Inhibition of glycogen synthase kinase 3beta (GSK3beta) decreases inflammatory responses in brain endothelial cells.

Immune mediators and leukocyte engagement of brain microvascular endothelial cells (BMVECs) contribute to blood-brain barrier impairment during neuroinflammation. Glycogen synthase kinase 3beta (GSK3beta) was recently identified as a potent regulator of immune responses in in vitro systems and animal models. However, the role of GSK3beta in regulation of immune endothelial functions remains undetermined. Here we evaluated the effect of GSK3beta inhibition on the regulation of inflammatory responses in BMVECs. A focused PCR gene array of 84 genes was performed to identify the cytokine and chemokine gene expression profile in tumor necrosis factor (TNF) alpha-stimulated BMVECs after GSK3beta inactivation by specific inhibitors. Fifteen of 39 genes induced by TNFalpha stimulation were down-regulated after GSK3beta inhibition. Genes known to contribute to neuroinflammation that were most negatively affected by GSK3beta inactivation included IP-10/CXCL10, MCP-1/CCL2, IL-8/CXCL8, RANTES/CCL5, and Groalpha/CXCL1. GSK3beta suppression resulted in diminished secretion of these proinflammatory mediators by inflamed BMVECs detected by ELISA. GSK3beta inhibition in BMVECs reduced adhesion molecule expression as well as monocyte adhesion to and migration across cytokine stimulated BMVEC monolayers. Interactions of monocytes with TNFalpha-activated BMVECs led to barrier disruption, and GSK3beta suppression in the endothelium restored barrier integrity. GSK3beta inhibition in vivo substantially decreased leukocyte adhesion to brain endothelium under inflammatory conditions. In summary, inhibition of GSK3beta emerges as an important target for stabilization of the blood-brain barrier in neuroinflammation.

Methamphetamine disrupts blood-brain barrier function by induction of oxidative stress in brain endothelial cells.

Methamphetamine (METH), a potent stimulant with strong euphoric properties, has a high abuse liability and long-lasting neurotoxic effects. Recent studies in animal models have indicated that METH can induce impairment of the blood-brain barrier (BBB), thus suggesting that some of the neurotoxic effects resulting from METH abuse could be the outcome of barrier disruption. In this study, we provide evidence that METH alters BBB function through direct effects on endothelial cells and explore possible underlying mechanisms leading to endothelial injury. We report that METH increases BBB permeability in vivo, and exposure of primary human brain microvascular endothelial cells (BMVEC) to METH diminishes the tightness of BMVEC monolayers in a dose- and time-dependent manner by decreasing the expression of cell membrane-associated tight junction (TJ) proteins. These changes were accompanied by the enhanced production of reactive oxygen species, increased monocyte migration across METH-treated endothelial monolayers, and activation of myosin light chain kinase (MLCK) in BMVEC. Antioxidant treatment attenuated or completely reversed all tested aspects of METH-induced BBB dysfunction. Our data suggest that BBB injury is caused by METH-mediated oxidative stress, which activates MLCK and negatively affects the TJ complex. These observations provide a basis for antioxidant protection against brain endothelial injury caused by METH exposure.

Peroxisome proliferator-activated receptor-gamma activation suppresses HIV-1 replication in an animal model of encephalitis.

OBJECTIVE: Poor penetration of antiretroviral therapy across the blood-brain barrier poses an impediment on control of HIV-1 infection in brain macrophages. Peroxisome proliferator-activated receptor (PPAR)-gamma, a member of the nuclear receptors family, regulates important physiological functions (including anti-inflammatory effects) in response to ligand-mediated activation. As PPARgamma agonists are rapidly absorbed by oral administration and efficiently permeate the blood-brain barrier, we hypothesized that PPARgamma stimulation may suppress HIV-1 replication. DESIGN AND METHODS: We investigated the effect of PPARgamma ligand (rosiglitazone) on HIV-1 replication in human monocyte-derived macrophages 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. RESULTS: Treatment with rosiglitazone caused a significant decrease of virus infection in macrophages. PPARgamma stimulation inhibited virus replication by modulating NF-kappaB activation in a receptor-dependent manner, leading to downregulation of HIV-1 long terminal repeat (LTR) promoter activity and suppression of HIV-1 replication. These effects were PPARgamma specific as PPARgamma silencing or addition of PPARgamma antagonist abolished effects of PPARgamma stimulation on HIV-1 LTR and virus replication. Using a murine model for HIV-1 encephalitis, we demonstrated that PPARgamma 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 PPARgamma activation suppresses HIV-1 replication, and in vivo findings underscored the ability of PPARgamma agonists to reduce HIV-1 replication in lymphocytes and brain macrophages, thus offering a new therapeutic intervention in brain and systemic infection.

The interaction between KSHV RTA and cellular RBP-Jkappa and their subsequent DNA binding are not sufficient for activation of RBP-Jkappa.

Kaposi's sarcoma-associated herpesvirus (KSHV) replication and transcription activator (RTA) is necessary and sufficient for the switch from KSHV latency to lytic replication. RTA activates promoters by several mechanisms. RTA can bind to sequences in viral promoters and activate transcription. In addition, RTA interacts with the cellular recombination signal sequence-binding protein-J kappa (RBP-Jkappa), a transcriptional repressor, converts the repressor into an activator and activates viral promoters via RBP-Jkappa. Because RBP-Jkappa is required for RTA to activate lytic replication, it is important to understand how RTA cooperates with RBP-Jkappa protein to activate KSHV lytic replication program. Previously, we identified an RTA mutant, RTA-K152E, which has a defect in its direct DNA-binding activity. In this report, the effect of the mutant RTA on KSHV activation via RBP-Jkappa protein is examined. We demonstrate that RTA-K152E interacts with RBP-Jkappa physically and the mutant RTA and RBP-Jkappa complex binds to target DNA properly in vivo and in vitro. However, the complex of RTA-K152E and RBP-Jkappa does not activate transcription. Furthermore, the RTA mutant (RTA-K12E) inhibits cellular Notch-mediated RBP-Jkappa activation. These data collectively suggest that the complex between KSHV RTA and cellular RBP-Jkappa and the subsequent DNA binding by the complex are not sufficient for the activation of RBP-Jkappa protein. Other factor(s) whether additional cofactor(s) in the complex or the intrinsic conformation of RTA, are predicted to be required for the activation of RBP-Jkappa protein by RTA.