| Background：Cell-to-cell communication is required to guarantee proper coordination amongdifferent cell types within tissues. There are multiple types of intercellular communication,including soluble factors, tunneling nanotubules, and extracellular vesicles (EVs), whichallow the transfer of surface molecules or cytoplasmic components from one cell to another.EVs are circular plasma membrane fragments that include exosomes and microparticles orshed vesicles，which are shed from almost all cell types under both physiological andpathological conditions[1,2]. The biological function of EVs is poorly understood, but mayinclude secretion, immunomodulation, coagulation, and intercellular communication[3-5].EVs may vary in their formation, abundance, size, and composition, but they oftencontain abundant molecules, which include functional transmembrane and cytosol proteins,message RNA (mRNA), and microRNA (miRNA)[6-9]. The components in EVs could betransferred from one cell to another by endocytosis or fusion with the recipient cell[10-13].Importantly, the transferred components in EVs are functional and can regulate thebiological functions of the recipient cells[8,9,14,15]. Moreover, The EV-mediatedintercellular communication plays an important role in pathogenesis of atherosclerosis[15].Recent studies have shown that both mitochondrial DNA (mtDNA) and chromosomalDNA were found in EVs[16,17]. Waldenstrom et al.[17]reported that chromosomal DNAsequences in EVs from cardiomyocytes could be transferred to the cytosol or nuclei oftarget cells. However, whether the transferred EV DNAs are functional or not is unclear. Inthis study, we investigated the function and potential mechanisms of transferrable EVgenomic DNAs (gDNAs) in the recipient cells. Finally, we also elucidated the critical roleof EV DNA that plays in pathogenesis of atherosclerosis. Methods：1. EVs from human plasma or cell culture supernatants were isolated through a seriesof ultracentrifugation steps as previously described[8,9,15]. To ensure that the EVs werecorrectly identified, electron microscopic, immunoblotting, and flow cytometry (FCM)analyses were used.2. We examined the characteristics of DNA in EVs derived from human plasma andsupernatants of vascular smooth muscle cells (VSMCs) in culture by fluorescence-activatedcell sorter (FACS), high performance liquid chromatography (HPLC), agarose gelelectrophoresis, Solexa sequencing and polymerase chain reaction (PCR).3. To determine the transportability and functionality of the DNA fragments in EVs,we studied the transport of gDNAs (AT1receptor DNA) in EVs from AT1receptortransfected-HEK293cells or VSMCs to non-transfected HEK293cells by PCR andimmunoblotting. To uncover the underlying mechanisms leading to the transcription oftransferred EV DNA, we investigated the effect of endogenous NF-κB in recipient cells, apromoter of the AT1receptor gene, on transferred DNAs including AT1receptor DNA byimmunofluorescence using laser confocal microscopy.4. The VSMC and HEK293cell studies only provided indirect evidence that theincreased AT1mRNA is due to the incorporation of DNA from EVs. To overcome thislimitation, we studied the transport of the BCR/ABL hybrid gene in EVs from K562cells toHEK293cells, which is not normally expressed in HEK293cells. To determine thepathophysiological significance of transferred gDNAs in EVs between cells, we examinedthe transfer of BCR/ABL hybrid gene in EVs from K562cells to normal human neutrophilsisolated from human peripheral blood by dual-color fluorescence in-situ hybridization(D-FISH).5. To investigate the critical role of EV DNA that plays in pathogenesis ofatherosclerosis, we screened out EV DNA fragments with differential gene copy numbers(GCNs) in plasma from patients with coronary artery disease (CAD) compared to controlhealthy subjects by Solexa sequencing. Then, we validated the screening EV DNAfragments in plasma by quantitative PCR. We also compared the levels of SRY GCNs,mRNA, and protein in WBCs between two groups by PCR and immunoblotting. Finally, wetransferred sex-determining region Y (SRY) gene-containing EVs or plasmids to monocytes or human umbilical vein endothelial cells (HUVECs) and observed adhesion functionbetween the two cells.Results：1. The electron micrographs of the EVs revealed rounded double-layer membranousvesicles of approximately30-1000nm in size, similar to previously described EVs[8,9,15].EVs more than200nm in size were confirmed by FCM analysis as this technology isunable to distinguish or enumerate single particles below200nm in size[18].Immunoblotting showed the presence of CD63, argonaute2(AGO2), tumor susceptibilitygene101(TSG101), Flotillin-1, and heat shock protein70(HSP70) proteins, commonlyused markers for EVs.2. Substantial amounts of DNA were detected by FACS, HPLC, agarose gelelectrophoresis, and PCR in EVs derived from human plasma and supernatants of VSMCsin culture. We found that EVs contained double-strand DNA (dsDNA) ranging in size from1to20kb, but mostly around17kb. Furtherly, Solexa sequencing indicated the presence ofat least16434genomic DNA (gDNA) fragments in the EVs from human plasma.3. We found the presence of AT1-EGFP DNA in EVs from AT1-HEK293cells thatstably expressed AT1protein by PCR and sequencing. After incubation of HEK293cellswith EVs from AT1-HEK293cells or VSMCs, immunofluorescence study showed directevidence that EV DNAs could be transferred into the recipient cells and localize to andinside the nuclear membrane. As an endogenous promoter of the AT1receptor, NF-κB,could be recruited to the transferred DNAs in the nucleus, and increased the transcription ofAT1receptor in the recipient cells. We also found the de-novo mRNA and protein of AT1receptor could be significantly inhibited by actinomycin D.4. Incubating HEK293cells with EVs from K562cells resulted in the expression ofBCR/ABL hybrid gene mRNA and protein in the recipient HEK293cells. Moreover, thisheterologous expression of BCR/ABL hybrid gene in the HEK293cells was prevented bythe concurrent incubation with actinomycin D. Using a dual-color fluorescence in situhybridization (D-FISH) method, we found that incubation of normal human neutrophilswith EVs from K562cells resulted in the expression of BCR/ABL hybrid gene in20%ofthe neutrophils by genomic analysis.5. Using Solexa sequencing followed by quantitative PCR, the GCNs of SRY DNA in EVs from plasma were significantly higher in patients with CAD than those in controlhealthy subjects. We also found the levels of SRY GCNs, mRNA, and protein in WBCswere higher in patients with CAD than those in control healthy subjects. Importantly,Transferred SRY gene-containing EVs or plasmids to monocytes and HUVECs enhancedadhesion function between the two cellsConclusions：In conclusion, we have shown that EV DNAs, which could be delivered from one cellto another, can increase the gDNA-coding mRNA and protein expressions in the recipientcells, and affect the physiological function in the recipient cells. Furtherly, we have foundSRY gene-containing EVs may play an critical role in atherosclerosis. EV-mediated transferof gDNAs may represent a new method of gene delivery and novel way of signaltransduction among cells. These findings would help in the discovery of novel mechanismsof cardiovascular disease and development of new therapeutic strategies. |
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