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Extracellular vesicles of the blood-brain barrier.

András IE, Toborek M - Tissue Barriers (2015)

Bottom Line: While there are a lot of reports on ECV/exosomes derived from a variety of cell types, there is limited information on ECV/exosomes originated from brain microvascular endothelial cells forming the blood-brain barrier (BBB).In this review, we summarize the literature data on brain endothelial ECV/exosomes and present our own data on BBB-derived ECV and their possible involvement in the brain's Aβ pathology.We propose that ECV/exosome release from brain endothelial cells associated with Aβ affects different cells of the neurovascular unit and may be an important contributor to the Aβ deposition in the central nervous system.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology; University of Miami School of Medicine ; Miami, FL USA.

ABSTRACT
Extracellular vesicles (ECV), like exosomes, gained recently a lot of attention as potentially playing a significant role in neurodegenerative diseases, particularly in Aβ pathology. While there are a lot of reports on ECV/exosomes derived from a variety of cell types, there is limited information on ECV/exosomes originated from brain microvascular endothelial cells forming the blood-brain barrier (BBB). In this review, we summarize the literature data on brain endothelial ECV/exosomes and present our own data on BBB-derived ECV and their possible involvement in the brain's Aβ pathology. We propose that ECV/exosome release from brain endothelial cells associated with Aβ affects different cells of the neurovascular unit and may be an important contributor to the Aβ deposition in the central nervous system.

No MeSH data available.


Related in: MedlinePlus

Extracellular vesicles from human brain microvascular endothelial cells (HBMEC). (A) Live fluorescence imaging of HBMEC transiently transfected with pT-CYTO-RFP and pT-CD63-GFP to visualize the brain endothelial cell (red) secreting green fluorescent CD63 positive extracellular vesicles (ECV). Scale bar: 2 μm. (B) HBMEC were transiently transfected with pT-CD63 GFP or pT-CD9 RFP. ECV were isolated from the cell culture media. Fluorescence microscopy images of isolated fluorescent ECV (CD63 GFP-green, upper panel; CD9 RFP-red, lower panel). Scale bar: 20 μm. (C) Differential interference contrast (DIC) image of isolated ECV. Scale bar: 2 μm.
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f0001: Extracellular vesicles from human brain microvascular endothelial cells (HBMEC). (A) Live fluorescence imaging of HBMEC transiently transfected with pT-CYTO-RFP and pT-CD63-GFP to visualize the brain endothelial cell (red) secreting green fluorescent CD63 positive extracellular vesicles (ECV). Scale bar: 2 μm. (B) HBMEC were transiently transfected with pT-CD63 GFP or pT-CD9 RFP. ECV were isolated from the cell culture media. Fluorescence microscopy images of isolated fluorescent ECV (CD63 GFP-green, upper panel; CD9 RFP-red, lower panel). Scale bar: 20 μm. (C) Differential interference contrast (DIC) image of isolated ECV. Scale bar: 2 μm.

Mentions: Because ECV are heterogeneous, several methods were proposed for their isolation. The methods have not been standardized and controversy exists about which isolation approach is most suitable.41 Therefore, it is important to take into account the isolation method when interpreting the results of individual research reports. In our laboratory, we use ExoQuickTC to isolate ECV/exosomes from the culture media of human brain microvascular endothelial cells (HBMEC).37 Exosomes are traced with CD63-GFP and/or CD9-RFP fusion Cyto-Tracers from SBI Biosciences. The tetraspanin CD63 and CD9 proteins are common biomarkers for exosomes. Using the CD63 and CD9 Cyto-Tracer constructs, the transiently transfected HBMEC secrete green or red fluorescent vesicles. For better visualization of individual cells secreting glowing exosomes, a double transfection is used. By cotransfecting HBMEC with pT-CYTO RFP (which gives the cytoplasm red fluorescence) and pT CD63 GFP (exosome tracer resulting in CD63 specific green fluorescence) we can follow green fluorescent ECV/exosome secretion by live cell imaging. Using this procedure, green fluorescent CD63 positive vesicles with different sizes budding from the cells can be observed (Fig. 1A). Secreted CD63-positive (green) or CD9-positive (red) ECV/exosomes can also be observed in the cell culture media following isolation using ExoQuickTC. Isolated vesicles are fixed, mounted and imaged by fluorescence microscopy (Fig. 1B). Visualization of ECV by differential interference contrast (DIC) imaging is presented in Figure 1C.Figure 1.


Extracellular vesicles of the blood-brain barrier.

András IE, Toborek M - Tissue Barriers (2015)

Extracellular vesicles from human brain microvascular endothelial cells (HBMEC). (A) Live fluorescence imaging of HBMEC transiently transfected with pT-CYTO-RFP and pT-CD63-GFP to visualize the brain endothelial cell (red) secreting green fluorescent CD63 positive extracellular vesicles (ECV). Scale bar: 2 μm. (B) HBMEC were transiently transfected with pT-CD63 GFP or pT-CD9 RFP. ECV were isolated from the cell culture media. Fluorescence microscopy images of isolated fluorescent ECV (CD63 GFP-green, upper panel; CD9 RFP-red, lower panel). Scale bar: 20 μm. (C) Differential interference contrast (DIC) image of isolated ECV. Scale bar: 2 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4836554&req=5

f0001: Extracellular vesicles from human brain microvascular endothelial cells (HBMEC). (A) Live fluorescence imaging of HBMEC transiently transfected with pT-CYTO-RFP and pT-CD63-GFP to visualize the brain endothelial cell (red) secreting green fluorescent CD63 positive extracellular vesicles (ECV). Scale bar: 2 μm. (B) HBMEC were transiently transfected with pT-CD63 GFP or pT-CD9 RFP. ECV were isolated from the cell culture media. Fluorescence microscopy images of isolated fluorescent ECV (CD63 GFP-green, upper panel; CD9 RFP-red, lower panel). Scale bar: 20 μm. (C) Differential interference contrast (DIC) image of isolated ECV. Scale bar: 2 μm.
Mentions: Because ECV are heterogeneous, several methods were proposed for their isolation. The methods have not been standardized and controversy exists about which isolation approach is most suitable.41 Therefore, it is important to take into account the isolation method when interpreting the results of individual research reports. In our laboratory, we use ExoQuickTC to isolate ECV/exosomes from the culture media of human brain microvascular endothelial cells (HBMEC).37 Exosomes are traced with CD63-GFP and/or CD9-RFP fusion Cyto-Tracers from SBI Biosciences. The tetraspanin CD63 and CD9 proteins are common biomarkers for exosomes. Using the CD63 and CD9 Cyto-Tracer constructs, the transiently transfected HBMEC secrete green or red fluorescent vesicles. For better visualization of individual cells secreting glowing exosomes, a double transfection is used. By cotransfecting HBMEC with pT-CYTO RFP (which gives the cytoplasm red fluorescence) and pT CD63 GFP (exosome tracer resulting in CD63 specific green fluorescence) we can follow green fluorescent ECV/exosome secretion by live cell imaging. Using this procedure, green fluorescent CD63 positive vesicles with different sizes budding from the cells can be observed (Fig. 1A). Secreted CD63-positive (green) or CD9-positive (red) ECV/exosomes can also be observed in the cell culture media following isolation using ExoQuickTC. Isolated vesicles are fixed, mounted and imaged by fluorescence microscopy (Fig. 1B). Visualization of ECV by differential interference contrast (DIC) imaging is presented in Figure 1C.Figure 1.

Bottom Line: While there are a lot of reports on ECV/exosomes derived from a variety of cell types, there is limited information on ECV/exosomes originated from brain microvascular endothelial cells forming the blood-brain barrier (BBB).In this review, we summarize the literature data on brain endothelial ECV/exosomes and present our own data on BBB-derived ECV and their possible involvement in the brain's Aβ pathology.We propose that ECV/exosome release from brain endothelial cells associated with Aβ affects different cells of the neurovascular unit and may be an important contributor to the Aβ deposition in the central nervous system.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology; University of Miami School of Medicine ; Miami, FL USA.

ABSTRACT
Extracellular vesicles (ECV), like exosomes, gained recently a lot of attention as potentially playing a significant role in neurodegenerative diseases, particularly in Aβ pathology. While there are a lot of reports on ECV/exosomes derived from a variety of cell types, there is limited information on ECV/exosomes originated from brain microvascular endothelial cells forming the blood-brain barrier (BBB). In this review, we summarize the literature data on brain endothelial ECV/exosomes and present our own data on BBB-derived ECV and their possible involvement in the brain's Aβ pathology. We propose that ECV/exosome release from brain endothelial cells associated with Aβ affects different cells of the neurovascular unit and may be an important contributor to the Aβ deposition in the central nervous system.

No MeSH data available.


Related in: MedlinePlus