| Part1:Circulating Exosomal Myo-miRs Regulate Mobilization of Bone Marrow Stem/Progenitor Cells after Acute Myocardial Infarction by Targeting on CXCR4Objective:Myo-miRs (myocardial microRNAs) are rapidly elevated in the circulation of patients with acute myocardial infarction (AMI). However, the target organs and biological activities of these circulating myo-miRs are rarely studied. Circulatory exosomes are small membranous vesicles of endocytic origin, with a40-100nm diameter, which could mediate communication of mRNAs and microRNAs between cells. It is well-known that AMI could induce a mobilization of bone marrow stem/progenitor cells. Hence, we hypothesize that the AMI-induced myo-miRs are associated with circulating exosomes and mediate a functional crosstalk between the ischemic heart and bone marrow stem/progenitor cells.Methods and Results:We established AMI animal model in8-10weeks old male C57BL/6mice, and executed these mice6hours after ligation of left anterior descending coronary artery. Then, we analyzed the levels of four myo-miRs (miR-la, miR-208a, miR-133a and miR-499-5p) in plasma, plasma exosomes, non-exosomal plasma components, bone marrow mononuclear cells (BM-MNCs) and certain tissues. The levels of circulating exosomal and non-exosomal myo-miRs were both significantly up-regulated after AMI. Among these four myo-miRs, miR-1a, miR-208a and miR-499-5p were mainly elevated in the exosomes, while miR-133a mainly elevated in the non-exosomal plasma components. To test whether exosomes mediate transfer of myo-miRs into BM-MNCs, we isolated circulating exosomes from the AMI mice and fluorescently labeled them with PKH67. We injected PKH67stained exosomes into normal mice in vivo,12h later, we executed these mice and obtained femurs. We observed PKH67-positive cells in metaphysis under a fluorescence microscope, indicating the circulating exosomes were transferred into bone marrow tissue especially in metaphysis. We detected the expression of myo-miRs in certain peripheral tissues after AMI, and observed a significant higher expression of myo-miRs in bone marrow than in kidney, liver and spleen, indicating the circulating exosomes were mainly transferred into bone marrow. Then, we divided the plasma into exosomes and non-exosomal plasma components after AMI and severally co-cultured them with BM-MNCs, interestingly, we discovered that only exosomes but not non-exosomal plasma components could induce the up-regulation of myo-miRs in BM-MNCs, indicating the transfer of myo-miRs into BM-MNCs was only mediated by exosomes. SDF-1/CXCR4signal pathway plays an important role on mobilization of bone marrow stem/progenitor cells. We transfected specific myo-miRs mimics into BM-MNCs respectively, and found a significant down-regulation of CXCR4after administration of any myo-miRs. Then, we co-cultured AMI exosomes with BM-MNCs in vitro, and also observed a down-regulation of CXCR4. However, specific myo-miRs inhibitors could prevent this down-regulation of CXCR4mediated by AMI exosomes. To further confirm whether AMI related exosomes could mediate a mobilization of BM-MNCs, we injected AMI exosomes into normal mice, and found a significant up-regulation of c-kit+, c-kit+Lin-and Lin-mononuclear cells in blood, indicating AMI exosomes could induce a mobilization of BM-MNCs into circulation.Conclusions:In conclusion, circulatory myo-miRs could induce a mobilization of bone marrow stem/progenitor cells after AMI by down-regulating the expression of CXCR4through an exosome-transport mechanism, which is a new therapeutic target of stem cells therapy for cardiovascular diseases. Part2:Acute Myocardial Infarction Related Circulatory Exosomal MiR-1has Potential to Regulate Chemotaxis of Bone Marrow Mesenchymal Stem Cells by Targeting on CX3CR1Objective:After acute myocardial infarction (AMI), ischemic or infarcted myocardium secretes cardiac specific microRNAs into circulation. It has been reported that miR-1is markedly elevated after AMI, while the transfer and biological effect of circulating miR-1has not been studied. Circulatory exosomes are small membranous vesicles of endocytic origin, with a40-100nm diameter, which could mediate communication of mRNAs and microRNAs between cells. CX3CR1is a chemokine receptor which abundantly expressed in bone marrow mesenchymal stem cells (BM-MSCs). CX3CR1/Fractalkine signal pathway plays an important role on chemotaxis of BM-MSCs to ischemic tissues. In this article, we hypothesize that the AMI-induced miR-1is associated with exosomes and has potential to regulate the expression of CX3CR1in BM-MSCs.Methods and Results:We established AMI and sham animal model in male SD rats. We collected plasma from these rats, and divided plasma into exosomes and non-exosomal plasma components. The levels of miR-1in plasma, exosomes and non-exosomal plasma components were detected by qRT-PCR. We observed a significant up-regulation of plasma miR-1after AMI, which has been generally reported. In addition, the elevation folders of circulating miR-1were higher in exosomes than in non-exosomal plasma components after AMI, indicating AMI related circulating miR-1is mainly existed in exosomes. To test whether exosomes were transferred into BM-MSCs, we cultured rat BM-MSCs in vitro, isolated exosomes from rats and fluorescently labeled exosomes with PKH67, then co-cultured PKH67labeled exosomes with BM-MSCs. Interestingly, we confirmed that exosomes could be uptake by BM-MSCs under a fluorescence microscope. Additionally, we severally co-cultured AMI related exosomes and AMI related non-exosomal plasma components with BM-MSCs, results found that miR-1increased in exosomes co-cultured BM-MSCs, but not in non-exosomal plasma components co-cultured BM-MSCs, indicating exosomes mediate the transfer of miR-1into BM-MSCs. To gain insights into the functional significance of the miR-1transfer, we transduced a specific miR-1mimic into BM-MSCs, and analyzed the expression of CX3CR1by Western blotting and qRT-PCR. Notably, we found a significant down-regulation of CX3CR1in miR-1transfected BM-MSCs, consequently, a decreased chemotaxis of miR-1transfected BM-MSCs to fractalkine.Conclusions:In conclusion, circulating exosomal miR-1has potential to down-regulate the CX3CR1expression in BM-MSCs and suppress CX3CR1mediated chemotaxis, which maybe a new therapeutic target of BM-MSCs therapy for heart ischemic injury. |
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