Combination of cGMP analogue and drug delivery system provides functional protection in hereditary retinal degeneration.

Inherited retinal degeneration (RD) is a devastating and currently untreatable neurodegenerative condition that leads to loss of photoreceptor cells and blindness. The vast genetic heterogeneity of RD, the lack of "druggable" targets, and the access-limiting blood-retinal barrier (BRB) present major hurdles toward effective therapy development. Here, we address these challenges (i) by targeting cGMP (cyclic guanosine- 3',5'-monophosphate) signaling, a disease driver common to different types of RD, and (ii) by combining inhibitory cGMP analogs with a nanosized liposomal drug delivery system designed to facilitate transport across the BRB. Based on a screen of several cGMP analogs we identified an inhibitory cGMP analog that interferes with activation of photoreceptor cell death pathways. Moreover, we found liposomal encapsulation of the analog to achieve efficient drug targeting to the neuroretina. This pharmacological treatment markedly preserved in vivo retinal function and counteracted photoreceptor degeneration in three different in vivo RD models. Taken together, we show that a defined class of compounds for RD treatment in combination with an innovative drug delivery method may enable a single type of treatment to address genetically divergent RD-type diseases.

Robust and Scalable Angiogenesis Assay of Perfused 3D Human iPSC-Derived Endothelium for Anti-Angiogenic Drug Screening.

To advance pre-clinical vascular drug research, in vitro assays are needed that closely mimic the process of angiogenesis in vivo. Such assays should combine physiological relevant culture conditions with robustness and scalability to enable drug screening. We developed a perfused 3D angiogenesis assay that includes endothelial cells (ECs) from induced pluripotent stem cells (iPSC) and assessed its performance and suitability for anti-angiogenic drug screening. Angiogenic sprouting was compared with primary ECs and showed that the microvessels from iPSC-EC exhibit similar sprouting behavior, including tip cell formation, directional sprouting and lumen formation. Inhibition with sunitinib, a clinically used vascular endothelial growth factor (VEGF) receptor type 2 inhibitor, and 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO), a transient glycolysis inhibitor, both significantly reduced the sprouting of both iPSC-ECs and primary ECs, supporting that both cell types show VEGF gradient-driven angiogenic sprouting. The assay performance was quantified for sunitinib, yielding a minimal signal window of 11 and Z-factor of at least 0.75, both meeting the criteria to be used as screening assay. In conclusion, we have developed a robust and scalable assay that includes physiological relevant culture conditions and is amenable to screening of anti-angiogenic compounds.

Regulation of brain endothelial barrier function by microRNAs in health and neuroinflammation.

Brain endothelial cells constitute the major cellular element of the highly specialized blood-brain barrier (BBB) and thereby contribute to CNS homeostasis by restricting entry of circulating leukocytes and blood-borne molecules into the CNS. Therefore, compromised function of brain endothelial cells has serious consequences for BBB integrity. This has been associated with early events in the pathogenesis of several disorders that affect the CNS, such as multiple sclerosis, HIV-associated neurologic disorder, and stroke. Recent studies demonstrate that brain endothelial microRNAs play critical roles in the regulation of BBB function under normal and neuroinflammatory conditions. This review will focus on emerging evidence that indicates that brain endothelial microRNAs regulate barrier function and orchestrate various phases of the neuroinflammatory response, including endothelial activation in response to cytokines as well as restoration of inflamed endothelium into a quiescent state. In particular, we discuss novel microRNA regulatory mechanisms and their contribution to cellular interactions at the neurovascular unit that influence the overall function of the BBB in health and during neuroinflammation.-Lopez-Ramirez, M. A., Reijerkerk, A., de Vries, H. E., Romero, I. A. Regulation of brain endothelial barrier function by microRNAs in health and neuroinflammation.

Glutathione conjugation dose-dependently increases brain-specific liposomal drug delivery in vitro and in vivo.

The blood-brain barrier (BBB) represents a major obstacle for the delivery and development of drugs curing brain pathologies. However, this biological barrier presents numerous endogenous specialized transport systems that can be exploited by engineered nanoparticles to enable drug delivery to the brain. In particular, conjugation of glutathione (GSH) onto PEGylated liposomes (G-Technology®) showed to safely enhance delivery of encapsulated drugs to the brain. Yet, understanding of the mechanism of action remains limited and full mechanistic understanding will aid in the further optimization of the technology. In order to elucidate the mechanism of brain targeting by GSH-PEG liposomes, we here demonstrate that the in vivo delivery of liposomal ribavirin is increased in brain extracellular fluid according to the extent of GSH conjugation onto the liposomes. In vitro, using the hCMEC/D3 human cerebral microvascular endothelial (CMEC) cell line, as well as primary bovine and porcine CMEC (and in contrast to non-brain derived endothelial and epithelial cells), we show that liposomal uptake occurs through the process of endocytosis and that the brain-specific uptake is also glutathione conjugation-dependent. Interestingly, the uptake mechanism is an active process that is temperature-, time- and dose-dependent. Finally, early endocytosis events rely on cytoskeleton remodeling, as well as dynamin- and clathrin-dependent endocytosis pathways. Overall, our data demonstrate that the glutathione-dependent uptake mechanism of the G-Technology involves a specific endocytosis pathway indicative of a receptor-mediated mechanism, and supports the benefit of this drug delivery technology for the treatment of devastating brain diseases.

MicroRNA-155 negatively affects blood-brain barrier function during neuroinflammation.

Blood-brain barrier (BBB) dysfunction is a hallmark of neurological conditions such as multiple sclerosis (MS) and stroke. However, the molecular mechanisms underlying neurovascular dysfunction during BBB breakdown remain elusive. MicroRNAs (miRNAs) have recently emerged as key regulators of pathogenic responses, although their role in central nervous system (CNS) microvascular disorders is largely unknown. We have identified miR-155 as a critical miRNA in neuroinflammation at the BBB. miR-155 is expressed at the neurovascular unit of individuals with MS and of mice with experimental autoimmune encephalomyelitis (EAE). In mice, loss of miR-155 reduced CNS extravasation of systemic tracers, both in EAE and in an acute systemic inflammation model induced by lipopolysaccharide. In cultured human brain endothelium, miR-155 was strongly and rapidly upregulated by inflammatory cytokines. miR-155 up-regulation mimicked cytokine-induced alterations in junctional organization and permeability, whereas inhibition of endogenous miR-155 partially prevented a cytokine-induced increase in permeability. Furthermore, miR-155 modulated brain endothelial barrier function by targeting not only cell-cell complex molecules such as annexin-2 and claudin-1, but also focal adhesion components such as DOCK-1 and syntenin-1. We propose that brain endothelial miR-155 is a negative regulator of BBB function that may constitute a novel therapeutic target for CNS neuroinflammatory disorders.

Retinoic acid induces blood-brain barrier development.

The blood-brain barrier (BBB) is crucial in the maintenance of a controlled environment within the brain to safeguard optimal neuronal function. The endothelial cells (ECs) of the BBB possess specific properties that restrict the entry of cells and metabolites into the CNS. The specialized BBB endothelial phenotype is induced during neurovascular development by surrounding cells of the CNS. However, the molecular differentiation of the BBB endothelium remains poorly understood. Retinoic acid (RA) plays a crucial role in the brain during embryogenesis. Because radial glial cells supply the brain with RA during the developmental cascade and associate closely with the developing vasculature, we hypothesize that RA is important for the induction of BBB properties in brain ECs. Analysis of human postmortem fetal brain tissue shows that the enzyme mainly responsible for RA synthesis, retinaldehyde dehydrogenase, is expressed by radial glial cells. In addition, the most important receptor for RA-driven signaling in the CNS, RA-receptor ß (RARß), is markedly expressed by the developing brain vasculature. Our findings have been further corroborated by in vitro experiments showing RA- and RARß-dependent induction of different aspects of the brain EC barrier. Finally, pharmacologic inhibition of RAR activation during the differentiation of the murine BBB resulted in the leakage of a fluorescent tracer as well as serum proteins into the developing brain and reduced the expression levels of important BBB determinants. Together, our results point to an important role for RA in the induction of the BBB during human and mouse development.

MicroRNAs regulate human brain endothelial cell-barrier function in inflammation: implications for multiple sclerosis.

Blood-brain barrier (BBB) dysfunction is a major hallmark of many neurological diseases, including multiple sclerosis (MS). Using a genomics approach, we defined a microRNA signature that is diminished at the BBB of MS patients. In particular, miR-125a-5p is a key regulator of brain endothelial tightness and immune cell efflux. Our findings suggest that repair of a disturbed BBB through microRNAs may represent a novel avenue for effective treatment of MS.

Cellular distribution of glucose and monocarboxylate transporters in human brain white matter and multiple sclerosis lesions.

To ensure efficient energy supply to the high demanding brain, nutrients are transported into brain cells via specific glucose (GLUT) and monocarboxylate transporters (MCT). Mitochondrial dysfunction and altered glucose metabolism are thought to play an important role in the progression of neurodegenerative diseases, including multiple sclerosis (MS). Here, we investigated the cellular localization of key GLUT and MCT proteins in human brain tissue of non-neurological controls and MS patients. We show that in control brain tissue GLUT and MCT proteins were abundantly expressed in a variety of central nervous system cells, particularly in microglia and endothelial cells. In active MS lesions, GLUTs and MCTs were highly expressed in infiltrating leukocytes and reactive astrocytes. Astrocytes manifest increased MCT1 staining and maintain GLUT expression in inactive lesions, whereas demyelinated axons exhibit significantly reduced GLUT3 and MCT2 immunoreactivity in inactive lesions. Finally, we demonstrated that the co-transcription factor peroxisome proliferator-activated receptor gamma co-activator 1-alpha (PGC-1α), an important protein involved in energy metabolism, is highly expressed in reactive astrocytes in active MS lesions. Overexpression of PGC-1α in astrocyte-like cells resulted in increased production of several GLUT and MCT proteins. In conclusion, we provide for the first time a comprehensive overview of key nutrient transporters in white matter brain samples. Moreover, our data demonstrate an altered expression of these nutrient transporters in MS brain tissue, including a marked reduction of axonal GLUT3 and MCT2 expression in chronic lesions, which may impede efficient nutrient supply to the hypoxic demyelinated axons thereby contributing to the ongoing neurodegeneration in MS.

Astrocyte-derived retinoic acid: a novel regulator of blood-brain barrier function in multiple sclerosis.

Multiple sclerosis (MS) lesions are characterized by the presence of activated astrocytes, which are thought to actively take part in propagating lesion progression by secreting pro-inflammatory mediators. Conversely, reactive astrocytes may exert disease-dampening effects through the production of trophic factors and anti-inflammatory mediators. Astrocytic control of the blood-brain barrier (BBB) is crucial for normal brain homeostasis and BBB disruption is a well-established early event in MS lesion development. Here, we set out to unravel potential protective effects of reactive astrocytes on BBB function under neuroinflammatory conditions as seen in MS, where we focus on the role of the brain morphogen retinoic acid (RA). Immunohistochemical analysis revealed that retinaldehyde dehydrogenase 2 (RALDH2), a key enzyme for RA synthesis, is highly expressed by reactive astrocytes throughout white matter lesions compared to control and normal appearing white matter. In vitro modeling of reactive astrocytes resulted in increased expression of RALDH2, enhanced RA synthesis, and a protective role for astrocyte-derived RA on BBB function during inflammation-induced barrier loss. Furthermore, RA induces endothelial immune quiescence and decreases monocyte adhesion under inflammatory conditions. Finally, we demonstrated that RA attenuated oxidative stress in inflamed endothelial cells, through activation of the antioxidant transcription factor nuclear factor E2 related factor 2. In summary, RA synthesis by reactive astrocytes represents an endogenous protective response to neuroinflammation, possibly aimed at protecting the BBB against inflammatory insult. A better understanding of RA signaling in MS pathophysiology may lead to the discovery of novel targets to halt disease progression.

P-glycoprotein regulates trafficking of CD8(+) T cells to the brain parenchyma.

The trafficking of cytotoxic CD8(+) T lymphocytes across the lining of the cerebral vasculature is key to the onset of the chronic neuro-inflammatory disorder multiple sclerosis. However, the mechanisms controlling their final transmigration across the brain endothelium remain unknown. Here, we describe that CD8(+) T lymphocyte trafficking into the brain is dependent on the activity of the brain endothelial adenosine triphosphate-binding cassette transporter P-glycoprotein. Silencing P-glycoprotein activity selectively reduced the trafficking of CD8(+) T cells across the brain endothelium in vitro as well as in vivo. In response to formation of the T cell-endothelial synapse, P-glycoprotein was found to regulate secretion of endothelial (C-C motif) ligand 2 (CCL2), a chemokine that mediates CD8(+) T cell migration in vitro. Notably, CCL2 levels were significantly enhanced in microvessels isolated from human multiple sclerosis lesions in comparison with non-neurological controls. Endothelial cell-specific elimination of CCL2 in mice subjected to experimental autoimmune encephalomyelitis also significantly diminished the accumulation of CD8(+) T cells compared to wild-type animals. Collectively, these results highlight a novel (patho)physiological role for P-glycoprotein in CD8(+) T cell trafficking into the central nervous system during neuro-inflammation and illustrate CCL2 secretion as a potential link in this mechanism.

Disturbed function of the blood-cerebrospinal fluid barrier aggravates neuro-inflammation.

Multiple sclerosis (MS) is a chronic neuro-inflammatory disorder, which is marked by the invasion of the central nervous system by monocyte-derived macrophages and autoreactive T cells across the brain vasculature. Data from experimental animal models recently implied that the passage of leukocytes across the brain vasculature is preceded by their traversal across the blood-cerebrospinal fluid barrier (BCSFB) of the choroid plexus. The correlation between the presence of leukocytes in the CSF of patients suffering from MS and the number of inflammatory lesions as detected by magnetic resonance imaging suggests that inflammation at the choroid plexus contributes to the disease, although in a yet unknown fashion. We here provide first insights into the involvement of the choroid plexus in the onset and severity of the disease and in particular address the role of the tight junction protein claudin-3 (CLDN3) in this process. Detailed analysis of human post-mortem brain tissue revealed a selective loss of CLDN3 at the choroid plexus in MS patients compared to control tissues. Importantly, mice that lack CLDN3 have an impaired BCSFB and experience a more rapid onset and exacerbated clinical signs of experimental autoimmune encephalomyelitis, which coincides with enhanced levels of infiltrated leukocytes in their CSF. Together, this study highlights a profound role for the choroid plexus in the pathogenesis of multiple sclerosis, and implies that CLDN3 may be regarded as a crucial and novel determinant of BCSFB integrity.

Roles for HB-EGF and CD9 in multiple sclerosis.

Early events in multiple sclerosis (MS) lesion formation are loss of blood-brain barrier (BBB) integrity, immune cell trafficking into the central nervous system, and demyelination. To date, the molecular mechanisms underlying these pathogenic events are poorly understood. Heparin-binding epidermal growth factor (HB-EGF) is a trophic factor that is induced by inflammatory stimuli and has previously been shown to interact with tetraspanins (TSPs), a family of transmembrane proteins that are involved in cellular migration and adhesion. Given the known roles of TSPs and HB-EGF, we hypothesized that HB-EGF and TSPs may play a role in the processes that underlie MS lesion formation. We examined the expression of HB-EGF and the TSPs CD9 and CD81 in MS brain and found that HB-EGF was highly induced in reactive astrocytes in active lesions. TSPs were constitutively expressed throughout normal appearing white matter and control white matter. In contrast, CD9 was reduced in demyelinated lesions and increased on blood vessels in lesion areas. In vitro studies revealed that expression of HB-EGF and TSPs is regulated during inflammation. Importantly, blocking either HB-EGF or CD9 significantly reduced the migration of monocytes across brain endothelial cell monolayers. Moreover, blocking CD9 strongly enhanced the barrier function of the BBB in vitro. Together, we demonstrate that these molecules are likely implicated in processes that are highly relevant for MS lesion formation, and therefore, HB-EGF and TSPs are promising therapeutic targets.

Characterization of immune response to neurofilament light in experimental autoimmune encephalomyelitis.

BACKGROUND: Autoimmunity to neuronal proteins occurs in several neurological syndromes, where cellular and humoral responses are directed to surface as well as intracellular antigens. Similar to myelin autoimmunity, pathogenic immune response to neuroaxonal components such as neurofilaments may contribute to neurodegeneration in multiple sclerosis. METHODS: We studied the immune response to the axonal protein neurofilament light (NF-L) in the experimental autoimmune encephalomyelitis animal model of multiple sclerosis. To examine the association between T cells and axonal damage, pathology studies were performed on NF-L immunized mice. The interaction of T cells and axons was analyzed by confocal microscopy of central nervous system tissues and T-cell and antibody responses to immunodominant epitopes identified in ABH (H2-Ag7) and SJL/J (H2-As) mice. These epitopes, algorithm-predicted peptides and encephalitogenic motifs within NF-L were screened for encephalitogenicity. RESULTS: Confocal microscopy revealed both CD4+ and CD8+ T cells alongside damaged axons in the lesions of NF-L immunized mice. CD4+ T cells dominated the areas of axonal injury in the dorsal column of spastic mice in which the expression of granzyme B and perforin was detected. Identified NF-L epitopes induced mild neurological signs similar to the observed with the NF-L protein, yet distinct from those characteristic of neurological disease induced with myelin oligodendrocyte glycoprotein. CONCLUSIONS: Our data suggest that CD4+ T cells are associated with spasticity, axonal damage and neurodegeneration in NF-L immunized mice. In addition, defined T-cell epitopes in the NF-L protein might be involved in the pathogenesis of the disease.

Reduced expression of PGC-1α partly underlies mitochondrial changes and correlates with neuronal loss in multiple sclerosis cortex.

There is growing evidence that mitochondrial dysfunction and associated reactive oxygen species (ROS) formation contribute to neurodegenerative processes in multiple sclerosis (MS). Here, we investigated whether alterations in transcriptional regulators of key mitochondrial proteins underlie mitochondrial dysfunction in MS cortex and contribute to neuronal loss. Hereto, we analyzed the expression of mitochondrial transcriptional (co-)factors and proteins involved in mitochondrial redox balance regulation in normal-appearing grey matter (NAGM) samples of cingulate gyrus and/or frontal cortex from 15 MS patients and nine controls matched for age, gender and post-mortem interval. PGC-1α, a transcriptional co-activator and master regulator of mitochondrial function, was consistently and significantly decreased in pyramidal neurons in the deeper layers of MS cortex. Reduced PGC-1α levels coincided with reduced expression of oxidative phosphorylation subunits and a decrease in gene and protein expression of various mitochondrial antioxidants and uncoupling proteins (UCPs) 4 and 5. Short-hairpin RNA-mediated silencing of PGC-1α in a neuronal cell line confirmed that reduced levels of PGC-1α resulted in a decrease in transcription of OxPhos subunits, mitochondrial antioxidants and UCPs. Moreover, PGC-1α silencing resulted in a decreased mitochondrial membrane potential, increased ROS formation and enhanced susceptibility to ROS-induced cell death. Importantly, we found extensive neuronal loss in NAGM from cingulate gyrus and frontal cortex of MS patients, which significantly correlated with the extent of PGC-1α decrease. Taken together, our data indicate that reduced neuronal PGC-1α expression in MS cortex partly underlies mitochondrial dysfunction in MS grey matter and thereby contributes to neurodegeneration in MS cortex.

Myc-associated zinc finger protein (MAZ) is regulated by miR-125b and mediates VEGF-induced angiogenesis in glioblastoma.

In patients with glioblastomas, vascular endothelial growth factor (VEGF) is a key mediator of tumor-associated angiogenesis. Glioblastomas are notorious for their capacity to induce neovascularization, driving continued tumor growth. Here we report that miR-125b is down-regulated in glioblastoma-associated endothelial cells, resulting in increased expression of its target, myc-associated zinc finger protein (MAZ), a transcription factor that regulates VEGF. The down-regulation of miR-125b was also observed on exposure of endothelial cells to glioblastoma-conditioned medium or VEGF, resulting in increased MAZ expression. Further analysis revealed that inhibition of MAZ accumulation by miR-125b, or by MAZ-specific shRNAs, attenuated primary human brain endothelial cell migration and tubule formation in vitro, phenomena considered to mimick angiogenic processes in vitro. Moreover, MAZ expression was elevated in brain blood vessels of glioblastoma patients. Altogether these results demonstrate a functional feed-forward loop in glioblastoma-related angiogenesis, in which VEGF inhibits the expression of miR-125b, resulting in increased expression of MAZ, which in its turn causes transcriptional activation of VEGF. This loop is functionally impeded by the VEGF receptor inhibitor vandetanib, and our results may contribute to the further development of inhibitors of tumor-angiogenesis.

Brain endothelial barrier passage by monocytes is controlled by the endothelin system.

Homeostasis of the brain is dependent on the blood-brain barrier (BBB). This barrier tightly regulates the exchange of essential nutrients and limits the free flow of immune cells into the CNS. Perturbations of BBB function and the loss of its immune quiescence are hallmarks of a variety of brain diseases, including multiple sclerosis (MS), vascular dementia, and stroke. In particular, diapedesis of monocytes and subsequent trafficking of monocyte-derived macrophages into the brain are key mediators of demyelination and axonal damage in MS. Endothelin-1 (ET-1) is considered as a potent pro-inflammatory peptide and has been implicated in the development of cardiovascular diseases. Here, we studied the role of different components of the endothelin system, i.e., ET-1, its type B receptor (ET(B)) and endothelin-converting enzyme-1 (ECE-1) in monocyte diapedesis of a human brain endothelial cell barrier. Our pharmacological inhibitory and specific gene knockdown studies point to a regulatory function of these proteins in transendothelial passage of monocytes. Results from this study suggest that the endothelin system is a putative target within the brain for anti-inflammatory treatment in neurological diseases.

Sphingosine 1-phosphate receptor 5 mediates the immune quiescence of the human brain endothelial barrier.

BACKGROUND: The sphingosine 1-phosphate (S1P) receptor modulator FTY720P (Gilenya®) potently reduces relapse rate and lesion activity in the neuroinflammatory disorder multiple sclerosis. Although most of its efficacy has been shown to be related to immunosuppression through the induction of lymphopenia, it has been suggested that a number of its beneficial effects are related to altered endothelial and blood-brain barrier (BBB) functionality. However, to date it remains unknown whether brain endothelial S1P receptors are involved in the maintenance of the function of the BBB thereby mediating immune quiescence of the brain. Here we demonstrate that the brain endothelial receptor S1P5 largely contributes to the maintenance of brain endothelial barrier function. METHODS: We analyzed the expression of S1P5 in human post-mortem tissues using immunohistochemistry. The function of S1P5 at the BBB was assessed in cultured human brain endothelial cells (ECs) using agonists and lentivirus-mediated knockdown of S1P5. Subsequent analyses of different aspects of the brain EC barrier included the formation of a tight barrier, the expression of BBB proteins and markers of inflammation and monocyte transmigration. RESULTS: We show that activation of S1P5 on cultured human brain ECs by a selective agonist elicits enhanced barrier integrity and reduced transendothelial migration of monocytes in vitro. These results were corroborated by genetically silencing S1P5 in brain ECs. Interestingly, functional studies with these cells revealed that S1P5 strongly contributes to brain EC barrier function and underlies the expression of specific BBB endothelial characteristics such as tight junctions and permeability. In addition, S1P5 maintains the immunoquiescent state of brain ECs with low expression levels of leukocyte adhesion molecules and inflammatory chemokines and cytokines through lowering the activation of the transcription factor NFκB. CONCLUSION: Our findings demonstrate that S1P5 in brain ECs contributes to optimal barrier formation and maintenance of immune quiescence of the barrier endothelium.

Fingolimod attenuates ceramide-induced blood-brain barrier dysfunction in multiple sclerosis by targeting reactive astrocytes.

Alterations in sphingolipid metabolism are described to contribute to various neurological disorders. We here determined the expression of enzymes involved in the sphingomyelin cycle and their products in postmortem brain tissue of multiple sclerosis (MS) patients. In parallel, we investigated the effect of the sphingosine-1 receptor agonist Fingolimod (Gilenya(®)) on sphingomyelin metabolism in reactive astrocytes and determined its functional consequences for the process of neuro-inflammation. Our results demonstrate that in active MS lesions, marked by large number of infiltrated immune cells, an altered expression of enzymes involved in the sphingomyelin cycle favors enhanced ceramide production. We identified reactive astrocytes as the primary cellular source of enhanced ceramide production in MS brain samples. Astrocytes isolated from MS lesions expressed enhanced mRNA levels of the ceramide-producing enzyme acid sphingomyelinase (ASM) compared to astrocytes isolated from control white matter. In addition, TNF-α treatment induced ASM mRNA and ceramide levels in astrocytes isolated from control white matter. Incubation of astrocytes with Fingolimod prior to TNF-α treatment reduced ceramide production and mRNA expression of ASM to control levels in astrocytes. Importantly, supernatants derived from reactive astrocytes treated with Fingolimod significantly reduced transendothelial monocyte migration. Overall, the present study demonstrates that reactive astrocytes represent a possible additional cellular target for Fingolimod in MS by directly reducing the production of pro-inflammatory lipids and limiting subsequent transendothelial leukocyte migration.

Adenosine triphosphate-binding cassette transporters mediate chemokine (C-C motif) ligand 2 secretion from reactive astrocytes: relevance to multiple sclerosis pathogenesis.

Adenosine triphosphate-binding cassette efflux transporters are highly expressed at the blood-brain barrier and actively hinder passage of harmful compounds, thereby maintaining brain homoeostasis. Since, adenosine triphosphate-binding cassette transporters drive cellular exclusion of potential neurotoxic compounds or inflammatory molecules, alterations in their expression and function at the blood-brain barrier may contribute to the pathogenesis of neuroinflammatory disorders, such as multiple sclerosis. Therefore, we investigated the expression pattern of different adenosine triphosphate-binding cassette efflux transporters, including P-glycoprotein, multidrug resistance-associated proteins-1 and -2 and breast cancer resistance protein in various well-characterized human multiple sclerosis lesions. Cerebrovascular expression of P-glycoprotein was decreased in both active and chronic inactive multiple sclerosis lesions. Interestingly, foamy macrophages in active multiple sclerosis lesions showed enhanced expression of multidrug resistance-associated protein-1 and breast cancer resistance protein, which coincided with their increased function of cultured foamy macrophages. Strikingly, reactive astrocytes display an increased expression of P-glycoprotein and multidrug resistance-associated protein-1 in both active and inactive multiple sclerosis lesions, which correlated with their enhanced in vitro activity on astrocytes derived from multiple sclerosis lesions. To investigate whether adenosine triphosphate-binding cassette transporters on reactive astrocytes can contribute to the inflammatory process, primary cultures of reactive human astrocytes were generated through activation of Toll-like receptor-3 to mimic the astrocytic phenotype as observed in multiple sclerosis lesions. Notably, blocking adenosine triphosphate-binding cassette transporter activity on reactive astrocytes inhibited immune cell migration across a blood-brain barrier model in vitro, which was due to the reduction of astrocytic release of the chemokine (C-C motif) ligand 2. Our data point towards a novel (patho)physiological role for adenosine triphosphate-binding cassette transporters, suggesting that limiting their activity by dampening astrocyte activation may open therapeutic avenues to diminish tissue damage during multiple sclerosis pathogenesis.

Sphingosine 1-phosphate receptor 1 and 3 are upregulated in multiple sclerosis lesions.

Sphingolipids are a class of biologically active lipids that have a role in multiple biological processes including inflammation. Sphingolipids exert their functions by direct signaling or through signaling by their specific receptors. Phosphorylated FTY720 (FTY720P) is a sphingosine 1-phosphate (S1P) analogue that is currently in trial for treatment of multiple sclerosis (MS), which targets all S1P receptors but S1P(2). To date, however, it remains unknown whether FTY720P may exert direct anti-inflammatory effects within the central nervous system (CNS), because data concerning S1P receptor expression and regulation under pathological conditions in the human brain are lacking. To investigate potential regulation of S1P receptors in the human brain during MS, we performed immunohistochemical analysis of S1P receptor 1 and 3 expression in well-characterized MS lesions. A strong increase in S1P receptor 1 and 3 expression on reactive astrocytes was detected in active and chronic inactive MS lesions. In addition, we treated primary cultures of human astrocytes with the proinflammatory cytokine tumor necrosis factor-alpha to identify the regulation of S1P(1/3) on astrocytes under pathological conditions. Importantly, we demonstrate that FTY720P exerts an anti-inflammatory action on human astrocytes by limiting secretion of proinflammatory cytokines. Our data demonstrate that reactive astrocytes in MS lesions and cultured under proinflammatory conditions strongly enhance expression of S1P receptors 1 and 3. Results from this study indicate that astrocytes may act as a yet-unknown target within the CNS for the anti-inflammatory effects observed after FTY720P administration in the treatment of MS.