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Transcriptomic profiling of cell-free and vesicular microRNAs from matched arterial and venous sera.

Author(s): Hermann S, Buschmann D, Kirchner B, Borrmann M, Brandes F, Kotschote S, Bonin M, Lindemann A, Reithmair M, Schelling G, Pfaffl MW

Publication: J Extracell Vesicles, 2019, Vol. 8, Page 1670935

PubMed ID: 31632620 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared miRNA yields and profiles in crude and SEC-purified extracellular vesicles from case-matched arterial and venous serum specimens. The subjects included 20 cardiac surgery patients (15 with coronary artery disease and five with combined heart valve disease) of which fifteen also had hypertension, seven also had diabetes, and five had minor renal function impairment.  Blood was collected into S-monovette serum tubes simultaneously from an arterial line introduced into the radial artery and a central venous line in the internal jugular vein after induction of anesthesia. After a 30 min clot time, serum was obtained by centrifugation at 3,400 x g for 10 min and then aliquoted and stored at -80°C. Crude EVs were obtained using the miRCURY Exosome Isolation Kit-Serum and Plasma kit.  To investigate the effects of further purification, EVs from 14 patients were further purified using SEC. RNA was extracted from the EVs using miRCURY RNA Isolation Kit-Biofluids kit for NGS analysis and the miRNeasy Micro Kit for real-time PCR analysis. The elution step was repeated for each kit and the eluates were further concentrated by vacuum-induced centrifugal evaporation. RNA yield and size distribution were determined by Bioanalyzer.  cDNA libraries were made using the NEBNext Multiplex Small RNA Library Prep Set for Illumina after dilution of adapters and primers and sequenced on a 2100 Bioanalyzer using the DNA 1000 Kit and a HiSeq2500. miRNAs found to differ between arterial and venous specimens were verified by real-time RT-PCR using the miRCURY LNA RT and SYBR Green PCR Kits.

   Summary of Findings:

   The amount of RNA obtained from crude and SEC-purified EVs was comparable in arterial and venous serum specimens. The mean proportion of miRNA reads was much lower for SEC-purified than crude EVs but were comparable among arterial and venous serum specimens (44.20% arterial in crude samples, 46.39% in venous crude samples, 9.51% in arterial purified EVs and 10.48% in venous purified EVs). miRNA profiles were very strongly correlated between venous and arterial specimens for both crude EVs (R²=0.9923) and SEC-purified EVs (R²=0.9992). Importantly, correlations between arterial and venous specimens remained very strong even when the lowest expressed (<10000 reads) miRNAs were considered (R²=0.9948 in crude EVS and R²=0.9943 in purified EVS). PCA analysis showed overlapping expression profiles in arterial and venous specimens regardless of EV SEC purification. However, while hierarchical clustering of crude EV specimens clustered by patient, clustering was less clear in SEC-purified EVs.  Among the ten most highly expressed miRNAs, variability was greater between patients than between arterial and venous specimens from the same patient indicating a limited effect of the location of blood sampling. Although no differentially expressed miRNAs were identified using the most stringent condition (baseMean≥50, absolute value of log2 fold change ≥1, and P-adjusted ≤0.05) regardless of whether EVs were crude or SEC-purified, when the least stringent conditions was applied miR-223-3p, miR-379-5p, miR-493-5p, miR-542-3p were higher in crude EVs isolated from venous as opposed to arterial specimens (baseMean ≥50, absolute value of log2 fold change ≥0.5, and P adjusted ≤0.1). However, this was not true for all specimens nor was it observed in SEC-purified EVs. Of theses 4 miRNAs, real-time PCR was only able to confirm higher average levels of miR-493-5p in arterial compared to venous specimens (2.37-fold, P=0.0014).

   Biospecimens

   • Bodily Fluid - Serum
   • Bodily Fluid - Blood

   Preservative Types

   • Frozen

   Diagnoses:

   • Coronary Artery Disease
   • Hypertension
   • Other diagnoses
   • Diabetes Type 2

   Platform:

   Analyte	Technology Platform
   RNA	Automated electrophoresis/Bioanalyzer
   RNA	Real-time qRT-PCR
   RNA	Next generation sequencing

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Anatomical location of blood draw	Radial artery Internal jugular vein
   Next generation sequencing Specific	Technology platform	Real-time PCR
   Biospecimen Aliquots and Components	Blood and blood products	Purified extracellular vesicles Crude extracellular vesicles

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2. Study Purpose

   The purpose of this study was to compare the size and morphology of EVs isolated from arterial and venous serum specimens before and after SEC purification. Subjects included 20 cardiac surgery patients (15 with coronary artery disease and five with combined heart valve disease) of which fifteen also had hypertension, seven also had diabetes, and five had minor renal function impairment.  Blood was collected into S-monovette serum tubes simultaneously from an arterial line introduced into the radial artery and a central venous line in the internal jugular vein after induction of anesthesia. After a 30 min clot time, serum was obtained by centrifugation at 3,400 x g for 10 min and then aliquoted and stored at -80°C. Crude EVs were obtained using the miRCURY Exosome Isolation Kit-Serum and Plasma kit.  To investigate the effects of additional purification, EVs from 14 patients were further purified by SEC. The size and concentration of extracellular particles were determined by nanoparticle tracking analysis and transmission electron microscopy. EV markers were evaluated by Western blot analysis using antibodies for syntenin, CD63, ALIX, CD81, apolipoprotein A1 (ApoA1), calnexin (CNX).

   Summary of Findings:

   While EVs isolated from arterial specimens were slightly smaller than those from venous serum specimens regardless of additional purification, the authors report that the difference between  means (113.20 versus 117.15 nm in crude, and 138.54 versus 147.54 nm in purified) and medians (97.85 versus 101.27 nm in crude, and 123.78 versus 132.00 nm in purified) were close to the margin of error. SEM confirmed the presence of EVs with diameters of approximately 100 nm in crude and purified arterial and venous specimens. Particle concentrations were also slightly lower in arterial than venous specimens, and much lower in purified compared to crude specimens (6.71E11 particles/ml in crude arterial, 7.27E11 particles/ml in crude venous, 1.30E+09 particles/ml in purified arterial and 1.38E+09 particles/ml in purified venous). Western blot analysis of SEC-purified EVs resulted in detection of alix, CD63, syntenin and CD81 in both arterial and venous specimens, and the expected lack of CNX indicated no evidence of cellular contamination. ApoA1 and albumin were detected at low levels in both arterial and venous specimens despite not being enriched in EVs.

    

   Biospecimens

   • Bodily Fluid - Serum
   • Bodily Fluid - Blood

   Preservative Types

   • Frozen

   Diagnoses:

   • Coronary Artery Disease
   • Other diagnoses
   • Hypertension
   • Diabetes Type 2

   Platform:

   Analyte	Technology Platform
   Protein	Western blot
   Morphology	Electron microscopy
   Morphology	Light scattering

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Anatomical location of blood draw	Internal jugular vein Radial artery
   Biospecimen Aliquots and Components	Blood and blood products	Crude extracellular vesicles Purified extracellular vesicles

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