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Astrocyte glutathione maintains endothelial barrier stability.

ABSTRACT: Blood-brain barrier (BBB) impairment clearly accelerates brain disease progression. As ways to prevent injury-induced barrier dysfunction remain elusive, better understanding of how BBB cells interact and modulate barrier integrity is needed. Our metabolomic profiling study showed that cell-specific adaptation to injury correlates well with metabolic reprogramming at the BBB. In particular we noted that primary astrocytes (AC) contain comparatively high levels of glutathione (GSH)-related metabolites compared to primary endothelial cells (EC). Injury significantly disturbed redox balance in EC but not AC motivating us to assess 1) whether an AC-EC GSH shuttle supports barrier stability and 2) the impact of GSH on EC function. Using an isotopic labeling/tracking approach combined with Time-of-Flight Mass Spectrometry (TOF-MS) we prove that AC constantly shuttle GSH to EC even under resting conditions - a flux accelerated by injury conditions in vitro. In correlation, co-culture studies revealed that blocking AC GSH generation and secretion via siRNA-mediated ?-glutamyl cysteine ligase (GCL) knockdown significantly compromises EC barrier integrity. Using different GSH donors, we further show that exogenous GSH supplementation improves barrier function by maintaining organization of tight junction proteins and preventing injury-induced tight junction phosphorylation. Thus the AC GSH shuttle is key for maintaining EC redox homeostasis and BBB stability suggesting GSH supplementation could improve recovery after brain injury.

SUBMITTER: Huang SF

PROVIDER: S-EPMC7267730 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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Astrocyte glutathione maintains endothelial barrier stability.

Huang Sheng-Fu SF Othman Alaa A Koshkin Alexey A Fischer Sabrina S Fischer David D Zamboni Nicola N Ono Katsuhiko K Sawa Tomohiro T Ogunshola Omolara O OO

Redox biology 20200519

Blood-brain barrier (BBB) impairment clearly accelerates brain disease progression. As ways to prevent injury-induced barrier dysfunction remain elusive, better understanding of how BBB cells interact and modulate barrier integrity is needed. Our metabolomic profiling study showed that cell-specific adaptation to injury correlates well with metabolic reprogramming at the BBB. In particular we noted that primary astrocytes (AC) contain comparatively high levels of glutathione (GSH)-related metabo ...[more]

PMID: 32502899

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Project description:BackgroundAstrocytes (AC) are essential for brain homeostasis. Much data suggests that AC support and protect the vascular endothelium, but increasing evidence indicates that during injury conditions they may lose their supportive role resulting in endothelial cell activation and BBB disturbance. Understanding the triggers that flip this switch would provide invaluable information for designing new targets to modulate the brain vascular compartment. Hypoxia-inducible factor-1 (HIF-1) has long been assumed to be a culprit for barrier dysfunction as a number of its target genes are potent angiogenic factors. Indeed AC themselves, reservoirs of an array of different growth factors and molecules, are frequently assumed to be the source of such molecules although direct supporting evidence is yet to be published. Being well known reservoirs of HIF-1 dependent angiogenic molecules, we asked if AC HIF-1 dependent paracrine signaling drives brain EC disturbance during hypoxia.MethodsFirst we collected conditioned media from control and siRNA-mediated HIF-1 knockdown primary rat AC that had been exposed to normoxic or hypoxic conditions. The conditioned media was then used to culture normoxic and hypoxic (1% O2) rat brain microvascular EC (RBE4) for 6 and 24 h. Various activation parameters including migration, proliferation and cell cycling were assessed and compared to untreated controls. In addition, tight junction localization and barrier stability per se (via permeability assay) was evaluated.ResultsAC conditioned media maintained both normoxic and hypoxic EC in a quiescent state by suppressing EC metabolic activity and proliferation. By FACs we observed reduced cell cycling with an increased number of cells in G0 phase and reduced cell numbers in M phase compared to controls. EC migration was also blocked by AC conditioned media and in correlation hypoxic tight junction organization and barrier functionality was improved. Surprisingly however, AC HIF-1 deletion did not impact EC responses or barrier stability during hypoxia.ConclusionsThis study demonstrates that AC HIF-1 dependent paracrine signaling does not contribute to AC modulation of EC barrier function under normoxic or hypoxic conditions. Thus other cell types likely mediate EC permeability in stress scenarios. Our data does however highlight the continuous protective effect of AC on the barrier endothelium. Exploring these protective mechanisms in more detail will provide essential insight into ways to prevent barrier disturbance during injury and disease.

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Astrocyte glutathione maintains endothelial barrier stability.

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Astrocyte glutathione maintains endothelial barrier stability.

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