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ADAM12 and ADAM17 are essential molecules for hypoxia-induced impairment of neural vascular barrier function.

Cui D, Arima M, Takubo K, Kimura T, Horiuchi K, Minagawa T, Matsuda S, Ikeda E - Sci Rep (2015)

Bottom Line: Therefore, the molecules involved in hypoxia-induced impairment of vascular barrier can be the targets to establish new therapies for intractable diseases.Hypoxic disappearance of claudin-5 from cell membranes and the consequent loss of barrier properties were completely suppressed by inhibition of the metalloproteinase activity which was found to be attributed to ADAM12 and ADAM17.This is the first report to specify the molecules which are responsible for hypoxia-induced impairment of neural vascular barrier and furthermore can be the targets of new therapeutic strategies for intractable neural diseases.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Yamaguchi University Graduate School of Medicine, 1-1-1 Minami-Kogushi, Ube, Yamaguchi 755-8505, Japan.

ABSTRACT
Neural vascular barrier is essential for the life of multicellular organisms, and its impairment by tissue hypoxia is known to be a central of pathophysiology accelerating the progression of various intractable neural diseases. Therefore, the molecules involved in hypoxia-induced impairment of vascular barrier can be the targets to establish new therapies for intractable diseases. Here, we demonstrate that a disintegrin and metalloproteinases (ADAMs) 12 and 17 expressed in endothelial cells are the molecules responsible for the impairment of neural vascular barrier by hypoxia. Brain microvascular endothelial cells in vitro lost their barrier properties immediately after hypoxic stimulation through diminished localization of claudin-5, a tight junction molecule, on cell membranes. Hypoxic disappearance of claudin-5 from cell membranes and the consequent loss of barrier properties were completely suppressed by inhibition of the metalloproteinase activity which was found to be attributed to ADAM12 and ADAM17. Inhibition of either ADAM12 or ADAM17 was sufficient to rescue the in vivo neural vasculature under hypoxia from the loss of barrier function. This is the first report to specify the molecules which are responsible for hypoxia-induced impairment of neural vascular barrier and furthermore can be the targets of new therapeutic strategies for intractable neural diseases.

No MeSH data available.


Related in: MedlinePlus

Hypoxia accelerates the disappearance of claudin-5 from cell membranes, and consequently impairs the barrier property of endothelial cells.(a) Immunofluorescence images for claudin-5 expression in bEnd.3 monolayers under normoxia or hypoxia for 30 minutes. (b) Quantitative analysis of claudin-5 levels on cell membranes corresponding to the images in a. (c) TEERs of bEnd.3 monolayers under normoxia or hypoxia. Levels of claudin-5 protein on cell membranes as well as the TEERs of bEnd.3 monolayers are decreased in response to hypoxia. (d and e) Confocal scanning microscopic images (d) and their corresponding quantitative analysis of claudin-5 levels on cell membranes of bEnd.3 cells under normoxia or hypoxia for 30, 50, 70 and 90 minutes after pretreatment with or without CHX. Although claudin-5 disappeared from cell membranes quickly even under normoxia, the disappearance of claudin-5 is shown to be accelerated under hypoxia. Data are presented as mean ± SD from 3 independent cultures. *P < 0.05.
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f1: Hypoxia accelerates the disappearance of claudin-5 from cell membranes, and consequently impairs the barrier property of endothelial cells.(a) Immunofluorescence images for claudin-5 expression in bEnd.3 monolayers under normoxia or hypoxia for 30 minutes. (b) Quantitative analysis of claudin-5 levels on cell membranes corresponding to the images in a. (c) TEERs of bEnd.3 monolayers under normoxia or hypoxia. Levels of claudin-5 protein on cell membranes as well as the TEERs of bEnd.3 monolayers are decreased in response to hypoxia. (d and e) Confocal scanning microscopic images (d) and their corresponding quantitative analysis of claudin-5 levels on cell membranes of bEnd.3 cells under normoxia or hypoxia for 30, 50, 70 and 90 minutes after pretreatment with or without CHX. Although claudin-5 disappeared from cell membranes quickly even under normoxia, the disappearance of claudin-5 is shown to be accelerated under hypoxia. Data are presented as mean ± SD from 3 independent cultures. *P < 0.05.

Mentions: Monolayers of bEnd.3, mouse brain microvascular endothelial cells, were cultured under normoxia and hypoxia, 21% O2 (atmospheric air) and 1% O2, respectively. Confocal imaging experiments with quantitative analysis demonstrated that claudin-5 molecules locate on cell membranes adjacent to neighboring cells under normoxia, and that the levels of claudin-5 on cell membranes significantly decrease, in parallel with a fall in the transendothelial electrical resistance (TEER) of cell monolayer, after exposure to hypoxia for 30 minutes (Fig. 1a–c). To monitor the turnover of claudin-5 molecules, the protein levels of claudin-5 on cell membranes were quantitatively analyzed in cells under normoxia or hypoxia for 30, 50, 70 and 90 minutes in the presence or absence of cycloheximide (CHX), a protein synthesis inhibitor. As demonstrated in Fig. 1d,e, the levels of claudin-5 on cell membranes of normoxic cells without CHX treatment were unchanged, while those of normoxic cells with CHX treatment decreased significantly already in 30 minutes and reached around 64.4 ± 2.2% (mean ± SD) of the control in 50 minutes, indicating the rapid turnover of claudin-5 under physiological condition. When the cells are exposure to hypoxia in the presence of CHX, claudin-5 disappeared from cell membranes more rapidly than under normoxia, and reached 37.2 ± 2.5% of the control in 50 minutes. Statistically, hypoxia accelerates the loss of claudin-5 from cell membranes.


ADAM12 and ADAM17 are essential molecules for hypoxia-induced impairment of neural vascular barrier function.

Cui D, Arima M, Takubo K, Kimura T, Horiuchi K, Minagawa T, Matsuda S, Ikeda E - Sci Rep (2015)

Hypoxia accelerates the disappearance of claudin-5 from cell membranes, and consequently impairs the barrier property of endothelial cells.(a) Immunofluorescence images for claudin-5 expression in bEnd.3 monolayers under normoxia or hypoxia for 30 minutes. (b) Quantitative analysis of claudin-5 levels on cell membranes corresponding to the images in a. (c) TEERs of bEnd.3 monolayers under normoxia or hypoxia. Levels of claudin-5 protein on cell membranes as well as the TEERs of bEnd.3 monolayers are decreased in response to hypoxia. (d and e) Confocal scanning microscopic images (d) and their corresponding quantitative analysis of claudin-5 levels on cell membranes of bEnd.3 cells under normoxia or hypoxia for 30, 50, 70 and 90 minutes after pretreatment with or without CHX. Although claudin-5 disappeared from cell membranes quickly even under normoxia, the disappearance of claudin-5 is shown to be accelerated under hypoxia. Data are presented as mean ± SD from 3 independent cultures. *P < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4525292&req=5

f1: Hypoxia accelerates the disappearance of claudin-5 from cell membranes, and consequently impairs the barrier property of endothelial cells.(a) Immunofluorescence images for claudin-5 expression in bEnd.3 monolayers under normoxia or hypoxia for 30 minutes. (b) Quantitative analysis of claudin-5 levels on cell membranes corresponding to the images in a. (c) TEERs of bEnd.3 monolayers under normoxia or hypoxia. Levels of claudin-5 protein on cell membranes as well as the TEERs of bEnd.3 monolayers are decreased in response to hypoxia. (d and e) Confocal scanning microscopic images (d) and their corresponding quantitative analysis of claudin-5 levels on cell membranes of bEnd.3 cells under normoxia or hypoxia for 30, 50, 70 and 90 minutes after pretreatment with or without CHX. Although claudin-5 disappeared from cell membranes quickly even under normoxia, the disappearance of claudin-5 is shown to be accelerated under hypoxia. Data are presented as mean ± SD from 3 independent cultures. *P < 0.05.
Mentions: Monolayers of bEnd.3, mouse brain microvascular endothelial cells, were cultured under normoxia and hypoxia, 21% O2 (atmospheric air) and 1% O2, respectively. Confocal imaging experiments with quantitative analysis demonstrated that claudin-5 molecules locate on cell membranes adjacent to neighboring cells under normoxia, and that the levels of claudin-5 on cell membranes significantly decrease, in parallel with a fall in the transendothelial electrical resistance (TEER) of cell monolayer, after exposure to hypoxia for 30 minutes (Fig. 1a–c). To monitor the turnover of claudin-5 molecules, the protein levels of claudin-5 on cell membranes were quantitatively analyzed in cells under normoxia or hypoxia for 30, 50, 70 and 90 minutes in the presence or absence of cycloheximide (CHX), a protein synthesis inhibitor. As demonstrated in Fig. 1d,e, the levels of claudin-5 on cell membranes of normoxic cells without CHX treatment were unchanged, while those of normoxic cells with CHX treatment decreased significantly already in 30 minutes and reached around 64.4 ± 2.2% (mean ± SD) of the control in 50 minutes, indicating the rapid turnover of claudin-5 under physiological condition. When the cells are exposure to hypoxia in the presence of CHX, claudin-5 disappeared from cell membranes more rapidly than under normoxia, and reached 37.2 ± 2.5% of the control in 50 minutes. Statistically, hypoxia accelerates the loss of claudin-5 from cell membranes.

Bottom Line: Therefore, the molecules involved in hypoxia-induced impairment of vascular barrier can be the targets to establish new therapies for intractable diseases.Hypoxic disappearance of claudin-5 from cell membranes and the consequent loss of barrier properties were completely suppressed by inhibition of the metalloproteinase activity which was found to be attributed to ADAM12 and ADAM17.This is the first report to specify the molecules which are responsible for hypoxia-induced impairment of neural vascular barrier and furthermore can be the targets of new therapeutic strategies for intractable neural diseases.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Yamaguchi University Graduate School of Medicine, 1-1-1 Minami-Kogushi, Ube, Yamaguchi 755-8505, Japan.

ABSTRACT
Neural vascular barrier is essential for the life of multicellular organisms, and its impairment by tissue hypoxia is known to be a central of pathophysiology accelerating the progression of various intractable neural diseases. Therefore, the molecules involved in hypoxia-induced impairment of vascular barrier can be the targets to establish new therapies for intractable diseases. Here, we demonstrate that a disintegrin and metalloproteinases (ADAMs) 12 and 17 expressed in endothelial cells are the molecules responsible for the impairment of neural vascular barrier by hypoxia. Brain microvascular endothelial cells in vitro lost their barrier properties immediately after hypoxic stimulation through diminished localization of claudin-5, a tight junction molecule, on cell membranes. Hypoxic disappearance of claudin-5 from cell membranes and the consequent loss of barrier properties were completely suppressed by inhibition of the metalloproteinase activity which was found to be attributed to ADAM12 and ADAM17. Inhibition of either ADAM12 or ADAM17 was sufficient to rescue the in vivo neural vasculature under hypoxia from the loss of barrier function. This is the first report to specify the molecules which are responsible for hypoxia-induced impairment of neural vascular barrier and furthermore can be the targets of new therapeutic strategies for intractable neural diseases.

No MeSH data available.


Related in: MedlinePlus