Functional phenotypes, which cells can acquire depending on the microenvironment, are currently the focus of investigations into new anti-inflammatory therapeutic approaches. Glial cells, microglia, ...and astrocytes are major participants in neuroinflammation, but their roles differ, as microglia are cells of mesodermal origin, while astrocytes are cells of ectodermal origin. The inflammatory phenotype of cells can be modulated by ω-6- and ω-3-polyunsaturated fatty acid-derived oxylipins, although data on changes in oxylipin profiles in different cell adaptations to pro- and anti-inflammatory stimuli are scarce. Our study aimed to compare UPLC-MS/MS-measured oxylipin profiles in various rat astrocyte adaptation states. We used cells treated for 24 h with lipopolysaccharide (LPS) for classical pro-inflammatory adaptation and with interleukin 4 (IL-4) or 10 (IL-10) for alternative anti-inflammatory adaptation, with the resulting phenotypes characterized by quantitative real-time PCR (RT-PCR). We also tested long-term, low-concentration LPS treatment (endotoxin treatment) as a model of astrocyte adaptations. The functional response of astrocytes was estimated by acute (4 h) LPS-induced cell reactivity, measured by gene expression markers and oxylipin synthesis. We discovered that, as well as gene markers, oxylipin profiles can serve as markers of pro- (A1-like) or anti-inflammatory (A2-like) adaptations. We observed predominant involvement of ω-6 polyunsaturated fatty acid (PUFA) and the cyclooxygenase branch for classical (LPS) pro-inflammatory adaptations and ω-3 PUFA and the lipoxygenase branch for alternative (IL-4) anti-inflammatory adaptations. Treatment with IL-4, but not IL-10, primes the ability of astrocytes to activate the innate immunity signaling pathways in response to LPS. Endotoxin-treated astrocytes provide an alternative anti-inflammatory adaptation, which makes cells less sensitive to acute LPS stimulation than the IL-4 induced adaptation. Taken together, the data reveal that oxylipin profiles associate with different states of polarization to generate a pro-inflammatory or anti-inflammatory phenotype. This association manifests itself both in native cells and in their responses to a pro-inflammatory stimulus.
Receptor-mediated transcytosis of the transferrin receptor has been suggested as a potential transport system to deliver therapeutic molecules into the brain. Recent studies have however shown that ...therapeutic antibodies, which have been reported to cross the brain endothelium, reach greater brain exposure when the affinity of the antibodies to the transferrin receptor is lowered. The lower affinity of the antibodies to the transferrin receptor facilitates the dissociation from the receptor within the endosomal compartments, which may indicate that the receptor itself does not necessarily move across the endothelial cells by transcytosis. The aim of the present study was to investigate transferrin receptor expression and role in transendothelial transferrin transport in cultured bovine brain endothelial cell monolayers.
Transferrin receptor mRNA and protein levels were investigated in endothelial mono-cultures and co-cultures with astrocytes, as well as in freshly isolated brain capillaries using qPCR, immunocytochemistry and Western blotting. Transendothelial transport and luminal association of holo-transferrin was investigated using 125Iholo-transferrin or 59Fe-transferrin.
Transferrin receptor mRNA expression in all cell culture configurations was lower than in freshly isolated capillaries, but the expression slightly increased during six days of culture. The mRNA expression levels were similar in mono-cultures and co-cultures. Immunostaining demonstrated comparable transferrin receptor localization patterns in mono-cultures and co-cultures. The endothelial cells demonstrated an up-regulation of transferrin receptor mRNA after treatment with the iron chelator deferoxamine. The association of 125Iholo-transferrin and 59Fe-transferrin to the endothelial cells was inhibited by an excess of unlabeled holo-transferrin, indicating receptor mediated association. However, over time the cell associated 59Fe-label exceeded that of 125Iholo-transferrin, which could indicate release of iron in the endothelial cells and receptor recycling. Luminal-to-abluminal transport of 125Iholo-transferrin across endothelial cell monolayers was low and not inhibited by unlabeled holo-transferrin. This indicated that transendothelial transferrin transport was independent of transferrin receptor-mediated transcytosis.
•The transferrin receptor (TfR) has been suggested to ferry drug compounds across the blood-brain barrier.•TfR expression and function is studied in cultured brain capillary endothelial cells.•Cultured endothelial cells express the TfR at both the mRNA and protein level.•Endothelial co-culture with astrocytes induces a tightening of the monolayers, but do not change TfR expression.•Endothelial monolayers bind transferrin, but transendothelial transferrin transport appears not to be receptor dependent.
Angiotensin (Ang) II and cannabinoids regulate physiologically relevant astroglial functions via receptor-mediated activation of Mitogen-activated protein kinases (MAPKs). In this study, we ...investigated the consequences of astroglial Ang II type 1 receptor (AT1R) and Cannabinoid type 1 receptor (CB1R) activation, alone and in combination, on MAPK activation in the presence and absence of hypertensive states. In addition, we also investigated a novel unidirectional crosstalk mechanism between AT1R and CB1R, that involves PKC-mediated phosphorylation of CB1R.
Astrocytes were isolated from the brainstem and cerebellum of Spontaneously hypertensive rats (SHRs) and normotensive Wistar rats. The cells were treated with either 100nM Ang II or 10nM Arachidonyl-2′-chloroethylamide (ACEA), both alone and in combination, for varying time periods, and the extent of phosphorylation of MAPKs, ERK and p38, and the phosphorylated forms of CB1R (p-CB1R), were measured using western blotting.
Ang II treatment resulted in a greater activation of MAPKs in SHR brainstem astrocytes, but not SHR cerebellar astrocytes when compared to Wistar rats. ACEA-mediated MAPK activation was significantly lower in brainstem astrocytes of SHRs when compared to Wistar rats. ACEA negatively modulates AT1R-mediated MAPK activation in both cerebellar and brainstem astrocytes of both models. The effect however was diminished in brainstem astrocytes. Ang II caused a significant increase in phosphorylation of CB1R in cerebellar astrocytes, while its effect was diminished in brainstem astrocytes of both models.
Both Ang II and ACEA-induced MAPK activation were significantly altered in SHR astrocytes when compared to Wistar astrocytes. A possible reduction in CB1R functionality, coupled with a hyperfunctional AT1R in the brainstem, could well be significant factors in the development of hypertensive states. AT1R-mediated phosphorylation of CB1R could be critical for impaired cerebellar development characterized by a hyperactive RAS.
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•Ang II enhanced MAPK activation in brainstem, but not cerebellar, astrocytes of SHRs when compared to Wistar rats.•ACEA-mediated MAPK activation is diminished in SHRs brainstem and cerebellar astrocytes, when compared to Wistar rats.•Ang II induced prominent CB1R phosphorylation in cerebellar, but not brainstem, astrocytes of both SHRs and Wistar rats.•Ang II-induced CB1R phosphorylation was mostly mediated via the AT1R-PKC axis.
Glioma survival is dismal, in part, due to an imbalance in antioxidant expression and activity. Peroxisome proliferator-activated receptor (PPAR) agonists have antineoplastic properties which present ...new redox-dependent targets for glioma anticancer therapies. Herein, we demonstrate that treatment of primary cultures of normal rat astrocytes with PPAR agonists increased the expression of catalase mRNA protein, and enzymatic activity. In contrast, these same agonists had no effect on catalase expression and activity in malignant rat glioma cells. The increase in steady-state catalase mRNA observed in normal rat astrocytes was due, in part, to de novo mRNA synthesis as opposed to increased catalase mRNA stability. Moreover, pioglitazone-mediated induction of catalase activity in normal rat astrocytes was completely blocked by transfection with a PPARγ-dominant negative plasmid. These data suggest that defects in PPAR-mediated signaling and gene expression may represent a block to normal catalase expression and induction in malignant glioma. The ability of PPAR agonists to differentially increase catalase expression and activity in normal astrocytes but not glioma cells suggests that these compounds might represent novel adjuvant therapeutic agents for the treatment of gliomas.
Astrocytes activation has been implicated in the inflammatory responses underlying brain injury and neurodegenerative diseases including bacterial infections, cerebral ischemia, and Parkinson's ...diseases. Acetylpuerarin is a newly modified isoflavone based on puerarin that has neuroprotective and antioxidant effects. In this study, we investigated the anti-inflammatory action of acetylpuerarin in regulating the eicosanoids generation and its underlying molecular mechanisms in lipopolysaccharide (LPS)-induced production of arachidonic acid (AA) metabolites in primary rat astrocytes. The results showed that acetylpuerarin concentration dependently inhibited the LPS-induced production of AA metabolites such as prostaglandin E
2
(PGE
2
) and leukotriene C
4
(LTC
4
), and acetylpuerarin significantly attenuated the expression and immunoreactivity of group V secretory phospholipase A
2
(sPLA
2
) protein induced by LPS in astrocytes. Furthermore, in astrocytes pretreated with acetylpuerarin, the time course of phosphorylation of extracellular signal-regulated kinase (ERK)1/2 and of cytosolic PLA
2
alpha (cPLA
2
α) and expression of transcription factors, nuclear factor kappa B (NF-κB), was markedly truncated. Acetylpuerarin concentration dependently abolished the LPS-induced expressions of AA-metabolizing enzymes including cyclooxygenase-2 (COX-2) and lipooxygenase-5 (LOX-5). This study indicates that acetylpuerarin inhibited LPS-induced AA-metabolizing enzymes and AA metabolites in astrocytes via downregulation expression of group V sPLA
2
and phosphorylation of ERK1/2, cPLA
2
α, and NF-κB. These findings reveal, in part, the molecular basis underlying the anti-inflammatory properties of acetylpuerarin.
Activation of protease‐activated receptors (PARs) is known to exert neuroprotection when low concentrations of the agonist protease thrombin are applied. However, the mechanism of protection is still ...unclear. Here, we showed that activation of multiple PARs, including PAR‐1, PAR‐2 and PAR‐4, was able to elevate the release of the chemokine cytokine‐induced neutrophil chemoattractant (CINC)‐3 from rat astrocytes, in addition to evoking CINC‐1 secretion. Different molecular mechanisms were identified as being involved in the secretion of CINC‐1 and CINC‐3, upon activation of different PARs. Importantly, we found that both CINC‐1 and CINC‐3 could signal to rat cortical neurons. Both chemokines acted via CXCR2 to prevent C2‐ceramide‐induced cytochrome c release from mitochondria. Consequently CINC‐1 and CINC‐3 protected neurons from apoptosis. We further revealed that conditioned media obtained from PAR‐activated astrocytes similarly protected cortical neurons against C2‐ceramide‐induced cell death. The neuroprotection was considerably suppressed by a CXCR2 antagonist. CXCR2 is the cognate receptor for CINC. Therefore, our findings demonstrate that PAR‐activated astrocytes are able to protect neurons against neurodegeneration and cell death via regulation of the secretion of chemokines CINC‐1 and CINC‐3. These data indicate a previously unknown mechanism for astrocyte‐mediated neuroprotection achieved by PAR activation.
Cysteinyl‐leukotrienes (cys‐LTs), potent mediators in inflammatory diseases, are produced by nervous tissue, but their cellular source and role in the brain are not very well known. In this report we ...have demonstrated that rat cultured astrocytes express the enzymes (5′‐lipoxygenase and LTC4 synthase) required for cys‐LT production, and release cys‐LTs in resting condition and, to a greater extent, in response to calcium ionophore A23187, 1 h combined oxygen–glucose deprivation or 2‐methyl‐thioATP, a selective P2Y1/ATP receptor agonist. MK‐886, a LT synthesis inhibitor, prevented basal and evoked cys‐LT release. In addition, 2‐methyl‐thioATP‐induced cys‐LT release was abolished by suramin, a P2 receptor antagonist, or by inhibitors of ATP binding cassette proteins involved in cys‐LT release. We also showed that astrocytes express cys‐LT1 and not cys‐LT2 receptors. The stimulation of these receptors by LTD4 activated the mitogen‐activated protein kinase (MAPK) pathway. This effect was: (i) insensitive to inhibitors of receptor‐coupled Gi protein (pertussis toxin) or tyrosine kinase receptors (genistein); (ii) abolished by MK‐571, a cys‐LT1 selective receptor antagonist, or PD98059, a MAPK inhibitor; (iii) reduced by inhibitors of calcium/calmodulin‐dependent kinase II (KN‐93), Ca2+‐dependent and ‐independent (GF102903X) or Ca2+‐dependent (Gö6976) protein kinase C isoforms. LTD4 also increased astrocyte proliferation and glial fibrillary acidic protein content, which are considered hallmarks of reactive astrogliosis. Both effects were counteracted by cell pretreatment with MK‐571 or PD98059. Thus, cys‐LTs released from astrocytes might play an autocrine role in the induction of reactive astrogliosis that, in brain injuries, contributes to the formation of a reparative glial scar.
Cytokine‐stimulated astrocytes produce nitric oxide, which can inhibit components of the mitochondrial respiratory chain. We have previously demonstrated that prolonged exposure (48 h) to rat ...astrocytic nitric oxide damages complexes II–III and IV of neighbouring rat neurons in coculture, resulting in neuronal death. Expanding on these observations, we have now shown that the NMDA receptor antagonist, MK‐801, prevents this damage, suggesting involvement of glutamate. We postulate that astrocyte‐derived nitric oxide stimulates release of neuronal glutamate. Indeed we demonstrate that neurons incubated with nitric oxide‐generating astrocytes display enhanced glutamate release. Furthermore, direct exposure to the nitric oxide donor, DETA‐NONOate resulted in a loss of activity of all the neuronal mitochondrial complexes, which was again prevented by MK‐801. Thus, nitric oxide, generated by both cytokine‐stimulated astrocytes and by a nitric oxide donor, causes activation of the NMDA receptor leading to damage to the neuronal mitochondrial respiratory chain. Glutamate exposure is known to damage the neuronal mitochondrial respiratory chain via neuronal nitric oxide synthase. Therefore, we propose that astrocyte‐derived nitric oxide is capable of eliciting neuronal glutamate release, which in turn activates the neuronal NMDA receptor and stimulates further formation of reactive nitrogen species via neuronal nitric oxide synthases, leading to mitochondrial damage and neuronal death. Our findings support the hypothesis that glutamate, reactive nitrogen species and mitochondrial dysfunction may have a role in the neurodegenerative process.
The brain is particularly vulnerable to oxygen free radicals, which have been implicated in the pathology of several neurological disorders. The antioxidant enzyme (AOE) system of the brain may play ...an important role in the protection against such oxidative stress. We investigated the influence of oxidative stress on the transcription of catalase and MnSOD mRNA. Primary rat astroglial cell cultures were treated either with hydrogen peroxide (H
2O
2), as a direct mediator of oxidative stress, or with the redox cycling compound paraquat. Both substances led to an increase of catalase and MnSOD mRNA levels. To further elucidate the mechanisms residing behind this increase, transfection experiments were performed. Transient transfection of primary astroglial cells with a reporter plasmid containing the upstream region of the catalase gene showed a decrease in reporter gene activity after exposure of transfected cells to either H
2O
2 or paraquat. In contrast, transfection experiments done with reporter plasmids for the MnSOD upstream region resulted in an increase of reporter gene activity after H
2O
2 as well as after paraquat treatment of transfected cells. These results indicate transcriptional regulation of MnSOD and post-transcriptional regulation of catalase gene expression after oxidative stress in primary rat astrocytes.
The release of ATP was studied in cultures of astrocytes derived from the brain hemispheres of newborn rats. There was a basal efflux of ATP, which was increased up to 19-fold by glutamate (300–1000
...μM),
N-methyl-
d-aspartate (20–500
μM),
α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA; 30–100
μM) and kainate (20
μM). The
N-methyl-
d-aspartate receptor-selective antagonist 2-amino-5-phosphonopentanoate (100
μM) blocked the effect of
N-methyl-
d-aspartate but not the effects of AMPA, kainate and glutamate. The AMPA receptor-selective antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline (30
μM) blocked the effect of AMPA and also of glutamate and
N-methyl-
d-aspartate, but not the effect of kainate. The kainate receptor-selective antagonist
d-glutamyl-amino-methanesulfonate (30
μM) blocked the effect of kainate but not of glutamate. Glutamate (1000
μM) did not increase the release of lactate dehydrogenase from astrocytes.
Excitatory amino acids are known to release adenyl compounds in the brain. The present results identify one adenyl compound thus released, namely ATP, and identify astrocytes as one source. The release is brought about by activation of any of the three ionotropic glutamate receptor types—
N-methyl-
d-aspartate, AMPA and kainate receptors. AMPA receptors seem to mediate at least a part of the effect of glutamate itself, but the involvement of other receptors cannot be ruled out. ATP and its degradation products, such as adenosine, once released, may exert acute as well as trophic effects on neurons and glial cells.
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