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This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Recently, mesenchymal stem cells (MSC) have been proved to be beneficial in acute respiratory distress syndrome (ARDS). Vascular endothelial growth factor (VEGF) is an important angiogenesis factor that MSC release. However, the precise role of VEGF- expressing character of MSC in the MSC treatment for ARDS remains obscure. Here, we firstly knocked down the gene VEGF in MSC (MSC- Sh. VEGF) with lentiviral transduction. Then we injected the MSC- Sh. VEGF to rats with lipopolysaccharide- induced acute lung injury (ALI) via the tail vein. Data showed that MSC transplantation significantly increased VEGF levels in the lung, reduced lung permeability, protected lung endothelium from apoptosis, facilitated VE- cadherin recovery, controlled inflammation, and attenuated lung injury. However, VEGF gene knockdown in MSC led to relatively insufficient VEGF expression in the injured lung and significantly diminished the therapeutic effects of MSC on ALI, suggesting an important role of VEGF- expressing behavior of MSC in the maintenance of VEGF in the lung and the MSC treatment for ALI. Hence, we conclude that MSC restores the lung permeability and attenuates lung injury in rats with ALI in part by maintaining a . Introduction. Acute respiratory distress syndrome (ARDS) is among the leading causes of mortality in intensive care units (ICUs). Although there is some progress in the mechanism and treatment of ARDS, including lung- protective ventilation . To date, pharmacologic therapies have not been able to improve clinical outcomes. Therefore, there is an urgent need for development of novel therapeutic strategies. ARDS is characterized by the injury to alveolar- capillary membrane which results in the increased vascular permeability with exudation of proteinaceous fluid and migration of inflammatory cells from the lung vascular compartment into the interstitial and alveolar space . Lung endothelium has been considered to provide the first and key barrier to protein and fluid flow into lung interstitial and alveolar space. Hence, the maintenance of the integrity of lung endothelium is critically important for the treatment of ARDS. Mesenchymal stem cells (MSC) are a kind of cell type which has a multipotent differentiation potential. In addition, MSC also display a strong paracrine capacity . MSC is able to secrete multiple soluble factors, like immunomodulation factors . However, the precise role of VEGF- releasing character of MSC in ARDS remains unknown. Hence, in this study, we aimed to display the role of VEGF- expressing character of MSC in lipopolysaccharide- (LPS- ) induced lung injury in rats. Materials and Methods. Ethics Statement. Male wild- type SD rats (Laboratory Animal Center, Shanghai, China) were maintained under specific pathogen- free condition (Animal Center, Nanjing, China). All experiments referring to the use of animals were approved by the Committee of Animal Care and Use of Southeast University. Rat Mesenchymal Stem Cells and Cell Culture. Rat MSC (Cyagen Bioscience, Inc., Guangzhou, China) was derived from normal rat bone marrow. Identification of rat MSC, including the cell surface phenotypes and multipotency, was performed by the supplier. Rat MSC expressed CD9. CD4. 4, and CD2. 9, but not CD3. CD4. 5, and CD1. 1b/c. This characterization was performed by fluorescence- activated cell sorting (FACS) analysis. The abilities of MSC to differentiate into the adipogenic, osteogenic, and chondrogenic lineages were determined by staining with oil red- O, alizarin red, or alcian blue, respectively, after culturing in adipogenic, osteogenic, or chondrogenic differentiation media supplied by Cyagen Bioscience, Inc., for 2- 3 weeks. The culture medium and subculture kits for rat MSC were purchased from Cyagen Bioscience, Inc. Briefly, the MSC culture medium was made of SD rat MSC basal medium supplemented with 1. SD rat MSC- qualified fetal bovine serum, 1% penicillin- streptomycin, and 1% glutamine. MSC used for in vitro studies were at the 1. MSC of passage number 9 were used in the rat experiments. Lentiviral Vectors Mediated VEGF Gene Knockdown in MSC and GFP Reporter Gene Detection. MSC of passage number 6 were used for VEGF gene knockdown experiment. Lentivirus carrying either GFP (LV3- GFP) or both GFP and Sh. RNA VEGF (LV3- Sh. RNA VEGF) was offered by Gene. Pharma (Shanghai, China). MSC were cultured in a 6- well plate (Corning, Inc., NY). After 2. 4 hours, we changed the culture medium, added LV3- GFP or LV3- Sh. RNA VEGF, and maintained the cells in a humidified incubator with 5% CO2 at 3. After 2. 4 hours, the old medium was aspirated and fresh culture medium was added. Then the cultures were performed by medium change every 2- 3 days. The transduction efficiency of the lentiviral vectors in passage 1. MSC was identified using fluorescence microscopy (Olympus Co., Tokyo, Japan). RNA Isolation and Quantitative Real- Time Polymerase Chain Reaction (q. RT- PCR)MSC treated by LV3- GFP (MSC- GFP) or LV3- Sh. RNA VEGF (MSC- Sh. VEGF) were obtained and cultured in cell culture medium, respectively. Total RNA was isolated from MSC, MSC- GFP, or MSC- Sh. VEGF at the 1. 0th passage, using TRIzol reagent (Takara Bio, Inc., Kyoto, Japan) according to the manufacturer. RNA was reverse transcribed to single- stranded c. DNA using c. DNA synthesis reagents (Gene. Pharma, Shanghai, China). Primers used for the RT- PCR were rat VEGF and GAPDH and were supplied by Gene. Pharma (Shanghai, China). The forward and reverse primer sequences for GADPH were 5. The forward and reverse primer sequences for VEGF were 5. The RT- PCR assays were performed following the instruction for One- Step RT- PCR described by FUNGLYN BIOTECH INC. Western Blotting Analysis. MSC, MSC- GFP, or MSC- Sh. VEGF at the 1. 0th passage were collected after transduction with lentivirus. Total cellular proteins from MSC, MSC- GFP, or MSC- Sh. VEGF were extracted and separated with the use of SDS- PAGE gels (8%) made by the same way as we previously described . Then, proteins were incubated with primary antibodies to VEGF (1. The blots were washed three times with tris- 1 buffer (Biosharp Biotechnology, Hefei, China) at p. H 7. 4 containing 0. Tween 2. 0 (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) (TBST) and then incubated with goat anti- rabbit Ig. G conjugated with horseradish peroxidase (Zhongshan Golden Bridge Biotechnology Co., Ltd., China). Immunoreactive complexes were visualized using chemiluminescence reagents (Thermo Scientific). Evaluation of VEGF Levels in the Cell Culture Medium by ELISAMSC, MSC- GFP, and MSC- Sh. VEGF were seeded in a 1. After 1. 2 hours, the old culture medium was replaced by fresh culture medium. MSC were maintained in an incubator at 3. Then the culture media of MSC, MSC- GFP, and MSC- Sh. VEGF were collected and centrifuged at 3. The supernatants were collected and stored in . The levels of VEGF protein in the medium were measured using ELISA kits (Ex. Cell. Bio, Shanghai, China) according to the manufacturer. When the cells achieved approximately 1. The cells were cultured in low- serum (1%) MDEM/1. The images of the wound healing were recorded by a light microscope at 0. The horizontal migration abilities of MSC were quantified by measuring the wound widths of five different wound surfaces in each group. The experiment was repeated three times. Transwell Migration Assay. The vertical migration abilities of MSC were assessed by Transwell migration assay. MSC were seeded on Transwell inserts (6. The cells were allowed to migrate at 3. The cells remaining on the upper surface of the insert were removed with cotton swabs. Then, the cells that migrated to the lower surface were fixed by 1% paraformaldehyde for 1. The stained cells from five randomly chosen fields were counted under a light microscope. LPS- Induced ALI in Rats. Six- to- eight- week- old wild- type SD rats (weighing 2. Louis, MO, USA) dissolved in 1. Rats (without LPS challenge) that received 1. Rats were sacrificed at 1 hour, 6 hours, and 2. PBS or cells (MSC, MSC- GFP, and MSC- Sh. VEGF) infusion. Lung lobes were obtained for further analysis. Assessment of Lung Edema. As we described previously . The lung- wet- weight- to- body- weight ratio (LWW/BW) was measured to determine the lung vascular permeability and lung edema. Evans Blue Dye Leakage Assay. To evaluate the protein permeability of pulmonary microvascular endothelium, Evans blue dye (Sigma- Aldrich, St. Louis, MO, USA) was used. For the control, ALI, MSC, MSC- GFP, and MSC- Sh. VEGF groups, Evans blue dye (2. Louis, MO, USA) for 2. Then, the concentration of Evans blue dye in lung tissue was measured by a spectrophotometer at 6. Immunofluorescence. To observe any changes of VE- cadherin expression in the lung, rat lungs were collected gently and then fixed in 4% paraformaldehyde at 4. OCT, Sakura Finetek USA, Inc., Torrance, CA, USA) and then frozen in . VE- cadherin was stained with anti- VE- cadherin primary antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and followed by a second antibody Fluorescein- (FITC- ) Affini. Pure Donkey Anti- Rabbit Ig. G (H + L) (Jackson Immuno. Research Inc., PA, USA). The nuclei were stained with 4,6- diamidino- 2- phenylindole (DAPI, Sigma- Aldrich). Fluorescence was monitored by an Olympus IX7. Olympus Co., Tokyo, Japan).
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