ABSTRACT
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.—Lopez‐Ramirez, M. A., Wu, D., Pryce, G., Simpson, J. E., Reijerkerk, A., King‐Robson, J., Kay, O, de Vries, H. E., Hirst, M. C., Sharrack, B., Baker D., Male, D. K., Michael, G. J., Romero, I. A. MicroRNA‐155 negatively affects blood–brain barrier function during neuroinflammation. FASEB J. 28, 2551–2565 (2014). www.fasebj.org
To advance pre-clinical vascular drug research,
assays are needed that closely mimic the process of angiogenesis
. 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.
Retinoic acid induces blood-brain barrier development Mizee, Mark R; Wooldrik, Desiree; Lakeman, Kim A M ...
The Journal of neuroscience,
2013-Jan-23, 2013-01-23, 20130123, Letnik:
33, Številka:
4
Journal Article
Recenzirano
Odprti dostop
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 β (RARβ), is markedly expressed by the developing brain vasculature. Our findings have been further corroborated by in vitro experiments showing RA- and RARβ-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.
Neuroinflammation contributes to a wide range of disorders of the central nervous system (CNS). Of the available anti-inflammatory drugs, only glucocorticoids have shown central efficacy in ...CNS-related disorders, such as multiple sclerosis (MS). However, their side effects are dose limiting. To optimally improve the therapeutic window of methylprednisolone, we enhanced its CNS delivery by using pegylated liposomes conjugated to the brain-targeting ligand glutathione. In healthy rats, plasma circulation and brain uptake were significantly increased after encapsulating methylprednisolone in glutathione pegylated (GSH-PEG) liposomes. Furthermore, the efficacy of GSH-PEG liposomal methylprednisolone was investigated in rats with acute experimental autoimmune encephalomyelitis (EAE), an animal model of MS; rats received treatment (10mg/kg; i.v. injection), before disease onset, at disease onset, or at the peak of disease. Free methylprednisolone and non-targeted pegylated (PEG) liposomal methylprednisolone served as control treatments. When treatment was initiated at disease onset, free methylprednisolone showed no effect, while GSH-PEG liposomal methylprednisolone significantly reduced the clinical signs to 42±6.4% of saline control. Moreover, treatment using GSH-PEG liposomes was significantly more effective compared to PEG liposomes. Our findings hold promise for MS treatment and warrant further investigations into this brain delivery system for the treatment of neuroinflammation.
Display omitted
Abstract
Partly due to poor blood-brain barrier drug penetration the treatment options for many brain diseases are limited. To safely enhance drug delivery to the brain, glutathione PEGylated ...liposomes (G-Technology®) were developed. In this study, in rats, we compared the pharmacokinetics and organ distribution of GSH-PEG liposomes using an autoquenched fluorescent tracer after intraperitoneal administration and intravenous administration. Although the appearance of liposomes in the circulation was much slower after intraperitoneal administration, comparable maximum levels of long circulating liposomes were found between 4 and 24 h after injection. Furthermore, 24 h after injection a similar tissue distribution was found. To investigate the effect of GSH coating on brain delivery in vitro uptake studies in rat brain endothelial cells (RBE4) and an in vivo brain microdialysis study in rats were used. Significantly more fluorescent tracer was found in RBE4 cell homogenates incubated with GSH-PEG liposomes compared to non-targeted PEG liposomes (1.8-fold, p < 0.001). In the microdialysis study 4-fold higher (p < 0.001) brain levels of fluorescent tracer were found after intravenous injection of GSH-PEG liposomes compared with PEG control liposomes. The results support further investigation into the versatility of GSH-PEG liposomes for enhanced drug delivery to the brain within a tolerable therapeutic window.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
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.
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.
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.
Our findings demonstrate that S1P5 in brain ECs contributes to optimal barrier formation and maintenance of immune quiescence of the barrier endothelium.
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.
The blood-brain barrier (BBB) prevents the entrance of circulating molecules and immune cells into the central nervous system. The barrier is formed by specialized brain endothelial cells that are ...interconnected by tight junctions (TJ). A defective function of the BBB has been described for a variety of neuroinflammatory diseases, indicating that proper regulation is essential for maintaining brain homeostasis. Under pathological conditions, reactive oxygen species (ROS) significantly contribute to BBB dysfunction and inflammation in the brain by enhancing cellular migration. However, a detailed study about the molecular mechanism by which ROS alter BBB integrity has been lacking. Here we demonstrate that ROS alter BBB integrity, which is paralleled by cytoskeleton rearrangements and redistribution and disappearance of TJ proteins claudin-5 and occludin. Specific signaling pathways, including RhoA and PI3 kinase, mediated observed processes and specific inhibitors of these pathways prevented ROS-induced monocyte migration across an in vitro model of the BBB. Interestingly, these processes were also mediated by protein kinase B (PKB/Akt), a previously unknown player in cytoskeleton and TJ dynamics that acted downstream of RhoA and PI3 kinase. Our study reveals new insights into molecular mechanisms underlying BBB regulation and provides novel opportunities for the treatment of neuroinflammatory diseases.--Schreibelt, G., Kooij. G., Reijerkerk, A., van Doorn, R., Gringhuis, S. I., van der Pol, S., Weksler, B. B., Romero, I. A., Couraud, P.-O., Piontek, J., Blasig, I. E., Dijkstra, C. D., Ronken, E., de Vries, H. E. Reactive oxygen species alter brain endothelial tight junction dynamics via RhoA, PI3 kinase and PKB signaling.
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. GLIA 2014;62:1125–1141
Main Points:
First comprehensive overview of glucose‐ and monocarboxylate transporter distribution in human CNS
PGC‐1α contributes to increased astrocytic expression of nutrient transporters in active MS lesions
Decreased GLUT3 and MCT2 expression in demyelinated axons in inactive MS lesions may contribute to axonal degeneration
PDF
