Brain Endothelial Cell Apoptosis Research Articles

Serum exosomal miRNA promote glioma progression by targeting SOS1 via abscopal effect of radiation

IntroductionLocal exposure to ionizing radiation (IR) can induce changes in biological processes in distant tissues and organs. Exosomes are nanoscale vesicles that transport biomolecules, mediate communication between cells and tissues, and can affect the abscopal effects of radiotherapy. MethodsMice were treated with 8.0 Gy doses of chest and abdomen IR, after which serum samples were taken 24 h after exposure. Their serum exosomes were then isolated via ultracentrifugation and the small RNA portions were extracted for sequencing and bioinformatic analysis. Exosomes were injected intravenously into the mice to assess their ability to cross the blood-brain barrier (BBB). Glioma cells and glioma stem cells (GSCs) were examined for malignant biological behaviors, stemness, and tumorigenic capacity after co-culturing with different groups of exosomes. ResultsWe found that serum exosomes crossed the BBB in mice after local IR exposure-which induced decreases in the expression of BBB tight-junction proteins and increased brain endothelial cell apoptosis. Exosomes from the exposed groups promoted malignant biological behaviors, stemness, and tumorigenic capacity in glioma cells and GSCs by upregulating the expression of SOS1. Phospho-MEK1/2 and Phospho-ERK1/2, of the MAPK signaling pathway, were found to be up-regulated in cells that were co-cultured with the exposing groups of the exosomes. Further analyses demonstrated that differentially expressed levels of miR-93–5p in mouse serum exosomes regulated the cellular expression of SOS1. ConclusionFollowing local IR exposure, serum exosomes cross the BBB to promote the progression of distant gliomas. Exosomal microRNAs play an important role in this process.

Antigen recognition detains CD8+ T cells at the blood-brain barrier and contributes to its breakdown

Blood-brain barrier (BBB) breakdown and immune cell infiltration into the central nervous system (CNS) are early hallmarks of multiple sclerosis (MS). High numbers of CD8+ T cells are found in MS lesions, and antigen (Ag) presentation at the BBB has been proposed to promote CD8+ T cell entry into the CNS. Here, we show that brain endothelial cells process and cross-present Ag, leading to effector CD8+ T cell differentiation. Under physiological flow in vitro, endothelial Ag presentation prevented CD8+ T cell crawling and diapedesis resulting in brain endothelial cell apoptosis and BBB breakdown. Brain endothelial Ag presentation in vivo was limited due to Ag uptake by CNS-resident macrophages but still reduced motility of Ag-specific CD8+ T cells within CNS microvessels. MHC class I-restricted Ag presentation at the BBB during neuroinflammation thus prohibits CD8+ T cell entry into the CNS and triggers CD8+ T cell-mediated focal BBB breakdown.

Abstract 111: Cerebrovascular Effects Of Pre/post-losartan Treatment In Humanized ACE2 Knock-in Mice After SARS-CoV-2 Spike Protein Injection

SARS-CoV-2 has killed millions of people globally and has left many with devastating health problems, including neurological deficits. SARS-CoV-2 invades endothelial cells via binding its Spike-protein with angiotensin-converting enzyme 2 (ACE2) receptors. Losartan, an angiotensin receptor blocker, upregulates ACE2 expression. We hypothesize that pre-treatment with losartan could increase the cerebrovascular dysfunction associated with SARS-CoV-2 spike-protein, while post-SARS-CoV-2 losartan would provide potential protective effects. Methods: Recombinant SARS-CoV-2 spike-protein was injected intravenously in transgenic hACE2 mice, humanized ACE2 receptors knock-in mice. Treatment with losartan was started either two weeks before or right after spike-protein injection. Cognitive function was assessed using Y-Maze. Vascular density and cerebral blood flow were evaluated in the hACE2 mice with/out spike protein injection. Western blot and RT-PCR analysis were used to measure protein/gene expression. Results: Spike-protein injection decreased cerebral vascular density and cerebral blood flow in hACE2 brains compared to control (*P<0.05), which was associated with increased cognitive dysfunction in h ACE2 mice (*P<0.05). Pre-losartan treatment exacerbated spike protein-induced cognitive dysfunction, while post-losartan treatment helped restore cognitive function (*P<0.05). Spike-protein decreased ACE2 expression and increased endothelial cell inflammation and apoptosis, as shown by the increased expression of TNF- α ; and caspase-3 (*P<0.05), which were prevented by post-losartan treatment. Conclusion: Spike-protein decreased vascular density, cerebral blood flow, and cognitive function via increasing brain endothelial cell apoptosis. Pre-losartan treatment exacerbated the detrimental effects of spike-protein, while post-losartan treatment restored ACE2 expression and decreased SARS-CoV-2 spike-protein damaging effects.

Role of NADPH Oxidases in Blood-Brain Barrier Disruption and Ischemic Stroke.

NADPH oxidases (Nox) are one of the main sources of reactive oxygen species (ROS) in the central nervous system (CNS). While these enzymes have been shown to be involved in physiological regulation of cerebral vascular tone, excessive ROS produced by Nox1-5 play a critical role in blood–brain barrier (BBB) dysfunction in numerous neuropathologies. Nox-derived ROS have been implicated in mediating matrix metalloprotease (MMP) activation, downregulation of junctional complexes between adjacent brain endothelial cells and brain endothelial cell apoptosis, leading to brain microvascular endothelial barrier dysfunction and consequently, increases in BBB permeability. In this review, we will highlight recent findings on the role played by these enzymes in BBB disruption induced by ischemic stroke.

Dysregulation of vascular endothelial growth factor receptor 2 phosphorylation is associated with disruption of the blood-brain barrier and brain endothelial cell apoptosis induced by plasma from women with preeclampsia

Disruption of the blood-brain barrier (BBB) is central in the pathophysiology of acute cerebral complications in women who have preeclampsia. Underling mechanisms are unclear.Using female human brain endothelial cells as an in vitro model of BBB, we show that plasma of women with preeclampsia increases cell apoptosis and permeability via activation of the vascular endothelial growth factor receptor 2 (VEGFR2).Since plasma of women with preeclampsia also enhanced VEGFR2 phosphorylation in the tyrosine 951 but decreased phosphorylation at the tyrosine 1175, we propose the former would be the more likely active form of VEGFR2 responsible for BBB alterations.

Metabolomic Profiling of Brain Protective Effect of Edaravone on Cerebral Ischemia-Reperfusion Injury in Mice.

Edaravone (EDA) injection has been extensively applied in clinics for treating stroke. Nevertheless, the metabolite signatures and underlying mechanisms associated with EDA remain unclear, which deserve further elucidation for improving the accurate usage of EDA. Ischemia stroke was simulated by intraluminal occlusion of the right middle cerebral artery for 1 h, followed by reperfusion for 24 h in mice. Brain infarct size, neurological deficits, and lactate dehydrogenase (LDH) levels were improved by EDA. Significantly differential metabolites were screened with untargeted metabolomics by cross-comparisons with pre- and posttreatment of EDA under cerebral ischemia/reperfusion (I/R) injury. The possibly involved pathways, such as valine, leucine, and isoleucine biosynthesis, and phenylalanine, taurine, and hypotaurine metabolisms, were enriched with differential metabolites and relevant regulatory enzymes, respectively. The network of differential metabolites was constructed for the integral exhibition of metabolic characteristics. Targeted analysis of taurine, an important metabolic marker, was performed for further validation. The level of taurine decreased in the MCAO/R group and increased in the EDA group. The inhibition of EDA on cerebral endothelial cell apoptosis was confirmed by TdT-mediated dUTP nick-end labeling (TUNEL) stain. Cysteine sulfinic acid decarboxylase (CSAD), the rate-limiting enzyme of taurine generation, significantly increased along with inhibiting endothelial cell apoptosis after treatment of EDA. Thus, CSAD, as the possible new therapeutic target of EDA, was selected and validated by Western blot and immunofluorescence. Together, this study provided the metabolite signatures and identified CSAD as an unrecognized therapeutic intervention for EDA in the treatment of ischemic stroke via inhibiting brain endothelial cell apoptosis.

Omi/HtrA2 Protease Associated Cell Apoptosis Participates in Blood-Brain Barrier Dysfunction.

Background: Omi/HtrA2 is a proapoptotic mitochondrial serine protease involved in caspase-dependent cell apoptosis, translocating from mitochondria to the cytosol after an apoptotic insult. Our previous study indicated pre-treatment with UCF-101, a specific inhibitor of Omi/HtrA2, could significantly reduce neuronal apoptosis and attenuate sepsis-induced cognitive dysfunction. Various hypotheses involving blood-brain-barrier (BBB) disruption have been proposed to account for sepsis-associated encephalopathy (SAE). Here, we attempted to explore whether interference of Omi/HtrA2 by RNA interference or UCF-101 pre-treatment can improve sepsis-induced disruption of BBB using human cerebral microvascular endothelial cell line (hCMEC/D3) in vitro and if so, to explore mechanisms involved Omi/HtrA2 protease mediates BBB disruption in SAE.Methods: hCMEC/D3 cell monolayers were intervened by different concentrations of LPS (0–50 μg/mL) over experimental period. Pharmacological or gene interventions (by silencing RNA of Omi/HtrA2) were used to study molecular mechanisms involved in sepsis-associated Omi/HtrA2 translocation, cell apoptosis and BBB dysfunction. BBB function was assessed by trans-endothelial electrical resistance (TEER) and permeability to labeled dextrans (FITC-4kDa). Tight junction (TJ) integrity was assessed by immunofluorescence, western blotting and transmission electron microscopic (TEM) analyses. Apoptosis was determined using flow cytometry and TUNEL assay. Mitochondrial membrane potential (MMP) and oxidative stress were also investigated.Results: LPS affects hCMEC/D3 TJ permeability in a concentration- and time-dependent manner. LPS intervention resulted in a significant disruption of BBB, as manifested by decreased TEER (by ~26%) and a parallel increased paracellular permeability to FITC- (4kDa) dextrans through hCMEC/D3 monolayers. The inhibition of Omi/HtrA2 by UCF-101 or Omi/HtrA2 shRNA reduced LPS-induced brain endothelial cell apoptosis, and resulted in significant improvement on LPS-induced BBB disruption as well as decreased occludin, claudin-5 and ZO-1 expressions. Omi/HtrA2 manipulated endothelial cell apoptosis by shifting into cytosol and inducing X-linked inhibitor of apoptosis protein (XIAP) degradation. UCF-101 administration or Omi/HtrA2 shRNA intervention did attenuate the degradation of XIAP, Poly ADP-ribose polymerase (PARP) cleavage, and caspase-3 cleavage. However, only UCF-101 partly prevented the mobilization of Omi/HtrA2 from the mitochondria to the cytosol after LPS intervention. That abrogation of Omi/HtrA2 by UCF-101 or Omi/HtrA2 shRNA resulted in a significant improvement on LPS-induced decrease of MMP. Oxidative stress was significantly increased in the LPS treated group compared to the control or NC-shRNA group. However, abrogation of Omi/HtrA2 by UCF-101 or Omi/HtrA2 shRNA did not significantly improve oxidative injury.Conclusions: Our study indicated an important role of Omi/HtrA2 in manipulating LPS-induced cell apoptosis and BBB integrity by translocating from mitochondria into cytosol in brain endothelial cells. Omi/HtrA2 induced mitochondrial pathway apoptosis, which involves inhibition of an important antiapoptotic protein XIAP and influence on MMP. Therapeutic methods that inhibit Omi/HtrA2 function may provide a novel therapeutic measure to septic encephalopathy.

C5a induces caspase-dependent apoptosis in brain vascular endothelial cells in experimental lupus.

Blood-brain barrier (BBB) dysfunction complicates central nervous system lupus, an important aspect of systemic lupus erythematosus. To gain insight into the underlying mechanism, vascular corrosion casts of brain were generated from the lupus mouse model, MRL/lpr mice and the MRL/MpJ congenic controls. Scanning electron microscopy of the casts showed loss of vascular endothelial cells in lupus mice compared with controls. Immunostaining revealed a significant increase in caspase 3 expression in the brain vascular endothelial cells, which suggests that apoptosis could be an important mechanism causing cell loss, and thereby loss of BBB integrity. Complement activation occurs in lupus resulting in increased generation of circulating C5a, which caused the endothelial layer to become 'leaky'. In this study, we show that C5a and lupus serum induced apoptosis in cultured human brain microvascular endothelial cells (HBMVECs), whereas selective C5a receptor 1 (C5aR1) antagonist reduced apoptosis in these cells, demonstrating C5a/C5aR1-dependence. Gene expression of initiator caspases, caspase 1 and caspase 8, and pro-apoptotic proteins death-associated protein kinase 1, Fas-associated protein (FADD), cell death-inducing DNA fragmentation factor 45 000 MW subunit A-like effector B (CIDEB) and BCL2-associated X protein were increased in HBMVECs treated with lupus serum or C5a, indicating that both the intrinsic and extrinsic apoptotic pathways could be critical mediators of brain endothelial cell apoptosis in this setting. Overall, our findings suggest that C5a/C5aR1 signalling induces apoptosis through activation of FADD, caspase 8/3 and CIDEB in brain endothelial cells in lupus. Further elucidation of the underlying apoptotic mechanisms mediating the reduced endothelial cell number is important in establishing the potential therapeutic effectiveness of C5aR1 inhibition that could prevent and/or reduce BBB alterations and preserve the physiological function of BBB in central nervous system lupus.

EF1A1/HSC70 Cooperatively Suppress Brain Endothelial Cell Apoptosis via Regulating JNK Activity.

In our previous study, eEF1A1 was identified to be a new target for protecting brain ischemia injury, but the mechanism remains largely unknown. In this study, we screened the downstream cellular protein molecules interacted with eEF1A1 and found mechanism of eEF1A1 in brain ischemia protection. Through co-immunoprecipitation and mass spectrometry for searching the interaction of proteins with eEF1A1 in bEnd3 cells, HSC70 was identified to be a binding protein of eEF1A1, which was further validated by Western blot and immunofluorescence. eEF1A1 or HSC70 knockdown, respectively, increased OGD-induced apoptosis of brain vascular endothelial cells, which was detected by Annexin V-FITC/PI staining. HSC70 or eEF1A1 knockdown enhances phosphorylated JNK, phosphorylation of c-JUN (Ser63, Ser73), cleaved caspase-9, and cleaved caspase-3 expression, which could be rescued by JNK inhibitor. In summary, our data suggest that the presence of chaperone forms of interaction between eEF1A1 and HSC70 in brain vascular endothelial cells, eEF1A1 and HSC70 can play a protective role in the process of ischemic stroke by inhibiting the JNK signaling pathway activation.

AI-03 * TARGETING ANGIOGENESIS WITHOUT INCREASING THE STROMAL CELL RESPONSE OR INVASION USING ABT-898, A THROMBOSPONDIN TYPE 1 REPEAT PEPTIDE

Anti-angiogenic therapy is a promising therapeutic approach for highly vascularized tumors including glioblastoma (GBM). Nonetheless, the efficacy of Bevacizumab - a monoclonal antibody to VEGF-A - is limited only to progression-free survival but not overall survival, due at least in part to the induction of a more invasive phenotype. Here, we investigated the anti-tumor effect of a thrombospondin-1&2 mimetic peptide, ABT-898, on intracerebral xenografts derived from primary patient GBM neurospheres in the nude mouse. ABT-898 treatment of xenograft tumors (80 mg/kg/day i.p.) prolonged mouse survival. Unlike Bevacizumab-treated mouse brain tumors, ABT-898-treated tumors did not exhibit a detectable increase in the number of invasive foci or phospho-Met expression, indicating no induction of an invasive phenotype. As stromal cells are thought to promote tumor cell invasion, we quantitated infiltration of Iba1+ activated microglial/macrophages into the tumor. Although Bevacizumab therapy significantly increased the number of recruited Iba1+ cells into the tumors, the number of Iba1+ cells in the ABT-898-treated tumors was not increased as compared to the vehicle-treated xenograft tumors. Supporting this finding, the migration of microglia cells (BV2), was significantly inhibited by ABT898 in a dose-dependent manner, and the inhibition was reversed by CD36-neutralizing antibody. This suggests that recruitment of tumor invasion-promoting microglia/macrophage can be regulated by ABT898 and this regulation is in part CD36-dependent. In addition, treatment with ABT898 significantly reduced vessel density in xenograft tumors. In vitro ABT898 induced apoptosis of brain endothelial cells (ECs) and inhibited tubulomorphogenesis in collagen gels, both in a dose-dependent manner, and both were reversed by treatment with CD36 neutralizing antibody, Collectively, these data suggest that ABT-898 offers the novel advantage over Bevacizumab of decreasing stromal cell recruitment to the tumor and thus should be considered as an alternative anti-angiogenic therapy for GBM.

Abstract 1024: T11TS impedes glioma angiogenesis by attenuating brain endothelial Angiopoietin-1/Tie2 signaling and inducing apoptosis of glioma associated brain endothelial cells

Abstract Malignant gliomas are highly invasive primary brain tumors resistant to conventional treatments primarily because of sustained angiogenesis and infiltrative nature. Despite multimodal therapeutic interventions high grade gliomas have a propensity for recurrence. We recently reported that T11-target structure (T11TS), a membrane glycopeptide isolated from sheep erythrocyte membrane inhibits VEGF signaling and prosurvival PI3K/Akt pathway in glioma associated brain endothelial cells. Angiopoietin signaling mediated through endothelial Tie2 receptors is a potent mediator of glioma angiogenesis. Apoptosis of tumor endothelial cells acts as a crucial regulator of tumor angiogenesis and some angiogenesis inhibitors mediate their anti-angiogenic effect through induction of endothelial cell apoptosis. The effect of T11TS on brain endothelial cell Angiopoietin-1/Tie2 signaling and endothelial cell apoptosis has not been investigated so far. The goal of this study is to determine the effects of T11TS administration on endothelial Angiopoietin-1/Tie 2 signaling and on apoptosis of brain endothelial cells in a chemically induced glioma model. Furthermore, this study investigated the mechanistic details of brain endothelial cell apoptosis induction caused by in vivo T11TS therapy. Expression of angiopoietin-1, Tie2, phospho-Tie2 proteins, apoptotic pathway regulator proteins and caspases were determined by flowcytometry, immunoblotting, immunofluorescence imaging and in situ immunofluorescent staining. Apoptosis in brain endothelial cells was directly evaluated by flowcytomeric Annexin V-PI assay and mitochondrial membrane JC-1 assay. T11TS administration in glioma induced rats significantly downregulate Angiopoietin-1 expression and Tie2 receptor activation in brain endothelial cells thereby blocking Angiopoietin-1/Tie2 signaling. T11TS therapy triggered dose-dependent externalization of membrane phosphatidylserine, reduction in mitochondrial membrane potential and cleavage of caspase 3 which strongly indicated induction of apoptosis in glioma associated brain endothelial cells. T11TS administration in glioma was found to activate both intrinsic and extrinsic pathways of apoptosis by modulating expression of apoptotic regulator proteins like p53, Bax, Bcl-2, Fas and FasL, triggering cytochorome c release into cytosol and activating caspases 8, 3 and 9 in brain endothelial cells. T11TS administration also stimulated cleavage of Bid in glioma associated brain endothelial cells. This preclinical study demonstrated that blockage of Angiopoietin-1/Tie2 signaling in glioma associated brain endothelial cells and induction of apoptosis in brain endothelial cells constitute the underlying mechanistic events that contribute to the anti-angiogenic efficacy of T11TS in malignant glioma. Citation Format: Debanjan Bhattacharya, Suhnrita Chaudhuri, Swapna Chaudhuri. T11TS impedes glioma angiogenesis by attenuating brain endothelial Angiopoietin-1/Tie2 signaling and inducing apoptosis of glioma associated brain endothelial cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1024. doi:10.1158/1538-7445.AM2014-1024

Abstract C5: T11TS arrests angiogenic signaling in malignant glioma by attenuating brain endothelial cell angiopoietin-1/Tie2 expression and inducing apoptosis in glioma associated brain endothelial cells.

Abstract Malignant gliomas are highly aggressive primary brain tumors resistant to conventional treatments mainly because of sustained uncontrolled angiogenesis. Limited efficacies of currently available treatments necessitate development of multi-level targeted anti-angiogenic therapy for treatment of malignant glioma. Previously we reported that sheep erythrocyte membrane glycopeptide T11 target structure (T11TS) therapy in a rodent glioma model inhibits brain endothelial cell VEGF signaling and downstream PI3K/Akt/eNOS pathway. The angiopoietin-1/Tie2 signaling operative in brain endothelial cells is implicated in glioma angiogenesis but the role of T11TS therapy on pro-angiogenic Ang-1/Tie2 axis has not been determined yet. Induction of endothelial cell apoptosis is a common mechanism of action of many anti-angiogenic agents that restrict tumor progression. The purpose of the present study is to investigate the effect of T11TS therapy in regulation of angiopoietin-1/Tie2 signaling in glioma associated brain endothelial cells and to identify whether T11TS therapy triggers apoptosis in glioma associated brain endothelial cells. Additionally, the study evaluated the apoptotic pathways and proteins involved in T11TS induced apoptosis of brain endothelial cells. Primary brain endothelial cells isolated from the cerebral cortices of glioma induced rats before and after T11TS therapy were used for experiments. Expression of angiopoietin-1, Tie2, phospho-Tie2, and apoptotic pathway regulator proteins and caspases in brain endothelial cells were determined by flowcytometry, immunoblotting, immunofluorescent imaging, and immunohistochemical staining of rat cerebral sections. Flow cytometric Annexin V-PI assay and JC-1 assay were employed for detection of apoptosis in glioma associated brain endothelial cells. Our findings revealed that T11TS therapy significantly downregulates angiopoietin-1 and Tie2 receptor expression and activation in glioma associated brain endothelial cells. T11TS therapy induced apoptotic death of glioma associated brain endothelial cells as evidenced from membrane phosphatidylserine translocation and reduction of mitochondrial membrane potential. T11TS therapy stimulated both mitochondrial and extrinsic pathways of apoptosis by activating caspases 9, 3, and 8, triggering cleavage of Bid and modulating expression of apoptotic regulator proteins like p53, Bax, Bcl-2, Fas, and FasL in brain endothelial cells. Blockage of Ang-1/Tie2 signaling in brain endothelial cells and induction of intrinsic and extrinsic pathway mediated apoptosis in glioma associated brain endothelial cells are the underlying mechanistic events contributing to the anti-angiogenic action of T11TS therapy in malignant glioma. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C5. Citation Format: Debanjan Bhattacharya, Swapna Chaudhuri. T11TS arrests angiogenic signaling in malignant glioma by attenuating brain endothelial cell angiopoietin-1/Tie2 expression and inducing apoptosis in glioma associated brain endothelial cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C5.

IL-10 Prevents Apoptosis of Brain Endothelium during Bacteremia

IL-10-deficient mice infected with the relapsing fever bacterium Borrelia turicatae rapidly succumb to a brain hemorrhage if they are unable to clear peak bacteremia. In this study, we investigated the protective role of IL-10 during relapsing-remitting bacteremia and explored the molecular events involved in the protection of brain endothelium by IL-10. Brain endothelial injury was measured with cytotoxicity and diverse apoptotic assays, whereas the signaling pathway analysis was done by quantitative PCR array. The results showed that severe endothelial cell injury leading to hemorrhage in the brain and other organs occurred in IL-10-deficient mice during relapsing-remitting infection. Human brain microvascular endothelial cells (HBMEC) produced abundant proinflammatory mediators upon exposure to whole bacteria or purified bacterial lipoprotein but did not produce any detectable IL-10. Whole bacteria and purified outer membrane lipoprotein rapidly killed HBMEC by apoptosis in a time- and concentration-dependent manner. Exogenous IL-10 protected HBMEC from apoptosis. HBMEC apoptosis during exposure to a low number of bacteria was associated with downregulation of TNF and TNFAIP3 and upregulation of BAX. In contrast, HBMEC apoptosis during exposure to high concentrations of purified outer membrane lipoprotein was associated with marked upregulation of FAS, FAS ligand, and the adaptor molecules RIPK1 and CFLAR. Exogenous IL-10 reversed all the apoptotic signaling changes induced by whole bacteria or its purified lipoprotein. The results indicate that prominent brain endothelial cell apoptosis occurs during relapsing-remitting bacteremia in the absence of IL-10 and point to a prominent role for bacterial lipoprotein-mediated activation of FAS and caspase-3 in this process.

Brain Endothelial Cell Death: Modes, Signaling Pathways, and Relevance to Neural Development, Homeostasis, and Disease

Emerging evidence indicates that brain microvascular endothelial cells play a critical role in brain development, maturation, and homeostasis. Acute or chronic insults, including oxidative stress, oxygen-glucose deprivation, trauma, infections, inflammatory cytokines, DNA damaging agents, beta-amyloid deposition, and endoplasmic reticulum stress induce brain endothelial cell dysfunction and damage, which can result in cell death. The homeostatic balance between endothelial cell survival and endothelial cell death is critical for brain development, remodeling, and repair. On the other hand, dysregulation of brain endothelial cell death exacerbates, or even initiates, several inflammatory, ischemic, and degenerative disorders of the central nervous system. In here, the morphological, biochemical, and functional characteristics of the brain endothelium and its contribution to brain homeostasis will be reviewed. Recent insights into modalities and regulatory pathways involved in brain endothelial cell death will be described. The effects of regulated and dysregulated endothelial cell death leading to angiogenesis will be outlined. The relevance of brain endothelial cell dysfunction and death to disease processes will be discussed with special reference to recent findings that could help translate current knowledge on brain endothelial cell apoptosis into new therapeutic strategies for the treatment of certain neurological disorders.

Preservation of Cellular Glutathione Status and Mitochondrial Membrane Potential by N-Acetylcysteine and Insulin Sensitizers Prevent Carbonyl Stress-Induced Human Brain Endothelial Cell Apoptosis

Oxidative stress-induced cerebral endothelial cell dysfunction is associated with cerebral microvascular complication of primary diabetic encephaolopathy, a neurodegenerative disorder of long-standing diabetes, but the injury mechanisms are poorly understood. This study sought to determine the contribution of carbonyl (methylglyoxal, MG) stress to human brain endothelial cell (IHEC) apoptosis, the relationship to cellular redox status and mitochondrial membrane potential, and the protection by thiol antioxidant and insulin sensitizers. MG exposure induced IHEC apoptosis in association with perturbed cellular glutathione (GSH) redox status, decreased mitochondrial membrane potential (Deltapsi(m)), activation of caspase-9 and -3, and cleavage of polyADP-ribose polymerase. Insulin sensitizers such as biguanides or AMP-activated protein kinase activator, but not glitazones, afforded cytoprotection through preventing (Deltapsi(m) collapse and activation of caspase-9 that was independent of cellular GSH. Similarly, cyclosporine A prevented Deltapsi(m) collapse, while N-acetylcysteine (NAC) mediated the recovery of cellular GSH redox balance that secondarily preserved Deltapsi(m). Collectively, these results provide mechanistic insights into the role of GSH redox status and mitochondrial potential in carbonyl stress-induced apoptosis of brain endothelial cells, with implications for cerebral microvascular complications associated with primary diabetic encephalopathy. The findings that thiol antioxidant and insulin sensitizers afforded cytoprotection suggest potential therapeutic approaches.

Nox4 NADPH oxidase mediates oxidative stress and apoptosis caused by TNF‐alpha in cerebral vascular endothelial cells.

Inflammatory brain disease may damage cerebral vascular endothelium leading to cerebral blood flow dysregulation. The pro‐inflammatory cytokine TNFα causes oxidative stress and apoptosis in cerebral microvascular endothelial cells (CMVEC) from newborn pigs. We investigated contribution of NADPH oxidase (Nox family), a cellular source of superoxide (O2·−) to the endothelial inflammatory response. NADPH oxidase inhibitors (DPI and apocynin) greatly reduced TNFα‐evoked O2·− generation and apoptosis. Among the Nox family members, Nox2 and Nox4 are expressed in CMVEC. Nox4 siRNA knockdown completely blocked oxidative stress and apoptosis caused by TNFα, while Nox2 siRNA knockdown only partially (by 20‐30%) attenuated apoptosis. Nox4 is co‐localized with HO‐2, the constitutive isoform of heme oxygenase (HO), which is critical for endothelial protection against TNFα toxicity. The product of HO activity, carbon monoxide (CO), inhibited TNFα induced Nox4 activity and apoptosis. We conclude that Nox4 is the primary source of inflammation‐ and TNFα‐induced oxidative stress leading to apoptosis in brain endothelial cells. The ability of CO to inhibit Nox4 activity, taken together with close intracellular compartmentalization of Nox4 and HO‐2 in cerebral vascular endothelium, may contribute to HO‐2 cytoprotection against inflammatory cerebrovascular disease.

Nox4 NADPH oxidase mediates oxidative stress and apoptosis caused by TNF-α in cerebral vascular endothelial cells

Inflammatory brain disease may damage cerebral vascular endothelium leading to cerebral blood flow dysregulation. The proinflammatory cytokine TNF-alpha causes oxidative stress and apoptosis in cerebral microvascular endothelial cells (CMVEC) from newborn pigs. We investigated contribution of major cellular sources of reactive oxygen species to endothelial inflammatory response. Nitric oxide synthase and xanthine oxidase inhibitors (N(omega)-nitro-l-arginine and allopurinol) had no effect, while mitochondrial electron transport inhibitors (CCCP, 2-thenoyltrifluoroacetone, and rotenone) attenuated TNF-alpha-induced superoxide (O(2)(*-)) and apoptosis. NADPH oxidase inhibitors (diphenylene iodonium and apocynin) greatly reduced TNF-alpha-evoked O(2)(*-) generation and apoptosis. TNF-alpha rapidly increased NADPH oxidase activity in CMVEC. Nox4, the cell-specific catalytic subunit of NADPH oxidase, is highly expressed in CMVEC, contributes to basal O(2)(*-) production, and accounts for a burst of oxidative stress in response to TNF-alpha. Nox4 small interfering RNA, but not Nox2, knockdown prevented oxidative stress and apoptosis caused by TNF-alpha in CMVEC. Nox4 is colocalized with HO-2, the constitutive isoform of heme oxygenase (HO), which is critical for endothelial protection against TNF-alpha toxicity. The products of HO activity, bilirubin and carbon monoxide (CO, as a CO-releasing molecule, CORM-A1), inhibited Nox4-generated O(2)(*-) and apoptosis caused by TNF-alpha stimulation. We conclude that Nox4 is the primary source of inflammation- and TNF-alpha-induced oxidative stress leading to apoptosis in brain endothelial cells. The ability of CO and bilirubin to combat TNF-alpha-induced oxidative stress by inhibiting Nox4 activity and/or by O(2)(*-) scavenging, taken together with close intracellular compartmentalization of HO-2 and Nox4 in cerebral vascular endothelium, may contribute to HO-2 cytoprotection against inflammatory cerebrovascular disease.

Cocaine opens the blood-brain barrier to HIV-1 invasion.

Cocaine abuse has been associated with vasculitis and stroke, and is suspected to influence the progression of AIDS dementia. Cocaine may enhance HIV-1 neuroinvasion by actions directed at the blood-brain barrier. HIV-1 appears to penetrate the human brain microvascular endothelial cell barrier by a paracellular route breached by tumor necrosis factor-alpha (TNF-alpha). Cocaine's effects on the blood-brain barrier were investigated using human brain microvascular endothelial cells and peripheral blood monocytes. Cocaine (10(-5) M and 10(-6) M) increased molecular permeability of the barrier and viral invasion by the macrophage-tropic HIV-1(JR-FL) into the brain chamber. Cocaine also augmented apoptosis of brain endothelial cells and monocytes, increased secretion of four chemokines (interleukin-8, interferon-inducible protein-10, macrophage inflammatory protein-1alpha, and monocyte chemoattractant protein-1) and the cytokine, TNF-alpha, by human monocytes. TNF-alpha enhanced invasion of the brain compartment by macrophage-tropic, lymphotropic, and bitropic HIV-1 strains. These data indicate that HIV-1 neuroinvasion can be increased by (a) cocaine's direct effects on brain microvascular endothelial cells and (b) paracrine effects of cocaine-induced pro-inflammatory cytokines and chemokines on the blood-brain barrier.