Abstract 523: Lipid-associated Plasminogen Is A Ribonucleoprotein Carrier Of Extracellular Small RNAs In Atherosclerosis

Abstract

Small RNAs (sRNA) are exchanged between cells and tissues by various lipid and protein carriers. Extracellular sRNAs regulate gene expression in recipient cells, including activation of pattern recognition receptors and inflammatory signaling pathways. Based on size-exclusion chromatography (SEC) fractionation of human plasma, a large proportion of extracellular sRNAs are detected in small lipid-associated ribonucleoproteins (50-150 kDa), a class of carriers that have been largely overlooked. Sequential fractionation approaches, mass spectrometry, enzymatic activity assays, and western blotting all support that lipid-associated human plasminogen (PLG) co-purifies with sRNAs in plasma. PLG transports approximately 10 μg total RNA/ mg protein in plasma. Depletion of PLG from plasma by lysine-sepharose resin was also found to reduce plasma miRNA levels. Based on microscale thermophoresis assays, PLG was determined to have moderate binding to single-stranded sRNAs (K d =1.8μM). Immune cells may be a source of sRNAs, as macrophages were found to export both host and non-host sRNAs to PLG in vitro . To identify PLG-associated sRNAs, sRNA sequencing was completed, and results showed that PLG host sRNA are predominantly derived from parent rRNAs (rDR), tRNAs (tDR), yRNA (yDRs), and microRNAs (miRNA). Furthermore, we found that host sRNAs on PLG are significantly altered in subjects with familial hypercholesterolemia (hetFH), including 22 miRNAs, 5 tDRs, and 10 yDRs. PLG was also found to transport many non-host microbial sRNAs derived from bacteria, fungi, and viruses in the microbiome and environment; and genome counts for many of these microbial species were significantly altered on PLG isolated from hetFH subjects. PLG was found to cause dramatic gene (mRNA) expression changes in treated-macrophages; however, it is unknown what fraction of these changes are associated with PLG-sRNA delivery. Collectively, results suggest that PLG transports high levels of extracellular sRNAs from host and non-host species that are sensitive to hypercholesterolemia and disease associated changes. Although the impact of PLG-sRNAs remains to be determined, they hold great potential as therapeutic targets for inflammation.