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Background: The efficacy of bone marrow mesenchymal stromal cells (BM-MSC) and its extracellular vesicles has been demonstrated for a broad spectrum of indications, including kidney diseases. However, BM-MSC donor characteristics and their potential are not usually considered. Therefore, the present work aims to evaluate the nephroprotective capacity of sEV secreted by BM-MSC from trained rats inunilateral ureteral obstruction (UUO) model. Methods: BM-MSC was characterized by their differentiation potential and immunophenotypic markers. The sEV were isolated by ultracentrifugation and characterized by nanoparticle tracking analysis and western blot. Its miRNA cargo was examined by quantitative PCR analysis for miR-26a, 126a, and 296. Wistar rats were submitted to UUO procedure and concomitantly treated with sEV secreted by BM-MSC from the untrained andtrained rats. The kidney tissue from all groups was evaluated for fibrosis mediators (transforming growth factor beta1 and collagen), CD34-angiogenesis marker, and hypoxia-inducible factor 1 alpha (HIF-1α). Results: Treadmill training stimulated in BM-MSC the production of sEV loaded with pro-angiogenic miR-296. The treatment with this sEVin UUO-rats was able to attenuate collagen accumulation and increase CD34 and HIF-1α in the kidney tissue when compared to untrained ones. Tubular proximal cells under hypoxia and exposed to BM-MSC sEV demonstrate accumulation in HIF-1α and NFR-2 (nuclear factor erythroid 2-related factor 2), possibly to mediate the response to hypoxia and oxidative stress, under these conditions. Conclusion: The BM-MSC sEV from trained animals presented an increased nephroprotective potential compared to untrained vesicles by carrying 296-angiomiR and contributing to angiogenesis in UUO model.(AU)
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Obstrucción Ureteral , Vesículas Extracelulares , Enfermedades Renales , Hipoxia , Estrés OxidativoRESUMEN
Abstract Background: The efficacy of bone marrow mesenchymal stromal cells (BM-MSC) and its extracellular vesicles has been demonstrated for a broad spectrum of indications, including kidney diseases. However, BM-MSC donor characteristics and their potential are not usually considered. Therefore, the present work aims to evaluate the nephroprotective capacity of sEV secreted by BM-MSC from trained rats inunilateral ureteral obstruction (UUO) model. Methods: BM-MSC was characterized by their differentiation potential and immunophenotypic markers. The sEV were isolated by ultracentrifugation and characterized by nanoparticle tracking analysis and western blot. Its miRNA cargo was examined by quantitative PCR analysis for miR-26a, 126a, and 296. Wistar rats were submitted to UUO procedure and concomitantly treated with sEV secreted by BM-MSC from the untrained andtrained rats. The kidney tissue from all groups was evaluated for fibrosis mediators (transforming growth factor beta1 and collagen), CD34-angiogenesis marker, and hypoxia-inducible factor 1 alpha (HIF-1). Results: Treadmill training stimulated in BM-MSC the production of sEV loaded with pro-angiogenic miR-296. The treatment with this sEVin UUO-rats was able to attenuate collagen accumulation and increase CD34 and HIF-1 in the kidney tissue when compared to untrained ones. Tubular proximal cells under hypoxia and exposed to BM-MSC sEV demonstrate accumulation in HIF-1 and NFR-2 (nuclear factor erythroid 2-related factor 2), possibly to mediate the response to hypoxia and oxidative stress, under these conditions. Conclusion: The BM-MSC sEV from trained animals presented an increased nephroprotective potential compared to untrained vesicles by carrying 296-angiomiR and contributing to angiogenesis in UUO model.
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Objective:To explore the therapeutic effects of intravenous injection of small extracellular vesicles (sEVs) derived from human umbilical cord mesenchymal stem cells (MSCs) on experimental autoimmune uveitis (EAU) in mice.Methods:MSCs from human umbilical cord were cultured and the supernatant was collected.The sEVs were isolated by ultracentrifugation method and a NanoSight instrument was used to analyze the particle size.The expression of surface markers sEVs, CD9, CD81 and CD63 was determined via Western blot.The morphology of sEVs was observed with a transmission electron microscope.Forty-eight 7-week-old female C57BL/6 mice were seclected to establish the EAU model through immunization with interphotoreceptor retinoid-binding protein peptide 651-670 (IRBP 651-670). The mice were divided into sEVs treatment group and phosphate buffer solution (PBS) control group using a random number table, with 24 mice in each group.The mice in the sEVs treatment group were injected with 50 μg of MSCs-derived sEVs via tail vein on the 11th day after modeling.In the PBS control group, the mice were injected with the same volume of PBS.Six mice in each group were randomly selected to observe the inflammation of the retina after mydriasis with an ophthalmoscope every other day from 8th day following modeling and the inflammation scores were evaluated.Six mice were randomly selected and sacrificed on the 14th day and 6 on the 18th day following modeling in each group, and both eyeballs of the mice were enucleated.Retinal tissue sections of the 6 mice sacrificed on the 18th day were stained with hematoxylin-eosin and the pathological scores were evaluated.The infiltration of helper T 1 (Th1) cells and Th17 cells in the eyeballs of the 6 mice sacrificed on the 18th day following modeling was detected by flow cytometry.T cells were isolated from spleen and lymph nodes of the 6 mice sacrificed on the 14th day, and the proliferation of T cells under different concentrations of IRBP 651-670 (0, 1, 10 and 20 μg/ml) was detected using a 5-bromodeoxyuridine (BrdU) method.To further study the effects of MSCs-derived sEVs on Th1/Th17 cells differentiation, naive T cells of spleen from another 3 normal mice were isolated by magnetic bead negative sorting and incubated with 10 μg/ml MSCs-derived sEVs or 10 μg/ml PBS, and then were cultured under Th1/Th17 cell differentiation conditions, respectively.Flow cytometry was used to measure the differentiation of naive T cells into Th1/Th17 cells.This study protocol complied with the regulations of the care and use of laboratory animals in China and was approved by an Ethics Committee of Tianjin Medical University (No.TJYY2019103022). Results:The isolated human MSCs-derived sEVs was with an average diameter of (102.4±33.6) nm and showed a double-layer membrane vesicle structure under the transmission electron microscope.The CD9, CD63 and CD81 proteins were highly expressed in sEVs.The inflammation scores of the sEVs treatment group were significantly lower than those in the control group on day 14, 16, 18, 20 and 22 after modeling (all at P<0.05). The pathological score of mice in the sEVs treatment group was significantly lower than that of PBS control group on the 18th day following modeling ( P<0.05). The flow cytometry results showed that on day 18 after modeling, the proportions of Th1 and Th17 cells in eyeballs in the sEVs treatment group were (15.55±2.03)% and (15.67±2.15)%, respectively, which were significantly lower than (21.35±0.72)% and (20.90±1.10)% in the PBS control group ( t=6.58, 5.31; both at P<0.01). BrdU results showed that when the IRBP 651-670 concentration was 20 μg/ml, the T cell proliferation ability in the sEVs treatment group was inhibited obviously compared with the control group ( P<0.05). The proportions of naive T cells differentiated into Th1 cells and Th17 cells in the sEVs treatment group were (28.15±1.32)% and (11.60±2.23)% respectively, which were significantly lower than (31.58±1.75)% and (23.52±1.76)% of the PBS control group, and the differences were statistically significant ( t=3.93, 10.26; both at P<0.05). Conclusions:Intravenous injection of human umbilical cord MSCs-derived sEVs can reduce the inflammation in EAU mice.The mechanism may be related to inhibiting the differentiation of naive T cells to Th1 and Th17 cells, and reducing the infiltration of Th1 and Th17 cells in the eyeballs.
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@#BACKGROUND: Small extracellular vesicles (sEVs) from bone marrow mesenchymal stem cells (BMSCs) have shown therapeutic potential for cerebral ischemic diseases. However, the mechanisms by which BMSC-derived sEVs (BMSC-sEVs) protect neurons against cerebral ischemia/reperfusion (I/R) injury remain unclear. In this study, we explored the neuroprotective effects of BMSC-sEVs in the primary culture of rat cortical neurons exposed to oxygen-glucose deprivation and reperfusion (OGD/R) injury. METHODS: The primary cortical neuron OGD/R model was established to simulate the process of cerebral I/R in vitro. Based on this model, we examined whether the mechanism through which BMSC-sEVs could rescue OGD/R-induced neuronal injury. RESULTS: BMSC-sEVs (20 μg/mL, 40 μg/mL) significantly decreased the reactive oxygen species (ROS) productions, and increased the activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx). Additionally, BMSC-sEVs prevented OGD/R-induced neuronal apoptosis in vivo, as indicated by increased cell viability, reduced lactate dehydrogenase (LDH) leakage, decreased terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) staining-positive cells, down-regulated cleaved caspase-3, and up-regulated Bcl-2/Bax ratio. Furthermore, Western blot and flow cytometry analysis indicated that BMSC-sEV treatment decreased the expression of phosphorylated calcium/calmodulin-dependent kinase II (p-CaMK II)/CaMK II, suppressed the increase of intracellular calcium concentration ([Ca2+]i) caused by OGD/R in neurons. CONCLUSIONS: These results demonstrate that BMSC-sEVs have significant neuroprotective effects against OGD/R-induced cell injury by suppressing oxidative stress and apoptosis, and Ca2+/CaMK II signaling pathways may be involved in this process.