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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 particle nature in fasting and postprandial blood plasma samples.(A,B) Representative FCM plots (A) and their quantification (B) determined from 10 µL fasting (black bars) and 10 µL 4 h postprandial (gray bars) PFP samples of healthy individuals (n = 9, FCM, mean + SEM, ***P < 0.001, Wilcoxon matched-pairs signed rank test). CD9, AX and CD41a were used to identify MVs, and only events both staining for EV markers and sensitive to 0.1% Tx-100 were considered EVs. (C) Number of apoB-positive events within the MV gate in fasting (0 h) and postprandial (4 h) states, treated with Tx-100 (mean + SEM, ***P < 0.001, Wilcoxon matched-pairs signed rank test). (D) TEM analysis of the top 200 μL fractions of 2.5 mL ultracentrifuged (100,000 g, 2 h, 4 °C) fasting and postprandial plasma samples using an “osmification-on-grid” approach. Scale bar: 500 nm. Note that highly electron-dense floating particles were detected in the postprandial sample.
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f2: Analysis of particle nature in fasting and postprandial blood plasma samples.(A,B) Representative FCM plots (A) and their quantification (B) determined from 10 µL fasting (black bars) and 10 µL 4 h postprandial (gray bars) PFP samples of healthy individuals (n = 9, FCM, mean + SEM, ***P < 0.001, Wilcoxon matched-pairs signed rank test). CD9, AX and CD41a were used to identify MVs, and only events both staining for EV markers and sensitive to 0.1% Tx-100 were considered EVs. (C) Number of apoB-positive events within the MV gate in fasting (0 h) and postprandial (4 h) states, treated with Tx-100 (mean + SEM, ***P < 0.001, Wilcoxon matched-pairs signed rank test). (D) TEM analysis of the top 200 μL fractions of 2.5 mL ultracentrifuged (100,000 g, 2 h, 4 °C) fasting and postprandial plasma samples using an “osmification-on-grid” approach. Scale bar: 500 nm. Note that highly electron-dense floating particles were detected in the postprandial sample.

Mentions: Next we addressed the question whether these particles that appeared in our FCM analysis postprandially, could be lipoproteins. Thus, we labelled fasting and 4 h postprandial PFPs (n = 9) with fluorescently labelled annexin V (AX) that binds phosphatidyl-serine, as well as anti-CD41a and anti-CD9 antibodies in order to detect EVs by FCM. Furthermore, we used an anti-apoB100/48 antibody to detect chylomicrons which have been reported to appear in blood ~4 h after food intake1622 (Fig. 2a, fluorescence-based gating strategy: Supplementary Fig. S2). Surprisingly, only a few events within the MV size range carried EV markers, and even this low percentage of events was reduced postprandially (Fig. 2b, **P < 0.01). In sharp contrast, most of the particles detected within the size range of MVs were recognized by an antibody against human apoB100/B48, even in fasting PFP samples. Importantly, the high number of apoB-positive events was significantly increased further in the postprandial state (Fig. 2b, ***P < 0.001). In order to identify EVs in our samples, we applied differential detergent lysis1923. Only EV marker positive events which were disrupted in the presence of 0.1% Tx-100 were considered EVs, and are presented here. Importantly, we found that some of the apoB-positive events were also sensitive to detergent lysis even in fasting conditions (Fig. 2c, ***P < 0.001). However, the remaining apoB carrying events still highly outnumbered the disappeared EV marker-bearing ones (up to 20×). To confirm that lipoproteins appearing postprandially were chylomicrons, we tested whether they float upon ultracentrifugation (UC) as described previously22. To this end we analyzed the top fractions of ultracentrifuged fasting and postprandial PFPs by transmission electron microscopy (TEM). As shown in Fig. 2d, floating, electron-dense lipoproteins appeared 4 h after food intake. To correlate our findings with routine laboratory measurements, we measured serum triglyceride, total cholesterol, LDL cholesterol, apoA1 and apoB100 content. As shown in Supplementary Table S1, the concentration of triglycerides increased significantly (P < 0.05) in postprandial state. This further confirmed that these floating particles were chylomicrons which are known to be enriched postprandially in triglycerides22.


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 particle nature in fasting and postprandial blood plasma samples.(A,B) Representative FCM plots (A) and their quantification (B) determined from 10 µL fasting (black bars) and 10 µL 4 h postprandial (gray bars) PFP samples of healthy individuals (n = 9, FCM, mean + SEM, ***P < 0.001, Wilcoxon matched-pairs signed rank test). CD9, AX and CD41a were used to identify MVs, and only events both staining for EV markers and sensitive to 0.1% Tx-100 were considered EVs. (C) Number of apoB-positive events within the MV gate in fasting (0 h) and postprandial (4 h) states, treated with Tx-100 (mean + SEM, ***P < 0.001, Wilcoxon matched-pairs signed rank test). (D) TEM analysis of the top 200 μL fractions of 2.5 mL ultracentrifuged (100,000 g, 2 h, 4 °C) fasting and postprandial plasma samples using an “osmification-on-grid” approach. Scale bar: 500 nm. Note that highly electron-dense floating particles were detected in the postprandial sample.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Analysis of particle nature in fasting and postprandial blood plasma samples.(A,B) Representative FCM plots (A) and their quantification (B) determined from 10 µL fasting (black bars) and 10 µL 4 h postprandial (gray bars) PFP samples of healthy individuals (n = 9, FCM, mean + SEM, ***P < 0.001, Wilcoxon matched-pairs signed rank test). CD9, AX and CD41a were used to identify MVs, and only events both staining for EV markers and sensitive to 0.1% Tx-100 were considered EVs. (C) Number of apoB-positive events within the MV gate in fasting (0 h) and postprandial (4 h) states, treated with Tx-100 (mean + SEM, ***P < 0.001, Wilcoxon matched-pairs signed rank test). (D) TEM analysis of the top 200 μL fractions of 2.5 mL ultracentrifuged (100,000 g, 2 h, 4 °C) fasting and postprandial plasma samples using an “osmification-on-grid” approach. Scale bar: 500 nm. Note that highly electron-dense floating particles were detected in the postprandial sample.
Mentions: Next we addressed the question whether these particles that appeared in our FCM analysis postprandially, could be lipoproteins. Thus, we labelled fasting and 4 h postprandial PFPs (n = 9) with fluorescently labelled annexin V (AX) that binds phosphatidyl-serine, as well as anti-CD41a and anti-CD9 antibodies in order to detect EVs by FCM. Furthermore, we used an anti-apoB100/48 antibody to detect chylomicrons which have been reported to appear in blood ~4 h after food intake1622 (Fig. 2a, fluorescence-based gating strategy: Supplementary Fig. S2). Surprisingly, only a few events within the MV size range carried EV markers, and even this low percentage of events was reduced postprandially (Fig. 2b, **P < 0.01). In sharp contrast, most of the particles detected within the size range of MVs were recognized by an antibody against human apoB100/B48, even in fasting PFP samples. Importantly, the high number of apoB-positive events was significantly increased further in the postprandial state (Fig. 2b, ***P < 0.001). In order to identify EVs in our samples, we applied differential detergent lysis1923. Only EV marker positive events which were disrupted in the presence of 0.1% Tx-100 were considered EVs, and are presented here. Importantly, we found that some of the apoB-positive events were also sensitive to detergent lysis even in fasting conditions (Fig. 2c, ***P < 0.001). However, the remaining apoB carrying events still highly outnumbered the disappeared EV marker-bearing ones (up to 20×). To confirm that lipoproteins appearing postprandially were chylomicrons, we tested whether they float upon ultracentrifugation (UC) as described previously22. To this end we analyzed the top fractions of ultracentrifuged fasting and postprandial PFPs by transmission electron microscopy (TEM). As shown in Fig. 2d, floating, electron-dense lipoproteins appeared 4 h after food intake. To correlate our findings with routine laboratory measurements, we measured serum triglyceride, total cholesterol, LDL cholesterol, apoA1 and apoB100 content. As shown in Supplementary Table S1, the concentration of triglycerides increased significantly (P < 0.05) in postprandial state. This further confirmed that these floating particles were chylomicrons which are known to be enriched postprandially in triglycerides22.

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