Brain microvascular endothelial cells (BMVEC) connected by tight junctions (TJ) form a tight monolayer at the blood-brain barrier (BBB). We investigated the idea that BBB dysfunction seen in alcohol abuse is associated with oxidative stress stemming from ethanol (EtOH) metabolism in BMVEC. Exposure to EtOH induced catalytic activity/expression of EtOH-metabolizing enzymes, which paralleled enhanced generation of reactive oxygen species (ROS). EtOH-mediated oxidative stress led to activation of myosin light chain (MLC) kinase, phosphorylation of MLC and TJ proteins, decreased BBB integrity, and enhanced monocyte migration across BBB. Acetaldehyde or ROS donors mimicked changes induced by EtOH in BMVEC. Thus, oxidative stress resulting from alcohol metabolism in BMVEC can lead to BBB breakdown in alcohol abuse, serving as an aggravating factor in neuroinflammatory disorders.
Human immunodeficiency virus-1 (HIV-1) encephalitis is characterized by brain infiltration of virus-infected monocytes and macrophages. Cellular products and viral proteins secreted by infected cells likely play an important role in blood-brain barrier (BBB) impairment and the development of HIV-1-associated dementia (HAD). We previously demonstrated that HIV-1 envelope glycoprotein gp120 induces toxicity and alters expression of tight junction proteins in human brain microvascular endothelial cells (HBMECs). Here, we delineate the mechanisms of gp120-induced BBB dysfunction. Human brain microvascular endothelial cells expressed HIV-1 co-receptors (CCR5 and CXCR4). Exposure of HBMECs to gp120 derived from macrophage (CCR5) or lymphocyte (CXCR4)-tropic viruses decreased BBB tightness, increased permeability, and enhanced monocyte migration across in vitro BBB models. Blood-brain barrier integrity was restored after gp120 removal. CCR5 antibodies and inhibitors of myosin light chain kinase or protein kinase C (PKC) blocked gp120-enhanced monocyte migration and permeability of BBB in vitro. Exposure of HBMECs to gp120 induced release of intracellular calcium ([Ca(2+)](i)) that was prevented by CCR5 antibody and partially blocked by CXCR4 antagonist. Human immunodeficiency virus-1 gp120 activated three PKC isoforms in HBMECs [PKC-alpha/betaII, PKC(pan)-betaII and PKC-zeta/lambda]. Furthermore, specific PKC inhibitors (acting at the ATP-binding and calcium release site) blocked gp120-induced PKC activation and prevented increase in BBB permeability, supporting the biologic significance of these results. Thus, gp120 can cause dysfunction of BBB via PKC pathways and receptor mediated [Ca(2+)](i) release leading to cytoskeletal alterations and increased monocyte migration.
These results suggest that EtOH activates MLCK leading to phosphorylation of MLC, occludin and claudin-5. Cytoskeletal alterations (MLC) and TJ changes (occludin and claudin-5 phosphorylation) result in BBB impairment (decrease in TEER). TJ compromise is associated with increased monocyte migration across the BBB.
Objective
Poor penetration of anti-retroviral therapy across the blood brain barrier (BBB) poses an impediment on control of HIV-1 infection in brain macrophages. PPARγ, a member of the nuclear receptors family, regulates important physiological functions (including anti-inflammatory effects) in response to ligand-mediated activation. Since PPARγ agonists are rapidly absorbed by oral administration and efficiently permeate the BBB we hypothesized that PPARγ stimulation may suppress HIV-1 replication.
Design & Methods
We investigated the effect of PPARγ ligand (rosiglitazone) on HIV-1 replication in human monocyte-derived macrophages (MDM) and in vivo using a murine model (immunodeficient mice reconstituted with human lymphocytes and intracerebrally inoculated with HIV-1 infected macrophages) of HIV-1 encephalitis (HIVE).
Results
Treatment with rosiglitazone caused a significant decrease of virus infection in macrophages. PPARγ stimulation inhibited virus replication by modulating NF-κB activation in a receptor-depended manner, leading to down-regulation of HIV-1 LTR promoter activity and suppression of HIV-1 replication. These effects were PPARγ specific as PPARγ silencing or addition of PPARγ antagonist abolished effects of PPARγ stimulation on HIV-1 LTR and virus replication. Using a murine model for HIVE, we demonstrated that PPARγ ligand suppressed HIV-1 replication in macrophages in brain tissue and reduced viremia by 50%.
Conclusion
In vitro data delineated the novel mechanism by which PPARγ activation suppresses HIV-1 replication, and in vivo findings underscored the ability of PPARγ agonists to reduce HIV-1 replication in lymphocytes and brain macrophages offering new therapeutic intervention in brain and systemic infection.
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