Following absorption, polychlorinated biphenyls (PCBs) bind to albumin and are transported via blood into the target tissues. PCBs then accumulate in tissues and induce a variety of harmful chronic and developmental effects. The aim of the present study is to determine binding parameters, such as binding constant, quenching constant, and number of binding sites for three PCB congeners (PCB118, PCB126 and PCB153) in complex with human serum albumin (HSA). The binding parameters for the complexes of HSA-PCB118, HSA-PCB126, and HSA-PCB153 excited at 280 nm were compared with those excited at 295 nm. Quenching (static and dynamic) of HSA fluorescence was analyzed based on the Stern-Volmer method. Binding (Ka) constants were calculated according to the Scatchard method and analysis of non-linear regression was based on a two-component model with the Lavenberg–Marquardt algorithm. For all analyzed complexes, a single independent class of binding site for PCB congeners was found in HSA subdomain IIA. Tyrosine residues appear to play the most prominent role in binding of PCB126 to HSA, while tryptophan-214 played a dominant role in interactions of PCB153 with HSA. Among studied PCB congeners, PCB118 formed the most stable complexes with HSA. These results illustrate the importance of studies targeting the binding of PCBs to serum albumin as part of the strategy to understand and protect against toxicity of these environmental toxicants.
PCB; albumin; fluorescence
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 (Aβ) nuclear entry in human brain endothelial cells (HBMEC), the main component of the blood-brain barrier (BBB). Treatment with HIV-1 and/or Aβ 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-β/Smad effectively attenuated HIV-1-induced Aβ accumulation in the nuclei of HBMEC. The present study indicates that nuclear uptake of Aβ involves the dynamin-dependent EEA1 and TGF-β/Smad signaling pathways. These results identify potential novel targets to protect against HIV-1-associated dysregulation of amyloid processes at the BBB level.
HIV-1; blood-brain barrier; amyloid beta
The strategies to protect against the disrupted blood–brain barrier (BBB) in HIV-1 infection are not well developed. Therefore, we investigated the potential of peroxisome proliferator-activated receptor (PPAR) agonists to prevent enhanced BBB permeability induced by HIV-1-specific protein Tat. Exposure to Tat via the internal carotid artery (ICA) disrupted permeability across the BBB; however, this effect was attenuated in mice treated with fenofibrate (PPARα agonist) or rosiglitazone (PPARγ agonist). In contrast, exposure to GW9662 (PPARγ antagonist) exacerbated Tat-induced disruption of the BBB integrity. Increased BBB permeability was associated with decreased tight junction (TJ) protein expression and activation of ERK1/2 and Akt in brain microvessels; these effects were attenuated by cotreatment with fenofibrate but not with rosiglitazone. Importantly, both PPAR agonists also protected against Tat-induced astrogliosis and neuronal loss. Because disruption of TJ integrity has been linked to matrix metalloproteinase (MMP) activity, we also evaluated Tat-induced effects in MMP-9-deficient mice. Tat-induced cerebrovascular toxicity, astrogliosis, and neuronal loss were less pronounced in MMP-9-deficient mice as compared with wild-type controls and were further attenuated by PPAR agonists. These results indicate that enhancing PPAR activity combined with targeting MMPs may provide effective therapeutic strategies in brain infection by HIV-1.
blood–brain barrier; human immunodeficiency virus-1; matrix metalloproteinase; peroxisome proliferator-activated receptor; Tat protein; neurotoxicity
Flavonoids, such as the tea catechin epigallocatechin-gallate (EGCG), can protect against atherosclerosis by decreasing vascular endothelial cell inflammation. Heme oxygenase (HO-1) is an enzyme that plays an important role in vascular physiology, and its induction may provide protection against atherosclerosis. HO-1 can be compartmentalized in caveolae in endothelial cells. Caveolae are plasma microdomains important in vesicular transport and the regulation of signaling pathways associated with the pathology of vascular diseases. We hypothesize that caveolae play a role in the uptake and transport of EGCG and mechanisms associated with the anti-inflammatory properties of this flavonoid. To test this hypothesis, we explored the effect of EGCG on the induction of Nrf2 and HO-1 in endothelial cells with or without functional caveolae. Treatment with EGCG activated Nrf2 and increased HO-1 expression and cellular production of bilirubin. In addition, EGCG rapidly accumulated in caveolae, which was associated with caveolin-1 displacement from the plasma membrane towards the cytosol. Similar to EGCG treatment, silencing of caveolin-1 by siRNA technique also resulted in upregulation of Nrf2, HO-1 and bilirubin production. These data suggest that EGCG-induced caveolin-1 displacement may reduce endothelial inflammation.
EGCG; Nrf2; Caveolae; Endothelial Cells; Atherosclerosis
The American Cancer Society estimated 1.5 million new cancer cases in the US in 2012. Although the exact number is not known, it is estimated that brain metastases occur in 20–40% of cancer patients (NCI). Due to the complexity of development and the variation in tumor etiology, therapy options have been limited for a number of cancers while progressive treatments have been successful for some malignancies. Combining treatment strategies has shown potential to increase positive outcomes, however cancer remains a formidable diagnosis with no true cure. Many researchers have focused on alternative forms of cancer prevention or treatment to slow cancer progression. Studies have shown that with moderate, regular exercise signaling pathways associated with increased antioxidant activity and cellular repair are upregulated in vascular tissue, however the physiological mechanisms are poorly understood. The purpose of this review is to examine the current literature in order to better understand the impact of exercise on cancer progression and tumor metastasis and discuss potential redox related signaling in the vasculature that may be involved.
Cancer; exercise; reactive oxygen species; brain microvasculature; metastasis; tight junction
Tumor cell extravasation into the brain requires passage through the blood-brain barrier (BBB). There is evidence that exercise can alter the oxidation status of the brain microvasculature and protect against tumor cell invasion into the brain, although the mechanisms are not well understood. In the current study, we focused on the role of microenvironment generated by exercise and metastasizing tumor cells at the levels of brain microvessels, influencing oxidative stress-mediated responses and activation of redox-sensitive small GTPases. Mature male mice were exercised for four weeks using a running wheel with the average voluntary running distance 9.0±0.3 km/day. Mice were then infused with 1.0×106 D122 (murine Lewis lung carcinoma) cells into the brain microvasculature, and euthanized either 48 hours (in short-term studies) or 2–3 weeks (in long-term studies) post tumor cell administration. A significant increase in the level of reactive oxygen species was observed following 48 hours or 3 weeks of tumor cells growth, which was accompanied by a reduction in MnSOD expression in the exercised mice. Activation of the small GTPase Rho was negatively correlated with running distance in the tumor cell infused mice. Together, these data suggest that exercise may play a significant role during aggressive metastatic invasion, especially at higher intensities in pre-trained individuals.
The current study focused on blood-brain barrier (BBB) disruption and neurovascular damage induced by engineered nanomaterials. Exposure to nanoalumina, but not to nanocarbon, induced a dose-dependent mitochondrial potential collapse, increased autophagy of brain endothelial cells, and decreased expression of tight junction proteins, occludin and claudin-5. Inhibition of autophagy by pretreatment with Wortmannin attenuated the effects of nanoalumina on decreased claudin-5 expression; however, it did not affect the disruption of occludin. These finding were confirmed in mice by administration of nanoalumina into the cerebral circulation. Systemic treatment with nanoalumina elevated autophagy-related genes and autophagic activity in the brain, decreased tight junction protein expression, and elevated BBB permeability. Finally, exposure to nanoalumina, but not to nanocarbon, increased brain infarct volume in mice subjected to a focal ischemic stroke model. Overall, our study reveals that autophagy constitutes an important mechanism involved in nanoalumina-induced neurovascular toxicity in the central nervous system.
nanoalumina; autophagy; blood-brain barrier; central nervous system
Exposure to polychlorinated biphenyls (PCBs) is associated with numerous adverse health effects. Although the main route of exposure to PCBs is through the gastrointestinal tract, little is known about the contribution of the gut to the health effects of PCBs. We hypothesize that PCBs can disrupt intestinal integrity, causing lipopolysaccharide (LPS) translocation into the bloodstream and potentiation of the systemic toxicity of PCBs. C57BL/6 mice were exposed to individual PCB congeners by oral gavage, followed by the assessment of small intestine morphology and plasma levels of proinflammatory mediators. In addition, mice and human brain endothelial cells were exposed to PCB118 in the presence or absence of LPS to evaluate the contribution of LPS to PCB-induced toxicity at the blood–brain barrier (BBB) level. Oral administration of PCB153, PCB118, or PCB126 disrupted intestinal morphology and increased plasma levels of LPS and proinflammatory cytokines. Direct injection of LPS and PCB118 into the cerebral microvasculature resulted in synergistic disruption of BBB integrity and decreased expression of tight junction proteins in brain microvessels. In vitro experiments confirmed these effects and indicated that stimulation of the toll-like receptor 4 (TLR4) pathway can be responsible for these effects via activation of interferon regulatory factor-3 (IRF-3). These results indicate that LPS may be a contributing factor in PCB-induced dysfunction of the brain endothelium via stimulation of the TLR4/IRF-3 pathway.
blood–brain barrier; inflammation; interferon regulatory factor-3; polychlorinated biphenyls; tight junctions; Toll-like receptor 4
Environmental polychlorinated biphenyls (PCBs) are frequently bound onto nanoparticles (NPs). However, the toxicity and health effects of PCBs assembled onto nanoparticles are unknown. The aim of this study was to study the hypothesis that binding PCBs to silica NPs potentiates PCB-induced cerebrovascular toxicity and brain damage in an experimental stroke model. Mice (C57BL/6, males, 12-week-old) were exposed to PCB153 bound to NPs (PCB153-NPs), PCB153, or vehicle. PCB153 was administered in the amount of 5 ng/g body weight. A group of treated animals was subjected to a 40 min ischemia, followed by a 24 h reperfusion. The blood-brain barrier (BBB) permeability, brain infarct volume, expression of tight junction (TJ) proteins, and inflammatory mediators were assessed. As compared to controls, a 24 h exposure to PCB153-NPs injected into cerebral vasculature resulted in significant elevation of the BBB permeability, disruption of TJ protein expression, increased proinflammatory responses, and enhanced monocyte transmigration in mouse brain capillaries. Importantly, exposure to PCB153-NPs increased stroke volume and potentiated brain damage in mice subjected to ischemia/reperfusion. A long-term (30 days) oral exposure to PCB153-NPs resulted in a higher PCB153 content in the abdominal adipose tissue and amplified adhesion of leukocytes to the brain endothelium as compared to treatment with PCB153 alone. This study provides the first evidence that binding to NPs increases cerebrovascular toxicity of environmental toxicants, such as PCB153.
blood-brain barrier; polychlorinated biphenyls; silica nanoparticles; stroke; tight junctions
Human immunodeficiency virus type 1 (HIV-1) infection of the central nervous system (CNS) affects cross-talk between the individual cell types of the neurovascular unit, which then contributes to disruption of the blood–brain barrier (BBB) and the development of neurological dysfunctions. While the toxicity of HIV-1 on neurons, astrocytes, and brain endothelial cells has been widely studied, there are no reports addressing the influence of HIV-1 on pericytes. Therefore, the purpose of this study was to evaluate whether pericytes can be infected with HIV-1 and how such an infection affects the barrier function of brain endothelial cells. Our results indicate that human brain pericytes express the major HIV-1 receptor CD4 and coreceptors CXCR4 and CCR5. We also determined that HIV-1 can replicate, though at a low level, in human brain pericytes as detected by HIV-1 p24 ELISA. Pericytes were susceptible to infection with both the X4-tropic NL4-3 and R5-tropic JR-CSF HIV-1 strains. Moreover, HIV-1 infection of pericytes resulted in compromised integrity of an in vitro model of the BBB. These findings indicate that human brain pericytes can be infected with HIV-1 and suggest that infected pericytes are involved in the progression of HIV-1-induced CNS damage.
HIV-1; pericytes; blood–brain barrier; neurovascular unit
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 (Aβ) 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 Aβ homeostasis. This review will describe the mechanisms by which the BBB may contribute to brain Aβ accumulation, and our findings in the context of HIV-1 infection will be discussed.
HIV-1; HIV-1-associated neurocognitive disorders; blood-brain barrier; brain endothelial cell; amyloid beta
Background: Methamphetamine is a drug of abuse that disrupts the blood-brain barrier.
Results: Blocking actin nucleation protects against methamphetamine-induced occludin internalization and disruption of blood-brain barrier integrity.
Conclusion: Methamphetamine-induced transendothelial breaches may result from actin-mediated redistribution of occludin.
Significance: Actin cytoskeletal dynamics modulates redistribution of occludin and blood-brain barrier integrity.
Methamphetamine (METH) is a drug of abuse with neurotoxic and neuroinflammatory effects, which include disruption of the blood-brain barrier (BBB) and alterations of tight junction protein expression. This study focused on the actin cytoskeletal rearrangement as a modulator of METH-induced redistribution of tight junction protein occludin in brain endothelial cells. Exposure to METH resulted in a shift of occludin localization from plasma membranes to endosomes. These changes were accompanied by activation of the actin-related protein 2/3 (Arp2/3) complex, which stimulates actin polymerization by promoting actin nucleation. In addition, METH-induced coronin-1b phosphorylation diminishes the inhibitory effect of nonphosphorylated coronin-1b on actin nucleation. Blocking actin nucleation with CK-666, a specific inhibitor of the Arp2/3 complex, protected against METH-induced occludin internalization and increased transendothelial monocyte migration. Importantly, treatment with CK-666 attenuated a decrease in occludin levels in brain microvessels and BBB permeability of METH-injected mice. These findings indicate that actin cytoskeletal dynamics is detrimental to METH-induced BBB dysfunction by increasing internalization of occludin.
Actin; Brain; Cytoskeleton; Endocytosis; Endothelium; Tight Junctions; Methamphetamine; Arp2/3 Complex; Actin; Nucleation; Blood-Brain Barrier; Occludin; Endocytosis; Endo
HIV-1-infected brains are characterized by elevated depositions of amyloid beta (Aβ); however, the interactions between Aβ and HIV-1 are poorly understood. In the present study, we administered specific HIV-1 protein Tat into the cerebral vasculature of 50–52 week old double transgenic (B6C3-Tg) mice that express a chimeric mouse/human amyloid precursor protein (Mo/HuAPP695swe) and a mutant human presenilin 1 (PS1-dE9) and are characterized by increased Aβ depositions in the brain. Exposure to Tat increased permeability across cerebral capillaries, enhanced disruption of zonula occludens (ZO)-1 tight junction protein, and elevated brain expression of matrix metalloproteinase-9 (MMP-9) in B6C3-Tg mice as compared to age-matched littermate controls. These changes were associated with increased leukocyte attachment and their transcapillary migration. The majority of Tat-induced effects were attenuated by treatment with a specific Rho inhibitor, hydroxyfasudil. The results of animal experiments were reproduced in cultured brain endothelial cells exposed to Aβ and/or Tat. The present data indicate that increased brain levels of Aβ can enhance vascular toxicity and proinflammatory responses induced by HIV-1 protein Tat.
HIV-1 infection; amyloid; HIV-1; aging; blood-brain barrier; tight junction proteins; inflammation
There is no effective therapeutic intervention developed targeting cerebrovascular toxicity of drugs of abuse, including methamphetamine (METH). We hypothesize that exercise protects against METH-induced disruption of the blood–brain barrier (BBB) by enhancing the antioxidant capacity of cerebral microvessels and modulating caveolae-associated signaling. Mice were subjected to voluntary wheel running for 5 weeks resembling the voluntary pattern of human exercise, followed by injection with METH (10 mg/kg). The frequency, duration, and intensity of each running session were monitored for each mouse via a direct data link to a computer and the running data are analyzed by Clock lab™ Analysis software. Controls included mice sedentary that did not have access to running wheels and/or injections with saline.
METH induced oxidative stress in brain microvessels, resulting in up regulation of caveolae-associated NAD(P)H oxidase subunits, and phosphorylation of mitochondrial protein 66Shc. Treatment with METH disrupted also the expression and colocalization of tight junction proteins. Importantly, exercise markedly attenuated these effects and protected against METH-induced disruption of the BBB integrity.
The obtained results indicate that exercise is an important modifiable behavioral factor that can protect against METH-induced cerebrovascular toxicity. These findings may provide new strategies in preventing the toxicity of drug of abuse.
Methamphetamine; Drug abuse; Exercise; Blood-brain; Oxidative stress; Tight junctions
Tea flavonoids such as epigallocatechin gallate (EGCG) protect against vascular diseases such as atherosclerosis via their antioxidant and anti-inflammatory functions. Persistent and widespread environmental pollutants, including polychlorinated biphenyls (PCB), can induce oxidative stress and inflammation in vascular endothelial cells. Even though PCBs are no longer produced, they are still detected in human blood and tissues and thus considered a risk for vascular dysfunction. We hypothesized that EGCG can protect endothelial cells against PCB-induced cell damage via its antioxidant and anti-inflammatory properties. To test this hypothesis, primary vascular endothelial cells were pretreated with EGCG, followed by exposure to the coplanar PCB 126. Exposure to PCB 126 significantly increased cytochrome P450 1A1 (Cyp1A1) mRNA and protein expression and superoxide production, events which were significantly attenuated following pretreatment with EGCG. Similarly, EGCG also reduced DNA binding of NF-κB and downstream expression of inflammatory markers such as monocyte chemotactic protein-1 (MCP-1) and vascular cell adhesion protein-1 (VCAM-1) after PCB exposure. Furthermore, EGCG decreased endogenous or base-line levels of Cyp1A1, MCP-1 and VCAM-1 in endothelial cells. Most of all, treatment of EGCG upregulated expression of NF-E2-related factor 2 (Nrf2)-controlled antioxidant genes, including glutathione S transferase (GST) and NAD(P)H:quinone oxidoreductase 1 (NQO1), in a dose-dependent manner. In contrast, silencing of Nrf2 increased Cyp1A1, MCP-1 and VCAM-1 and decreased of GST and NQO1 expression, respectively. These data suggest that EGCG can inhibit AhR regulated genes and induce Nrf2-regulated antioxidant enzymes, thus providing protection against PCB-induced inflammatory responses in endothelial cells.
PCB; EGCG; polyphenol; endothelial cell; inflammation; atherosclerosis
Background: The gut microbiome, a dynamic bacterial community that interacts with the host, is integral to human health because it regulates energy metabolism and immune functions. The gut microbiome may also play a role in risks from environmental toxicants.
Objectives: We investigated the effects of polychlorinated biphenyls (PCBs) and exercise on the composition and structure of the gut microbiome in mice.
Methods: After mice exercised voluntarily for 5 weeks, they were treated by oral gavage with a mixture of environmentally relevant PCB congeners (PCB153, PCB138, and PCB180; total PCB dose, 150 µmol/kg) for 2 days. We then assessed the microbiome by determination of 16S rRNA using microarray analysis.
Results: Oral exposure to PCBs significantly altered the abundance of the gut microbiome in mice primarily by decreasing the levels of Proteobacteria. The activity level of the mice correlated with a substantial shift in abundance, biodiversity, and composition of the microbiome. Importantly, exercise attenuated PCB-induced changes in the gut microbiome.
Conclusions: Our results show that oral exposure to PCBs can induce substantial changes in the gut microbiome, which may then influence their systemic toxicity. These changes can be attenuated by behavioral factors, such as voluntary exercise.
environmental toxicants; exercise; gut microbiome; polychlorinated biphenyls; PhyloChip
PCBs bind to environmental particles; however, potential toxicity exhibited by such complexes is not well understood. The aim of the present study is to study the hypothesis that assembling onto nanoparticles can influence the PCB153-induced brain endothelial toxicity via interaction with the toll-like receptor 4 (TLR4). To address this hypothesis, TLR4-deficient and wild type control mice (males, 10 week old) were exposed to PCB153 (5 ng/g body weight) bound to chemically inert silica nanoparticles (PCB153-NPs), PCB153 alone, silica nanoparticles (NPs; diameter, 20 nm), or vehicle. Selected animals were also subjected to 40 min ischemia, followed by a 24 h reperfusion. As compared to exposure to PCB153 alone, treatment with PCB153-NP potentiated the brain infarct volume in control mice. Importantly, this effect was attenuated in TLR4-deficient mice. Similarly, PCB153-NP-induced proinflammatory responses and disruption of tight junction integrity were less pronounced in TLR4-deficient mice as compared to control animals. Additional in vitro experiments revealed that TLR4 mediates toxicity of PCB153-NP via recruitment of tumor necrosis factor-associated factor 6 (TRAF6). The results of current study indicate that binding to seemingly inert nanoparticles increase cerebrovascular toxicity of PCBs and suggest that targeting the TLR4/TRAF6 signaling may protect against these effects.
Amyloid beta (Aβ) 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 Aβ accumulation in HBMEC. Both silencing of caveolin-1 (cav-1) and disruption of lipid rafts by pretreatment with beta-methyl-cyclodextrin (MCD) protected against Aβ accumulation in HBMEC. Exposure to HIV-1 and Aβ activated caveolae-associated Ras and p38. While inhibition of Ras by farnesylthiosalicylic acid (FTS) effectively protected against HIV-1-induced accumulation of Aβ, 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 Aβ 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 Aβ accumulation in HBMEC is lipid raft and caveolae dependent and involves the caveolae-associated Ras signaling.
HIV-1; blood-brain barrier; amyloid beta
Polychlorinated biphenyls (PCBs) are environmental toxicants that cause vascular inflammation and facilitate the development of brain metastases. The crucial event in metastasis formation is adhesion of blood-borne tumor cells to the vascular endothelium, followed by their transcapillary migration. The aim of the present study was to examine the mechanisms of PCB118-induced brain metastasis formation at the blood-brain barrier level with the focus on tumor cell adhesion to the brain endothelium. PCB118 was administered orally to wild-type or intercellular cell adhesion molecule-1 (ICAM-1)–deficient mice, followed by an injection of Lewis lung carcinoma cells into the carotid artery. Treatment with PCB118 resulted in enhanced development of brain metastases. Injection of tumor cells induced overexpression of ICAM-1 and vascular endothelial cell adhesion molecule-1 (VCAM-1) in brain endothelium that was further potentiated in mice exposed to PCB118. PCB118 did not affect the number of adhered and extravasated tumor cells in ICAM-1–deficient mice. Additional in vitro studies indicated that VCAM-1–neutralizing antibody protected against PCB118-induced adhesion of tumor cells to cultured brain endothelial cells. These results indicate that exposure to selected PCB congeners, such as PCB118, induces adhesion and transcapillary migration of tumor cells. This process is facilitated by proinflammatory adhesion molecules and results in potentiation of brain metastasis formation.
blood-brain barrier; ICAM-1; VCAM-1; tumor cell adhesion; vascular inflammation
The HIV specific protein 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 (CREB), binding sites which 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.
Blood-brain barrier; brain endothelial cells; cell signaling; HIV-1; tight junctions; transcription factors
Methamphetamine (METH) is a drug of abuse with neurotoxic and vascular effects that may be mediated by reactive oxygen species (ROS). However, potential sources of METH-induced generation of ROS are not fully understood. The present study is focused on the role of NAD(P)H oxidase (NOX) in METH-induced dysfunction of brain endothelial cells. Treatment with METH induced a time-dependent increase in phosphorylation of NOX subunit p47, followed by its binding with gp91 and p22, and the formation of an active NOX complex. An increase in NOX activity was associated with elevated production of ROS, alterations of occludin levels, and increased transendothelial migration of monocytes. Inhibition of NOX by NSC 23766 attenuated METH-induced ROS generation, changes in occludin protein levels, and monocyte migration. Because an active NOX complex is localized to caveolae, we next evaluated the role of caveolae in METH-mediated toxicity to brain endothelial cells. Treatment with METH induced phosphorylation of ERK1/2 and caveolin-1 protein. Inhibition of ERK1/2 activity or caveolin-1 silencing protected against METH-induced alterations of occludin levels. These findings indicate an important role of NOX and functional caveolae in METH-induced oxidative stress in brain endothelial cells that contribute to the subsequent alterations of occludin levels and transendothelial migration of inflammatory cells.
blood-brain barrier; brain endothelial cells; drug abuse; methamphetamine; oxidative stress; tight junctions
Proteins on cell surface play important roles during cancer progression and metastasis via their ability to mediate cell-to-cell interactions and navigate the communication between cells and the microenvironment.
In this study a targeted proteomic analysis was conducted to identify the differential expression of cell surface proteins in human benign (BPH-1) vs. malignant (LNCaP and PC-3) prostate epithelial cells. We identified EMMPRIN (extracellular matrix metalloproteinase inducer) as a key candidate and shRNA functional approaches were subsequently applied to determine the role of EMMPRIN in prostate cancer cell adhesion, migration, invasion as well as cytoskeleton organization.
EMMPRIN was found to be highly expressed on the surface of prostate cancer cells compared to BPH-1 cells, consistent with a correlation between elevated EMMPRIN and metastasis found in other tumors. No significant changes in cell proliferation, cell cycle progression or apoptosis were detected in EMMPRIN knockdown cells compared to the scramble controls. Furthermore, EMMPRIN silencing markedly decreased the ability of PC-3 cells to form filopodia, a critical feature of invasive behavior, while it increased expression of cell-cell adhesion and gap junction proteins.
Our results suggest that EMMPRIN regulates cell adhesion, invasion and cytoskeleton reorganization in prostate cancer cells. This study identifies a new function for EMMPRIN as a contributor to prostate cancer cell-cell communication and cytoskeleton changes towards metastatic spread, and suggests its potential value as a marker of prostate cancer progression to metastasis.
Prostate Cancer; EMMPRIN; Cytoskeleton; shRNA; Filopodia
Human immunodeficiency virus type 1 (HIV-1) infection of the central nervous system (CNS) affects cross-talk between the individual cell types of the neurovascular unit, which then contributes to disruption of the blood–brain barrier (BBB) and the development of neurological dysfunctions. Although the toxicity of HIV-1 on neurons, astrocytes and brain endothelial cells has been widely studied, there are no reports addressing the influence of HIV-1 on pericytes. Therefore, the purpose of this study was to evaluate whether or not pericytes can be infected with HIV-1 and how such an infection affects the barrier function of brain endothelial cells. Our results indicate that human brain pericytes express the major HIV-1 receptor CD4 and co-receptors CXCR4 and CCR5. We also determined that HIV-1 can replicate, although at a low level, in human brain pericytes as detected by HIV-1 p24 ELISA. Pericytes were susceptible to infection with both the X4-tropic NL4-3 and R5-tropic JR-CSF HIV-1 strains. Moreover, HIV-1 infection of pericytes resulted in compromised integrity of an in vitro model of the BBB. These findings indicate that human brain pericytes can be infected with HIV-1 and suggest that infected pericytes are involved in the progression of HIV-1-induced CNS damage.
HIV-1; pericytes; blood–brain barrier; neurovascular unit
Occlusions of bilateral common carotid arteries (bi-CCA) in mice are popular models for the investigation of transient forebrain ischemia. Currently available technologies for assessing cerebral blood flow (CBF) and oxygenation in ischemic mice have limitations. This study tests a novel near-infrared diffuse correlation spectroscopy (DCS) flow-oximeter for monitoring both CBF and cerebral oxygenation in mice undergoing repeated transient forebrain ischemia. Concurrent flow measurements in a mouse brain were first conducted for validation purposes; DCS measurement was found highly correlated with laser Doppler measurement (R2 = 0.94) and less susceptible to motion artifacts. With unique designs in experimental protocols and fiber-optic probes, we have demonstrated high sensitivities of DCS flow-oximeter in detecting the regional heterogeneity of CBF responses in different hemispheres and global changes of both CBF and cerebral oxygenation across two hemispheres in mice undergoing repeated 2-minute bi-CCA occlusions over 5 days. More than 75% CBF reductions were found during bi-CCA occlusions in mice, which may be considered as a threshold to determine a successful bi-CCA occlusion. With the progress of repeated 2-minute bi-CCA occlusions over days, a longitudinal decline in the magnitudes of CBF reduction was observed, indicating the brain adaptation to cerebral ischemia through the repeated preconditioning.
(170.0170) Medical optics and biotechnology; (170.3660) Light propagation in tissues; (170.3880) Medical and biological imaging; (170.6480) Spectroscopy, speckle