Effects of Electronic Nicotine Delivery Systems and Cigarettes on Systemic Circulation and Blood-Brain Barrier: Implications for Cognitive Decline.

Electronic nicotine delivery systems (often known as e-cigarettes) are a novel tobacco product with growing popularity, particularly among younger demographics. The implications for public health are twofold, as these products may represent a novel source of tobacco-associated disease but may also provide a harm reduction strategy for current tobacco users. There is increasing recognition that e-cigarettes impact vascular function across multiple organ systems. Herein, we provide a comparison of evidence regarding the role of e-cigarettes versus combustible tobacco in vascular disease and implications for blood-brain barrier dysfunction and cognitive decline. Multiple non-nicotinic components of tobacco smoke have been identified in e-cigarette aerosol, and their involvement in vascular disease is discussed. In addition, nicotine and nicotinic signaling may modulate peripheral immune and endothelial cell populations in a highly context-dependent manner. Direct preclinical evidence for electronic nicotine delivery system-associated neurovascular impairment is provided, and a model is proposed in which non-nicotinic elements exert a proinflammatory effect that is functionally antagonized by the presence of nicotine.

Author Correction: Hyperglycemia and advanced glycation end products disrupt BBB and promote occludin and claudin-5 protein secretion on extracellular microvesicles.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Combination of HIV-1 and Diabetes Enhances Blood Brain Barrier Injury via Effects on Brain Endothelium and Pericytes.

Despite combined antiretroviral therapy (ART) achieving efficient HIV replication control, HIV-associated neurocognitive disorders (HAND) continue to be highly prevalent in HIV-infected patients. Diabetes mellitus (DM) is a well-known comorbidity of HAND in HIV-infected patients. Blood brain barrier (BBB) dysfunction has been linked recently to dementia development, specifically in DM patients. BBB injury exists both in HIV and DM, likely contributing to cognitive decline. However, its extent, exact cellular targets and mechanisms are largely unknown. In this report, we found a decrease in pericyte coverage and expression of tight junction proteins in human brain tissues from HIV patients with DM and evidence of HAND when compared to HIV-infected patients without DM or seronegative DM patients. Using our in vitro BBB models, we demonstrated diminution of barrier integrity, enhanced monocyte adhesion, changes in cytoskeleton and overexpression of adhesion molecules in primary human brain endothelial cells or human brain pericytes after exposure to HIV and DM-relevant stimuli. Our study demonstrates for the first-time evidence of impaired BBB function in HIV-DM patients and shows potential mechanisms leading to it in brain endothelium and pericytes that may result in poorer cognitive performance compared to individuals without HIV and DM.

Hyperglycemia and advanced glycation end products disrupt BBB and promote occludin and claudin-5 protein secretion on extracellular microvesicles.

Cognitive impairment is a well-known complication of diabetes mellitus (DM). Microvascular compromise was described one DM complication. Recently we showed blood brain barrier (BBB) permeability and memory loss are associated with diminution of tight junctions (TJ) in brain endothelium and pericyte coverage and inflammation in cerebral microvessels and brain tissue paralleling hyperglycemia in mice of both DM types. The current study demonstrates that exposure of brain microvessels to hyperglycemic conditions or advanced glycation end products (AGEs) ex vivo resulted in significant abnormalities in membranous distribution of TJ proteins. We found significant increase in the amount of extracellular vesicles (EVs) isolated from DM mice and enhanced presence of TJ proteins, occludin and claudin-5, on EVs. Exposure of BMVECs to high glucose and AGEs led to significant augmentation of ICAM and VCAM expression, elevated leukocyte adhesion to and migration across BMVEC monolayers, and increased BBB permeability in vitro. Pericytes exposed to hyperglycemia and AGEs displayed diminished expression of integrin α1, PDGF-R1ß and connexin-43. Our findings indicate BBB compromise in DM ex vivo, in vitro and in vivo models in association with BMVEC/pericyte dysfunction and inflammation. Prevention of BBB injury may be a new therapeutic approach to avert cognitive demise in DM.

Electronic cigarette exposure disrupts blood-brain barrier integrity and promotes neuroinflammation.

Electronic cigarette (e-cigarette) use has grown substantially since inception, particularly among adolescents and combustible tobacco users. Several cigarette smoke constituents with known neurovascular effect are present in e-cigarette liquids or formed during the vapor generation. The present study establishes inhaled models of cigarette and e-cigarette use with normalized nicotine delivery, then characterizes the impact on blood-brain barrier (BBB) function. Sequencing of microvessel RNA following exposure revealed downregulation of several genes with critical roles in BBB function. Reduced protein expression of Occludin and Glut1 is also observed at the tight junction in all groups following exposure. Pro-inflammatory changes in leukocyte-endothelial cell interaction are also noted, and mice exposed to nicotine-free e-cigarettes have impaired novel object recognition performance. On this basis, it is concluded that long term e-cigarette use may adversely impact neurovascular health. The observed effects are noted to be partly independent of nicotine content and nicotine may even serve to moderate the effects of non-nicotinic components on the blood-brain barrier.

let-7g counteracts endothelial dysfunction and ameliorating neurological functions in mouse ischemia/reperfusion stroke model.

Stroke is a debilitating disease, accounting for almost 20% of all hospital visits, and 8% of all fatalities in the United States in 2017. Following an ischemic attack, inflammatory processes originating from endothelial cells within the brain microvasculature can induce many toxic effects into the impacted area, from both sides of the blood brain barrier (BBB). In addition to increased BBB permeability, impacted brain microvascular endothelial cells can recruit macrophages and other immune cells from the periphery and can also trigger the activation of microglia and astrocytes within the brain. We have identified a key microRNA, let-7g, which levels were drastically diminished as consequence of transient middle cerebral artery occlusion (tMCAO) in vivo and oxygen-glucose deprivation (OGD) in vitro ischemia/reperfusion conditions, respectively. We have observed that let-7g* liposome-based delivery is capable of attenuating inflammation after stroke, reducing BBB permeability, limiting brain infiltration by CD3+CD4+ T-cells and Ly6G+ neutrophils, lessening microglia activation and neuronal death. These effects consequently improved clinical outcomes, shown by mitigating post-stroke gait asymmetry and extremity motor function. Due to the role of the endothelium in propagating the effects of stroke and other inflammation, treatments which can reduce endothelial inflammation and limit ischemic damage and improving recovery after a stroke are required. Our findings demonstrate a critical link between the CNS inflammation and the immune system reaction and lay important groundwork for future stroke pharmacotherapies.

miR-98 reduces endothelial dysfunction by protecting blood-brain barrier (BBB) and improves neurological outcomes in mouse ischemia/reperfusion stroke model.

Most neurological diseases, including stroke, lead to some degree of blood-brain barrier (BBB) dysfunction. A significant portion of BBB injury is caused by inflammation, due to pro-inflammatory factors produced in the brain, and by leukocyte engagement of the brain endothelium. Recently, microRNAs (miRNAs) have appeared as major regulators of inflammation-induced changes to gene expression in the microvascular endothelial cells (BMVEC) that comprise the BBB. However, miRNAs' role during cerebral ischemia/reperfusion is still underexplored. Endothelial levels of miR-98 were significantly altered following ischemia/reperfusion insults, both in vivo and in vitro, transient middle cerebral artery occlusion (tMCAO), and oxygen-glucose deprivation (OGD), respectively. Overexpression of miR-98 reduced the mouse's infarct size after tMCAO. Further, miR-98 lessened infiltration of proinflammatory Ly6CHI leukocytes into the brain following stroke and diminished the prevalence of M1 (activated) microglia within the impacted area. miR-98 attenuated BBB permeability, as demonstrated by changes to fluorescently-labeled dextran penetration in vivo and improved transendothelial electrical resistance (TEER) in vitro. Treatment with miR-98 improved significantly the locomotor impairment. Our study provides identification and functional assessment of miRNAs in brain endothelium and lays the groundwork for improving therapeutic approaches for patients suffering from ischemic attacks.

Secoisolariciresinol diglucoside is a blood-brain barrier protective and anti-inflammatory agent: implications for neuroinflammation.

BACKGROUND: Secoisolariciresinol diglucoside (SDG), the main lignan in flaxseed, is known for its beneficial effects in inflammation, oxidative stress, heart disease, tumor progression, atherosclerosis, and diabetes. SDG might be an attractive natural compound that protects against neuroinflammation. Yet, there are no comprehensive studies to date investigating the effects of SDG on brain endothelium using relevant in vivo and in vitro models. METHODS: We evaluated the effects of orally administered SDG on neuroinflammatory responses using in vivo imaging of the brain microvasculature during systemic inflammation and aseptic encephalitis. In parallel, the anti-inflammatory actions of SDG on brain endothelium and monocytes were evaluated in vitro blood-brain barrier (BBB) model. Multiple group comparisons were performed by one-way analysis of variance with Dunnet's post hoc tests. RESULTS: We found that SDG diminished leukocyte adhesion to and migration across the BBB in vivo in the setting of aseptic encephalitis (intracerebral TNFα injection) and prevented enhanced BBB permeability during systemic inflammatory response (LPS injection). In vitro SDG pretreatment of primary human brain microvascular endothelial cells (BMVEC) or human monocytes diminished adhesion and migration of monocytes across brain endothelial monolayers in conditions mimicking CNS inflammatory responses. Consistent with our in vivo observations, SDG decreased expression of the adhesion molecule, VCAM1, induced by TNFα, or IL-1ß in BMVEC. SDG diminished expression of the active form of VLA-4 integrin (promoting leukocyte adhesion and migration) and prevented the cytoskeleton changes in primary human monocytes activated by relevant inflammatory stimuli. CONCLUSION: This study indicates that SDG directly inhibits BBB interactions with inflammatory cells and reduces the inflammatory state of leukocytes. Though more work is needed to determine the mechanism by which SDG mediates these effects, the ability of SDG to exert a multi-functional response reducing oxidative stress, inflammation, and BBB permeability makes it an exciting potential therapeutic for neuroinflammatory diseases. SDG can serve as an anti-inflammatory and barrier-protective agent in neuroinflammation.

Characterization of human fetal brain endothelial cells reveals barrier properties suitable for in vitro modeling of the BBB with syngenic co-cultures.

Endothelial cells (ECs) form the basis of the blood-brain barrier (BBB), a physical barrier that selectively restricts transport into the brain. In vitro models can provide significant insight into BBB physiology, mechanisms of human disease pathology, toxicology, and drug delivery. Given the limited availability of primary human adult brain microvascular ECs ( aBMVECs), human fetal tissue offers a plausible alternative source for multiple donors and the opportunity to build syngenic tri-cultures from the same host. Previous efforts to culture fetal brain microvascular ECs ( fBMVECs) have not been successful in establishing mature barrier properties. Using optimal gestational age for isolation and flow cytometry cell sorting, we show for the first time that fBMVECs demonstrate mature barrier properties. fBMVECs exhibited similar functional phenotypes when compared to aBMVECs for barrier integrity, endothelial activation, and gene/protein expression of tight junction proteins and transporters. Importantly, we show that tissue used to culture fBMVECs can also be used to generate a syngenic co-culture, creating a microfluidic BBB on a chip. The findings presented provide a means to overcome previous challenges that limited successful barrier formation by fBMVECs. Furthermore, the source is advantageous for autologous reconstitution of the neurovascular unit for next generation in vitro BBB modeling.

PARP inhibition in leukocytes diminishes inflammation via effects on integrins/cytoskeleton and protects the blood-brain barrier.

BACKGROUND: Blood-brain barrier (BBB) dysfunction/disruption followed by leukocyte infiltration into the brain causes neuroinflammation and contributes to morbidity in multiple sclerosis, encephalitis, traumatic brain injury, and stroke. The identification of pathways that decreases the inflammatory potential of leukocytes would prevent such injury. Poly(ADP-ribose) polymerase 1 (PARP) controls various genes via its interaction with myriad transcription factors. Selective PARP inhibitors have appeared lately as potent anti-inflammatory tools. Their effects are outside the recognized PARP functions in DNA repair and transcriptional regulation. In this study, we explored the idea that selective inhibition of PARP in leukocytes would diminish their engagement of the brain endothelium. METHODS: Cerebral vascular changes and leukocyte-endothelium interactions were surveyed by intravital videomicroscopy utilizing a novel in vivo model of localized aseptic meningitis when TNFα was introduced intracerebrally in wild-type (PARP+/+) and PARP-deficient (PARP-/-) mice. The effects of selective PARP inhibition on primary human monocytes ability to adhere to or migrate across the BBB were also tested in vitro, employing primary human brain microvascular endothelial cells (BMVEC) as an in vitro model of the BBB. RESULTS: PARP suppression in monocytes diminished their adhesion to and migration across BBB in vitro models and prevented barrier injury. In monocytes, PARP inactivation decreased conformational activation of integrins that plays a key role in their tissue infiltration. Such changes were mediated by suppression of activation of small Rho GTPases and cytoskeletal rearrangements in monocytes. In vitro observations were confirmed in vivo showing diminished leukocyte-endothelial interaction after selective PARP suppression in leukocytes accompanied by BBB protection. PARP knockout animals demonstrated a substantial diminution of inflammatory responses in brain microvasculature and a decrease in BBB permeability. CONCLUSIONS: These results suggest PARP inhibition in leukocytes as a novel approach to BBB protection in the setting of endothelial dysfunction caused by inflammation-induced leukocyte engagement.

Dysfunction of brain pericytes in chronic neuroinflammation.

Brain pericytes are uniquely positioned within the neurovascular unit to provide support to blood brain barrier (BBB) maintenance. Neurologic conditions, such as HIV-1-associated neurocognitive disorder, are associated with BBB compromise due to chronic inflammation. Little is known about pericyte dysfunction during HIV-1 infection. We found decreased expression of pericyte markers in human brains from HIV-1-infected patients (even those on antiretroviral therapy). Using primary human brain pericytes, we assessed expression of pericyte markers (α1-integrin, α-smooth muscle actin, platelet-derived growth factor-B receptor ß, CX-43) and found their downregulation after treatment with tumor necrosis factor-α (TNFα) or interleukin-1 ß (IL-1ß). Pericyte exposure to virus or cytokines resulted in decreased secretion of factors promoting BBB formation (angiopoietin-1, transforming growth factor-ß1) and mRNA for basement membrane components. TNFα and IL-1ß enhanced expression of adhesion molecules in pericytes paralleling increased monocyte adhesion to pericytes. Monocyte migration across BBB models composed of human brain endothelial cells and pericytes demonstrated a diminished rate in baseline migration compared to constructs composed only of brain endothelial cells. However, exposure to the relevant chemokine, CCL2, enhanced the magnitude of monocyte migration when compared to BBB models composed of brain endothelial cells only. These data suggest an important role of pericytes in BBB regulation in neuroinflammation.

The dual action of poly(ADP-ribose) polymerase -1 (PARP-1) inhibition in HIV-1 infection: HIV-1 LTR inhibition and diminution in Rho GTPase activity.

Multifactorial mechanisms comprising countless cellular factors and virus-encoded transactivators regulate the transcription of HIV-1 (HIV). Since poly(ADP-ribose) polymerase 1 (PARP-1) regulates numerous genes through its interaction with various transcription factors, inhibition of PARP-1 has surfaced recently as a powerful anti-inflammatory tool. We suggest a novel tactic to diminish HIV replication via PARP-1 inhibition in an in vitro model system, exploiting human primary monocyte-derived macrophages (MDM). PARP-1 inhibition was capable to lessen HIV replication in MDM by 60-80% after 7 days infection. Tat, tumor necrosis factor α (TNFα), and phorbol 12-myristate 13-acetate (PMA) are known triggers of the Long Terminal Repeat (LTR), which can switch virus replication. Tat overexpression in MDM transfected with an LTR reporter plasmid resulted in a 4.2-fold increase in LTR activation; PARP inhibition caused 70% reduction of LTR activity. LTR activity, which increased 3-fold after PMA or TNFα treatment, was reduced by PARP inhibition (by 85-95%). PARP inhibition in MDM exhibited 90% diminution in NFκB activity (known to mediate TNFα- and PMA-induced HIV LTR activation). Cytoskeleton rearrangements are important in effective HIV-1 infection. PARP inactivation reduced actin cytoskeleton rearrangements by affecting Rho GTPase machinery. These discoveries suggest that inactivation of PARP suppresses HIV replication in MDM by via attenuation of LTR activation, NFκB suppression and its effects on the cytoskeleton. PARP appears to be essential for HIV replication and its inhibition may provide an effective approach to management of HIV infection.

miR-98 and let-7g* protect the blood-brain barrier under neuroinflammatory conditions.

Pathologic conditions in the central nervous system, regardless of the underlying injury mechanism, show a certain level of blood-brain barrier (BBB) impairment. Endothelial dysfunction is the earliest event in the initiation of vascular damage caused by inflammation due to stroke, atherosclerosis, trauma, or brain infections. Recently, microRNAs (miRNAs) have emerged as a class of gene expression regulators. The relationship between neuroinflammation and miRNA expression in brain endothelium remains unexplored. Previously, we showed the BBB-protective and anti-inflammatory effects of glycogen synthase kinase (GSK) 3ß inhibition in brain endothelium in in vitro and in vivo models of neuroinflammation. Using microarray screening, we identified miRNAs induced in primary human brain microvascular endothelial cells after exposure to the pro-inflammatory cytokine, tumor necrosis factor-α, with/out GSK3ß inhibition. Among the highly modified miRNAs, let-7 and miR-98 were predicted to target the inflammatory molecules, CCL2 and CCL5. Overexpression of let-7 and miR-98 in vitro and in vivo resulted in reduced leukocyte adhesion to and migration across endothelium, diminished expression of pro-inflammatory cytokines, and increased BBB tightness, attenuating barrier 'leakiness' in neuroinflammation conditions. For the first time, we showed that miRNAs could be used as a therapeutic tool to prevent the BBB dysfunction in neuroinflammation.

Activation of Cannabinoid Type Two Receptors (CB2) Diminish Inflammatory Responses in Macrophages and Brain Endothelium.

Chronic neuroinflammatory disorders (such as HIV associated neurodegeneration) require treatment that decreases production of inflammatory factors by activated microglia and macrophages and protection of blood brain barrier (BBB) injury secondary to activation of brain endothelium. Cannabioid type 2 receptor (CB2) is highly expressed on macrophages and brain microvasular enndothelial cells (BMVEC) and is upregulated in inflammation and HIV infection. It has been shown that CB2 activation dampened inflammatory responses in macrophages and BMVEC. In this study, we assessed by PCR array the expression of a wide range of genes increased in macrophages and BMVEC in inflammation. TNFα treatment upregulated 33 genes in primary human BMVEC, and two highly selective CB2 agonists diminished expression of 31 and 32 genes. These results were confirmed by functional assays (BBB protection after inflammatory insult and decreased migration of monocytes across BMVEC monolayers after CB2 stimulation). Similarly, CB2 stimulation in primary human macrophages led to the suppression of 35 genes out of the 50 genes upregulated by LPS. Such changes in gene expression paralleled diminished secretion of proinflammatory factors. These results indicate the potential utility of CB2 agonists for the treatment of neuroinflammation.

Heroin use promotes HCV infection and dysregulates HCV-related circulating microRNAs.

Hepatitis C virus (HCV) infection is common among injection drug users (IDUs). There is accumulating evidence that circulating microRNAs (miRNAs) are associated with HCV infection and disease progression. The present study was undertaken to determine the in vivo impact of heroin use on HCV infection and HCV-related circulating miRNA expression. Using the blood specimens from four groups of the study subjects (HCV-infected individuals, heroin users with/without HCV infection, and healthy volunteers), we found that HCV-infected heroin users had significantly higher viral load than HCV-infected non-heroin users (p = 0.0004). Measurement of HCV-related circulating miRNAs in plasma showed that miRs-122, 141, 29a, 29b, and 29c were significantly increased in the heroin users with HCV infection, whereas miR-351, an HCV inhibitory miRNA, was significantly decreased in heroin users as compared to control subjects. Further investigation identified a negative correlation between the plasma levels of miR-29 family members and severity of HCV infection based on aspartate aminotransferase to platelet ratio index (APRI). In addition, heroin use and/or HCV infection also dysregulated a panel of plasma miRNAs. Taken together, these data for the first time revealed in vivo evidence that heroin use and/or HCV infection alter circulating miRNAs, which provides a novel mechanism for the impaired innate anti-HCV immunity among IDUs.

Poly(ADP-ribose) polymerase-1 inhibition in brain endothelium protects the blood-brain barrier under physiologic and neuroinflammatory conditions.

Blood-brain barrier (BBB) dysfunction seen in neuroinflammation contributes to mortality and morbidity in multiple sclerosis, encephalitis, traumatic brain injury, and stroke. Identification of molecular targets maintaining barrier function is of clinical relevance. We used a novel in vivo model of localized aseptic meningitis where tumor necrosis factor alpha (TNFα) was introduced intracerebrally and surveyed cerebral vascular changes and leukocyte-endothelium interactions by intravital videomicroscopy. Poly(ADP-ribose) polymerase-1 (PARP) inhibition significantly reduced leukocyte adhesion to and migration across brain endothelium in cortical microvessels. PARP inactivation diminished BBB permeability in an in vivo model of systemic inflammation. PARP suppression in primary human brain microvascular endothelial cells (BMVEC), an in vitro model of BBB, enhanced barrier integrity and augmented expression of tight junction proteins. PARP inhibition in BMVEC diminished human monocyte adhesion to TNFα-activated BMVEC (up to 65%) and migration (80-100%) across BBB models. PARP suppression decreased expression of adhesion molecules and decreased activity of GTPases (controlling BBB integrity and monocyte migration across the BBB). PARP inhibitors down-regulated expression of inflammatory genes and dampened secretion of pro-inflammatory factors increased by TNFα in BMVEC. These results point to PARP suppression as a novel approach to BBB protection in the setting of endothelial dysfunction caused by inflammation.

Selective activation of cannabinoid receptor 2 in leukocytes suppresses their engagement of the brain endothelium and protects the blood-brain barrier.

Cannabinoid receptor 2 (CB2) is highly expressed in immune cells and stimulation decreases inflammatory responses. We tested the idea that selective CB2 activation in human monocytes suppresses their ability to engage the brain endothelium and migrate across the blood-brain barrier (BBB), preventing consequent injury. Intravital videomicroscopy was used to quantify adhesion of leukocytes to cortical vessels in lipopolysaccharide-induced neuroinflammation, after injection of ex vivo CB2-activated leukocytes into mice; CB2 agonists markedly decreased adhesion of ex vivo labeled cells in vivo. In an in vitro BBB model, CB2 activation in monocytes largely attenuated adhesion to and migration across monolayers of primary human brain microvascular endothelial cells and diminished BBB damage. CB2 stimulation in monocytes down-regulated active forms of integrins, lymphocyte function-associated antigen 1 (LFA-1), and very late antigen 4 (VLA-4). Cells treated with CB2 agonists exhibited increased phosphorylation levels of inhibitory sites of the actin-binding proteins cofilin and VASP, which are upstream regulators of conformational integrin changes. Up-regulated by relevant stimuli, Rac1 and RhoA were suppressed by CB2 agonists in monocytes. CB2 stimulation decreased formation of lamellipodia, which play a key role in monocyte migration. These results indicate that selective CB2 activation in leukocytes decreases key steps in monocyte-BBB engagement, thus suppressing inflammatory leukocyte responses and preventing neuroinflammation.

Attenuation of HIV-1 replication in macrophages by cannabinoid receptor 2 agonists.

Infiltrating monocytes and macrophages play a crucial role in the progression of HIV-1 infection in the CNS. Previous studies showed that activation of the CB2 can attenuate inflammatory responses and affect HIV-1 infectivity in T cells and microglia. Here, we report that CB2 agonists can also act as immunomodulators on HIV-1-infected macrophages. First, our findings indicated the presence of elevated levels of CB2 expression on monocytes/macrophages in perivascular cuffs of postmortem HIV-1 encephalitic cases. In vitro analysis by FACS of primary human monocytes revealed a step-wise increase in CB2 surface expression in monocytes, MDMs, and HIV-1-infected MDMs. We next tested the notion that up-regulation of CB2 may allow for the use of synthetic CB2 agonist to limit HIV-1 infection. Two commercially available CB2 agonists, JWH133 and GP1a, and a resorcinol-based CB2 agonist, O-1966, were evaluated. Results from measurements of HIV-1 RT activity in the culture media of 7 day-infected cells showed a significant decrease in RT activity when the CB2 agonist was present. Furthermore, CB2 activation also partially inhibited the expression of HIV-1 pol. CB2 agonists did not modulate surface expression of CXCR4 or CCR5 detected by FACS. We speculate that these findings indicate that prevention of viral entry is not a central mechanism for CB2-mediated suppression in viral replication. However, CB2 may affect the HIV-1 replication machinery. Results from a single-round infection with the pseudotyped virus revealed a marked decrease in HIV-1 LTR activation by the CB2 ligands. Together, these results indicate that CB2 may offer a means to limit HIV-1 infection in macrophages.

Inhibition of glycogen synthase kinase 3ß promotes tight junction stability in brain endothelial cells by half-life extension of occludin and claudin-5.

Neuroinflammatory conditions often involve dysfunction of the Blood-Brain Barrier (BBB). Therefore, identifying molecular targets that can maintain barrier fidelity is of clinical importance. We have previously reported on the anti-inflammatory effects that glycogen synthase kinase 3ß (GSK3ß) inhibition has on primary human brain endothelial cells. Here we show that GSK3ß inhibitors also promote barrier tightness by affecting tight junction (TJ) protein stability. Transendothelial electrical resistance (TEER) was used to evaluate barrier integrity with both pharmacological inhibitors and mutants of GSK3ß. Inhibition of GSK3ß produced a gradual and sustained increase in TEER (as much as 22% over baseline). Analysis of subcellular membrane fractions revealed an increase in the amount of essential tight junction proteins, occludin and claudin-5, but not claudin-3. This phenomenon was attributed to a decrease in TJ protein turnover and not transcriptional regulation. Using a novel cell-based assay, inactivation of GSK3ß significantly increased the half-life of occludin and claudin-5 by 32% and 43%, respectively. A correlation was also established between the enhanced association of ß-catenin with ZO-1 as a function of GSK3ß inhibition. Collectively, our findings suggest the possibility of using GSK3ß inhibitors as a means to extend the half-life of key tight junction proteins to promote re-sealing of the BBB during neuroinflammation.

Glycogen synthase kinase 3ß inhibition prevents monocyte migration across brain endothelial cells via Rac1-GTPase suppression and down-regulation of active integrin conformation.

Glycogen synthase kinase (GSK) 3ß has been identified as a regulator of immune responses. We demonstrated previously that GSK3ß inhibition in human brain microvascular endothelial cells (BMVECs) reduced monocyte adhesion/migration across BMVEC monolayers. Herein, we tested the idea that GSK3ß inhibition in monocytes can diminish their ability to engage the brain endothelium and migrate across the blood-brain barrier. Pretreatment of primary monocytes with GSK3ß inhibitors resulted in a decrease in adhesion (60%) and migration (85%), with similar results in U937 monocytic cells. Monocyte-BMVEC interactions resulted in diminished barrier integrity that was reversed by GSK3ß suppression in monocytic cells. Because integrins mediate monocyte rolling/adhesion, we detected the active conformational form of very late antigen 4 after stimulation with a peptide mimicking monocyte engagement by vascular cell adhesion molecule-1. Peptide stimulation resulted in a 14- to 20-fold up-regulation of the active form of integrin in monocytes that was suppressed by GSK3ß inhibitors (40% to 60%). Because small GTPases, such as Rac1, control leukocyte movement, we measured active Rac1 after monocyte activation with relevant stimuli. Stimulation enhanced the level of active Rac1 that was diminished by GSK3ß inhibitors. Monocytes treated with GSK3ß inhibitors showed increased levels of inhibitory sites of the actin-binding protein, cofilin, and vasodilator-stimulated phosphoprotein-regulating conformational changes of integrins. These results indicate that GSK3ß inhibition in monocytes affects active integrin expression, cytoskeleton rearrangement, and adhesion via suppression of Rac1-diminishing inflammatory leukocyte responses.