Manganese Superoxide Dismutase Gene–Modified Mesenchymal Stem Cells Attenuate Acute Radiation-Induced Lung Injury

Abstract

Radiation-induced lung injury (RILI) is a major clinical complication for radiotherapy in thoracic tumors. An immediate effect of lung irradiation is the generation of reactive oxygen that can produce oxidative damage to DNA, lipids, and proteins resulting in lung cell injury or death. Currently, the medical management of RILI remains supportive. Therefore, there is an urgent need for the development of countermeasures. The present study aimed to evaluate the protective effect of manganese superoxide dismutase (MnSOD) gene–modified mesenchymal stem cells (MSCs) to facilitate the improved recovery of RILI. Here, nonobese diabetic/severe combined immunodeficiency mice received a 13 Gy dose of whole-thorax irradiation, and were then transfused intravenously with MnSOD-MSCs and monitored for 30 days. Lung histopathologic analysis, plasma levels of inflammatory cytokines (interleukin [IL]-1, IL-6, IL-10, and tumor necrosis factor-α), profibrotic factor transforming growth factor-β1, and the oxidative stress factor (hydroxyproline) were evaluated after MnSOD-MSC transplant. Apoptotic rates were evaluated by terminal deoxynucleotidyl transferase-mediated nick-end labeling immunohistochemical method. Colonization and differentiation of MnSOD-MSCs in the irradiated lung were analyzed by immunofluorescence staining. Consequently, systemic administration of MnSOD-MSCs significantly attenuated lung inflammation, ameliorated lung damage, and protected the lung cells from apoptosis. MnSOD-MSCs could differentiate into epithelial-like cells in vivo. MnSOD-MSCs were effective in modulating RILI in mice and had great potential for accelerating from bench to bedside.

Introduction

Radiation-induced lung injury (RILI) occurs in patients with thoracic malignancies such as lung cancer, breast cancer, and various lymphomas.^1–2 RILI is associated with the increased generation of reactive oxygen species (ROS) and the production of biological mediators such as cytokines. Acute radiation (RT) induced lung toxicity and subsequently resulting in inflammation associated pneumonitis, ultimately leading to impaired lung function and respiratory failure.^3
–5 Thus, it is important to alleviate RILI to improve the patient's quality of life. Currently there are no effective protective strategies that definitely prevent or repair RILI.^6–7

To this end, stem cell–based therapies have raised the possibility of improving lung repair. Mesenchymal stem cells (MSCs) derived from bone marrow have been attractive cell therapy vehicles for the delivery of agents due to their convenient isolation and expansion, regenerative and multiple differentiation abilities, low immunogenicity, and potent immunosuppressive effects.^8

–13 Several studies have demonstrated that the MSCs have beneficial effects in reducing lung injury in experimental models, indicating the potential for repair of injured lung.^14
–16 Moreover, MSC antifibrotic functions have been described in various organs.^17–18 Accordingly, stem cell therapy using genetically modified MSCs would be an attractive candidate for RILI.

Manganese superoxide dismutase (MnSOD) is the major antioxidant enzyme localized in the mitochondria catalyzing the dismutation of O₂ into oxygen and hydrogen peroxide and protecting mitochondria against ROS.^19–20 Recently, we have reported that overexpression of MnSOD in MSCs appears protect against tert-butyl hydroperoxide induced apoptosis in vitro ²¹ and ameliorate radiation-induced intestinal syndrome in vivo.²² In the present study, we sought to determine whether administration of MnSOD-MSCs could attenuate RILI.

Materials and Methods

Establishment and characterization of transgenic human MSCs that overexpress MnSOD

Human bone marrow–derived MSCs that stably express MnSOD were established as previously described.²² Briefly, MnSOD cDNA was cloned from human fetal liver tissue using RT-PCR. Then the amplified human MnSOD fragments were ligated into a Pgc-lV lentiviral vector plasmid containing enhanced green fluorescent protein (eGFP).²¹ The lentiviruses were assembled in 293-FT cells lines (Invitrogen). Human MSCs were seeded at 2.0 × 10⁴ cells/cm² one day before transduction. The medium was replaced with virus-containing supernatant supplemented with 4 mg/mL polybrene (Sigma-Aldrich), and incubated for 48 h. After 48 h of transfection, the eGFP-positive MSCs that constitutively expressed MnSOD were sorted by fluorescence-activated cell sorting.

Experimental animals

Six-week-old male nonobese diabetic/severe combined immunodeficiency mice were purchased from Beijing Vital-River Lab Animal Technology Co. Ltd (SCXK JING 2012-0001). All mice were bred and maintained under specific pathogen-free conditions in the experimental animal unit with a 12 h light–dark cycle. All animal handling and experimental procedures were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee of the General Hospital of the Chinese People's Liberation Army (Beijing, China).

Local radiation and cell administration

Radiation was performed on anesthetized mice (intraperitoneal pentobarbital sodium 50 mg/kg). Mice were placed in specifically designed, well-ventilated plastic box and given 13-Gy gamma radiation from a cobalt-60 source to the whole thorax at a dose rate of 1 Gy/min. Then each recipient mouse was given a tail vein injection of 200 μl phosphate-buffered saline (PBS), 1.0 × 10⁶ GFP-MSCs/200 μl PBS, and 1.0 × 10⁶ MnSOD-MSCs/200 μl PBS within 4 h. Animal survival was monitored for 30 days.

Lung wet/dry weight ratio

The left lung was removed immediately after euthanasia, weighed to obtain the wet weight, and then placed in an oven at 60°C for 72 h for measurement of the dry weight. The ratio of wet weight to dry weight was calculated to assess the tissue edema.

Lipid peroxidation assay

Malondialdehyde (MDA) content in lung homogenate was measured using commercialized chemical assay kits according to the manufacturer's protocol (Nanjing Jiancheng Bioenginneering Institute, Nanjing, China). Thiobarbituric acid reacts with MDA in the acidic medium at 95°C for 30 min to form phenobarbitone acid–reactive product, and the absorbance of the resultant pink product were measured at 534 nm.

Broncho alveolar lavage fluid analysis

For broncho alveolar lavage fluid (BALF) analysis, the mice were sacrificed, an open tracheotomy was performed, and a small plastic tube was inserted into the trachea. The BALF was extracted three times with 2 mL physiological saline and centrifuged (400 g, 15 min) in mL modified Hank's balanced salt solution. Total nucleated cell and neutrophils counts were performed on cellular monolayers prepared by cytocentrifugation at 800 rpm for 10 min, followed by Giemsa-Wright's staining.

Lung histopathology analysis

For histological examination, the lung tissue samples in each group at each time were fixed in 4% paraformaldehyde for 24 h, dehydrated in a graded ethanol series, embedded in paraffin, and sectioned at an average thickness of 5 μm. The tissue sections were stained with hematoxylin-eosin. The severity of lung pathology was evaluated blindly by light microscopy depending on the following criteria: alveolar congestion, hemorrhage, interstitial edema, inflammatory cell infiltration, alveolar wall thickening, and small airway epithelial injury.²³ Collagen deposition was detected by Masson staining.

Enzyme-linked immunosorbent assay

Plasma from the peripheral blood was harvested by centrifugation. Interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, IL-10, and transforming growth factor (TGF)-β1 levels were measured by enzyme-linked immunosorbent assay kits (all kits from R&D Systems Inc., Minneapolis) according to the manufactures' instructions. For hydroxyproline analysis, the lung tissues were homogenized and assayed by use of hydroxyproline enzyme-linked immunosorbent assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's protocol.

Apoptosis assay

Identification of apoptosis was confirmed using terminal deoxynucleotidyl transferase-mediated nick-end labeling immunohistochemical staining (ApopTag Peroxidase In Situ Apoptosis Detection Kit). Identification of apoptosis was confirmed according to the manufacturer's instructions. Terminal deoxynucleotidyl transferase-mediated nick-end labeling–positive cells were quantified by morphological criteria in five random images per group.

Immunofluorescence staining

Lung tissues were removed, frozen in optimum cutting temperature medium, and sectioned at 7 μm. The sections were fixed in ice cold acetone, permeabilized with 1% Triton X-100 in PBS for 15 min, and incubated in blocking buffer containing 10% goat serum for 30 min. For double-immunofluorescence staining, the sections were incubated with mouse antibodies against vimentin (Santa Cruz, 1:200) or E-cadherin (Santa Cruz, 1:100) and rabbit antibody against GFP (Proteintech Group, 1:500) overnight at 4°C, followed by incubation with secondary antibodies for 1 h at room temperature. Nuclei were visualized by 4′,6-diamino-2-phenylindole (Sigma). Fluorescence images were analyzed by inverted light microscopy (IX70, Olympus Optical Co.). Confocal images were collected on a LSM 510 META confocal system (Zeiss, Carl Zeiss Micro Imaging).

Statistical methods

All data were expressed as means ± of standard deviation and analyzed using SPSS 16.0 software (SPSS, Chicago, IL). For comparisons between multiple groups, repeated measures analysis of variance tests were applied. Kaplan-Meier survival curve was used for survival analysis. A probability value of less than 0.05 or 0.01 was considered statistically significant.

Results

MnSOD-MSCs improved survival and lung histopathology injury

Of the mice receiving radiation, 48.6% survived to 30 days (the period of observation). The survival rates at 30 days after radiation in mice receiving GFP-MSCs and MnSOD-MSCs were 63.5% and 81.8% respectively. As shown, the MnSOD-MSCs group appeared to be superior to the GFP-MSCs group (Fig. 1B).

Figure 1.

Protective effects of MnSOD-MSCs on histological changes after radiation. (A) Schematic representation of the therapeutic potential of MnSOD-MSCs on RILI. (B) Kaplan-Meier survival rate of mice. (C) HE staining of lung tissues in sham control, RT, GFP-MSCs and MnSOD-MSCs groups. Administration of MnSOD-MSCs reduced the level of lung oedema and inflammation compared with the RT and GFP-MSCs group. These photographs are representative of three mice in each group. Scale bar represents 100 μm. (D) Microscopic injury of the lung tissue was statistically scored. Data were expressed as the means ± SD. (*p < 0.05, ^** p < 0.01, n = 3). HE: hematoxylin-eosin; GFP, green fluorescent protein; IL, interleukin; MDA, malondialdehyde; MSCs: mesenchymal stem cells; MnSOD: manganese superoxide dismutase; PBS, phosphate-buffered saline; RILI: radiation-induced lung injury; RT: radiation; SD, standard deviation; TGF, tumor growth factor. Color images available online at www.liebertpub.com/hum

Representative lung histological sections from each group are presented in Fig. 1. Lung tissue from animals without radiation showed lung architecture, respiratory bronchiole, alveolar duct, alveolar sac, and alveoli appearing histologically normal. In contrast, radiation significantly damaged lung architecture, with the remarkable hemorrhage, alveoli collapse, and inflammatory cell infiltrates. GFP-MSCs group showed relatively normal alveolar spaces in some areas. MnSOD-MSCs treatment attenuated alveolar wall thickening and minimal interstitial edema. And there seemed no significant difference compared with the sham control group. The evaluation of the lung histopathology injury was approved by lung score on day 15 after radiation. The lung score was highest in the PBS group. In contrast, the histological changes and the lung injury score were alleviated in GFP-MSCs group. The results suggested that MnSOD-MSCs alleviated the injuries and regenerated the lung structure and integrity.

MnSOD-MSCs treatment alleviated inflammation after RILI

Radiation exhibited a progressive increase in wet/dry ratio. GFP-MSCs administration showed significantly less increase in wet/dry ratio. Treatment with MnSOD-MSCs reduced the wet/dry ratio, which is an index of pulmonary edema, compared with the PBS group and GFP-MSCs group (Fig. 2A). In addition, the numbers of total cells and neutrophils in BALF was measured. The total cell counts and neutrophils counts of the BALF were increased significantly in RT group. In the GFP-MSCs group and MnSOD-MSCs group, the numbers of total cells and neutrophils in the BALF were significantly reduced, and MnSOD-MSCs group appeared to be superior to GFP-MSCs (Supplementary Fig. S1; Supplementary Data are available online at www.liebertpub.com/hum).

Figure 2.

Anti-inflammatory effects of MnSOD-MSCs on RILI. (A) Lung wet/dry weight ratio. (B–E) IL-1β, IL-6, TNF-α, and IL-10 plasma levels were detected by means of ELISA assay. The data represent the mean ± SD (*p < 0.05, **p < 0.01, n = 3). (F) MDA in lung homogenates (*p < 0.05, **p < 0.01, n = 3). ELISA, enzyme-linked immunosorbent assay; TNF, tumor necrosis factor.

A number of cytokines and chemokines have been involved in radiation-induced lung pathology.²⁴ Radiation induced obvious increases in pro-inflammatory cytokines in plasma levels, including interleukin-1β (IL-1β), IL-6, and TNF-α. Treatment with either GFP-MSCs or MnSOD-MSCs decreased plasma concentrations of these cytokines (Fig. 2B–D), indicating that the treatments attenuate the radiation-induced acute inflammatory response. Meanwhile, IL-10 is a highly anti-inflammatory cytokine that inhibits macrophage production.²¹ The level of the anti-inflammatory cytokine IL-10 in MnSOD-MSCs group was higher than the RT and GFP-MSCs group (Fig. 2E).

MDA is a marker of lipid peroxidation.²⁵ Compared with the PBS group, GFP-MSC and MnSOD-MSC administration decreased MDA concentrations in the lung tissues (Fig. 2F). Our data indicated that MnSOD-MSCs treatment might promote the recovery of the balance between pro-inflammatory and anti-inflammatory cytokines, which suggesting that MnSOD-MSCs have an anti-inflammation effect in RILI.

MnSOD-MSCs exert an antifibrotic effect on RILI

Fibrosis in lung tissues was assessed by Masson staining and hydroxyproline detection. Radiation caused marked collagen deposition in the lung interstitium as well as around blood vessels and bronchi. Such changes were attenuated by GFP-MSCs treatment and significantly alleviated by MnSOD-MSCs treatment (Fig. 3A).

Figure 3.

Improvements of lung fibrosis by MnSOD-MSCs administration. (A) Masson staining of lung sections. Scale bar represents 100μm. The concentration of Transforming growth factor-β1 (TGF-β1) in plasma (B) and hydroxyproline content in lung homogenates (C) were assayed using ELISA. (*p < 0.05, **p < 0.01, n = 5). Color images available online at www.liebertpub.com/hum

TGF-β1 contributes to the maintenances of tissue architecture by influencing the production of extracellular matrix components. TGF-β1 is proposed to be a potent inducer in the lung tissues fibrosis. The level of TGF-β1 in plasma was compared with that of normal mice, with 5 mice at day 30 after radiation. As shown in Fig. 3B, the expression of TGF-β1 in the plasma of the PBS group and GFP-MSCs group significantly increased compared with sham-exposed mice. MnSOD-MSC treatment significantly reduced TGF-β1 level compared with the PBS group and GFP-MSCs group, although they were still higher than those in the sham control group. Apoptotic cells were studied on histological slides of lung tissue by TUNEL assay (Fig. 4A). We also analyzed the percentage of TUNEL-positive cells (Fig. 4B). At day 7 and day 15, a large number of apoptotic cells were observed in the lung tissue of irradiated mice and GFP-MSCs transplanted mice. In contrast, only a small number of apoptotic cells were observed in the lung tissue of MnSOD-MSCs transplanted mice.

Figure 4.

MnSOD-MSCs protected lung tissue cells from apoptosis after radiation. (A) Apoptosis in lung sections was assayed by terminal deoxynucleotidyl transferase-mediated nick-end labeling staining. These photographs are representative of three mice in each group. Scale bar represents 100 μm. (B) The apoptosis index was quantified of five random images per group. The results were expressed as means ±SD (*p < 0.05, **p < 0.01). Color images available online at www.liebertpub.com/hum

Location and differentiation of MnSOD-MSCs in vivo

We first evaluated the engraftment of MnSOD-MSCs in the lungs. Immunofluorescence assay revealed that the GFP positive cells were mostly located around the epithelia of bronchiole/small airway (Fig. 5). And only few MnSOD-MSCs were detected in the lung tissues (data not shown). We observed the mesenchymal cell marker and epithelial marker, including vimentin and E-cadherin expression. The results showed that most of the engrafted MnSOD-MSCs and their offspring were positive for vimentin (Fig. 6A), and a small proportion of infused MnSOD-MSCs showed E-cadherin expression (Fig. 6B). These data suggested that the infused MnSOD-MSCs could differentiate into epithelial-like cells in vivo.

Figure 5.

Chemotactic homing of MnSOD-MSCs. MnSOD-MSCs were located in the areas of alveolar duct and alveoli septa, and only a few MnSOD-MSCs were detected. Nuclei were stained with DAPI. Scale bar represents 100 μm. DAPI, 4′,6-diamino-2-phenylindole. Color images available online at www.liebertpub.com/hum

Figure 6.

MnSOD-MSCs enhance repair of RILI. (A–B) showed MnSOD-MSCs of interstitial and epithelial markers, such as vimentin and E-cadherin. Most of cytokines treated MnSOD-MSCs were positive for vimentin and E-cadherin. Scale bar represents 100 μm. Data from three mice are expressed as means ± SD. Color images available online at www.liebertpub.com/hum

Discussion

Radiation-induced pulmonary injury is part of the major dose-limiting factors of radiotherapy for thoracic malignancies such as lung and breast cancers.^26–27 Ionizing radiation–induced cellular and tissue damage includes mediated radiochemical reactions and oxidative stress. RILI develop through a complex pathological process involving an acute inflammation, increased collagen production, and ultimately pathological fibrosis.^28–29

MnSOD, one of the subtypes of natural superoxide dismutases, acts by catalyzing the dismutation of superoxide radicals into oxygen and hydrogen peroxide to protect cells against ROS.³⁰ Use of human MnSOD plasmid liposomes has provided significant protection against RILI in mice.³¹

Furthermore, stem cell–based gene delivery using MSCs are promising for the development of future therapy. MSCs possess potent lineages differentiation capacity, and anti oxidative stress properties.^32

–35 Available data also suggested that MSCs suppressed the excessive immune responses from T cells, B cells, dendritic cells, macrophages, and natural killer cells.³⁶ Various investigations have evaluated the therapeutic effect of MSCs in preclinical animal models.^37

–40 Our group has previously demonstrated that overexpression of MnSOD gene in MSCs appears to protect against ionizing radiation–induced apoptosis in vitro. In addition, intravenous MnSOD-MSC therapy enhances the recovery of radiation-induced intestinal injury.²² We wish to determine the therapeutic efficacy of MnSOD-MSCs on RILI. Inflammatory cytokines play a critical role in mediating, amplifying, and maintaining the RILI process. The radiation induced obvious increases in representative proinflammatory and profibrotic cytokines in plasma, including IL-1β, TNF-α, and IL-6. Our results demonstrate that MnSOD-MSCs markedly decreased plasma concentrations of these cytokines. Moreover, it inhibited the secretion of pro-inflammatory cytokines, such as IL-1β, IL-6, and TNF-α, and strengthened the production of anti-inflammatory cytokine, IL-10. Therefore, MnSOD-MSCs play a major role in the regulation of excessive inflammatory responses in RILI.

Lung irradiation–induced fibrosis is a complex process involving an activation of numerous profibrotic cytokines, which are produced by damaged alveolar epithelial cells, endothelial cells and activated interstitial cells. MSCs have been shown to alleviate fibrosis in various experimental models. Increased TGF-β1 expression leads to recruitment of macrophages and monocytes to the inflammatory site, which could enhance the maturation and activation of fibroblasts.⁴¹ TGF-β1 has been recognized as the key cytokine in the development of lung fibrosis.^42–43 We investigated TGF-β1 level at day 30 after radiation. MnSOD-MSC treatment significantly reduced TGF-β1 level compared with the PBS and GFP-MSCs groups.

MSCs have a tendency to home to damaged tissue sites. The precise molecular mechanisms by which MSCs are able to migrate and home into sites of injury are not yet fully understood.⁴⁴ The chemokine stromal—derived factor 1 chemokine receptor 4 axis is a signaling pathway associated with active stem cell recruitment in MSC homing.⁴⁵ Systemically administered MSCs are found to migrate specifically to damaged tissue sites with inflammation.^22,46 In present study, we evaluated the engraftment of MnSOD-MSCs in the lungs. Immunofluorescence assay revealed that the GFP positive cells were mostly located around the epithelia of bronchiole/small airway. However, only a few MnSOD-MSCs were found in the injured lungs. The low rate of MnSOD-MSCs in injured lungs seemed insufficient to explain the therapeutic effects. We speculate that the therapeutic benefit of MSCs may be mediated by a paracrine action including MSC migration to the irradiation exposed sites, MSC production of vital growth factors and antioxidants.⁴⁷ In addition, MnSOD-MSCs could differentiate into fibroblasts and some could differentiate into epithelial-like cells in vivo.

In conclusion, this study showed that administration of MnSOD-MSCs and their secreted products strikingly reduced systemic and lung inflammation, ameliorated radiation-induced pulmonary fibrosis, improved the survival of irradiated mice, and protected the lung cells from apoptosis. The mechanism of MnSOD-MSCs repairing RILI is through secreting anti-inflammatory and reparative growth factors and also engraftment and differentiation. We speculate that MSCs overexpressing MnSOD gene represent a promising therapeutic option for RILI in patients who undergo radiotherapy of thoracic tumors.

Footnotes

Acknowledgments

The authors gratefully acknowledge financial support from the Natural Science Foundation of China (Nos. 31440039, 31600681, 81402345, 81673873, 81400274, 31570854, and 81670496), Beijing National Science Foundation of China (No. 5154035), and Beijing Nova Program of Science and Technology (xx2017103).

Author Disclosure

No competing financial interests exist. All authors declare that there are no conflicts of interest with regard to this study or in reporting the findings described in this article.

References

Bradley

, Movsas

. Radiation pneumonitis and esophagitis in thoracic irradiation. Cancer Treat Res, 2006; 128:43–64.

Madani

, De Ruyck

, Goeminne

, et al. Predicting risk of radiation-induced lung injury. J Thorac Oncol, 2007; 2:864–874.

, Xue

, and Lu

. [Current situation and prospect of treatment for radiation-induced lung injury]. Sheng Wu Yi Xue Gong Cheng Xue Za Zhi, 2010; 27:937–940.

Giroux Leprieur

, Fernandez

, Chatellier

, et al. Non-small cell lung cancer: risk factors of radiation pneumonitis. Cancer Radiother, 2012; 16:257–262.

Graves

, Siddiqui

, Anscher

, et al. Radiation pulmonary toxicity: from mechanisms to management. Semin Radiat Oncol, 2010; 20:201–207.

Williams

, Johnston

, Finkelstein

. Treatment for radiation-induced pulmonary late effects: spoiled for choice or looking in the wrong direction?. Curr Drug Targets, 2010; 11:1386–1394.

Giridhar

, Mallick

, Rath

, et al. Radiation induced lung injury: prediction, assessment and management. Asian Pac J Cancer Prev, 2015; 16:2613–2617.

Penfornis

, Pochampally

. Isolation and expansion of mesenchymal stem cells/multipotential stromal cells from human bone marrow. Methods Mol Biol, 2011; 698:11–21.

Rasini

, Dominici

, Kluba

, et al. Mesenchymal stromal/stem cells markers in the human bone marrow. Cytotherapy, 2013; 15:292–306.

10.

Lindner

, Kramer

, Rohwedel

, et al. Mesenchymal stem or stromal cells: toward a better understanding of their biology?. Transfus Med Hemother, 2010; 37:75–83.

11.

Jacobs

, Roobrouck

, Verfaillie

, et al. Immunological characteristics of human mesenchymal stem cells and multipotent adult progenitor cells. Immunol Cell Biol, 2013; 91:32–39.

12.

Golemovic

, Skific

, Cepulic

. [Mesenchymal stem cells: immunomodulatory properties and clinical application]. Lijec Vjesn, 2012; 134:42–49.

13.

Singh

, Sen

. Mesenchymal stem cells in cardiac regeneration: a detailed progress report of the last 6 years (2010–2015). Stem Cell Res Ther, 2016; 7:82.

14.

Epperly

, Guo

, Gretton

, et al. Bone marrow origin of myofibroblasts in irradiation pulmonary fibrosis. Am J Respir Cell Mol Biol, 2003; 29:213–224.

15.

Mouiseddine

, Francois

, Semont

, et al. Human mesenchymal stem cells home specifically to radiation-injured tissues in a non-obese diabetes/severe combined immunodeficiency mouse model. Br J Radiol, 2007; 80:S49–55.

16.

Moodley

, Sturm

, Shaw

, et al. Human mesenchymal stem cells attenuate early damage in a ventilated pig model of acute lung injury. Stem Cell Res, 2016; 17:25–31.

17.

Usunier

, Benderitter

, Tamarat

, et al. Management of fibrosis: the mesenchymal stromal cells breakthrough. Stem Cells Int, 2014; 2014:340257.

18.

Ueno

, Nakashima

, Doi

, et al. Mesenchymal stem cells ameliorate experimental peritoneal fibrosis by suppressing inflammation and inhibiting TGF-beta1 signaling. Kidney Int, 2013; 84:297–307.

19.

Halliwell

. Free radicals and antioxidants: updating a personal view. Nutr Rev, 2012; 70:257–265.

20.

Kim

, Kil

, Seok

, et al. Orchiectomy attenuates post-ischemic oxidative stress and ischemia/reperfusion injury in mice. A role for manganese superoxide dismutase. J Biol Chem, 2006; 281:20349–20356.

21.

Yang

, Dai

, Zhang

, et al. Protective effects of overexpression of MnSOD on t-BHP induced apoptosis in mesenchymal stem cells. Prog Biochem Biophys, 2013; 40:556–564.

22.

Yang

, Chen

, Zhou

, et al. Manganese superoxide dismutase gene therapy protects against irradiation- induced intestinal injury. Curr Gene Ther, 2013; 13:305–314.

23.

Arkovitz

, Garcia

, Szabo

, et al. Decreased pulmonary compliance is an early indicator of pulmonary oxygen injury. J Surg Res, 1997; 67:193–198.

24.

Sohn

, Lee

, Park

, et al. The inflammasome accelerates radiation-induced lung inflammation and fibrosis in mice. Environ Toxicol Pharmacol, 2015; 39:917–926.

25.

Gawel

, Wardas

, Niedworok

, et al. Malondialdehyde (MDA) as a lipid peroxidation marker. Wiad Lek, 2004; 57:453–455.

26.

Testolin

, Favretto

, Cora

, et al. Stereotactic body radiation therapy for a new lung cancer arising after pneumonectomy: dosimetric evaluation and pulmonary toxicity. Br J Radiol, 2015; 88:20150228.

27.

Chow

, Louie

, Palma

, et al. Radiation-induced lung injury after concurrent neoadjuvant chemoradiotherapy for locally advanced breast cancer. Acta Oncol, 2014; 53:697–701.

28.

Osterreicher

, Vavrova

, Knizek

, et al. Radiation pneumonitis: the model of interstitial edema. Exp Toxicol Pathol, 1999; 51:491–493.

29.

Kharofa

and Gore

. Symptomatic radiation pneumonitis in elderly patients receiving thoracic irradiation. Clin Lung Cancer, 2013; 14:283–287.

30.

Holley

, Bakthavatchalu

, Velez-Roman

, et al. Manganese superoxide dismutase: guardian of the powerhouse. Int J Mol Sci, 2011; 12:7114–7162.

31.

Epperly

, Bray

, Krager

, et al. Intratracheal injection of adenovirus containing the human MnSOD transgene protects athymic nude mice from irradiation-induced organizing alveolitis. Int J Radiat Oncol Biol Phys, 1999; 43:169–181.

32.

Fang

, Zheng

, Liao

, et al. Identification of human chronic myelogenous leukemia progenitor cells with hemangioblastic characteristics. Blood, 2005; 105:2733–2740.

33.

Xishan

, Guangyu

, Yuguang

, et al. The research on the immuno-modulatory defect of mesenchymal stem cell from chronic myeloid leukemia patients. J Exp Clin Cancer Res, 2011; 30:47.

34.

, Hynes

, White

, et al. Immunomodulatory properties of induced pluripotent stem cell-derived mesenchymal cells. J Cell Biochem, 2016; 117:2844–2853.

35.

Ciuffreda

, Malpasso

, Musaro

, et al. Protocols for in vitro differentiation of human mesenchymal stem cells into osteogenic, chondrogenic and adipogenic lineages. Methods Mol Biol, 2016; 1416:149–158.

36.

Salem

, Thiemermann

. Mesenchymal stromal cells: current understanding and clinical status. Stem Cells, 2010; 28:585–596.

37.

Meirelles Lda

and Nardi

. Methodology, biology and clinical applications of mesenchymal stem cells. Front Biosci, 2009; 14:4281–4298.

38.

Voswinkel

, Francois

, Simon

, et al. Use of mesenchymal stem cells (MSC) in chronic inflammatory fistulizing and fibrotic diseases: a comprehensive review. Clin Rev Allergy Immunol, 2013; 45:180–192.

39.

Xia

, Chang

, Zhang

, et al. Therapeutic effects of bone marrow-derived mesenchymal stem cells on radiation-induced lung injury. Oncol Rep, 2016; 35:731–738.

40.

Maria

, Maria

, Ybarra

, et al. Mesenchymal stem cells adopt lung cell phenotype in normal and radiation-induced lung injury conditions. Appl Immunohistochem Mol Morphol, 2016; 24:283–295.

41.

Kasai

, Allen

, Mason

, et al. TGF-beta1 induces human alveolar epithelial to mesenchymal cell transition (EMT). Respir Res, 2005; 6:56.

42.

Ueha

, Shand

, and Matsushima

. Cellular and molecular mechanisms of chronic inflammation-associated organ fibrosis. Front Immunol, 2012; 3:71.

43.

Oka

, Komuro

. [Molecular and cellular mechanisms of organ fibrosis]. Nihon Rinsho, 2012; 70:1510–1516.

44.

, Liu

, Lin

, et al. SDF-1alpha/CXCR4 axis mediates the migration of mesenchymal stem cells to the hypoxic-ischemic brain lesion in a rat model. Cell J, 2015; 16:440–447.

45.

, Wu

, Gao

, et al. Stromal cell-derived factor-1-directed bone marrow mesenchymal stem cell migration in response to inflammatory and/or hypoxic stimuli. Cell Adh Migr, 2016; 8:1–18.

46.

Horwitz

, Gordon

, Koo

, et al. Isolated allogeneic bone marrow-derived mesenchymal cells engraft and stimulate growth in children with osteogenesis imperfecta: implications for cell therapy of bone. Proc Natl Acad Sci U S A, 2002; 99:8932–8937.

47.

Gnecchi

, Danieli

, Malpasso

, et al. Paracrine mechanisms of mesenchymal stem cells in tissue repair. Methods Mol Biol, 2016; 1416:123–146.

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.00 MB

0.06 MB