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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

LDL binds onto isolated MVs and EXOs in vitro.(A,B) TEM analysis of commercial LDL (A) and cell line-derived MVs (B) using the “osmification-on-grid” method. LDL particles appear as highly electron-dense, round structures and show aggregation (A) (scale bars: 500 nm). MVs isolated from the conditioned media of a cell line had vesicular morphology (B) (scale bars: 500 nm). (C) TEM images obtained after in vitro mixing of commercial LDL with cell-derived MVs for 1 h at room temperature (scale bar: 500 nm). Electron-dense LDL was observed to bind extensively onto MVs, to cover their surfaces, and to form aggregates in the size range of the vesicles. Note that almost all MVs were covered with LDL particles to various extents. (D) TEM picture of cell line-derived EXOs (scale bar: 500 nm). (E) Images of cell line-derived EXOs in vitro mixed with commercial LDL. Note that the surfaces of the EXOs are covered with LDL particles. The electron dense particles in the size range of EXOs represent LDL aggregates (scale bar: 500 nm).
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f7: LDL binds onto isolated MVs and EXOs in vitro.(A,B) TEM analysis of commercial LDL (A) and cell line-derived MVs (B) using the “osmification-on-grid” method. LDL particles appear as highly electron-dense, round structures and show aggregation (A) (scale bars: 500 nm). MVs isolated from the conditioned media of a cell line had vesicular morphology (B) (scale bars: 500 nm). (C) TEM images obtained after in vitro mixing of commercial LDL with cell-derived MVs for 1 h at room temperature (scale bar: 500 nm). Electron-dense LDL was observed to bind extensively onto MVs, to cover their surfaces, and to form aggregates in the size range of the vesicles. Note that almost all MVs were covered with LDL particles to various extents. (D) TEM picture of cell line-derived EXOs (scale bar: 500 nm). (E) Images of cell line-derived EXOs in vitro mixed with commercial LDL. Note that the surfaces of the EXOs are covered with LDL particles. The electron dense particles in the size range of EXOs represent LDL aggregates (scale bar: 500 nm).

Mentions: Next, we investigated further the capacity of LDL to associate with EVs. Using the “osmification-on-grid” approach, TEM revealed that commercial LDL was highly similar to the structures that we found in our PFPs previously (highly electron-dense round particles) (Fig. 7a). In contrast, MVs isolated from a cell line in serum free conditions, showed the typical vesicular morphology (Fig. 7b). Upon in vitro mixing LDL at a concentration close to physiological with the cell line-derived MVs, we found that LDL bound extensively onto MVs, covered their surfaces, and formed aggregates within the size range of the vesicles (Fig. 7c). Importantly almost all MVs were covered with lipoproteins to various extents. We also analyzed the LDL-association of cell-line derived EXOs. Upon in vitro mixing the EXOs with LDL, we detected the same phenomenon as with the MVs. The LDL particles associated with EXOs, and formed aggregates in the EXO size range as well (Fig. 7d,e). In these experiments we did not perform any additional washing step after mixing LDL with EVs (MVs or EXOs); therefore the association of LDL with EVs was not a result of co-pelleting.


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)

LDL binds onto isolated MVs and EXOs in vitro.(A,B) TEM analysis of commercial LDL (A) and cell line-derived MVs (B) using the “osmification-on-grid” method. LDL particles appear as highly electron-dense, round structures and show aggregation (A) (scale bars: 500 nm). MVs isolated from the conditioned media of a cell line had vesicular morphology (B) (scale bars: 500 nm). (C) TEM images obtained after in vitro mixing of commercial LDL with cell-derived MVs for 1 h at room temperature (scale bar: 500 nm). Electron-dense LDL was observed to bind extensively onto MVs, to cover their surfaces, and to form aggregates in the size range of the vesicles. Note that almost all MVs were covered with LDL particles to various extents. (D) TEM picture of cell line-derived EXOs (scale bar: 500 nm). (E) Images of cell line-derived EXOs in vitro mixed with commercial LDL. Note that the surfaces of the EXOs are covered with LDL particles. The electron dense particles in the size range of EXOs represent LDL aggregates (scale bar: 500 nm).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f7: LDL binds onto isolated MVs and EXOs in vitro.(A,B) TEM analysis of commercial LDL (A) and cell line-derived MVs (B) using the “osmification-on-grid” method. LDL particles appear as highly electron-dense, round structures and show aggregation (A) (scale bars: 500 nm). MVs isolated from the conditioned media of a cell line had vesicular morphology (B) (scale bars: 500 nm). (C) TEM images obtained after in vitro mixing of commercial LDL with cell-derived MVs for 1 h at room temperature (scale bar: 500 nm). Electron-dense LDL was observed to bind extensively onto MVs, to cover their surfaces, and to form aggregates in the size range of the vesicles. Note that almost all MVs were covered with LDL particles to various extents. (D) TEM picture of cell line-derived EXOs (scale bar: 500 nm). (E) Images of cell line-derived EXOs in vitro mixed with commercial LDL. Note that the surfaces of the EXOs are covered with LDL particles. The electron dense particles in the size range of EXOs represent LDL aggregates (scale bar: 500 nm).
Mentions: Next, we investigated further the capacity of LDL to associate with EVs. Using the “osmification-on-grid” approach, TEM revealed that commercial LDL was highly similar to the structures that we found in our PFPs previously (highly electron-dense round particles) (Fig. 7a). In contrast, MVs isolated from a cell line in serum free conditions, showed the typical vesicular morphology (Fig. 7b). Upon in vitro mixing LDL at a concentration close to physiological with the cell line-derived MVs, we found that LDL bound extensively onto MVs, covered their surfaces, and formed aggregates within the size range of the vesicles (Fig. 7c). Importantly almost all MVs were covered with lipoproteins to various extents. We also analyzed the LDL-association of cell-line derived EXOs. Upon in vitro mixing the EXOs with LDL, we detected the same phenomenon as with the MVs. The LDL particles associated with EXOs, and formed aggregates in the EXO size range as well (Fig. 7d,e). In these experiments we did not perform any additional washing step after mixing LDL with EVs (MVs or EXOs); therefore the association of LDL with EVs was not a result of co-pelleting.

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