarba P, Olivotto M. The role of hypoxia in the maintenance of hematopoietic stem cells. Blood. 1993;82:2031–7.PubMedGoogle Scholar
- Leroux L, Descamps B, Tojais NF, Seguy B, Oses P, Moreau C, et al. Hypoxia preconditioned mesenchymal stem cells improve vascular and skeletal muscle fiber regeneration after ischemia through a Wnt4-dependent pathway. Mol Ther. 2010;18:1545–52.PubMed CentralPubMedView ArticleGoogle Scholar
- Martin-Rendon E, Hale SJ, Ryan D, Baban D, Forde SP, Roubelakis M, et al. Transcriptional profiling of human cord blood CD133+ and cultured bone marrow mesenchymal stem cells in response to hypoxia. Stem Cells. 2007;25:1003–12.PubMedView ArticleGoogle Scholar
- Yue Y, Zhang P, Liu D, Yang JF, Nie C, Yang D. Hypoxia preconditioning enhances the viability of ADSCs to increase the survival rate of ischemic skin flaps in rats. Aesthetic Plast Surg. 2013;37:159–70.PubMedView ArticleGoogle Scholar
- Linge A, Morishima N, Kasper M, Barth K. Bleomycin induces caveolin-1 and -2 expression in epithelial lung cancer A549 cells. Anticancer Res. 2007;27:1343–51.PubMedGoogle Scholar
- Lee VY, Schroedl C, Brunelle JK, Buccellato LJ, Akinci OI, Kaneto H, et al. Bleomycin induces alveolar epithelial cell death through JNK-dependent activation of the mitochondrial death pathway. Am J Physiol Lung Cell Mol Physiol. 2005;289:L521–8.PubMedView ArticleGoogle Scholar
- Rojas M, Xu J, Woods CR, Mora AL, Spears W, Roman J, et al. Bone marrow-derived mesenchymal stem cells in repair of the injured lung. Am J Respir Cell Mol Biol. 2005;33:145–52.PubMed CentralPubMedView ArticleGoogle Scholar
- Zhao F, Zhang YF, Liu YG, Zhou JJ, Li ZK, Wu CG, et al. Therapeutic effects of bone marrow-derived mesenchymal stem cells engraftment on bleomycin-induced lung injury in rats. Transplant Proc. 2008;40:1700–5.PubMedView ArticleGoogle Scholar
- Zhen G, Liu H, Gu N, Zhang H, Xu Y, Zhang Z. Mesenchymal stem cells transplantation protects against rat pulmonary emphysema. Front Biosci. 2008;13:3415–22.PubMedView ArticleGoogle Scholar
- Uji M, Nakada A, Nakamura T. Intravenous administration of adipose-derived stromal cells does not ameliorate bleomycin-induced lung injury in rats. Open J Regen Med. 2013;2:39–45.View ArticleGoogle Scholar
- Aguilar S, Scotton CJ, McNulty K, Nye E, Stamp G, Laurent G, et al. Bone marrow stem cells expressing keratinocyte growth factor via an inducible lentivirus protects against bleomycin-induced pulmonary fibrosis. PLoS One. 2009;4, e8013.PubMed CentralPubMedView ArticleGoogle Scholar
- Moodley Y, Vaghjiani V, Chan J, Baltic S, Ryan M, Tchongue J, et al. Anti-inflammatory effects of adult stem cells in sustained lung injury: a comparative study. PLoS One. 2013;8, e69299.PubMed CentralPubMedView ArticleGoogle Scholar
- Cargnoni A, Gibelli L, Tosini A, Signoroni PB, Nassuato C, Arienti D, et al. Transplantation of allogeneic and xenogeneic placenta-derived cells reduces bleomycin-induced lung fibrosis. Cell Transplant. 2009;18:405–22.PubMedView ArticleGoogle Scholar
- Nakamura T, Mizuno S. The discovery of hepatocyte growth factor (HGF) and its significance for cell biology, life sciences and clinical medicine. Proc Jpn Acad Ser B Phys Biol Sci. 2010;86:588–610.PubMed CentralPubMedView ArticleGoogle Scholar
- Panganiban RA. Hepatocyte growth factor in lung repair and pulmonary fibrosis. Acta Pharmacol Sin. 2011;32:12–20.PubMed CentralPubMedView ArticleGoogle Scholar
- Crestani B, Marchand-Adam S, Quesnel C, Plantier L, Borensztajn K, Marchal J, et al. Hepatocyte growth factor and lung fibrosis. Proc Am Thorac Soc. 2012;9:158–63.PubMedView ArticleGoogle Scholar
- Ishikawa H, Jo JI, Tabata Y. Liver anti-fibrosis therapy with mesenchymal stem cells secreting hepatocyte growth factor. J Biomater Sci Polym Ed. 2012;23:2259–72.Google Scholar
- Lu F, Zhao X, Wu J, Cui Y, Mao Y, Chen K, et al. MSCs transfected with hepatocyte growth factor or vascular endothelial growth factor improve cardiac function in the infarcted porcine heart by increasing angiogenesis and reducing fibrosis. Int J Cardiol. 2013;167:2524–32.PubMedView ArticleGoogle Scholar
- Wang H, Yang YF, Zhao L, Xiao FJ, Zhang QW, Wen ML, et al. Hepatocyte growth factor gene-modified mesenchymal stem cells reduce radiation-induced lung injury. Hum Gene Ther. 2013;24:343–53.PubMedView ArticleGoogle Scholar
- Yu Y, Lu L, Qian X, Chen N, Yao A, Pu L, et al. Antifibrotic effect of hepatocyte growth factor-expressing mesenchymal stem cells in small-for-size liver transplant rats. Stem Cells Dev. 2010;19:903–14.PubMedView ArticleGoogle Scholar
- Cui Q, Fu S, Li Z. Hepatocyte growth factor inhibits TGF-beta1-induced myofibroblast differentiation in tendon fibroblasts: role of AMPK signaling pathway. J Physiol Sci. 2013;63:163–70.PubMedView ArticleGoogle Scholar
- Mizuno S, Matsumoto K, Li MY, Nakamura T. HGF reduces advancing lung fibrosis in mice: a potential role for MMP-dependent myofibroblast apoptosis. FASEB J. 2005;19:580–2.PubMedGoogle Scholar
- Cui Q, Wang Z, Jiang D, Qu L, Guo J, Li Z. HGF inhibits TGF-beta1-induced myofibroblast differentiation and ECM deposition via MMP-2 in Achilles tendon in rat. Eur J Appl Physiol. 2011;111:1457–63.PubMedView ArticleGoogle Scholar
- Jiang D, Jiang Z, Han F, Zhang Y, Li Z. HGF suppresses the production of collagen type III and alpha-SMA induced by TGF-beta1 in healing fibroblasts. Eur J Appl Physiol. 2008;103:489–93.PubMedView ArticleGoogle Scholar
- Shukla MN, Rose JL, Ray R, Lathrop KL, Ray A, Ray P. Hepatocyte growth factor inhibits epithelial to myofibroblast transition in lung cells via Smad7. Am J Respir Cell Mol Biol. 2009;40:643–53.PubMed CentralPubMedView ArticleGoogle Scholar
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