| Cardiovascular disease is one of the leading pathological causes of mortality worldwide. Cardiovascular ischemic diseases such as heart failure (HF), acute myocardial infarction (AMI) and myocardial ischemia/reperfusion (MI/R) injury produce plenty reactive oxygen species (ROS) in ischemic zone, which is a major contributor to cardiomyocytes apoptosis and death, and accelerate the evolvement of cardiac disease. So it’s urgent to find an effective way to restore the cardiovascular system under oxidative stress.Stem cell transplantation is an effective way to replace the apoptotic or dead cardiomyocytes, but the mechanism of this repair process has not been fully explained. Cardiac progenitor cells (CPCs) resident in adult heart have emerged as one of the most promising stem cell types for cardiac regeneration and repair. The mechanism of post transplantation has always been predicated on the hypothesis that these cells would engraft, differentiate and replace damaged cardiac tissues. Although both direct cell differentiation and indirect paracrine effect mechanisms have been implicated in the therapeutic benefit, accumulating evidence suggests predominant roles of the paracrine secretion by CPCs. Furthermore, many researchers indicate that transplanted CPCs secrete a lot of factors to reduce tissue injury and/or enhance tissue repair.Over the last few years, several experimental evidences have demonstrated the CPCs released a specialized membranous nano-sized vesicles termed exosomes improved cardiac function in the damaged heart. Exosomes are small (30-100 nm) membrane vesicles, merging their membrane contents into the recipient cell plasma membrane and delivering effectors including transcription factors, oncogenes, small and large non-coding regulatory RNAs (such as miRNAs), mRNAs and infectious particles into recipient cells. In this way, exosomes secreted by CPCs were considered to participate in cardiac protection and repair. But exosomes contents vary from different pathological condition, the difference might cause completely revered fate of target cells. Hence, it’s necessary to investigate exosomes’function under a specific pathological condition, especially oxidative stress, it’s meaningful for prediction and treatment of myocardium injury.Among the contents of exosomes, miRNAs have been shown to control important processes that contribute to the pathophysiological consequences of acute myocardial infarction. It is a class of short (about 22 nucleotides), single-stranded non-coding RNAs that play key roles in the regulation of gene expression. MiRNAs can either promote or inhibit cardiomyocyte cell apoptosis, and also regulate ROS-mediated heart disease. But whether miRNAs from CPC-derived exosomes have some important role in ROS-induced cardiomyocytes still stayed unclear. Here we investigated whether exosomes secreted by CPCs have myocardial protection under ROS stress, and what role of the miRNAs contained in exosomes played in this process.Methods:1. CPCs were isolated from mouse heart using Magnetic Cell Sorting(MACS) system. Flow Cytometry(FC) determined the purity of stem cell surface antigen-1 positiveCSca-1+) CPCs, and the expression of Sca-1 protein in cell was measured by immunofluorescence; Exosome were purified from 48h CPC cultured-conditional medium by using ExoQuick reagent, Nanoparticle Trafficking Analyzed(NTA) the diameters and concentration of exosomes, then ultrastructure of exosomes was determined by transmission electron microscope, and confirmed by Western blot using CD63, CD9, Alix as biomarkers;2. using different concentration of H2O2 induced the cardiomyocytes injury to mimic the oxidative stress in some cardiovascular disease, find the best concentration in our experiment; 12 miRNAs were chosen according to the previous literatures, which is related to our research, the potential miRNAs contained in exosomes were detected by RT-qPCR, and compared the fold change of detectable* miRNAs between normal exosomes and H2O2-induced exosomes, the significant changed miRNAs named as miR-X.3. RT-qPCR detected the expression levels of miR-X in normal/oxidative stress induced H9C2. Target genes of miR-X were predicted by using TargetScan, Western Blotting measured expression levels of the target protein after transfection of miR-X mimics, inhibitors and negative control, to confirm the interaction of miR-X and target protein in H2O2 injured H9C2. Luciferase activity assay determined the direct binding effect of miR-X and 3’UTR of target protein mRNA.4. RT-qPCR detected the expression of miR-X and Western blotting detected the target protein and caspase-3 in CPC co-culture, normal exosomes and H2O2-induced exosomes pre-treated oxidative stress injured H9C2 cells, the apoptotic rate of H9C2 cells after Annexin V/PI dual staining was estimated with Flow cytometry.Results:1. The Sca-1+cells isolated from adult mouse heart presented as long spindle-shaped fibrocyte-like adherent cells. The percentage of Sca-1+CPCs was determined with Flow Cytometry, and the results showed that up to 95.04±4.29 of population were Sca-1+ cells after MACS sorting. To obtain the CPC-derived exosomes particles, the culture medium of CPCs was collected and precipitated. NTA results demonstrated that the concentration of the particles was 1.31 x 109 ± 0.29 x 109 particles/ml, transmission electron microscope(TEM) revealed particles were round-shaped vesicles with double-layer membrane structure and diameters about 100 nm (Figure Id). Finally, the protein levels of exosomes markers CD63, CD9 and Alix were detectable with western blotting. After incubating the labeled exosome with cardiomyocytes for 12 hours, the exosomes pellet show strong red fluorescence in the cytoplasm of H9C2, CPC-exosomes can be taken up by cardiomyocytes.2. After treated with H2O2 on indicated concentrations for 6 hours, the H9C2 cells were harvested for protein collection and western blotting. The results showed that 100μM H2O2 increased the level of cleaved caspase-3 (the active type of caspase-3, NTA results showed that the exosomes concentrations increased from 1.31±0.29 ×109 particles/ml to 3.36±0.66×109 particles/ml from CPCs were treated with 100μM H2O2 for 6 hours.13 miRNAs in CPC-derived exosomes was estimated through quantitative PCR. There were six miRNAs (miR-21,24,214,132,195,210) detected in both h2o2-induced exosomes and non-induced ones. Among these miRNAs, miR-21 in CPC-derived exosomes was significantly up-regulated (>5-fold change) after the H2O2 induction. The level of miR-21 was examined in H2O2-treated H9C2 cells, and the results showed that miR-21 were significantly down-regulated in H9C2 cells under H2O2 treatment, the gain-of-and loss-of function experiments were performed using the mimic/inhibitor of miR-21. MiR-21 mimic or inhibitor obviously increase or decrease miR-21 expression in H9C2 specifically. The levels of caspase-3/cleaved caspase-3 were detected by western blotting on the H2O2-induced apoptosis of cardiomyocytes. miR-21 mimic obviously decreased cleaved caspase-3 expression, while inhibitor increased the expression in H9C2 under oxidative stress(100uM H2O2).3. There are conserved binding sites in 3’UTR of PDCD4 mRNA in different species. Western blotting results showed that PDCD4 were significantly up-regulated in H9C2 cells under H2O2 treatment, consistent with the decrease of miR-21 under the same treatment. The gain-of-and loss-of function experiments showed that miR-21 inhibitor increased, whereas miR-21 mimic decreased PDCD4 mRNA and protein expression in cardiomyocytes. Furthermore miR-21 able to directly bind to PDCD4 and inhibit PDCD4 expression, miR-21 mimic, but not inhibitor or negative control, inhibited wild type, not mutant type construct of 3’-UTR of PDCD4 luciferase activity.4. When the cells were pre-treated with CPC-derived exosomes, the decreased level of miR-21 was rescued under oxidative stress and the increasing of PDCD4 and cleaved caspase-3 were accordingly suppressed. Consistent with the higher yields of exosomes and exosomal miR-21 under oxidative stress, the exosomes derived from H2O2-treated CPCs showed even stronger effects on increasing miR-21 levels and decreasing PDCD4 expression in the receptor cells. An Annexin V/PI analysis results showed that, the H2O2-pre-treated exosomes decreased the percentage of the apoptotic cells to 13.58%, compared to the 33.29% in H2O2 control group, while the normal exosomes (non-H2O2 induced) could only reduce the apoptotic percentage to 17.39%.Conclusion:1. The number of CPCs in adult mouse is limited, the immune magnetic bead sorting method could isolate the high purity of specific cell types. CPCs, like most types of cells, can secrete exosomes, and the production and contents of secreted exosomes are differed from source cells types to microenvironment.2. CPCs have potential to secrete more exosomes under the stimulus of oxidative stress, the expression levels of miR-21 is significantly up-regulated in H2O2-exosomes,contained in exosomes. Oxidative stress can cause myocardial cell apoptosis, and reduce the levels of miR-21, suggesting that miR-21 is possibly related to the apoptosis of myocardial cells caused by oxidative stress.3. PDCD4 as target genes of miR-21, involved in the apoptosis process regulated by miR-21. The miR-21 and PDCD4 negative regulation exist in the normal cardiomyocytes, in the event of the stimulation of oxidative stress, miR-21 decreases and consistent with the increase of PDCD4, thus cells prone to apoptosis.4. Exosomes pre-protection can obviously relieve myocardial cell apoptosis caused by oxidative stress, and the exosome contains a large number of miR-21, which could transfer to the myocardial cell to restore the miR-21/PDCD4 pathway, reduce the expression of PDCD4 in cardiomyocytes, thereby alleviating cardiomyocytes apoptosis.Therefore, this research mainly illuminates protective effect of the CPC-derived exosomes’for myocardial cells in ischemic myocardial injury model, the first time put forward the systemic mechanisms of stem cell transplantation, which is mainly through passing on exosomal miR-21 inhibit PDCD4 in myocardial cells. The fruitful work provides a potential cell therapy strategy for myocardial ischemic diseases. |
