[Show abstract][Hide abstract] ABSTRACT: Nerve and blood vessel ingrowth during intervertebral disc degeneration, is thought to be a major cause of low back pain, however the regulation of this process is poorly understood. Here, we investigated the expression and regulation of a subclass of axonal guidance molecules known as the class 3 semaphorins, and their receptors; plexins and neuropilins within human NP tissue and their regulation by pro-inflammatory cytokines. Importantly this determined whether semaphorin expression was associated with the presence of nerves and blood vessels in tissues from human intervertebral discs. The study demonstrated that semaphorin3A, 3C, 3D, 3E and 3F and their receptors were expressed by native NP cells and further demonstrated their expression was regulated by IL-1β but to a lesser extent by IL-6 and TNFα. This is the first study to identify sema3C, sema3D and their receptors within the nucleus pulposus of intervertebral discs. Immunopositivity shows significant increases in semaphorin3C, 3D and their receptor neuropilin-2 in degenerate samples which were shown to contain nerves and blood vessels, compared to non-degenerate samples without nerves and blood vessels. Therefore data presented here suggests that semaphorin3C may have a role in promoting innervation and vascularisation during degeneration, which may go on to cause low back pain.
[Show abstract][Hide abstract] ABSTRACT: IntroductionThe degenerate intervertebral disc (IVD) becomes innervated by sensory nerve fibres, and vascularised by blood vessels. This study aimed to identify neurotrophins, neuropeptides and angiogenic factors within native IVD tissue and to further investigate whether pro-inflammatory cytokines are involved in the regulation of expression levels within nucleus pulposus (NP) cells, nerve and endothelial cells.Methods
Quantitative real-time PCR (qRT-PCR) was performed on 53 human IVDs from 52 individuals to investigate native gene expression of neurotrophic factors and their receptors, neuropeptides and angiogenic factors. The regulation of these factors by cytokines was investigated in NP cells in alginate culture, and nerve and endothelial cells in monolayer using RT-PCR and substance P (SP) protein expression in interleukin-1 (IL-1ß) stimulated NP cells.ResultsInitial investigation on uncultured NP cells identified expression of all neurotrophins by native NP cells, whilst the nerve growth factor (NGF) receptor was only identified in severely degenerate and infiltrated discs, and brain derived neurotrophic factor (BDNF) receptor expressed by more degenerate discs. BDNF expression was significantly increased in infiltrated and degenerate samples. SP and vascular endothelial growth factor (VEGF) were higher in infiltrated samples. In vitro stimulation by IL-1ß induced NGF in NP cells. Neurotropin-3 was induced by tumour necrosis factor alpha in human dermal microvascular endothelial cells (HDMECs). SP gene and protein expression was increased in NP cells by IL-1ß. Calcitonin gene related peptide was increased in SH-SY5Y cells upon cytokine stimulation. VEGF was induced by IL-1ß and interleukin-6 in NP cells, whilst pleiotrophin was decreased by IL-1ß. VEGF and pleiotrophin were expressed by SH-SY5Y cells, and VEGF by HDMECs, but were not modulated by cytokines.Conclusions
The release of cytokines, in particular IL-1ß during IVD degeneration, induced significant increases in NGF and VEGF which could promote neuronal and vascular ingrowth. SP which is released into the matrix could potentially up regulate the production of matrix degrading enzymes and also sensitise nerves, resulting in nociceptive transmission and chronic low back pain. This suggests that IL-1ß is a key regulatory cytokine, involved in the up regulation of factors involved in innervation and vascularisation of tissues.
[Show abstract][Hide abstract] ABSTRACT: Multiple sclerosis (MS) is a chronic inflammatory neurodegenerative disease, considered to be autoimmune in origin. Post-translational modification of central nervous system proteins, including glial fibrillary acidic protein (GFAP) and myelin basic protein (MBP), through citrullination of arginine residues, may lead to exposure of neoepitopes, triggering autoimmunity. Here we investigated the expression of citrullinated proteins in active MS lesions, MS normal appearing white matter and control brain white matter. We demonstrate increased citrullinated GFAP and MBP by immunohistochemistry and western blotting in areas of ongoing demyelination, suggesting a pivotal role for deimination of GFAP and MBP in MS pathogenesis MS.
Journal of Neuroimmunology 05/2014; 273:85-95. DOI:10.1016/j.jneuroim.2014.05.007 · 2.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The aims of these studies were to identify the cytokine and chemokine expression profile of nucleus pulposus (NP) cells and to determine the relationships between NP cell cytokine and chemokine production and the characteristic tissue changes seen during intervertebral disc (IVD) degeneration.
Real-time q-PCR cDNA Low Density Array (LDA) was used to investigate the expression of 91 cytokine and chemokine associated genes in NP cells from degenerate human IVDs. Further real-time q-PCR was used to investigate 30 selected cytokine and chemokine associated genes in NP cells from non-degenerate and degenerate IVDs and those from IVDs with immune cell infiltrates ('infiltrated'). Immunohistochemistry (IHC) was performed for four selected cytokines and chemokines to confirm and localize protein expression in human NP tissue samples.
LDA identified the expression of numerous cytokine and chemokine associated genes including 15 novel cytokines and chemokines. Further q-PCR gene expression studies identified differential expression patterns in NP cells derived from non-degenerate, degenerate and infiltrated IVDs. IHC confirmed NP cells as a source of IL-16, CCL2, CCL7 and CXCL8 and that protein expression of CCL2, CCL7 and CXCL8 increases concordant with histological degenerative tissue changes.
Our data indicates that NP cells are a source of cytokines and chemokines within the IVD and that these expression patterns are altered in IVD pathology. These findings may be important for the correct assessment of the 'degenerate niche' prior to autologous or allogeneic cell transplantation for biological therapy of the degenerate IVD.
[Show abstract][Hide abstract] ABSTRACT: The objective of the study was to investigate how inflammatory cytokines, IL-1β and TNF-α control Notch signaling activity in nucleus pulposus (NP) cells. An increase in expression of selective Notch receptors (Notch1, 2), ligand (Jagged 2) and target genes (Hes1, Hey1 and Hey2) was observed in NP cells following cytokine treatment. A concomitant increase in Notch signaling as evidenced by induction in activity of target gene Hes1 and Hey1 promoters and reporter 12xCSL was seen. Moreover, treatment increased activity of a 2 kb Notch2 promoter. Treatment of cells with NF-κB and MAPK inhibitors abolished the inductive effect of cytokines on Notch2 promoter and its expression. Gain and loss-of-function studies confirmed the inductive effect of p65 on Notch2 promoter activity. In contrast, p50 blocked the cytokine induction of promoter activity. Supporting the promoter studies, lentiviral delivery of sh-p65, and sh-IKKβ significantly decreased cytokine dependent change in Notch2 expression. Interestingly, MAPK signaling showed an isoform specific control of Notch2 promoter, p38α/β2/δ, ERK1 and ERK2 contributed to cytokine dependent induction while p38γ played no role. Analysis of human NP tissues showed that Notch 1, 2 and Hey2 expression correlated with each other. Moreover, expression of Notch2 and IL-1β as well as the number of cells immunopositive for Notch2 significantly increased in histologically degenerate discs compared to non-degenerate discs. Taken together these results explain the observed dysregulated expression of Notch genes in degenerative disc disease. Thus controlling IL-1β and TNF-αactivities during disc disease may restore Notch signaling and nucleus pulposus cell function.
[Show abstract][Hide abstract] ABSTRACT: Objective
To investigate tumor necrosis factor α (TNFα) and interleukin-1β (IL-1β) regulation of CCL3 expression in nucleus pulposus (NP) cells and in macrophage migration. Methods
Quantitative reverse transcription–polymerase chain reaction and immunohistochemistry were used to measure CCL3 expression in NP cells. Transfections were used to determine the role of NF-κB, CCAAT/enhancer binding protein (C/EBPβ), and MAPK on cytokine-mediated CCL3 promoter activity. The effect of NP-conditioned medium on macrophage migration was measured using a Transwell system. ResultsAn increase in CCL3 expression and promoter activity was observed in NP cells after TNFα or IL-1β treatment. Treatment of cells with NF-κB and MAPK inhibitors abolished the effect of the cytokines on CCL3 expression. The inductive effect of p65 and C/EBPβ on the CCL3 promoter was confirmed through gain-of-function and loss-of-function studies. Notably, cotransfection with p50 completely blocked cytokine- and p65-dependent induction. In contrast, c-Rel and RelB had little effect on promoter activity. Lentiviral transduction with short hairpin RNA for p65 (shp65) and shIKKβ significantly decreased the TNFα-dependent increase in CCL3 expression. Analysis of degenerated human NP tissue samples showed that CCL3, but not CCL4, expression correlated positively with the grade of tissue degeneration. Importantly, treatment of macrophages with conditioned medium of NP cells treated with TNFα or IL-1β promoted their migration. Pretreatment of macrophages with an antagonist of CCR1, the primary receptor for CCL3 and CCL4, blocked cytokine-mediated migration. Conclusion
Our findings indicate that TNFα and IL-1β modulate the expression of CCL3 in NP cells by controlling the activation of MAPK, NF-κB, and C/EBPβ signaling. The CCL3–CCR1 axis may play an important role in promoting macrophage infiltration in degenerated, herniated discs.
[Show abstract][Hide abstract] ABSTRACT: ADAMTS-13 is the Von Willebrand factor (vWF) cleaving protease, responsible for the cleavage and down-regulation of the pro-thrombotic properties of ultra large VWF multimers. It is expressed predominantly by the hepatic stellate cells of the liver, but is also found to be expressed in other tissues, including brain. Reduced ADAMTS-13 is associated with a variety of thrombotic microangiopathies. Since the cellular origin and regulation of ADAMTS-13 expression in the brain is unknown, we aimed to investigate this in four different central nervous system (CNS)-derived cell lines, SHSY-5Y (human neuroblastoma), U373 (human astroglioma), CHME-3 (human foetal microglia) and hCMEC/D3 (adult human brain endothelial cells). All cell lines expressed ADAMTS-13 mRNA constitutively with neuroblastoma cells showing the highest expression. Interleukin (IL)-1β down-regulated ADAMTS-13 mRNA expression in astroglioma cells and microglial cells whereas TNF and IL-6 treatment showed no significant differences in ADAMTS-13 mRNA expression in any cell line tested. ADAMTS-13 protein expression was reduced in a dose-dependent manner only in astroglioma cells following stimulation by IL-1β. The ability of IL-1β to significantly reduce ADAMTS-13 mRNA expression in human microglia and astroglioma cells suggests a role in the haemostasis of the local microenvironment under inflammatory conditions. This is the first report of ADAMTS-13 expression in cells of the CNS; however, its function remains to be determined.
[Show abstract][Hide abstract] ABSTRACT: Multiple sclerosis is a chronic immune-mediated disease of the CNS. Although it is a predominantly T-cell mediated condition, B cells and autoreactive antibodies play an important role in its pathogenesis, with the presence of oligoclonal immunoglobulins in the cerebrospinal fluid being an important diagnostic indicator. The target of these immunoglobulins has not yet been fully characterized. However, post-translational modifications of CNS-specific proteins are thought to contribute to their production through the generation of novel epitopes. One post-translational modification in particular, the conversion of the amino acid arginine to the nonstandard amino acid, citrulline, has been increasingly described in the literature as a factor in the pathogenesis of this condition. In this article, we summarize and discuss the current knowledge on citrullination in multiple sclerosis, the importance of this in relation to its pathogenesis and, potentially, its diagnosis.
[Show abstract][Hide abstract] ABSTRACT: Clarification of the pathophysiological mechanisms of intra-arterial thromboembolism may lead to novel treatments for cerebrovascular disease. There is increasing evidence for the role of von Willebrand factor (VWF) cleaving protease (ADAMTS-13) in modulating the thrombotic cascade in high flow arterial settings. VWF multimers are rich in ultra large forms (ULVWF) which can rapidly bind its primary platelet receptor resulting in spontaneous aggregation of platelets. Under normal conditions, these ULVWF are regulated by rapid proteolysis converting them to smaller, less active forms. The protease responsible for cleavage of ULVWF is ADAMTS-13. We measured the ADAMTS-13 antigen and ADAMTS-13 activity levels in the plasma of consecutive patients with transient ischaemic attack (TIA), compared with nonstroke controls, in a hospital based TIA clinic. This was compared with VWF levels. Samples were analysed in the acute phase and at 3 months postevent. In our pilot study patients with partial anterior circulation type TIAs had significantly reduced ADAMTS-13 activity compared to controls (p=0.0394). No significant differences were seen between high- and low-risk ABCD2 scored patients. We did not observe any significant changes in VWF levels in our study sample. Our pilot study suggests a potential role for altered ADAMTS-13 activity in the pathophysiology of TIA.
Journal of neurology, neurosurgery, and psychiatry 11/2010; 81(11):e69. DOI:10.1136/jnnp.2010.226340.207 · 6.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The ADAMTS enzymes (a disintegrin and metalloproteinase with thrombospondin type 1-like motifs) have important roles in central nervous system (CNS) physiology and pathology. This current study aimed to analyse the expression of ADAMTS-9 following transient middle cerebral artery occlusion (tMCAo) in the rat, a model of focal cerebral ischaemia. Using real-time RT-PCR, ADAMTS-9 mRNA was demonstrated to be significantly up-regulated in tMCAo brain tissue compared to sham-operated at 24h post-ischaemia. The mature form of the ADAMTS-9 protein was only detected by Western blotting in brains subjected to tMCAo at 24h. In situ hybridisation demonstrated that ADAMTS-9 mRNA was expressed by neurones in tMCAo tissue. This study indicates that ADAMTS-9 expression is modulated in response to cerebral ischaemia in vivo and further research will resolve whether it plays a role in the subsequent degenerative or repair processes.
[Show abstract][Hide abstract] ABSTRACT: Multiple sclerosis (MS) is a chronic inflammatory neurodegenerative disease, which predominantly affects the white matter of the central nervous system (CNS). It is the most common neurological disease in young adults with a mean age of onset of 30 years (Trapp and Nave, 2008). The disease is characterized by destruction of the myelin sheath, which insulates the axons of nerves, resulting in lesions of demyelinated axon fibres (Lassman, 2004). Within these lesions there is infi ltration of T and B lymphocytes and macrophages, as well as locally activated CNS resident cells, astrocytes and microglia (Musse and Harauz, 2007).
[Show abstract][Hide abstract] ABSTRACT: The ECM (extracellular matrix) is a complex molecular framework that provides physical support to cells and tissues, while also providing signals for cell growth, migration, differentiation and survival. The ECM of the CNS (central nervous system) is unusual in that it is rich in CSPGs (chondroitin sulfate proteoglycans), hyaluronan and tenascins. The CSPGs are widely expressed throughout the developing and adult CNS and have a role in guiding or limiting neurite outgrowth and cell migration. Alterations in the synthesis or breakdown of the ECM may contribute to disease processes. Here, we examine changes in the brain-specific CSPGs, brevican and phosphacan, following transient middle cerebral artery occlusion, a model of stroke in the rat. We have investigated their expression at various time points as well as their spatial relationship with ADAMTS-4 (a disintegrin and metalloprotease with thrombospondin motifs 4). The co-localization of ADAMTS or its activity may indicate a functional role for this matrix-protease pair in degeneration/regeneration processes that occur in stroke.
[Show abstract][Hide abstract] ABSTRACT: ADAM-17, a disintegrin and metalloproteinase, is the major proteinase responsible for the cleavage of membrane-bound tumour necrosis factor (TNF) as well as being an active sheddase of other cytokines, cytokine receptors, growth factors and adhesion molecules. TNF is a major proinflammatory cytokine that has been identified as having a pathogenic role in inflammatory diseases within the CNS including multiple sclerosis (MS). Here we report the cellular origin and distribution of ADAM-17 expression within clinically and neuropathologically confirmed MS and normal control white matter, assessed by immunohistochemistry, western blotting and PCR. ADAM-17 expression was associated with the blood vessel endothelium, activated macrophages/microglia and parenchymal astrocytes in MS white matter. Increased levels of ADAM-17 immunoreactivity were displayed in active lesions with evidence of recent myelin breakdown. Further studies into the functional role of ADAM-17 in the pathogenesis of MS and other inflammatory conditions are required.
[Show abstract][Hide abstract] ABSTRACT: ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) -1, -4 and -5 proteases have been identified in the CNS at the mRNA level. These glutamyl endopeptidases, inhibited by tissue inhibitor of metalloproteinases (TIMP)-3, are key enzymes in the degradation of the aggregating chondroitin sulphate proteoglycans (CSPGs), and may therefore play a role in CNS extracellular matrix (ECM) changes in multiple sclerosis (MS). We have investigated ADAMTS and TIMP-3 expression in normal and MS CNS white matter by real-time RT-PCR, western blotting and immunohistochemistry. We report for the first time the presence of ADAMTS-1, -4 and -5 in normal and MS white matter. Levels of ADAMTS-1 and -5 mRNA were decreased in MS compared to normal tissue, with no significant change in ADAMTS-4 mRNA levels. Protein levels of ADAMTS-4 were significantly higher in MS tissue compared to normal tissue. Immunohistochemical studies demonstrated that ADAMTS-4 was associated predominantly with astrocytes with increased expression within MS lesions. TIMP-3 mRNA was significantly decreased in MS compared to controls. These studies suggest a role for ADAMTS-4 in the pathogenesis of MS. Further studies on the activity of ADAMTS-4 will enable a better understanding of its role in the turnover of the ECM of white matter in MS.