Nancy L. Reichenbach

Activation of Cannabinoid Receptor 2 Attenuates Leukocyte–Endothelial Cell Interactions and Blood–Brain Barrier Dysfunction under Inflammatory Conditions

Previous studies have shown that modulation of the receptor-mediated cannabinoid system during neuroinflammation can produce potent neuroprotective and anti-inflammatory effects. However, in this context, little is known about how selective activation of the cannabinoid type-2 receptor (CB2R) affects the activated state of the brain endothelium and blood–brain barrier (BBB) function. Using human brain tissues and primary human brain microvascular endothelial cells (BMVECs), we demonstrate that the CB2R is highly upregulated during inflammatory insult. We then examined whether the CB2R agonists could attenuate inflammatory responses at the BBB using a mouse model of LPS-induced encephalitis and highly selective CB2R agonists. Visualization by intravital microscopy revealed that administration of JWH133 [(6aR,10aR)-3-(1,1-dimethylbutyl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyran] or a novel resorcinol-based compound, O-1966 (1-[4-(1,1-dimethyl-heptyl)-2,6-dimethoxy-phenyl]-3-methyl-cyclohexanol), greatly attenuated leukocyte adhesion in surface pial vessels and in deep ascending cortical postcapillary venules. BBB permeability assessments with small and large fluorescent tracers showed that CB2R agonists were effective at preventing barrier leakiness after LPS administration. To determine whether the effects by CB2R agonists on barrier protection are not only due to the CB2R modulation of immune cell function, we tested the agonists in vitro with barrier-forming primary BMVECs. Remarkably, the addition of CB2R agonist increased transendothelial electrical resistance and increased the amount of tight junction protein present in membrane fractions. Furthermore, CB2R agonists decreased the induction of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 surface expression in BMVECs exposed to various proinflammatory mediators. Together, these results suggest that pharmacological CB2R ligands offer a new strategy for BBB protection during 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.

Inhibition of Glycogen Synthase Kinase 3β (GSK3β) 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.

Dyad of CD40/CD40 Ligand Fosters Neuroinflammation at the Blood–Brain Barrier and Is Regulated via JNK Signaling: Implications for HIV-1 Encephalitis

Human immunodeficiency virus 1 (HIV-1) infection may result in activation of peripheral monocytes followed by their infiltration into the CNS, where the release of proinflammatory mediators causes neurologic disease. Previously, we detected high levels of soluble CD40 ligand (CD40L) in CSF and plasma of HIV-infected patients with cognitive impairment. We now show that CD40, a receptor for CD40L, is highly expressed in brain endothelial cells of patients affected by HIV-1 encephalitis (HIVE), suggesting an important role for the CD40/CD40L dyad in regulating blood–brain barrier (BBB) functions. This concept was further supported by in vitro experiments. Exposure of primary human brain microvascular endothelial cells (BMVECs) to CD40L upregulated the expression of adhesion molecules intracellular adhesion molecule-1 and vascular cell adhesion molecule-1, which caused a fourfold increase in monocyte adhesion to BMVECs and stimulated migration across an in vitro BBB model. Investigations into the intracellular signaling pathways that govern these events revealed that cJUN-N-terminal kinase (JNK) is critical to CD40 activation in the BMVECs. CD40L induced activation of mixed-lineage-kinase-3 and JNK, leading to the subsequent activation of cJUN/AP-1 (activating-protein-1). JNK inhibition in the BMVECs prevented CD40L-mediated induction of adhesion molecules, monocyte adhesion, and transendothelial migration. These new findings support the concept that the CD40/CD40L dyad plays an important role in HIVE neuroinflammation.

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.

Inhibition of Glycogen Synthase Kinase 3β 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 3β (GSK3β) inhibition has on primary human brain endothelial cells. Here we show that GSK3β 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 GSK3β. Inhibition of GSK3β 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 GSK3β 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 β-catenin with ZO-1 as a function of GSK3β inhibition. Collectively, our findings suggest the possibility of using GSK3β inhibitors as a means to extend the half-life of key tight junction proteins to promote re-sealing of the BBB during neuroinflammation.

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) 3β 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 GSK3β 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.

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.

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.

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 β, CX-43) and found their downregulation after treatment with tumor necrosis factor-α (TNFα) or interleukin-1 β (IL-1β). Pericyte exposure to virus or cytokines resulted in decreased secretion of factors promoting BBB formation (angiopoietin-1, transforming growth factor-β1) and mRNA for basement membrane components. TNFα and IL-1β 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.