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Evaluating early and delayed cardioprotection by plasma exosomes in simulated ischaemiareperfusion injury

Evaluating early and delayed cardioprotection by plasma exosomes in simulated... BioscienceHorizons Volume 8 2015 10.1093/biohorizons/hzv001 Research article Evaluating early and delayed cardioprotection by plasma exosomes in simulated ischaemia–reperfusion injury Jiawen Liu*, Derek M. Yellon and Sean M. Davidson Hatter Cardiovascular Institute, 67 Chenies Mews, London WC1E 6HX, England *Corresponding author: Division of Biosciences, University College London, Gower Street, London WC1E 6BT, England. Tel: +44 756 321 3012. Email: jiawen.liu.11@ucl.ac.uk Supervisors: Prof. Derek M. Yellon, Dr. Sean M. Davidson, Hatter Cardiovascular Institute, University College London, 67 Chenies Mews, London WC1E 6HX, England. Tel: +44 203 447 5732; Fax: +44 203 447 9505. Email: s.davidson@ucl.ac.uk Reductions in cardiac infarct size can be achieved in ischaemic preconditioning, a procedure which subjects the heart to intermit- tent, non-lethal cycles of ischaemia and reperfusion. Similar cardioprotection can be induced upon preconditioning distal tissues such as the upper limb, and this procedure is called remote ischaemic preconditioning. Nano-sized, cell-derived vesicles known as exosomes have been shown previously to be capable of inducing cardioprotection when administered acutely, and we hypothesized that exosomes produced after remote ischaemic preconditioning (RIPC) enhances the observed cardioprotection. We also investigated whether exosomes can confer cardioprotection 24 h later. We isolated exosomes from the plasma of three healthy male volunteers before and after they underwent four cycles of 5 min upper limb ischaemia and 5 min reperfusion, and then administered the exosomes to primary rat ventricular cardiomyocytes isolated from male Sprague Dawley rats. Cardiomyocytes were subjected to 2.5 h of simulated ischaemia and 1 h simulated reperfusion. We assessed cardiomyocyte via- bility with propidium iodide staining and observed significant reductions in cardiomyocyte death when control exosomes were administered either 30 min or 24 h before hypoxia. Exosomes obtained after RIPC induced a similar degree of cardioprotection at both time points. As miRNA-144/451 have been proposed to mediate RIPC, we investigated whether introducing antagomiRs of miRNA-144/451 with the exosomes would attenuate cardioprotection after 24 h. No significant differences in cardioprotection were observed, suggesting that miRNA-144/451 may not be directly involved in this model. Our findings suggest that regardless of their origin from control or RIPC hearts, exosomes per se can be used to induce cardioprotection either acutely or after 24 h. Key words: exosomes, remote, ischaemic, preconditioning, reperfusion, cardioprotection Submitted on 5 November 2014; accepted on 21 January 2015 Introduction in a dog after ligation of its coronary vessels, and findings from subsequent experiments conducted in the 19th century Between the 16th and 18th centuries, the concepts of angina supported the idea that obstructions within the coronary cir- pain, vessel degeneration and sudden death due to a blockage culation starved the myocardium of oxygen and inflicted in the heart gained currency following the contributions of damage (Leibowitz, 1970a,b). ideas and anatomical findings from Benivieni (1507), Amatus Lusitanus (1560), Petrus Salius Diversus (1586), Bellini Coronary angioplasty is the modern-day cornerstone (1683) and Thebesius (1708) (Leibowitz, 1970a,b). In 1698, procedure to promptly liberate the occluded vasculature and to Pierre Chirac first described his observations of cardiac arrest reperfuse the ischaemic myocardium, but despite improvements © The Author 2015. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.  Research article Bioscience Horizons • Volume 8 2015 in technologies and quality of care, the number of individuals COX-2 signalling pathway, and these effects were reversed living with coronary heart disease (CHD) remains high and is when the 2 miRNAs were knocked down. increasing. The British Heart Foundation (BHF) reported that One variation of IPC is known as remote ischaemic precon- there are still at least 2.3 million people in the UK living with ditioning (RIPC), whereby the ischaemic bouts are performed CHD and more than £7 billion has been spent within England on distant tissues such as the upper limb rather than the myo- alone to treat cardiovascular diseases (BHF Cardiovascular cardium (Fig. 1). RIPC has been demonstrated in animal mod- Disease Statistics, Townsend, 2014). The number of coronary els to reduce infarct size and oxidative stress (Hausenloy and angioplasties performed in the UK in 2012 has more than dou- Yellon, 2007), but the underlying mechanism(s) has yet to be bled from a decade ago, but acute myocardial infarctions fully elucidated. Various groups have proposed the role of the (AMIs) still accounted for at least 175 000 inpatient episodes autonomic (Redington et al., 2012) nervous system (Basalay (BHF Cardiovascular Disease Statistics, Townsend, 2014), and et al., 2012; Mastitskaya et al., 2012; Donato et al., 2013) there is an ~10% mortality rate associated with AMIs and/or the secretion of extracellular vesicles that range from 30 (Hausenloy and Yellon, 2007). A plausible cause may be ‘lethal to 1000 nM in diameter. Previously characterized as unimport- reperfusion injury’, a phenomenon that arises when the sudden ant cell debris, extracellular vesicles have attracted waves of influx of oxygenated blood into the ischaemic region initiates renewed interest as emerging studies unravel their roles in various signalling pathways that culminate in widespread car- transporting mRNA, miRNA, plasma membrane receptors diomyocyte necrosis (Hausenloy and Yellon, 2011; Sanada and signalling ligands across cells as well as their involvement et al., 2011). This overwhelms endogenous myocardium repair in angiogenesis, inflammation and cytopro tection (Anderson mechanisms and results in the formation of scars that subse- et al., 2010; Park et al., 2010; Lee et al., 2012; Yellon and quently compromise cardiac function. Preventing reperfusion Davidson, 2014). Several studies have reported associations of injury thus represents a possible strategy to reduce the eco- nomic burden and prevalence of AMIs. In 1986, Murry et al. observed that bouts of ischaemic stresses impeded the rate of ATP depletion, and they postu- lated that with these bouts, harmful catabolites could simul- taneously be expelled and this would delay the onset of irreversible cardiomyocyte injury (Murry et al., 1986). Using canine hearts, the group experimented with four cycles of 5 min circumflex artery occlusion and 5 min reperfusion fol- lowed by a sustained 40 min occlusion and observed marked improvements in cardiomyocyte viability compared with control hearts (Murry et al., 1986). Murry et al. termed their procedure of ischaemic bouts as ischaemic preconditioning (IPC), and IPC has been repeatedly demonstrated over the subsequent two decades to improve cardiac function in humans and other animal models (For review, see Hausenloy and Yellon, 2011). Classical IPC-induced protection is biphasic—the early phase occurs immediately and lasts for 2–3 h, and the delayed phase occurs 12–24 h later and persists for 48–72 h (Hausenloy and Yellon, 2010). Since early protection is immediate, the underlying mechanism is more likely to involve secreted proteins as opposed to lengthy gene tran- scription and regeneration processes, of which would be more prominent during the delayed phase of protection. De novo protein synthesis of distal cardioprotective factors could be triggered by small non-coding nucleic acids known Figure 1. Schematic illustration of possible underlying mechanisms of as microRNAs (miRNAs), and specific miRNAs have already RIPC. RIPC is a procedure involving cycles of non-lethal ischaemia and been suggested to be involved—for example, Wang et al. reperfusion of distant tissues such as the upper limb instead of the myocardium. RIPC has been demonstrated in animal models to reduce (2012) observed significant elevations of miR-144/451 in IPC infarct size and oxidative stress after a prolonged and lethal ischaemic hearts compared with sham hearts, and IPC-mediated cardio- challenge. The intermittent ischaemic bouts could stimulate the protection was absent in miR-144/451 knock-out mice. autonomic nervous system and/or triggers the secretion of, as yet, These findings were complementary with those from Zhang unidentified humoral cardioprotective factors which can subsequently et al. (2010), who reported that the ectopic expression of activate the reperfusion injury salvage kinase (RISK), JAK-STAT, miR-144/451 enhanced cardiomyocyte survival via the acti- ERK/MAPK and Akt/PI3 K kinase pathways in the myocardium and lead vation of the CUG triplet repeat-binding protein 2 (CUGBP2)/ to tissue salvage. 2 Bioscience Horizons • Volume 8 2015 Research article circulating microvesicles (100–1000 nM in diameter) with the 2012; Yellon and Davidson, 2014) and whether the cell is dis- progression of cardiovascular diseases (Chung et al., 2007; eased or healthy (Taylor and Taylor, 2008; Huang et al., 2013). Fichtlscherer et al., 2010; Kanhai et al., 2013), but relatively Minor cellular stresses can also alter exosomal content, as less is known about the roles of nano-sized (30–100 nM in observed in exosomes derived from endothelial cells undergo- diameter) extracellular vesicles known as exosomes. Exosomes ing non-lethal hypoxia and inflammation ( de Jong et al., are formed when the luminal membranes of multivesicular 2012), and in patients who underwent RIPC, their exosomes bodies (formed within the late endosome) invaginate, and contained a greater proportion of miR-144 and 451 than fusion of these multivesicular bodies with the cell membrane before undergoing RIPC (D.M. Yellon et al., unpublished releases exosomes into the circulation (Fig. 2). Exosomes are data). known to contain tetraspanins (CD9, CD81), fusion proteins (Annexins, GTPases), lipid rafts, heat-shock proteins (Hsp70, Recent evidence suggests that exosomes protect against Hsp90) and ongoing studies speculate the ability of exosomes simulated ischaemia–reperfusion injury when administered to enclose humoral cardioprotective factors (Calzolari et al., acutely (Lai et al., 2010; Chen et al., 2013), but it is not 2006; Jeanneteau et al., 2012; Hirsch et al., 2013; Przyklenk, known whether exosomes can induce protection in a delayed 2014) that activate the reperfusion injury salvage kinase setting. Delayed cardioprotection might be more relevant in (RISK), JAK-STAT, ERK/MAPK and Akt/PI3 K kinase clinical procedures where ischaemia–reperfusion injury can signalling pathways (Kristiansen et al., 2005; Serejo et al., be predicted, such as in organ transplantation, coronary 2007; Hausenloy and Yellon, 2008; Lim et al., 2010) in the artery bypass graft (CABG) surgery or planned coronary myocardium to mitigate cardiomyocyte damage during an angioplasty (Hausenloy and Yellon, 2010). Hence in this AMI. Variations of exosomal content can arise depending on study, our primary aim was to investigate whether human the type of cell the exosome originates from (Vlassov et al., plasma exosomes could confer delayed cardioprotection in Figure 2. Schematic illustration of the formation of exosomes and their release into the circulation. Exosomes are formed from the invagination of the late endosome (the resultant entity is known as a multivesicular body) and released into the circulation when the multivesicular body fuses with the plasma membrane. Exosomes have been characterized to contain tetraspanins (CD9, CD81), fusion proteins (Annexins, GTPases), lipid rafts and heat-shock proteins (Hsp70, Hsp 90). Variations in exosomal content can arise depending on the type of cell the exosomes originate from. 3 Research article Bioscience Horizons • Volume 8 2015 primary rat ventricular cardiomyocytes undergoing simu- Preparation of RIPC human plasma lated ischaemia–reperfusion injury in vitro. If delayed cardio- exosomes protection is observed, we hypothesized that this process was mediated by exosomal delivery of miRNAs into cardiomyo- After collection of the first blood sample, the same volunteers cytes. As mentioned previously, RIPC significantly up- underwent RIPC. A standard blood pressure cuff was used to regulates cardioprotective miR-144 and 451. Therefore, the perform four cycles of 5 min upper limb ischaemia and 5 min secondary aim of this study was to investigate whether incu- reperfusion (Fig. 3). A second blood sample was collected bating exosomes with antagomiRs (miRNA-inhibitors) of from each volunteer and centrifuged (as per above) to obtain miR-144 and 451 would abolish any cardioprotection a sample of RIPC plasma exosomes. conferred. Characterization of exosomes with Methods nanoparticle tracking analysis Statement of ethical approval We used nanoparticle tracking analysis (NTA) to confirm the presence of exosomes. The rate of Brownian particle motion in Animals were treated in accordance with the Animals solution is dependent on particle size, and particle movements (Scientific Procedure) act 1986 published by the UK Home are monitored by directing a laser beam (405 nm) towards the Office and the Guide for the Care and Use of Laboratory solution and using a high-sensitivity camera (Nanosight Animals published by the US National Institutes of Health LM10-HS) to capture the scattered light. The NTA software (Publication No. 95-23, revised 1996). For human samples, will then determine the diameter of particles based on their these experiments were conducted according to Declaration movement over time. of Helsinki principles. All human and animal studies were approved by the appropriate institutional review boards, spe- Preparation of rat cardiomyocyte isolation cifically in accordance with the animal project license PPL No. 70/7140. The study of human samples was performed buffer according to ethics approval reference 13/LO/0222. The rat cardiomyocyte isolation buffer was made up with 130 M NaCl, 5.4 M KCl, 1.4 M MgCl , 0.4 M Na HPO and Preparation of human plasma exosomes 2 2 4 4.2 M HEPES. The buffer was maintained at 37°C and pH After written consent, three healthy male volunteers (30–45 7.4. Next, 10 mM glucose, 20 mM taurine and 10 mM cre- years old) each provided one blood sample. Blood was drawn atine were added, and the solution was filtered with a sterile from the antecubital vein via a butterfly syringe into citrated vacuum filter pump. vaccutainers™. The blood samples were centrifuged at 1600 × g for 20 min at 4°C to obtain plasma, followed by centrifugation Preparation of perfusate solutions at 10 000 × g for 30 min at 4°C to separate extraneous cell rem- nants and platelets. The final sample of exosomes was then iso - The filtered rat cardiomyocyte isolation buffer was divided lated following two additional centrifugations at 100 000 × g into five tubes and labelled solutions 1–5. Supplementary for 60 min at 4°C and diluted into aliquots with Dulbecco’s material online in Table 1 summarizes the additions made to phosphate- buffered saline (PBS). each solution. Figure 3. Schematic illustration of exosome extraction. Blood samples were collected from healthy volunteers before and after they underwent four cycles of 5 min upper limb ischaemia and 5 min reperfusion. Blood samples were centrifuged at 1600 × g for 20 min at 4°C to obtain plasma, followed by centrifugation at 10 000 × g for 30 min at 4°C to separate unwanted cell remnants and platelets. The final sample of exosomes was then acquired after two further centrifugations at 100 000 × g for 60 min at 4°C and diluted into aliquots with Dulbecco’s PBS. 4 Bioscience Horizons • Volume 8 2015 Research article for 8–10 min and filtered through iron mesh to obtain the final Isolation of primary rat ventricular solution of ventricular cardiomyocytes. After leaving the solu- cardiomyocytes tion to pellet for 10 min, the supernatant was discarded and Male Sprague Dawley rats (weights 250–300 g, n = 14) were solution 4 maintained at 37°C was added. This was repeated anaesthetised and had their hearts excised. The heart was placed with solution 5 after an additional 10 min. Finally, the isolated in a weighing boat containing ice-cold isolation buffer as the cardiomyocytes were resuspended in Medium 199 (M4530; aortic opening was located and cleared of excess tissue. Next, Sigma) containing 5 mM creatine, 2 mM carnitine, 5 mM tau- the aorta was secured onto the cannula of a Langendorff col- rine, 1% penicillin-streptomycin. Figure 4 is an illustrated sum- umn with a suture before rapid retrograde perfusion with solu- mary of the overall isolation protocol. tion 1 maintained at 37°C and continuous bubbling with 95% O /5% CO for a constant pH of 7.4. After 3.5 min, solution 1 2 2 Incubation of antagomiR-144/451 with was replaced with solution 2. Four minutes later, solution 2 was exosomes replaced with solution 3, and following 1 min of perfusion, the effluent was collected and recycled. After 8 min, the ventricles Two microlitres of each antagomiR (AM11051, AM10285, were excised and carefully minced in a weighing boat containing AM20299; Life Technologies) was added to 25 µ l of exo- leftover solution 3. The minced tissue was bubbled with pure O somes and incubated over ice for 30 min. Figure 4. Schematic illustration of primary rat cardiomyocyte isolation. Healthy male Sprague Dawley rats (n = 14) were anaesthetised and had their hearts excised. The heart was attached to a Langendorff column and different solutions were perfused across various time points to arrest and digest the heart. Ventricles were extracted and bubbled to obtain a pellet of live ventricular cardiomyocytes. Pelleted cardiomyocytes were resuspended in medium 199 and distributed onto sterile culture wells that have been pre-coated with laminin. 5 Research article Bioscience Horizons • Volume 8 2015 comparison tests for post hoc comparison of multiple treated Experimental design wells against the control group (Hypoxia). A p-value of Sterile culture wells were pre-coated for 1 h with laminin <0.05 was regarded as statistically significant. obtained from a 150 µ l stock diluted in 10 ml Dulbecco’s PBS. Isolated cardiomyocytes were evenly distributed across Results seven treatment groups (Supplementary material online in Table 2), and 2 ml of Medium 199 was added to each well. Characterization of purified exosome All wells were incubated overnight at 95% O and 37°C. The samples next day, dead and unattached cardiomyocytes were washed away with a fresh replacement of Medium 199 maintained Through NTA, we determined the modal particle size in our at 37°C. exosome samples to be 84 ± 2 nm (control exosomes) and 79 ± 2 nm (RIPC exosomes). Given that these values are con- Preparation of hypoxic and normoxic sistent with the expected size of exosomes (diameter <100 nm) buffers and that NTA obtained 1 distinct peak signal (multiple sig- nals indicate interference from other particles), our exosome To simulate ischaemia, a hypoxic buffer was made up with samples are sufficiently purified. The baseline concentration 0.13 M NaCl, 15 mM KCl, 1.2 mM KH PO , 2.5 mM 2 4 8 of control exosomes was (4 ± 0.08) × 10 per ml, and this MgSO , 2.2 mM NaHCO , 1.7 M lactate, 1 M CaCl and 4 3 2 concentration of exosomes increased after RIPC by ~70% to bubbled with CO /N until a pH of 6.4 and P <10% (relative 2 2 O2 8 (7 ± 0.20) × 10 per ml. to total gas composition) were achieved. We defined hypoxia as a state of glucose deprivation and reduced oxygen relative Control exosomes confer early and delayed to the cell’s metabolic demand. To simulate reperfusion, a nor- cardioprotection moxic buffer was made up with 0.12 M NaCl, 2.6 mM KCl, 1.2 mM KH PO , 2.5 mM MgSO , 21 mM NaHCO , 10 mM Control exosomes conferred both early and delayed 2 4 4 3 glucose and bubbled with CO /O until a pH of 7.4 and P cardioprotection to primary rat ventricular cardiomyo- 2 2 O2 68–80% (relative to total gas composition) were achieved. cytes undergoing simulated ischaemia–reperfusion (Fig. 5). Administration of control exosomes 30 min before hypoxia Simulation of ischaemia/reperfusion reduced cardiomyocyte death by 35.5% (from 62 ± 6 to 40 ± 4%), whereas control exosomes applied 24 h before Two millilitres of normoxic buffer was added to control groups ‘Normoxia’ and ‘Normoxia + antagomiRs’. These wells were incubated for 2.5 h at 37°C and 95% O . Two mil- lilitres of hypoxic buffer was added to the remaining wells and gassed continuously with CO /N for 15 min before relocat- 2 2 ing to the hypoxic chamber for 2.5 h at 37°C and 5% CO /0% O . When simulated ischaemia was over, hypoxic buffers were replaced with 2 ml of normoxic buffers to simulate reperfu- sion and the wells were incubated at 37°C and 95% O . Assessing cardiomyocyte viability After 1 h of simulated reperfusion, cells were stained with 4 µ l of fluorescent propidium iodide (PI). Static snapshots of each well were captured and unviable cardiomyocytes were manually counted based on their PI fluorescence and physical morphology. The degree of cardiomyocyte death was then calculated by the percentage of unviable cardiomyocytes over the total number of cardiomyocytes. Data analysis Data for all experiments are presented as mean ± SEM cor- Figure 5. Administration of human plasma exosomes significantly reduced death of primary rat ventricular cardiomyocytes undergoing rected to 1 significant figure and analysed using GraphPad simulated ischaemia–reperfusion (n = 5). Exosomes were extracted Prism version 6.00 for Windows (GraphPad Software, San from healthy male volunteers and administered to cardiomyocytes Diego, CA, USA). Calculated differences in the proportion of 24 h and 30 min before hypoxia. Cardiomyocyte death was cardiomyocyte death are corrected to 3 significant figures. To determined by PI staining and cell morphology after 2.5 h of hypoxia evaluate differences in cardioprotection across treatment and 1 h of reoxygenation. Data values are mean ± SEM. (*P < 0.05 vs. groups, a repeated-measures one-way analysis of variance hypoxia, repeated-measures one-way ANOVA with Dunnett’s multiple comparison tests). (ANOVA) was used, followed by Dunnett’s multiple 6 Bioscience Horizons • Volume 8 2015 Research article hypoxia reduced cardiomyocyte death by 30.6% (from control or RIPC exosomes, no significant attenuations of 62 ± 6 to 43 ± 7%). No significant differences between delayed cardioprotection were observed, indicating that miR- early and delayed cardioprotection were observed. 144/451 may not be directly involved in this model. RIPC exosomes confer early and delayed Discussion cardioprotection RIPC may be an effective and convenient method of interven- RIPC exosomes were just as capable of conferring early and tion to prevent widespread cardiomyocyte death following delayed cardioprotection (Fig. 5). RIPC exosomes adminis- reperfusion of ischaemic vasculature. In the present study, we tered 30 min before hypoxia reduced cardiomyocyte death demonstrate that regardless of their origin from control or by 43.5% (from 62 ± 6 to 35 ± 5%) and RIPC exosomes RIPC hearts, exosomes per se appear to confer cardioprotec- administered 24 h before hypoxia reduced cardiomyocyte tion to primary rat ventricular cardiomyocytes undergoing death by 32.3% (from 62 ± 6 to 42 ± 7%). No significant dif - simulated ischaemia–reperfusion in vitro, and RIPC appears ferences were observed between the degrees of early and to enhance the number of circulating exosomes. Our findings delayed cardioprotection. are consistent with that of our and other groups who intro- duced human exosomes to isolated rat hearts or intrave- Applied antagomiRs of miR-144 and 451 nously via the tail vein (Lai et al., 2010; Chen et al., 2013; did not affect delayed cardioprotection Yellon et al., 2013; Przyklenk, 2014; Vicencio et al., 2014; Zheng et al., 2014). by exosomes We demonstrated that exosomes administered 24 h before Consistent with our first set of experiments, both control and hypoxia significantly reduced cardiomyocyte death, and this RIPC exosomes conferred delayed cardioprotection (Fig. 6). finding bolsters the hypothesis that exosomes contain a fac - Control exosomes reduced cardiomyocyte death by 24.6% tor that stimulates the de novo synthesis of cardioprotective (from 61 ± 4 to 46 ± 6%), whereas RIPC exosomes reduced mediators. We speculated that miRNA-144 and 451 may be cardiomyocyte death by 27.9% (from 61 ± 4 to 44 ± 6%). responsible and incubated antagomiR-144/451 with exo- When antagomiRs of miR-144/451 were applied with either somes to attempt to attenuate this effect. We did not observe any significant reductions in cardiomyocyte survival, and this result suggests the preclusion of miR-144/451 in the underlying mechanism. Other miRNAs or humoral factors such as adenosine, cytokines or opioids could be involved instead (Hausenloy and Yellon, 2010). We anticipated a greater degree of cardiomyocyte survival in the group treated with RIPC exosomes compared with control exosomes, as a recent and as yet unpublished study by Yellon’s group reflected a trend of enhanced protection by exosomes derived from preconditioned HUVEC cells as opposed to exosomes derived from unconditioned cells (Zheng et al., 2014). We did not observe any significant differences between the con - trol and RIPC exosomes (Figs 5 and 6), and this could be related to our applied doses of exosomes. From NTA, our applied doses translate to ~1.06 × 10 control exosome par- ticles and 1.80 × 10 RIPC exosome particles per group, and these doses could have saturated underlying mechanisms of protection. In addition to measuring infarct size, other parameters of cardiomyocyte function can be used as surrogate end points. Cohen et al. (1991) showed that IPC in rabbits did not solely reduce infarct size 24 h later but also restored systolic func- tion and Bolli et al. (1997) observed that delayed IPC-induced Figure 6. AntagomiRs did not affect delayed cardioprotection by protection against myocardial stunning in conscious rabbits exosomes (n = 9). Exosomes extracted from healthy male volunteers was independent of cardimoyocyte necrosis as the applied were incubated with antagomiRs of miR-144/451 for 30 min over ice ischaemic stimulation was insufficient to induce an infarct. and added to corresponding wells 24 h before hypoxia. Cardiomyocyte Damage from ischaemia–reperfusion injury is also known to death was determined by PI staining and cell morphology after 2.5 h spill over to the endothelium, and endothelial cells can of hypoxia and 1 h of reoxygenation. Data values are mean ± SEM. become desensitized to vasodilating stimuli, leading to the (*P < 0.05 vs. hypoxia, repeated-measures one-way ANOVA with aggregation of platelets, increased vasospasm and thrombosis Dunnett’s multiple comparison tests). 7 Research article Bioscience Horizons • Volume 8 2015 (Laude et al., 2002). Laude et al. (2002) subjected rat hearts higher infarct sizes but compensate with faster contractile to classical IPC, and after simulating ischaemia–reperfusion recovery (Cohen et al., 1991; Bolli et al., 1997), and in ongo- injury, they noted that coronary arteries from sham hearts ing human studies, surrogate end points such as ischaemic had an impaired ability of relaxation in the presence of ace- electrocardiogram changes are correlated with precondition- tylcholine, whereas coronary arteries from preconditioned ing effects (Hausenloy and Yellon, 2010). hearts retained this ability. No common theory about the Limitations of the study underlying mechanism has been proposed and groups such as Kaeffer et al. (1997) have suggested the involvement of NO Our in vitro rat model may not be representative of an and ROS, whereas Loktionova et al. (1998) have provided actual ischaemia–reperfusion setting in vivo since it cannot some evidence for the involvement of heat-shock proteins. recapitulate certain ongoing physiological processes such as The fact that exosomes can contain multiple cardioprotective cardiac hypercontracture, sarcolemmal disruptions and vas- factors and/or vary depending on their cell origin alludes to cular plugging, and it ignores the influence of the autonomic their versatility in activating multiple mechanisms and in nervous system, of which has been exhibited to be essential salvaging different aspects of cardiac function. before the humoral arm of RIPC-induced protection in vivo can be induced (Gourine and Gourine, 2014). Certain Future research can explore the role of exosomes in modu- experiments exhibited poor PI staining, and dead cardio- lating cardiac mitochondrial function. The role of the mito- myocytes had to be counted based on their physical mor- chondria in ischaemia–reperfusion injury has been extensively phology. Future experiments could thus consider the usage studied, and several studies have reported that determinants of a more objective and sensitive cytotoxicity assay such as of mitochondrial function such as membrane potential and the lactate dehydrogenase (LDH) cytotoxicity assay, which energy metabolism become dysfunctional due to ischaemia- relates irreversible cell death to the amount of LDH pro- induced calcium overload and overproduction of free radi- duced by cells (Hoek et al., 1996; Adderley and Fitzgerald, cals (Jassem et al., 2002; Di Lisa and Bernardi, 2006). The 1999). Our study provides some evidence that miR-144/451 general consensus is that mitochondrial K channels are ATP may not be involved in delayed cardioprotection, but there more prominent than sarcolemmal K channels in the pres- ATP could have been poor antagomiR uptake by the exosomes. ervation of mitochondrial function, and interrupting the For a successful inhibition of cardioprotection, the applied opening of the mitochondrial permeability transition pore antagomiRs have to first traverse the exosomal membrane, during reperfusion is pivotal to overall cardiomyocyte sur- firmly bind to its complementary miRNA sequence and vival (Hausenloy et al., 2004; Liu et al., 1998; Fryer et al., then block subsequent translation of the cardioprotective 2000; Jassem et al., 2002; Di Lisa and Bernardi, 2006). factor(s). We did not confirm whether the expression Slagsvold et al. (2014) reported that RIPC preserved mito- levels of miR-144/451 and their target genes had been chondrial function of atrial tissue during coronary bypass down- regulated before administration of exosomes to the surgery and suggest that the underlying mechanism of RIPC- cardiomyocytes. induced protection could originate from the mitochondria. It is not known whether exosomes behave differently in Conclusion the presence of other cell types, and pathologic cells them- selves are known to produce exosomes with antagonistic fac- We present novel evidence that control and RIPC exosomes tors; stressed cells have been reported to secrete exosomes are capable of conferring delayed cardioprotection in our containing HSP60, which upon release triggers cellular in vitro rat model, and miR-144 and 451 may not be involved inflammatory pathways ( Gupta and Knowlton, 2007; in the underlying mechanism. Our study highlights the Merendino et al., 2010). Experimental models should be immense potential of exosomes in alleviating ischaemia– designed to mimic human ischaemia–reperfusion injury with reperfusion injury and opens the door for further studies to the greatest fidelity and several advantages accompany the investigate the cardioprotective properties of other factors use of cardiomyocyte cultures in studying RIPC, including stored within exosomes. the lack of interference from other cell types such as fibro - blasts and the convenience of conducting visual and micros- copy analyses. The aforementioned in vitro studies including Author’s biography ours performed a simulation of ischaemia–reperfusion and J.L. graduated with a first-class honours BSc in Biomedical each group has carefully defined their experimental conditions Sciences from University College London in 2014 and aspires based on their selected animal species and parameter of inter- to become a cardiologist. Her other research interests include est. While certain groups have chosen to simulate ischaemia translational medicine and the biochemistry of complex dis- by pelleting freshly isolated cardiomyocytes under oil to com- eases. Under the supervision of Prof. D.M.Y. and Dr S.M.D., bine severe hypoxia with accumulation of metabolites, lac- who together designed the research as part of a final-year tate and hydrogen ions (Diaz and Wilson, 2006), we elected dissertation project, J.L. conducted the experiments, wrote to simulate ischaemia in culture with hypoxic buffers. Groups the present paper and has primary responsibility for the final investigating myocardial stunning have a preference for rab- content. bit hearts, which compared with rats and canines, have 8 Bioscience Horizons • Volume 8 2015 Research article cell-derived exosomes, Journal of Extracellular Vesicles, 1, http:// Supplementary data dx.doi.org/10.3402/jev.v1i0.18396. Supplementary data is available at Bioscience Horizons Diaz, R. J. and Wilson, G. J. 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(2014) 27 exosomes released from 4-mediated miR-144/451 cluster in protection against simulated endothelial cells are cardioprotective, Heart, 100 (Suppl. 1), A10–A10. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Bioscience Horizons Oxford University Press

Evaluating early and delayed cardioprotection by plasma exosomes in simulated ischaemiareperfusion injury

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BioscienceHorizons Volume 8 2015 10.1093/biohorizons/hzv001 Research article Evaluating early and delayed cardioprotection by plasma exosomes in simulated ischaemia–reperfusion injury Jiawen Liu*, Derek M. Yellon and Sean M. Davidson Hatter Cardiovascular Institute, 67 Chenies Mews, London WC1E 6HX, England *Corresponding author: Division of Biosciences, University College London, Gower Street, London WC1E 6BT, England. Tel: +44 756 321 3012. Email: jiawen.liu.11@ucl.ac.uk Supervisors: Prof. Derek M. Yellon, Dr. Sean M. Davidson, Hatter Cardiovascular Institute, University College London, 67 Chenies Mews, London WC1E 6HX, England. Tel: +44 203 447 5732; Fax: +44 203 447 9505. Email: s.davidson@ucl.ac.uk Reductions in cardiac infarct size can be achieved in ischaemic preconditioning, a procedure which subjects the heart to intermit- tent, non-lethal cycles of ischaemia and reperfusion. Similar cardioprotection can be induced upon preconditioning distal tissues such as the upper limb, and this procedure is called remote ischaemic preconditioning. Nano-sized, cell-derived vesicles known as exosomes have been shown previously to be capable of inducing cardioprotection when administered acutely, and we hypothesized that exosomes produced after remote ischaemic preconditioning (RIPC) enhances the observed cardioprotection. We also investigated whether exosomes can confer cardioprotection 24 h later. We isolated exosomes from the plasma of three healthy male volunteers before and after they underwent four cycles of 5 min upper limb ischaemia and 5 min reperfusion, and then administered the exosomes to primary rat ventricular cardiomyocytes isolated from male Sprague Dawley rats. Cardiomyocytes were subjected to 2.5 h of simulated ischaemia and 1 h simulated reperfusion. We assessed cardiomyocyte via- bility with propidium iodide staining and observed significant reductions in cardiomyocyte death when control exosomes were administered either 30 min or 24 h before hypoxia. Exosomes obtained after RIPC induced a similar degree of cardioprotection at both time points. As miRNA-144/451 have been proposed to mediate RIPC, we investigated whether introducing antagomiRs of miRNA-144/451 with the exosomes would attenuate cardioprotection after 24 h. No significant differences in cardioprotection were observed, suggesting that miRNA-144/451 may not be directly involved in this model. Our findings suggest that regardless of their origin from control or RIPC hearts, exosomes per se can be used to induce cardioprotection either acutely or after 24 h. Key words: exosomes, remote, ischaemic, preconditioning, reperfusion, cardioprotection Submitted on 5 November 2014; accepted on 21 January 2015 Introduction in a dog after ligation of its coronary vessels, and findings from subsequent experiments conducted in the 19th century Between the 16th and 18th centuries, the concepts of angina supported the idea that obstructions within the coronary cir- pain, vessel degeneration and sudden death due to a blockage culation starved the myocardium of oxygen and inflicted in the heart gained currency following the contributions of damage (Leibowitz, 1970a,b). ideas and anatomical findings from Benivieni (1507), Amatus Lusitanus (1560), Petrus Salius Diversus (1586), Bellini Coronary angioplasty is the modern-day cornerstone (1683) and Thebesius (1708) (Leibowitz, 1970a,b). In 1698, procedure to promptly liberate the occluded vasculature and to Pierre Chirac first described his observations of cardiac arrest reperfuse the ischaemic myocardium, but despite improvements © The Author 2015. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.  Research article Bioscience Horizons • Volume 8 2015 in technologies and quality of care, the number of individuals COX-2 signalling pathway, and these effects were reversed living with coronary heart disease (CHD) remains high and is when the 2 miRNAs were knocked down. increasing. The British Heart Foundation (BHF) reported that One variation of IPC is known as remote ischaemic precon- there are still at least 2.3 million people in the UK living with ditioning (RIPC), whereby the ischaemic bouts are performed CHD and more than £7 billion has been spent within England on distant tissues such as the upper limb rather than the myo- alone to treat cardiovascular diseases (BHF Cardiovascular cardium (Fig. 1). RIPC has been demonstrated in animal mod- Disease Statistics, Townsend, 2014). The number of coronary els to reduce infarct size and oxidative stress (Hausenloy and angioplasties performed in the UK in 2012 has more than dou- Yellon, 2007), but the underlying mechanism(s) has yet to be bled from a decade ago, but acute myocardial infarctions fully elucidated. Various groups have proposed the role of the (AMIs) still accounted for at least 175 000 inpatient episodes autonomic (Redington et al., 2012) nervous system (Basalay (BHF Cardiovascular Disease Statistics, Townsend, 2014), and et al., 2012; Mastitskaya et al., 2012; Donato et al., 2013) there is an ~10% mortality rate associated with AMIs and/or the secretion of extracellular vesicles that range from 30 (Hausenloy and Yellon, 2007). A plausible cause may be ‘lethal to 1000 nM in diameter. Previously characterized as unimport- reperfusion injury’, a phenomenon that arises when the sudden ant cell debris, extracellular vesicles have attracted waves of influx of oxygenated blood into the ischaemic region initiates renewed interest as emerging studies unravel their roles in various signalling pathways that culminate in widespread car- transporting mRNA, miRNA, plasma membrane receptors diomyocyte necrosis (Hausenloy and Yellon, 2011; Sanada and signalling ligands across cells as well as their involvement et al., 2011). This overwhelms endogenous myocardium repair in angiogenesis, inflammation and cytopro tection (Anderson mechanisms and results in the formation of scars that subse- et al., 2010; Park et al., 2010; Lee et al., 2012; Yellon and quently compromise cardiac function. Preventing reperfusion Davidson, 2014). Several studies have reported associations of injury thus represents a possible strategy to reduce the eco- nomic burden and prevalence of AMIs. In 1986, Murry et al. observed that bouts of ischaemic stresses impeded the rate of ATP depletion, and they postu- lated that with these bouts, harmful catabolites could simul- taneously be expelled and this would delay the onset of irreversible cardiomyocyte injury (Murry et al., 1986). Using canine hearts, the group experimented with four cycles of 5 min circumflex artery occlusion and 5 min reperfusion fol- lowed by a sustained 40 min occlusion and observed marked improvements in cardiomyocyte viability compared with control hearts (Murry et al., 1986). Murry et al. termed their procedure of ischaemic bouts as ischaemic preconditioning (IPC), and IPC has been repeatedly demonstrated over the subsequent two decades to improve cardiac function in humans and other animal models (For review, see Hausenloy and Yellon, 2011). Classical IPC-induced protection is biphasic—the early phase occurs immediately and lasts for 2–3 h, and the delayed phase occurs 12–24 h later and persists for 48–72 h (Hausenloy and Yellon, 2010). Since early protection is immediate, the underlying mechanism is more likely to involve secreted proteins as opposed to lengthy gene tran- scription and regeneration processes, of which would be more prominent during the delayed phase of protection. De novo protein synthesis of distal cardioprotective factors could be triggered by small non-coding nucleic acids known Figure 1. Schematic illustration of possible underlying mechanisms of as microRNAs (miRNAs), and specific miRNAs have already RIPC. RIPC is a procedure involving cycles of non-lethal ischaemia and been suggested to be involved—for example, Wang et al. reperfusion of distant tissues such as the upper limb instead of the myocardium. RIPC has been demonstrated in animal models to reduce (2012) observed significant elevations of miR-144/451 in IPC infarct size and oxidative stress after a prolonged and lethal ischaemic hearts compared with sham hearts, and IPC-mediated cardio- challenge. The intermittent ischaemic bouts could stimulate the protection was absent in miR-144/451 knock-out mice. autonomic nervous system and/or triggers the secretion of, as yet, These findings were complementary with those from Zhang unidentified humoral cardioprotective factors which can subsequently et al. (2010), who reported that the ectopic expression of activate the reperfusion injury salvage kinase (RISK), JAK-STAT, miR-144/451 enhanced cardiomyocyte survival via the acti- ERK/MAPK and Akt/PI3 K kinase pathways in the myocardium and lead vation of the CUG triplet repeat-binding protein 2 (CUGBP2)/ to tissue salvage. 2 Bioscience Horizons • Volume 8 2015 Research article circulating microvesicles (100–1000 nM in diameter) with the 2012; Yellon and Davidson, 2014) and whether the cell is dis- progression of cardiovascular diseases (Chung et al., 2007; eased or healthy (Taylor and Taylor, 2008; Huang et al., 2013). Fichtlscherer et al., 2010; Kanhai et al., 2013), but relatively Minor cellular stresses can also alter exosomal content, as less is known about the roles of nano-sized (30–100 nM in observed in exosomes derived from endothelial cells undergo- diameter) extracellular vesicles known as exosomes. Exosomes ing non-lethal hypoxia and inflammation ( de Jong et al., are formed when the luminal membranes of multivesicular 2012), and in patients who underwent RIPC, their exosomes bodies (formed within the late endosome) invaginate, and contained a greater proportion of miR-144 and 451 than fusion of these multivesicular bodies with the cell membrane before undergoing RIPC (D.M. Yellon et al., unpublished releases exosomes into the circulation (Fig. 2). Exosomes are data). known to contain tetraspanins (CD9, CD81), fusion proteins (Annexins, GTPases), lipid rafts, heat-shock proteins (Hsp70, Recent evidence suggests that exosomes protect against Hsp90) and ongoing studies speculate the ability of exosomes simulated ischaemia–reperfusion injury when administered to enclose humoral cardioprotective factors (Calzolari et al., acutely (Lai et al., 2010; Chen et al., 2013), but it is not 2006; Jeanneteau et al., 2012; Hirsch et al., 2013; Przyklenk, known whether exosomes can induce protection in a delayed 2014) that activate the reperfusion injury salvage kinase setting. Delayed cardioprotection might be more relevant in (RISK), JAK-STAT, ERK/MAPK and Akt/PI3 K kinase clinical procedures where ischaemia–reperfusion injury can signalling pathways (Kristiansen et al., 2005; Serejo et al., be predicted, such as in organ transplantation, coronary 2007; Hausenloy and Yellon, 2008; Lim et al., 2010) in the artery bypass graft (CABG) surgery or planned coronary myocardium to mitigate cardiomyocyte damage during an angioplasty (Hausenloy and Yellon, 2010). Hence in this AMI. Variations of exosomal content can arise depending on study, our primary aim was to investigate whether human the type of cell the exosome originates from (Vlassov et al., plasma exosomes could confer delayed cardioprotection in Figure 2. Schematic illustration of the formation of exosomes and their release into the circulation. Exosomes are formed from the invagination of the late endosome (the resultant entity is known as a multivesicular body) and released into the circulation when the multivesicular body fuses with the plasma membrane. Exosomes have been characterized to contain tetraspanins (CD9, CD81), fusion proteins (Annexins, GTPases), lipid rafts and heat-shock proteins (Hsp70, Hsp 90). Variations in exosomal content can arise depending on the type of cell the exosomes originate from. 3 Research article Bioscience Horizons • Volume 8 2015 primary rat ventricular cardiomyocytes undergoing simu- Preparation of RIPC human plasma lated ischaemia–reperfusion injury in vitro. If delayed cardio- exosomes protection is observed, we hypothesized that this process was mediated by exosomal delivery of miRNAs into cardiomyo- After collection of the first blood sample, the same volunteers cytes. As mentioned previously, RIPC significantly up- underwent RIPC. A standard blood pressure cuff was used to regulates cardioprotective miR-144 and 451. Therefore, the perform four cycles of 5 min upper limb ischaemia and 5 min secondary aim of this study was to investigate whether incu- reperfusion (Fig. 3). A second blood sample was collected bating exosomes with antagomiRs (miRNA-inhibitors) of from each volunteer and centrifuged (as per above) to obtain miR-144 and 451 would abolish any cardioprotection a sample of RIPC plasma exosomes. conferred. Characterization of exosomes with Methods nanoparticle tracking analysis Statement of ethical approval We used nanoparticle tracking analysis (NTA) to confirm the presence of exosomes. The rate of Brownian particle motion in Animals were treated in accordance with the Animals solution is dependent on particle size, and particle movements (Scientific Procedure) act 1986 published by the UK Home are monitored by directing a laser beam (405 nm) towards the Office and the Guide for the Care and Use of Laboratory solution and using a high-sensitivity camera (Nanosight Animals published by the US National Institutes of Health LM10-HS) to capture the scattered light. The NTA software (Publication No. 95-23, revised 1996). For human samples, will then determine the diameter of particles based on their these experiments were conducted according to Declaration movement over time. of Helsinki principles. All human and animal studies were approved by the appropriate institutional review boards, spe- Preparation of rat cardiomyocyte isolation cifically in accordance with the animal project license PPL No. 70/7140. The study of human samples was performed buffer according to ethics approval reference 13/LO/0222. The rat cardiomyocyte isolation buffer was made up with 130 M NaCl, 5.4 M KCl, 1.4 M MgCl , 0.4 M Na HPO and Preparation of human plasma exosomes 2 2 4 4.2 M HEPES. The buffer was maintained at 37°C and pH After written consent, three healthy male volunteers (30–45 7.4. Next, 10 mM glucose, 20 mM taurine and 10 mM cre- years old) each provided one blood sample. Blood was drawn atine were added, and the solution was filtered with a sterile from the antecubital vein via a butterfly syringe into citrated vacuum filter pump. vaccutainers™. The blood samples were centrifuged at 1600 × g for 20 min at 4°C to obtain plasma, followed by centrifugation Preparation of perfusate solutions at 10 000 × g for 30 min at 4°C to separate extraneous cell rem- nants and platelets. The final sample of exosomes was then iso - The filtered rat cardiomyocyte isolation buffer was divided lated following two additional centrifugations at 100 000 × g into five tubes and labelled solutions 1–5. Supplementary for 60 min at 4°C and diluted into aliquots with Dulbecco’s material online in Table 1 summarizes the additions made to phosphate- buffered saline (PBS). each solution. Figure 3. Schematic illustration of exosome extraction. Blood samples were collected from healthy volunteers before and after they underwent four cycles of 5 min upper limb ischaemia and 5 min reperfusion. Blood samples were centrifuged at 1600 × g for 20 min at 4°C to obtain plasma, followed by centrifugation at 10 000 × g for 30 min at 4°C to separate unwanted cell remnants and platelets. The final sample of exosomes was then acquired after two further centrifugations at 100 000 × g for 60 min at 4°C and diluted into aliquots with Dulbecco’s PBS. 4 Bioscience Horizons • Volume 8 2015 Research article for 8–10 min and filtered through iron mesh to obtain the final Isolation of primary rat ventricular solution of ventricular cardiomyocytes. After leaving the solu- cardiomyocytes tion to pellet for 10 min, the supernatant was discarded and Male Sprague Dawley rats (weights 250–300 g, n = 14) were solution 4 maintained at 37°C was added. This was repeated anaesthetised and had their hearts excised. The heart was placed with solution 5 after an additional 10 min. Finally, the isolated in a weighing boat containing ice-cold isolation buffer as the cardiomyocytes were resuspended in Medium 199 (M4530; aortic opening was located and cleared of excess tissue. Next, Sigma) containing 5 mM creatine, 2 mM carnitine, 5 mM tau- the aorta was secured onto the cannula of a Langendorff col- rine, 1% penicillin-streptomycin. Figure 4 is an illustrated sum- umn with a suture before rapid retrograde perfusion with solu- mary of the overall isolation protocol. tion 1 maintained at 37°C and continuous bubbling with 95% O /5% CO for a constant pH of 7.4. After 3.5 min, solution 1 2 2 Incubation of antagomiR-144/451 with was replaced with solution 2. Four minutes later, solution 2 was exosomes replaced with solution 3, and following 1 min of perfusion, the effluent was collected and recycled. After 8 min, the ventricles Two microlitres of each antagomiR (AM11051, AM10285, were excised and carefully minced in a weighing boat containing AM20299; Life Technologies) was added to 25 µ l of exo- leftover solution 3. The minced tissue was bubbled with pure O somes and incubated over ice for 30 min. Figure 4. Schematic illustration of primary rat cardiomyocyte isolation. Healthy male Sprague Dawley rats (n = 14) were anaesthetised and had their hearts excised. The heart was attached to a Langendorff column and different solutions were perfused across various time points to arrest and digest the heart. Ventricles were extracted and bubbled to obtain a pellet of live ventricular cardiomyocytes. Pelleted cardiomyocytes were resuspended in medium 199 and distributed onto sterile culture wells that have been pre-coated with laminin. 5 Research article Bioscience Horizons • Volume 8 2015 comparison tests for post hoc comparison of multiple treated Experimental design wells against the control group (Hypoxia). A p-value of Sterile culture wells were pre-coated for 1 h with laminin <0.05 was regarded as statistically significant. obtained from a 150 µ l stock diluted in 10 ml Dulbecco’s PBS. Isolated cardiomyocytes were evenly distributed across Results seven treatment groups (Supplementary material online in Table 2), and 2 ml of Medium 199 was added to each well. Characterization of purified exosome All wells were incubated overnight at 95% O and 37°C. The samples next day, dead and unattached cardiomyocytes were washed away with a fresh replacement of Medium 199 maintained Through NTA, we determined the modal particle size in our at 37°C. exosome samples to be 84 ± 2 nm (control exosomes) and 79 ± 2 nm (RIPC exosomes). Given that these values are con- Preparation of hypoxic and normoxic sistent with the expected size of exosomes (diameter <100 nm) buffers and that NTA obtained 1 distinct peak signal (multiple sig- nals indicate interference from other particles), our exosome To simulate ischaemia, a hypoxic buffer was made up with samples are sufficiently purified. The baseline concentration 0.13 M NaCl, 15 mM KCl, 1.2 mM KH PO , 2.5 mM 2 4 8 of control exosomes was (4 ± 0.08) × 10 per ml, and this MgSO , 2.2 mM NaHCO , 1.7 M lactate, 1 M CaCl and 4 3 2 concentration of exosomes increased after RIPC by ~70% to bubbled with CO /N until a pH of 6.4 and P <10% (relative 2 2 O2 8 (7 ± 0.20) × 10 per ml. to total gas composition) were achieved. We defined hypoxia as a state of glucose deprivation and reduced oxygen relative Control exosomes confer early and delayed to the cell’s metabolic demand. To simulate reperfusion, a nor- cardioprotection moxic buffer was made up with 0.12 M NaCl, 2.6 mM KCl, 1.2 mM KH PO , 2.5 mM MgSO , 21 mM NaHCO , 10 mM Control exosomes conferred both early and delayed 2 4 4 3 glucose and bubbled with CO /O until a pH of 7.4 and P cardioprotection to primary rat ventricular cardiomyo- 2 2 O2 68–80% (relative to total gas composition) were achieved. cytes undergoing simulated ischaemia–reperfusion (Fig. 5). Administration of control exosomes 30 min before hypoxia Simulation of ischaemia/reperfusion reduced cardiomyocyte death by 35.5% (from 62 ± 6 to 40 ± 4%), whereas control exosomes applied 24 h before Two millilitres of normoxic buffer was added to control groups ‘Normoxia’ and ‘Normoxia + antagomiRs’. These wells were incubated for 2.5 h at 37°C and 95% O . Two mil- lilitres of hypoxic buffer was added to the remaining wells and gassed continuously with CO /N for 15 min before relocat- 2 2 ing to the hypoxic chamber for 2.5 h at 37°C and 5% CO /0% O . When simulated ischaemia was over, hypoxic buffers were replaced with 2 ml of normoxic buffers to simulate reperfu- sion and the wells were incubated at 37°C and 95% O . Assessing cardiomyocyte viability After 1 h of simulated reperfusion, cells were stained with 4 µ l of fluorescent propidium iodide (PI). Static snapshots of each well were captured and unviable cardiomyocytes were manually counted based on their PI fluorescence and physical morphology. The degree of cardiomyocyte death was then calculated by the percentage of unviable cardiomyocytes over the total number of cardiomyocytes. Data analysis Data for all experiments are presented as mean ± SEM cor- Figure 5. Administration of human plasma exosomes significantly reduced death of primary rat ventricular cardiomyocytes undergoing rected to 1 significant figure and analysed using GraphPad simulated ischaemia–reperfusion (n = 5). Exosomes were extracted Prism version 6.00 for Windows (GraphPad Software, San from healthy male volunteers and administered to cardiomyocytes Diego, CA, USA). Calculated differences in the proportion of 24 h and 30 min before hypoxia. Cardiomyocyte death was cardiomyocyte death are corrected to 3 significant figures. To determined by PI staining and cell morphology after 2.5 h of hypoxia evaluate differences in cardioprotection across treatment and 1 h of reoxygenation. Data values are mean ± SEM. (*P < 0.05 vs. groups, a repeated-measures one-way analysis of variance hypoxia, repeated-measures one-way ANOVA with Dunnett’s multiple comparison tests). (ANOVA) was used, followed by Dunnett’s multiple 6 Bioscience Horizons • Volume 8 2015 Research article hypoxia reduced cardiomyocyte death by 30.6% (from control or RIPC exosomes, no significant attenuations of 62 ± 6 to 43 ± 7%). No significant differences between delayed cardioprotection were observed, indicating that miR- early and delayed cardioprotection were observed. 144/451 may not be directly involved in this model. RIPC exosomes confer early and delayed Discussion cardioprotection RIPC may be an effective and convenient method of interven- RIPC exosomes were just as capable of conferring early and tion to prevent widespread cardiomyocyte death following delayed cardioprotection (Fig. 5). RIPC exosomes adminis- reperfusion of ischaemic vasculature. In the present study, we tered 30 min before hypoxia reduced cardiomyocyte death demonstrate that regardless of their origin from control or by 43.5% (from 62 ± 6 to 35 ± 5%) and RIPC exosomes RIPC hearts, exosomes per se appear to confer cardioprotec- administered 24 h before hypoxia reduced cardiomyocyte tion to primary rat ventricular cardiomyocytes undergoing death by 32.3% (from 62 ± 6 to 42 ± 7%). No significant dif - simulated ischaemia–reperfusion in vitro, and RIPC appears ferences were observed between the degrees of early and to enhance the number of circulating exosomes. Our findings delayed cardioprotection. are consistent with that of our and other groups who intro- duced human exosomes to isolated rat hearts or intrave- Applied antagomiRs of miR-144 and 451 nously via the tail vein (Lai et al., 2010; Chen et al., 2013; did not affect delayed cardioprotection Yellon et al., 2013; Przyklenk, 2014; Vicencio et al., 2014; Zheng et al., 2014). by exosomes We demonstrated that exosomes administered 24 h before Consistent with our first set of experiments, both control and hypoxia significantly reduced cardiomyocyte death, and this RIPC exosomes conferred delayed cardioprotection (Fig. 6). finding bolsters the hypothesis that exosomes contain a fac - Control exosomes reduced cardiomyocyte death by 24.6% tor that stimulates the de novo synthesis of cardioprotective (from 61 ± 4 to 46 ± 6%), whereas RIPC exosomes reduced mediators. We speculated that miRNA-144 and 451 may be cardiomyocyte death by 27.9% (from 61 ± 4 to 44 ± 6%). responsible and incubated antagomiR-144/451 with exo- When antagomiRs of miR-144/451 were applied with either somes to attempt to attenuate this effect. We did not observe any significant reductions in cardiomyocyte survival, and this result suggests the preclusion of miR-144/451 in the underlying mechanism. Other miRNAs or humoral factors such as adenosine, cytokines or opioids could be involved instead (Hausenloy and Yellon, 2010). We anticipated a greater degree of cardiomyocyte survival in the group treated with RIPC exosomes compared with control exosomes, as a recent and as yet unpublished study by Yellon’s group reflected a trend of enhanced protection by exosomes derived from preconditioned HUVEC cells as opposed to exosomes derived from unconditioned cells (Zheng et al., 2014). We did not observe any significant differences between the con - trol and RIPC exosomes (Figs 5 and 6), and this could be related to our applied doses of exosomes. From NTA, our applied doses translate to ~1.06 × 10 control exosome par- ticles and 1.80 × 10 RIPC exosome particles per group, and these doses could have saturated underlying mechanisms of protection. In addition to measuring infarct size, other parameters of cardiomyocyte function can be used as surrogate end points. Cohen et al. (1991) showed that IPC in rabbits did not solely reduce infarct size 24 h later but also restored systolic func- tion and Bolli et al. (1997) observed that delayed IPC-induced Figure 6. AntagomiRs did not affect delayed cardioprotection by protection against myocardial stunning in conscious rabbits exosomes (n = 9). Exosomes extracted from healthy male volunteers was independent of cardimoyocyte necrosis as the applied were incubated with antagomiRs of miR-144/451 for 30 min over ice ischaemic stimulation was insufficient to induce an infarct. and added to corresponding wells 24 h before hypoxia. Cardiomyocyte Damage from ischaemia–reperfusion injury is also known to death was determined by PI staining and cell morphology after 2.5 h spill over to the endothelium, and endothelial cells can of hypoxia and 1 h of reoxygenation. Data values are mean ± SEM. become desensitized to vasodilating stimuli, leading to the (*P < 0.05 vs. hypoxia, repeated-measures one-way ANOVA with aggregation of platelets, increased vasospasm and thrombosis Dunnett’s multiple comparison tests). 7 Research article Bioscience Horizons • Volume 8 2015 (Laude et al., 2002). Laude et al. (2002) subjected rat hearts higher infarct sizes but compensate with faster contractile to classical IPC, and after simulating ischaemia–reperfusion recovery (Cohen et al., 1991; Bolli et al., 1997), and in ongo- injury, they noted that coronary arteries from sham hearts ing human studies, surrogate end points such as ischaemic had an impaired ability of relaxation in the presence of ace- electrocardiogram changes are correlated with precondition- tylcholine, whereas coronary arteries from preconditioned ing effects (Hausenloy and Yellon, 2010). hearts retained this ability. No common theory about the Limitations of the study underlying mechanism has been proposed and groups such as Kaeffer et al. (1997) have suggested the involvement of NO Our in vitro rat model may not be representative of an and ROS, whereas Loktionova et al. (1998) have provided actual ischaemia–reperfusion setting in vivo since it cannot some evidence for the involvement of heat-shock proteins. recapitulate certain ongoing physiological processes such as The fact that exosomes can contain multiple cardioprotective cardiac hypercontracture, sarcolemmal disruptions and vas- factors and/or vary depending on their cell origin alludes to cular plugging, and it ignores the influence of the autonomic their versatility in activating multiple mechanisms and in nervous system, of which has been exhibited to be essential salvaging different aspects of cardiac function. before the humoral arm of RIPC-induced protection in vivo can be induced (Gourine and Gourine, 2014). Certain Future research can explore the role of exosomes in modu- experiments exhibited poor PI staining, and dead cardio- lating cardiac mitochondrial function. The role of the mito- myocytes had to be counted based on their physical mor- chondria in ischaemia–reperfusion injury has been extensively phology. Future experiments could thus consider the usage studied, and several studies have reported that determinants of a more objective and sensitive cytotoxicity assay such as of mitochondrial function such as membrane potential and the lactate dehydrogenase (LDH) cytotoxicity assay, which energy metabolism become dysfunctional due to ischaemia- relates irreversible cell death to the amount of LDH pro- induced calcium overload and overproduction of free radi- duced by cells (Hoek et al., 1996; Adderley and Fitzgerald, cals (Jassem et al., 2002; Di Lisa and Bernardi, 2006). The 1999). Our study provides some evidence that miR-144/451 general consensus is that mitochondrial K channels are ATP may not be involved in delayed cardioprotection, but there more prominent than sarcolemmal K channels in the pres- ATP could have been poor antagomiR uptake by the exosomes. ervation of mitochondrial function, and interrupting the For a successful inhibition of cardioprotection, the applied opening of the mitochondrial permeability transition pore antagomiRs have to first traverse the exosomal membrane, during reperfusion is pivotal to overall cardiomyocyte sur- firmly bind to its complementary miRNA sequence and vival (Hausenloy et al., 2004; Liu et al., 1998; Fryer et al., then block subsequent translation of the cardioprotective 2000; Jassem et al., 2002; Di Lisa and Bernardi, 2006). factor(s). We did not confirm whether the expression Slagsvold et al. (2014) reported that RIPC preserved mito- levels of miR-144/451 and their target genes had been chondrial function of atrial tissue during coronary bypass down- regulated before administration of exosomes to the surgery and suggest that the underlying mechanism of RIPC- cardiomyocytes. induced protection could originate from the mitochondria. It is not known whether exosomes behave differently in Conclusion the presence of other cell types, and pathologic cells them- selves are known to produce exosomes with antagonistic fac- We present novel evidence that control and RIPC exosomes tors; stressed cells have been reported to secrete exosomes are capable of conferring delayed cardioprotection in our containing HSP60, which upon release triggers cellular in vitro rat model, and miR-144 and 451 may not be involved inflammatory pathways ( Gupta and Knowlton, 2007; in the underlying mechanism. Our study highlights the Merendino et al., 2010). Experimental models should be immense potential of exosomes in alleviating ischaemia– designed to mimic human ischaemia–reperfusion injury with reperfusion injury and opens the door for further studies to the greatest fidelity and several advantages accompany the investigate the cardioprotective properties of other factors use of cardiomyocyte cultures in studying RIPC, including stored within exosomes. the lack of interference from other cell types such as fibro - blasts and the convenience of conducting visual and micros- copy analyses. The aforementioned in vitro studies including Author’s biography ours performed a simulation of ischaemia–reperfusion and J.L. graduated with a first-class honours BSc in Biomedical each group has carefully defined their experimental conditions Sciences from University College London in 2014 and aspires based on their selected animal species and parameter of inter- to become a cardiologist. Her other research interests include est. While certain groups have chosen to simulate ischaemia translational medicine and the biochemistry of complex dis- by pelleting freshly isolated cardiomyocytes under oil to com- eases. Under the supervision of Prof. D.M.Y. and Dr S.M.D., bine severe hypoxia with accumulation of metabolites, lac- who together designed the research as part of a final-year tate and hydrogen ions (Diaz and Wilson, 2006), we elected dissertation project, J.L. conducted the experiments, wrote to simulate ischaemia in culture with hypoxic buffers. Groups the present paper and has primary responsibility for the final investigating myocardial stunning have a preference for rab- content. bit hearts, which compared with rats and canines, have 8 Bioscience Horizons • Volume 8 2015 Research article cell-derived exosomes, Journal of Extracellular Vesicles, 1, http:// Supplementary data dx.doi.org/10.3402/jev.v1i0.18396. Supplementary data is available at Bioscience Horizons Diaz, R. J. and Wilson, G. J. 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Bioscience HorizonsOxford University Press

Published: Feb 26, 2015

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