Extracellular vesicles regulate gap junction-mediated intercellular communication and HIV-1 infection of human neural progenitor cells.

Human immunodeficiency virus-1 (HIV-1) has been shown to cross the blood-brain barrier and cause HIV-associated neurocognitive disorders (HAND) through a process that may involve direct or indirect interactions with the central nervous system (CNS) cells and alterations of amyloid ß (Aß) homeostasis. The present study focused on the mechanisms of HIV-1 infecting human neural progenitor cells (hNPCs) and affecting NPC intercellular communications with human brain endothelial cells (HBMEC). Despite the lack of the CD4 receptor, hNPCs were effectively infected by HIV-1 via a mechanism involving the chemokine receptors, CXCR4 and CCR5. HIV-1 infection increased expression of connexin-43 (Cx43), phosphorylated Cx43 (pCx43), and pannexin 2 (Panx2) protein levels in hNPCs, suggesting alterations in gap-junction (GJ) and pannexin channel communication. Indeed, a functional GJ assay indicated an increase in communication between HIV-infected hNPCs and non-infected HBMEC. We next analyzed the impact of HBMEC-derived extracellular vesicles (EVs) and EVs carrying Aß (EV-Aß) on the expression of Cx43, pCx43, and Panx2 in HIV-1 infected and non-infected hNPCs. Exposure to EV-Aß resulted in significant reduction of Cx43 and pCx43 protein expression in non-infected hNPCs when compared to EV controls. Interestingly, EV-Aß treatment significantly increased levels of Cx43, pCx43, and Panx2 in HIV-1-infected hNPCs when compared to non-infected controls. These results were confirmed in a GJ functional assay and an ATP release assay, which is an indicator of connexin hemichannel and/or pannexin channel functions. Overall, the current study demonstrates the importance of hNPCs in HIV-1 infection and indicates that intercellular communications between infected hNPCs and HBMEC can be effectively modulated by EVs carrying Aß as their cargo.

HIV-1 and Amyloid Beta Remodel Proteome of Brain Endothelial Extracellular Vesicles.

Amyloid beta (Aß) depositions are more abundant in HIV-infected brains. The blood-brain barrier, with its backbone created by endothelial cells, is assumed to be a core player in Aß homeostasis and may contribute to Aß accumulation in the brain. Exposure to HIV increases shedding of extracellular vesicles (EVs) from human brain endothelial cells and alters EV-Aß levels. EVs carrying various cargo molecules, including a complex set of proteins, can profoundly affect the biology of surrounding neurovascular unit cells. In the current study, we sought to examine how exposure to HIV, alone or together with Aß, affects the surface and total proteomic landscape of brain endothelial EVs. By using this unbiased approach, we gained an unprecedented, high-resolution insight into these changes. Our data suggest that HIV and Aß profoundly remodel the proteome of brain endothelial EVs, altering the pathway networks and functional interactions among proteins. These events may contribute to the EV-mediated amyloid pathology in the HIV-infected brain and may be relevant to HIV-1-associated neurocognitive disorders.

Extracellular vesicles of the blood-brain barrier: Role in the HIV-1 associated amyloid beta pathology.

HIV-infected brains are characterized by increased amyloid beta (Aß) deposition. It is believed that the blood-brain barrier (BBB) is critical for Aß homeostasis and contributes to Aß accumulation in the brain. Extracellular vesicles (ECV), like exosomes, recently gained a lot of attention as potentially playing a significant role in Aß pathology. In addition, HIV-1 hijacks the exosomal pathway for budding and release. Therefore, we investigated the involvement of BBB-derived ECV in the HIV-1-induced Aß pathology in the brain. Our results indicate that HIV-1 increases ECV release from brain endothelial cells as well as elevates their Aß cargo when compared to controls. Interestingly, brain endothelial cell-derived ECV transferred Aß to astrocytes and pericytes. Infusion of brain endothelial ECV carrying fluorescent Aß into the internal carotid artery of mice resulted in Aß fluorescence associated with brain microvessels and in the brain parenchyma. These results suggest that ECV carrying Aß can be successfully transferred across the BBB into the brain. Based on these observations, we conclude that HIV-1 facilitates the shedding of brain endothelial ECV carrying Aß; a process that may increase Aß exposure of cells of neurovascular unit, and contribute to amyloid deposition in HIV-infected brain.

Lipid rafts regulate PCB153-induced disruption of occludin and brain endothelial barrier function through protein phosphatase 2A and matrix metalloproteinase-2.

Occludin is an essential integral transmembrane protein regulating tight junction (TJ) integrity in brain endothelial cells. Phosphorylation of occludin is associated with its localization to TJ sites and incorporation into intact TJ assembly. The present study is focused on the role of lipid rafts in polychlorinated biphenyl (PCB)-induced disruption of occludin and endothelial barrier function. Exposure of human brain endothelial cells to 2,2',4,4',5,5'-hexachlorobiphenyl (PCB153) induced dephosphorylation of threonine residues of occludin and displacement of occludin from detergent-resistant membrane (DRM)/lipid raft fractions within 1h. Moreover, lipid rafts modulated the reduction of occludin level through activation of matrix metalloproteinase 2 (MMP-2) after 24h PCB153 treatment. Inhibition of protein phosphatase 2A (PP2A) activity by okadaic acid or fostriecin markedly protected against PCB153-induced displacement of occludin and increased permeability of endothelial cells. The implication of lipid rafts and PP2A signaling in these processes was further defined by co-immunoprecipitation of occludin with PP2A and caveolin-1, a marker protein of lipid rafts. Indeed, a significant MMP-2 activity was observed in lipid rafts and was increased by exposure to PCB153. The pretreatment of MMP-2 inhibitors protected against PCB153-induced loss of occludin and disruption of lipid raft structure prevented the increase of endothelial permeability. Overall, these results indicate that lipid raft-associated processes, such as PP2A and MMP-2 activation, participate in PCB153-induced disruption of occludin function in brain endothelial barrier. This study contributes to a better understanding of the mechanisms leading to brain endothelial barrier dysfunction in response to exposure to environmental pollutants, such as ortho-substituted PCBs.

HIV-1 stimulates nuclear entry of amyloid beta via dynamin dependent EEA1 and TGF-ß/Smad signaling.

Clinical evidence indicates increased amyloid deposition in HIV-1-infected brains, which contributes to neurocognitive dysfunction in infected patients. Here we show that HIV-1 exposure stimulates amyloid beta (Aß) nuclear entry in human brain endothelial cells (HBMEC), the main component of the blood-brain barrier (BBB). Treatment with HIV-1 and/or Aß resulted in concurrent increase in early endosomal antigen-1 (EEA1), Smad, and phosphorylated Smad (pSmad) in nuclear fraction of HBMEC. A series of inhibition and silencing studies indicated that Smad and EEA1 closely interact by influencing their own nuclear entry; the effect that was attenuated by dynasore, a blocker of GTP-ase activity of dynamin. Importantly, inhibition of dynamin, EEA1, or TGF-ß/Smad effectively attenuated HIV-1-induced Aß accumulation in the nuclei of HBMEC. The present study indicates that nuclear uptake of Aß involves the dynamin-dependent EEA1 and TGF-ß/Smad signaling pathways. These results identify potential novel targets to protect against HIV-1-associated dysregulation of amyloid processes at the BBB level.

Disruption of epithelial barrier by quorum-sensing N-3-(oxododecanoyl)-homoserine lactone is mediated by matrix metalloproteinases.

The intestinal epithelium forms a selective barrier maintained by tight junctions (TJs) and separating the luminal environment from the submucosal tissues. N-acylhomoserine lactone (AHL) quorum-sensing molecules produced by gram-negative bacteria in the gut can influence homeostasis of the host intestinal epithelium. In the present study, we evaluated the regulatory mechanisms affecting the impact of two representative long- and short-chain AHLs, N-3-(oxododecanoyl)-homoserine lactone (C12-HSL) and N-butyryl homoserine lactone (C4-HSL), on barrier function of human intestinal epithelial Caco-2 cells. Treatment with C12-HSL, but not with C4-HSL, perturbed Caco-2 barrier function; the effect was associated with decreased levels of the TJ proteins occludin and tricellulin and their delocalization from the TJs. C12-HSL also induced matrix metalloprotease (MMP)-2 and MMP-3 activation via lipid raft- and protease-activated receptor (PAR)-dependent signaling. Pretreatment with lipid raft disruptors, PAR antagonists, or MMP inhibitors restored the C12-HSL-induced loss of the TJ proteins and increased permeability of Caco-2 cell monolayers. These results indicate that PAR/lipid raft-dependent MMP-2 and -3 activation followed by degradation of occludin and tricellulin are involved in C12-HSL-induced alterations of epithelial paracellular barrier functions.

Amyloid beta accumulation in HIV-1-infected brain: The role of the blood brain barrier.

In recent years, we face an increase in the aging of the HIV-1-infected population, which is not only due to effective antiretroviral therapy but also to new infections among older people. Even with the use of the antiretroviral therapy, HIV-associated neurocognitive disorders represent an increasing problem as the HIV-1-infected population ages. Increased amyloid beta (Aß) deposition is characteristic of HIV-1-infected brains, and it has been hypothesized that brain vascular dysfunction contributes to this phenomenon, with a critical role suggested for the blood-brain barrier in brain Aß homeostasis. This review will describe the mechanisms by which the blood-brain barrier may contribute to brain Aß accumulation, and our findings in the context of HIV-1 infection will be discussed.

Extracellular vesicle-Serpine-1 affects neural progenitor cell mitochondrial functions and synaptic density: modulation by amyloid beta and HIV-1.

Brain endothelial extracellular vesicles carrying amyloid beta (EV-Aß) can be transferred to neural progenitor cells (NPCs) leading to NPC dysfunction. However, the events involved in this EV-mediated Aß pathology are unclear. EV-proteomics studies identified Serpine-1 (plasminogen activator inhibitor 1, PAI-1) as a major connecting "hub" on several protein-protein interaction maps. Serpine-1 was described as a key player in Aß pathology and was linked to HIV-1 infection as well. Therefore, the aim of this work was to address the hypothesis that Serpine-1 can be transferred via EVs from brain endothelial cells to NPCs and contribute to NPC dysfunction. HBMEC concentrated and released Serpine-1 via EVs, the effect that was potentiated by HIV-1 and Aß. EVs loaded with Serpine-1 were readily taken up by NPCs, and HIV-1 enhanced this event. Interestingly, a highly specific Serpine-1 inhibitor PAI039 increased EV-Aß transfer to NPCs in the presence of HIV-1. PAI039 also partially blocked mitochondrial network morphology and mitochondrial function alterations in the recipient NPCs, which developed mainly after HIV + Aß-EV transfer. PAI039 partly attenuated HIV-EV-mediated decreased synaptic protein levels in NPCs, while increased synaptic protein levels in NPC projections. These findings contribute to a better understanding of the complex mechanisms underlying EV-Serpine-1 related Aß pathology in the context of HIV infection. They are relevant to HIV-1 associated neurocognitive disorders (HAND) in an effort to elucidate the mechanisms of neuropathology in HIV infection.

Extracellular Vesicle-Serpine-1 Affects Neural Progenitor Cell Mitochondrial Networks and Synaptic Density: Modulation by Amyloid Beta and HIV-1.

Brain endothelial extracellular vesicles carrying amyloid beta (EV-Aß) can be transferred to neural progenitor cells (NPCs) leading to NPC dysfunction. However, the events involved in this EV-mediated Aß pathology are unclear. EV-proteomics studies identified Serpine-1 (plasminogen activator inhibitor 1, PAI-1) as a major connecting "hub" on several protein-protein interaction maps. Serpine-1 was described as a key player in Aß pathology and was linked to HIV-1 infection as well. Therefore, the aim of this work was to address the hypothesis that Serpine-1 can be transferred via EVs from brain endothelial cells (HBMEC) to NPCs and contribute to NPC dysfunction. HBMEC concentrated and released Serpine-1 via EVs, the effect that was potentiated by HIV-1 and Aß. EVs loaded with Serpine-1 were readily taken up by NPCs, and HIV-1 enhanced this event. Interestingly, a highly specific Serpine-1 inhibitor PAI039 increased EV-Aß transfer to NPCs in the presence of HIV-1. PAI039 also partially blocked mitochondrial network morphology alterations in the recipient NPCs, which developed mainly after HIV + Aß-EV transfer. PAI039 partly attenuated HIV-EV-mediated decreased synaptic protein levels in NPCs, while increased synaptic protein levels in NPC projections. These findings contribute to a better understanding of the complex mechanisms underlying EV-Serpine-1 related Aß pathology in the context of HIV infection. They are relevant to HIV-1 associated neurocognitive disorders (HAND) in an effort to elucidate the mechanisms of neuropathology in HIV infection.

Lipid rafts and functional caveolae regulate HIV-induced amyloid beta accumulation in brain endothelial cells.

Amyloid beta (Aß) levels are increased in HIV-1 infected brains due to not yet fully understood mechanisms. In the present study, we investigate the role of lipid rafts, functional caveolae, and caveolae-associated signaling in HIV-1-induced Aß accumulation in HBMEC. Both silencing of caveolin-1 (cav-1) and disruption of lipid rafts by pretreatment with beta-methyl-cyclodextrin (MCD) protected against Aß accumulation in HBMEC. Exposure to HIV-1 and Aß activated caveolae-associated Ras and p38. While inhibition of Ras by farnesylthiosalicylic acid (FTS) effectively protected against HIV-1-induced accumulation of Aß, blocking of p38 did not have such an effect. We also evaluated the role of caveolae in HIV-1-induced upregulation of the receptor for advanced glycation end products (RAGE), which regulates Aß transfer from the blood stream into the central nervous system. HIV-1-induced RAGE expression was prevented by infecting HBMEC with cav-1 specific shRNA lentiviral particles or by pretreatment of cells with FTS. Overall, the present results indicate that Aß accumulation in HBMEC is lipid raft and caveolae dependent and involves the caveolae-associated Ras signaling.

PPARα and PPARγ protect against HIV-1-induced MMP-9 overexpression via caveolae-associated ERK and Akt signaling.

Activation of matrix metalloproteinase-9 (MMP-9) is involved in HIV-1-induced disruption of the blood-brain barrier (BBB). In the present study, we hypothesize that peroxisome proliferator-activated receptor (PPAR)-α or PPARγ can protect against HIV-1-induced MMP-9 overexpression in brain endothelial cells (hCMEC cell line) by attenuating cellular oxidative stress and down-regulation of caveolae-associated redox signaling. Exposure to HIV-1-infected monocytes induced phosphorylation of ERK1/2 and Akt in hCMEC by 2.5- and 3.6-fold, respectively; however, these effects were attenuated by overexpression of PPARα or PPARγ and by silencing of caveolin-1 (cav-1). Coculture of hCMEC with HIV-1-infected monocytes significantly induced MMP-9 promoter and enzyme activity by 3- to 3.5-fold. Promoter mutation studies indicated that SP-1 (g1940t_g1941t) is an essential transcription factor involved in induction of MMP-9 promoter by HIV-1. In addition, HIV-1-stimulated activity of MMP-9 promoter was inhibited by mutation of AP-1 site 2 (c1918t_a1919g) and both (but not individual) NF-κB binding sites (g1389c and g1664c). PPAR overexpression, ERK1/2 or Akt inhibition, and silencing of cav-1 all effectively protected against HIV-1-induced MMP-9 promoter activity, indicating a close relationship among HIV-1-induced cerebrovascular toxicity, redox-regulated mechanisms, and functional caveolae. Such a link was further confirmed in MMP-9-deficient mice exposed to PPARα or PPARγ agonist and injected with the HIV-1-specific protein Tat into cerebral vasculature. Overall, our results indicate that ERK1/2, Akt, and cav-1 are involved in the regulatory mechanisms of PPAR-mediated protection against HIV-1-induced MMP-9 expression in brain endothelial cells.

Brain endothelium-derived extracellular vesicles containing amyloid-beta induce mitochondrial alterations in neural progenitor cells.

Aim: Elevated brain deposits of amyloid beta (Aß40) contribute to neuropathology and cognitive dysfunction in Alzheimer's disease (AD). However, the role of the blood-brain barrier (BBB) as an interface for the transfer of Aß40 from the periphery into the brain is not well characterized. In addition, a substantial population of neural progenitor cells (NPCs) resides in close proximity to brain capillaries that form the BBB. The aim of this study is to understand the impact of brain endothelium-derived extracellular vesicles (EV) containing Aß40 on metabolic functions and differentiation of NPCs. Methods: Endothelial EVs were derived from an in vitro model of the brain endothelium treated with 100 nM Aß40 or PBS. We then analyzed the impact of these EVs on mitochondrial morphology and bioenergetic disruption of NPCs. In addition, NPCs were differentiated and neurite development upon exposure to EVs was assessed using the IncuCyte Zoom live cell imaging system. Results: We demonstrate that physiological concentrations of Aß40 can be transferred to accumulate in NPCs via endothelial EVs. This transfer results in mitochondrial dysfunction, disrupting crista morphology, metabolic rates, fusion and fission dynamics of NPCs, as well as their neurite development. Conclusion: Intercellular transfer of Aß40 is carried out by brain endothelium-derived EVs, which can affect NPC differentiation and induce mitochondrial dysfunction, leading to aberrant neurogenesis. This has pathological implications because NPCs growing into neurons are incorporated into cerebral structures involved in learning and memory, two common phenotypes affected in AD and related dementias.

HIV-1-induced amyloid beta accumulation in brain endothelial cells is attenuated by simvastatin.

HIV-1-infected brains are characterized by increased amyloid deposition. To study the influence of HIV-1 on amyloid beta (Abeta) homeostasis at the blood-brain barrier (BBB) level, we employed a model of brain microvascular endothelial cells exposed to HIV-1 in the presence or absence of Abeta. HIV-1 markedly increased endogenous Abeta levels and elevated accumulation of exogenous Abeta. Simvastatin, the HMG-CoA reductase inhibitor, blocked these effects. We next evaluated the effects of HIV-1 and/or simvastatin on expression of the receptor for lipoprotein related protein (LRP1) and the receptor for advanced glycation end products (RAGE), known to regulate Abeta transport across the BBB. LRP1 expression was not affected by HIV-1; however, it was increased by simvastatin. Importantly, simvastatin attenuated HIV-1-induced RAGE expression. These results suggest that HIV-1 may directly contribute to Abeta accumulation at the BBB level. In addition, statins may protect against increased Abeta levels associated with HIV-1 infection in the brain.

Inhibition of telomerase activity alters tight junction protein expression and induces transendothelial migration of HIV-1-infected cells.

Telomerase, via its catalytic component telomerase reverse transcriptase (TERT), extends telomeres of eukaryotic chromosomes. The importance of this reaction is related to the fact that telomere shortening is a rate-limiting mechanism for human life span that induces cell senescence and contributes to the development of age-related pathologies. The aim of the present study was to evaluate whether the modulation of telomerase activity can influence human immunodeficiency virus type 1 (HIV-1)-mediated dysfunction of human brain endothelial cells (hCMEC/D3 cells) and transendothelial migration of HIV-1-infected cells. Telomerase activity was modulated in hCMEC/D3 cells via small interfering RNA-targeting human TERT (hTERT) or by using a specific pharmacological inhibitor of telomerase, TAG-6. The inhibition of hTERT resulted in the upregulation of HIV-1-induced overexpression of intercellular adhesion molecule-1 via the nuclear factor-kappaB-regulated mechanism and induced the transendothelial migration of HIV-1-infected monocytic U937 cells. In addition, the blocking of hTERT activity potentiated a HIV-induced downregulation of the expression of tight junction proteins. These results were confirmed in TERT-deficient mice injected with HIV-1-specific protein Tat into the cerebral vasculature. Further studies revealed that the upregulation of matrix metalloproteinase-9 is the underlying mechanisms of disruption of tight junction proteins in hCMEC/D3 cells with inhibited TERT and exposed to HIV-1. These results indicate that the senescence of brain endothelial cells may predispose to the HIV-induced upregulation of inflammatory mediators and the disruption of the barrier function at the level of the brain endothelium.

PPARalpha and PPARgamma attenuate HIV-induced dysregulation of tight junction proteins by modulations of matrix metalloproteinase and proteasome activities.

The blood-brain barrier (BBB) plays an important role in HIV trafficking into the brain and the development of the central nervous system complications in HIV infection. Tight junctions are the main structural and functional elements that regulate the BBB integrity. Exposure of human brain microvascular endothelial cells (hCMEC/D3 cell line) to HIV-infected monocytes resulted in decreased expression of tight junction proteins, such as junctional adhesion molecule-A (JAM)-A, occludin, and zonula occludens (ZO)-1. Control experiments involved exposure to uninfected monocytes. Alterations of tight junction protein expression were associated with increased endothelial permeability and elevated transendothelial migration of HIV-infected monocytes across an in vitro model of the BBB. Notably, overexpression of the peroxisome proliferator-activated receptor (PPAR)alpha or PPARgamma attenuated HIV-mediated dysregulation of tight junction proteins. With the use of exogenous PPARgamma agonists and silencing of PPARalpha or PPARgamma, these protective effects were connected to down-regulation of matrix metalloproteinase (MMP) and proteasome activities. Indeed, the HIV-induced decrease in the expression of JAM-A and occludin was restored by inhibition of MMP activity. Moreover, both MMP and proteasome inhibitors attenuated HIV-mediated altered expression of ZO-1. The present data indicate that down-regulation of MMP and proteasome activities constitutes a novel mechanism of PPAR-induced protections against HIV-induced disruption of brain endothelial cells.

Extracellular vesicle-mediated amyloid transfer to neural progenitor cells: implications for RAGE and HIV infection.

Amyloid beta (Aß) deposition was demonstrated to be elevated in the brains of HIV-infected patients and associated with neurocognitive decline; however, the mechanisms of these processes are poorly understood. The goal of the current study was to address the hypothesis that Aß can be transferred via extracellular vesicles (ECVs) from brain endothelial cells to neural progenitor cells (NPCs) and that this process can contribute to abnormal NPC differentiation. Mechanistically, we focused on the role of the receptor for advanced glycation end products (RAGE) and activation of the inflammasome in these events. ECVs loaded with Aß (Aß-ECVs) were readily taken up by NPCs and Aß partly colocalized with the inflammasome markers ASC and NLRP3 in the nuclei of the recipient NPCs. This colocalization was affected by HIV and RAGE inhibition by a high-affinity specific inhibitor FPS-ZM1. Blocking RAGE resulted also in an increase in ECV number produced by brain endothelial cells, decreased Aß content in ECVs, and diminished Aß-ECVs transfer to NPC nuclei. Interestingly, both Aß-ECVs and RAGE inhibition altered NPC differentiation. Overall, these data indicate that RAGE inhibition affects brain endothelial ECV release and Aß-ECVs transfer to NPCs. These events may modulate ECV-mediated amyloid pathology in the HIV-infected brain and contribute to the development of HIV-associated neurocognitive disorders.

Simvastatin protects against amyloid beta and HIV-1 Tat-induced promoter activities of inflammatory genes in brain endothelial cells.

Increased deposition of amyloid beta (Abeta) is characteristic for normal aging and human immunodeficiency virus-1 (HIV-1)-associated alterations of the central nervous system. In addition, both Abeta and HIV-1 are known to induce cellular oxidative stress and disruption of the blood-brain barrier (BBB). Therefore, we hypothesize that Abeta and HIV-1 protein Tat can potentiate their proinflammatory effects at the brain endothelium level. To address this hypothesis, we studied promoter activity of three proinflammatory genes in an in vitro BBB model of human brain microvascular endothelial cells (HBMEC) cocultured with a human astrocyte cell line producing Tat (SVGA-Tat cells) and exposed to Abeta. Treatment of HBMEC with Abeta(1-40) in the presence of SVGA-Tat cells resulted in a significant up-regulation of E-selectin, CC chemokine ligand-2, and interleukin-6 promoter activities and protein levels compared with the individual effects of Abeta or Tat. In addition, Abeta markedly amplified E-selectin promoter activity in HBMEC cocultured with HIV-1-infected Jurkat T cells. Simvastatin, the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, effectively blocked proinflammatory reactions induced by Abeta in cocultures with SVGA-Tat cells or with HIV-1-infected Jurkat cells. The present study indicates that a combined exposure to Abeta and Tat or HIV-1 can synergistically potentiate the expression of inflammatory genes in brain endothelial cells. In addition, simvastatin may provide a beneficial influence by reducing these effects at the BBB level.

PPARalpha and PPARgamma effectively protect against HIV-induced inflammatory responses in brain endothelial cells.

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors which down-regulate inflammatory signaling pathways. Therefore, we hypothesized that alterations of PPAR functions can contribute to human immunodeficiency virus-1 (HIV-1)-induced dysfunction of brain endothelial cells. Indeed, treatment with HIV-1 transactivator of transcription (Tat) protein decreased PPAR transactivation in brain endothelial cells. We next stably over-expressed PPARalpha and PPARgamma in a newly developed cell line of human brain endothelial cells (hCMEC/D3 cells). Tat-induced up-regulation of inflammatory mediators, such as interleukin (IL)-1beta, tumor necrosis factor-alpha, CCL2, and E-selectin were markedly attenuated in hCMEC/D3 over-expressing PPARalpha or PPARgamma. These results were confirmed in CCL2 and E-selectin promoter activity studies. Similar protective effects were observed in hCMEC/D3 after activation of PPARgamma by exogenous PPAR agonists (dPGJ(2) and rosiglitazone). PPAR over-expression also prevented Tat-induced binding activity and transactivation of nuclear factor-kappaB. Importantly, increased PPAR activity attenuated induction of IL-1beta, tumor necrosis factor-alpha, CCL2, and E-selectin in hCMEC/D3 cells co-cultured with HIV-1-infected Jurkat cells. The protective effects of PPAR over-expression were reversed by the antagonists of PPARalpha (MK886) or PPARgamma (GW9662). The present data suggest that targeting PPAR signaling may provide a novel therapeutic approach to attenuate HIV-1-induced local inflammatory responses in brain endothelial cells.

Pcbs and tight junction expression.

Polychlorinated biphenyl (PCB) congeners exhibit a broad range of adverse biological effects including neurotoxicity. The mechanisms by which PCBs cause neurotoxic effects are still not completely understood. The blood-brain barrier (BBB) is a physical and metabolic barrier separating brain microenvironment from the peripheral circulation and is mainly composed of endothelial cells connected by tight junctions. We examined the effects of several highly-chlorinated PCB congeners on expression of tight junction proteins in human brain endothelial cells. Treatment for 24 h with selective PCB congeners disrupted expression of the cytosolic scaffold proteins of tight junctions, such as zonula occludens (ZO)-1, ZO-2, and AF6. In contrast, PCB exposure did not alter expression of integral membrane proteins, junctional adhesion molecule-A (JAM-A), and claudin-1. Based on these data, we suggest that PCB-mediated selective alterations of tight junction protein expression may contribute to their neurotoxic effects in the central nervous system.

The NMDA and AMPA/KA receptors are involved in glutamate-induced alterations of occludin expression and phosphorylation in brain endothelial cells.

Glutamate levels increase dramatically in cerebral ischemia and stroke. This may lead to opening of the blood-brain barrier (BBB) and induce further brain damage. Because endothelial tight junctions are critical elements of the BBB integrity, the aim of this study was to investigate the mechanisms of glutamate-induced alterations of the tight-junction protein occludin in cultured brain microvascular endothelial cells (BMECs). Transient exposure to glutamate resulted in cellular redistribution of occludin, followed by a decrease in the total level of this protein and diminished barrier function of BMECs. Inhibition of the N-methyl-D-aspartate (NMDA) or alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate (AMPA/KA) receptors attenuated glutamate-induced changes in occludin redistribution but not in the total protein levels. Treatment with glutamate also increased tyrosine phosphorylation and decreased threonine phosphorylation of occludin. Inhibition of the NMDA receptors by MK-801 partially protected against glutamate-induced elevation of occludin tyrosine phosphorylation. In addition, pretreatment with MK-801-attenuated glutamate-mediated disruption of endothelial barrier function. Blocking of the AMPA/KA receptors by 6,7-dinitroquinoxaline-2.3-dione (DNQX) protected against hypophosphorylation of threonine residues of occludin; however, it did not affect disruption of endothelial integrity. These findings indicate the opposite effects of the NMDA and AMPA/KA receptors on occludin phosphorylation and disruption of the BBB functions.