DNA Content in Extracellular Vesicles Isolated from Porcine Coronary Venous Blood Directly after Myocardial Ischemic Preconditioning

Abstract

BackgroundExtracellular vesicles (EV) are nano-sized membranous structures released from most cells. They have the capacity to carry bioactive molecules and gene expression signals between cells, thus mediating intercellular communication. It is believed that EV confer protection after ischemic preconditioning (IPC). We hypothesize that myocardial ischemic preconditioning will lead to rapid alteration of EV DNA content in EV collected from coronary venous effluent.Materials and MethodsIn a porcine myocardial ischemic preconditioning model, EV were isolated from coronary venous blood before and after IPC by differential centrifugation steps culminating in preparative ultracentrifugation combined with density gradient ultracentrifugation. The EV preparation was validated, the DNA was extracted and further characterized by DNA sequencing followed by bioinformatics analysis.ResultsPorcine genomic DNA fragments representing each chromosome, including mitochondrial DNA sequences, were detected in EV isolated before and after IPC. There was no difference detected in the number of sequenced gene fragments (reads) or in the genomic coverage of the sequenced DNA fragments in EV isolated before and after IPC. Gene ontology analysis showed an enrichment of genes coding for ion channels, enzymes and proteins for basal metabolism and vesicle biogenesis and specific cardiac proteins.ConclusionsThis study demonstrates that porcine EV isolated from coronary venous blood plasma contain fragments of DNA from the entire genome, including the mitochondria. In this model we did not find specific qualitative or quantitative changes of the DNA content in EV collected immediately after an in vivo myocardial IPC provocation. This does not rule out the possibility that EV DNA content changes in response to myocardial IPC which could occur in a later time frame.

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

“…In another IPC study, the authors investigated whether this treatment might promote a change in the DNA content in EVs. The authors could not detect any difference in the number of sequenced gene fragments between treatment and control 16 . In a rat model, rIPC resulted in an increase of miR-29a in serum-derived EVs 40 .…”

“…Others investigated the capabilities and characteristics of EVs secreted under regular conditions 7 – 10 , 13 – 36 , 39 – 42 , 44 – 51 . Different treatments of the EVs source also enhanced EV properties 8 , 10 , 14 – 16 , 21 , 22 , 28 – 33 , 39 , 40 , 48 , 49 . Researchers investigating fibroblast-derived EVs postulated that they might not contribute to cardioprotection in the same extent 13 , 17 , 35 , 45 , 47 whereas other groups observed enhanced proliferation/migration after treatment with fibroblast-derived EVs 33 .…”

“… 8 Coronary perfusates UC 10–1000 nm I/R injury NSI HSP60 Svennerholm et al . 16 Plasma UC, sucrose gradient 30–350 nm NSI NSI CD81 Yamaguchi et al . 40 Serum UC NSI MI (miR-29a) CD9, HSP90, GAPDH X. Wang et al .…”

The p66ShcA adaptor protein regulates healing after myocardial infarction

“…Circulating exDNA has been studied as a biomarker in ischemic heart disease (115) and heart transplant rejection (116). Extracellular vesicles isolated directly from porcine venous blood after myocardial ischemic preconditioning contained both exDNA of nuclear and mitochondrial origin (117). The severity of coronary atherosclerosis and susceptibility to thrombosis correlated with exDNA levels (118).…”

Emerging role of non-coding RNAs and extracellular vesicles in cardioprotection by remote ischemic conditioning of the heart

“…Interestingly, in addition to proposed pivotal role of certain miRNAs in remote cardioprotection mediated via exosomal transfer, rapid changes in mRNA content was found in cardiac extracellular vesicles due to IPC (Svennerholm et al, 2015). On the contrary, no specific qualitative or quantitative changes in DNA content were found in these particles after an in vivo myocardial IPC (Svennerholm et al, 2016).…”