Deletion of astroglial connexins weakens the blood-brain barrier.
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ABSTRACT:
SUBMITTER: Ezan P
PROVIDER: S-EPMC3421093 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The blood-brain barrier is formed by capillary endothelial cells expressing connexin 37 (Cx37), Cx40, and Cx43 and is joined by closely apposed astrocytes expressing Cx43 and Cx30. We investigated whether connexin-targeting peptides could limit barrier leakage triggered by LPS-induced systemic inflammation in mice. Intraperitoneal LPS administration increased endothelial and astrocytic Cx43 expression; elevated TNF-α, IL-1β, IFN-γ, and IL-6 in plasma and IL-6 in the brain; and induced barrier leakage recorded over 24 hours. Barrier leakage was largely prevented by global Cx43 knockdown and Cx43/Cx30 double knockout in astrocytes, slightly diminished by endothelial Cx43 knockout, and not protected by global Cx30 knockout. Intravenous administration of Gap27 or Tat-Gap19 peptides just before LPS also prevented barrier leakage, and intravenously administered BAPTA-AM to chelate intracellular calcium was equally effective. Patch-clamp experiments demonstrated LPS-induced Cx43 hemichannel opening in endothelial cells, which was suppressed by Gap27, Gap19, and BAPTA. LPS additionally triggered astrogliosis that was prevented by intravenous Tat-Gap19 or BAPTA-AM. Cortically applied Tat-Gap19 or BAPTA-AM to primarily target astrocytes also strongly diminished barrier leakage. In vivo dye uptake and in vitro patch-clamp showed Cx43 hemichannel opening in astrocytes that was induced by IL-6 in a calcium-dependent manner. We conclude that targeting endothelial and astrocytic connexins is a powerful approach to limit barrier failure and astrogliosis.
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Project description:Hypothalamic glucose detection participates in maintaining glycemic balance, food intake, and thermogenesis. Although hypothalamic neurons are the executive cells involved in these responses, there is increasing evidence that astrocytes participate in glucose sensing (GS); however, it is unknown whether astroglial networking is required for glucose sensitivity. Astroglial connexins 30 and 43 (Cx30 and Cx43) form hexameric channels, which are apposed in gap junctions, allowing for the intercellular transfer of small molecules such as glucose throughout the astroglial networks. Here, we hypothesized that hypothalamic glucose sensitivity requires these connexins. First, we showed that both Cxs are enriched in the rat hypothalamus, with highly concentrated Cx43 expression around blood vessels of the mediobasal hypothalamus (MBH). Both fasting and high glycemic levels rapidly altered the protein levels of MBH astroglial connexins, suggesting cross talk within the MBH between glycemic status and the connexins' ability to dispatch glucose. Finally, the inhibition of MBH Cx43 (by transient RNA interference) attenuated hypothalamic glucose sensitivity in rats, which was demonstrated by a pronounced decreased insulin secretion in response to a brain glucose challenge. These results illustrate that astroglial connexins contribute to hypothalamic GS.
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Project description:In the brain, a unique homeostatic equilibrium strictly controls immune reactions. In healthy conditions, the immune system poorly communicates with the central nervous system (CNS), immune cell migration across the blood brain barrier (BBB) is kept at a very low level and the unique CNS microenvironment strictly restricts immune reactions. Here, we identified the astroglial gap junction protein Connexin 43 has a new actor of this homeostasis. We showed that genetic inactivation of astroglial Cx43 in the mouse induces expression of a specific set of chemokines and endothelial adhesion molecules leading to neutrophils, macrophages, B and T lymphocytes and mature plasmocytes infiltration at the BBB, and to the development of antigen presentation-related mechanisms, in sterile conditions. Thus, astroglial Cx43 by decreasing the expression of leukocytes adhesion and migration mediators, promotes the immune quiescence of the BBB
2015-03-16 | GSE54458 | GEO
Project description:The[Q3] gelatinases, matrix metalloproteinase 2 (MMP-2) and MMP-9, are key for leukocyte penetration of the brain parenchymal border in neuroinflammation and the functional integrity of this barrier; however, it is unclear which MMP substrates are involved. Using a tailored, sensitive, label-free mass spectrometry–based secretome approach, not previously applied to nonimmune cells, we identified 119 MMP-9 and 21 MMP-2 potential substrates at the cell surface of primary astrocytes, including known substrates (β-dystroglycan) and a broad spectrum of previously unknown MMP-dependent events involved in cell-cell and cell-matrix interactions. Using neuroinflammation as a model of assessing compromised astroglial barrier function, a selection of the potential MMP substrates were confirmed in vivo and verified in human samples, including vascular cell adhesion molecule–1 and neuronal cell adhesion molecule. We provide a unique resource of potential MMP-2/MMP-9 substrates specific for the astroglia barrier. Our data support a role for the gelatinases in the formation and maintenance of this barrier but also in astrocyte-neuron interactions.
2023-10-24 | PXD043521 | Pride
Project description:AimsIschemic stroke is a life-threatening disease with limited therapeutic strategies. Blood-brain barrier (BBB) disruption is a critical pathological process that contributes to poor outcomes in ischemic stroke. We previously showed that the microglial inhibition of the inflammasome sensor absent in melanoma 2 (AIM2) suppressed the inflammatory response and protected against ischemic stroke. However, whether AIM2 is involved in BBB disruption during cerebral ischemia is unknown.MethodsMiddle cerebral artery occlusion (MCAO) and oxygen-glucose deprivation/reoxygenation (OGD/R) were used to mimic cerebral ischemia in mice and brain microvascular endothelial cells (HBMECs), respectively. The infarct volume, neurological deficits, and BBB permeability were measured in mice after MCAO. Transendothelial electrical resistance (TEER) and neutrophil adhesion to the HBMEC monolayer were assessed after OGD/R treatment. Western blot and immunofluorescence analyses were conducted to evaluate the expression of related proteins.ResultsAIM2 was shown to be expressed in brain endothelial cells and upregulated after ischemic stroke in the mouse brain. AIM2 deletion reduced the infarct volume, improved neurological and motor functions, and decreased BBB disruption. In vitro, OGD/R significantly increased the protein levels of AIM2 and ICAM-1 and decreased those of the tight junction (TJ) proteins ZO-1 and occludin. AIM2 knockdown effectively protected BBB integrity by promoting the expression of TJ proteins and decreasing ICAM-1 expression and neutrophil adhesion. Mechanistically, AIM2 knockdown reversed the OGD/R-induced increases in ICAM-1 expression and STAT3 phosphorylation in brain endothelial cells. Furthermore, treatment with the p-STAT3 inhibitor AG490 mitigated the effect of AIM2 on BBB breakdown.ConclusionOur findings indicated that inhibiting AIM2 preserved the BBB integrity after ischemic stroke, at least partially by modulating STAT3 activation and that AIM2 may be a promising therapeutic target for cerebral ischemic stroke.
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Project description:The mechanism of copper (Cu) transport into the brain is unclear. This study evaluated the main species and route of Cu transport into the brain using in situ brain perfusion technique, and assessed the levels of mRNA encoding Cu transporters using real time RT-PCR. Free (64)Cu uptake in rat choroid plexus (CP), where the blood-cerebrospinal fluid barrier (BCB) is primarily located, is about 50 and 1000 times higher than (64)Cu-albumin and (64)Cu-ceruloplasmin uptake, respectively. The unidirectional transport rate constants (K(in)) for Cu in the CP and brain capillaries of the blood-brain barrier (BBB) were 1034 and 319 microl/s/g, respectively, while K(in) in CSF and capillary-depleted parenchyma were much reduced, 0.8 and 112 microl/s/g, respectively. The K(in) in cerebellum was significantly lower than that in hippocampus. The mRNAs encoding Cu transporter-1 (Ctr1) and ATP7A were higher in the CP than those in brain capillaries and parenchyma, whereas ATP7B mRNA was higher in brain capillaries than those in the CP and brain parenchyma. Taken together, these data suggest that the expression of Cu transporters is higher in brain barriers than in brain parenchyma; the Cu transport into the brain is mainly achieved through the BBB as a free Cu ion and the BCB may serve as a main regulatory site of Cu in the CSF.
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Project description:Potential therapeutic actions of the cannabinoids delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD) are based on their activity as analgesics, anti-emetics, anti-inflammatory agents, anti-seizure compounds. THC and CBD lipophilicity and their neurological actions makes them candidates as new medicinal approaches to treat central nervous system (CNS) diseases. However, they show differences about penetrability and disposition in the brain. The present article is an overview about THC and CBD crossing the blood-brain barrier (BBB) and their brain disposition. Several findings indicate that CBD can modify the deleterious effects on BBB caused by inflammatory cytokines and may play a pivotal role in ameliorating BBB dysfunction consequent to ischemia. Thus supporting the therapeutic potential of CBD for the treatment of ischemic and inflammatory diseases of CNS. Cannabinoids positive effects on cognitive function could be also considered through the aspect of protection of BBB cerebrovascular structure and function, indicating that they may purchase substantial benefits through the protection of BBB integrity. Delivery of these cannabinoids in the brain following different routes of administration (subcutaneous, oral, and pulmonary) is illustrated and commented. Finally, the potential role of cannabinoids in drug-resistance in the clinical management of neurological or psychiatric diseases such as epilepsy and schizophrenia is discussed on the light of their crossing the BBB.
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Project description:Recent advances in maximum safe glioma resection have included the introduction of a host of visualization techniques to complement intraoperative white-light imaging of tumors. However, barriers to the effective use of these techniques within the central nervous system remain. In the healthy brain, the blood-brain barrier ensures the stability of the sensitive internal environment of the brain by protecting the active functions of the central nervous system and preventing the invasion of microorganisms and toxins. Brain tumors, however, often cause degradation and dysfunction of this barrier, resulting in a heterogeneous increase in vascular permeability throughout the tumor mass and outside it. Thus, the characteristics of both the blood-brain and blood-brain tumor barriers hinder the vascular delivery of a variety of therapeutic substances to brain tumors. Recent developments in fluorescent visualization of brain tumors offer improvements in the extent of maximal safe resection, but many of these fluorescent agents must reach the tumor via the vasculature. As a result, these fluorescence-guided resection techniques are often limited by the extent of vascular permeability in tumor regions and by the failure to stain the full volume of tumor tissue. In this review, we describe the structure and function of both the blood-brain and blood-brain tumor barriers in the context of the current state of fluorescence-guided imaging of brain tumors. We discuss features of currently used techniques for fluorescence-guided brain tumor resection, with an emphasis on their interactions with the blood-brain and blood-tumor barriers. Finally, we discuss a selection of novel preclinical techniques that have the potential to enhance the delivery of therapeutics to brain tumors in spite of the barrier properties of the brain.
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Project description:Gliomas generally infiltrate the surrounding normal brain parenchyma, a process associated with increased vascular permeability (VP) and dysregulation of the blood-brain barrier (BBB). However, the molecular mechanisms underlying glioma-induced VP in the brain remain poorly understood. Using a conditional, endothelium-specific deletion of the focal adhesion kinase (FAK) in the mouse (FAK CKO), we show that FAK is critical for destabilization of the tumor endothelium in tumor-bearing mice, with mutant mice exhibiting a relatively normalized vasculature compared with wild-type mice (FAK WT). Tumor vessels in the FAK CKO mice displayed reduced VP compared with FAK WT mice, resulting in reduced tumor growth. Additionally, FAK CKO mice displayed partial restoration of cell-cell junction proteins in the tumor vessels and astrocyte-endothelium interactions in tumors, revealing an additional role of astrocytes in mediating tumor-induced VP. Together, these results provide genetic evidence that FAK is a mediator of tumor-induced VP in the brain. Our findings may help understand how therapeutics might be used to regulate specific cell-type interactions to restore BBB structure/function in cancer and perhaps other pathologic conditions.
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Project description:Cyclooxygenase (COX)-2 and matrix metalloproteinase (MMP)-9 are two crucial mediators contributing to blood-brain barrier (BBB) damage during cerebral ischemia. However, it is not known whether MMP-9 activation is involved in COX-2-mediated BBB disruption in ischemic stroke. In this study, we hypothesized that genetic deletion or pharmacological inhibition of COX-2 reduces BBB damage by reducing MMP-9 activity in a mouse model of ischemic stroke. Male COX-2 knockout (COX-2-/-) and wild-type (WT) mice were subjected to 60 min of middle cerebral artery occlusion (MCAO) followed by 24 h of reperfusion. Genetic deletion of COX-2 or post-ischemic treatment with CAY10404, a highly selective COX-2 inhibitor, significantly reduced BBB damage and hemorrhagic transformation, as assessed by immunoglobulin G (IgG) extravasation and brain hemoglobin (Hb) levels, respectively. Immunoblotting analysis showed that tight junction proteins (TJPs) zonula occludens (ZO)-1 and occludin as well as junctional adhesion molecule-A (JAM-A) and the basal lamina protein collagen IV were dramatically reduced in the ischemic brain. Stroke-induced loss of these BBB structural proteins was significantly attenuated in COX-2-/- mice. Similarly, stroke-induced loss of ZO-1 and occludin was significantly attenuated by CAY10404 treatment. Ischemia-induced increase in MMP-9 protein levels in the ipsilateral cerebral cortex was significantly reduced in COX-2-/- mice. Stroke induced a dramatic increase in MMP-9 enzymatic activity in the ischemic cortex, which was markedly reduced by COX-2 gene deficiency or pharmacological inhibition with CAY10404. Levels of myeloperoxidase (MPO, an indicator of neutrophil infiltration into the brain parenchyma), neutrophil elastase (NE), and lipocalin-2 (LCN2, also known as neutrophil gelatinase-associated lipocalin), measured by western blot and specific ELISA kits, respectively, were markedly increased in the ischemic brain. Increased levels of markers for neutrophil infiltration were significantly reduced in COX-2-/- mice compared with WT controls following stroke. Altogether, neurovascular protective effects of COX-2 blockade are associated with reduced BBB damage, MMP-9 expression/activity and neutrophil infiltration. Our study shows for the first time that MMP-9 is an important downstream effector contributing to COX-2-mediated neurovascular damage in ischemic stroke. Targeting the COX-2/MMP-9 pathway could represent a promising strategy to reduce neuroinflammatory events in order to preserve the BBB integrity and ameliorate ischemic stroke injury.
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