Murine Cerebral Endothelial Cells Research Articles

Identification of protein targets in cerebral endothelial cells for brain arteriovenous malformation (AVMs) molecular therapies

BackgroundTo develop a new molecular targeted treatment for brain (AVMs), identification of membrane proteins that are localised on the AVM endothelium is crucial. Current treatment methods are surgery and radiosurgery. However, complete occlusion post radiosurgery are achieved within 3 years, while patient remain at risk of haemorrhage. This study aims to identify potential protein targets in AVM endothelial cells that discriminate these vessels from normal vessels; these proteins targets will be investigated for the molecular therapy of brain AVMs to promote rapid thrombosis after radiosurgery.MethodsWe employed in vitro biotinylation that we developed, and mass spectrometry to detect cell surface-exposed proteins in cultures of murine cerebral endothelial cells (bEnd.3). Two forms of mass spectrometry were applied (iTRAQ-MS and MSE) to identify and quantify membrane protein expression at various time-points following irradiation which simulates a radiosurgical treatment approach. Immunocytochemistry was used to confirm the expression of selected membrane proteins. ProteinPilot V4.0 software was used to analyse the iTRAQ-MS data and the MSE data was analysed using ProteinLynx Global Server version 2.5 software.ResultsThe proteomics data revealed several differentially expressed membrane proteins between irradiated and non-irradiated cells at specific time points, e.g. PECAM-1, cadherin-5, PDI, EPCR and integrins. Immunocytochemistry data confirmed the expression of these proteins.ConclusionCell surface protein biotinylation and proteomics analysis successfully identified membrane proteins from murine brain endothelial cells in response to irradiation. This work suggests potential target protein molecules for evaluation in animal models of brain-AVM.

Differential effects of indoxyl sulfate and inorganic phosphate in a murine cerebral endothelial cell line (bEnd.3).

Endothelial dysfunction plays a key role in stroke in chronic kidney disease patients. To explore the underlying mechanisms, we evaluated the effects of two uremic toxins on cerebral endothelium function. bEnd.3 cells were exposed to indoxyl sulfate (IS) and inorganic phosphate (Pi). Nitric oxide (NO), reactive oxygen species (ROS) and O2•– were measured using specific fluorophores. Peroxynitrite and eNOS uncoupling were evaluated using ebselen, a peroxide scavenger, and tetrahydrobiopterin (BH4), respectively. Cell viability decreased after IS or Pi treatment (p < 0.01). Both toxins reduced NO production (IS, p < 0.05; Pi, p < 0.001) and induced ROS production (p < 0.001). IS and 2 mM Pi reduced O2•– production (p < 0.001). Antioxidant pretreatment reduced ROS levels in both IS- and Pi-treated cells, but a more marked reduction of O2•– production was observed in Pi-treated cells (p < 0.001). Ebselen reduced the ROS production induced by the two toxins (p < 0.001); suggesting a role of peroxynitrite in this process. BH4 addition significantly reduced O2•– and increased NO production in Pi-treated cells (p < 0.001), suggesting eNOS uncoupling, but had no effect in IS-treated cells. This study shows, for the first time, that IS and Pi induce cerebral endothelial dysfunction by decreasing NO levels due to enhanced oxidative stress. However, Pi appears to be more deleterious, as it also induces eNOS uncoupling.

Homocysteine induces cerebral endothelial cell death by activating the acid sphingomyelinase ceramide pathway

Homocysteine (Hcy) levels may rise after a stroke, but the mechanism of Hcy-induced cerebral endothelial cell (CEC) dysfunction has not been explored. In this study we examined the role of the acid sphingomyelinase (Asm)-ceramide pathway in the molecular mechanism of Hcy-induced CEC dysfunction. Murine CECs were prepared from fresh mouse brains. CECs were treated with 50-500 μM Hcy and 30-100 μM C2-ceramide for 48 h. Sphingomyelinase assays were performed to determine Asm activity. Quantitative assessments of cell survival and death by the MTT reduction and LDH release were conducted. Treatment of murine CECs with Hcy and ceramide caused cell death in a dose-dependent manner as determined by LDH and MTT assays. 250 μM Hcy and 50 μM C2-ceramide caused 50% cell death. Hcy induced murine CEC death also occurred in a time-dependant manner with substantial cell death noted as early as 24h after Hcy exposure. C2-ceramide-induced murine CEC death occurred earlier than Hcy-induced cell death by about 18h. Hcy treatment increased Asm activity and intracellular ceramide accumulation. This study demonstrated that Hcy and C2-ceramide can cause murine CEC death. Hcy induces CEC death possibly by activating the Asm-ceramide pathway.

In Vitro and In Vivo Biotinylation of Endothelial Cell Surface Proteins in the Pursuit of Targets for Vascular Therapies for Brain AVMs

Identification of membrane proteins that are expressed on the arteriovenous malformation (AVM) endothelium post radiosurgery is of fundamental importance in developing a new treatment of brain AVMs. We successfully optimized and then employed in vitro and in vivo biotinylation methodology of murine cerebral endothelial cell cultures (bEnd.3) and the rat model of AVM to label membrane proteins. Those membrane proteins were then captured on streptavidin resin and identified using proteomics analysis. This is the first time that proteomics has been employed in the study of AVMs.

The epigenetic effects of amyloid-β 1–40 on global DNA and neprilysin genes in murine cerebral endothelial cells

Amyloid-β (Aβ) is the core component of senile plaques, which are the pathological markers for Alzheimer’s disease and cerebral amyloid angiopathy. DNA methylation/demethylation plays a crucial role in gene regulation and could also be responsible for presentation of senescence. Oxidative stress, which may be induced by Aβ, is thought to be an important contributor of DNA hyper-methylation; however, contradicting this is the fact that global DNA hypo-methylation has been found in aging brains. It therefore remains largely unknown as to whether Aβ does in fact cause DNA methylation/demethylation. Neprilysin (NEP) is one of the enzymes responsible for Aβ degradation, with its expression decreasing in both Alzheimer and aging brains. Using high-performance liquid chromatography (HPLC), we explore whether Aβ is responsible for alteration of the global DNA methylation status on a murine cerebral endothelial cells model, and also use methylation-specific PCR (MSPCR) to examine whether DNA methylation status is altered on the NEP promoter region. We find that Aβ reduces global DNA methylation whilst increasing NEP DNA methylation and further suppressing the NEP expression in mRNA and protein levels. Our results support that Aβ induces epigenetic effects, implying that DNA methylation may be part of a vicious cycle involving the reduction in NEP expression along with a resultant increase in Aβ accumulation, and that Aβ may induce global DNA hypo-methylation.

Responses of Endothelial Cell and Astrocyte Matrix-Integrin Receptors to Ischemia Mimic Those Observed in the Neurovascular Unit

Apposition of endothelial cells and astrocyte foot processes to the basal lamina matrix is postulated to underlie the cerebral microvessel permeability barrier. Focal cerebral ischemia induces rapid loss of select matrix-binding integrins from both cell compartments in the nonhuman primate. This study is the first to examine the conditions underlying integrin loss from these cell-types during ischemia in vitro and their relation to the changes in vivo. The impact of normoxia or standardized oxygen-glucose deprivation on integrin expression by murine primary cerebral endothelial cells and astrocytes grown on matrix substrates (collagen IV, laminin, and perlecan) of the basal lamina were quantitatively assessed by flow cytometry. Endothelial cell expression of the beta1 and alpha 5 subunits significantly increased on all matrix ligands, whereas astrocytes displayed modest significant decreases in alpha 5 and alpha 6 subunits. Oxygen-glucose deprivation produced a further significant increase in subunit beta1 expression by both cell types, but a clear decrease in both alpha1 and alpha 6 subunits by murine astrocytes. Ischemia in vitro significantly increased endothelial cell beta1 expression, which is consistent with the increase in beta1 transcription by microvessels peripheral to the ischemic core. The loss of alpha1 and alpha 6 integrins from murine astrocytes is identical to that seen in the nonhuman primate in vivo. These findings establish both isolated murine cerebral endothelial cells and astrocytes as potential integrin response cognates of microvascular cells of the neurovascular unit in primates, and allow determination of the mechanisms of their changes to ischemia.

Apoptosis Signal-Regulating Kinase 1 in Amyloid β Peptide-Induced Cerebral Endothelial Cell Apoptosis

A pathological hallmark of Alzheimer's disease is accumulation of amyloid-beta peptide (Abeta) in senile plaques. Abeta has also been implicated in vascular degeneration in cerebral amyloid angiopathy because of its cytotoxic effects on non-neuronal cells, including cerebral endothelial cells (CECs). We explore the role of apoptosis signal-regulating kinase 1 (ASK1) in Abeta-induced death in primary cultures of murine CECs. Abeta induced ASK1 dephosphorylation, which could be prevented by selective inhibition of protein phosphatase 2A (PP2A) but not PP2B. ASK1 dephosphorylation resulted in its dissociation from 14-3-3. ASK1, released from 14-3-3 inhibition, activated p38 mitogen-activated protein kinase (p38MAPK), leading to p53 phosphorylation. p53, a proapoptotic transcription factor, in turn transactivated the expression of Bax, a proapoptotic protein. Transfection with various dominant-negative mutants (DNs), including ASK1 DN and p38MAPK DN, suppressed Abeta-induced p38MAPK activation, p53 phosphorylation, and Bax upregulation and partially prevented CEC death. Bax knockdown using a bax small interfering RNA strategy also reduced Bax expression and subsequent CEC death. These results suggest that Abeta activates the ASK1-p38MAPK-p53-Bax cascade to cause CEC death in a PP2A-dependent manner.

Promoter Region Methylation and Reduced Expression of Thrombospondin-1 after Oxygen—Glucose Deprivation in Murine Cerebral Endothelial Cells

Angiogenesis is induced in response to ischemia. Thrombospondin-1 (TSP-1) is a potent angiostatic factor. Silencing of TSP-1 expression may contribute to the postischemic angiogenesis. Upregulation of TSP-1, in contrast, may terminate the postischemic angiogenesis. A possible mechanism that silences TSP-1 expression is the DNA methylation of its promoter region. DNA methylation has been reported following cerebral ischemia. The present study aimed to explore whether methylation of the promoter region of TSP-1 regulates its expression after oxygen-glucose deprivation (OGD) in murine cerebral endothelial cells (CECs) in vitro. Sublethal OGD increased the extent of methylation of the promoter region of TSP-1 with a concurrent decrease in TSP-1 mRNA and protein expression in CECs. After reoxygenation, demethylation of the TSP-1 promoter region led to the restoration of TSP-1 mRNA and protein expression. The extent of methylation of the promoter region of TSP-1 was inversely correlated with the extent of TSP-1 gene expression at mRNA and protein levels after OGD. Oxygen-glucose deprivation-induced reduction in the TSP-1 mRNA level was not accompanied by a change in mRNA stability. These findings raise the possibility that OGD downregulation of TSP-1 expression is at least in part due to methylation of its promoter region.

Matrix metalloproteinase-9 in cerebral-amyloid-angiopathy-related hemorrhage

Spontaneous intracerebral hemorrhage (ICH) is one of the most recognized complications of cerebral amyloid angiopathy (CAA), but little is known about the molecular pathogenesis of this life-threatening complication. In this review, we present preliminary evidence which suggests that the extracellular-matrix-degrading protease, matrix metalloproteinase-9 (MMP-9), may play a role in the development of spontaneous ICH resulting from CAA. The amyloid-beta peptide (Aβ) induced the synthesis, cellular release, and activation of MMP-9 in murine cerebral endothelial cells (CECs), resulting in increased extracellular matrix (ECM) degradation. Furthermore, in a mouse model of CAA (APPsw transgenic mice), MMP-9 immunoreactivity was observed in amyloid-laden cerebral vessels in aged APPsw mice but not in young APPsw or aged wild-type mice. More extensive MMP-9 immunostaining was present in amyloid-laden vessels with evidence of microhemorrhage. These results suggest that increased vascular MMP-9 expression, stimulated by Aβ, may play a role in the pathogenesis of spontaneous intracerebral hemorrhage (ICH) in patients with CAA.

Neutral sphingomyelinase activation in endothelial and glial cell death induced by amyloid beta-peptide

We have explored the molecular mechanism underlying amyloid beta-peptide (Aβ)-mediated cytotoxicity in vitro. Exposure of murine cerebral endothelial cells (CECs) or C6 glioma cells to Aβ25-35 resulted in dose-dependent cell death. Ceramide is a pro-apoptotic lipid mediator. Forced elevation of cellular ceramide levels, either by application of an exogenous C2 ceramide analogue or bacterial sphingomyelinase that induces endogenous ceramide release from sphingomyelin, mimicked Aβ25–35 cytotoxicity in both CECs and C6 glioma cells. Aβ25–35-induced synthesis of ceramide was selectively mediated by activation of neutral sphingomyelinase (nSMase), but not acidic sphingomyelinase (aSMase) or ceramide synthase. Both 3-O-Me-SM and N-acetyl- l-cysteine, the selective and nonselective pharmacological inhibitors of nSMase, respectively, suppressed nSMase activation, ceramide production, and cytotoxic action induced by Aβ25–35 in CECs. Furthermore, genetic knockdown of nSMase by an antisense strategy rendered C6 glioma cells specifically resistant to Aβ25–35 cytotoxicity without affecting their vulnerability to serum deprivation. Together, nSMase activation with subsequent ceramide production may contribute, at least partially, to Aβ25–35 cytotoxicity in cell types with cerebral endothelial and glial lineage.

Matrix metalloproteinase-9 and spontaneous hemorrhage in an animal model of cerebral amyloid angiopathy.

We examined the potential role of the extra-cellular matrix-degrading enzyme, matrix metalloproteinase-9 (MMP-9), in the pathogenesis of cerebral amyloid angiopathy (CAA)-induced spontaneous hemorrhage. The amyloid-beta peptide (Abeta) induced the synthesis, release and activation of MMP-9 in murine cerebral endothelial cells, resulting in increased extracellular matrix degradation. Furthermore, extensive MMP-9 immunoreactivity was observed in CAA-vessels with evidence of microhemorrhage in aged APPsw transgenic mice, but not detected in aged wild type or young APPsw mice. These results suggest that increased vascular MMP-9 expression, stimulated by Abeta, may play a role in the pathogenesis of spontaneous intracerebral hemorrhage in patients with CAA.

Amyloid-β Induces Smac Release via AP-1/Bim Activation in Cerebral Endothelial Cells

Insoluble fibrils of amyloid-beta peptide (Abeta) are the major component of senile and vascular plaques found in the brains of Alzheimer's disease (AD) patients. Abeta has been implicated in neuronal and vascular degeneration because of its toxicity to neurons and endothelial cells in vitro; some of these cells die with characteristic features of apoptosis. We used primary cultures of murine cerebral endothelial cells (CECs) to explore the mechanisms involved in Abeta-induced cell death. We report here that Abeta(25-35), a cytotoxic fragment of Abeta, induced translocation of the apoptosis regulator termed second-mitochondria-derived activator of caspase (Smac) from the intramembranous compartment of the mitochondria to the cytosol 24 hr after exposure. In addition, we demonstrated that X chromosome-linked inhibitor-of-apoptosis protein (XIAP) coimmunoprecipitated with Smac, suggesting that the two proteins bound to one another subsequent to the release of Smac from the mitochondria. Abeta(25-35) treatment also led to rapid AP-1 activation and subsequent expression of Bim, a member of the BH3-only family of proapoptotic proteins. Bim knockdown using an antisense oligonucleotide strategy suppressed Abeta(25-35)-induced Smac release and resulted in attenuation of CEC death. Furthermore, AP-1 inhibition, with curcumin or c-fos antisense oligonucleotide, reduced bim expression. These results suggest that Abeta activates an apoptotic cascade involving AP-1 DNA binding, subsequent bim induction, followed by Smac release and binding to XIAP, resulting in CEC death.

Amyloid beta peptide-induced cerebral endothelial cell death involves mitochondrial dysfunction and caspase activation.

Amyloid beta peptide (A beta), a 39 to 43 amino acid fragment of the beta-amyloid precursor protein (betaAPP), forms insoluble fibrillar accumulation in neurofibrillary tangles and vascular plaques. A beta has been implicated in neuronal and vascular degeneration in brain regions susceptible to plaque formation because of its cytotoxic effect on neurons and endothelial cells (ECs). The authors used a murine cerebral endothelial cell (CEC) line and primary cultures of bovine CECs to explore the cytotoxic mechanism of A beta. A beta 1-40 and A beta 25-35 peptides caused cell death in a dose-dependent and time-dependent manner. Exposure to either A beta 25-35 or A beta 1-40 at 10 micromol/L for 48 hours caused at least 40% cell death. Cerebral endothelial cell death was characterized by nuclear condensation, mitochondrial dysfunction, and nuclear and mitochondrial DNA damage. A beta 25-35 activated both caspase-8 and caspase-3 in murine CECs. zVAD-fmk, a broad-spectrum caspase inhibitor, prevented A beta 25-35-induced increase in caspase-3 activity and CEC death. N-acetyl-cysteine, an antioxidant, also prevented A beta-induced cell death. Together, these findings indicate that A beta-mediated CEC death is an apoptotic process that is characterized by increased oxidative stress, caspase activation, mitochondrial dysfunction, and nuclear and mitochondrial DNA damage.

Regulation of Cytokine-Induced iNOS Expression by a Hairpin Oligonucleotide in Murine Cerebral Endothelial Cells

Inducible nitric oxide synthase (iNOS) is expressed in response to cytokines by a number of cell types participating in CNS inflammation, including brain cerebral endothelial cells. NF-κB, a transcription factor, mediates effector actions of pro-inflammatory cytokines. A combination of tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) enhanced the expression of iNOS in murine cerebral endothelial cells (MCECs). In an attempt to modulate TNF-α+IFN-γ induced expression of iNOS in MCECs, we designed a double-strand hairpin (hp) oligonucleotide carrying the NF-κB motif. This hp oligonucleotide inhibited NF-κB binding activity and decreased both iNOS mRNA and protein expression induced by TNF-α+IFN-γ. As a control, a mutant hp oligonucleotide was without effect. The present study confirms the role of transcription factor NF-κB in iNOS expression induced by TNF-α+IFN-γ in MCECs. More importantly, it demonstrates that an appropriately designed hp oligonucleotide is an effective tool to modulate iNOS expression and may be of potential pharmacological use.

Regulation of the expression of intercellular adhesion molecule-1 (ICAM-1) and the putative adhesion molecule Basigin on murine cerebral endothelial cells by MHV-4 (JHM).

Cerebral vascular endothelial cells are integral components of the blood-brain barrier and are believed to be an important site which can restrict infection of the central nervous system by viruses and other pathogens. A potential role of cerebral vascular endothelial cells in resisting CNS infection relates to the expression of lymphocyte-endothelial adhesion molecules which could impact on the generation of anti-viral immune reactivity.KeywordsEndothelial CellNorthern AnalysisBrain Endothelial CellImmunoglobulin SuperfamilyCerebral Endothelial CellThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Receptor-linked hydrolysis of phosphoinositides and production of prostacyclin in cerebral endothelial cells.

The receptor agonist-mediated hydrolysis of phosphoinositides and production of prostacyclin were studied in murine cerebral endothelial cells (MCEC). Of 11 neurotransmitters and neuromodulators examined, carbachol, noradrenaline (NE), bradykinin, and thrombin significantly increased 3H-inositol phosphate accumulation in the presence of LiCl (20 mM). The maximal stimulation of [3H]inositol monophosphate ([3H]IP1) reached approximately 11, 11, seven, and four times the basal levels for carbachol, NE, bradykinin, and thrombin, respectively. The EC50 values of IP1 accumulation for carbachol and NE were 34 and 0.16 microM, respectively. The muscarinic antagonists, atropine and pirenzepine, blocked the carbachol-induced IP1 accumulation with Ki values of 0.3 and 30 nM, respectively. The adrenergic antagonist, prazosin, blocked NE-induced IP1 accumulation with a Ki of 0.1 nM. The calcium ionophore A23187, histamine, glutamate, vasopressin, serotonin, platelet activating factor, and substance P did not stimulate IP1 accumulation. A23187, bradykinin, and thrombin stimulated prostacyclin release to approximately four, four, and two times the basal levels, respectively, whereas carbachol and NE had little effect upon prostacyclin release. These results suggest that the activation of phospholipase C and of phospholipase A2 in MCEC are regulated separately.

Mouse hepatitis virus (MHV-4, JHM) blocks γ-interferon-induced major histocompatibility complex class II antigen expression on murine cerebral endothelial cells

The regulation of γ-interferon-induced major histocompatibility complex (MHC) class II antigen expression on mouse cerebral endothelial cells by the neurotropic mouse hepatitis virus (MHV-4, JHM) was studied in vitro. The results presented demonstrate that MHV-4 can selectively block γ-interferon-induced class II antigen expression on cerebral endothelial cells. The blocking effect of class II expression occurs in a strain-dependent manner, and is limited to virus-susceptible mouse strains. Virus replication is not required to obtain the blocking effect since UV-inactivated MHV-4 produces the same result. MHV-4 blocking of γ-interferon-induced class II antigen expression is observed at both the cell surface (flow cytometry) and transcriptional level (Norhtern analysis).

Mouse hepatitis virus (MHV-4, JHM) blocks γ-interferon-induced MHC class II antigen expression on murine cerebral endothelial cells

Mouse hepatitis virus (MHV-4, JHM) blocks γ-interferon-induced MHC class II antigen expression on murine cerebral endothelial cells

Mouse hepatitis virus (MHV-4, JHM) blocks γ-interferon-induced MHC class II antigen expression on murine cerebral endothelial cells

Mouse hepatitis virus (MHV-4, JHM) blocks γ-interferon-induced MHC class II antigen expression on murine cerebral endothelial cells