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Endocytosis and intracellular processing of platelet microparticles by brain endothelial cells.

Faille D, El-Assaad F, Mitchell AJ, Alessi MC, Chimini G, Fusai T, Grau GE, Combes V - J. Cell. Mol. Med. (2012)

Bottom Line: Platelet-derived microparticle uptake was greatly diminished by treatment of HBEC with cytochalasin D, an inhibitor of microfilament formation required for both phagocytosis and macropinocytosis, with methyl-β-cyclodextrin that depletes membrane cholesterol needed for macropinocytosis and with amiloride that inhibits the Na(+)/H(+) exchanger involved in macropinocytosis.In conclusion, PMP are taken up by active endocytosis in HBEC, involving mechanisms consistent with both phagocytosis and macropinocytosis.These findings identify new processes by which PMP could modify endothelial cell phenotype and functions.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Sydney, Camperdown, Australia. dorothee.faille@libertysurf.fr

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Related in: MedlinePlus

Co-localization of PMP cytoplasmic content and PMP-derived membranes inside the target cell. PMP were double-labelled with membrane dye PKH26 (red) and calcein AM that only becomes green fluorescent once hydrolysed by cytoplasmic esterases. (A) Analysis of PMP double-labelling by flow cytometry. Number in the upper right quadrant represents the percentage of double-labelled PMP. (B) PMP were then incubated with HBEC for 90 min. at 37°C. PMP supernatant was used as a negative control. Cells were washed, fixed with PFA 2% (v/v) and analysed by confocal microscopy. Merged images showing transmitted light and green and red channels are represented. Insert of the merged image is represented in the right column. Bar: 10 μm.
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fig02: Co-localization of PMP cytoplasmic content and PMP-derived membranes inside the target cell. PMP were double-labelled with membrane dye PKH26 (red) and calcein AM that only becomes green fluorescent once hydrolysed by cytoplasmic esterases. (A) Analysis of PMP double-labelling by flow cytometry. Number in the upper right quadrant represents the percentage of double-labelled PMP. (B) PMP were then incubated with HBEC for 90 min. at 37°C. PMP supernatant was used as a negative control. Cells were washed, fixed with PFA 2% (v/v) and analysed by confocal microscopy. Merged images showing transmitted light and green and red channels are represented. Insert of the merged image is represented in the right column. Bar: 10 μm.

Mentions: To search for PMP fusion with the endothelial membrane and to detect a possible PMP fragmentation once internalized, PMP content was stained with soluble calcein AM (green) in addition to the membranous PKH26-labelling (red). After staining, 48% of PMP were double labelled for both calcein and PKH26 (Fig. 2A). Figure 2B shows that, after PMP internalization by HBEC, no PKH26 was transferred from PMP to endothelial membrane and no entrapped calcein was released within the cytosol, suggesting that PMP content did not diffuse inside the target cell but remained co-localized with PMP membranes. This observation clearly indicates that PMP internalization by HBEC does not involve the fusion of PMP lipids with the endothelial plasma membrane but, rather, an uptake of PMP material with subsequent localization in intracellular vesicles.


Endocytosis and intracellular processing of platelet microparticles by brain endothelial cells.

Faille D, El-Assaad F, Mitchell AJ, Alessi MC, Chimini G, Fusai T, Grau GE, Combes V - J. Cell. Mol. Med. (2012)

Co-localization of PMP cytoplasmic content and PMP-derived membranes inside the target cell. PMP were double-labelled with membrane dye PKH26 (red) and calcein AM that only becomes green fluorescent once hydrolysed by cytoplasmic esterases. (A) Analysis of PMP double-labelling by flow cytometry. Number in the upper right quadrant represents the percentage of double-labelled PMP. (B) PMP were then incubated with HBEC for 90 min. at 37°C. PMP supernatant was used as a negative control. Cells were washed, fixed with PFA 2% (v/v) and analysed by confocal microscopy. Merged images showing transmitted light and green and red channels are represented. Insert of the merged image is represented in the right column. Bar: 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3822686&req=5

fig02: Co-localization of PMP cytoplasmic content and PMP-derived membranes inside the target cell. PMP were double-labelled with membrane dye PKH26 (red) and calcein AM that only becomes green fluorescent once hydrolysed by cytoplasmic esterases. (A) Analysis of PMP double-labelling by flow cytometry. Number in the upper right quadrant represents the percentage of double-labelled PMP. (B) PMP were then incubated with HBEC for 90 min. at 37°C. PMP supernatant was used as a negative control. Cells were washed, fixed with PFA 2% (v/v) and analysed by confocal microscopy. Merged images showing transmitted light and green and red channels are represented. Insert of the merged image is represented in the right column. Bar: 10 μm.
Mentions: To search for PMP fusion with the endothelial membrane and to detect a possible PMP fragmentation once internalized, PMP content was stained with soluble calcein AM (green) in addition to the membranous PKH26-labelling (red). After staining, 48% of PMP were double labelled for both calcein and PKH26 (Fig. 2A). Figure 2B shows that, after PMP internalization by HBEC, no PKH26 was transferred from PMP to endothelial membrane and no entrapped calcein was released within the cytosol, suggesting that PMP content did not diffuse inside the target cell but remained co-localized with PMP membranes. This observation clearly indicates that PMP internalization by HBEC does not involve the fusion of PMP lipids with the endothelial plasma membrane but, rather, an uptake of PMP material with subsequent localization in intracellular vesicles.

Bottom Line: Platelet-derived microparticle uptake was greatly diminished by treatment of HBEC with cytochalasin D, an inhibitor of microfilament formation required for both phagocytosis and macropinocytosis, with methyl-β-cyclodextrin that depletes membrane cholesterol needed for macropinocytosis and with amiloride that inhibits the Na(+)/H(+) exchanger involved in macropinocytosis.In conclusion, PMP are taken up by active endocytosis in HBEC, involving mechanisms consistent with both phagocytosis and macropinocytosis.These findings identify new processes by which PMP could modify endothelial cell phenotype and functions.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Sydney, Camperdown, Australia. dorothee.faille@libertysurf.fr

Show MeSH
Related in: MedlinePlus