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  • In the present study we examined the above mentioned propert

    2018-10-20

    In the present study, we examined the above-mentioned properties of MSC-derived exosomes to limit cell death after myocardial I/R injury in vivo. We show that intact exosomes directly target cardiac auda to reduce infarct size. Furthermore, we demonstrate that, in line with our in vitro data, exosomes exert a therapeutic effect via increased ATP production, decreased oxidative stress and induced PI3K/Akt signaling.
    Materials and methods
    Results
    Discussion Previous studies have shown that MSC transplantation improves cardiac function after infarction. The initial hypothesis that this efficacy was mediated by the engraftment and differentiation of transplanted MSCs to replace injured tissues is increasingly untenable. Most studies suggest that MSCs mediate their therapeutic efficacy through the secretion of paracrine factors (Chimenti et al., 2010; Gnecchi et al., 2005, 2006, 2008). Like others (Deuse et al., 2009; Matsuura et al., 2009; Rogers et al., 2011), we have demonstrated that culture medium conditioned by MSCs reduces I/R injury in a pig and mouse model (Timmers et al., 2007). We identified the active component in this CM as an exosome, a 50–100ηm bi-lipid membrane secreted microvesicle (Lai et al., 2010a, 2010b). In this study, we demonstrated that exosomes not only reduced infarct size, but also resulted in a long-term preservation of cardiac function and reduced adverse remodeling. Interestingly, the therapeutic effect of MSC-derived exosomes was dependent on their physical integrity such that vigorous agitation and homogenization, likely to disrupt the bi-lipid membrane, prevented cardioprotection. These observations are consistent with the reported uptake of exosomes by cells via endocytosis or phagocytosis (Feng et al., 2010; Tian et al., 2010). More specifically, we have also demonstrated that MSC-derived exosomes are taken up by H9C2 cardiomyocytes (Chen et al., 2010). Furthermore, ex vivo Langendorff experiments revealed that exosomes are able to reduce infarct size to the same extent as in the in vivo situation. These findings strongly indicate that exosomes exert their therapeutic effect via viability enhancement of cardiac tissue and do not require the presence of circulating blood cells. Unfortunately, we were not able to follow exosomes in vivo after administration. Both fluorescent (GFPpos-MSC secreted GFPpos-exosomes and ex vivo protein labeling) and radioactive (111In-oxinate) labeling proved to be too insensitive for detection (data not shown). The main issue is that the efficacious dosage is relatively low at 0.1–0.4μg per mouse. More sensitive labeling techniques are needed to explore the dynamics of exosomes after in vivo administration. Our recent proteomic profiling of the ischemic/reperfused heart (Li et al., 2012) and MSC-derived exosome (Li et al., 2012; www.exocarta.org) demonstrated that many of the hallmark biochemical features of reperfusion injury, namely ATP deficit, oxidative stress and cell death are underpinned by either depletion or accumulation of proteins in the reperfused ischemic heart tissues. MSC-derived exosomes have either complementary or compensating proteins to revert or circumvent these biochemical features. Based on the proteomic composition of the ischemic heart and exosomes, we hypothesized that MSC-derived exosomes elicited protection against myocardial I/R injury. The mode of action may be by replenishing depleted glycolytic enzymes to increase ATP production and supplementing the reperfused cardiomyocytes with additional protein components of the cellular antioxidant system (such as the peroxiredoxins and glutathione S-transferases) to reduce oxidative stress and activating adenosine-mediated RISK pathway to reduce cell death (Li et al., 2012). Consistent with our hypothesis, we observed that within an hour of reperfusion, exosome treatment significantly increased tissue level of ATP and NADH in the heart. We presumed that this increase resulted from increased glycolysis through replenishment by glycolytic enzymes from exosomes. It should be noted that in ischemic/reperfused myocardium, only glycolytic but not the ETC proteins were depleted (Li et al., 2012). In fact, the level of several ETC proteins was increased. Therefore increasing glycolytic flux in the reperfused ischemic myocardium would increase ATP production not only by glycolysis but also by oxidative phosphorylation. Similarly, we hypothesized that anti-oxidants such as peroxiredoxins and glutathione S-transferases in exosomes could supplement depleted cellular antioxidants in ischemic/reperfused myocardium. In line with our hypothesis, oxidative stress was reduced in exosome-treated mice. Finally, we also predicted that the presence of enzymatically active CD73 on exosomes would produce adenosine from extracellular ATP to activate RISK and enhance myocardial viability (Hausenloy and Yellon, 2004). The importance of RISK such as PI3 kinase–Akt in limiting reperfusion injury was recently highlighted in a position paper from the European Society of Cardiology (Ovize et al., 2010). Adenosine has been shown to be efficacious in limiting infarct size in animal models and human trials (Headrick and Lasley, 2009). More importantly, CD73 has been shown to be the major enzyme responsible for the formation of extracellular adenosine from released adenine nucleotides (Zimmermann, 2000). Consistent with the observed infarct size reduction, exosome treatment significantly increased Akt and GSK3 phosphorylation. Incidentally, pro-apoptotic phosphorylation of c-JNK was reduced. The cause of this reduction remains to be determined.