Targeted Nanocarriers Co-Opting Pulmonary Intravascular Leukocytes for Drug Delivery to the Injured Brain.

Ex vivo-loaded white blood cells (WBC) can transfer cargo to pathological foci in the central nervous system (CNS). Here we tested affinity ligand driven in vivo loading of WBC in order to bypass the need for ex vivo WBC manipulation. We used a mouse model of acute brain inflammation caused by local injection of tumor necrosis factor alpha (TNF-α). We intravenously injected nanoparticles targeted to intercellular adhesion molecule 1 (anti-ICAM/NP). We found that (A) at 2 h, >20% of anti-ICAM/NP were localized to the lungs; (B) of the anti-ICAM/NP in the lungs >90% were associated with leukocytes; (C) at 6 and 22 h, anti-ICAM/NP pulmonary uptake decreased; (D) anti-ICAM/NP uptake in brain increased up to 5-fold in this time interval, concomitantly with migration of WBCs into the injured brain. Intravital microscopy confirmed transport of anti-ICAM/NP beyond the blood-brain barrier and flow cytometry demonstrated complete association of NP with WBC in the brain (98%). Dexamethasone-loaded anti-ICAM/liposomes abrogated brain edema in this model and promoted anti-inflammatory M2 polarization of macrophages in the brain. In vivo targeted loading of WBC in the intravascular pool may provide advantages of coopting WBC predisposed to natural rapid mobilization from the lungs to the brain, connected directly via conduit vessels.

Centrally Acting Angiotensin-Converting Enzyme Inhibitor Suppresses Type I Interferon Responses and Decreases Inflammation in the Periphery and the CNS in Lupus-Prone Mice.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by multi-organ damage. Neuropsychiatric lupus (NPSLE) is one of the most common manifestations of human SLE, often causing depression. Interferon-α (IFNα) is a central mediator in disease pathogenesis. Administration of IFNα to patients with chronic viral infections or cancers causes depressive symptoms. Angiotensin-converting enzyme (ACE) is part of the kallikrein-kinin/renin-angiotensin (KKS/RAS) system that regulates many physiological processes, including inflammation, and brain functions. It is known that ACE degrades bradykinin (BK) into inactive peptides. We have previously shown in an in vitro model of mouse bone-marrow-derived dendritic cells (BMDC) and human peripheral blood mononuclear cells that captopril (a centrally acting ACE inhibitor-ACEi) suppressed Type I IFN responsive gene (IRG) expression. In this report, we used the MRL/lpr lupus-prone mouse model, an established model to study NPSLE, to determine the in vivo effects of captopril on Type I IFN and associated immune responses in the periphery and brain and effects on behavior. Administering captopril to MRL/lpr mice decreased expression of IRGs in brain, spleen and kidney, decreased circulating and tissue IFNα levels, decreased microglial activation (IBA-1 expression) and reduced depressive-like behavior. Serotonin levels that are decreased in depression were increased by captopril treatment. Captopril also reduced autoantibody levels in plasma and immune complex deposition in kidney and brain. Thus, ACEi's may have potential for therapeutic use for systemic and NPSLE.

Tobacco smoke and morphine alter peripheral and CNS inflammation following HIV infection in a humanized mouse model.

Tobacco smoking is common in HIV-infected patients, and is prevalent among intravenous opiate abusers. Conversely, intravenous opiate abusers are more likely HIV-infected, and opiate abuse is associated with more severe neuroinflammation. Given the coincident use of tobacco smoking among HIV-infected intravenous drug users (IVDUs), we set out to study the effects of smoke exposure, chronic morphine administration, and HIV infection using the NSG humanized mouse model. Our results show that smoke, morphine, and the combination promotes the decline in CD4+ T cells in HIV-infected mice. Further, chronic morphine administration increases the numbers of circulating CD8+ T cells which express the inhibitory receptor PD-1, as well as the cytolytic proteins perforin and granzyme B in the infected mice. We also found that the combination of smoke and morphine inhibited the expression of IL-1α, IL-4 and IL-17A. Finally, the combination of smoke and morphine exposure induces microglial activation following infection, as well as in the absence of HIV infection. To our knowledge, this is the first report to assess the combined effects of smoke and chronic morphine exposure on the inflammation associated with HIV infection, and demonstrate that these two insults exert significant neuroinflammatory activity.

Selective targeting of nanomedicine to inflamed cerebral vasculature to enhance the blood-brain barrier.

Drug targeting to inflammatory brain pathologies such as stroke and traumatic brain injury remains an elusive goal. Using a mouse model of acute brain inflammation induced by local tumor necrosis factor alpha (TNFα), we found that uptake of intravenously injected antibody to vascular cell adhesion molecule 1 (anti-VCAM) in the inflamed brain is >10-fold greater than antibodies to transferrin receptor-1 and intercellular adhesion molecule 1 (TfR-1 and ICAM-1). Furthermore, uptake of anti-VCAM/liposomes exceeded that of anti-TfR and anti-ICAM counterparts by ∼27- and ∼8-fold, respectively, achieving brain/blood ratio >300-fold higher than that of immunoglobulin G/liposomes. Single-photon emission computed tomography imaging affirmed specific anti-VCAM/liposome targeting to inflamed brain in mice. Intravital microscopy via cranial window and flow cytometry showed that in the inflamed brain anti-VCAM/liposomes bind to endothelium, not to leukocytes. Anti-VCAM/LNP selectively accumulated in the inflamed brain, providing de novo expression of proteins encoded by cargo messenger RNA (mRNA). Anti-VCAM/LNP-mRNA mediated expression of thrombomodulin (a natural endothelial inhibitor of thrombosis, inflammation, and vascular leakage) and alleviated TNFα-induced brain edema. Thus VCAM-directed nanocarriers provide a platform for cerebrovascular targeting to inflamed brain, with the goal of normalizing the integrity of the blood-brain barrier, thus benefiting numerous brain pathologies.

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.

Combining vascular targeting and the local first pass provides 100-fold higher uptake of ICAM-1-targeted vs untargeted nanocarriers in the inflamed brain.

New advances in intra-arterial (IA) catheters offer clinically proven local interventions in the brain. Here we tested the effect of combining local IA delivery and vascular immunotargeting. Microinjection of tumor necrosis factor alpha (TNFα) in the brain parenchyma causes cerebral overexpression of Inter-Cellular Adhesion Molecule-1 (ICAM-1) in mice. Systemic intravenous injection of ICAM-1 antibody (anti-ICAM-1) and anti-ICAM-1/liposomes provided nearly an order of magnitude higher uptake in the inflamed vs normal brain (from ~0.1 to 0.8%ID/g for liposomes). Local injection of anti-ICAM-1 and anti-ICAM-1/liposomes via carotid artery catheter provided an additional respective 2-fold and 5-fold elevation of uptake in the inflamed brain vs levels attained by IV injection. The uptake in the inflamed brain of respective untargeted IgG counterparts was markedly lower (e.g., uptake of anti-ICAM-1/liposomes was 100-fold higher vs IgG/liposomes). These data affirm the specificity of the combined effect of the first pass and immunotargeting. Intravital real-time microscopy via cranial window revealed that anti-ICAM-1/liposomes, but not IgG/liposomes bind to the lumen of blood vessels in the inflamed brain within minutes after injection. This straightforward framework provides the basis for translational efforts towards local vascular drug targeting to the brain.

Chronic Intrahippocampal Infusion of HIV-1 Neurotoxic Proteins: A Novel Mouse Model of HIV-1 Associated Inflammation and Neural Stem Cell Dysfunction.

HIV-1 infection causes chronic neuroinflammation resulting in cognitive decline associated with diminution of survival of neural stem cells (NSC). In part, this is attributable to production of toxic viral proteins (gp120 and tat) by infected cells in the brain that can activate microglia. Here, we evaluated a novel model for HIV-1 neuropathogenesis by direct administration of viral proteins into the hippocampus. Chronic administration of either HIV-1 gp120 or tat over 14 days significantly decreased NSC proliferation, survival and neuroblast formation (by 32-37%) within the hippocampal subgranular zone as detected by doublecortin/BrdU or Ki67-positive cells. Intrahippocampal administration of gp120 or tat induced microglial activation within the hippocampus as determined by increases in microglial number and increases in the volume of the microglia (2.5-3-fold, evaluated by double IBA-1/CD68 staining). We further assessed inflammatory responses within the hippocampus by RNAseq and Ingenuity Pathway Analysis. There was a significant mRNA upregulation of numerous inflammatory mediators including Il1b, Icam1, Il12a, Ccl2, and Ccl4. These data suggest that chronic administration induces a prolonged inflammatory state within the hippocampus that negatively affects NSC survival potentially leading to cognitive dysfunction. Graphical Abstract.

Activation of GPR55 induces neuroprotection of hippocampal neurogenesis and immune responses of neural stem cells following chronic, systemic inflammation.

New neurons are continuously produced by neural stem cells (NSCs) within the adult hippocampus. Numerous diseases, including major depressive disorder and HIV-1 associated neurocognitive disorder, are associated with decreased rates of adult neurogenesis. A hallmark of these conditions is a chronic release of neuroinflammatory mediators by activated resident glia. Recent studies have shown a neuroprotective role on NSCs of cannabinoid receptor activation. Yet, little is known about the effects of GPR55, a candidate cannabinoid receptor, activation on reductions of neurogenesis in response to inflammatory insult. In the present study, we examined NSCs exposed to IL-1ß in vitro to assess inflammation-caused effects on NSC differentiation and the ability of GPR55 agonists to attenuate NSC injury. NSC differentiation and neurogenesis was determined via immunofluorescence and flow cytometric analysis of NSC markers (Nestin, Sox2, DCX, S100ß, ßIII Tubulin, GFAP). GPR55 agonist treatment protected against IL-1ß induced reductions in neurogenesis rates. Moreover, inflammatory cytokine receptor mRNA expression was down regulated by GPR55 activation in a neuroprotective manner. To determine inflammatory responses in vivo, we treated C57BL/6 and GPR55-/- mice with LPS (0.2 mg/kg/day) continuously for 14 days via osmotic mini-pump. Reductions in NSC survival (as determined by BrdU incorporation), immature neurons, and neuroblast formation due to LPS were attenuated by concurrent direct intrahippocampal administration of the GPR55 agonist, O-1602 (4 µg/kg/day). Molecular analysis of the hippocampal region showed a suppressed ability to regulate immune responses by GPR55-/- animals manifesting in a prolonged inflammatory response (IL-1ß, IL-6, TNFα) after chronic, systemic inflammation as compared to C57BL/6 animals. Taken together, these results suggest a neuroprotective role of GPR55 activation on NSCs in vitro and in vivo and that GPR55 provides a novel therapeutic target against negative regulation of hippocampal neurogenesis by inflammatory insult.

Hyperglycemia-Driven Neuroinflammation Compromises BBB Leading to Memory Loss in Both Diabetes Mellitus (DM) Type 1 and Type 2 Mouse Models.

End organ injury in diabetes mellitus (DM) is driven by microvascular compromise (including diabetic retinopathy and nephropathy). Cognitive impairment is a well-known complication of DM types 1 and 2; however, its mechanism(s) is(are) not known. We hypothesized that blood-brain barrier (BBB) compromise plays a key role in cognitive decline in DM. Using a DM type 1 model (streptozotocin injected C57BL/6 mice) and type 2 model (leptin knockout obese db/db mice), we showed enhanced BBB permeability and memory loss (Y maze, water maze) that are associated with hyperglycemia. Gene profiling in isolated microvessels from DM type 1 animals demonstrated deregulated expression of 54 genes related to angiogenesis, inflammation, vasoconstriction/vasodilation, and platelet activation pathways by at least 2-fold (including eNOS, TNFα, TGFß1, VCAM-1, E-selectin, several chemokines, and MMP9). Further, the magnitude of gene expression was linked to degree of cognitive decline in DM type 1 animals. Gene analysis in brain microvessels of DM type 2 db/db animals showed alterations of similar genes as in DM 1 model, some to an even greater extent. Neuropathologic analyses of brain tissue derived from DM mice showed microglial activation, expression of ICAM-1, and attenuated coverage of pericytes compared to controls. There was a significant upregulation of inflammatory genes in brain tissue in both DM models. Taken together, our findings indicate that BBB compromise in DM in vivo models and its association with memory deficits, gene alterations in brain endothelium, and neuroinflammation. Prevention of BBB injury may be a new therapeutic approach to prevent cognitive demise in DM.

Activation of GPR55 increases neural stem cell proliferation and promotes early adult hippocampal neurogenesis.

BACKGROUND AND PURPOSE: The cannabinoid system exerts functional regulation of neural stem cell (NSC) proliferation and adult neurogenesis, yet not all effects of cannabinoid-like compounds seen can be attributed to the cannabinoid 1 (CB1 ) or CB2 receptor. The recently de-orphaned GPR55 has been shown to be activated by numerous cannabinoid ligands suggesting that GPR55 is a third cannabinoid receptor. Here, we examined the role of GPR55 activation in NSC proliferation and early adult neurogenesis. EXPERIMENTAL APPROACH: The effects of GPR55 agonists (LPI, O-1602, ML184) on human (h) NSC proliferation in vitro were assessed by flow cytometry. Human NSC differentiation was determined by flow cytometry, qPCR and immunohistochemistry. Immature neuron formation in the hippocampus of C57BL/6 and GPR55-/- mice was evaluated by immunohistochemistry. KEY RESULTS: Activation of GPR55 significantly increased proliferation rates of hNSCs in vitro. These effects were attenuated by ML193, a selective GPR55 antagonist. ML184 significantly promoted neuronal differentiation in vitro while ML193 reduced differentiation rates as compared to vehicle treatment. Continuous administration of O-1602 into the hippocampus via a cannula connected to an osmotic pump resulted in increased Ki67+ cells within the dentate gyrus. O-1602 increased immature neuron generation, as assessed by DCX+ and BrdU+ cells, as compared to vehicle-treated animals. GPR55-/- animals displayed reduced rates of proliferation and neurogenesis within the hippocampus while O-1602 had no effect as compared to vehicle controls. CONCLUSIONS AND IMPLICATIONS: Together, these findings suggest GPR55 activation as a novel target and strategy to regulate NSC proliferation and adult neurogenesis.

Kallikrein-Kinin System Suppresses Type I Interferon Responses: A Novel Pathway of Interferon Regulation.

The Kallikrein-Kinin System (KKS), comprised of kallikreins (klks), bradykinins (BKs) angiotensin-converting enzyme (ACE), and many other molecules, regulates a number of physiological processes, including inflammation, coagulation, angiogenesis, and control of blood pressure. In this report, we show that KKS regulates Type I IFN responses, thought to be important in lupus pathogenesis. We used CpG (TLR9 ligand), R848 (TLR7 ligand), or recombinant IFN-α to induce interferon-stimulated genes (ISGs) and proteins, and observed that this response was markedly diminished by BKs, klk1 (tissue kallikrein), or captopril (an ACE inhibitor). BKs significantly decreased the ISGs induced by TLRs in vitro and in vivo (in normal and lupus-prone mice), and in human PBMCs, especially the induction of Irf7 gene (p < 0.05), the master regulator of Type I IFNs. ISGs induced by IFN-α were also suppressed by the KKS. MHC Class I upregulation, a classic response to Type I IFNs, was reduced by BKs in murine dendritic cells (DCs). BKs decreased phosphorylation of STAT2 molecules that mediate IFN signaling. Among the secreted pro-inflammatory cytokines/chemokines analyzed (IL-6, IL12p70, and CXCL10), the strongest suppressive effect was on CXCL10, a highly Type I IFN-dependent cytokine, upon CpG stimulation, both in normal and lupus-prone DCs. klks that break down into BKs, also suppressed CpG-induced ISGs in murine DCs. Captopril, a drug that inhibits ACE and increases BK, suppressed ISGs, both in mouse DCs and human PBMCs. The effects of BK were reversed with indomethacin (compound that inhibits production of PGE2), suggesting that BK suppression of IFN responses may be mediated via prostaglandins. These results highlight a novel regulatory mechanism in which members of the KKS control the Type I IFN response and suggest a role for modulators of IFNs in the pathogenesis of lupus and interferonopathies.

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.

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.

Craniula: A cranial window technique for prolonged imaging of brain surface vasculature with simultaneous adjacent intracerebral injection.

BACKGROUND: Imaging of the brain surface vasculature following inflammatory insults is critical to study structural and functional changes in the living brain under normal and pathological conditions. Although there have been published reports relating to the changes that occur in the blood brain barrier (BBB) during the inflammatory process, the ability to visualize and track such changes in vivo and over time has proven to be problematic. Different techniques have been used to achieve visualization of pial vessels, but the approach has limits, which can jeopardize the well-being of the animals. Development of the cranial window technique provided a major advance in the acquisition of live images of the brain vasculature and its response to different insults and treatments. METHODS: We describe in detail a protocol for delivery of a localized inflammatory insult to the mouse brain via a craniula (cranial window and adjacent cannula) and subsequent imaging of the mouse brain vasculature by intravital microscopy and two-photon laser scanning microscopy. The surgical implantation of the craniula can be completed in 30-45 min and images can be acquired immediately and for several months thereafter. The technique is minimally invasive and permits serial injections directly to the brain, thereby allowing longitudinal imaging studies. The craniula technique permits the study of structural and functional changes of the BBB following inflammatory insult and as such has wide application to neuroscience research.

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.

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.