Circadian disruption alters gut barrier integrity via a ß-catenin-MMP-related pathway.

We evaluated the mechanistic link between circadian rhythms and gut barrier permeability. Mice were subjected to either constant 24-h light (LL) or 12-h light/dark cycles (LD). Mice housed in LL experienced a significant increase in gut barrier permeability that was associated with dysregulated ß-catenin expression and altered expression of tight junction (TJ) proteins. Silencing of ß-catenin resulted in disruption of barrier function in SW480 cells, with ß-catenin appearing to be an upstream regulator of the core circadian components, such as Bmal1, Clock, and Per1/2. In addition, ß-catenin silencing downregulated ZO-1 and occludin TJ proteins with only limited or no changes at their mRNA levels, suggesting post transcriptional regulation. Indeed, silencing of ß-catenin significantly upregulated expression of matrix metallopeptidase (MMP)-2 and MMP-9, and blocking MMP-2/9 activity attenuated epithelial disruption induced by ß-catenin silencing. These results indicate the regulatory role of circadian disruption on gut barrier integrity and the associations between TJ proteins and circadian rhythms, while demonstrating the regulatory role of ß-catenin in this process.

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.

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.

HIV-1 Tat triggers nuclear localization of ZO-1 via Rho signaling and cAMP response element-binding protein activation.

The human immunodeficiency virus (HIV)-specific protein trans-activator of transcription (Tat) can contribute to the dysfunction of brain endothelial cells and HIV trafficking into the brain by disrupting tight junction (TJ) integrity at the blood-brain barrier (BBB) level. Specific TJ proteins, such as zonula occludens (ZO) proteins, localize not only at the cell-cell borders but are also present in the nuclei. The objective of the present study was to evaluate the mechanisms and significance of Tat-induced nuclear localization of ZO-1. Treatment of a brain endothelial cell line (hCMEC/D3 cells) with Tat resulted in a decrease in total levels of ZO-1 but significantly upregulated ZO-1 protein expression in the nuclei. In addition, exposure to Tat stimulated Rho signaling and induced phosphorylation and activity of transcription factor cAMP response element-binding protein (CREB), binding sites that have been identified in the proximal region of the ZO-1 promoter. Interestingly, inhibition of the Rho cascade protected against Tat-induced upregulation of ZO-1 in the nuclei and activation of CREB. Depletion of CREB by infection of cells with specific shRNA lentiviral particles attenuated both Tat-induced Rho signaling and nuclear targeting of ZO-1. A decrease in CREB levels also attenuated Tat-induced endothelial and BBB hyperpermeability as well as transendothelial migration of monocytic cells. The role of CREB in Tat-mediated alterations of ZO-1 was confirmed in brain microvessels in mice with CREB shRNA lentiviral particles injected into the cerebral circulation. The present results indicate the crucial role of Rho signaling and CREB in modulation of nuclear localization of ZO-1 and maintaining the integrity of endothelial monolayers.

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.

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.

Deficiency of telomerase activity aggravates the blood-brain barrier disruption and neuroinflammatory responses in a model of experimental stroke.

Epidemiology and genetic studies indicate that patients with telomere length shorter than average are at higher risk of dying from heart disease or stroke. Telomeres are located at the ends of eukaryotic chromosomes, which demonstrate progressive length reduction in most somatic cells during aging. The enzyme telomerase can compensate for telomere loss during cell replication. The present study sought to investigate the contribution of telomerase to stroke and blood-brain barrier (BBB) dysfunction. Telomerase reverse transcriptase knockout (TERT(-/-)) mice and littermate controls with normal TERT expression were subjected to a 24-hr permanent middle cerebral artery occlusion (pMCAO). The stroke outcomes were assessed in terms of neurological scores and infarct volumes. In addition, we evaluated oxidative stress, permeability across the BBB, and integrity of tight junctions in brain microvessels. Neurological testing revealed that TERT(-/-) mice showed enhanced deficits compared with controls. These changes were associated with a greater infarct volume. The expression of tight junction protein ZO-1 decreased markedly in ischemic hemispheres of TERT(-/-) mice. The brain microvessels of TERT(-/-) mice also were more susceptible to oxidative stress, revealing higher superoxide and lower glutathione levels compared with mice with normal TERT expression. Importantly, TERT deficiency potentiated the production of inflammatory mediators, such as tumor necrosis factor-alpha, interleukin-1 beta, and intercellular adhesion molecule-1, in the ischemic hemispheres of mice with pMCAO. Our study suggests that TERT deficiency can predispose to the development of stroke in an experimental model of this disease.

Polychlorinated biphenyl-induced VCAM-1 expression is attenuated in aortic endothelial cells isolated from caveolin-1 deficient mice.

Exposure to environmental contaminants, such as polychlorinated biphenyls (PCBs), is a risk factor for the development of cardiovascular diseases such as atherosclerosis. Vascular cell adhesion molecule-1 (VCAM-1) is a critical mediator for adhesion and uptake of monocytes across the endothelium in the early stages of atherosclerosis development. The upregulation of VCAM-1 by PCBs may be dependent on functional membrane domains called caveolae. Caveolae are particularly abundant in endothelial cell membranes and involved in trafficking and signal transduction. The objective of this study was to investigate the role of caveolae in PCB-induced endothelial cell dysfunction. Primary mouse aortic endothelial cells (MAECs) isolated from caveolin-1-deficient mice and background C57BL/6 mice were treated with coplanar PCBs, such as PCB77 and PCB126. In addition, siRNA gene silencing technique was used to knockdown caveolin-1 in porcine vascular endothelial cells. In MAECs with functional caveolae, VCAM-1 protein levels were increased after exposure to both coplanar PCBs, whereas expression levels of VCAM-1 were not significantly altered in cells deficient of caveolin-1. Furthermore, PCB-induced monocyte adhesion was attenuated in caveolin-1-deficient MAECs. Similarly, siRNA silencing of caveolin-1 in porcine endothelial cells confirmed the caveolin-1-dependent VCAM-1 expression. Treatment of cells with PCB77 and PCB126 resulted in phosphorylation of extracellular signal-regulated kinase-1/2 (ERK1/2), and pharmacological inhibition of ERK1/2 diminished the observed PCB-induced increase in monocyte adhesion. These findings suggest that coplanar PCBs induce adhesion molecule expression, such as VCAM-1, in endothelial cells, and that this response is regulated by caveolin-1 and functional caveolae. Our data demonstrate a critical role of functional caveolae in the activation and dysfunction of endothelial cells by coplanar PCBs.

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.

NADPH oxidase and lipid raft-associated redox signaling are required for PCB153-induced upregulation of cell adhesion molecules in human brain endothelial cells.

Exposure to persistent organic pollutants, such as polychlorinated biphenyls (PCBs), can lead to chronic inflammation and the development of vascular diseases. Because cell adhesion molecules (CAMs) of the cerebrovascular endothelium regulate infiltration of inflammatory cells into the brain, we have explored the molecular mechanisms by which ortho-substituted polychlorinated biphenyls (PCBs), such as PCB153, can upregulate CAMs in brain endothelial cells. Exposure to PCB153 increased expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), as well as elevated adhesion of leukocytes to brain endothelial cells. These effects were impeded by inhibitors of EGFR, JAKs, or Src activity. In addition, pharmacological inhibition of NADPH oxidase or disruption of lipid rafts by cholesterol depleting agents blocked PCB153-induced phosphorylation of JAK and Src kinases and upregulation of CAMs. In contrast, silencing of caveolin-1 by siRNA interference did not affect upregulation of ICAM-1 and VCAM-1 in brain endothelial cells stimulated by PCB153. Results of the present study indicate that lipid raft-dependent NADPH oxidase/JAK/EGFR signaling mechanisms regulate the expression of CAMs in brain endothelial cells and adhesion of leukocytes to endothelial monolayers. Due to its role in leukocyte infiltration, induction of CAMs may contribute to PCB-induced cerebrovascular disorders and neurotoxic effects in the CNS.

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.

ORAL ADMINISTRATION OF PCBs INDUCES PROINFLAMMATORY AND PROMETASTATIC RESPONSES.

Exposure to specific congeners of polychlorinated biphenyls (PCBs) can induce proinflammatory alterations, which may contribute to the formation of blood-borne tumor metastasis. The main aim of the present study was to establish an experimental model of PCB exposure in which PCBs are administered by oral gavage, which resembles the human exposure through the food chain. To determine structure-function relationship, we studied induction of inflammatory responses in the livers, lungs and brains of mice treated with PCB77 (a major coplanar PCB), PCB104 (a non-coplanar PCB with multiple ortho-chlorine substituents), and PCB153 (a major non-coplanar PCB) after a single gavage dose (150 µmol/kg body weight). The strongest expression of proinflammatory proteins occurred 24 h following the PCB administration independent of the class of PCB congeners. These data indicate that food-chain exposure to PCBs can induce proinflammatory mediators in organs that are potential targets for PCB-induced toxicity.

Interplay between epidermal growth factor receptor and Janus kinase 3 regulates polychlorinated biphenyl-induced matrix metalloproteinase-3 expression and transendothelial migration of tumor cells.

We hypothesize that environmental toxicants, such as polychlorinated biphenyl congeners, can activate vascular endothelial cells and thus increase formation of blood-borne metastases. This study indicates that exposure of human microvascular endothelial cells to 2,2',4,6,6'-pentachlorobiphenyl can stimulate transendothelial migration of tumor cells through up-regulation of matrix metalloproteinase (MMP)-3. In a series of experiments with specific small interfering RNA and pharmacologic inhibitors, we provide evidence that 2,2',4,6,6'-pentachlorobiphenyl can activate epidermal growth factor receptor (EGFR) and Janus kinase 3 (JAK3) in a closely coordinated and cross-dependent fashion. Activated EGFR and JAK3 stimulate in concert c-Jun NH(2)-terminal kinase and extracellular signal-regulated kinase 1/2 as well as increase DNA-binding activity of transcription factors activator protein-1 and polyomavirus enhancer activator protein 3, leading to transcriptional up-regulation of MMP-3 expression. These results indicate that the interplay among EGFR, JAK3, and mitogen-activated protein kinases, such as c-Jun NH(2)-terminal kinase and extracellular signal-regulated kinase 1/2, is critical for polychlorinated biphenyl-induced MMP-3 expression and accelerated transendothelial migration of tumor cells.

Limited role of COX-2 in HIV Tat-induced alterations of tight junction protein expression and disruption of the blood-brain barrier.

Tat protein released from HIV-infected blood-borne leukocytes can contribute to the breakdown of the blood-brain barrier (BBB) and induction of inflammatory responses and can provide entry for HIV into the brain. To mimic this pathology, Tat was injected into the tail vein of C57BL/6 mice. Treatment with Tat markedly upregulated expression of cyclooxygenase-2 (COX-2) and decreased expression of tight junction proteins, occludin and zonula occludens-1 (ZO-1). These alterations were associated with the disruption of the BBB integrity as quantified by extravasation of Evans blue dye into the brain tissue. In addition, direct treatment of brain microvessels with prostaglandin E(2), a product of COX-2 activity, resulted in decreased expression of both occludin and ZO-1. To determine if upregulation of COX-2 is involved in the disruption of tight junction proteins and BBB integrity, mice were pretreated with rofecoxib, a specific inhibitor of COX-2, prior to Tat treatment. COX-2 inhibition attenuated Tat-induced alterations of occludin expression. However, rofecoxib was ineffective in preventing downregulation of ZO-1 expression and increased BBB permeability. These results suggest only a limited role of COX-2 overexpression in the loss of tight junction integrity and the BBB breakdown in HIV-related brain diseases.

HIV-TAT protein upregulates expression of multidrug resistance protein 1 in the blood-brain barrier.

Central nervous system (CNS) complications of human immunodeficiency virus (HIV) infection remain a serious health risk in HIV/acquired immunodeficiency syndrome despite significant advances in highly active antiretroviral therapy (HAART). Specific drugs used for HAART are substrates for the efflux transport systems, such as the multidrug resistance-associated proteins (MRPs), which are present on brain microvascular endothelial cells (BMEC) and astrocytes, that is, the main cell types that form the blood-brain barrier (BBB). Thus, drugs employed in HAART are actively removed from the CNS and do not efficiently inhibit HIV replication in the brain. To study the potential mechanisms of this process, the aim of the present research was to address the hypothesis that HIV Tat protein can contribute to upregulation of MRP expression at the BBB level. Tat is a protein produced and released by HIV-infected cells, which may play an important role in brain vascular pathology in the course of HIV infection. Among the family of MRPs, exposure to Tat specifically induced MRP1 messenger ribonucleic acid and protein expression both in BMEC and astrocytes. These alterations were accompanied by enhanced MRP1-mediated efflux functions. Furthermore, activation of the mitogen-activated protein kinase signaling cascade was identified as the mechanism involved in Tat-mediated overexpression of MRP1. These results indicate that Tat exposure can lead to alterations of the BBB functions and decrease HAART efficacy in the CNS through overexpression of drug efflux transporters.

c-Src is the primary signaling mediator of polychlorinated biphenyl-induced interleukin-8 expression in a human microvascular endothelial cell line.

Interleukin-8/CXCL8 (IL-8) is a prominent factor that modulates endothelial cell proliferation, migration, and angiogenesis. Therefore, the present study focused on the regulatory mechanisms of IL-8 expression induced by environmental pollutants such as polychlorinated biphenyls (PCBs). Treatment of human microvascular endothelial cells (HMECs) with specific PCB congener, 2,2',4,6,6'-pentachlorobiphenyl (PCB 104), dose dependently increased levels of IL-8 mRNA and secreted protein. IL-8-neutralizing antibody inhibited migration of endothelial cells stimulated by conditioned media derived from PCB 104-treated HMECs. Site-directed mutagenesis of the IL-8 promoter- and DNA-binding assays revealed that activator protein 1 (AP-1) and nuclear factor kappaB (NF-kappaB) sites are required for PCB 104-induced IL-8 transcription. Most importantly, pharmacological inhibition of Src kinase activity or overexpression of dominant-negative c-src in HMECs resulted in a significant decrease in IL-8 expression and promoter activity. In contrast, ectopic expression of activated c-Src markedly increased promoter activity of IL-8. These stimulatory effects of dominant-positive c-src were abrogated by mutagenesis of AP-1- and NF-kappaB-binding sites in the IL-8 promoter.

Estrogen-mediated protection against HIV Tat protein-induced inflammatory pathways in human vascular endothelial cells.

OBJECTIVE: It has been proposed that human immunodeficiency virus (HIV) infection-induced inflammatory environment may contribute to the pathogenesis of cardiovascular diseases. Recent studies have also demonstrated the potential role of estrogen as therapeutic agents in the prevention or treatment of cardiovascular diseases. In the present study, we assessed the hypothesis that estrogen may attenuate the HIV Tat protein-induced inflammatory pathways in human vascular endothelium. METHODS: Expression of inflammatory mediators in human endothelial cells was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). Electrophoretic mobility shift assay (EMSA) also was performed to investigate the DNA-binding activities of several transcription factors, which are known to regulate expression of these inflammatory genes. RESULTS: Acute exposure of human endothelial cells to Tat markedly induced the mRNA and protein expression of IL-1beta, MCP-1, VCAM-1, and E-selectin. Tat also stimulated the adherence of inflammatory cells to endothelial cell monolayers. Significant and dose-dependent increases in NF-kappaB DNA-binding activity were observed in human endothelial cells treated with Tat. However, Tat did not affect DNA-binding activities of AP-1, CREB, and STAT1. Pretreatment with 17beta-estradiol dramatically blocked the activation of NF-kappaB in human endothelial cells exposed to Tat. In addition, 17beta-estradiol selectively inhibited the Tat-induced expression of IL-1beta. CONCLUSION: Our results suggest that estrogen may protect against Tat-induced inflammatory reactions in human vascular endothelium via blocking the NF-kappaB-mediated molecular signaling pathways. These data may contribute to understanding the pathogenesis of cardiovascular complications and development of therapeutic strategies for HIV-infected patients.

Selenoglycoproteins attenuate adhesion of tumor cells to the brain microvascular endothelium via a process involving NF-κB activation.

Selenium-containing compounds and selenized yeast have anticancer properties. In order to address possible mechanisms involved in these effects, selenoglycoproteins (SGPs) were extracted from selenium-enriched yeast at pH 4.0 and 6.5 (the fractions are called SGP40 and SGP65, respectively), followed by evaluation of their impact on the interactions of lung and breast tumor cells with human brain microvascular endothelial cells (HBMECs). Extracted SGPs, especially SGP40, significantly inhibited adhesion of tumor cells to HBMECs and their transendothelial migration. Because the active components of SGPs are unknown, small selenium-containing compounds [leucyl-valyl-selenomethionyl-arginine (LVSe-MR) and methylselenoadenosine (M-Se-A)], which are normally present in selenized yeast, were introduced as additional treatment groups. Treatment of HBMECs with SGP40, LVSe-MR and M-Se-A induced changes in gene signatures, which suggested a central involvement of nuclear factor (NF)-κB-dependent pathway. These observations were confirmed in the subsequent analysis of NF-κB DNA binding activity, quantitative measurements of the expression of selected genes and proteins, and tumor cell adhesion assay with a specific NF-κB inhibitor as the additional treatment factor. These findings indicate that specific organic selenium-containing compounds have the ability to inhibit tumor cell adhesion to brain endothelial cells via down-regulation of NF-κB. SGPs appear to be more effective than small selenium-containing compounds, suggesting the role of not only selenium but also the glycoprotein component in the observed protective impact.

PCB 104-induced proinflammatory reactions in human vascular endothelial cells: relationship to cancer metastasis and atherogenesis.

Polychlorinated biphenyls (PCBs) are widespread environmental contaminants that are known to induce carcinogenic and possibly atherogenic events. Recent evidence suggests that selected PCBs may be potent developmental agents of vascular inflammatory responses by inducing cellular oxidative stress and activating redox-responsive transcription factors. Therefore, the aim of this paper is to investigate PCB-induced proinflammatory reactions in human vascular endothelial cells. To determine the proinflammatory effects, cellular oxidative stress and expression of genes encoding for monocyte chemoattractant protein-1 (MCP-1) and adhesion molecules, such as E-selectin and intercellular adhesion molecule-1 (ICAM-1), were assessed in human umbilical vein endothelial cells (HUVEC) exposed to 2,2',4,6,6'-pentachlorobiphenyl (PCB 104), a representative of ortho-substituted, non-coplanar PCB congeners. PCB 104 increased the oxidative stress in endothelial cells, as determined by the increased 2',7'-dichlorofluorescein (DCF) and rhodamine 123 fluorescence. In addition, PCB 104 markedly upregulated the expression of MCP-1, E-selectin, and ICAM-1 at both the mRNA and protein levels. These effects were time- and concentration-dependent. The maximum expression of inflammatory genes was observed in endothelial cells exposed to 20 microM of PCB 104 for 1 or 2 h, depending on the specific gene. In addition, PCB 104 elevated the adhesion of THP-1 cells (a human acute monocytic leukemia cell line) to endothelial cell monolayers. These results indicate that PCB 104 is a potent stimulant of inflammatory mediators in human vascular endothelial cells. We hypothesize that these proinflammatory processes may contribute to the development of cancer metastasis and/or atherogenesis in patients exposed to PCBs.