Therapeutic Mechanism Of BMSCs Combined With EPO Against High Oxygen Induced Bronchopulmonary Dysplasia Model In Neonatal Mice

Background:Bronchopulmonary dysplasia(BPD)is a chronic respiratory disease in preterm infants requiring mechanical ventilation and oxygen therapy,and is characterized by restricted lung growth and subdued alveolar and blood vessel development;the structural development of the alveoli is blunted as a consequence of inflammation and oxygen toxicity.To date,there is no effective therapy for preventing or treating lung injury,and new therapies are urgently needed.A large number of studies have shown that stem cell-based therapies,such as mesenchymal stem/stromal cells(MSCs),are promising approaches in preclinical models for the prevention and/or treatment of BPD and other major sequelae of preterm birth.However,there are still some crucial problems that largely restrict the efficacy of these treatments,such as cell survival,homing,and differentiation.Erythropoietin(EPO)is a glycoprotein hormone produced primarily by the adult kidney.Over the last decade,EPO has been shown to be an important cytoprotective cytokine against various forms of stress in many organs,including pulmonary disease,and the mechanism is through the repair of alveolar structure,enhancement of angiogenesis,and suppression of fibrosis.The proper formation of the microvascular system is necessary for normal alveolar development,and an abnormal microvascular system has been observed in the lung of infants with BPD and in BPD-like animal models.Vascular endothelial growth factor(VEGF)plays an essential role in stimulating angiogenesis and the survival of endothelial cells.Reports have showed that VEGF expression and pulmonary capillary density are significantly decreased in the lungs of BPD patients and animal models.In addition,increased VEGF expression inhibits high oxygen(HO)-induced alveolar disruption.However,the influence of combination therapy with EPO and MSCs on angiogenesis in BPD and the underlying mechanism are not fully clear;therefore,the present study was performed to examine these issues.Objective:To explore the mechanism of combination of EPO and MSCs in the treatment of BPD,and try to find a new method to treat BPD.Method:MSCs cultureC57BL/6-green fluorescent protein(GFP)transgenic mice(6-8 weeks old)were bought from the experimental animal center of the Fourth Military Medical University(Xian,China).MSCs were isolated from tibias and femurs using a whole bone marrow culture.Characteristic immunoreactivity for cell markers was detected using fluorescence-activated cell sorting(FACS)analysis.MSCs were suspended with trypsin and 5×105 cells and were washed twice with PBS and then incubated with primary antibodies against rabbit CD34,CD44,CD45,CD90,CD106and CD117.The second polyclonal antibody was added and incubated at 4℃ for an additional 30 min in a dark room.BPD model and cell transplantationNeonatal C57BL/6 mice(24 h old;1-2 g)were obtained from the Animal Experiment Center of Shandong University(Shandong,China).All animal procedures were approved by the animal ethics committee.BPD model was established as previously described with some modifications,briefly,BPD model was established through placed the pups animals in Plexiglas chamber,in which the oxygen concentration was maintained at a FiO2=0.21(normoxia)or FiO2=0.60(hyperoxia)for 14 days.Exposure to hyperoxia was continuous,and mice were maintained in a hypoxic environment,with a brief interruption for animal care(less than 10 min/day).Cultivated MSCs(1-5 × 106)in 50 μl phosphate-buffered saline(PBS)was injected via intravenous administration or/and 5000U/kg recombinant human EPO by intraperitoneal injection respectively at 1 h before and at 7 days after HO exposure.HistologyAt 2 weeks post-treatment,the lungs were harvested and fixed in 4%paraformaldehyde for 24 h.The tissue was embedded in paraffin and cross-sections were cut with a microtome at 4-5 μm.Hematoxylin and eosin staining was used to analyze radial alveolar counts and alveolar septum thickness.Briefly,from the center of the respiratory bronchiole a perpendicular was drawn to the edge of the acinus(as defined by a connective tissue septum or the pleura),and the number of septa intersected by this line was counted.A total of five counts were performed for each animal;the average count was determined from five randomly selected high-power fields(magnification× 100).Immunohistochemical analysisImmunohistochemistry was used to evaluate microvascular growth and the fibrosis of lung.Briefly,the tissue was incubated with a primary anti-platelet endothelial cell adhesion molecule 1(PECAM-1)antibody,polyclonal collagen type Ⅰ(COL1),transforming growth factor-β1(TGF-β1)overnight,or a nonspecifific IgG antibody for 1 h at room temperature,and then a 2-step Plus(?)poly-horseradish peroxidase anti-mouse/rabbit IgG detection system was used.Double immunofluorescence stainingThe proliferative activity of microvascular endothelium was determined by double immunofluorescence staining with rabbit anti-mouse PECAM.1 and Ki-67 antibodies,and differentiation was detected by staining with a rabbit anti-mouse type Ⅱ alveolar epithelial cell(AECⅡ)-specific marker surfactant protein-C(SP-C)antibody and an AECIspecific marker aquaporin 5(AQP5)antibody in vivo.The cryosections were first blocked with 5%goat serum for 30 min and incubated with primary antibodies overnight at 4℃,which was followed by 1-h incubation in the dark with tetramethylrhodamine isothiocyanate and a DyLight 350-conjugated goat anti-rabbit IgG(H+L)secondary antibody at a dilution of 1:200.Immunofluorescent images were taken with an Eclipse 90i microscope.Staining was quantified using Image Pro Plus 6.0 image analysis software as described previously.Quantitative real-time reverse transcription PCR analysisThe mRNA levels of VEGF,C-X-C chemokine receptor type 4(CXCR4),and stromal cell-derived factor-1(SDF-1)were detected by quantitative real-time reverse transcription PCR(qRT-PCR).Total RNA was extracted from lung tissue using TRIzol(?)reagent(Invitrogen,arlsbad,CA)according to the manufacturer’s instructions.The following primers were used:CXCR4 forward,5’-GGCTGACCTCC TCTTTGT-3’ and reverse,5’-GTTTCCTTGGCCTTTGAC-3’;SDF-1forward,5’-CCC TGCCGATTCTTTGAC-3’andreverse,5’-GTCCTTTGGGCTGTTGTG-3’;VEGF forward,5’-CTGCTCTCCTGGGTGCATTG-3’ and reverse,5’-ACTCCTGGAAGAT GTCCACCA-3’;and β-action forward,5’-AACAGTCCGCCTAGAAGCAC-3’ and reverse,5’-CGTTGACATCCGTAAAGACC-3’.The cycling conditions comprised of 10 min polymerase activation at 95℃ and 40 cycles at 95℃ for 15 s and 60℃ for 60 s.The values of the genes were first normalized against β-action,and then compared with controls.Western blot analysisProtein expression was detected by western blot analysis.Total protein was electrophoresed on 4-20%gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and transferred to a nitrocellulose membrane.Anti-CXCR4,anti-SDF-1,and anti-VEGF primary antibodies were used,and the membrane was incubated at 37℃ for 1 h with a secondary goat antirabbit IgG antibody.Immunoreactions were visualized using an electrochemiluminescence kit according to the manufacturer’s instructions.MSC proliferation analysis in vitroThe proliferation of MSCs was analyzed using a Cell Counting Kit-8(CCK-8)assay.Briefly,1.0 × 104 subconfluent MSCs were detached by trypsinization,resuspended,and seeded into 96-well plates in a room air(RA,21%O2 and 5%CO2)or HO(95%O2 and 5%CO2)environment.After 24-h incubation,the CCK-8 reagent was added to each well for 3 h at 37℃.Absorbance was measured at 450 nm using a microplate reader.MSC migration analysis in vitroMigration assays were performed using Transwell cell culture chambers.Briefly,8-μm pore polycarbonate filters were placed between the upper and lower chamber pore filters.A total of 5.0×104 cells in 200 μL medium containing 1%(v/v)fetal bovine serum(FBS)were plated in the upper chamber,and the lower chamber was filled with complete medium containing 10%(v/v)FBS with or without 2 U/mL EPO and cultured at 37℃ for 24 h.The upper surface of the Transwell membrane was wiped clean with a cotton swab,and the cells that had migrated to the lower surface were fixed and stained with crystal violet.The magnitude of MSC migration was evaluated by counting the number of migrated cells under a Nikon Eclipse 90i microscope at a magnification of 200×,Apoptosis stainingAnnexin V-FITC/propidium iodide(PI)double staining was used to evaluate apoptosis by flow cytometry according to the manufacturer’s instructions.Cells(1.0× 106)were collected and suspended in 500 μL binding buffer,and 5 μL annexin V-FITC and 5 μL PI were added to each sample and incubated in the dark for 15 min.The cell surface levels of phosphatidylserine in apoptotic cells were estimated quantitatively using flow cytometry.Statistical analysisComparisons of parameters between two groups were made with an unpaired Student’s t test.Statistical analysis was carried out using SPSS 13.0 software.Statistical analyses between the control and other groups were performed with 1-way analysis of variance(ANOVA)with a Dunnett’s post-hoc test.All data are expressed as the mean±standard deviation(SD).P<0.05 was regarded as a significant statistical difference.Results:Characterization of MSCsCharacteristic immunoreactivity for cell surface markers was detected using FACS analysis with rat monoclonal anti-mouse antibodies.The cells were positive for the expression of the surface markers CD44,CD90,and CD 106,but negative for the expression of the hematopoietic markers CD34,CD45,and CD 117.Effect of MSCs+EPO combination therapy on body weight and lung injuryThe body weight of neonatal mice was measured at 3,7and 14 days post-treatment.Average body weight was significantly increased in the EPO,MSCs and MSCs+EPO groups as compared with the BPD model group;moreover,body weight was increased more in the MSCs+EPO group than in the MSCs or EPO group alone(P<0.05).Hematoxylin and eosin-stained sections of lung tissue demonstrated that alveolar structure was markedly abnormal after neonatal mice were exposed to high oxygen for 14 days,furthermore,degree of alveolarization measured by radial alveolar counts and alveolar septum thickness were significantly improved in the MSCs,EPO and MSCs+EPO groups compared with BPD group,especially in the MSCs+EPO group(P<0.05).Effect of MSCs+EPO combination therapy on vessel densityThe number of new vessels was significantly increased in both treatment groups compared with the BPD group(P<0.05).Moreover,the number of new vessels was higher in the MSCs+EPO group than in the MSCs or EPO group alone(P<0.05).Effect of MSCs+EPO combination therapy on the fibrosis of lungThe fibrosis of lung was detected by assessment the level of collagen type I(COL1)and transforming growth factor β1(TGF-β1).The results indicated that positive expression of COL1 and TGF-β1 was increased in the neonatal mice exposure to hyperoxia induced BPD group than in control,but reduced in MSCs,EPO,particularly MSCs+EPO group in comparison with BPD mice(P<0.05).Effect of combination therapy on cell proliferationDouble immunofluorescence staining for Ki-67 and PEC AM-1 was used to determine the proliferative activity of microvascular endothelium.The optical density(OD)value of representative sections was significantly increased in the MSCs+EPO group compared with the MSCs or EPO group alone(P<0.05).Effect of combination therapy on differentiation in vivoThe transplanted cells were tracked through GFP;MSCs were marked with green and the AECII-specific marker SP-C was stained red,and the results indicated a higher differentiation rate in the MSCs+EPO group than in the MSCs group(P<0.05).Effect of MSCs+EPO combination therapy on the expression of CXCR4,SDF-1,and VEGF in vivoEffect of MSCs+EPO combination therapy on the expression of CXCR4,SDF-1,and VEGF in vivo qRT-PCR and western blot analyses indicated that the mRNA and protein levels of CXCR4,SDF-1,and VEGF were significantly decreased in the lung tissue of three treatment groups when compared with the BPD group;moreover,a greater improvement was observed in the MSCs+EPO group compared with the MSCs or EPO group alone(P<0.05).Effect of EPO on MSC proliferation,migration,and apoptosis in vitroThe proliferation and migration of MSCs were significantly decreased in the high oxygen(HO)group compared with the room air(RA,control)group;however,proliferation and migration were significantly increased in the cells in the HO environment with complete medium containing 2 U/mL EPO.Analysis of apoptosis by flow cytometry showed that there was no difference between the RA and RA EPO groups(6.64%vs.6.87%,respectively),but apoptosis was increased(45.16%)when the cells wereexposed to an HO environment than to an RA environment(P<0.05);moreover,a clearly lower rate of apoptosis was observed when the complete medium contained 2 U/mL EPO(26.58%,P<0.05).Conclusion:This study indicated that combination treatment with MSCs and EPO could significantly repair HO-induced alveoli dysplasia damage and the fibrosis of lung.Moreover,the density of new vessels and the expression of VEGF,SDF-1,and its receptor CXCR4 were significantly higher in the MSCs+EPO group than in the MSCs or EPO group alone.Furthermore,our data confirmed that EPO improved the proliferation,migration,and antiapoptosis ability of MSCs in vitro and in vivo.