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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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Perivascular glioma cells can displace astrocytic endfeet along the vasculatureImmunofluorescence of CD31 (PECAM), aquaporin-4 (AQ4) and eGFP-expressing human glioma cells (D54) implanted in the cerebrum of immunodeficient mice show displacement of the astrocyte endfeet by tumor cells from the vessels in general (a) and from arterioles/arteries labeled with either alpha smooth muscle action (αSMA) (b) or Alexa Fluor 633 hydrazide (633 hyd) (c). This could be confirmed by implanting patient-derived xenograft tumors labeled with Human Nuclei (HuN) (d) or TdTomato-expressing human glioma cells (D54) (e) into the cerebrum of Aldh1l1-eGFP immunodeficient mice allowing visualization astrocyte endfeet independent of AQ4 expression. Perivascular glioma cells can intercalate between endothelial cells and astrocytic endfeet (a,c) or completely displace the astrocytic endfoot from the vascular surface (b). Electron microscopy (g-i) shows that perivascular astrocytes (brown) are less electron-dense than perivascular human glioma cells (green), which can displace astrocytic endfeet, allowing for physical contact with the endothelial cell (g-i) or can sometimes sit on top of astrocyte endfeet (i). Black font was used to describe white labels in confocal images. See panel f for quantification (n=34 vessels for D54; n=44 for GBM22; n=38 for GBM14). Scale, 20 μm (a-e); 2 μm (g), 4 μm (h,i). Statistical data: a-cn=18 animals; dn=12; en=5 animals; gn=3 animals, hn=2 animals, in=2 animals.
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Figure 2: Perivascular glioma cells can displace astrocytic endfeet along the vasculatureImmunofluorescence of CD31 (PECAM), aquaporin-4 (AQ4) and eGFP-expressing human glioma cells (D54) implanted in the cerebrum of immunodeficient mice show displacement of the astrocyte endfeet by tumor cells from the vessels in general (a) and from arterioles/arteries labeled with either alpha smooth muscle action (αSMA) (b) or Alexa Fluor 633 hydrazide (633 hyd) (c). This could be confirmed by implanting patient-derived xenograft tumors labeled with Human Nuclei (HuN) (d) or TdTomato-expressing human glioma cells (D54) (e) into the cerebrum of Aldh1l1-eGFP immunodeficient mice allowing visualization astrocyte endfeet independent of AQ4 expression. Perivascular glioma cells can intercalate between endothelial cells and astrocytic endfeet (a,c) or completely displace the astrocytic endfoot from the vascular surface (b). Electron microscopy (g-i) shows that perivascular astrocytes (brown) are less electron-dense than perivascular human glioma cells (green), which can displace astrocytic endfeet, allowing for physical contact with the endothelial cell (g-i) or can sometimes sit on top of astrocyte endfeet (i). Black font was used to describe white labels in confocal images. See panel f for quantification (n=34 vessels for D54; n=44 for GBM22; n=38 for GBM14). Scale, 20 μm (a-e); 2 μm (g), 4 μm (h,i). Statistical data: a-cn=18 animals; dn=12; en=5 animals; gn=3 animals, hn=2 animals, in=2 animals.

Mentions: Next, we asked whether glioma cells preferentially associate with vessels carrying arterial as opposed to venous blood. Arteries (35-100 μm) and arterioles (7-35 μm) contain contractile vascular smooth muscle cells (VSMCs) that regulate vascular tone, and larger arteries have an internal elastic lamina23, 24. Cerebral venules and large veins lack VSMCs and the ability to actively constrict and dilate. All vessels are non-selectively labeled with the endothelial marker CD31 (Fig. 1a-i). To identify arterioles/arteries, we used either alpha-smooth muscle actin (αSMA), present in the VSMCs, or Alexa Fluor 633 hydrazide dye, which binds to the elastin found only in arteries/arterioles equal or greater than 10 μm in diameter25. Representative examples of each vessel type associated with glioma cells are depicted in Fig. 1g-I, k-m and Fig. 2a-c. Quantitative analysis showed 54.46% covering venules/veins over 10 μm and 36.28% associating with arterioles/arteries over 10 μm (Fig. 1j). For the 9.25% of vessels between 7 and 10 μm, we were unable to differentiate between arterioles and venules since they lack elastin that is bound by the hydrazide dye (Fig. 1j).


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)

Perivascular glioma cells can displace astrocytic endfeet along the vasculatureImmunofluorescence of CD31 (PECAM), aquaporin-4 (AQ4) and eGFP-expressing human glioma cells (D54) implanted in the cerebrum of immunodeficient mice show displacement of the astrocyte endfeet by tumor cells from the vessels in general (a) and from arterioles/arteries labeled with either alpha smooth muscle action (αSMA) (b) or Alexa Fluor 633 hydrazide (633 hyd) (c). This could be confirmed by implanting patient-derived xenograft tumors labeled with Human Nuclei (HuN) (d) or TdTomato-expressing human glioma cells (D54) (e) into the cerebrum of Aldh1l1-eGFP immunodeficient mice allowing visualization astrocyte endfeet independent of AQ4 expression. Perivascular glioma cells can intercalate between endothelial cells and astrocytic endfeet (a,c) or completely displace the astrocytic endfoot from the vascular surface (b). Electron microscopy (g-i) shows that perivascular astrocytes (brown) are less electron-dense than perivascular human glioma cells (green), which can displace astrocytic endfeet, allowing for physical contact with the endothelial cell (g-i) or can sometimes sit on top of astrocyte endfeet (i). Black font was used to describe white labels in confocal images. See panel f for quantification (n=34 vessels for D54; n=44 for GBM22; n=38 for GBM14). Scale, 20 μm (a-e); 2 μm (g), 4 μm (h,i). Statistical data: a-cn=18 animals; dn=12; en=5 animals; gn=3 animals, hn=2 animals, in=2 animals.
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Related In: Results  -  Collection

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Figure 2: Perivascular glioma cells can displace astrocytic endfeet along the vasculatureImmunofluorescence of CD31 (PECAM), aquaporin-4 (AQ4) and eGFP-expressing human glioma cells (D54) implanted in the cerebrum of immunodeficient mice show displacement of the astrocyte endfeet by tumor cells from the vessels in general (a) and from arterioles/arteries labeled with either alpha smooth muscle action (αSMA) (b) or Alexa Fluor 633 hydrazide (633 hyd) (c). This could be confirmed by implanting patient-derived xenograft tumors labeled with Human Nuclei (HuN) (d) or TdTomato-expressing human glioma cells (D54) (e) into the cerebrum of Aldh1l1-eGFP immunodeficient mice allowing visualization astrocyte endfeet independent of AQ4 expression. Perivascular glioma cells can intercalate between endothelial cells and astrocytic endfeet (a,c) or completely displace the astrocytic endfoot from the vascular surface (b). Electron microscopy (g-i) shows that perivascular astrocytes (brown) are less electron-dense than perivascular human glioma cells (green), which can displace astrocytic endfeet, allowing for physical contact with the endothelial cell (g-i) or can sometimes sit on top of astrocyte endfeet (i). Black font was used to describe white labels in confocal images. See panel f for quantification (n=34 vessels for D54; n=44 for GBM22; n=38 for GBM14). Scale, 20 μm (a-e); 2 μm (g), 4 μm (h,i). Statistical data: a-cn=18 animals; dn=12; en=5 animals; gn=3 animals, hn=2 animals, in=2 animals.
Mentions: Next, we asked whether glioma cells preferentially associate with vessels carrying arterial as opposed to venous blood. Arteries (35-100 μm) and arterioles (7-35 μm) contain contractile vascular smooth muscle cells (VSMCs) that regulate vascular tone, and larger arteries have an internal elastic lamina23, 24. Cerebral venules and large veins lack VSMCs and the ability to actively constrict and dilate. All vessels are non-selectively labeled with the endothelial marker CD31 (Fig. 1a-i). To identify arterioles/arteries, we used either alpha-smooth muscle actin (αSMA), present in the VSMCs, or Alexa Fluor 633 hydrazide dye, which binds to the elastin found only in arteries/arterioles equal or greater than 10 μm in diameter25. Representative examples of each vessel type associated with glioma cells are depicted in Fig. 1g-I, k-m and Fig. 2a-c. Quantitative analysis showed 54.46% covering venules/veins over 10 μm and 36.28% associating with arterioles/arteries over 10 μm (Fig. 1j). For the 9.25% of vessels between 7 and 10 μm, we were unable to differentiate between arterioles and venules since they lack elastin that is bound by the hydrazide dye (Fig. 1j).

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