Emerging Roles of Extracellular Vesicles in Cardiac Repair and Rejuvenation

Am J Physiol Heart Circ Physiol 315: H733–H744, 2018. First published June 27, 2018; doi:10.1152/ajpheart.00100.2018.

REVIEW Cardiac Regeneration and Repair

Emerging roles of extracellular vesicles in cardiac repair and rejuvenation

X Faisal J. Alibhai,1 Stephanie W. Tobin,1 Azadeh Yeganeh,1 Richard D. Weisel,1,2 and Ren-Ke Li1,2 1Division of Cardiovascular Surgery, Toronto General Hospital Research Institute, University Health Network, Toronto, Ontario, Canada; and 2Division of Cardiac Surgery, Department of Surgery, University of Toronto, Toronto, Ontario, Canada Submitted 5 February 2018; accepted in ﬁnal form 17 June 2018

Alibhai FJ, Tobin SW, Yeganeh A, Weisel RD, Li R-K. Emerging roles of extracellular vesicles in cardiac repair and rejuvenation. Am J Physiol Heart Circ Physiol 315: H733–H744, 2018. First published June 27, 2018; doi:10.1152/ ajpheart.00100.2018.—Cell therapy has received signiﬁcant attention as a therapeutic approach to restore cardiac function after myocardial infarction. Accumu- lating evidence supports that beneﬁcial effects observed with cell therapy are due to paracrine secretion of multiple factors from transplanted cells, which alter the tissue microenvironment and orchestrate cardiac repair processes. Of these paracrine factors, extracellular vesicles (EVs) have emerged as a key effector of cell therapy. EVs regulate cellular function through the transfer of cargo, such as microRNAs and proteins, which act on multiple biological pathways within recipient cells. These discoveries have led to the development of cell-free therapies using EVs to improve cardiac repair after a myocardial infarction. Here, we present an overview of the current use of EVs to enhance cardiac repair after myocardial infarction. We also discuss the emerging use of EVs for rejuvenation-based therapies. Finally, future directions for the use of EVs as therapeutic agents for cardiac regenerative medicine are also discussed. aging; cell therapy; extracellular vesicles; myocardial infarction; rejuvenation

INTRODUCTION EXTRACELLULAR VESICLES After myocardial infarction (MI), the heart undergoes mal- Classification of EVs adaptive changes in shape and size in a process termed ventricular remodeling. The extent of ventricular remodeling di- EV is a broad term referring to membrane-bound vesicles rectly influences the development of cardiac dysfunction and that are released by cells. These vesicles fall into three main 1 2 3 the progression to heart failure. The heart has limited regener- categories: ) exosomes, ) microvesicles, and ) apoptotic bodies. Traditionally, EVs have been classified on the basis of ative capacity; therefore, interventions are needed to prevent size and mode of secretion (Fig. 1). Exosomes are vesicles the loss of contractile elements and enhance endogenous car- ~50–150 nm in size that arise from the endosome and are diac repair to limit detrimental ventricular remodeling. Al- released through multivesicular body (MVB) fusion with the though much attention has been given to cell therapy as a plasma membrane (45). Microvesicles range from ~20 nm to 1 treatment for MI, the therapeutic benefits achieved in clinical ␮m and are formed by direct budding from the cell membrane trials have been limited. This has led a number of research (75). Apoptotic bodies range from ~50 nm to 5 ␮m and are groups to develop new approaches to improve cardiac repair released by blebbing of cells undergoing apoptosis (5). As a post-MI. One promising approach is to supply the factors result of the increasingly recognized complexity and overlap- secreted by transplanted stem cells in a cell-free therapy. ping size ranges of EVs, more stringent classification criteria Extracellular vesicles (EVs) have emerged as key mediators of for EVs have recently been proposed (65, 90). For example, the stem cell function and are currently being studied by many International Society for Extracellular Vesicles has proposed investigators to prevent cardiac dysfunction after a MI. More- that isolated vesicle populations should be characterized using over, there is emerging interest in the use of EVs as mediators a combination of Western blot analysis, flow cytometry, nano- of rejuvenation, whereby EVs restore biological processes in particle tracking analysis, and transmission electron micros- aged individuals to that of a younger state. copy (65). Moreover, protein markers in four categories should be assessed in each vesicle preparation: 1) transmembrane or lipid-bound proteins, 2) cytosolic proteins, 3) intracellular proteins, and 4) extracellular proteins (65). Proper character- Address for reprint requests and other correspondence: R.-K. Li, Univ. Health Network, PMCRT, Rm. 3-702, 101 College St., Toronto, ON, Canada ization of isolated EVs is essential as a change in EV compo- M5G 1L7 (e-mail: [email protected]). sition can impact their therapeutic potential. Although a num- http://www.ajpheart.org 0363-6135/18 Copyright © 2018 the American Physiological Society H733 Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (142.150.190.039) on October 11, 2018. Copyright © 2018 American Physiological Society. All rights reserved. H734 EVs IN CARDIAC REPAIR AND REJUVENATION

2 Microvesicles

Fig. 1. Schematic of extracellular vesicle secretion. 1) Sorting of early endosomal cargo in endosomal sorting complex required for transport (ESCRT)-dependent and 1 Exosomes -independent manners leads to the formation of intraluminal vesicles (ILVs) and multive- Early sicular bodies (MVBs). Fusion of MVBs endosome with the lysosome releases ILVs into the compartment, where they are degraded. Fu- MVB sion of MVBs with the plasma membrane leads to the release of ILVs into the extracellular space where they are termed exosomes. 2) In contrast, microvesicles arise ILV from direct budding and pinching from the ESCRT dependent and plasma membrane to release the vesicle into independent sorting the extracellular space. 3) Finally, apoptotic bodies are formed during cell apoptosis as a result of cell blebbing, which releases cellular contents into the extracellular space. Lysosome Degradation

3 Apoptotic body ber of studies have used the term exosome to describe isolated lipid content in secreted EVs (73). Induction of autophagy EVs, no isolation method currently can distinguish exosomes increases fusion of MVBs with autophagosomes, whereas a and microvesicles of similar sizes, and, therefore, the broad reduction in autophagy increases secretion to compensate for term EV is used in this article (35, 57). lysosome dysfunction to dispose of cellular cargo.

REGULATION OF EV BIOGENESIS AND SECRETION ESCRT-Independent Regulation Endosomal Sorting Complex Required for Transport- A number of pathways regulate EV biogenesis in an Dependent Regulation ESCRT-independent manner. Neutral sphingomyelinase (N- SMase) controls intraendosomal transport through regula- The endosomal sorting complex required for transport tion of lipid microdomains; inhibition of N-SMase with (ESCRT) comprises more than 30 different proteins that as- GW4869 reduces exosome secretion (99). Rab-GTPases, semble into four complexes (ESCRT-0, ESCRT -I, ESCRT-II, such as Rab27a/b (80) and Rab35 (42), regulate EV secre- and ESCRT-III) and play a major role in regulating EV tion through regulation of vesicle docking to the plasma biogenesis (43). The ESCRT plays an important role in the membrane. Finally, cellular oxygen tension influences EV formation of intraluminal vesicles (ILVs) and MVBs from the biogenesis, as hypoxia increases EV secretion in a hypoxia- endosome (18). ILVs are subsequently released as exosomes inducible factor (HIF)-1␣-dependent manner (34, 105). Col- into the extracellular space after MVB fusion with the plasma lectively, these studies have demonstrated a number of membrane (Fig. 1). Knockdown of ESCRT components tumor cellular pathways that influence EV production through susceptibility gene 101, hepatocyte growth factor receptor ESCRT-dependent and -independent mechanisms. substrate (HRS), or signal transducing adaptor molecule 1 reduces MVB formation and exosome secretion (18). Another EV Cargo mechanism by which ESCRT regulates EV production is through an interaction with the autophagy pathway. Starvation- EVs carry diverse cargo, including mRNAs (101), noncod- induced autophagy reduces EV secretion, as shown using the ing RNAs (14, 15), proteins (33, 46), lipids (40), chromosomal K562 cancer cell line (28). In contrast, blockade of autophagy DNA (94), and mitochondrial DNA (84). In light of this with bafilomycin A1 increases EV secretion in SH-SY5Y diverse cargo, a number of databases have been developed for neuroblastoma cells (6). Mechanistically, the autophagy ma- researchers to search EV cargo. These include ExoCarta (89), chinery and ESCRT components interact to regulate EV bio- Vesiclepedia (46), and EVpedia (52). High-throughput analy- genesis; autophagy related (Atg)12-Atg3 interacts with Alix, ses of EVs have been essential in identifying the factors carried an ESCRT component, to regulate endolysosomal trafficking by EVs. For example, next-generation sequencing studies have (76). These studies support that a balance exists between revealed that EVs carry a diverse set of noncoding RNAs, autophagy and EV secretion. Changes in autophagy flux also including transfer RNAs, Y-RNAs, microRNAs (miRNAs), affect EV composition, as impaired autophagy increases amy- and piRNAs (14, 88). Mass spectrometry studies have also loid precursor protein COOH-terminal fragments and sphingo- identified a large number of proteins associated with EVs (9,

AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00100.2018 • www.ajpheart.org Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (142.150.190.039) on October 11, 2018. Copyright © 2018 American Physiological Society. All rights reserved. EVs IN CARDIAC REPAIR AND REJUVENATION H735 20, 87). Given this diverse cargo, it is important to understand stimulated cells (4). Moreover, EVs derived from endothelial how different factors affect EV cargo, as this will ultimately cells subjected to hypoxia or activated with TNF-␣ exhibit affect their therapeutic effect. Factors that influence EV cargo different RNA and protein profiles compared with unstimu- include the EV subpopulations present after isolation, the cell lated cells (25). This approach has also been applied to CPCs, source from which the EVs are derived, and the cellular as EVs isolated from CPCs subjected to hypoxia exhibit activity of EV-producing cells. different miRNA expression compared with control cells (3). Changes in EV cargo as a result of cell activation alter EV EV Subpopulations Carry Different Cargo function. HIF-1␣ overexpression in MSCs alters EV miRNA EV subpopulations separated by distinct biological charac- and protein content, and isolated EVs exhibit a greater proan- teristics carry different cargo (1, 58, 102, 113). Subpopulations giogenic potential compared with EVs isolated from normoxic of EVs isolated by differential centrifugation exhibit different MSCs (34). EVs isolated from CPCs subjected to hypoxia RNA profiles compared using Bioanalyzer (21). Low-density more effectively enhanced cardiac repair compared with con- EV RNA content closely correlates with cellular content, trol CPC-EVs (3). whereas high-density EVs do not (56). Proteomic signatures Collectively, these studies have demonstrated a number of also differ between EV subpopulations, as shown by mass factors that influence EV secretion and cargo. Moreover, these spectrometry analysis of proteins isolated from microvesicles, studies raise a number of questions for the clinical implemen- low-density EVs, and high-density EVs (108). Moreover, sep- tation of EVs. As recently reviewed by Reiner et al. (82), there aration of EV subpopulations based on cell surface markers is a need for the standardization of methods used to produce revealed that CD9-, CD63-, and CD81-positive EVs have and purify EVs. Different EV isolation methods may prefer- different protein content when examined using mass spectrom- entially enrich different EV subpopulations leading to the etry (53). Separation of B16-F10-EV subpopulations by asym- enrichment of different cargo. Moreover, the conditions in metric flow field-flow fractionation revealed that EVs of dis- which cells are cultured will affect EV secretion and cargo. tinct size (60–80 nm, EXO-S; and 90–120 nm, EXO-L) Together, these differences can lead to discrepancies in the exhibit different expression of cell surface makers and cargo therapeutic effects of isolated EVs. There is also a need to when examined by mass spectrometry (113). Notably, EXO-S standardize EV quality; this can be achieved through the were enriched in proteins involved in the endosome/protein development of toxicology tests, potency assays, and consis- secretion, whereas EXO-L were enriched in proteins involved tency assays to assess batch to batch variability (82). in mitotic spindle and IL-2/STAT5 signaling (113). Differ- ences in EV subpopulations are important functionally, as they EV THERAPY FOR THE TREATMENT OF MI affect recipient cells differently. H5V endothelial cells exhibit different mRNA expression signatures when treated with high- Cell therapy has been used for the treatment of MI to limit versus low-density EVs isolated by differential centrifugation ventricular remodeling and heart failure progression. Cell ther- (108). apy was first demonstrated in the 1990s, whereby a number of different cell types were shown to improve repair when trans- EV Cargo and Cell Sources planted into the injured heart (59, 79, 98). Since these initial reports, cell therapy has become an active area of research with EVs from different cellular origins carry different cargo and, numerous clinical trials investigating the therapeutic use of thus, can have different effects on recipient cells. Comparison stem cells for promoting cardiac repair post-MI (for reviews, of human mesenchymal stem cell (MSC) and cardiosphere- see Refs. 13 and 78). One major challenge facing cell therapy derived cell (CDC)-derived EVs using miRNA arrays demon- is that the transplanted cells do not differentiate into functional strated that EV miRNA profiles differ on the basis of cell cardiomyocytes and do not persist long term. Instead, trans- source (37). Protein signatures also differ between EVs derived planted cells secrete a number of paracrine factors that enhance from different cell sources as a comparison of cardiac progen- cardiac repair; this was first demonstrated using conditioned itor cells, and human bone MSCs revealed a number of differ- media from cultured MSCs (32, 96). Subsequent studies have entially expressed proteins using a semiquantitatively label- demonstrated that EVs are the key effectors of conditioned free proteomic approach (9). Notably, cardiac progenitor cells media for enhancing cardiac repair post-MI (7, 44, 55). Since (CPC)-derived EVs were enriched in proteins involved in these initial reports, EVs have emerged as important mediators regulating redox balance, whereas MSC-EVs were enriched in of cell therapy, and a number of groups have shown that EVs extracellular matrix-associated proteins (9). Cell source can can be used to improve cardiac repair post-MI. also affect EV uptake, as EVs isolated from myogenic C2C12, EVs have received attention as a potentially powerful cell- melanoma B16F10, and bone marrow-derived dendritic cells free therapy to prevent remodeling after a MI. Using a variety exhibit different tissue distribution when injected intrave- of cell sources, isolation methods, administration routes, and nously (107). Differences in EV cargo, due to differences in preclinical models, EVs have been shown to be beneficial in cell source, have important physiological effects as cardiac the treatment of MI. EVs derived from MSCs (Table 1), progenitor cell-EVs were found to be more effective than cardiac stem cells (Table 2), induced pluripotent stem cell/ MSC-EVs for improving cardiac repair post-MI (9). embryonic stem cells (Table 3), and nonstem cell sources Cell Activity Influences EV Secretion and Cargo (Table 4) have been shown to be beneficial in the treatment of MI. The mechanisms by which EVs improve infarct healing Cellular activity also influences EV secretion and cargo, are similar to those that have been shown to occur with cell which, in turn, affect EV function. Stimulation of endothelial therapy. EVs act at multiple levels of infarct healing to reduce cells with TNF-␣ increases EV secretion compared with un- cell death, increase resident stem/progenitor cell proliferation,

EV Source (Isolation Method) Experimental Design (Species, MI Model) Outcome Identified Factors Reference Rat adipose-derived MSCs 400 ␮g of EVs injected intravenously Reduced infarct expansion miR-126 67 overexpressing miR-126 (PC) after LAD ligation (rat, P/O) at 4 wk post-MI Rat bone marrow MSCs (PC) 5 ␮g injected into the ischemic Reduced infarct size and None identified; increased recipient 63 myocardium 5 min before improved EF at 7 days cell autophagy reperfusion (rat, I/R) post-MI Rat adipose-derived MSCs (UC) 400 ␮g injected intravenously 5 min Reduced infarct size 3 h None identified; increased recipient 22 after reperfusion (rat, I/R) post-MI cell Wnt Signaling Mouse bone marrow MSCs EVs derived from 2 ϫ 107 MSCs Reduced infarct expansion miR-210 116 stimulated with hypoxia for injected in the border zone after and improved EF at 4 24 h (UC) LAD ligation (mouse, P/O) wk post-MI Rat bone marrow MSCs (PC) 20 ␮g injected in the infarct border Improved EF at 7 days No specific factor studied; identified 86 (rat, P/O) post-MI high expression of miR-29 and miR-24 in EVs Human umbilical cord-derived 400 ␮g injected intravenously after Improved EF at 5 wk Platelet-derived growth factor D 69 MSCs transfected with LAD ligation (rat, P/O) post-MI activated Akt (UC-SG) Mouse bone marrow MSCs EVs were injected intravenously at Reduced left ventricular miR-210 104 (UC) day 0 and day 6 post-MI (mouse, dilation and improved P/O) EF at 4 wk post-MI Rat bone marrow MSCs (PC) 80 ␮g injected in the border zone 1 h Improved EF and reduced None identified 95 after LAD ligation (rat, P/O) infarct expansion at 4 wk post-MI Human umbilical cord-derived 400 ␮g injected intravenously after Improved EF and reduced None identified 115 MSCs (UC-SG) ligation (rat, P/O) fibrosis 4 wk post-MI Rat bone marrow MSCs EVs harvested from 4 ϫ 106 MSCs Improved EF and reduced miR-19a 111 overexpressing GATA4 (PC) injected intramyocardially after infarct expansion at 4 LAD ligation (rat, P/O) wk post-MI Mouse bone marrow MSCs 1 ␮g injected in the border zone after Reduced infarct expansion miR-22 29 given IPC (PC) LAD ligation (mouse, P/O) 4 wk post-MI Human bone marrow MSCs 80 ␮g injected in the border zone Improved EF and reduced None identified; increased 12 stimulated with hypoxia for after LAD ligation (rat, P/O) infarct expansion at 4 angiogenesis in the infarct region 72 h (UC) wk post-MI Mouse bone marrow MSCs 12.5 ␮g injected into the pericardial Reduced infarct size 1 day miR-21 68 (UC) sac 24 h before I/R (mouse, I/R) post-MI EF, ejection fraction; EVs, excellular vesicles; I/R, ischemia-reperfusion; MI, myocardial infarction; PC, precipitation; P/O, permanent occlusion of the left anterior descending coronary artery (LAD); MSCs, mesenchymal stem cells; SEC, size exclusion chromatography; UF, ultrafiltration; UC, ultracentrifuge; UC-SG, ultracentrifuge-sucrose gradient. modulate inflammatory responses, and enhance angiogenesis damaged tissue and participate in cardiac repair (30, 77). (Fig. 2). Cardiac repair mediated by immune cells is influenced by cell polarization (toward a proinflammatory or proreparative state) EV Therapy to Reduce Cellular Apoptosis and associated cytokines secreted by these cells. EVs interact EVs reduce infarct size post-MI by limiting cardiomyocyte with infiltrating immune cells to influence immune cell polar- death. For example, EVs derived from CDCs (24, 31), MSCs ization and cytokine secretion. For example, CDC-EVs induce (63), induced pluripotent stem cells (106), and plasma (103) macrophage polarization to a reparative phenotype through the decrease infarct size post-MI by reducing cardiomyocyte apo- transfer of miR-181b (24). CDC-EVs have also been shown to ptosis. Regulation of autophagy by EVs is one mechanism by deliver a Y-RNA fragment to increase IL-10 secretion in the which EVs limit cardiomyocyte death. EV transfer of miR-30a infarcted myocardium and improve cardiac repair (14). EVs limits cardiomyocyte death in response to hypoxia due to can also affect T cell function as dendritic cell EVs increase the increase of the autophagy core regulators beclin-1, Atg12, and activation of CD4-positive T cells, which, in turn, alters the light chain (LC)3II/LC3I (110). MSC-EVs increase host car- expression of proinflammatory cytokines in the infarcted myo- diomyocyte autophagy through modulation of AMP-activated cardium (62). Further supporting a role of EVs in regulating protein kinase/mechanistic target of rapamycin (mTOR) sig- immune responses post-MI, endogenously derived EVs have naling to limit cell death after ischemia-reperfusion (I/R) injury been recently shown to regulate infarct healing. Locally se- (63). EVs can also interact with cell surface receptors to creted EVs in the infarcted myocardium were shown to be influence cell survival; EV-associated heat shock protein 70 taken up by infiltrating immune cells and reduce IL-6, chemo- binding to cardiomyocyte Toll-like receptor 4 increases cell kine (C-C motif) ligand (CCL)2, and CCL7 secretion (66). survival after I/R in rats (103). Taken together, EVs act to modulate inflammatory responses toward a reparative state to enhance cardiac repair. EV Therapy to Target Inflammatory Responses EV Therapy Increases Angiogenesis Another mechanism by which EVs confer cardioprotection is through modulation of immune responses post-MI. Immune EVs derived from MSCs (12), induced pluripotent stem cells cells infiltrate the infarcted myocardium post-MI to remove (2), CDCs (31), CPCs (3), and fetal rat cardiomyocytes (83)

EV Source (Isolation Method) Experimental Design (Species, MI Model) Outcome Identified Factors Reference CDCs Human CDCs (UF and PC) 1) In rats, 105 ␮g and 305 ␮g of CDC-EVs Reduced infarct size 48 h miR-181b 24 were injected into the LV cavity over a post-MI period of 20 s with an aortic cross clamp 20 min after reperfusion 2) In pigs, 7.5 mg of CDC-EVs were injected in the ischemic myocardium 30 min after reperfusion Human CDCs (UF and PC) 1) 15 mg of EVs injected intramyocardially Reduced infarct size; None identified; reduced cardiac 31 30 min after reperfusion (pig) improved LV function inflammation, reduced 2) 7.5 mg of EVs injected intramyocarially 4 fibrosis, and increased infarct wk post-MI (pig, I/R) vascular density Human CDCs transfected 10 min after reperfusion 10 ␮gofEVs Reduced infarct size 48 h Y-RNA fragment 14 with Y-RNA (PC) injected into the LV cavity over a period post-MI of 20 s with an aortic cross clamp (rat, I/R) Human CDCs (PC) 2.8 ϫ 109 EVs were injected IM immediately Improved EF and reduced miR-146a 44 after LAD ligation or at 3 wk post-MI infarct expansion (mouse, I/R) CPCs Human CPCs (UC) 80 ␮g/kg injected intramyocardially 30 min Reduced infarct size 1 No specific factor studied; 3 after reperfusion (rat, I/R) day post-MI; improved identified 32 different EF and reduced LV miRNAs targeting multiple fibrosis 4 wk post-MI biological pathways Human CPCs (SEC) EVs derived from 1 ϫ 106 CPCs injected Improved EF and No specific factor studied; heat 87 intramyocardially (rat, P/O) increased infarct vessel shock factor-1 shown to density 4 wk post-MI regulate the CPC secretome Human ESC-derived CPCs EVs derived from ~5 ϫ 105 cells injected by Improved EF and reduced None identified 50 (UC) guided closed-chest transcutaneous LV dilation echocardiography at 2–3 wk post-MI (mouse, P/O) Rat CPCs subjected to 12 h 5 ␮g/kg injected in the border zone after Improved EF and reduced miR-292-5p, miR-20a, miR- 36 of hypoxia (UC) reperfusion (rat, I/R) cardiac fibrosis 21 days 199a-5p, miR-17, miR-103, post-MI miR-210, and miR-15b Human CPCs (PC and UC) 30 ␮gor300␮g of EVs injected Improved EF and reduced miR-210, miR-132, and 10 intramyocardially 1 h after ligation (rat, infarct expansion 7 miR-146a P/O) days post-MI Human CPCs (UC) EVs injected after reperfusion at three Improved EF and reduced Pregnancy-associated plasma 9 different sites totalling 1 ϫ 1011 EVs in scar size 4 wk post-MI protein-A 100 ␮l (rat, I/R) CDCs, cardiosphere-derived cells; CPCs, cardiac progenitor cells; EF, ejection fraction; EVs, extracellular vesicles; I/R, ischemia-reperfusion; LV, left ventricular; MI, myocardial infarction; PC, precipitation; P/O, permanent occlusion of the left anterior descending coronary artery (LAD); SEC, size exclusion chromatography; UC, ultracentrifuge; UF, ultrafiltration. administered post-MI enhance angiogenesis. The transfer of addressed by future studies. First, no perfect isolation method proangiogenic miRNAs is one mechanism by which EVs exists as non-EV factors are often coisolated (64, 92, 100). enhance angiogenesis. Transfer of miR-126-3p from CD34- These potential contaminating factors make it difficult to de- positive-derived EVs to host endothelial cells increases angio- termine the exact role of EV versus non-EV factors with genesis after hindlimb ischemia (72). Injection of EVs over- respect to the mechanism of action. A number of different expressing miR-126 increases capillary density and reduces controls have been used to account for nonspecific EV isola- infarct expansion in rats post-MI (67). Transfer of miR-222 tion, such as inhibition of secretion using the N-SMase inhib- and miR-143 from EVs derived from fetal cardiomyocytes itor GW4869 (44). However, because of the broad role of subjected to ischemic preconditioning increases the angiogenic N-SMase in cellular functions (17), it is possible that GW4869 activity of recipient endothelial cells in vitro (83). Transfer of affects more than EV secretion. Additional studies inhibiting miR-31 from adipose cell-derived stem cell-derived EVs to EV biogenesis and secretion at multiple levels (ESCRT depen- endothelial cells increases angiogenesis in vitro and in vivo dent and independent) are needed to better define the contri- (49). EVs are also capable of transferring proangiogenic pro- bution of EVs to cardiac repair. Moreover, additional studies teins; transfer of Jagged1 from HIF-1␣-overexpressing MSC- are needed to define the components of the EV and EV-free EVs increases tube formation in vitro and Matrigel plug an- fractions. Improved fractionation of EVs using next-generation giogenesis in vivo (34). Collectively, these studies have dem- methods, such as those using microfluid-based platforms (19, onstrated a number of mechanisms by which EVs can enhance 109, 113), may help better define the factors carried by EVs. cardiac repair post-MI. A number of different factors have been suggested to me- Although numerous studies have demonstrated beneficial diate the beneficial effects of EVs; these include transfer of effects of EVs, there are a number of limitations that need to be EV-associated miRNAs, Y-RNA fragments and proteins, in-

EV Source (Isolation Method) Experimental Design (Species, MI Model) Outcome Identified Factors Reference iPSCs Mouse fibroblasts derived 100 ␮g injected in the peri-infarct region Improved EF and reduced infarct No specific factor studied; 2 iPSCs (UC) 48 h after I/R (mouse, I/R) expansion at 35 days post-MI identified 33 miRNAs and ~200 proteins enriched in iPSC-EVs Mouse cardiac fibroblast- EVs derived from 2.5 ϫ 106 iPSCs Reduced cardiomyocyte miR-21 and miR-210 106 derived iPSCs (PC) injected intramyocardially after LAD apoptosis at 24 h post-MI ligation (mice, I/R) Human iPSC-derived EVs secreted by 1.4 ϫ 106 cells over 24 h Improved EF and reduced infarct 16 different conserved miRNAs 27 cardiac progenitor cells injected intramyocardially by guided expansion by 7 wk after EV enriched in EVs (UC) closed-chest transcutaneous administration echocardiography 3 wk post-MI (mouse, P/O) Embryonic stem cells HuES9.E1-derived MSCs 4or16␮g/kg injected intravenously 5 Reduced infarct size 1 day post- None identified; EVs increased Akt 7 (SEC) min before reperfusion (mouse, I/R) MI; reduced LV dilation activation by 1 h post-I/R improved EF at 4 wk post-MI HuES9.E1-derived MSCs 0.4 ␮g injected intravenously 5 min before Reduced infarct size 3 h after None identified 55 (SEC) reperfusion (mouse) reperfusion Mouse embryonic stem Two 10-␮l EV doses injected in the Reduced LV dilation, improved miR-294 51 cells (UC-SG) border zone after LAD ligation (mouse, EF at 4 wk post-MI P/O) EF, ejection fraction; EVs, extracellular vesicles; iPSCs, induced puripotent stem cells; I/R, ischemia-reperfusion; LV, left ventricular; MSCs, mesenchymal stem cells; PC, precipitation; P/O, permanent occlusion of the left anterior descending coronary artery (LAD); SEC, size exclusion chromatography; UC, ultracentrifuge; UC-SG, ultracentrifuge-sucrose gradient. teraction of EV surface proteins with cardiomyocyte cell sur- shown in Tables 1–4. Standardized methods for EV therapy face receptors, and interaction of EV-associated enzymes with (EV production, isolation, and treatment) will help ensure IGF-binding protein 4, which increase local IGF-1 (Tables reproducibility of results across independent research facilities. 1–4). Moreover, EVs have been suggested to interact with a These challenges in developing best practices for EV therapy number of different cells in the remodeling heart, including have been recently reviewed (82). resident stem cells (51), infiltrating immune cells (24), cardiomyocytes (103), and endothelial cells (83). On the basis of the REJUVENATION FOR THE TREATMENT OF MI array of mechanisms proposed, it is more likely that the mechanisms by which EVs confer cardioprotection are multi- Aging is associated with an increase in the occurrence of MI, factorial. Improved methods of tracking EVs in vivo are and prevalence is greater in those over 65 yr of age (16, 41). needed to determine which cell types interact with adminis- Moreover, age is also a strong predictor of mortality after MI, tered EVs. Moreover, future single cell high-throughput studies both in hospital and after discharge (70, 85). Although many examining the cellular pathways affected by administered EVs clinical trials have demonstrated the feasibility and safety of may help better define the mechanisms by which EVs regulate cell therapy for treatment of MI, the functional benefits ob- cellular functions to improve cardiac repair. served in clinical trials have not matched the results obtained in Finally, improved standardization of EV therapy is needed. preclinical studies (for a review, see Ref. 78). Our group has Although some studies have investigated different treatment demonstrated that both host and donor age significantly im- regimens (31, 44), there is high variability between studies, as pacts the beneficial effects of cell therapy for cardiac repair

Table 4. Preclinical studies demonstrating beneﬁcial effects of EV therapy post-MI using nonstem cell sources

EV Source (Isolation Method) Experimental Design (Species, MI Model) Outcome Identiﬁed Factors Reference Dendritic cells Mouse bone marrow-derived 10 ␮g of EVs injected intravenously 1 day Improved ejection fraction at 7 None identiﬁed 62 dendritic cells (PC) post-MI (mouse, P/O) days post-MI Plasma/serum Rat plasma (UC) ~5 ϫ 1010 EVs injected intravenously 15 Reduced infarct size 2 h after Heat shock 103 min before thoracotomy (rat, I/R) reperfusion protein-70 Mouse Serum (PC) 10 ␮g of EVs injected in the left ventricular Reduced infarct size 24 h post-MI miR-21 39 free wall before ligation (mouse, P/O) Cardiomyocytes Rat fetal cardiomyocytes 15 ␮g of EVs injected in to the ischemic Increased capillary density in the miR-222 and 83 subjected to ischemia myocardium after LAD ligation (mouse, peri-infarct region 4 wk miR-143 (UC) P/O) post-MI EV, extracellular vesicles; I/R, ischemia-reperfusion; MI, myocardial infarction; PC, precipitation; P/O, permanent occlusion of the left anterior descending coronary artery (LAD); UC, ultracentrifuge.

Stem cells Differentiated cells rejuvenation of aged cells is due to rejuvenation of the cell EV secretion profile; future studies should investigate how cellular rejuvenation affects EV function. Another approach is to rejuvenate the repair capacity of aged individuals before MI. Our laboratory has shown that reconstitution of aged mice with young bone marrow cells reduces infarct expansion and improves cardiac function post-MI by repopulating the heart with cardiac resident bone marrow-derived progenitor cells (61). Mechanistically, we found that after young bone marrow reconstitution, stem cell antigen 1 (Sca-1)-positive cells EVs home to the heart and improve cardiac repair responses, suggestive of rejuvenation (60, 97). Collectively, these studies demonstrate the feasibility and potential approaches of Cardiac tissue rejuvenation for the treatment of MI. Cardiomyocytes Endothelial cells EVs AS MEDIATORS OF REJUVENATION Although this is a new area of research with a limited number of studies, stem cell-derived EVs show promise as a rejuvenation therapy. Stem cell-derived EVs are capable of Resident stem cells Immune cells enhancing or restoring the function of a number of biological systems ex vivo and in vivo. Notably, EVs have been shown to limit or reverse age-associated dysfunction of the heart, skeletal muscle, hematopoietic stem cells, and brain. Marbán and colleagues (38) have shown that CDC implantation into aged rats improves diastolic function and reduces cardiomyocyte hypertrophy. This is, in part, mediated through CDC secretion of EVs that act to reduce the number of Cardiomyocyte apoptosis Angiogenesis Stem cell proliferation Immunomodulation senescent cells in the heart. Consistent with a role of EVs in skeletal muscle rejuvenation, implantation of CDCs in the aged rat heart improves exercise capacity; mechanistically, the authors suggested that secretion of EVs from implanted CDCs Improved cardiac repair results in systemic rejuvenation (38). EVs also show promise for the rejuvenation of hematopoietic stem cell (HSC) function. Fig. 2. Stem cells and differentiated cells secrete extracellular vesicles (EVs), which can be isolated for therapeutic uses. EVs administered before or after Ex vivo treatment of aged HSCs with young MSC-EVs in- myocardial infarction (MI) interact with immune cells, resident cardiac stem creases aged HSC engraftment and reduces myeloid lineage cells, cardiomyocytes, and endothelial cells to enhance infarct healing. Inter- skewing (54). Finally, EVs have also been used to counteract action of EVs with these cell types modulates immune responses, increases the cognitive decline associated with aging. Injection of hypo- resident stem cell proliferation, reduces cardiomyocyte apoptosis, and en- hances angiogenesis. Together, these actions help to limit ventricular dysfunc- thalamic stem/progenitor cell-derived EVs into the hypotha- tion and improve cardiac repair post-MI. lamic third ventricle for 4 mo improves the cognitive perfor- mance of aged mice (114). Although these studies support that EVs can be used to rejuvenate processes that decline with age, post-MI (47, 112). Thus, there is a need to identify ap- the mechanisms by which EVs rejuvenate aged cells are not proaches to rejuvenate aged cells or individuals to the fully understood and are multifactorial. For example, CDC- functional state found in younger individuals to improve EV-mediated rejuvenation increases telomerase activity to re- cardiac repair post-MI. duce the number of senescent cells in the heart (38). However, One approach that has been used is to rejuvenate aged stem the factors transferred by CDC-EVs to increase telomerase activ- cells ex vivo before transplant. Our group has shown that ity were not investigated. Rejuvenation of aged HSCs by MSC- MSCs derived from aged patients can be rejuvenated by EVs is attributed to the transfer of autophagy-associated mRNAs, seeding aged MSCs on a collagen scaffold containing immo- which increase the autophagy of aged HSCs (54). Finally, reju- bilized VEGF and basic fibroblast growth factor (48) or by venation using hypothalamic stem/progenitor cell-derived EVs overexpressing neuron-derived neurotrophic factor (NDNF) was achieved through the transfer of miRNAs, which act to (91). Rejuvenation using both approaches improved the repair reduce hypothalamic inflammation and maintain neural stem cell capacity of aged MSCs compared with nonrejuvenated cells. populations (114). Further studies are needed to better define the Rejuvenation has also been investigated using cardiac stem cell mechanisms by which EVs mediate rejuvenation. Moreover, populations. Treatment of cardiac stem cells from decompen- whether rejuvenation using EVs improves cardiac repair post-MI sated hearts with rapamycin and resveratrol ex vivo restores the has not yet been investigated. cardiac stem cell repair capacity in a mouse MI model (8). Sussman and colleagues (74) demonstrated that overexpression EVs as Targets for Rejuvenation of Pim-1 kinase increases telomere length and the proliferative capacity of aged cardiac progenitor cells. With respect to EVs, A number of investigators have demonstrated that EV cargo it is not known whether the functional benefits achieved with changes with aging; currently, these changes are being used as

AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00100.2018 • www.ajpheart.org Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (142.150.190.039) on October 11, 2018. Copyright © 2018 American Physiological Society. All rights reserved. H740 EVs IN CARDIAC REPAIR AND REJUVENATION biomarkers of aging (81, 93). In addition to changes in cargo, 4. Akbar N, Digby JE, Cahill TJ, Tavare AN, Corbin AL, Saluja S, the concentration of circulating EVs declines with aging in Dawkins S, Edgar L, Rawlings N, Ziberna K, McNeill E, Johnson E, Aljabali AA, Dragovic RA, Rohling M, Belgard TG, Udalova IA, both rats and humans (11, 26). Changes in cargo and concen- Greaves DR, Channon KM, Riley PR, Anthony DC, Choudhury RP; tration are associated with changes in EV function. Monocytes Oxford Acute Myocardial Infarction (OxAMI) Study. Endothelium- and B cells exhibit greater activation after incubation with aged derived extracellular vesicles promote splenic monocyte mobilization in plasma EVs ex vivo compared with young plasma EVs (26). myocardial infarction. JCI Insight 2: 93344, 2017. doi:10.1172/jci. Increased miR-183-5p in bone-EVs impairs bone marrow stem insight.93344. 5. Akers JC, Gonda D, Kim R, Carter BS, Chen CC. Biogenesis of cell function and induces senescence (23). Given that EVs may extracellular vesicles (EV): exosomes, microvesicles, retrovirus-like ves- be playing a pathophysiological role in the aging process, it is icles, and apoptotic bodies. J Neurooncol 113: 1–11, 2013. doi:10.1007/ possible that EVs may also be a target for rejuvenation. One s11060-013-1084-8. interesting approach to target EVs for rejuvenation could be to 6. Alvarez-Erviti L, Seow Y, Schapira AH, Gardiner C, Sargent IL, selectively remove pathogenic vesicles in vivo by hemofiltra- Wood MJ, Cooper JM. Lysosomal dysfunction increases exosome- mediated alpha-synuclein release and transmission. Neurobiol Dis 42: tion of EVs from the blood (71). Removal of “toxic” EVs may 360–367, 2011. doi:10.1016/j.nbd.2011.01.029. be beneficial in a number of models and may contribute to 7. Arslan F, Lai RC, Smeets MB, Akeroyd L, Choo A, Aguor EN, rejuvenation. Timmers L, van Rijen HV, Doevendans PA, Pasterkamp G, Lim SK, de Kleijn DP. Mesenchymal stem cell-derived exosomes increase ATP CONCLUSIONS levels, decrease oxidative stress and activate PI3K/Akt pathway to enhance myocardial viability and prevent adverse remodeling after myo- EVs show great promise for the treatment of MI; however, cardial ischemia/reperfusion injury. Stem Cell Res (Amst) 10: 301–312, much is still unknown. Improved understanding of the factors 2013. doi:10.1016/j.scr.2013.01.002. carried by EVs and how these factors are transferred to recip- 8. Avolio E, Gianfranceschi G, Cesselli D, Caragnano A, Athanasakis ient cells to elicit cardioprotection are needed. Moreover, E, Katare R, Meloni M, Palma A, Barchiesi A, Vascotto C, Toffoletto B, Mazzega E, Finato N, Aresu G, Livi U, Emanueli C, Scoles G, although preclinical studies have shown that EVs improve Beltrami CA, Madeddu P, Beltrami AP. Ex vivo molecular rejuvena- cardiac repair post-MI, no human studies have been conducted. tion improves the therapeutic activity of senescent human cardiac stem In the future, clinical studies will be essential to ensure that this cells in a mouse model of myocardial infarction. Stem Cells 32: 2373– therapy is safe, feasible, and potentially effective in humans. 2385, 2014. doi:10.1002/stem.1728. Although much remains unknown about EVs, promising pre- 9. Barile L, Cervio E, Lionetti V, Milano G, Ciullo A, Biemmi V, Bolis S, Altomare C, Matteucci M, Di Silvestre D, Brambilla F, Fertig TE, clinical studies support the use of EVs as a cell-free approach Torre T, Demertzis S, Mauri P, Moccetti T, Vassalli G. Cardiopro- to improve cardiac repair and to rejuvenate aged individuals by tection by cardiac progenitor cell-secreted exosomes: role of pregnancy- preventing ventricular remodeling after MI. associated plasma protein-A. Cardiovasc Res 114: 992–1005, 2018. doi:10.1093/cvr/cvy055. ACKNOWLEDGMENTS 10. Barile L, Lionetti V, Cervio E, Matteucci M, Gherghiceanu M, Popescu LM, Torre T, Siclari F, Moccetti T, Vassalli G. Extracellular We thank Dr. Leigh Botly for help with manuscript preparation and editing. vesicles from human cardiac progenitor cells inhibit cardiomyocyte GRANTS apoptosis and improve cardiac function after myocardial infarction. Cardiovasc Res 103: 530–541, 2014. doi:10.1093/cvr/cvu167. F. J. Alibhai is a recipient of a Canadian Institutes of Health Research 11. Bertoldi K, Cechinel LR, Schallenberger B, Corssac GB, Davies S, Postdoctoral Fellowship. This work was supported by Canadian Institutes of Guerreiro ICK, Bello-Klein A, Araujo ASR, Siqueira IR. Circulating Health Research Grant 332652 (to R.-K. Li). R.-K. Li holds a Tier 1 Canada extracellular vesicles in the aging process: impact of aerobic exercise. Research Chair in Cardiac Regeneration. Mol Cell Biochem 440: 115–125, 2018. doi:10.1007/s11010-017-3160-4. 12. Bian S, Zhang L, Duan L, Wang X, Min Y, Yu H. Extracellular DISCLOSURES vesicles derived from human bone marrow mesenchymal stem cells No conflicts of interest, financial or otherwise, are declared by the authors. promote angiogenesis in a rat myocardial infarction model. J Mol Med (Berl) 92: 387–397, 2014. doi:10.1007/s00109-013-1110-5. AUTHOR CONTRIBUTIONS 13. Brunt KR, Weisel RD, Li RK. Stem cells and regenerative medicine— future perspectives. 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