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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

Analysis of apoB-positivity in blood plasma and PLT concentrate-derived EXOs.(A,B) FCM detection of the indicated markers in EXOs conjugated onto latex beads. The EXOs were isolated from fasting PFP (A) or PLT concentrate (B) by differential UC and gravity size filtration (gray histograms: blocked beads incubated with antibody, empty histograms: EXO sample). (C) EXOs from fasting PFP purified on an OptiprepTM density-gradient. Distribution of CD9 positive events was determined by FCM (upper panel, n = 3, mean + SEM) and CD63 positivity of fractions was determined by Western blotting (lower panel). Of note, Western blotting only shows one of the analyzed samples while the FCM shows the average ± SEM of 3 measurements. (D) The distribution of apoB-positive events determined by FCM (upper panel, n = 3) and by Western blotting (lower panel) from the same samples as in (C). (E) PLT-derived EXOs were also purified on a density-gradient. The EXO containing FR7-8 was analyzed by FCM for CD9-, CD63- and apoB-positivity (gray histograms: blocked beads incubated with antibody, empty histograms: EXO sample).
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f5: Analysis of apoB-positivity in blood plasma and PLT concentrate-derived EXOs.(A,B) FCM detection of the indicated markers in EXOs conjugated onto latex beads. The EXOs were isolated from fasting PFP (A) or PLT concentrate (B) by differential UC and gravity size filtration (gray histograms: blocked beads incubated with antibody, empty histograms: EXO sample). (C) EXOs from fasting PFP purified on an OptiprepTM density-gradient. Distribution of CD9 positive events was determined by FCM (upper panel, n = 3, mean + SEM) and CD63 positivity of fractions was determined by Western blotting (lower panel). Of note, Western blotting only shows one of the analyzed samples while the FCM shows the average ± SEM of 3 measurements. (D) The distribution of apoB-positive events determined by FCM (upper panel, n = 3) and by Western blotting (lower panel) from the same samples as in (C). (E) PLT-derived EXOs were also purified on a density-gradient. The EXO containing FR7-8 was analyzed by FCM for CD9-, CD63- and apoB-positivity (gray histograms: blocked beads incubated with antibody, empty histograms: EXO sample).

Mentions: Since we detected the co-purification of lipoproteins with MVs, next we tested whether they co-purified with EXOs as well. EXOs isolated by differential UC and gravity size filtration from fasting PFP (Fig. 5a) and PLT concentrate (Fig. 5b), were bound onto latex beads for FCM analysis. As expected, they stained for EXO markers such as CD9 and CD63. Importantly, they also showed very high apoB-positivity and a weaker staining for apoCII and ApoE. We also analyzed EXOs isolated form postprandial PFPs. We found that even in fasting condition apoB-positive events covered the surface of beads predominantly (>95% was apoB-positive compared to the <0.5% CD9/CD63/AX-positivity; data not shown). Postprandially we could not detect a significant further increase in the apoB signal. Given the predominant apoB-positivity already present in fasting EXO samples, similarly to the analysis of MVs, in our further purification studies we focused on EXOs derived from fasting PFPs only.


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)

Analysis of apoB-positivity in blood plasma and PLT concentrate-derived EXOs.(A,B) FCM detection of the indicated markers in EXOs conjugated onto latex beads. The EXOs were isolated from fasting PFP (A) or PLT concentrate (B) by differential UC and gravity size filtration (gray histograms: blocked beads incubated with antibody, empty histograms: EXO sample). (C) EXOs from fasting PFP purified on an OptiprepTM density-gradient. Distribution of CD9 positive events was determined by FCM (upper panel, n = 3, mean + SEM) and CD63 positivity of fractions was determined by Western blotting (lower panel). Of note, Western blotting only shows one of the analyzed samples while the FCM shows the average ± SEM of 3 measurements. (D) The distribution of apoB-positive events determined by FCM (upper panel, n = 3) and by Western blotting (lower panel) from the same samples as in (C). (E) PLT-derived EXOs were also purified on a density-gradient. The EXO containing FR7-8 was analyzed by FCM for CD9-, CD63- and apoB-positivity (gray histograms: blocked beads incubated with antibody, empty histograms: EXO sample).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Analysis of apoB-positivity in blood plasma and PLT concentrate-derived EXOs.(A,B) FCM detection of the indicated markers in EXOs conjugated onto latex beads. The EXOs were isolated from fasting PFP (A) or PLT concentrate (B) by differential UC and gravity size filtration (gray histograms: blocked beads incubated with antibody, empty histograms: EXO sample). (C) EXOs from fasting PFP purified on an OptiprepTM density-gradient. Distribution of CD9 positive events was determined by FCM (upper panel, n = 3, mean + SEM) and CD63 positivity of fractions was determined by Western blotting (lower panel). Of note, Western blotting only shows one of the analyzed samples while the FCM shows the average ± SEM of 3 measurements. (D) The distribution of apoB-positive events determined by FCM (upper panel, n = 3) and by Western blotting (lower panel) from the same samples as in (C). (E) PLT-derived EXOs were also purified on a density-gradient. The EXO containing FR7-8 was analyzed by FCM for CD9-, CD63- and apoB-positivity (gray histograms: blocked beads incubated with antibody, empty histograms: EXO sample).
Mentions: Since we detected the co-purification of lipoproteins with MVs, next we tested whether they co-purified with EXOs as well. EXOs isolated by differential UC and gravity size filtration from fasting PFP (Fig. 5a) and PLT concentrate (Fig. 5b), were bound onto latex beads for FCM analysis. As expected, they stained for EXO markers such as CD9 and CD63. Importantly, they also showed very high apoB-positivity and a weaker staining for apoCII and ApoE. We also analyzed EXOs isolated form postprandial PFPs. We found that even in fasting condition apoB-positive events covered the surface of beads predominantly (>95% was apoB-positive compared to the <0.5% CD9/CD63/AX-positivity; data not shown). Postprandially we could not detect a significant further increase in the apoB signal. Given the predominant apoB-positivity already present in fasting EXO samples, similarly to the analysis of MVs, in our further purification studies we focused on EXOs derived from fasting PFPs only.

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