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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

ADAM12 and ADAM17 are essential molecules for the impairment of barrier function of retinal vasculature under hypoxia.(a) Immunofluorescence images showing the oxygenation of retinas from mice under normoxia or hypoxia. Intraperitoneally injected pimonidazole hydrochloride (green) is incorporated in retinal cells from mice under hypoxia, as compared with minimal incorporation in retinal cells from mice under normoxia. Retinal vasculature is visualized by immunostaining for CD31 (red). (b) Immunofluorescence images, for claudin-5 expression, of flat mounts of retinas from mice under normoxia or hypoxia, with prior intravitreous injection of non-silencing siRNA (NC siRNA, for negative control), siRNA for ADAM12 or siRNA for ADAM17. Intravitreous injection of siRNA either for ADAM12 or ADAM17 rescues the retinal vasculature from the hypoxia-induced decrease in claudin-5 expression, in contrast to the hypoxic vasculature with the injection of non-silencing siRNA (NC siRNA). (c and d) Tracer experiment to evaluate the permeability of retinal vasculature under normoxia or hypoxia, with prior intravitreous injection of siRNAs (c) or neutralizing antibodies (d) specific for ADAM12 or ADAM17. For negative controls, non-silencing siRNA (NC siRNA) (c) and non-immune goat immunoglobulins (Non-immune IgG) (d) were injected intravitreously. Injected tracers, Hoechst stain (blue) and dextran (red), in flat mounts of retinas were detected under confocal microscopy, and merged views are presented. Nuclear staining of retinal cells by the extravasated Hoechst stain is noted in hypoxic retinas of negative controls, while the staining is minimal in hypoxic retinas with pre-injection of siRNAs or neutralizing antibodies for ADAM12 or ADAM17.
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f4: ADAM12 and ADAM17 are essential molecules for the impairment of barrier function of retinal vasculature under hypoxia.(a) Immunofluorescence images showing the oxygenation of retinas from mice under normoxia or hypoxia. Intraperitoneally injected pimonidazole hydrochloride (green) is incorporated in retinal cells from mice under hypoxia, as compared with minimal incorporation in retinal cells from mice under normoxia. Retinal vasculature is visualized by immunostaining for CD31 (red). (b) Immunofluorescence images, for claudin-5 expression, of flat mounts of retinas from mice under normoxia or hypoxia, with prior intravitreous injection of non-silencing siRNA (NC siRNA, for negative control), siRNA for ADAM12 or siRNA for ADAM17. Intravitreous injection of siRNA either for ADAM12 or ADAM17 rescues the retinal vasculature from the hypoxia-induced decrease in claudin-5 expression, in contrast to the hypoxic vasculature with the injection of non-silencing siRNA (NC siRNA). (c and d) Tracer experiment to evaluate the permeability of retinal vasculature under normoxia or hypoxia, with prior intravitreous injection of siRNAs (c) or neutralizing antibodies (d) specific for ADAM12 or ADAM17. For negative controls, non-silencing siRNA (NC siRNA) (c) and non-immune goat immunoglobulins (Non-immune IgG) (d) were injected intravitreously. Injected tracers, Hoechst stain (blue) and dextran (red), in flat mounts of retinas were detected under confocal microscopy, and merged views are presented. Nuclear staining of retinal cells by the extravasated Hoechst stain is noted in hypoxic retinas of negative controls, while the staining is minimal in hypoxic retinas with pre-injection of siRNAs or neutralizing antibodies for ADAM12 or ADAM17.

Mentions: To validate in vivo the involvement of ADAM12 and ADAM17 in hypoxic disruption of neural vascular barrier, the permeability of retinal vasculature from mice kept under either normoxic (atmospheric air) or hypoxic (4 to 7% oxygen concentration) condition for 36 hours was monitored by intracardiac injection of fluorescence dyes, Hoechst stain H33258 (molecular mass, 534 Da) and tetramethylrhodamine-conjugated lysine-fixable dextran (molecular mass, 10,000 Da). Tissue hypoxia of retinas from mice kept under hypoxia was confirmed by increased incorporation of intraperitoneally injected pimonidazole hydrochloride (Hypoxyprobe-1) (Fig. 4a). In hypoxic retinas, the immunofluorescence signal of cell membrane-localized claudin-5 was diminished (Fig. 4b; left panels). In parallel with the decrease in claudin-5 expression, the vascular barrier function in hypoxic retinas was impaired, showing the increase in permeability against a smaller molecule Hoechst stain H33258 (Fig. 4c,d; left panels). It is noteworthy that the prior injection of ADAM12-specific or ADAM17-specific siRNA into the vitreous cavities clearly rescued the retinal vasculature from the decrease in claudin-5 expression as well as the enhanced leakage of dye (Fig. 4b,c; center and right panels). The same inhibitory effect on hypoxia-induced impairment of vascular barrier was obtained with the prior intravitreous injection of a neutralizing antibody against either ADAM12 or ADAM17 (Fig. 4d; center and right panels). These in vivo findings support the in vitro data demonstrating the indispensable roles of ADAM12 and ADAM17 in hypoxia-induced impairment of neural vascular barrier function, and furthermore indicate the availability of ADAM12 and ADAM17 for therapeutic targets in the intractable neural diseases with vascular barrier dysfunction.


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)

ADAM12 and ADAM17 are essential molecules for the impairment of barrier function of retinal vasculature under hypoxia.(a) Immunofluorescence images showing the oxygenation of retinas from mice under normoxia or hypoxia. Intraperitoneally injected pimonidazole hydrochloride (green) is incorporated in retinal cells from mice under hypoxia, as compared with minimal incorporation in retinal cells from mice under normoxia. Retinal vasculature is visualized by immunostaining for CD31 (red). (b) Immunofluorescence images, for claudin-5 expression, of flat mounts of retinas from mice under normoxia or hypoxia, with prior intravitreous injection of non-silencing siRNA (NC siRNA, for negative control), siRNA for ADAM12 or siRNA for ADAM17. Intravitreous injection of siRNA either for ADAM12 or ADAM17 rescues the retinal vasculature from the hypoxia-induced decrease in claudin-5 expression, in contrast to the hypoxic vasculature with the injection of non-silencing siRNA (NC siRNA). (c and d) Tracer experiment to evaluate the permeability of retinal vasculature under normoxia or hypoxia, with prior intravitreous injection of siRNAs (c) or neutralizing antibodies (d) specific for ADAM12 or ADAM17. For negative controls, non-silencing siRNA (NC siRNA) (c) and non-immune goat immunoglobulins (Non-immune IgG) (d) were injected intravitreously. Injected tracers, Hoechst stain (blue) and dextran (red), in flat mounts of retinas were detected under confocal microscopy, and merged views are presented. Nuclear staining of retinal cells by the extravasated Hoechst stain is noted in hypoxic retinas of negative controls, while the staining is minimal in hypoxic retinas with pre-injection of siRNAs or neutralizing antibodies for ADAM12 or ADAM17.
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f4: ADAM12 and ADAM17 are essential molecules for the impairment of barrier function of retinal vasculature under hypoxia.(a) Immunofluorescence images showing the oxygenation of retinas from mice under normoxia or hypoxia. Intraperitoneally injected pimonidazole hydrochloride (green) is incorporated in retinal cells from mice under hypoxia, as compared with minimal incorporation in retinal cells from mice under normoxia. Retinal vasculature is visualized by immunostaining for CD31 (red). (b) Immunofluorescence images, for claudin-5 expression, of flat mounts of retinas from mice under normoxia or hypoxia, with prior intravitreous injection of non-silencing siRNA (NC siRNA, for negative control), siRNA for ADAM12 or siRNA for ADAM17. Intravitreous injection of siRNA either for ADAM12 or ADAM17 rescues the retinal vasculature from the hypoxia-induced decrease in claudin-5 expression, in contrast to the hypoxic vasculature with the injection of non-silencing siRNA (NC siRNA). (c and d) Tracer experiment to evaluate the permeability of retinal vasculature under normoxia or hypoxia, with prior intravitreous injection of siRNAs (c) or neutralizing antibodies (d) specific for ADAM12 or ADAM17. For negative controls, non-silencing siRNA (NC siRNA) (c) and non-immune goat immunoglobulins (Non-immune IgG) (d) were injected intravitreously. Injected tracers, Hoechst stain (blue) and dextran (red), in flat mounts of retinas were detected under confocal microscopy, and merged views are presented. Nuclear staining of retinal cells by the extravasated Hoechst stain is noted in hypoxic retinas of negative controls, while the staining is minimal in hypoxic retinas with pre-injection of siRNAs or neutralizing antibodies for ADAM12 or ADAM17.
Mentions: To validate in vivo the involvement of ADAM12 and ADAM17 in hypoxic disruption of neural vascular barrier, the permeability of retinal vasculature from mice kept under either normoxic (atmospheric air) or hypoxic (4 to 7% oxygen concentration) condition for 36 hours was monitored by intracardiac injection of fluorescence dyes, Hoechst stain H33258 (molecular mass, 534 Da) and tetramethylrhodamine-conjugated lysine-fixable dextran (molecular mass, 10,000 Da). Tissue hypoxia of retinas from mice kept under hypoxia was confirmed by increased incorporation of intraperitoneally injected pimonidazole hydrochloride (Hypoxyprobe-1) (Fig. 4a). In hypoxic retinas, the immunofluorescence signal of cell membrane-localized claudin-5 was diminished (Fig. 4b; left panels). In parallel with the decrease in claudin-5 expression, the vascular barrier function in hypoxic retinas was impaired, showing the increase in permeability against a smaller molecule Hoechst stain H33258 (Fig. 4c,d; left panels). It is noteworthy that the prior injection of ADAM12-specific or ADAM17-specific siRNA into the vitreous cavities clearly rescued the retinal vasculature from the decrease in claudin-5 expression as well as the enhanced leakage of dye (Fig. 4b,c; center and right panels). The same inhibitory effect on hypoxia-induced impairment of vascular barrier was obtained with the prior intravitreous injection of a neutralizing antibody against either ADAM12 or ADAM17 (Fig. 4d; center and right panels). These in vivo findings support the in vitro data demonstrating the indispensable roles of ADAM12 and ADAM17 in hypoxia-induced impairment of neural vascular barrier function, and furthermore indicate the availability of ADAM12 and ADAM17 for therapeutic targets in the intractable neural diseases with vascular barrier dysfunction.

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