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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

Detection of apoB-positive particles in isolated MV preparations.(A,B) MVs were isolated by differential centrifugation and gravity driven size filtration from 500 μL of fasting PFPs (black bars) and 4 h postprandial PFPs (gray bars) and analyzed by FCM. (A) The percentage of events detected within the MV gate increased significantly in the postprandial state (n = 9, mean + SEM, *P < 0.05, Wilcoxon matched-pairs signed rank test). (b) The isolated MVs were stained with anti-apoB and AX. For the AX labeling only 0.1% Tx-100 sensitive event s (which we considered EVs) were shown. The difference between the fasting and postprandial apoB-positive events was not significant, however, the number AX-positive events decreased significantly upon food intake (n = 9, mean + SEM, ****P < 0.0001, paired t-test). (C) Representative TRPS measurement of isolated fasting and postprandial MV preparations (continuous line: fasting MVs, dotted line: 4 h postprandial MVs). Note that the mean particle size was not affected. (D) TEM image of an ultrathin section prepared from a postprandial PFP-derived MV pellet (scale bar: 500 nm). Asterisk indicates MVs. (E) The same sample analyzed in suspension by an “osmification-on-grid” approach (scale bar: 500 nm). Note the highly electron-dense, round particles reminiscent to the TEM morphology of lipoproteins upon osmification (arrowheads). Membrane enclosed MVs (asterisk) showed association with these electron-dense particles.
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f3: Detection of apoB-positive particles in isolated MV preparations.(A,B) MVs were isolated by differential centrifugation and gravity driven size filtration from 500 μL of fasting PFPs (black bars) and 4 h postprandial PFPs (gray bars) and analyzed by FCM. (A) The percentage of events detected within the MV gate increased significantly in the postprandial state (n = 9, mean + SEM, *P < 0.05, Wilcoxon matched-pairs signed rank test). (b) The isolated MVs were stained with anti-apoB and AX. For the AX labeling only 0.1% Tx-100 sensitive event s (which we considered EVs) were shown. The difference between the fasting and postprandial apoB-positive events was not significant, however, the number AX-positive events decreased significantly upon food intake (n = 9, mean + SEM, ****P < 0.0001, paired t-test). (C) Representative TRPS measurement of isolated fasting and postprandial MV preparations (continuous line: fasting MVs, dotted line: 4 h postprandial MVs). Note that the mean particle size was not affected. (D) TEM image of an ultrathin section prepared from a postprandial PFP-derived MV pellet (scale bar: 500 nm). Asterisk indicates MVs. (E) The same sample analyzed in suspension by an “osmification-on-grid” approach (scale bar: 500 nm). Note the highly electron-dense, round particles reminiscent to the TEM morphology of lipoproteins upon osmification (arrowheads). Membrane enclosed MVs (asterisk) showed association with these electron-dense particles.

Mentions: Next we addressed the question whether lipoproteins carrying apoB were also present in isolated MV preparations. To test this, we isolated MVs from 500 μL PFP with differential centrifugation and gravity-driven size-filtration111924. As shown in Fig. 3a, the percentage of events within the SSC-FSC based MV gate increased significantly in the postprandial state (P: 0.0195). However, there was no significant difference in the anti-apoB-staining of fasting and postprandial isolated MVs (Fig. 3b). This was most likely due to the fact that the already high amount of antibody (optimized for PFP staining) was still not sufficient to label all of the postprandially increased number of particles in MV preparations (Supplementary Fig S3). The AX-positive events decreased significantly upon food intake (P < 0.0001).


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)

Detection of apoB-positive particles in isolated MV preparations.(A,B) MVs were isolated by differential centrifugation and gravity driven size filtration from 500 μL of fasting PFPs (black bars) and 4 h postprandial PFPs (gray bars) and analyzed by FCM. (A) The percentage of events detected within the MV gate increased significantly in the postprandial state (n = 9, mean + SEM, *P < 0.05, Wilcoxon matched-pairs signed rank test). (b) The isolated MVs were stained with anti-apoB and AX. For the AX labeling only 0.1% Tx-100 sensitive event s (which we considered EVs) were shown. The difference between the fasting and postprandial apoB-positive events was not significant, however, the number AX-positive events decreased significantly upon food intake (n = 9, mean + SEM, ****P < 0.0001, paired t-test). (C) Representative TRPS measurement of isolated fasting and postprandial MV preparations (continuous line: fasting MVs, dotted line: 4 h postprandial MVs). Note that the mean particle size was not affected. (D) TEM image of an ultrathin section prepared from a postprandial PFP-derived MV pellet (scale bar: 500 nm). Asterisk indicates MVs. (E) The same sample analyzed in suspension by an “osmification-on-grid” approach (scale bar: 500 nm). Note the highly electron-dense, round particles reminiscent to the TEM morphology of lipoproteins upon osmification (arrowheads). Membrane enclosed MVs (asterisk) showed association with these electron-dense particles.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Detection of apoB-positive particles in isolated MV preparations.(A,B) MVs were isolated by differential centrifugation and gravity driven size filtration from 500 μL of fasting PFPs (black bars) and 4 h postprandial PFPs (gray bars) and analyzed by FCM. (A) The percentage of events detected within the MV gate increased significantly in the postprandial state (n = 9, mean + SEM, *P < 0.05, Wilcoxon matched-pairs signed rank test). (b) The isolated MVs were stained with anti-apoB and AX. For the AX labeling only 0.1% Tx-100 sensitive event s (which we considered EVs) were shown. The difference between the fasting and postprandial apoB-positive events was not significant, however, the number AX-positive events decreased significantly upon food intake (n = 9, mean + SEM, ****P < 0.0001, paired t-test). (C) Representative TRPS measurement of isolated fasting and postprandial MV preparations (continuous line: fasting MVs, dotted line: 4 h postprandial MVs). Note that the mean particle size was not affected. (D) TEM image of an ultrathin section prepared from a postprandial PFP-derived MV pellet (scale bar: 500 nm). Asterisk indicates MVs. (E) The same sample analyzed in suspension by an “osmification-on-grid” approach (scale bar: 500 nm). Note the highly electron-dense, round particles reminiscent to the TEM morphology of lipoproteins upon osmification (arrowheads). Membrane enclosed MVs (asterisk) showed association with these electron-dense particles.
Mentions: Next we addressed the question whether lipoproteins carrying apoB were also present in isolated MV preparations. To test this, we isolated MVs from 500 μL PFP with differential centrifugation and gravity-driven size-filtration111924. As shown in Fig. 3a, the percentage of events within the SSC-FSC based MV gate increased significantly in the postprandial state (P: 0.0195). However, there was no significant difference in the anti-apoB-staining of fasting and postprandial isolated MVs (Fig. 3b). This was most likely due to the fact that the already high amount of antibody (optimized for PFP staining) was still not sufficient to label all of the postprandially increased number of particles in MV preparations (Supplementary Fig S3). The AX-positive events decreased significantly upon food intake (P < 0.0001).

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