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.
Highlights
Extracellular vesicles (EV) are nano-sized membranous structures released from most cells
In 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
There was no difference detected in the number of sequenced gene fragments or in the genomic coverage of the sequenced DNA fragments in EV isolated before and after IPC
Summary
Extracellular vesicles (EV) are nano-sized membranous structures released from most cells. The term EV includes different membrane enclosed vesicles, exosomes, microvesicles and apoptotic bodies. While these different subgroups are different in size, there have until recently not been specific markers that distinguish subsets of EV from each other [4]. EV from a broad panel of cancer cell lines and from plasma of patients with pancreatic cancer have been shown to contain DNA fragments which represent the entire genome, including the mutation of the cellular tumor source [8, 14]. They have the capacity to carry bioactive molecules and gene expression signals between cells, mediating intercellular communication. We hypothesize that myocardial ischemic preconditioning will lead to rapid alteration of EV DNA content in EV collected from coronary venous effluent
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