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Intravenous mesenchymal stem cell-derived exosomes ameliorate myocardial inflammation in the dilated cardiomyopathy
Biochemical and Biophysical Research Communications xxx (2018) 1e8
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Intravenous mesenchymal stem cell-derived exosomes ameliorate myocardial inflammation in the dilated cardiomyopathy Xuan Sun, Anqi Shan, Zilun Wei, Biao Xu* Department of Cardiology, Drum Tower Hospital, Nanjing University Medical School, Nanjing, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 July 2018 Accepted 1 August 2018 Available online xxx
Mesenchymal stem cells (MSCs) have been shown to be efficacy to attenuating cardiovascular inflammation; however, there are many limitations to stem cell treatment. Present study was to prove MSCderived exosomes (MSC-Exos) could alleviating inflammatory cardiomyopathy by improving the inflammatory microenvironment of myocardium, especially by regulating the activity of macrophages. Mice were intraperitoneal injected of doxorubicin (DOX) to establish a dilated cardiomyopathy (DCM) model, and then received intravenous injection of either MSC-Exos or PBS as control. Mice receiving MSC-Exos showed improved cardiac function via echocardiography and attenuated cardiac dilation via HE staining, as well as reduced cardiomyocytes apoptosis. Expression levels of inflammatory factors were reduced. And there was a significant decrease of the inflammatory cells infiltration in the MSC-Exos treatment group comparing to the PBS group. Meanwhile, MSC-Exos could remarkably attenuate the pro-inflammatory macrophages amount in both blood and heart, which was proved that MSC-Exos relied on the JAK2-STAT6 pathway mediating macrophages activation. MSC-Exos improved the inflammatory microenvironment of dilated cardiomyopathy by regulating the polarization of the macrophage, which may hold promise for dilated cardiomyopathy clinical therapy. © 2018 Elsevier Inc. All rights reserved.
Keywords: Mesenchymal stem cell exosome Dilated cardiomyopathy Myocardial inflammation Macrophages
1. Introduction Dilated cardiomyopathy (DCM) is the most common complication heart disease of myocardial inflammation. The prevalence of DCM is estimated to be 36.5 per 100,000 and each year 7 people per 100,000 people are affected in global [1,2]. DCM is characterized by ventricular enlargement, systolic dysfunction and usually accompany with myocardial inflammation [3]. Traditionally, dilated cardiomyopathy is mainly caused by genic inheritance. And so far it is remained the most common indication of heart transplant [4,5]. However, recent studies have shown that myocardial inflammation is the main initial link and risk factor of DCM, but the underlying mechanism has not been fully elucidated [2,6]. Therefore, given the devastating consequences of this disease in a large number of patients, regulating the myocardial inflammation effectively and understanding the etiology of DCM and has a large clinical relevance. Mesenchymal stem cells (MSCs) derived from bone marrow
have emerged as one of the most promising stem cell types for treating cardiovascular disease [7]. They have multiple biological functions such as pluripotent differentiation, immune regulation, neuroprotection and so on [8,9]. Although MSC exert their therapeutic effects, but more concerns like teratoma formation, limited cell homing and differentiation lead to the restriction of stem cell therapy. Recent reports have suggested that MSC reparative effects are mediated by secreted paracrine factors [10,11]. In support of this paracrine hypothesis, many studies have observed that stem cells secrete cytokines, chemokines, and growth factors that could potentially repair injured myocardium [12,13]. Stem cell-derived exosomes are macrovesicles secreted from the endosomal membrane and have been shown to act as regulators of cell-cell communication [14,15]. An increasing number of reports have suggested that MSC-derived exosome is an attractive candidate for cell therapy in several diseases [16e18]. Exosomes range in size from 10 to 100 nm and contain proteins, mRNA and miRNA molecules, which can be functionally
* Corresponding author. Department of Cardiology, Drum Tower Hospital, Nanjing University Medical School, Zhongshan Road, Nanjing, 210008, China. E-mail address: [email protected] (B. Xu). https://doi.org/10.1016/j.bbrc.2018.08.012 0006-291X/© 2018 Elsevier Inc. All rights reserved.
Please cite this article in press as: X. Sun, et al., Intravenous mesenchymal stem cell-derived exosomes ameliorate myocardial inflammation in the dilated cardiomyopathy, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.08.012
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transferred between cells and affect target cells [19e21]. Besides, exosomes have the advantage of no teratogenic and less vascular occlusion compared with stem cells [22]. Hence, exosomes have great clinical prospects. A growing number of studies have shown that monocytemacrophages are involved in the regulation of cardiovascular diseases, including atherosclerosis [23], ischemic cardiomyopathy [24], and diabetes-induced arrhythmia [25]. Recruitment of mononuclear-macrophages cause myocardial inflammation, and locally enriched macrophages exhibit a magnifying effect which is aggravating myocardial inflammation and ultimately leading to cardiomyopathy [26]. Macrophages have the characteristics of function and phenotypic diversity according to influence of different tissue microenvironments [27]. In general, inflammatory damage can mobilize monocytes of circulating blood, and then monocytes infiltrate into the damaged heart to differentiate to mature macrophages. Infiltrated macrophages have pivotal roles in inflammation, angiogenesis and cardiac remodeling. Currently, macrophages are divided into two polarized phenotypes including classical activated macrophages (M1) and alternative activated macrophages (M2). M1-like macrophages participate in positive immune responses by secreting various pro-inflammatory cytokines. M2-like macrophages are mainly involved in tissue cell regeneration and homeostasis of tissues, thereby exert antagonistic effects on inflammation [28]. It is believed that Ly6Chigh monocytes always differentiate into the pro-inflammatory M1-like macrophages and Ly6Clow monocytes always differentiate into the antiinflammatory M2-like macrophages. Previous studies have found that local injection of MSCs have a role in regulating the phenotype of macrophages during myocardial infarction [29,30]. Moreover, these reparative effects are possibly mediated by exosomes. Therefore, it is important to realize the role of macrophages regulated by MSC-derived exosomes during DCM. In this study, we established the murine model of doxorubicin
induced dilated cardiomyopathy to investigate the protective role of intravenous injected MSCs-derived exosome in improving the inflammatory microenvironment of myocardium, especially by regulating the activity of macrophages. 2. Materials and methods MSCeExos were purified by ultra-centrifugation. Male mice were intraperitoneally administered with DOX 15 mg/kg to induce dilated cardiomyopathy. After 7 days, the mice were injected with either 200ul PBS containing 300ug MSC-Exos or 200ul PBS via tail vein. Histological analyses, TUNEL assay, flow cytometry, Realtime PCR, Immunobiological analysis and western blot were employed to evaluate the effect of MSC-Exos on DCM mice. Detailed materials and methods are described in Supplementary materials. 3. Results 3.1. The extraction and identification of MSC-derived exosomes The exosomes were obtained from the conditioned media of MSCs using ultracentrifugation method and identified by the transmission electron microscopy. The majority of them presented a typical cup-shaped structure with diameters within 100 nm (Fig. 1A). Subsequent microparticle analysis of the exosomes sample using a DLS detector revealed that the extracted MSC exosome displayed a physically homogeneous population with a peak at 35.21 nm (Fig. 1B). Then we identified the surface markers of MSCs exosomes using western blot. The results showed that isolated precipitates expressed exosome-specific membrane proteins: NALIX, TSG101, CD9 and CD63, indicating their cytoplasmic origin (Fig. 1C). These above results demonstrated that MSC exosomes were purified successfully in our experiments.
Fig. 1. Characterization of MSC-derived exosomes. A. The ultrastructure of exosomes were analyzed by transmission electron microscopy (scale bar, 100 nm). B. Representative DLS number distribution measurement of isolated exosome population demonstrated a single peak of 35.21 nm diameter. C. Western blot showed characteristic protein markers of exosomes such as N-ALIX, TSG101, CD9 and CD63.
Please cite this article in press as: X. Sun, et al., Intravenous mesenchymal stem cell-derived exosomes ameliorate myocardial inflammation in the dilated cardiomyopathy, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.08.012
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3.2. MSC-Exos improve cardiac function in DCM Dilated cardiomyopathy (DCM) is characterized by ventricular dilatation, systolic dysfunction (reduced LVEF), abnormalities of diastolic filling, and either normal or reduced wall thickness (i.e., pathological ventricular remodeling; eccentric hypertrophy). To investigate the effect of MSC-Exosomes (MSC-Exos) on DCM, we induced murine DCM in C57BL6 mice by doxorubicin (DOX) injection, and then modeling mice were randomly divided into MSCs exosomes treatment group and PBS control group after DOX injection 7 days. Four weeks after exosomes treatment, the images of
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murine echocardiography were showed as Fig. 2A. The analysis of the LVEF and LVFS pre 1 week or post 1,2,3,4 weeks MSC-Exo injection were showed in Fig. 2B.There was a decrease of LVEF and LVFS after DOX injection, which indicated that the model of doxorubicin-induced dilated cardiomyopathy was successful. And then LVEF and LVFS of exosomes-treated group were significantly improved compared with the PBS group, which run through to the 4 weeks endpoint. At the 4th week after exosomes treatment, both the LVEDD and LVESD in the MSC-Exos group were significantly lower than those in the PBS group (Fig. 2C). At the same time, HE staining of the heart at the 4th week revealed that the MSC-Exos
Fig. 2. Analysis of mice myocardial function and apoptosis of cardiomyotes after MSC-Exos or PBS treatment. A. Representative echocardiograms of mice were showed 4 weeks after given MSC-Exos or PBS. (n ¼ 10 each group).B. Quantitative group data for every week echocardiographic measurements: a, Left ventricular ejection fraction (LVEF); b, left ventricular fractional shortening (LVFS). (n ¼ 10 each group).C.Quantitative group data for echocardiographic measurements:a, Left ventricular end-diastolic diameter (LVEDD);b, left ventricular end-systolic diameter (LVESD) at 4 weeks after given MSC-Exos or PBS. (n ¼ 10 each group). D. Representative images of hematoxylin and eosin (H&E) were showed 4 weeks after MSC-Exos or PBS injection. (n ¼ 6 each group).E. Cardiac tissue apoptosis was detected by flow cytometry after 4 weeks exosomes treatment. F. Western blot showed the cardiac tissues expressions of Bax, Bcl-2 after treatment of MSC-Exos or PBS at the 4-week endpoint. Theses protein expressions were qualified and normalized to the levels of GAPDH (n ¼ 6 each group). *p < 0.05 between groups; **p < 0.01 between groups; ***p < 0.001 between groups; and ****p < 0.0001 between groups.
Please cite this article in press as: X. Sun, et al., Intravenous mesenchymal stem cell-derived exosomes ameliorate myocardial inflammation in the dilated cardiomyopathy, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.08.012
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treatment group had higher myocardial thickness and less cardiac chambers diameter than the PBS group (Fig. 2D). Together, these findings demonstrated that MSC-derived exosomes has a major protective effect on cardiac contractile function in the DOX induced DCM. 3.3. MSC-Exos alleviate the apoptosis of cardiomyocytes during DCM progression The above results showed an improvement of murine DCM due to exosomes treatment. Then we wonder if exosomes could decrease the apoptosis of cardiomyocytes. The flow cytometry results showed that the number of the Annexin V and PI double positive apoptosis cells were significantly decreased in exosomes treatment group compared with PBS group (Fig. 2E). But there was no significant difference in the Annexin Vþ and PI apoptotic cells in these two groups, suggesting that MSC-exosomes could apparently decrease the later apotosis or necrotic cardiomyocytes rather than the early apoptosis cardiomyocytes. Moreover, the antiapoptotic effect of MSC-exosomes was further confirmed by Western blotting (Fig. 2F). The results showed that classic endogenous apoptotic pathway, pro-apoptotic protein Bax expression was decreased and pro-survival protein Bcl-2 expression was increased in the MSC-Exos treatment group comparing to the PBS
group. These results indicated that exosomes reduced cardiomyocyte apoptosis in dilated cardiomyopathy. In addition to this, we cultured the neonatal mice ventricular myocytes (NMVMs) in vitro. DOX-stimulated NMVMs were then given exosome or PBS treatment. The apoptosis of NMVMs were detected by staining of TUNEL immunofluorescence (Fig. S1). The statistical data showed that the ratio of TUNEL is obviously reduced in the MSC-Exos treatment group. Moreover, MSC-Exos could also reduce the oxidative stress in the murine DCM (Fig. S2). Thereby, the above results indicated that MSC-Exosomes could retard DCM progression through inhibiting the apoptosis of cardiomyocytes. 3.4. Intravenous-delivered MSC-Exos suppressed circulating inflammation response of DCM Immune inflammatory responses take a great role in the pathological process of dilated cardiomyopathy. DCM associate with inflammation, documented by increased inflammatory cytokines and circulating innate immune cells. To define the effect of MSCExos on inflammation of DCM, we first detected the inflammatory cytokines such as IL-1, IL-6 and TNF-a in the mice serum. Interestingly, ELISA analysis showed that injection of MSCs exosomes resulted in a robust decrease of these inflammatory factors compared with the PBS group (Fig. 3A). Hence the former results
Fig. 3. MSC-exos exerts anti-inflammation function on DCM. A. ELISA analysis of inflammatory factors IL-1, IL-6, and TNF-a levels in murine plasma after treatment of MSC-Exos or PBS at the 4-week endpoint (n ¼ 6 each group). B. Representative flow cytometry analysis of circulating blood F4/80 þ CD11b þ macrophages in DCM mice with or without exosomes treatment at the 4-week endpoint (n ¼ 6 each group). C. Dot plots show the percentages of Ly6Chigh expression and Ly6Clow expression macrophages in circulating blood of DCM mice with or without exosomes treatment at the 4-week endpoint (n ¼ 6 each group).*p < 0.05 between groups; and ****p < 0.0001 between groups.
Please cite this article in press as: X. Sun, et al., Intravenous mesenchymal stem cell-derived exosomes ameliorate myocardial inflammation in the dilated cardiomyopathy, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.08.012
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indicated that DCM is closely related to inflammation. We then focus on the key regulator of inflammation, macrophages. Flow cytometry analysis showed that the MSC-Exos treatment group has less F4/80þCD11bþ-labelled macrophages induced by doxorubicin comparing to the PBS group (Fig. 3B). The above results suggested that MSC-Exos could retard the mobilization of macrophages in the circulating. To probe into the polarization of the macrophages, we detected the phenotype of macrophages. There was remarkably less pro-inflammatory Ly6Chigh macrophages amount in the MSC-Exos treatment group than PBS group, correspondingly, antiinflammatory Ly6Clow macrophages amount was significantly exceeding (Fig. 3C). Taken together, these results indicated that MSC-Exos could lower cardiac inflammation cytokines production, reduce circulating macrophages amount in DCM. And it may promote the conversion of macrophages from pro-inflammatory to antiinflammatory. 3.5. MSC-Exos regulate macrophages polarization through JAK2STAT6 pathway Macrophages as the main immune effector cells, play an important role in regulating myocardial inflammation. As former study mentioned, MSCs-derived exosomes could significantly reduce circulating macrophages amount in DCM. To future investigate the inflammation responses of MSCs-derived exosomes in the myocardial tissues, we explored the detection of inflammatory factors. The Q-PCR analysis first showed that there was lower mRNA level of IL-1, IL-6, and TNF-a in MSC-Exos treatment group than those in PBS group (Fig. 4A). Besides, the immunohistochemistry results showed that the number of CD68þ cells were obviously decreased in the MSC-Exos group comparing to the PBS group (Fig. 4B). Moreover, the myocardial tissues flow cytometry analysis indicated that there is a observably less F4/80þCD11bþ macrophages in the MSC-Exos treatment group than PBS group (Fig. 4C). Combined with previous findings that MSCs-derived exosomes could significantly reduce circulating macrophages mobilization, we considered MSC-Exos could further result in macrophages infiltration reduction. Not only the quantity but also the phenotypic switch of macrophage is critical for inflammation in DCM progression. Hence the previous study showed that MSC-Exos could there was a decrease Ly6Chi macrophages as well as a enhance Ly6Clow macrophages amount in circulating blood, then we would to clarify the effect of MSC-Exos on macrophages polarization in myocardial tissues. The flow cytometry results showed that M1-like F4/ 80þCD11cþ macrophages were significantly reduced but M2-like F4/80þCD206þ macrophages were elevated in the MSC-Exos treatment group than the PBS group (Fig. 4D). These data were consistent with previous study as shown in Fig. 3C.Then we conducted Q-PCR to further verify and the results showed that the expression of M1-like macrophages markers iNOS, CD32 and CD11c were markably reduced but the expression of M2-like macrophages markers Arg1, Mgl2 and CD206 were markably hoisted in the MSCExos treatment group comparing to the PBS group (Fig. 4E). Together, the above results indicated that MSC-Exos could refrain from inflammation deterioration by promoting the conversion of macrophages from pro-inflammatory to anti-inflammatory and thereby exert a protective role during the DCM progression. Furthermore, in order to explore the molecular mechanism underlying MSC-Exos regulating macrophages polarization. We performed the western blot experiments to analyze classical macrophages polarization signaling pathway JAK2-STAT6 in the cardiac tissues. The western analysis showed that JAK2 along with its downstream STAT6 were significantly activated in the MSC-Exos
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treatment group rather than the PBS group (Fig. 4F). These results suggested that MSC-derived exosomes suppressed inflammation responses and promote the conversion of macrophages to antiinflammatory in myocardial tissue through a JAK2-STAT6 mediated pathway. 4. Discussion In this study, the main finding is that systematically infused MSC-derived exosomes could enhance alternatively activated M2like anti-inflammatory macrophages phenotype polarization in murine DCM, which may play a protective role during DCM progression. Our study firstly reveals that the injection of MSC-Exos via tail vein can lessen the cardiac inflammation through regulating macrophages functions and exert the protection on non-ischemic inflammatory cardiomyopathy. Moreover, we also discover that MSC-Exos modulate macrophages phenotype polarization through interaction with JAK2-STAT6 signal pathway. Dilated cardiomyopathy has been recognized as the main cause of heart failure in human under the age of 40. Nevertheless, there is no effective therapy except heart transplantation. Stem cell-based therapy has become an attractive and promising approach for the treatment of cardiovascular diseases. However, the use of stem cells is often limited by series shortages of tumorigenicity, immunogenicity, low survival, poor targeting and so on. Interestingly, MSCs have been found to secrete exosomes, which are considered as vital mediators of cellular communication and may regulate various physiological and pathological processes by transferring membrane protein, mRNAs, miRNAs to recipient cells. Compared to stem cell therapy, exosomes can be delivered more readily by less sophisticated and more conventional delivery devices. In this study, MSCderived exosomes injection performed on the murine dilated cardiomyopathy is therapeutically effective and helpful in improving myocardial injury. As shown in Fig. 2, MSC-Exos treatment could significantly reduce ventricular dilatation and improve cardiac function during DCM progression. Moreover, myocardial apoptosis is the main form of myocytes loss and the key link of ventricular remodeling. Indeed, our studies showed that Annexin VþPIþ cardiomyocytes were decreased in the MSC-Exos treatment group rather than PBS group (Fig. 2). And these findings were further concluded by the in vitro experiment using TUNEL staining (Fig. S1). At the same time, our previous results have proved that the amount of oxidative stress cardiomyocytes were rescued by MSC-Exos injection (Fig. S2). Together, MSC-Exos play a role of cardiac protection suggesting a paracrine effect of MSC. Inflammation response to heart tissue is often involved in the pathogenesis of dilated cardiomyopathy. DCM associate with inflammation, documented by increased levels of inflammatory cytokines such as IL-1, IL-6 and TNF-a and innate immune cells. Consistent with it, our results proved that MSC-Exos could significantly reduce expression of inflammatory factors in circulating blood of DCM mice (Fig. 3A). Macrophages are central mediators for inflammation response. As such, damaged myocardium always leads to monocytes recruitment from the vasculature to generate new macrophages travel to the injured myocardium for tissue repair. We found that MSC-Exos treatment group decreased the proportion of F4/80þCD11bþ macrophages in the circulating blood compared with the PBS group (Fig. 3B). Several reports suggested that two subsets of monocytes, pro-inflammatory Ly6Chigh and anti-inflammatory Ly6Clow cells are participated in the process of DCM. In our study, the amount of classically activated Ly6Chigh macrophages were reduced by MSC-Exos injection. And the presence of increased circulating Ly6Clow expression macrophages suggested that the increase in alternatively activated macrophages infiltrating in the heart due to a phenotype switch in circulating
Please cite this article in press as: X. Sun, et al., Intravenous mesenchymal stem cell-derived exosomes ameliorate myocardial inflammation in the dilated cardiomyopathy, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.08.012
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Fig. 4. MSC-exos suppressed the cardiac inflammation responses in the DCM mice. A. Inflammatory cytokine IL-1, IL-6, and TNF-a expressions in the DCM myocardium were assessed by qRT-PCR at the 4-week endpoint. These genes expression were normalized to the GAPDH expression and expressed as fold change relative to sham group (n ¼ 6 each group).B. Representative images and quantification of immunohistochemical staining for CD68 in the DCM hearts at the 4-week endpoint (n ¼ 4 each group). Scale bar ¼ 40 mm. C. Representative flow cytometry analysis of cardiac tissue F4/80 þ CD11b þ macrophages in DCM mice with or without exosomes treatment at the 4-week endpoint (n ¼ 6 each group).D.Dot plots show the percentages of M1-like macrophages as F4/80 þ CD11c þ macrophages expression and M2-like macrophages as F4/80 þ CD206 þ macrophages expression in the cardiac tissues with or without exosomes treatment at the 4-week endpoint (n ¼ 6 each group).E. Real-time quantitative PCR for the relative mRNA expression of M1-like macrophages markers such as iNOS,CD32, CD11c and M2-like macrophages markers such as Arg1, Mgl2, CD206 in the cardiac tissues with or without exosomes treatment at the 4-week endpoint (n ¼ 4 each group).F. Western blot analysis of JAK2 -STAT6 in the cardiac tissues with or without exosomes treatment at the 4-week endpoint (n ¼ 4 each group).*p < 0.05 between groups; **p < 0.01 between groups; ***p < 0.001 between groups; and ****p < 0.0001 between groups.
Please cite this article in press as: X. Sun, et al., Intravenous mesenchymal stem cell-derived exosomes ameliorate myocardial inflammation in the dilated cardiomyopathy, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.08.012
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monocytes (Fig. 3C). These above data provide preliminary evidence that MSC-Exos injection by tail vein significantly attenuated circulating inflammation during DCM process by mediating a switch of monocytes to an anti-inflammatory state. We further investigated the effect of MSC-Exos on improving the inflammatory microenvironment of myocardium. The data presented in this study showed that the levels of inflammatory factors were limited by MSC-Exos injection (Fig. 4A). As an important effector during inflammation, the contribution of infiltrated macrophages in myocardium were also restrained after MSCExos treatment (Fig. 4B-C). Nevertheless, macrophages could either promote cardiac injury or cardiac protection due to its own phenotypic switch. We found that MSC-Exos decreased the number of M1-like macrophages but increased the number of M2-like macrophages (Fig. 4D). We propose the increase of M2-like macrophages among the cells infiltrating the myocardium to be due to a higher proportion of Ly6clow circulating monocytes in MSC-Exos group. Similarly, these data could further explain the lower expression of M1-like macrophages markers as well as higher levels of M2-like macrophage markers after MSC-Exos treatment (Fig. 4E). The treatment changed the cytokine profile of macrophages and increased the elaboration of macrophages-derived factors involved in injured myocardium healing. Further, to explore the key regulators responsible for MSC-Exos regulating macrophages polarization, we detected the classical macrophages polarization signal pathway JAK2/STATA6 in the injured myocardium. Our results showed that MSC-Exos promote macrophages polarizing towards an anti-inflammatory phenotype by activating the expression of transcription factors JAK2 and its downstream STAT6 (Fig. 4F). Firstly, it was proved that treatment with MSC-derived exosomes has great benefits in inflammatory dilated cardiomyopathy by restricting cardiac inflammation response. Secondly, there is increasing support in that exosomes can be delivered more readily by less sophisticated and more conventional delivery devices compared to stem cell therapy. It's the first time that we demonstrate a significant beneficial effect from MSCs-derived exosomes intravenous administration on DOX induced dilated cardiomyopathy, which presents similar results in progenitor cells. Furthermore, we delivered exosomes via tail vein-injection approach which is convenient for repeated injections, although more relevant researches are need to be addressed in the future. With these advances, we believe MSC-derived exosomes treatment would be a huge clinical gain in the future. In summary, the present studies provide evidence that intravenous injection of MSC-Exos could regulate the balance between M1 and M2 in macrophages via JAK2/STAT6 axis, which may result in a favorable anti-inflammatory environment to cardiac repairment during DCM progression. Acknowledgements This work was supported by grants from the Natural Science Foundation of China (81470371), the Funds for Jiangsu Provincial Key Medical Discipline (ZDXKB2016013), the Key Projects of Science and Technology of Jiangsu Province (BE2016607), the Funds for Jiangsu Provincial Medical Youth Talent (QNRC2016033), the Postgraduate Research & Practice Innovation Program of Jiangsu Province (KYCX18_1462) and the Programs of the Science Foundation in Nanjing (JQX15002 and ZKX17011). Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.bbrc.2018.08.012.
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Intravenous mesenchymal stem cell-derived exosomes ameliorate myocardial inflammation in the dilated cardiomyopathy Xuan Sun, Anqi Shan, Zilun Wei, Biao Xu* Department of Cardiology, Drum Tower Hospital, Nanjing University Medical School, Nanjing, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 July 2018 Accepted 1 August 2018 Available online xxx
Mesenchymal stem cells (MSCs) have been shown to be efficacy to attenuating cardiovascular inflammation; however, there are many limitations to stem cell treatment. Present study was to prove MSCderived exosomes (MSC-Exos) could alleviating inflammatory cardiomyopathy by improving the inflammatory microenvironment of myocardium, especially by regulating the activity of macrophages. Mice were intraperitoneal injected of doxorubicin (DOX) to establish a dilated cardiomyopathy (DCM) model, and then received intravenous injection of either MSC-Exos or PBS as control. Mice receiving MSC-Exos showed improved cardiac function via echocardiography and attenuated cardiac dilation via HE staining, as well as reduced cardiomyocytes apoptosis. Expression levels of inflammatory factors were reduced. And there was a significant decrease of the inflammatory cells infiltration in the MSC-Exos treatment group comparing to the PBS group. Meanwhile, MSC-Exos could remarkably attenuate the pro-inflammatory macrophages amount in both blood and heart, which was proved that MSC-Exos relied on the JAK2-STAT6 pathway mediating macrophages activation. MSC-Exos improved the inflammatory microenvironment of dilated cardiomyopathy by regulating the polarization of the macrophage, which may hold promise for dilated cardiomyopathy clinical therapy. © 2018 Elsevier Inc. All rights reserved.
Keywords: Mesenchymal stem cell exosome Dilated cardiomyopathy Myocardial inflammation Macrophages
1. Introduction Dilated cardiomyopathy (DCM) is the most common complication heart disease of myocardial inflammation. The prevalence of DCM is estimated to be 36.5 per 100,000 and each year 7 people per 100,000 people are affected in global [1,2]. DCM is characterized by ventricular enlargement, systolic dysfunction and usually accompany with myocardial inflammation [3]. Traditionally, dilated cardiomyopathy is mainly caused by genic inheritance. And so far it is remained the most common indication of heart transplant [4,5]. However, recent studies have shown that myocardial inflammation is the main initial link and risk factor of DCM, but the underlying mechanism has not been fully elucidated [2,6]. Therefore, given the devastating consequences of this disease in a large number of patients, regulating the myocardial inflammation effectively and understanding the etiology of DCM and has a large clinical relevance. Mesenchymal stem cells (MSCs) derived from bone marrow
have emerged as one of the most promising stem cell types for treating cardiovascular disease [7]. They have multiple biological functions such as pluripotent differentiation, immune regulation, neuroprotection and so on [8,9]. Although MSC exert their therapeutic effects, but more concerns like teratoma formation, limited cell homing and differentiation lead to the restriction of stem cell therapy. Recent reports have suggested that MSC reparative effects are mediated by secreted paracrine factors [10,11]. In support of this paracrine hypothesis, many studies have observed that stem cells secrete cytokines, chemokines, and growth factors that could potentially repair injured myocardium [12,13]. Stem cell-derived exosomes are macrovesicles secreted from the endosomal membrane and have been shown to act as regulators of cell-cell communication [14,15]. An increasing number of reports have suggested that MSC-derived exosome is an attractive candidate for cell therapy in several diseases [16e18]. Exosomes range in size from 10 to 100 nm and contain proteins, mRNA and miRNA molecules, which can be functionally
* Corresponding author. Department of Cardiology, Drum Tower Hospital, Nanjing University Medical School, Zhongshan Road, Nanjing, 210008, China. E-mail address: [email protected] (B. Xu). https://doi.org/10.1016/j.bbrc.2018.08.012 0006-291X/© 2018 Elsevier Inc. All rights reserved.
Please cite this article in press as: X. Sun, et al., Intravenous mesenchymal stem cell-derived exosomes ameliorate myocardial inflammation in the dilated cardiomyopathy, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.08.012
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transferred between cells and affect target cells [19e21]. Besides, exosomes have the advantage of no teratogenic and less vascular occlusion compared with stem cells [22]. Hence, exosomes have great clinical prospects. A growing number of studies have shown that monocytemacrophages are involved in the regulation of cardiovascular diseases, including atherosclerosis [23], ischemic cardiomyopathy [24], and diabetes-induced arrhythmia [25]. Recruitment of mononuclear-macrophages cause myocardial inflammation, and locally enriched macrophages exhibit a magnifying effect which is aggravating myocardial inflammation and ultimately leading to cardiomyopathy [26]. Macrophages have the characteristics of function and phenotypic diversity according to influence of different tissue microenvironments [27]. In general, inflammatory damage can mobilize monocytes of circulating blood, and then monocytes infiltrate into the damaged heart to differentiate to mature macrophages. Infiltrated macrophages have pivotal roles in inflammation, angiogenesis and cardiac remodeling. Currently, macrophages are divided into two polarized phenotypes including classical activated macrophages (M1) and alternative activated macrophages (M2). M1-like macrophages participate in positive immune responses by secreting various pro-inflammatory cytokines. M2-like macrophages are mainly involved in tissue cell regeneration and homeostasis of tissues, thereby exert antagonistic effects on inflammation [28]. It is believed that Ly6Chigh monocytes always differentiate into the pro-inflammatory M1-like macrophages and Ly6Clow monocytes always differentiate into the antiinflammatory M2-like macrophages. Previous studies have found that local injection of MSCs have a role in regulating the phenotype of macrophages during myocardial infarction [29,30]. Moreover, these reparative effects are possibly mediated by exosomes. Therefore, it is important to realize the role of macrophages regulated by MSC-derived exosomes during DCM. In this study, we established the murine model of doxorubicin
induced dilated cardiomyopathy to investigate the protective role of intravenous injected MSCs-derived exosome in improving the inflammatory microenvironment of myocardium, especially by regulating the activity of macrophages. 2. Materials and methods MSCeExos were purified by ultra-centrifugation. Male mice were intraperitoneally administered with DOX 15 mg/kg to induce dilated cardiomyopathy. After 7 days, the mice were injected with either 200ul PBS containing 300ug MSC-Exos or 200ul PBS via tail vein. Histological analyses, TUNEL assay, flow cytometry, Realtime PCR, Immunobiological analysis and western blot were employed to evaluate the effect of MSC-Exos on DCM mice. Detailed materials and methods are described in Supplementary materials. 3. Results 3.1. The extraction and identification of MSC-derived exosomes The exosomes were obtained from the conditioned media of MSCs using ultracentrifugation method and identified by the transmission electron microscopy. The majority of them presented a typical cup-shaped structure with diameters within 100 nm (Fig. 1A). Subsequent microparticle analysis of the exosomes sample using a DLS detector revealed that the extracted MSC exosome displayed a physically homogeneous population with a peak at 35.21 nm (Fig. 1B). Then we identified the surface markers of MSCs exosomes using western blot. The results showed that isolated precipitates expressed exosome-specific membrane proteins: NALIX, TSG101, CD9 and CD63, indicating their cytoplasmic origin (Fig. 1C). These above results demonstrated that MSC exosomes were purified successfully in our experiments.
Fig. 1. Characterization of MSC-derived exosomes. A. The ultrastructure of exosomes were analyzed by transmission electron microscopy (scale bar, 100 nm). B. Representative DLS number distribution measurement of isolated exosome population demonstrated a single peak of 35.21 nm diameter. C. Western blot showed characteristic protein markers of exosomes such as N-ALIX, TSG101, CD9 and CD63.
Please cite this article in press as: X. Sun, et al., Intravenous mesenchymal stem cell-derived exosomes ameliorate myocardial inflammation in the dilated cardiomyopathy, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.08.012
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3.2. MSC-Exos improve cardiac function in DCM Dilated cardiomyopathy (DCM) is characterized by ventricular dilatation, systolic dysfunction (reduced LVEF), abnormalities of diastolic filling, and either normal or reduced wall thickness (i.e., pathological ventricular remodeling; eccentric hypertrophy). To investigate the effect of MSC-Exosomes (MSC-Exos) on DCM, we induced murine DCM in C57BL6 mice by doxorubicin (DOX) injection, and then modeling mice were randomly divided into MSCs exosomes treatment group and PBS control group after DOX injection 7 days. Four weeks after exosomes treatment, the images of
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murine echocardiography were showed as Fig. 2A. The analysis of the LVEF and LVFS pre 1 week or post 1,2,3,4 weeks MSC-Exo injection were showed in Fig. 2B.There was a decrease of LVEF and LVFS after DOX injection, which indicated that the model of doxorubicin-induced dilated cardiomyopathy was successful. And then LVEF and LVFS of exosomes-treated group were significantly improved compared with the PBS group, which run through to the 4 weeks endpoint. At the 4th week after exosomes treatment, both the LVEDD and LVESD in the MSC-Exos group were significantly lower than those in the PBS group (Fig. 2C). At the same time, HE staining of the heart at the 4th week revealed that the MSC-Exos
Fig. 2. Analysis of mice myocardial function and apoptosis of cardiomyotes after MSC-Exos or PBS treatment. A. Representative echocardiograms of mice were showed 4 weeks after given MSC-Exos or PBS. (n ¼ 10 each group).B. Quantitative group data for every week echocardiographic measurements: a, Left ventricular ejection fraction (LVEF); b, left ventricular fractional shortening (LVFS). (n ¼ 10 each group).C.Quantitative group data for echocardiographic measurements:a, Left ventricular end-diastolic diameter (LVEDD);b, left ventricular end-systolic diameter (LVESD) at 4 weeks after given MSC-Exos or PBS. (n ¼ 10 each group). D. Representative images of hematoxylin and eosin (H&E) were showed 4 weeks after MSC-Exos or PBS injection. (n ¼ 6 each group).E. Cardiac tissue apoptosis was detected by flow cytometry after 4 weeks exosomes treatment. F. Western blot showed the cardiac tissues expressions of Bax, Bcl-2 after treatment of MSC-Exos or PBS at the 4-week endpoint. Theses protein expressions were qualified and normalized to the levels of GAPDH (n ¼ 6 each group). *p < 0.05 between groups; **p < 0.01 between groups; ***p < 0.001 between groups; and ****p < 0.0001 between groups.
Please cite this article in press as: X. Sun, et al., Intravenous mesenchymal stem cell-derived exosomes ameliorate myocardial inflammation in the dilated cardiomyopathy, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.08.012
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treatment group had higher myocardial thickness and less cardiac chambers diameter than the PBS group (Fig. 2D). Together, these findings demonstrated that MSC-derived exosomes has a major protective effect on cardiac contractile function in the DOX induced DCM. 3.3. MSC-Exos alleviate the apoptosis of cardiomyocytes during DCM progression The above results showed an improvement of murine DCM due to exosomes treatment. Then we wonder if exosomes could decrease the apoptosis of cardiomyocytes. The flow cytometry results showed that the number of the Annexin V and PI double positive apoptosis cells were significantly decreased in exosomes treatment group compared with PBS group (Fig. 2E). But there was no significant difference in the Annexin Vþ and PI apoptotic cells in these two groups, suggesting that MSC-exosomes could apparently decrease the later apotosis or necrotic cardiomyocytes rather than the early apoptosis cardiomyocytes. Moreover, the antiapoptotic effect of MSC-exosomes was further confirmed by Western blotting (Fig. 2F). The results showed that classic endogenous apoptotic pathway, pro-apoptotic protein Bax expression was decreased and pro-survival protein Bcl-2 expression was increased in the MSC-Exos treatment group comparing to the PBS
group. These results indicated that exosomes reduced cardiomyocyte apoptosis in dilated cardiomyopathy. In addition to this, we cultured the neonatal mice ventricular myocytes (NMVMs) in vitro. DOX-stimulated NMVMs were then given exosome or PBS treatment. The apoptosis of NMVMs were detected by staining of TUNEL immunofluorescence (Fig. S1). The statistical data showed that the ratio of TUNEL is obviously reduced in the MSC-Exos treatment group. Moreover, MSC-Exos could also reduce the oxidative stress in the murine DCM (Fig. S2). Thereby, the above results indicated that MSC-Exosomes could retard DCM progression through inhibiting the apoptosis of cardiomyocytes. 3.4. Intravenous-delivered MSC-Exos suppressed circulating inflammation response of DCM Immune inflammatory responses take a great role in the pathological process of dilated cardiomyopathy. DCM associate with inflammation, documented by increased inflammatory cytokines and circulating innate immune cells. To define the effect of MSCExos on inflammation of DCM, we first detected the inflammatory cytokines such as IL-1, IL-6 and TNF-a in the mice serum. Interestingly, ELISA analysis showed that injection of MSCs exosomes resulted in a robust decrease of these inflammatory factors compared with the PBS group (Fig. 3A). Hence the former results
Fig. 3. MSC-exos exerts anti-inflammation function on DCM. A. ELISA analysis of inflammatory factors IL-1, IL-6, and TNF-a levels in murine plasma after treatment of MSC-Exos or PBS at the 4-week endpoint (n ¼ 6 each group). B. Representative flow cytometry analysis of circulating blood F4/80 þ CD11b þ macrophages in DCM mice with or without exosomes treatment at the 4-week endpoint (n ¼ 6 each group). C. Dot plots show the percentages of Ly6Chigh expression and Ly6Clow expression macrophages in circulating blood of DCM mice with or without exosomes treatment at the 4-week endpoint (n ¼ 6 each group).*p < 0.05 between groups; and ****p < 0.0001 between groups.
Please cite this article in press as: X. Sun, et al., Intravenous mesenchymal stem cell-derived exosomes ameliorate myocardial inflammation in the dilated cardiomyopathy, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.08.012
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indicated that DCM is closely related to inflammation. We then focus on the key regulator of inflammation, macrophages. Flow cytometry analysis showed that the MSC-Exos treatment group has less F4/80þCD11bþ-labelled macrophages induced by doxorubicin comparing to the PBS group (Fig. 3B). The above results suggested that MSC-Exos could retard the mobilization of macrophages in the circulating. To probe into the polarization of the macrophages, we detected the phenotype of macrophages. There was remarkably less pro-inflammatory Ly6Chigh macrophages amount in the MSC-Exos treatment group than PBS group, correspondingly, antiinflammatory Ly6Clow macrophages amount was significantly exceeding (Fig. 3C). Taken together, these results indicated that MSC-Exos could lower cardiac inflammation cytokines production, reduce circulating macrophages amount in DCM. And it may promote the conversion of macrophages from pro-inflammatory to antiinflammatory. 3.5. MSC-Exos regulate macrophages polarization through JAK2STAT6 pathway Macrophages as the main immune effector cells, play an important role in regulating myocardial inflammation. As former study mentioned, MSCs-derived exosomes could significantly reduce circulating macrophages amount in DCM. To future investigate the inflammation responses of MSCs-derived exosomes in the myocardial tissues, we explored the detection of inflammatory factors. The Q-PCR analysis first showed that there was lower mRNA level of IL-1, IL-6, and TNF-a in MSC-Exos treatment group than those in PBS group (Fig. 4A). Besides, the immunohistochemistry results showed that the number of CD68þ cells were obviously decreased in the MSC-Exos group comparing to the PBS group (Fig. 4B). Moreover, the myocardial tissues flow cytometry analysis indicated that there is a observably less F4/80þCD11bþ macrophages in the MSC-Exos treatment group than PBS group (Fig. 4C). Combined with previous findings that MSCs-derived exosomes could significantly reduce circulating macrophages mobilization, we considered MSC-Exos could further result in macrophages infiltration reduction. Not only the quantity but also the phenotypic switch of macrophage is critical for inflammation in DCM progression. Hence the previous study showed that MSC-Exos could there was a decrease Ly6Chi macrophages as well as a enhance Ly6Clow macrophages amount in circulating blood, then we would to clarify the effect of MSC-Exos on macrophages polarization in myocardial tissues. The flow cytometry results showed that M1-like F4/ 80þCD11cþ macrophages were significantly reduced but M2-like F4/80þCD206þ macrophages were elevated in the MSC-Exos treatment group than the PBS group (Fig. 4D). These data were consistent with previous study as shown in Fig. 3C.Then we conducted Q-PCR to further verify and the results showed that the expression of M1-like macrophages markers iNOS, CD32 and CD11c were markably reduced but the expression of M2-like macrophages markers Arg1, Mgl2 and CD206 were markably hoisted in the MSCExos treatment group comparing to the PBS group (Fig. 4E). Together, the above results indicated that MSC-Exos could refrain from inflammation deterioration by promoting the conversion of macrophages from pro-inflammatory to anti-inflammatory and thereby exert a protective role during the DCM progression. Furthermore, in order to explore the molecular mechanism underlying MSC-Exos regulating macrophages polarization. We performed the western blot experiments to analyze classical macrophages polarization signaling pathway JAK2-STAT6 in the cardiac tissues. The western analysis showed that JAK2 along with its downstream STAT6 were significantly activated in the MSC-Exos
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treatment group rather than the PBS group (Fig. 4F). These results suggested that MSC-derived exosomes suppressed inflammation responses and promote the conversion of macrophages to antiinflammatory in myocardial tissue through a JAK2-STAT6 mediated pathway. 4. Discussion In this study, the main finding is that systematically infused MSC-derived exosomes could enhance alternatively activated M2like anti-inflammatory macrophages phenotype polarization in murine DCM, which may play a protective role during DCM progression. Our study firstly reveals that the injection of MSC-Exos via tail vein can lessen the cardiac inflammation through regulating macrophages functions and exert the protection on non-ischemic inflammatory cardiomyopathy. Moreover, we also discover that MSC-Exos modulate macrophages phenotype polarization through interaction with JAK2-STAT6 signal pathway. Dilated cardiomyopathy has been recognized as the main cause of heart failure in human under the age of 40. Nevertheless, there is no effective therapy except heart transplantation. Stem cell-based therapy has become an attractive and promising approach for the treatment of cardiovascular diseases. However, the use of stem cells is often limited by series shortages of tumorigenicity, immunogenicity, low survival, poor targeting and so on. Interestingly, MSCs have been found to secrete exosomes, which are considered as vital mediators of cellular communication and may regulate various physiological and pathological processes by transferring membrane protein, mRNAs, miRNAs to recipient cells. Compared to stem cell therapy, exosomes can be delivered more readily by less sophisticated and more conventional delivery devices. In this study, MSCderived exosomes injection performed on the murine dilated cardiomyopathy is therapeutically effective and helpful in improving myocardial injury. As shown in Fig. 2, MSC-Exos treatment could significantly reduce ventricular dilatation and improve cardiac function during DCM progression. Moreover, myocardial apoptosis is the main form of myocytes loss and the key link of ventricular remodeling. Indeed, our studies showed that Annexin VþPIþ cardiomyocytes were decreased in the MSC-Exos treatment group rather than PBS group (Fig. 2). And these findings were further concluded by the in vitro experiment using TUNEL staining (Fig. S1). At the same time, our previous results have proved that the amount of oxidative stress cardiomyocytes were rescued by MSC-Exos injection (Fig. S2). Together, MSC-Exos play a role of cardiac protection suggesting a paracrine effect of MSC. Inflammation response to heart tissue is often involved in the pathogenesis of dilated cardiomyopathy. DCM associate with inflammation, documented by increased levels of inflammatory cytokines such as IL-1, IL-6 and TNF-a and innate immune cells. Consistent with it, our results proved that MSC-Exos could significantly reduce expression of inflammatory factors in circulating blood of DCM mice (Fig. 3A). Macrophages are central mediators for inflammation response. As such, damaged myocardium always leads to monocytes recruitment from the vasculature to generate new macrophages travel to the injured myocardium for tissue repair. We found that MSC-Exos treatment group decreased the proportion of F4/80þCD11bþ macrophages in the circulating blood compared with the PBS group (Fig. 3B). Several reports suggested that two subsets of monocytes, pro-inflammatory Ly6Chigh and anti-inflammatory Ly6Clow cells are participated in the process of DCM. In our study, the amount of classically activated Ly6Chigh macrophages were reduced by MSC-Exos injection. And the presence of increased circulating Ly6Clow expression macrophages suggested that the increase in alternatively activated macrophages infiltrating in the heart due to a phenotype switch in circulating
Please cite this article in press as: X. Sun, et al., Intravenous mesenchymal stem cell-derived exosomes ameliorate myocardial inflammation in the dilated cardiomyopathy, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.08.012
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Fig. 4. MSC-exos suppressed the cardiac inflammation responses in the DCM mice. A. Inflammatory cytokine IL-1, IL-6, and TNF-a expressions in the DCM myocardium were assessed by qRT-PCR at the 4-week endpoint. These genes expression were normalized to the GAPDH expression and expressed as fold change relative to sham group (n ¼ 6 each group).B. Representative images and quantification of immunohistochemical staining for CD68 in the DCM hearts at the 4-week endpoint (n ¼ 4 each group). Scale bar ¼ 40 mm. C. Representative flow cytometry analysis of cardiac tissue F4/80 þ CD11b þ macrophages in DCM mice with or without exosomes treatment at the 4-week endpoint (n ¼ 6 each group).D.Dot plots show the percentages of M1-like macrophages as F4/80 þ CD11c þ macrophages expression and M2-like macrophages as F4/80 þ CD206 þ macrophages expression in the cardiac tissues with or without exosomes treatment at the 4-week endpoint (n ¼ 6 each group).E. Real-time quantitative PCR for the relative mRNA expression of M1-like macrophages markers such as iNOS,CD32, CD11c and M2-like macrophages markers such as Arg1, Mgl2, CD206 in the cardiac tissues with or without exosomes treatment at the 4-week endpoint (n ¼ 4 each group).F. Western blot analysis of JAK2 -STAT6 in the cardiac tissues with or without exosomes treatment at the 4-week endpoint (n ¼ 4 each group).*p < 0.05 between groups; **p < 0.01 between groups; ***p < 0.001 between groups; and ****p < 0.0001 between groups.
Please cite this article in press as: X. Sun, et al., Intravenous mesenchymal stem cell-derived exosomes ameliorate myocardial inflammation in the dilated cardiomyopathy, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.08.012
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monocytes (Fig. 3C). These above data provide preliminary evidence that MSC-Exos injection by tail vein significantly attenuated circulating inflammation during DCM process by mediating a switch of monocytes to an anti-inflammatory state. We further investigated the effect of MSC-Exos on improving the inflammatory microenvironment of myocardium. The data presented in this study showed that the levels of inflammatory factors were limited by MSC-Exos injection (Fig. 4A). As an important effector during inflammation, the contribution of infiltrated macrophages in myocardium were also restrained after MSCExos treatment (Fig. 4B-C). Nevertheless, macrophages could either promote cardiac injury or cardiac protection due to its own phenotypic switch. We found that MSC-Exos decreased the number of M1-like macrophages but increased the number of M2-like macrophages (Fig. 4D). We propose the increase of M2-like macrophages among the cells infiltrating the myocardium to be due to a higher proportion of Ly6clow circulating monocytes in MSC-Exos group. Similarly, these data could further explain the lower expression of M1-like macrophages markers as well as higher levels of M2-like macrophage markers after MSC-Exos treatment (Fig. 4E). The treatment changed the cytokine profile of macrophages and increased the elaboration of macrophages-derived factors involved in injured myocardium healing. Further, to explore the key regulators responsible for MSC-Exos regulating macrophages polarization, we detected the classical macrophages polarization signal pathway JAK2/STATA6 in the injured myocardium. Our results showed that MSC-Exos promote macrophages polarizing towards an anti-inflammatory phenotype by activating the expression of transcription factors JAK2 and its downstream STAT6 (Fig. 4F). Firstly, it was proved that treatment with MSC-derived exosomes has great benefits in inflammatory dilated cardiomyopathy by restricting cardiac inflammation response. Secondly, there is increasing support in that exosomes can be delivered more readily by less sophisticated and more conventional delivery devices compared to stem cell therapy. It's the first time that we demonstrate a significant beneficial effect from MSCs-derived exosomes intravenous administration on DOX induced dilated cardiomyopathy, which presents similar results in progenitor cells. Furthermore, we delivered exosomes via tail vein-injection approach which is convenient for repeated injections, although more relevant researches are need to be addressed in the future. With these advances, we believe MSC-derived exosomes treatment would be a huge clinical gain in the future. In summary, the present studies provide evidence that intravenous injection of MSC-Exos could regulate the balance between M1 and M2 in macrophages via JAK2/STAT6 axis, which may result in a favorable anti-inflammatory environment to cardiac repairment during DCM progression. Acknowledgements This work was supported by grants from the Natural Science Foundation of China (81470371), the Funds for Jiangsu Provincial Key Medical Discipline (ZDXKB2016013), the Key Projects of Science and Technology of Jiangsu Province (BE2016607), the Funds for Jiangsu Provincial Medical Youth Talent (QNRC2016033), the Postgraduate Research & Practice Innovation Program of Jiangsu Province (KYCX18_1462) and the Programs of the Science Foundation in Nanjing (JQX15002 and ZKX17011). Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.bbrc.2018.08.012.
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Please cite this article in press as: X. Sun, et al., Intravenous mesenchymal stem cell-derived exosomes ameliorate myocardial inflammation in the dilated cardiomyopathy, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.08.012