Evaluation of the cardioprotective potential of extracellular vesicles – a systematic review and meta-analysis

Cardiovascular diseases are the main cause of death worldwide, demanding new treatments and interventions. Recently, extracellular vesicles (EVs) came in focus as important carriers of protective molecules such as miRNAs and proteins which might contribute to e.g. improved cardiac function after myocardial infarction. EVs can be secreted from almost every cell type in the human body and can be transferred via the bloodstream in almost every compartment. To provide an all-encompassing overview of studies investigating these beneficial properties of EVs we performed a systematic review/meta-analysis of studies investigating the cardioprotective characteristics of EVs. Forty-three studies were investigated and catalogued according to the EV source. We provide an in-depth analysis of the purification method, size of the EVs, the conducted experiments to investigate the beneficial properties of EVs as well as the major effector molecule encapsulated in EVs mediating protection. This study provides evidence that EVs from different cell types and body fluids provide cardioprotection in different in vivo and in vitro studies. A meta-analysis was performed to estimate the underlying effect size. In conclusion, we demonstrated that EVs from different sources might serve as a promising tool for treating cardiovascular diseases in the future.

In the following, we will sort the publications by the main source of EVs investigated in the studies and extracted the EV purification method (detailed description in supplemental part), size, injury model, if applicable the main effector in EVs as well as the investigated EV marker.
Results per EV source. Cardiomyocytes. Cardiomyocytes are, next to fibroblasts and endothelial cells, one of the most abundant cell types in the mammalian heart. Due to their importance in cardiac function, researchers are extensively studying their physiological properties 52 as well as their capabilities to secrete EVs 33 . Publications investigating cardiomyocyte derived EVs are summarized in Table 2. Garcia et al. showed that starvation of the immortalized cardiomyocyte cell line H9c2 increased the secretion of EVs with altered composition and enhanced capability to induce tube formation 21  the EV composition of primary cardiomyocytes and H9c2 cells after preconditioning with hypoxia or isoflurane which resulted in significantly altered cargo composition of the cell-derived EVs 33 . A similar study confirmed that EVs from ischemic cardiomyocytes protected against oxidative-induced lesion, promoted angiogenesis and proliferation of endothelial cells in vitro. The authors suggested that miR-222 and miR-143, encapsulated in hypoxic EVs, are partially responsible for the pro-angiogenic effects. In vivo experiments confirmed enhanced angiogenesis due to hypoxic EV treatment after MI but no reduction of fibrosis was observed 30 . Zhang et al. identified HSP20, as a possible mediator of cardioprotection transferred by EVs 43 . The authors postulated that HSP20-overexpressing primary cardiomyocytes secrete EVs with elevated levels of HSP20 compared to EVs from control cells. HSP20 additionally promoted proliferation, migration and tube formation. Unfortunately, due to methodological limitations in this study, not all observed effects can be attributed to HSP20 in EVs. Basic EV-related experiments such as EM-images and testing for EV markers were not conducted in this study 43 .
In a following publication, the authors investigated whether cellular HSP20 overexpression and thereby elevated HSP20 levels in EVs might protect the myocardium in diabetes 24 . Compared to EVs from control cells, EVs secreted from cardiomyocytes HSP20 exhibited elevated levels of p-protein kinase B (pAkt), survivin and superoxiddismutase 1 (SOD1) and protected against in vitro hyperglycemia-triggered cell death 24 .
Cardiac progenitor cells. Cardiac progenitor cells (CPCs) represent a heterogeneous group of cells throughout the heart and the surrounding vessels which can be activated upon injury and contribute to the cardiac renewal 53,54 . Recent findings indicated that CPC-derived EVs might have a predominant role in transmitting cardioprotective mediators to the damaged heart. With our defined search criteria, we found four articles investigating the protectivity of CPC-derived EVs (Table 3).
Barile et al. isolated CPCs from patients who underwent heart valve surgery 13 . Apoptosis was reduced in the starved and reperfused immortalized cardiomyocyte HL-1 cell line, which were treated with CPC-derived EVs. Additionally, tube formation in in human umbilical vein endothelial cells (HUVECs) and angiogenesis in vivo were enhanced by those EVs. In vivo experiments indicated that a treatment with EVs improved the left ventricular ejection fraction (LVEF) and reduced scar tissue after MI. Levels of miR-210 and miR-132, were elevated in CPC-derived EVs compared to EVs from fibroblasts. The authors suggested that these miRNAs down-regulate ephrin A3, protein-tyrosine phosphatase 1 (PTP1) and RasGTPase-activating protein (RasGap)-p120 and thereby transduced their beneficial effects in the recipient cells and tissue 13 .
In a similar study, the authors challenged H9c2 cells with H 2 O 2 and performed an in vivo model of I/R injury 34 . CPC-derived EVs were again able to attenuate apoptosis, reduced the amount of terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) positive cells and pro-apoptotic caspase 3/7 activation. Additionally, the transcription factor GATA4-responsive miR-451 was overexpressed in CPC-derived EVs 34    programmed cell death protein 4 (PDCD4), which is involved in apoptosis. The authors could show that H9c2 cells, which were pre-treated with CPC-derived EVs, were more resistant to H 2 O 2 treatment. Interestingly, EVs from pre-treated source were even more protective, presumably due to the elevated miR-21 levels and thereby reduced PDCD4 levels in the recipient cells 39 . Gray et al. investigated EVs derived from hypoxia stimulated CPCs, that were able to promote tube formation and decreased profibrotic gene expression 22 . Hypoxic treatment of the cells indeed altered the EV composition. 11 miRNAs were upregulated in EVs derived from hypoxic CPCs compared to EVs, which were isolated from normoxic cells. Additionally, those EVs were able to improve cardiac function in a model of I/R-injury. EVs from CPCs, which were previously subjected to 12 h hypoxia, were able to reduce fibrosis in vivo. EVs from cells, which were not treated with hypoxia or experienced a shorter treatment, had attenuated effects 22 .
Cardiosphere derived cells. Cardiac surgical biopsy specimens exhibit the potential of secreting a heterogeneous population of cardiac cells called cardiosphere derived cells (CDCs) 55 . Publications matching our search criteria and investigating EVs from these cells are stated in Table 4.
EVs from CDCs inhibited apoptosis and promoted proliferation in neonatal cardiomyocytes 35 . In vivo data also demonstrated reduced scar mass, resulting in elevated contractility and increased viable mass in a MI-injury model upon treatment with CDC-derived EVs. Blocking the generation of EVs with GW4869 in vitro and in vivo resulted in enhanced apoptosis, diminished cardiomyocytes proliferation, increased scar mass and reduced function of the heart. The authors identified miR-146a as key mediator of cardioprotection 35 . GW4869 is able to inhibit sphingomyelinases, thereby blocking the ceramide-dependent budding of intraluminal vesicles into the lumen of MVBs which reduces biogenesis of EVs 56,57 . CDC-derived EVs additionally decreased caspase 3/7 activity in human embryonic stem cell-derived cardiomyocytes upon cobalt chloride treatment 25 and enhanced tube formation in HUVECs 28 . The in vivo relevance of CDC derived EVs was confirmed by a study conducted in 2017 showing that EV associated miR-181b could decrease the infarct size after I/R injury 47 .
Fibroblasts. The main function of fibroblasts is to produce extracellular matrix and thereby stabilize the surrounding tissue 58 . New studies attribute those cells to far more complex signalling within the heart which is in part based on EVs. Recent findings indicated that fibroblast-derived EVs might contribute to cell migration and proliferation of cardiac fibroblasts whereas others demonstrated a detrimental impact 17,33 . Publications matching our search criteria and investigated fibroblast-derived EVs are summarized in Table 5.
Cardiac fibroblast-derived EVs induced pathological hypertrophy in cardiomyocytes in vitro 17 . miR-21-3p was identified as specific mediator in those EVs which targets sorbin and SH3 domain-containing protein 2 (SORBS2) and PDZ and LIM domain 5 (PDLIM5) inducing hypertrophy 17 . Others showed that EVs from fibroblasts had diminished protective capabilities. Ibrahim et al. demonstrated the benefits of EVs from cardiosphere derived cells (CDCs) by different in vivo and in vitro experiments. In contrast, normal human dermal fibroblasts (NHDFs) were not able to transmit comparable protection 35 . These findings were supported by others 47 . Wang et al. 45 additionally supported this notion by demonstrating that EVs from induced pluripotent stem cells (iPS) were able to protect against myocardial I/R-injury while EVs from cardiac fibroblast had a diminished effect. Nevertheless, cardiac fibroblast-derived EVs significantly reduced caspase 3/7 activity after H 2 O 2 treatment in H9c2 cells compared to control 45 and enhanced proliferation/migration in cardiac fibroblasts 33 .

Mesenchymal stem cells.
Mesenchymal stem cells (MSCs) are adult stem cells with the potential to differentiate into multiple other cell types. The tremendous capabilities of MSCs are also attributed to their great potential of secreting important factors for the control of haematopoiesis or immunomodulation 59 . We found several publications fitting our search criteria and investigated whether MSC-derived EVs promote protection (Table 6).
Feng and Co-workers identified miR-22 as potential cardioprotectant which was secreted via EVs from ischemic-preconditioned MSCs 10 . EVs from these cells were able to reduce fibrosis in infarcted hearts. Methyl CpG binding protein (Mecp2) is a direct target of miR-22, which was enriched in the investigated EVs and contributed to reduced cardiac damage 10 .
MSC-derived EVs also contributed to cardioprotection in a sepsis model induced by cecal ligation and puncture. EV treatment increased the ejection fraction of mice and improved the survival in polymicrobial sepsis 9 . The authors could demonstrate that tumor necrosis factor-α (TNFα), interleukin 6 (IL-6) and IL-1β secretion was reduced in macrophages after the treatment with MSC-derived EVs in vitro. The authors attributed the cardioprotective properties to miR-223 from WT-MSC derived EVs and thereby targeting Semaphorin-3A (Sema3A)  and Signal transducer and activator of transcription 3 (Stat3) 9 . In addition to enhanced HUVEC tube formation and reduced fibrosis, Teng and Co-workers could show that MSC-derived EVs reduced the inflammation in the infarcted area 44 . These results were supported by an in vivo model of MI where MSC-derived EVs increased cardiac stem cell tube formation and reduced fibrosis. The authors suggested that this distinct miRNA cargo might be the reason for the cardioprotective properties of MSC-derived EVs 46 . Additionally, in a model of MI, administration of EVs reduced cardiac damage and improved systolic function 23 .
In comparison to EVs from wild types, transfection enforced expression of specific proteins in host cells and might further enhance the resulting EV capabilities. EVs derived from MSC CXCR4 were able to reduce caspase 3 activity and induced upregulation of IGF-1α and pAkt in neonatal cardiomyocytes 36 . Implantation of a cell patch, which was treated with EVs from MSC CXCR4 , was significantly more potent to reduce the infarct size compared to cell patches with control EVs. These data provided evidence that the protectivity from MSC-derived EVs may be enhanced by specific cargo loading 36 . In addition, miR-221 was investigated as EV-mediated cardioprotective factor. Rat ventricle cardiomyocytes which were cultivated under hypoxic conditions were more robust against this stimulus when incubated with supernatant from MSC GATA-4 41 . However, the experiments conducted in this study do not allow the conclusion that MSC-derived EVs are protective. For instance, isolated EVs were not transferred to other cells to investigate their ability of cytoprotection 41 . In a study conducted two years later, EVs from MSC GATA-4 protected neonatal cardiomyocytes from hypoxia-induced cell death 42 . The cardiac function, after ligation of the left anterior descending coronary artery, was also improved. miR-19a, which was enriched in EVs from MSC GATA-4 , was identified as the effector mediating the protection 42 . miR-19a targets phosphatase and tensin homolog (PTEN), inhibiting cell proliferation and induces apoptosis 60 . Even though the authors showed that EVs from MSC GATA-4 mediated improved cell function and protection, EVs from control MSCs were still protective as well 42 . In an early study conducted in 2009, Lai and co-workers investigated the protective effects of EVs secreted from human embryonic stem cell-derived mesenchymal stem cells (HuES9.E1). Isolated EVs were able to reduce the infarct size after myocardial I/R injury 20 . The underlying signalling pathways include decreased oxidative stress as well as increased Akt and glucogen synthase kinase-3α/β phosphorylation (GSK-3α/β) 50 . EVs from, previously with H 2 O 2 treated, MSCs additionally contributed to reduced oxidative stress induced cell death by inhibition of PTEN. miR-21 was identified as key mediator of those protective properties 48 .   Body fluids. In contrast to the previously investigated EV sources, the original sources of EVs in body fluids are diverse. Numerous publications were identified by our search criteria, studying EVs from different body fluids and are further investigated in the following (Table 7). Vicencio et al. hypothesized that an established cardioprotective treatment has an impact on EVs and their cargo 7 . The authors analysed whether blood derived EVs from a remote ischemic preconditioned (rIPC) donor were more protective than those from an untreated source. Surprisingly, several in vitro and in vivo experiments revealed that EVs from treated and untreated source were protective in a similar fashion. EVs in general were able to reduce cell death and ultimately the infarct size. The authors suggested that HSP70 on the EV surface, might interacted with toll-like receptor 4 (TLR-4) on the recipient cells, thereby triggering a signal cascade which activates intracellular HSP27 which further promotes cardioprotection 7 . In a similar study, EVs from a rIPC group and the corresponding control group were isolated from serum and analysed 37 . The predicted effector, miR-144 was not upregulated upon rIPC treatment in EVs but in the serum of the treated animals. In contrast, the precursor form of miR-144 was enriched in EVs. The authors suggested that miR-144 is important for cardioprotection but EVs are probably not the main carrier and mediator of this protective miRNA 37 . A similar study revealed that EVs, isolated from rIPC-rats, could reduce the infarct size in a model of in vivo I/R injury 15 . Minghua et al. supported these findings by demonstrating that EVs from rIPC rats decreased apoptosis in an in vitro H 2 O 2 stress model as well as decreased infarct size in an I/R injury in vivo model. The authors suggested that miR-24 encapsulated in EVs is able to transduce the protective properties 49 . A different approach investigated EV mediated protection in an ex vivo IPC Langendorff model. The perfusates from preconditioned rat hearts were collected and used to treat hearts prior to infarction. EV-depleted perfusates caused increased infarct size compared to the EV-containing perfusates 8 . In another IPC study, the authors investigated whether this treatment might promote a change in the DNA content in EVs. The authors could not detect any difference in the number of sequenced gene fragments between treatment and control 16 . In a rat model, rIPC resulted in an increase of miR-29a in serum-derived EVs 40 . miR-29a is a key regulator of tissue fibrosis and the increase of this miRNA might contribute to the finding of reduced fibrosis after rIPC treatment. Nevertheless, the exclusive protectivity of EVs was not investigated 40 . As mentioned previously, HSPs, in or on the surface of EVs, might mediate cardioprotection. Wang et al. developed a transgenic mouse model with cardiac specific overexpression of HSP20 24 . EVs isolated from mouse HSP20 serum had higher HSP20 levels compared to EVs from control mice. The cardiac contractile function of diabetic mice HSP20 was also improved compared to control mice. Attenuating the release of EVs by GW4869 in vivo 61 resulted in reduced HSP20-mediated cardiac function, evaluated by left ventricular internal dimension-diastole (LVIDd) and LVEF in diabetic mice 24 .
EVs from diabetic rats or patients were not able to protect cardiomyocytes from hypoxia/reoxygenation injury in vitro. EVs from healthy donors instead did 27 . Similar results were obtained in a study subjecting healthy and diabetic rats to rIPC and evaluating the protectivity in an in vitro model of hypoxia reoxygenation. Cell death of HL-1 cells was reduced if treated with EVs from healthy rats but no effects were observed with EVs from diabetic rats 32 .
An observative study was conducted in 2016, comparing the plasma EV cargo of patients with MI and patients with stable angina. Indeed, the authors identified several EV proteins which were upregulated upon MI 18 . A similar study investigated in a porcine in vivo model the influence of ischemic preconditioning on EV cargo. EVs from preconditioned animals had an altered mRNA cargo related to proteins which are commonly associated with the protective effects of ischemic preconditioning 31 .
Other cell types. Several studies which were found by our search criteria did not fit in the previously described groups. We will therefore describe the benefits of EVs from these sources in Table 8.

Ref.
Source

IPS cells transduce their beneficial properties also through EVs.
In vitro experiments indicated that iPS-derived EVs inhibit proapoptotic caspase 3/7 activation after H 2 O 2 treatment of H9c2 cells 45 . The conducted experiments also identified two specific miRNAs miR-21 and miR-210 which potentially transmitted the cardioprotective properties of iPS cell-derived EVs although no confirmation experiments were performed. In an in vivo model of I/R injury, apoptosis of cardiomyocytes was additionally reduced after treatment with iPS-derived EVs 45 .
Recently adipose tissue has proven to be a reliable source of stem cells 62 . Kang et al. were able to show that EVs from adipose-derived stem cells (ASCs), preconditioned with endothelial differentiation medium, induced HUVEC tube formation. miR-31 was identified as mediator of these pro-angiogenic effects by targeting the factor-inhibiting hypoxia inducible factor-1 (HIF-1) (FIH1) 14 .
Gu and co-workers performed several in vitro experiments to investigate whether EVs from endothelial progenitor cells (EPCs) might protect H9c2 cells from angiotensin II induced hypertrophy 19 . Apoptosis and cell viability were improved by EPC-derived EVs. Additionally, the isolated EVs induced phosphorylation of Akt and endothelial nitric oxide synthase (eNOS) in angiotensin II treated H9c2 cells 19 .
The beneficial effects of EVs from cardiac stem cells were investigated in a mouse model of doxorubicin induced dilated cardiomyopathy. Mice received cardiac stem cell-derived EVs which were able to improve cardiac function, reduce fibrosis in the myocardium as well as TUNEL positive cells, respectively DNA fragmentation 51 .
Endothelial cells, overexpressing HIF-1 secreted EVs with higher contents of miR-126 and miR-210. The specific cargo of these EVs resulted in an activation of pro-survival kinases and induced a glycolytic switch in the recipient CPCs. EVs additionally reduced the cellular damage during hypoxic conditions in vitro 38 . Human amniotic fluid stem cells (hAFS) secreted EVs which were able to mediate antiapoptotic effects in vitro. Hypoxic preconditioning of hAFS additionally enhanced the protectivity of EVs and furthermore modulated the miRNA cargo of those EVs 26 . Surprisingly, EVs from HUVECs, cultivated under hyperglycaemic conditions, were not able to protect primary adult cardiomyocytes from hypoxia-reoxygenation whereas EVs from regular cultivated cells were protective 27 . In a recent study, Obata demonstrated that adiponectin is able to stimulate ceramide secretion by EVs, reducing the intracellular level of ceramides in vitro and in vivo 29 .
To evaluate whether the described properties of EVs are indeed cardioprotective we performed a meta-analysis. Pooling two independent studies 44,46 which investigated the number of capillaries after EV treatment (Fig. 3) and two studies investigating the protective effect of EVs in a setting of hypoxia-reoxygenation (Fig. 4) 7,27 . Both analysis indicated significant effects favouring EV treatment.

Discussion
This is the first all-encompassing systematic review/meta-analysis investigating the cardioprotective effects of EVs. 43 studies were chosen for analysis and data extraction. We found that EVs derived from different cell types as well as from different body fluids, mediated beneficial properties. Only EVs from fibroblasts had, as described in some investigated studies, harmful effects and mediated hypertrophy. We evaluated the EV specific experiments, investigating the predominant mediators of protection carried by EVs and categorized the studies by  EV source and the experiments performed to investigate the positive capabilities of EVs. We finally conducted a meta-analysis and verified the positive properties of EVs by combining the results of independent studies. Investigating EVs and their beneficial properties is often challenging and several basic experiments are needed to ensure that the described effects in the corresponding investigation are transferred by EVs. Recommendations, first described in 2013, what kind of experiments are needed or which isolation or purifications methods are suitable to ensure an appropriate EV preparation were given by several publications 11,12,[63][64][65] . Nevertheless, some of these guidelines have been published only recently and several of the here included publications were published before these guidelines. These guidelines are constantly changing due to novel developments and new insights in the field of EV research, making it impossible to introduce guidelines for a broader group of researchers. Applying those, partly strict, criteria to all investigated publications in our review may therefore not be suitable. Nevertheless, EM images and identification of EV-marker are in our understanding mandatory in EV research.
EVs from numerous sources were successfully isolated by the included studies. Few studies did not perform the necessary experiments to ensure an appropriate EV preparation as described previously 11,63,64 . We have to mention that the existing methods to verify EV properties are far from absolute. For instance, techniques to measure the concentration of EVs or to describe the morphological properties might not distinguish between EVs and particles with a similar size range, as reviewed by others 66 . The indicated sizes, stated in the results part, have also to be considered as a range since EVs with only one size cannot be isolated so far. Only a variety of different experiments is suitable to absolutely ensure that the isolated particles are indeed EVs. Additionally, precipitation and basic ultracentrifugation methods might result in co-isolation of non-EV particles and thereby hold the risk for impurities 67,68 . A study considering these problems was performed in 2016, investigating EV marker after different centrifugation steps in the resulting pellets 64 . Due to the great variety and no gold standard EV purification protocol, we therefore only distinguished between UC, precipitation-based and other protocols in our systematic review. Further research and development of more appropriate methods for EV purification and detection of multiple EV sources are needed to ensure comparable results from different studies. We would like to point out that in some of the described studies immortalized cell lines such as H9c2 and HL-1 cells were used to investigate EV properties. For instance, undifferentiated H9c2 cells might not represent a cardiac specific phenotype and the results might therefore be met with caution 69,70 .
The cardioprotective properties of EVs from different sources have been investigated by several studies included in this review. The main effector of these benefits are miRNAs such as miR-210 or miR-132, inhibiting apoptosis or enhancing tube formation 13 . Especially miR-21 and miR-210 are encapsulated in EVs from numerous sources and mediated protection in different ways. The beneficial effects of EV-derived miRNAs in other diseases such as autoimmune hepatitis 71 or sepsis 72 has additionally shown by other groups. The conclusions whether EVs from genetically engineered cells, from a treated donor or regularly secreted are cardioprotective, are inconsistent. In several studies which investigated EVs from a genetically engineered origin, the EVs from control cells had also beneficial capabilities, even if the effects were not that distinct. These observations were also made in studies investigating the EVs from previously treated sources such as ischemic preconditioning. We therefore conclude that EVs in general are protective and that these properties might be enhanced by an appropriate treatment of the EV source or transfection of the host cells. EVs from several body fluids have also been proven to mediate positive properties. These EVs might originate from different sources making it difficult to identify specific molecules mediating the protective effects. The results have therefore to be met with caution and further in vitro analysis might be needed to investigate which treatment triggers the release of EVs from a distinct cell type mediating the protection.
Even though the same cell types were investigated in different studies and similar experiments were performed to evaluate the EV mediated protection, a stringent meta-analysis was not possible due to the lack of consistency. Tube formation or cell survival experiments were conducted by numerous studies. But these, for example, were performed with MSC-derived EVs either from genetically engineered or wild type cells or with different tube forming cells 36,44,46 . To evaluate exemplarily the protective properties of EVs, we combined the data of two studies investigating the formation of capillaries in the heart after EV treatment. One publication evaluated the number of capillaries after direct EV treatment whereas the second article investigated if cells, previously treated with EVs, promote angiogenesis after implantation into the heart 44,46 . Combination of both data sets revealed significant difference favouring EV treatment. The meta-analysis from two different studies conducted by the same group indicated that EVs protect cardiomyocytes from hypoxia/reoxygenation injury 7,27 .
Taken together, these findings demonstrate the urgent need for more consistency and adequately designed studies, not only for the EV-purification methods but also for the performed experiments investigating the effect of EVs.
Even though the protectivity of EVs has been proven in several in vitro and in vivo models the translation to humans will be a major challenge in the future. Unlike other approaches which failed to accomplish the translation from bench to bedside, the conserved mechanism of EV release and uptake in many species has the great potential that EVs might be of special use in the near future 65,73 .

Conclusion
EVs are important mediators of cardiac protection and deliver specific molecules such as proteins and miRNAs to the recipient cells. The great majority of the investigated publications could proof the benefit of EVs especially by reducing cardiac damage or induction of angiogenesis. The inconsistency, in EV purification methods and experiments investigating EV mediated benefits, made it difficult to recapitulate data from different studies. Our evident conclusion is that EVs are important mediators of protection in cardiovascular diseases. These findings substantiate the assumption that EVs can serve as a potent therapeutic in the future. An urgent need, especially for a general EV-purification protocol, remains and has to be addressed in the future. Criteria for considering studies for this review. Types of studies. We included experimental research studies, which investigated EVs in in vitro or in vivo models (animal and human). We did not differentiate between exosomes, microvesicles or apoptotic bodies as long as the vesicular origin and effect matched our inclusion criteria.

Types of interventions.
We included all studies, which investigated the protective effect of EVs. We did not further restrict type of intervention as long as the assumed protective effects of EVs or EV-derived components were the main focus of the study.
Search methods for identification of studies. We identified trials through systematic searches of the following bibliographic databases on May 24 th , 2018: The following search strategy was applied to identify matching studies: #1 extracellular vesicle* OR EV OR exosome* OR microvesicle* #2 cardio OR cardiac OR heart OR cardioprotection #3 protection OR *conditioning #4 #1 AND #2 AND #3. Reference lists of all primary studies and review articles were checked for additional references. We imported citations from each database into a reference management software (EndNote X8, PA, USA) and removed duplicates. Titles and abstracts of the selected articles were screened independently by two authors (SW, SK) and coded "suitable" or "not suitable". In case of a disagreement a third author (CS) was questioned.
Data extraction and quality assessment. Data from all suitable publications were reviewed, rated and extracted by two authors independently (SW, SK). In case of a disagreement a third author was questioned and the issue was discussed until the authors reached an agreement. The following information were extracted from every article: first author, year of publication, EV purification method, size of the detected EVs, damage model, mediator in EVs which transmitted the protectivity, assay to measure the beneficial effect of EVs and investigated EV marker. The EV purification methods were distinguished in methods based on ultracentrifugation, precipitation or others.