Cytotherapy, 2014; 0: 1e12

Mesenchymal stem cell injection protects against oxidative stress in Escherichia colieinduced acute lung injury in mice SALLY M. SHALABY1, AMAL S. EL-SHAL1, SOMIA H. ABD-ALLAH1, ASSMAA O. SELIM2, SALLY A. SELIM2, ZIENAB A. GOUDA2, DALIA M. ABD EL MOTTELEB3, HALA E. ZANFALY4, HEBA M. EL-ASSAR4 & SHYMAA ABDELAZIM5 1

Medical Biochemistry Department, 2Histology and Cell Biology Department, 3Clinical Pharmacology Department, Anesthesia and Intensive Care Department, and 5Microbiology and Immunology Department, Faculty of Medicine, Zagazig University, Zagazig, Egypt

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Abstract Background aims. Stem cells may be a promising therapy for acute respiratory distress syndrome. Recent in vivo and in vitro studies suggested that the mesenchymal stromal cells (MSCs) have anti-oxidative stress properties. We hypothesized that intravenous injection of bone marrowederived mesenchymal stem cells (MSCs) could attenuate Escherichia colieinduced acute lung injury (ALI) in mice by controlling the oxidative stress status. Methods. Eighty mice were randomly divided into four groups: group 1 (control group) received 25 mL of saline as a vehicle; group 2 contained E colieinduced ALI mice; group 3 included mice that received MSCs before induction of ALI; group 4 included mice that received MSCs after induction of ALI. Lung samples were isolated and assayed for oxidative stress variables and histopathologic analysis. Total anti-oxidant capacity was measured in broncho-alveolar lavage. Results. Pre- and post-injury MSC injection increased survival, reduced pulmonary edema and attenuated lung injuries in ALI mice. Histologically, MSCs exhibited a considerable degree of preservation of the pulmonary alveolar architecture. An increase of anti-oxidant enzyme activities and a decrease of myeloperoxidase activity and malondialdehyde levels in the MSC recipient groups versus the ALI group were found. Furthermore, the total anti-oxidant capacity and reduced glutathione levels were significantly increased in MSCs recipient groups versus the ALI group. Weak þve inducible nitric oxide synthase immuno-expression in groups that received MSCs was detected. Pre-injury MSC injection showed better effects than did post-injury MSC injection. Conclusions. Systemic bone marrowederived MSC injection was effective in modulating the oxidative stress status in E colieinduced acute lung injury in mice. Key Words: acute lung injury, Escherichia coli, mesenchymal stromal cells, oxidative stress markers, mice

Introduction Acute lung injury (ALI) and its severe form, acute respiratory distress syndrome (ARDS), are lung diseases characterized by a severe inflammatory process causing alveolar damage and resulting in a variable degree of ventilation perfusion mismatch, severe hypoxemia, poor lung compliance and noncardiogenic pulmonary edema (1). Patients with ALI have a ratio of arterial oxygen tension to fraction of inspired oxygen (PaO2/FiO2) of 201 to 300 mm Hg, whereas patients with ARDS have worse hypoxemia, with a PaO2/FiO2 of 200 mm Hg (2). Acute respiratory distress syndrome is a major cause of acute respiratory failure in critically ill patients. Several

etiological factors associated with the development of ALI as sepsis, pneumonia and trauma with multiple transfusions accounting for most cases. A major cause for the development of ALI is sepsis, wherein Gram-negative bacteria are a prominent cause (3). There are no therapies for ARDS until now, yet management remains supportive (2). Improvements in these measures, such as protective mechanical ventilation strategies (4), restrictive intravenous fluid management (5) and prone positioning of severely hypoxemic patients (6) have decreased mortality rates from ARDS. Despite all innovations in intensive care medicine, the mortality of acute respiratory failure from ARDS remains up to 40% (1). More

Correspondence: Sally Shalaby, MD, Medical Biochemistry Department, Faculty of Medicine, Zagazig University, Zagazig, Egypt. E-mail: sallyshalaby@ hotmail.com (Received 3 October 2013; accepted 12 December 2013) ISSN 1465-3249 Copyright Ó 2014, International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jcyt.2013.12.006

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complex strategies, aimed to reduce early injury and/ or enhance repair after injury, are needed. These insights have led to a renewed interest in the therapeutic potential of mesenchymal stromal cells (MSCs). MSCs are multipotent cells derived from adult tissues that are capable of self-renewal and differentiation into chondrocytes, osteocytes and adipocytes. The derivation of MSCs from adult tissues, their relative ease of isolation and enormous expansion potential in culture make them attractive therapeutic candidates (7). They are immunologically well tolerated (8) and therefore can be transplanted from an individual to another, an important advantage for acute illnesses such as ARDS. Recent studies have demonstrated that bone marrow (BM)derived MSCs reduce lung injury in experimental models of lipopolysaccharide (LPS)-induced ALI in mouse (9e11), even though the mechanisms underlying the therapeutic effect of MSCs on ALI has yet to be elucidated. One of the main mechanisms involved in the pathogenesis of ALI is through an increase in oxidative stress, resulting from an imbalance between the production of reactive oxygen species (ROS) and their elimination (12). Intracellular ROS accumulation can result in nitration and/ or oxidation of cellular biomolecules including proteins, lipids and DNA (12). Therefore, the antioxidative enzymes such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) can prevent lung injury induced by LPS from ROS exposure (13). We hypothesized that intravenous injection of BM-MSCs could attenuate Escherichia colieinduced ALI in mice, and, if so, the protective mechanism may be mediated by controlling the oxidative stress status. We examined the effects of MSCs in two groups, one with the injection of MSCs before injury and the other after ALI injury, to evaluate whether MSCs had a protective rather than a therapeutic effect. The anti-oxidant markers as SOD, CAT, GPx and glutathione reductase (GR) activities, reduced glutathione (GSH) levels in lung homogenates and total anti-oxidant capacity (TAC) in broncho-alveolar lavage (BAL) were estimated; malondialdehyde (MDA) level as an indicator of lipid peroxidation, myeloperoxidase (MPO) activity as an indicator of neutrophils recruitment and inducible nitric oxide synthase (iNOS) expression were evaluated to elucidate the potential anti-oxidative responses of the MSCs. Methods This study was performed in a stem cell research laboratory in the Medical Biochemistry Department in collaboration with Clinical Pharmacology, Histology

and Cell Biology, Microbiology and Immunology, and Anesthesia and Intensive Care Departments, Faculty of Medicine, Zagazig University, Egypt.

Preparation of BM-MSCs from mice Soft tissue was gently dissected away from tibiae and femora, and the bones were crushed gently with the use of a pestle to collect BM cells (14). Bone marrow cells were plated at a density of 4  106 cells per cm2 in Roswell Park Memorial Institute medium supplemented with 10% fetal bovine serum and 1% penicillinestreptomycineamphotericin B mixture (10 IU/10 IU/25 mg, 100 mL, (Lonza Bioproducts, Verviers, Belgium). The culture medium was changed on day 2 to remove nonadherent cells. The whole medium was subsequently replaced weekly. The cells were grown for 3e4 weeks until almost confluent. Adherent cells were then detached by 0.25% trypsine ethylenediaminetetraacetic acid (EDTA) and replated. Subsequent passaging and seeding of the cells were performed at a density of 5000 cells per cm2. MSC were used after four passages (15).

Bacterial preparation Escherichia coli were used as the source of infection because it is a common cause of Gram-negative bacterial lung infection (16). E coli was cultured overnight in 10 mL of brain-heart infusion broth (Oxoid, Basingstoke, United Kingdom) at 37 C. The bacteria were then diluted in brain-heart infusion media and grown for 1 h to mid-logarithmic phase. The suspension was centrifuged for 10 min at 7000 rpm and washed in phosphate-buffered saline (PBS) (Lonza Bioproducts, Verviers, Belgium). Optical density was measured, and E coli preparations were adjusted to the final concentration: 107 colony-forming units in 0.05 mL of PBS (17).

Animal model Eighty healthy male Balb c mice of matched age and weight (22  3 g) were included in this study. Animals were inbred in pathogen-free conditions at the experimental animal unit, Faculty of Medicine, Zagazig University. Mice were maintained according to the standard guidelines of Institutional Animal Care and Use Committee and after institutional review board approval. Before commencing the experiment, all animals were subjected to a 2-week period of passive preliminaries to adapt themselves to their new environment, to ascertain their physical well-being and to exclude any diseased or infected animals. Mice were maintained on a stock diet and

Mechanistic action of MSCs in prevention of ALI kept under fixed appropriate conditions of housing and handling. Experimental design Mice were randomly divided into four groups (with 20 animals each): group 1 (control group) received 25 mL of saline as a vehicle; group 2 (ALI group) contained E colieinduced ALI mice; group 3 included mice that received MSCs before induction of ALI; group 4 included mice that received MSCs after induction of ALI. To induce ALI, mice were lightly anesthetized with 2.0% isoflurane (Abbott, Cham, Switzerland) delivered in a box; 107 E coli was then introduced intratracheally. Injection of MSCs into mice The formed colonies of the fourth-generation MSCs were washed twice with PBS, and cells were trypsinized with 0.25% trypsin in 1 mmol/L EDTA for 5 min at 37 C. MSCs (5  105 cells/mL/22 g) were injected intravenously in mice (through the tail vein) (11). Mice of group 3 received MSCs 24 h before induction of ALI; mice of group 4 received MSCs injection 12 h after injury. Mice from all groups were anesthetized under thiopental anesthesia (50 mg/kg) and euthanized at post-injury day 2.

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Measurement of MPO activity in lung homogenate The reaction buffer contained 50 mmol/L potassium phosphate (pH 6.0), 0.0005% H2O2 and 0.167 mg/mL O-dianisidine dihydrochloride (Sigma-Aldrich, St Louis, MO, USA). After addition of 0.167 mg/mL of O-dianisidine hydrochloride and 0.0005% hydrogen peroxide to each sample, their absorbances were measured by means of spectrophotometry at 460 nm. Results are expressed as units of MPO per gram of wet tissue (18). Lipid peroxidation assay MDA, as a marker of lipid peroxidation, was measured colorimetrically in lung homogenate according to the method of Okhawa et al. (19) with the use of a commercially available kit (Biodiagnostic, Cairo, Egypt). Thiobarbituric acid reacts with MDA in acidic medium at 95 C for 30 min to form thiobarbituric acidereactive product, and the absorbance of the resultant pink product can be measured at 534 nm. Anti-oxidant measurements in lung homogenate

The thoracic cage was opened; the left lung was processed for BAL and the right upper and middle lung lobes were used for biochemical assays. The right lower lung lobes were used for histological evaluation. Dry/wet weight measurements were performed on whole lungs from a separate set of animals. For preparation of the lung homogenate, the lung tissue was homogenized with 5e10 mL of cold buffer (50 mmol/L potassium phosphate, pH 7.4, 1 mmol/L EDTA) per gram of tissue. The homogenate was centrifuged at 4000 rpm for 15 min at 4 C, and the supernatant was stored on ice. If not assayed on the same day, the samples were frozen at 80 C.

The SOD activity (in U/g lung tissue) was determined by use of the method of Nishikimi et al. (20). CAT activity (in U/g tissue) was assessed by means of the method of Aebi (21), as catalase reacts with a known quantity of hydrogen peroxide and the reaction is stopped after 1 min with catalase inhibitor. In the presence of peroxidase, the remaining hydrogen peroxide reacts with 3,5-dichloro-2-hydroxybenzene sulfonic acid and 4-aminophenazone to form a chromophore with a color intensity inversely proportional to the amount of catalase in the sample. The absorbance was measured at 510 nm. Reduced GSH was determined according to the method of Beutler et al. (22). GSH determination is based on the reduction of 5,50 -dithiobis (2-nitrobenzoic acid) with GSH to produce a yellow compound. The reduced chromogen is directly proportional to GSH concentration, and its absorbance can be measured at 405 nm. GPx and GR activities were also determined (23,24). All the above-mentioned kits were obtained from Biodiagnostic, Cairo, Egypt.

Lung wet/dry weight ratio

Estimation of TAC in BAL

Lungs were removed immediately after euthanasia and the wet weight was recorded. Lungs were then placed in an incubator at 60 C for 72 h, and the dry weight was detected. The wet lung mass divided by the dry lung mass represented the wet-dry lung ratio, which indicates the fraction of wet lung weight caused by water.

BAL fluid was obtained by placing a 20-gauge catheter into the trachea through which 3 mL of cold PBS was flushed back and forth three times. The BAL fluid was centrifuged at 3000 rpm for 20 min at 4 C. The resulting cell pellet was used to determine the TAC (Biodiagnostic, Cairo, Egypt), according to Koracevic et al. (25). The method is

Tissue preparation

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based on determination of the ability to eliminate added hydrogen peroxide. The remaining H2O2 is determined colorimetrically by an enzymatic reaction converting 3,5-dichloro-2-hydroxyl benzenesulfonate to a colored product that is measured at 532 nm. Light microscopic study The specimens were fixed in 10% neutral-buffered formalin, dehydrated and embedded in paraffin; sections were then cut at 5 mm and stained with hematoxylin and eosin for routine histological examination (26). The immunohistochemical staining iNOS was demonstrated through the use of the labeled streptavidinebiotin immunoperioxidase technique. In brief, the sections were deparaffinized in xylene, rehydrated in ethyl alcohol and washed twice with distilled water. For better antigen retrieval, the samples were boiled two times for 5 min in a microwave oven in a citrate buffer (pH 6.0). Endogenous peroxidases were blocked by 5% H2O2 treatment for 5 min. The samples were washed with PBS (pH 7.2). Immunoperoxidase stain for iNOS (1:200 dilution; BD Biosciences, San Diego, CA, USA) was used to recognize iNOS in the cytoplasm. The samples were incubated with the primary antibody for 60 min at 4 C. Before the secondary antibody was applied and slides were washed twice with PBS, the slides were incubated for 45 min with the biotinylated secondary antibody, followed by washing and 50-min incubation in an avidine biotinylated peroxidase complex reagent (Vectastain Rabbit ABC Elite Kit; Vector Laboratories, Burlingame, CA, USA). Expressions were visualized through the use of a 5-min diaminobenzidine tetrahydrochloride (Sigma-Aldrich, St. Louis, MO, USA) treatment. The slides were counterstained with Mayer’s hematoxylin, dehydrated and mounted with DPX (BDH Ltd, Poole, United Kingdom) (27). The image analyzer computer system, Leica Qwin 500, Microsystems Imaging Solutions Ltd (Cambridge, United Kingdom), in the image-analyzing unit of the Pathology Department, Faculty of Dentistry, Cairo University, Egypt, was used to evaluate the alveolar diameter, thickening of interalveolar septa and optical density of iNOS immunoexpression. It was measured with the use of the interactive measure menu. The area percentage and standard measuring frame of a standard area equal to 118,476.6 mm2 were chosen from the parameters measuring 10 readings from five fields from each slide. The alveolar diameter and thickness of inter-alveolar septa measurements were evaluated at magnification 100; optical density of iNOS at was evaluated at magnification 400.

Electron microscopic study Small pieces of lower lobes of right lungs were immediately fixed in 2.5% glutaraldehyde buffered with 0.1 mol/L PBS at pH 7.4 for 2 h at 4 C and postfixed in 1% osmium tetroxide in the same buffer for 1 h at 4 C. The specimens were processed and embedded in Embded-812 resin in BEEM capsules at 60 C for 24 h. Ultrathin sections were obtained with the use of Lecia ultracut UCT (Germany), stained with uranyl acetate and lead citrate (28) and were then examined with the use of a JEOL-JEM 1010 electron microscope (Tokyo, Japan) at the Mycology and Regional Biotechnology Center, Al Azhar University, Cairo, Egypt. Statistical analysis Statistical differences between the groups were tested by analysis of variance and least significant differences tests. Survival rates were compared by means of the Kaplan-Meier analysis followed by a log rank test. Data were presented as mean  standard deviation, and values of P < 0.05 were considered significant. Statistical analyses were performed with the use of SPSS software (version 17; Chicago, IL, USA). Results MSC characterization Mouse MSCs were obtained from bone marrow of Balb c mice by adherence to plastic culture flasks. Morphologically, these cells had a spindled, fibroblast appearance after expansion. Cells that had been passaged four times were used in all experiments. Flow cytometric analysis demonstrated that MSCs did not express CD34 (0.2%) but expressed CD105, CD73 (99.5% and 96.4%, respectively). Survival rate E colieinduced ALI significantly reduced the survival rate (70%, 14/20 mice surviving) at postinjury day 2 compared with the 100% survival rate (20/20) of the control group (P < 0.05). The reduced survival rate improved in the MSC injection before induction of ALI group (95%, 19/20 mice surviving, P < 0.05 versus ALI group) and also improved in MSC injection after induction of ALI (85%, 17/20 mice surviving, P < 0.05 versus ALI group) but the survival rate was higher in preinjury MSC injection than in post injury injection (P < 0.05).

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group as compared with the control group (P < 0.001). A significant decrease of MPO activity in MSCs recipient groups (groups 3 and 4) versus ALI mice was detected. Moreover, there was a significantly greater decrease of MPO activity in group 3 than in group 4 (P < 0.001) (Figure 1B). Oxidativeeanti-oxidative parameters results

Figure 1. (A) Wet-dry lung ratio in all studied groups (n ¼ 8 per group), *P < 0.05 when compared with control group, MSC recipient before ALI group or MSCs recipient after ALI group. (B) Lung MPO activity, *P < 0.001 compared with control group, #P < 0.001 compared with ALI group, þP < 0.001 compared with MSC recipient before ALI group.

Lung water content E colieinduced ALI increased lung wet/dry weight ratio when compared with the control group (P < 0.001). Treatment with BM-MSCs significantly decreased pulmonary edema in groups 3 and 4 in comparison with the ALI group (P ¼ 0.002, 0.006, respectively) (Figure 1A). MPO activity MPO activity as an indicator of neutrophil recruitment was high and significantly increased in the ALI

The activities of SOD, CAT, GR and GPx in group 2 (ALI group) decreased significantly in the lung homogenate as compared with the controls (P ¼ 0.001 for SOD; P < 0.001 for catalase, GR and GPx). TAC and GSH levels also significantly decreased in the ALI group when compared with the control group (P < 0.001 and 0.004, respectively). MDA level as an indicator of lipid peroxidation was high and significantly increased in ALI group as compared with the control group. No significant difference of the same parameters was detected in groups 3 and 4 (ALI þ MSCs injection) when compared with group 1 (control group) (P > 0.05) except for MDA levels of group 4 (MSC injection after ALI injury). Interestingly, we found a significant increase of anti-oxidant enzyme activities, GSH and TAC levels and a significant decrease of MDA levels in MSC recipient groups versus ALI mice. When we compared between groups 3 and 4, there was a greater decrease of the anti-oxidant parameters levels in group 4 than in group 3 (nonsignificant), whereas there was a significantly greater decrease of MDA levels in group 3 than in group 4 (P ¼ 0.01) (Table I). Histopathological results Examination of hematoxylin and eosinestained sections of the control group (group 1) showed normal lung architecture, respiratory bronchiole, alveolar duct, alveolar sac and alveoli and a relatively thin inter-alveolar septum (Figures 2A and 3A). In contrast, at posteacute lung injury day 2 (group 2), the lungs

Table I. Oxidative and anti-oxidative markers in all studied groups. Control group Acute lung injury group (n ¼ 20) (n ¼ 14) CAT (U/g) MDA (nmol/g) GSH (mg/g) GR (U/g) GPx (U/g) SOD (U/g) TAC(mmol/L) a

39.7 3.2 8.3 5.6 118.7 30.4 0.7

      

4.3 1.2 2.8 1.5 15.4 2.5 0.03

When compared with control group.

25.5 9.4 5.4 3.4 72.4 27.4 0.02

      

1.9 3.4 2.5 1.7 9.8 2.3 0.01

Pa 0.05) (Figure 2F).

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Figure 3. Hematoxylin and eosinestained lung sections of different groups at magnification 400. (A) Control lung with alveolar sac (C), alveoli (A), relatively thin inter-alveolar septum (thin arrow) and relatively thick inter-alveolar septum (thick arrow). (BeE) Lung of E colieinduced ALI group; (B) Collapsed alveoli (CA), interstitial exudates (*) and very thick inter-alveolar septa (4) with numerous blood capillaries (/). (C, D) Extensive inflammatory cells (IF) with fatty (F) cellular infiltration and also area of hemorrhage (Hg) are seen. (E) Thickened muscular wall (M) of congested blood vessel (BV) is also observed. (F) Lung of MSC recipient group before ALI shows nearly apparently normal alveoli with relatively thin (/), thick (thick arrow) and also very thick primary inter-alveolar septa (4) with blood vessel (BV). (G) Lung of MSC recipient group after ALI with relatively normal alveoli (A); collapsed alveoli (CA) has relatively thin (thin arrow), thick (thick arrow) and very thick inter-alveolar septa (4).

Immunohistochemical staining for iNOS in the control group revealed a weak positive reaction (brown color) in some interstitial cells (Figure 4A). Lungs of the ALI group showed strong positive reaction of iNOS in numerous cells that infiltrated the lung (Figure 4B). However, a weak positive iNOS expression was detected in some interstitial cells in the lung of the MSC recipient group before and after ALI (Figure 4C,D). The E colieinduced ALI group induced highly significantly increased in the optical density of iNOS immuno-expression when compared with the control group (P < 0.001). No significant difference in this density was detected in group 3 (MSC injection before ALI) (P > 0.05) but recorded a significant increase in group 4 (MSC injection after ALI) when compared with the control group (P < 0.05) (Figure 4E). Ultrastructural examination revealed the identical layers of the air-blood barrier: attenuated cytoplasm of

pneumocyte type I, fused basal laminae of both endothelial cell and pneumocyte type I, and cytoplasm of capillary endothelial cells in the control group (Figure 5A). The ALI group showed deformed air-blood barrier: extensive swollen cytoplasm of both pneumocyte type I and capillary endothelium (Figure 5B). However, a nearly normal barrier but with little swelling in cytoplasm of pneumocyte type I of the barrier was observed in the MSC recipient group before ALI (Figure 5C). A slight swelling in cytoplasm of pneumocyte type I and irregularity in the fused basal lamina of the barrier were detected in the MSC recipient group after ALI (Figure 5D).

Discussion ALI/ARDS account for 10e15% intensive care unit admissions; even with the current advances in

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Figure 4. Immunohistochemical staining of iNOS at magnification 400. (A) Lung of control group shows weak positive reacted cells (/). (B) Lung of E colieinduced ALI group shows strong positive reaction in numerous cells (/). (C) Lung of MSC recipient before ALI group shows mild positive reaction (/) in some cells. (D) Lung of MSC recipient after ALI group shows moderate positive reaction in a few cells (/). (E) Histogram shows that E colieinduced ALI (group 2) induced a significant increase in the optical density of iNOS immunoexpression when compared with control group (group 1) (P < 0.001). No significant difference in this density is detected in group 3 (MSCs injection before ALI) (P > 0.05). A significant increase is recorded in group 4 (MSCs injection after ALI) when compared with the control group (P < 0.05). *Group 2 highly significantly different from group 1, #group 4 significantly different from group 1.

lung-protective ventilation and fluid management, there is still a high mortality rate (29). They are followed by poor survival and diminished quality of lung function. In addition, survivors often have longterm neuromuscular, cognitive and psychological symptoms (2). Probable reasons for the failure to find a successful therapy include deficits in our understanding of the disease, coupled with a focus on strategies that inhibit one aspect of a multi-faceted injury process. LPS-induced lung injury has been used as an animal model to mimic infectious human ALI. Transplantation of E coli into mouse lungs resulted in ALI with a presentation similar to that of human ALI, including severe pneumonia (30). The present data about the E colieinduced ALI group revealed vascular congestion, hemorrhage, alveolar collapse, very thick inter-alveolar septa and fatty cellular infiltration of the lungs. It was shown that the

infected bacteria may release ciliary toxins, pneumolysin, endotoxin and immunoglobulin A proteases, which compromise mucociliary clearance and activate alveolar macrophages, neutrophils and epithelial cells through toll-like receptors to recognize pathogen-associated molecular patterns of the bacteria (31). After the administration of LPS, the physiological and structural alterations in the lung can be best observed between 6 and 48 h (32); therefore mice of group 3 received MSCs 24 h before induction of ALI to prevent the occurrence of the disease. Mice of group 4 received injections of MSCs 12 h after injury as treatment of the disease. All mice from all groups were euthanized after 2 days of injury. ALI is a disease characterized by dramatic perturbation in the systemic redox environment. As the results of the current study, in the ALI group there was an increase of MDA levels, a decrease of the

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Figure 5. Uranyl acetateestained and lead citrateestained ultra-thin sections of lungs of different groups show the air-blood barrier. (A) Lung of the control group shows normal air-blood barrier. It is formed of the attenuated cytoplasm of pneumocyte type I (P), fused basal lamina (arrow) and cytoplasm of capillary endothelial cells (E) with red blood cells (R) in its lumen (magnification 30,000). (B) Lung of E colieinduced ALI with deformed barrier, extensive swollen cytoplasm of pneumocyte type I (P), thin fused basal lamina (arrow) and swollen cytoplasm of capillary endothelium (E) (magnification 20,000). (C) Lung of MSC recipient group before ALI with nearly normal barrier; capillary endothelium (E) with indented nucleus (N), fuses basal lamina (arrow) and little swelling in cytoplasm of pneumocyte type I (P) are observed (magnification 30,000). (D) Lung of MSC recipient group after ALI shows this barrier with irregular, little swelling in cytoplasm of pneumocyte type I (P) and with irregularity in the fused basal lamina (arrow) (magnification 30,000).

activity of key anti-oxidant enzymes in the lung (SOD, GPx, CAT and GR) and last, a decrease of GSH and TAC. Immunohistochemical staining of ALI mice showed a highly significant increased immuno-reaction for iNOS in most interstitial cells compared with those of the controls. These results were in agreement with results of da Cunha et al. (33), Melo et al. (34) and Gokakin et al. (33e35), as they found that oxidative stress and oxidative damage are closely related to the development of ALI. Li et al. (36) suggested that the iNOS expression in ALI mice was caused by the increase production of NO. In the current study, ultrastructure changes of the blood-air barrier were in agreement with others (36e38). They suggested that neutrophils accumulated in the lung may cause mechanical obstruction of the pulmonary capillary bed, leading to microcirculation disturbance. In addition, its metabolic products can destroy the alveolar capillary barrier and increase its permeability. This causes proteinrich fluid to leak into the alveolar lumen and interstitial lung tissue, which results in pulmonary edema (36e38). One theory to account for ROS production is through neutrophil recruitment because neutrophils produce vast quantities of ROS and nitrogen species such as O2, N2 and NO through their oxidantgenerating systems such as phagocyte nicotinamide adenine dinucleotide phosphate oxidase and NOS,

respectively (39). Neutrophil-derived oxidants promote deleterious pro-inflammatory effects, thus being a major cause of neutrophil-dependent tissue injury in ALI (38). The current study showed an increase of MPO activity in the lung of ALI mice. MPO enzyme is the major indicator of neutrophil infiltration or activity. This result was confirmed by Li et al. (36) and Haegens et al. (40). In the current study, intravenous injection of BM-MSCs into E colieinduced ALI mice significantly increased survival, reduced pulmonary edema and attenuated lung injuries. The mechanisms by which systemically administered stem cells are recruited to the lung remain poorly understood. After venous administration, many cells initially migrate to the lungs as the first major capillary bed encountered. Lung injury can result in increased localization and retention of these cells in the lung (41). Although the precise mechanisms of action of MSCs in pre-clinical models of ALI were unclear, previous studies suggested that MSCs could favorably modulate the immune response to reduce lung injury (42,43). Cell-to-cell contact appears to be one of mechanisms by which MSCs modulate immune effector cells such as macrophages and T cells. MSCs appear to stimulate macrophages to produce more interleukin 10 (IL-10) through the release of prostaglandin E2, which acts on EP2 and EP4

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macrophage receptors (44). MSCs could downregulate the inflammatory process by decreasing the pro-inflammatory cytokine expression such as tumor necrosis factor-a (TNF-a) and macrophage inflammatory protein 2 (31,45) and secreting antiinflammatory agents such as interlukin-10, IL-1RA, IL-13, angiopoietin-1 and keratinocyte growth factor (45,46). MSCs also secrete anti-microbial peptides such as LL-37, which may directly retard bacterial growth (10). Additionally, MSCs could facilitate lung regeneration and repair through the secretion of cytoprotective agents (11,46). Our study elucidated another mechanism that treatment with BM-MSCs tended to normalize all the investigated markers of oxidative stress upregulated by ALI as MDA levels, MPO activity and iNOS immune expression. Also, MSCs normalize the antioxidant variables downregulated by the disease as, SOD, GPx, CAT and GR activities, important findings not previously reported in other literature. This work is therefore a proof that the anti-oxidative properties of MSCs may contribute to their protective role in ALI. Previous studies demonstrated the relation of MSCs and the oxidativeeanti-oxidative status. Kim et al. (47) found that the anti-oxidant capacity of adipose tissueederived MSC-conditioned medium is comparable to 100 mmol/L of ascorbic acid. MSCs cultured at 32 C expressed lower ROS levels and demonstrated decreased NO and protein carbonyl content and increase of SOD and GPx levels (48). Additionally, studies in a mouse model of LPSinduced ARDS infused with MSCs showed an improvement in cysteine and GSH homeostasis (49). Many reports studied the transfer of mitochondria, which are a major source of ROS, from MSCs to the epithelium. Spees et al. (50) found that human MSCs could rescue mitochondrial function in epithelial cells containing nonfunctional mitochondria. This was confirmed recently in a mouse model of LPS-induced ARDS, in which mitochondria from instilled human MSCs were observed optically to be transferred to the mouse lung epithelium. In this study, lung protection was canceled when MSCs with defective mitochondria were used (51). In clinical view, we suggested that MSCs could be tested for their ability to prevent ALI in very high-risk groups and their efficacy when administered during the progression of lung injury so that improved clinical outcomes can be ascertained. Therefore, in the current study, we tested the efficacy of MSCs when administered before the induction of ALI and during the progression of the disease. Surprisingly, pre-injury MSC injection showed better effects than did post-injury MSC injection, according to the biochemical and the histological results.

Conclusions Our study suggests that BM-MSC injection in mice with E colieinduced ALI improves survival and attenuates ALI through anti-oxidative mechanisms. To date, although few mesenchymal stem cells clinical trials in critically ill patients have not reported adverse immune side effects; the reported data were very limited. Therefore, Mesenchymal stem cells may be a promising therapy for patients suffering from ARDS but the gaps in our knowledge regarding the optimal mesenchymal stem cell administration, dosage regimens, and their safety in critically ill patients should be studied. We hope that these remaining deficits in knowledge will be addressed in the future and that progression from pre-clinical studies to clinical trials in patients with ALI is likely in the near future. Disclosure of interests: The authors have no commercial, proprietary, or financial interest in the products or companies described in this article. References 1. Rubenfeld GD, Caldwell E, Peabody E, Weaver J, Martin DP, Neff M, et al. Incidence and outcomes of acute lung injury. N Engl J Med. 2005;353:1685e93. 2. Walkey AJ, Summer R, Ho V, Alkana P. Acute respiratory distress syndrome: epidemiology and management approaches. Clin Epidemiol. 2012;4:159e69. 3. Sheu CC, Gong MN, Zhai R, Chen F, Bajwa EK, Clardy PF, et al. Clinical characteristics and outcomes of sepsis-related vs non-sepsis-related ARDS. Chest. 2010;138:449e67. 4. Ware LB, Matthay MA. The acute respiratory distress syndrome. N Engl J Med. 2000;342:1334e49. 5. Wiedemann HP, Wheeler AP, Bernard GR, Thompson BT, Hayden D, deBoisblanc B, et al. Comparison of two fluidmanagement strategies in acute lung injury. N Engl J Med. 2006;354:2564e75. 6. Abroug F, Ouanes-Besbes L, Elatrous S, Brochard L. The effect of prone positioning in acute respiratory distress syndrome or acute lung injury: a meta-analysis: areas of uncertainty and recommendations for research. Intensive Care Med. 2008;34:1002e11. 7. Prockop DJ, Kota DJ, Bazhanov N, Reger RL. Evolving paradigms for repair of tissues by adult stem/progenitor cells (MSCs). J Cell Mol Med. 2010;14:2190e9. 8. Moodley Y, Manuelpillai U, Weiss DJ. Cellular therapies for lung disease: a distant horizon. Respirology. 2011;16:223e37. 9. Gupta N, Su X, Popov B, Lee JW, Serikov V, Matthay MA. Intrapulmonary delivery of bone marrow-derived mesenchymal stem cells improves survival and attenuates endotoxininduced acute lung injury in mice. J Immunol. 2007;179: 1855e63. 10. Krasnodembskaya A, Song Y, Fang X, Gupta N, Serikov V, Lee JW, Matthay MA. Antibacterial effect of human mesenchymal stem cells is mediated in part from secretion of the antimicrobial peptide LL-37. Stem Cells. 2010;28:2229e38. 11. Danchuk S, Ylostalo JH, Hossain F, Sorge R, Ramsey A, Bonvillain RW, et al. Human multipotent stromal cells attenuate lipopolysaccharide-induced acute lung injury in

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