Gareth R. John

Multiple sclerosis: Re-expression of a developmental pathway that restricts oligodendrocyte maturation

During mammalian central nervous system (CNS) development, contact-mediated activation of Notch1 receptors on oligodendrocyte precursors by the ligand Jagged1 induces Hes5, which inhibits maturation of these cells. Here we tested whether the Notch pathway is re-expressed in the adult CNS in multiple sclerosis (MS), an inflammatory demyelinating disease in which remyelination is typically limited. We found that transforming growth factor-β1 (TGF-β1), a cytokine upregulated in MS, specifically re-induced Jagged1 in primary cultures of human astrocytes. Within and around active MS plaques lacking remyelination, Jagged1 was expressed at high levels by hypertrophic astrocytes, whereas Notch1 and Hes5 localized to cells with an immature oligodendrocyte phenotype, and TGF-β1 was associated with perivascular extracellular matrix in the same areas. In contrast, there was negligible Jagged1 expression in remyelinated lesions. Experiments in vitro showed that Jagged1 signaling inhibited process outgrowth from primary human oligodendrocytes. These data are the first to implicate the Notch pathway in the limited remyelination in MS. Thus, Notch may represent a potential target for therapeutic intervention in this disease.

VEGF-mediated disruption of endothelial CLN-5 promotes blood-brain barrier breakdown

Breakdown of the blood-brain barrier (BBB) is an early and significant event in CNS inflammation. Astrocyte-derived VEGF-A has been implicated in this response, but the underlying mechanisms remain unresolved. Here, we identify the endothelial transmembrane tight junction proteins claudin-5 (CLN-5) and occludin (OCLN) as targets of VEGF-A action. Down-regulation of CLN-5 and OCLN accompanied up-regulation of VEGF-A and correlated with BBB breakdown in experimental autoimmune encephalomyelitis, an animal model of CNS inflammatory disease. In cultures of brain microvascular endothelial cells, VEGF-A specifically down-regulated CLN-5 and OCLN protein and mRNA. In mouse cerebral cortex, microinjection of VEGF-A disrupted CLN-5 and OCLN and induced loss of barrier function. Importantly, functional studies revealed that expression of recombinant CLN-5 protected brain microvascular endothelial cell cultures from a VEGF-induced increase in paracellular permeability, whereas recombinant OCLN expressed under the same promoter was not protective. Previous studies have shown CLN-5 to be a key determinant of trans-endothelial resistance at the BBB. Our findings suggest that its down-regulation by VEGF-A constitutes a significant mechanism in BBB breakdown.

Astrocyte-derived VEGF-A drives blood-brain barrier disruption in CNS inflammatory disease

In inflammatory CNS conditions such as multiple sclerosis (MS), current options to treat clinical relapse are limited, and more selective agents are needed. Disruption of the blood-brain barrier (BBB) is an early feature of lesion formation that correlates with clinical exacerbation, leading to edema, excitotoxicity, and entry of serum proteins and inflammatory cells. Here, we identify astrocytic expression of VEGF-A as a key driver of BBB permeability in mice. Inactivation of astrocytic Vegfa expression reduced BBB breakdown, decreased lymphocyte infiltration and neuropathology in inflammatory and demyelinating lesions, and reduced paralysis in a mouse model of MS. Knockdown studies in CNS endothelium indicated activation of the downstream effector eNOS as the principal mechanism underlying the effects of VEGF-A on the BBB. Systemic administration of the selective eNOS inhibitor cavtratin in mice abrogated VEGF-A-induced BBB disruption and pathology and protected against neurologic deficit in the MS model system. Collectively, these data identify blockade of VEGF-A signaling as a protective strategy to treat inflammatory CNS disease.

Cytokines: Powerful Regulators of Glial Cell Activation

It is now clear that cytokines function as powerful regulators of glial cell function in the central nervous system (CNS), either inhibiting or promoting their contribution to CNS pathology. Although these interactions are complex, the availability of animals with targeted deletions of these genes and/or their receptors, as well as transgenic mice in which cytokine expression has been targeted to specific cell types, and the availability of purified populations of glia that can be studied in vitro, has provided a wealth of interesting and frequently surprising data relevant to this activity. A particular feature of many of these studies is that it is the nature of the receptor that is expressed, rather than the cytokine itself, that regulates the functional properties of these cytokines. Because cytokine receptors are themselves modulated by cytokines, it becomes evident that the effects of these cytokines may change dramatically depending upon the cytokine milieu present in the immediate environment. An additional exciting aspect of these studies is the previously underappreciated role of these factors in repair to the CNS. In this review, we focus on current information that has helped to define the role of cytokines in regulating glial cell function as it relates to the properties of microglia and astrocytes.

IL-1β Regulates Blood-Brain Barrier Permeability via Reactivation of the Hypoxia-Angiogenesis Program

Loss of blood-brain barrier (BBB) integrity is believed to be an early and significant event in lesion pathogenesis in the inflammatory demyelinating disease multiple sclerosis (MS), and understanding mechanisms involved may lead to novel therapeutic avenues for this disorder. Well-differentiated endothelium forms the basis of the BBB, while astrocytes control the balance between barrier stability and permeability via production of factors that restrict or promote vessel plasticity. In this study, we report that the proinflammatory cytokine IL-1β, which is prominently expressed in active MS lesions, causes a shift in the expression of these factors to favor plasticity and permeability. The transcription factor, hypoxia inducible factor-1 (HIF-1), plays a significant role in this switch. Using a microarray-based approach, we found that in human astrocytes, IL-1β induced the expression of genes favoring vessel plasticity, including HIF-1α and its target, vascular endothelial growth factor-A (VEGF-A). Demonstrating relevance to MS, we showed that HIF-1α and VEGF-A were expressed by reactive astrocytes in active MS lesions, while the VEGF receptor VEGFR2/flk-1 localized to endothelium and IL-1 to microglia/macrophages. Suggesting functional significance, we found that expression of IL-1β in the brain induced astrocytic expression of HIF-1α, VEGF-A, and BBB permeability. In addition, we confirmed VEGF-A to be a potent inducer of BBB permeability and angiogenesis, and demonstrated the importance of IL-1β-induced HIF-1α in its regulation. These results suggest that IL-1β contributes to BBB permeability in MS via reactivation of the HIF–VEGF axis. This pathway may represent a potential therapeutic target to restrict lesion formation.

IL-1-regulated responses in astrocytes: relevance to injury and recovery.

In the central nervous system (CNS), the cellular processes of astrocytes make intimate contact with essentially all areas of the brain. They have also been shown to be functionally coupled to neurons, oligodendrocytes, and other astrocytes via both contact‐dependent and non‐contact‐dependent pathways. These observations have led to the suggestion that a major function of astrocytes in the CNS is to maintain the homeostatic environment, thus promoting the proper functioning of the neuronal network. Inflammation in the CNS disrupts this process either transiently or permanently and, as such, is thought to be tightly regulated by both astrocytes and microglia. The remarkable role that single cytokines, such as TNF and IL‐1, may play in this process has now been well accepted, but the extent of the reprogramming of the transcriptional machinery initiated by these factors remains to be fully appreciated. With the advent of microarray technology, a more comprehensive analysis of this process is now available. In this report we review data obtained with this technology to provide an overview of the extent of changes induced in astrocytes by the cytokine IL‐1.

Inhibition of Endothelial Cell Migration, Intercellular Communication, and Vascular Tube Formation by Thromboxane A2

The eicosanoid thromboxane A2 (TXA2) is released by activated platelets, monocytes, and the vessel wall and interacts with high affinity receptors expressed in several tissues including endothelium. Whether TXA2 might alter endothelial migration and tube formation, two determinants of angiogenesis, is unknown. Thus, we investigated the effect of the TXA2 mimetic [1S-(1α,2β(5Z),3α(1E,3R),4α]-7-[3-(3-hydroxy-4-(4′-iodophenoxy)-1-butenyl)-7-oxabicyclo-[2.2.1]heptan-2-yl]-5′-heptenoic acid (IBOP) on human endothelial cell (HEC) migration and angiogenesis in vitro. IBOP stimulation inhibited HEC migration by 50% and in vitro capillary formation by 75%. These effects of IBOP were time- and concentration-dependent with an IC50 of 25 nm. IBOP did not affect integrin expression or cytoskeletal morphology of HEC. Since gap junction-mediated intercellular communication increases in migrating HEC, we determined whether IBOP might inhibit coupling or connexin expression in HEC. IBOP reduced the passage of microinjected dyes between HEC by 50%, and the effects of IBOP on migration and tube formation were mimicked by the gap junction inhibitor 18β-glycyrrhetinic acid (1 μm) with a similar time course and efficacy. IBOP (24 h) did not affect the expression or phosphorylation of connexin 43 in whole HEC lysates. Immunohistologic examination of HEC suggested that IBOP may impair functional coupling by altering the cellular distribution of gap junctions, leading to increased connexin 43 internalization. Thus, this finding that TXA2 mimetics can prevent HEC migration and tube formation, possibly by impairing intercellular communication, suggests that antagonizing TXA2 signaling might enhance vascularization of ischemic tissue.

Interleukin-1β Induces a Reactive Astroglial Phenotype via Deactivation of the Rho GTPase–Rock Axis

The cytokine interleukin-1β (IL-1β) is critical to the formation of an astrocytic scar after CNS injury, but the mechanisms by which it induces a reactive phenotype remain unresolved. Here, we show that IL-1β regulates the phenotype of astrocytes via deactivation of the Rho GTPase–Rho kinase (ROCK) pathway, which governs cellular morphology and migration via effects on F-actin and its interactions with focal adhesions, nonmuscle myosin, and microvillar adapter proteins of the ezrin–radixin–moesin (ERM) family. We found that IL-1β induced cortical reorganization of F-actin and dephosphorylation of focal adhesion kinase, myosin light chain 2, and myosin phosphatase targeting subunit 1 in primary human astrocytes, and that all of these effects were mimicked by Rho-ROCK pathway blockade. We also found that IL-1β conversely potentiated ERM phosphorylation, and that this effect was mediated via a Rho–ROCK-independent mechanism. Next, we used a rhotekin pulldown assay to confirm directly that IL-1β deactivates Rho, and further demonstrated that a constitutively active Rho construct rescued astrocytes from developing an IL-1β-induced reactive phenotype. These data implicate cytokine regulation of the Rho–ROCK pathway in the generation of a reactive astrogliosis, and we suggest that interventions targeted at this level may facilitate manipulation of the glial scar in inflammatory disorders of the human CNS.

Notch1 signaling plays a role in regulating precursor differentiation during CNS remyelination

In the developing CNS, Notch1 and its ligand, Jagged1, regulate oligodendrocyte differentiation and myelin formation, but their role in repair of demyelinating lesions in diseases such as multiple sclerosis remains unresolved. To address this question, we generated a mouse model in which we targeted Notch1 inactivation to oligodendrocyte progenitor cells (OPCs) using Olig1Cre and a floxed Notch1 allele, Notch112f. During CNS development, OPC differentiation was potentiated in Olig1Cre:Notch112f/12f mice. Importantly, in adults, remyelination of demyelinating lesions was also accelerated, at the expense of proliferation within the progenitor population. Experiments in vitro confirmed that Notch1 signaling was permissive for OPC expansion but inhibited differentiation and myelin formation. These studies also revealed that astrocytes exposed to TGF-β1 restricted OPC maturation via Jagged1-Notch1 signaling. These data suggest that Notch1 signaling is one of the mechanisms regulating OPC differentiation during CNS remyelination. Thus, Notch1 may represent a potential therapeutical avenue for lesion repair in demyelinating disease.

Role for CXCR2 and CXCL1 on glia in multiple sclerosis

As part of a need to understand myelin repair mechanisms, molecular pathways underlying oligodendrocyte behavior and central nervous system (CNS) remyelination are currently key topics in multiple sclerosis (MS). In the present study, we report expression of a chemoattractant receptor of the immune system, the chemokine receptor, CXCR2, on normal and proliferating oligodendrocytes in active MS lesions. Proliferating oligodendrocytes were occasionally associated with reactive astrocytes positive for CXCL1 (GRO‐α), the ligand for CXCR2. CXCL1 expression was not seen on astrocytes in control and normal CNS tissue, while CXCR2 expression was constitutive on oligodendrocytes. At the functional level, following stimulation with the proinflammatory cytokine, interleukin‐1β (IL‐1β), we found high‐level synthesis of CXCL1 by human fetal astrocytes in vitro. In contrast, human oligodendrocytes in culture expressed the receptor, CXCR2, constitutively. We propose that the concurrence of CXCR2 on oligodendrocytes and induced CXCL1 on hypertrophic astrocytes in MS provides a novel mechanism for recruitment of oligodendrocytes to areas of damage, an essential prerequisite for lesion repair in this devastating human condition.