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Low-density lipoprotein mimics blood plasma-derived exosomes and microvesicles during isolation and detection.

Sódar BW, Kittel Á, Pálóczi K, Vukman KV, Osteikoetxea X, Szabó-Taylor K, Németh A, Sperlágh B, Baranyai T, Giricz Z, Wiener Z, Turiák L, Drahos L, Pállinger É, Vékey K, Ferdinandy P, Falus A, Buzás EI - Sci Rep (2016)

Bottom Line: Here we studied human pre-prandial and 4 hours postprandial platelet-free blood plasma samples as well as human platelet concentrates.Based on biophysical properties of LDL this finding was highly unexpected.Current state-of-the-art extracellular vesicle isolation and purification methods did not result in lipoprotein-free vesicle preparations from blood plasma or from platelet concentrates.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Cell- and Immunobiology, Semmelweis University, Budapest, 1085, Hungary.

ABSTRACT
Circulating extracellular vesicles have emerged as potential new biomarkers in a wide variety of diseases. Despite the increasing interest, their isolation and purification from body fluids remains challenging. Here we studied human pre-prandial and 4 hours postprandial platelet-free blood plasma samples as well as human platelet concentrates. Using flow cytometry, we found that the majority of circulating particles within the size range of extracellular vesicles lacked common vesicular markers. We identified most of these particles as lipoproteins (predominantly low-density lipoprotein, LDL) which mimicked the characteristics of extracellular vesicles and also co-purified with them. Based on biophysical properties of LDL this finding was highly unexpected. Current state-of-the-art extracellular vesicle isolation and purification methods did not result in lipoprotein-free vesicle preparations from blood plasma or from platelet concentrates. Furthermore, transmission electron microscopy showed an association of LDL with isolated vesicles upon in vitro mixing. This is the first study to show co-purification and in vitro association of LDL with extracellular vesicles and its interference with vesicle analysis. Our data point to the importance of careful study design and data interpretation in studies using blood-derived extracellular vesicles with special focus on potentially co-purified LDL.

No MeSH data available.


Related in: MedlinePlus

Purification of PLT concentrate-derived MVs with OptiprepTM density gradient UC and SEC.(A,B) Quantification of FCM data and Western-blotting of PLT concentrate-derived MV fractions purified on an OptiprepTM density gradient (n = 3, mean + SEM). The event number was detected within the MV-gate. MVs were detected by AX (FCM) and CD63 (Western blotting) (A) and lipoproteins by apoB (550 kDa) (B). Of note, Western blotting only shows one of the analyzed samples while the FCM shows the average ± SEM of 3 measurements. (C) Comparison of the apoB-positive events (black bars) and the AX-positives (gray bars) (FCM, n = 2, mean + SEM). (D) SEC purification of MVs isolated from PLT concentrates, fractions analyzed by FCM. (apoB: black; AX: gray; CD41a: light gray bars, n = 2, mean + SEM) Note that the apoB-positivity was co-purified with the EV markers.
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f4: Purification of PLT concentrate-derived MVs with OptiprepTM density gradient UC and SEC.(A,B) Quantification of FCM data and Western-blotting of PLT concentrate-derived MV fractions purified on an OptiprepTM density gradient (n = 3, mean + SEM). The event number was detected within the MV-gate. MVs were detected by AX (FCM) and CD63 (Western blotting) (A) and lipoproteins by apoB (550 kDa) (B). Of note, Western blotting only shows one of the analyzed samples while the FCM shows the average ± SEM of 3 measurements. (C) Comparison of the apoB-positive events (black bars) and the AX-positives (gray bars) (FCM, n = 2, mean + SEM). (D) SEC purification of MVs isolated from PLT concentrates, fractions analyzed by FCM. (apoB: black; AX: gray; CD41a: light gray bars, n = 2, mean + SEM) Note that the apoB-positivity was co-purified with the EV markers.

Mentions: We found apoB-positivity in MVs isolated from fasting PFP (Fig. 3a,b), so we decided to investigate whether lipoproteins also co-purified with PLT concentrate-derived MVs. For purification we used an OptiprepTM gradient (Fig. 4a,b). Using FCM and Western blotting, AX and CD63 labelling indicated that MVs were enriched in the 6th fraction of the gradient (Fig. 4a) (1.05–1.10 g/mL, Supplementary Fig. S6). Surprisingly, apoB-positive events were detectable in almost all fractions (Fig. 4b). Western blot analysis proved that this apoB-positivity was due to the presence apoB100 (550 kDa) suggesting that the co-purified particles were mostly LDLs, not chylomicrons (bearing apoB48, 260 kDa). Even in the most MV-enriched fraction (FR6), apoB-positive events outnumbered the AX-positive ones up to 5× (Fig. 4c). Next, we tested whether SEC resulted in pure PLT-MVs (Fig. 4d). The third fraction that contained the most pure population of EVs according to the manufacturer contained still high amount of apoB-positive particles measured by FCM.


Low-density lipoprotein mimics blood plasma-derived exosomes and microvesicles during isolation and detection.

Sódar BW, Kittel Á, Pálóczi K, Vukman KV, Osteikoetxea X, Szabó-Taylor K, Németh A, Sperlágh B, Baranyai T, Giricz Z, Wiener Z, Turiák L, Drahos L, Pállinger É, Vékey K, Ferdinandy P, Falus A, Buzás EI - Sci Rep (2016)

Purification of PLT concentrate-derived MVs with OptiprepTM density gradient UC and SEC.(A,B) Quantification of FCM data and Western-blotting of PLT concentrate-derived MV fractions purified on an OptiprepTM density gradient (n = 3, mean + SEM). The event number was detected within the MV-gate. MVs were detected by AX (FCM) and CD63 (Western blotting) (A) and lipoproteins by apoB (550 kDa) (B). Of note, Western blotting only shows one of the analyzed samples while the FCM shows the average ± SEM of 3 measurements. (C) Comparison of the apoB-positive events (black bars) and the AX-positives (gray bars) (FCM, n = 2, mean + SEM). (D) SEC purification of MVs isolated from PLT concentrates, fractions analyzed by FCM. (apoB: black; AX: gray; CD41a: light gray bars, n = 2, mean + SEM) Note that the apoB-positivity was co-purified with the EV markers.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4834552&req=5

f4: Purification of PLT concentrate-derived MVs with OptiprepTM density gradient UC and SEC.(A,B) Quantification of FCM data and Western-blotting of PLT concentrate-derived MV fractions purified on an OptiprepTM density gradient (n = 3, mean + SEM). The event number was detected within the MV-gate. MVs were detected by AX (FCM) and CD63 (Western blotting) (A) and lipoproteins by apoB (550 kDa) (B). Of note, Western blotting only shows one of the analyzed samples while the FCM shows the average ± SEM of 3 measurements. (C) Comparison of the apoB-positive events (black bars) and the AX-positives (gray bars) (FCM, n = 2, mean + SEM). (D) SEC purification of MVs isolated from PLT concentrates, fractions analyzed by FCM. (apoB: black; AX: gray; CD41a: light gray bars, n = 2, mean + SEM) Note that the apoB-positivity was co-purified with the EV markers.
Mentions: We found apoB-positivity in MVs isolated from fasting PFP (Fig. 3a,b), so we decided to investigate whether lipoproteins also co-purified with PLT concentrate-derived MVs. For purification we used an OptiprepTM gradient (Fig. 4a,b). Using FCM and Western blotting, AX and CD63 labelling indicated that MVs were enriched in the 6th fraction of the gradient (Fig. 4a) (1.05–1.10 g/mL, Supplementary Fig. S6). Surprisingly, apoB-positive events were detectable in almost all fractions (Fig. 4b). Western blot analysis proved that this apoB-positivity was due to the presence apoB100 (550 kDa) suggesting that the co-purified particles were mostly LDLs, not chylomicrons (bearing apoB48, 260 kDa). Even in the most MV-enriched fraction (FR6), apoB-positive events outnumbered the AX-positive ones up to 5× (Fig. 4c). Next, we tested whether SEC resulted in pure PLT-MVs (Fig. 4d). The third fraction that contained the most pure population of EVs according to the manufacturer contained still high amount of apoB-positive particles measured by FCM.

Bottom Line: Here we studied human pre-prandial and 4 hours postprandial platelet-free blood plasma samples as well as human platelet concentrates.Based on biophysical properties of LDL this finding was highly unexpected.Current state-of-the-art extracellular vesicle isolation and purification methods did not result in lipoprotein-free vesicle preparations from blood plasma or from platelet concentrates.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Cell- and Immunobiology, Semmelweis University, Budapest, 1085, Hungary.

ABSTRACT
Circulating extracellular vesicles have emerged as potential new biomarkers in a wide variety of diseases. Despite the increasing interest, their isolation and purification from body fluids remains challenging. Here we studied human pre-prandial and 4 hours postprandial platelet-free blood plasma samples as well as human platelet concentrates. Using flow cytometry, we found that the majority of circulating particles within the size range of extracellular vesicles lacked common vesicular markers. We identified most of these particles as lipoproteins (predominantly low-density lipoprotein, LDL) which mimicked the characteristics of extracellular vesicles and also co-purified with them. Based on biophysical properties of LDL this finding was highly unexpected. Current state-of-the-art extracellular vesicle isolation and purification methods did not result in lipoprotein-free vesicle preparations from blood plasma or from platelet concentrates. Furthermore, transmission electron microscopy showed an association of LDL with isolated vesicles upon in vitro mixing. This is the first study to show co-purification and in vitro association of LDL with extracellular vesicles and its interference with vesicle analysis. Our data point to the importance of careful study design and data interpretation in studies using blood-derived extracellular vesicles with special focus on potentially co-purified LDL.

No MeSH data available.


Related in: MedlinePlus