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Disruption of astrocyte-vascular coupling and the blood-brain barrier by invading glioma cells.

Watkins S, Robel S, Kimbrough IF, Robert SM, Ellis-Davies G, Sontheimer H - Nat Commun (2014)

Bottom Line: Malignant gliomas are highly invasive tumours that use the perivascular space for invasion and co-opt existing vessels as satellite tumour form.This causes a focal breach in the BBB.These findings have important clinical implications regarding blood flow in the tumour-associated brain and the ability to locally deliver chemotherapeutic drugs in disease.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Neurobiology, Center for Glial Biology in Medicine, University of Alabama at Birmingham, 1719 6th Avenue South, CIRC 425, Birmingham, Alabama 35294, USA [2].

ABSTRACT
Astrocytic endfeet cover the entire cerebral vasculature and serve as exchange sites for ions, metabolites and energy substrates from the blood to the brain. They maintain endothelial tight junctions that form the blood-brain barrier (BBB) and release vasoactive molecules that regulate vascular tone. Malignant gliomas are highly invasive tumours that use the perivascular space for invasion and co-opt existing vessels as satellite tumour form. Here we use a clinically relevant mouse model of glioma and find that glioma cells, as they populate the perivascular space of preexisting vessels, displace astrocytic endfeet from endothelial or vascular smooth muscle cells. This causes a focal breach in the BBB. Furthermore, astrocyte-mediated gliovascular coupling is lost, and glioma cells seize control over the regulation of vascular tone through Ca(2+)-dependent release of K(+). These findings have important clinical implications regarding blood flow in the tumour-associated brain and the ability to locally deliver chemotherapeutic drugs in disease.

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Loss of astrocyte-vascular coupling following vessel co-option by gliomas(a) DIC images of vessels without (top) or with (bottom) perivascular glioma cells before and after application of 100 μM trans-ACPD (t-ACPD). Perivascular glioma cell presence was verified by eGFP-fluorescence. (b) Changes in vessel diameter for an arteriole associated (grey) and not associated (black) with perivascular glioma cells over the course of one experiment when exposed to 100 μM trans-ACPD (arrowhead). (c) Average change in vessel diameters observed at high (h) (95%) and low (l) (20%) oxygen for vessels associated (grey) and not associated (black) with glioma cells when exposed to 100 μM trans-ACPD. (d) DIC images of vessels without (top) or with (bottom) perivascular glioma cells before and after application of 10 μM norepinephrine (NE). Perivascular glioma cell presence was verified by eGFP-fluorescence. (e) Changes in vessel diameter for an arteriole associated (grey) and not associated (black) with perivascular glioma cells over the course of one experiment when exposed to 10 μM NE (arrowhead). (f) Average change in vessel diameters observed at high (h) (95%) and low (l) (20%) oxygen for vessels associated (grey) and not associated (black) with glioma cells when exposed to 10 μM NE. For experiments performed at low oxygen concentrations, arterioles were preconstricted with 125 nM U46619 for 20 min. Statistical data provided in results section, error bars refer to SEM. Scale, 20 μm.
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Figure 4: Loss of astrocyte-vascular coupling following vessel co-option by gliomas(a) DIC images of vessels without (top) or with (bottom) perivascular glioma cells before and after application of 100 μM trans-ACPD (t-ACPD). Perivascular glioma cell presence was verified by eGFP-fluorescence. (b) Changes in vessel diameter for an arteriole associated (grey) and not associated (black) with perivascular glioma cells over the course of one experiment when exposed to 100 μM trans-ACPD (arrowhead). (c) Average change in vessel diameters observed at high (h) (95%) and low (l) (20%) oxygen for vessels associated (grey) and not associated (black) with glioma cells when exposed to 100 μM trans-ACPD. (d) DIC images of vessels without (top) or with (bottom) perivascular glioma cells before and after application of 10 μM norepinephrine (NE). Perivascular glioma cell presence was verified by eGFP-fluorescence. (e) Changes in vessel diameter for an arteriole associated (grey) and not associated (black) with perivascular glioma cells over the course of one experiment when exposed to 10 μM NE (arrowhead). (f) Average change in vessel diameters observed at high (h) (95%) and low (l) (20%) oxygen for vessels associated (grey) and not associated (black) with glioma cells when exposed to 10 μM NE. For experiments performed at low oxygen concentrations, arterioles were preconstricted with 125 nM U46619 for 20 min. Statistical data provided in results section, error bars refer to SEM. Scale, 20 μm.

Mentions: Central to astrocyte-mediated control of blood flow is the presence of endfeet on the vasculature. Since we found astrocytic endfeet along arterioles frequently displaced from the VSMCs by perivascular glioma cells (Fig. 2b,c; Fig. 3g, Supplementary Fig. 2-5), we questioned whether vascular coupling was altered. To answer this, we first used a pharmacological approach to selectively stimulate astrocytes and assessed responses of arterioles. We bath applied two drugs known to selectively increase [Ca2+]i in astrocytes, namely trans-ACPD and norepinephrine (NE)11, 34, 35 to acute brain slices and measured the vessel diameter change by video-microscopy for arterioles identified by a thickened vessel wall indicating the presence of a VSMC layer with D54 ((+)Tumor cell(s)) or without perivascular glioma cells ((-)Tumor cell(s)). For all experiments, diameter changes in response to trans-ACPD and NE were assessed at high (95%) and low (20%) oxygen concentrations as these oxygen concentrations have been shown to cause opposite vascular responses due to the limitation of 20-HETE and nitric oxide production by limited oxygen amounts7, 34. In agreement with previous studies11, 34, application of trans-ACPD caused vasodilatation (O2low, n=12) or vasoconstriction (O2high, n=20) in tumor-free vessels (Fig. 4a-c). Similarly, NE application induced constriction (O2high, n=18) or dilation (O2low, n=22) in glioma-free arterioles (Fig. 4d-f). In contrast, arterioles encased by glioma cells showed a significant decrease in vessel response after trans-ACPD (O2low: n=9, two-tailed unpaired t-test, p=0.012; O2high: n=16, two-tailed Mann-Whitney test, p=0.0032) (Fig. 4a-c) or NE application (O2high: n=18, two-tailed unpaired t-test, p=0.0049; O2low: n=21, two-tailed unpaired t-test, p=0.01) (Fig. 4d-f).


Disruption of astrocyte-vascular coupling and the blood-brain barrier by invading glioma cells.

Watkins S, Robel S, Kimbrough IF, Robert SM, Ellis-Davies G, Sontheimer H - Nat Commun (2014)

Loss of astrocyte-vascular coupling following vessel co-option by gliomas(a) DIC images of vessels without (top) or with (bottom) perivascular glioma cells before and after application of 100 μM trans-ACPD (t-ACPD). Perivascular glioma cell presence was verified by eGFP-fluorescence. (b) Changes in vessel diameter for an arteriole associated (grey) and not associated (black) with perivascular glioma cells over the course of one experiment when exposed to 100 μM trans-ACPD (arrowhead). (c) Average change in vessel diameters observed at high (h) (95%) and low (l) (20%) oxygen for vessels associated (grey) and not associated (black) with glioma cells when exposed to 100 μM trans-ACPD. (d) DIC images of vessels without (top) or with (bottom) perivascular glioma cells before and after application of 10 μM norepinephrine (NE). Perivascular glioma cell presence was verified by eGFP-fluorescence. (e) Changes in vessel diameter for an arteriole associated (grey) and not associated (black) with perivascular glioma cells over the course of one experiment when exposed to 10 μM NE (arrowhead). (f) Average change in vessel diameters observed at high (h) (95%) and low (l) (20%) oxygen for vessels associated (grey) and not associated (black) with glioma cells when exposed to 10 μM NE. For experiments performed at low oxygen concentrations, arterioles were preconstricted with 125 nM U46619 for 20 min. Statistical data provided in results section, error bars refer to SEM. Scale, 20 μm.
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Related In: Results  -  Collection

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Figure 4: Loss of astrocyte-vascular coupling following vessel co-option by gliomas(a) DIC images of vessels without (top) or with (bottom) perivascular glioma cells before and after application of 100 μM trans-ACPD (t-ACPD). Perivascular glioma cell presence was verified by eGFP-fluorescence. (b) Changes in vessel diameter for an arteriole associated (grey) and not associated (black) with perivascular glioma cells over the course of one experiment when exposed to 100 μM trans-ACPD (arrowhead). (c) Average change in vessel diameters observed at high (h) (95%) and low (l) (20%) oxygen for vessels associated (grey) and not associated (black) with glioma cells when exposed to 100 μM trans-ACPD. (d) DIC images of vessels without (top) or with (bottom) perivascular glioma cells before and after application of 10 μM norepinephrine (NE). Perivascular glioma cell presence was verified by eGFP-fluorescence. (e) Changes in vessel diameter for an arteriole associated (grey) and not associated (black) with perivascular glioma cells over the course of one experiment when exposed to 10 μM NE (arrowhead). (f) Average change in vessel diameters observed at high (h) (95%) and low (l) (20%) oxygen for vessels associated (grey) and not associated (black) with glioma cells when exposed to 10 μM NE. For experiments performed at low oxygen concentrations, arterioles were preconstricted with 125 nM U46619 for 20 min. Statistical data provided in results section, error bars refer to SEM. Scale, 20 μm.
Mentions: Central to astrocyte-mediated control of blood flow is the presence of endfeet on the vasculature. Since we found astrocytic endfeet along arterioles frequently displaced from the VSMCs by perivascular glioma cells (Fig. 2b,c; Fig. 3g, Supplementary Fig. 2-5), we questioned whether vascular coupling was altered. To answer this, we first used a pharmacological approach to selectively stimulate astrocytes and assessed responses of arterioles. We bath applied two drugs known to selectively increase [Ca2+]i in astrocytes, namely trans-ACPD and norepinephrine (NE)11, 34, 35 to acute brain slices and measured the vessel diameter change by video-microscopy for arterioles identified by a thickened vessel wall indicating the presence of a VSMC layer with D54 ((+)Tumor cell(s)) or without perivascular glioma cells ((-)Tumor cell(s)). For all experiments, diameter changes in response to trans-ACPD and NE were assessed at high (95%) and low (20%) oxygen concentrations as these oxygen concentrations have been shown to cause opposite vascular responses due to the limitation of 20-HETE and nitric oxide production by limited oxygen amounts7, 34. In agreement with previous studies11, 34, application of trans-ACPD caused vasodilatation (O2low, n=12) or vasoconstriction (O2high, n=20) in tumor-free vessels (Fig. 4a-c). Similarly, NE application induced constriction (O2high, n=18) or dilation (O2low, n=22) in glioma-free arterioles (Fig. 4d-f). In contrast, arterioles encased by glioma cells showed a significant decrease in vessel response after trans-ACPD (O2low: n=9, two-tailed unpaired t-test, p=0.012; O2high: n=16, two-tailed Mann-Whitney test, p=0.0032) (Fig. 4a-c) or NE application (O2high: n=18, two-tailed unpaired t-test, p=0.0049; O2low: n=21, two-tailed unpaired t-test, p=0.01) (Fig. 4d-f).

Bottom Line: Malignant gliomas are highly invasive tumours that use the perivascular space for invasion and co-opt existing vessels as satellite tumour form.This causes a focal breach in the BBB.These findings have important clinical implications regarding blood flow in the tumour-associated brain and the ability to locally deliver chemotherapeutic drugs in disease.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Neurobiology, Center for Glial Biology in Medicine, University of Alabama at Birmingham, 1719 6th Avenue South, CIRC 425, Birmingham, Alabama 35294, USA [2].

ABSTRACT
Astrocytic endfeet cover the entire cerebral vasculature and serve as exchange sites for ions, metabolites and energy substrates from the blood to the brain. They maintain endothelial tight junctions that form the blood-brain barrier (BBB) and release vasoactive molecules that regulate vascular tone. Malignant gliomas are highly invasive tumours that use the perivascular space for invasion and co-opt existing vessels as satellite tumour form. Here we use a clinically relevant mouse model of glioma and find that glioma cells, as they populate the perivascular space of preexisting vessels, displace astrocytic endfeet from endothelial or vascular smooth muscle cells. This causes a focal breach in the BBB. Furthermore, astrocyte-mediated gliovascular coupling is lost, and glioma cells seize control over the regulation of vascular tone through Ca(2+)-dependent release of K(+). These findings have important clinical implications regarding blood flow in the tumour-associated brain and the ability to locally deliver chemotherapeutic drugs in disease.

Show MeSH
Related in: MedlinePlus