Hypoxia-elicited mesenchymal stem cell-derived exosomes facilitates cardiac repair through miR-125b-mediated prevention of cell death in myocardial infarction.
ABSTRACT: Exosomes (Exo) secreted from hypoxia-conditioned bone marrow mesenchymal stem cells (BM-MSCs) were found to be protective for ischemic disease. However, the role of exosomal miRNA in the protective effect of hypoxia-conditioned BM-MSCs-derived Exo (Hypo-Exo) remains largely uncharacterized and the poor specificity of tissue targeting of Exo limits their clinical applications. Therefore, the objective of this study was to examine the effect of miRNA in Hypo-Exo on the repair of ischemic myocardium and its underlying mechanisms. We further developed modified Hypo-Exo with high specificity to the myocardium and evaluate its therapeutic effects. Methods: Murine BM-MSCs were subjected to hypoxia or normoxia culture and Exo were subsequently collected. Hypo-Exo or normoxia-conditioned BM-MSC-derived Exo (Nor-Exo) were administered to mice with permanent condition of myocardial infarction (MI). After 28 days, to evaluate the therapeutic effects of Hypo-Exo, infarction area and cardio output in Hypo-Exo and Nor-Exo treated MI mice were compared through Masson's trichrome staining and echocardiography respectively. We utilized the miRNA array to identify the significantly differentially expressed miRNAs between Nor-Exo and Hypo-Exo. One of the most enriched miRNA in Hypo-Exo was knockdown by applying antimiR in Hypoxia-conditioned BM-MSCs. Then we performed intramyocardial injection of candidate miRNA-knockdown-Hypo-Exo in a murine MI model, changes in the candidate miRNA's targets expression of cardiomyocytes and the cardiac function were characterized. We conjugated Hypo-Exo with an ischemic myocardium-targeted (IMT) peptide by bio-orthogonal chemistry, and tested its targeting specificity and therapeutic efficiency via systemic administration in the MI mice. Results: The miRNA array revealed significant enrichment of miR-125b-5p in Hypo-Exo compared with Nor-Exo. Administration of miR-125b knockdown Hypo-Exo significantly increased the infarction area and suppressed cardiomyocyte survival post-MI. Mechanistically, miR-125b knockdown Hypo-Exo lost the capability to suppress the expression of the proapoptotic genes p53 and BAK1 in cardiomyocytes. Intravenous administration of IMT-conjugated Hypo-Exo (IMT-Exo) showed specific targeting to the ischemic lesions in the injured heart and exerted a marked cardioprotective function post-MI. Conclusion: Our results illustrate a new mechanism by which Hypo-Exo-derived miR125b-5p facilitates ischemic cardiac repair by ameliorating cardiomyocyte apoptosis. Furthermore, our IMT- Exo may serve as a novel drug carrier that enhances the specificity of drug delivery for ischemic disease.
Project description:Macrophages are re-educated and polarized in response to myocardial infarction (MI). The M2 anti-inflammatory phenotype is a known dominator of late stage MI. Mesenchymal stem cells (MSCs) represent a promising tool for cell therapy, particularly heart related diseases. In general, MSCs induce alteration of the macrophage subtype from M1 to M2, both in vitro and in vivo. We conjectured that hypoxic conditions can promote secretome productivity of MSCs. Hypoxia induces TGF-?1 expression, and TGF-?1 mediates M2 macrophage polarization for anti-inflammation and angiogenesis in infarcted areas. We hypothesized that macrophages undergo advanced M2 polarization after exposure to MSCs in hypoxia. Treatment of MSCs derived hypoxic conditioned medium (hypo-CM) promoted M2 phenotype and neovascularization through the TGF-?1/Smad3 pathway. In addition, hypo-CM derived from MSCs improved restoration of ischemic heart, such as attenuating cell apoptosis and fibrosis, and ameliorating microvessel density. Based on our results, we propose a new therapeutic method for effective MI treatment using regulation of macrophage polarization. [BMB Reports 2020; 53(11): 600-604].
Project description:Exosomes (Exo) secreted from mesenchymal stem cells (hMSCs) are protective against myocardial injury. The purpose of the study was to investigate the role and mechanisms by which exosomes promote cardiomyocyte survival and function following myocardial infarction (MI). hMSCs were cultured under hypoxic and normoxic conditions. Hypoxia-conditioned hMSC-derived exosomes (Hypo-Exo) and normoxic-conditioned hMSC-derived exosomes (Nor-Exo) were collected and intramyocardially injected into rats with MI. The therapeutic effects of Hypo-Exo and Nor-Exo were evaluated after 4 weeks. Quantitative real-time PCR (qRT-PCR) was used to detect the expression of candidate long noncoding RNA urothelial carcinoma associated 1 (lncRNA-UCA1) in Nor-Exo and Hypo-Exo. Intramyocardial injection of lncRNA-UCA1-knockdown-Hypo-Exo in a rat model of MI was then performed and the cardiac function was characterized. The target and downstream of the molecular mechanism lncRNA-UCA1 was disclosed by luciferase reporter assays and western blot. Circulating exosomal lncRNA-UCA1 level in AMI patients and healthy volunteers was assessed. We found that (1) hMSC exosomal (from hypoxic and normoxic conditions) cardioprotection in vitro and in vivo correlated with the presence of encapsulated lncRNA-UCA1 in exosomes; (2) lncRNA-UCA1 targeted miR-873 via sponging, reducing the latter's suppressive effects on its target XIAP, and this translated into AMPK phosphorylation and increased level of the antiapoptotic protein BCL2; and (3) plasma derived from patients with AMI contained exosomes enriched with the lncRNA-UCA1, unlike that from normal subjects. This study demonstrates that Hypo-Exo lncRNA-UCA1 plays a cardioprotective role via the miR-873-5p/XIAP axis and circulating exosomal lncRNA-UCA1 may be a promising novel biomarker for the diagnosis of AMI.
Project description:Rationale: Mesenchymal stem cells (MSCs) play important roles in tissue repair and regeneration. However, the molecular mechanisms underlying MSCs activation remain largely unknown, thus hindering their clinical translation. Exosomes are small vesicles that act as intercellular messengers, and their potential for stem cell activation in pathological conditions has not been fully characterized yet. Here, we aim to investigate whether serum exosomes are involved in the remote activation of MSCs after myocardial infarction (MI). Methods: We established MI mouse model by ligating the left anterior descending branch of the coronary artery. Afterwards, serum exosomes were isolated from control (Con Exo) and MI mice (MI Exo) by differential centrifugation. Exosomes were characterized through transmission electron microscopy and nanoparticle tracking analysis. The cell proliferation rate was evaluated by CCK-8 and EdU incorporation assays. Exosomal miRNA and protein levels were assessed using qRT-PCR and western blotting, respectively. VEGF levels in the supernatant and serum were quantified by ELISA. Matrigel plug and tube formation assays were used to evaluate angiogenesis. To explore miR-1956 roles, overexpression and knock-down experiments were performed using mimic and inhibitor, respectively. Finally, miR-1956 target genes were confirmed using the luciferase reporter assay. Results: Both types of exosomes exhibited typical characteristics and could be internalized by adipose-derived MSCs (ADMSCs). MI Exo enhanced ADMSCs proliferation through the activation of ERK1/2. Gain- and loss-of-function studies allowed the validation of miR-1956 (enriched in MI Exo) as the functional messenger that stimulates ADMSCs-mediated angiogenesis and paracrine VEGF signaling, by downregulating Notch-1. Finally, we found that the ischemic myocardium and kidney may be the main sources that release serum exosomes after MI. Conclusions: Cardio-renal exosomes deliver miR-1956 and activate paracrine proangiogenic VEGF signaling in ADMSCs after MI; this process also involves Notch-1, which functions as the core mediator.
Project description:Bone marrow-mesenchymal stem cell (BM-MSC) therapy improves the recovery of cardiac function after myocardial infarction (MI); however, the underlying molecular mechanisms are not completely understood. Recent studies have shown that microRNAs (miRNAs) modulate the pathophysiology of cardiovascular diseases. Here, we investigated the mechanisms underlying the effects of BM-MSC-derived paracrine factors and cardiac miRNAs on myocardial regeneration after MI. In our study, MI was induced by permanent ligation of the left anterior descending (LAD) coronary artery. BM-MSCs transplanted in infarcted rats significantly downregulated the expression of miRNA-23a and miRNA-92a and inhibited apoptosis in the myocardium. An in vitro experiment showed that supernatant from BM-MSCs cultured under hypoxia contained higher levels of vascular endothelial growth factor (VEGF) than that from BM-MSCs under normoxia. In addition, inhibition of miRNA-23a and miRNA-92a reduced cardiac apoptosis. Moreover, the VEGF-containing BM-MSC supernatant inhibited miRNA-23a and miRNA-92a expression and reduced apoptotic signaling in cardiomyocytes under hypoxia. These effects were inhibited when the supernatant was treated with neutralizing antibodies against VEGF. Our results indicate that the paracrine factor, VEGF, derived from transplanted BM-MSCs, regulated the expression of miRNAs such as miRNA-23a and miRNA-92a and exerted anti-apoptotic effects in cardiomyocytes after MI.
Project description:The therapeutic potential of mesenchymal stem cells (MSCs) may be attributed partly to humoral factors such as growth factors, cytokines, and chemokines. Human term placental tissue-derived MSCs (PlaMSCs), or conditioned medium left over from cultures of these cells, have been reported to enhance angiogenesis. Recently, the exosome, which can transport a diverse suite of macromolecules, has gained attention as a novel intercellular communication tool. However, the potential role of the exosome in PlaMSC therapeutic action is not well understood. The purpose of this study was to evaluate PlaMSC-derived exosome angiogenesis promotion in vitro and in vivo.MSCs were isolated from human term placental tissue by enzymatic digestion. Conditioned medium was collected after 48-h incubation in serum-free medium (PlaMSC-CM). Angiogenic factors present in PlaMSC-CM were screened by a growth factor array. Exosomes were prepared by ultracentrifugation of PlaMSC-CM, and confirmed by transmission electron microscopy, dynamic light scattering, and western blot analyses. The proangiogenic activity of PlaMSC-derived exosomes (PlaMSC-exo) was assessed using an endothelial tube formation assay, a cell migration assay, and reverse transcription-PCR analysis. The in-vivo angiogenic activity of PlaMSC-exo was evaluated using a murine auricle ischemic injury model.PlaMSC-CM contained both angiogenic and angiostatic factors, which enhanced endothelial tube formation. PlaMSC-exo were incorporated into endothelial cells; these exosomes stimulated both endothelial tube formation and migration, and enhanced angiogenesis-related gene expression. Laser Doppler blood flow analysis showed that PlaMSC-exo infusion also enhanced angiogenesis in an in-vivo murine auricle ischemic injury model.PlaMSC-exo enhanced angiogenesis in vitro and in vivo, suggesting that exosomes play a role in the proangiogenic activity of PlaMSCs. PlaMSC-exo may be a novel therapeutic approach for treating ischemic diseases.
Project description:Background:Myocardial infarction (MI) is a common cause of mortality in people. Mesenchymal stem cell (MSC) has been shown to exert therapeutic potential to treat myocardial infarction (MI). However, in patients with diabetes, the diabetic environment affected MSCs activity and could impair the efficacy of treatment. Interleukin-10 (IL-10) has been shown to attenuate MI by suppressing inflammation. In current study, the combination of MSC transplantation with IL-10 was evaluated in a diabetic mice model with MI. Methods:We engineered bone marrow derived MSCs (BM-MSCs) to overexpress IL-10 by using CRISPR activation. We established the diabetic mice model with MI and monitored the IL-10 expression after BM-MSCs transplantation. We also evaluated the effects of BM-MSCs transplantation on inflammatory response, cell apoptosis, cardiac function and angiogenesis. Results:CRISPR activation system enabled overexpression of IL-10 in BM-MSCs. Transplantation of BM-MSCs overexpressing IL-10 resulted in IL-10 expression in heart after transplantation. Transplantation of BM-MSCs overexpressing IL-10 inhibited inflammatory cell infiltration and pro-inflammatory cytokines production, improved cardiac functional recovery, alleviated cardiac injury, decreased apoptosis of cardiac cells and increased angiogenesis. Conclusion:In summary, we have demonstrated the therapeutic potential of IL-10 overexpressed BM-MSCs in the treatment of MI in diabetic mice.
Project description:Bone marrow-derived mesenchymal stem cells (BM-MSCs) are important precursors of tumor stromal cells. Previously, we have demonstrated that miR-155-5p inhibition directly induced transition of BM-MSCs into gastric cancer-associated MSCs. Whether miR-155-5p is involved in the education of BM-MSCs by gastric cancer cells has not been established. Murine BM-MSCs (mMSCs) were isolated and grown in conditioned medium derived from gastric cancer cell line MFC (MFC-CM). The tumor-promoting phenotype and function of mMSCs were detected by immunofluorescence staining, quantitative reverse transcription-polymerase chain reaction (qRT-PCR), cell colony formation assay, transwell migration, and invasion assays. Luciferase reporter assays and western blot analyses were conducted to reveal the relationship between nuclear factor kappa-light-chain-enhancer of activated B cells (NF-?B) p65 and mmu-miR-155-5p. miRNA mimics, inhibitor, and the NF-?B inhibitor pyrrolidine dithiocarbamic acid (PDTC) were used to evaluate the role of miR-155-5p-NF-?B signaling in the education of mMSCs by MFC-CM. We successfully established the education model of mMSCs by MFC-CM and found that mmu-miR-155-5p expression levels were reduced in mMSCs. Mimicking this deregulation by transfecting miRNA inhibitor into mMSCs produced a similar effect as that of MFC-CM on mMSCs. NF-?B p65 was validated as a target of mmu-miR-155-5p, which also negatively regulated NF-?B activation. Inhibition of NF-?B activation by PDTC abolished the effect of the miRNA inhibitor on mMSCs. mmu-miR-155-5p overexpression partially blocked the effect of MFC-CM in educating mMSCs, while PDTC treatment completely eliminated MFC-CM activity. These results indicate that miR-155-5p is not the sole miRNA mediating the education of BM-MSCs by gastric cancer cells, but downstream NF-?B signaling is indispensable for this process.
Project description:BACKGROUND:The beneficial functions of bone marrow mesenchymal stem cells (BM-MSCs) decline with decreased cell survival, limiting their therapeutic efficacy for myocardial infarction (MI). Irisin, a novel myokine which is cleaved from its precursor fibronectin type III domain-containing protein 5 (FNDC5), is believed to be involved in a cardioprotective effect, but little was known on injured BM-MSCs and MI repair yet. Here, we investigated whether FNDC5 or irisin could improve the low viability of transplanted BM-MSCs and increase their therapeutic efficacy after MI. METHODS:BM-MSCs, isolated from dual-reporter firefly luciferase and enhanced green fluorescent protein positive (Fluc+-eGFP+) transgenic mice, were exposed to normoxic condition and hypoxic stress for 12?h, 24?h, and 48?h, respectively. In addition, BM-MSCs were treated with irisin (20?nmol/L) and overexpression of FNDC5 (FNDC5-OV) in serum deprivation (H/SD) injury. Furthermore, BM-MSCs were engrafted into infarcted hearts with or without FNDC5-OV. RESULTS:Hypoxic stress contributed to increased apoptosis, decreased cell viability, and paracrine effects of BM-MSCs while irisin or FNDC5-OV alleviated these injuries. Longitudinal in vivo bioluminescence imaging and immunofluorescence results illustrated that BM-MSCs with overexpression of FNDC5 treatment (FNDC5-MSCs) improved the survival of transplanted BM-MSCs, which ameliorated the increased apoptosis and decreased angiogenesis of BM-MSCs in vivo. Interestingly, FNDC5-OV elevated the secretion of exosomes in BM-MSCs. Furthermore, FNDC5-MSC therapy significantly reduced fibrosis and alleviated injured heart function. CONCLUSIONS:The present study indicated that irisin or FNDC5 improved BM-MSC engraftment and paracrine effects in infarcted hearts, which might provide a potential therapeutic target for MI.
Project description:The clinical use of human bone marrow-derived mesenchymal stem cells (BM-MSCs) has been hampered by their poor performance after transplantation into failing hearts. Here, to improve the therapeutic potential of BM-MSCs, we developed a strategy termed in vivo priming in which BM-MSCs are primed in vivo in myocardial infarction (MI)-induced hearts through genetically engineered hepatocyte growth factor-expressing MSCs (HGF-eMSCs) that are encapsulated within an epicardially implanted 3D cardiac patch. Primed BM-MSCs through HGF-eMSCs exhibited improved vasculogenic potential and cell viability, which ultimately enhanced vascular regeneration and restored cardiac function to the MI hearts. Histological analyses further demonstrated that the primed BM-MSCs survived longer within a cardiac patch and conferred cardioprotection evidenced by substantially higher numbers of viable cardiomyocytes in the MI hearts. These results provide compelling evidence that this in vivo priming strategy can be an effective means to enhance the cardiac repair of MI hearts.
Project description:Mesenchymal stem cells (MSCs) repair infarcted heart through paracrine mechanism. We sought to compare the effectiveness of MSCs and MSC-derived exosomes (MSC-Exo) in repairing infarcted hearts and to identify how MSC-Exo mediated cardiac repair is regulated. In a rat myocardial infarction model, we found that MSC-Exo inhibited cardiac fibrosis, inflammation, and improved cardiac function. The beneficial effects of MSC-Exo were significantly superior compared to that of MSCs. To explore the potential mechanisms underlying MSC-Exo's effects, we performed several in vitro experiments and miRNA-sequence analysis. MSC-Exo stimulated cardiomyocyte H9C2 cell proliferation, inhibited apoptosis induced by H<sub>2</sub>O<sub>2</sub>, and inhibited TGF-<i>?</i> induced transformation of fibroblast cell into myofibroblast. Importantly, novel miRNA sequencing results indicated that MSC-Exo and MSCs have similar miRNA expression profile, which could be one of the reasons that MSC-Exo can replace MSCs for cardiac repair. In addition, the expression of several miRNAs from MSC-Exo was significantly different from that of MSCs, which may explain why MSC-Exo has better therapeutic effect than MSCs. In conclusion, this study demonstrates that MSC-Exo could be used alone to promote cardiac repair and are superior to MSCs in repairing injured myocardium.