Characterization of extracellular vesicles derived from cardiac cells in an in vitro model of preconditioning

Abstract

Preconditioning is a promising technique to protect the heart from ischaemia-reperfusion injury. In this context, the crosstalk between different cardiac cell types and especially the exchange of cardioprotective mediators has come into the focus of current research. Recently, extracellular vesicles (EVs), nano-sized structures, emerged as possible communication mediators. They are taken up by recipient cells and can alter gene expression or activate intracellular signal cascades. It has been shown that all cardiac cell types are able to secrete EVs, but so far the influence of an in vitro preconditioning stimulus on EV concentration and composition has not been investigated. Therefore, we stimulated primary cardiac myocytes and fibroblasts from neonatal rats, as well as H9c2 cells, with two known in vitro preconditioning stimuli: hypoxia or isoflurane. EVs were isolated from cell culture supernatants 48 h after stimulation by differential centrifugation and size exclusion chromatography. They were characterized by transmission electron microscopy, tunable resistive pulse sensing, miRNA array and Western blot analysis. The detected EVs had the typical cup-shaped morphology and a size of about 150 nm. No significant differences in EV concentration were observed between the different groups. The protein and miRNA load was affected by in vitro preconditioning with isoflurane or hypoxia. EV markers like Alix, CD63, flotillin-1 and especially heat shock protein 70 were significantly up-regulated by the treatments. Several miRNAs like miR-92b-3p, miR-761 and miR-101a-5p were also significantly affected. A migration assay confirmed the physiological benefit of these EVs. Taken together, our findings show that a model of in vitro preconditioning of cardiac cells does not influence EV concentration but strongly regulates the EV cargo and affects migration. This might indicate a role for EV-mediated communication in isoflurane- and hypoxia-induced in vitro preconditioning.

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

“…The main purification procedures were based on ultracentrifugation 7 – 10 , 13 – 33 and precipitation methods 10 , 13 , 18 , 21 , 24 , 29 , 34 – 49 . Six publications used both methods for EV purification 10 , 13 , 18 , 21 , 24 , 29 and five publications used other methods or explicit different additional methods, to isolate EVs 20 , 29 , 33 , 50 , 51 .…”

“…The main purification procedures were based on ultracentrifugation 7 – 10 , 13 – 33 and precipitation methods 10 , 13 , 18 , 21 , 24 , 29 , 34 – 49 . Six publications used both methods for EV purification 10 , 13 , 18 , 21 , 24 , 29 and five publications used other methods or explicit different additional methods, to isolate EVs 20 , 29 , 33 , 50 , 51 . Of all investigated articles only nine publications did not perform EM pictures of the isolated EVs 9 , 19 , 24 , 32 , 40 , 43 , 47 , 50 , 51 and three publications did not investigate if the isolated particles contained typical EV markers 19 , 43 , 50 .…”

“…Cardiomyocytes are, next to fibroblasts and endothelial cells, one of the most abundant cell types in the mammalian heart. Due to their importance in cardiac function, researchers are extensively studying their physiological properties 52 as well as their capabilities to secrete EVs 33 . Publications investigating cardiomyocyte derived EVs are summarized in Table 2 .…”

Therapeutic Strategies of Secretome of Mesenchymal Stem Cell

“…preconditioning. An experimental model of in vitro preconditioning of cardiac cells concluded that it does not influence the concentration of ExMVs, but regulates their load and affects migration [69].…”

MiR-133a-3p transferred by circulating microvesicles derived from myocardial ischemic preconditioning protects cardiomyocytes against hypoxia/reoxygenation injury

“…Giving blood reperfusion and oxygen supply to ischemic myocardium, at the same time, aggravated myocardial injury, that is called ischemia/reperfusion (I/R) injury [1]. It is wellknown that ischemic preconditioning (IPC) is one of the most powerful cardioprotective interventions to reduce myocardial I/R injury [2], which could induce extracellular vesicles (EVs) such as microvesicles (MVs) and exosomes release and changes in microRNA (miRNA) expression in cardiomyocytes [3,4], but the underlying mechanism remains elusive. Recently, it has been shown in our study that circulating MVs released from the heart after IPC (IPC-MVs) are required for cardioprotection by IPC [5] and that IPC-MVs could protect against myocardial I/R injury by regulating GRP78 and CHOP expression in endoplasmic reticulum pathway [6].…”