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Background: Cardiovascular disease is a major comorbidity of rheumatoid arthritis (RA) and a leading cause of death. Chronic systemic inflammation involving tumour necrosis factor alpha (TNF) could contribute to endothelial activation and atherogenesis. A number of anti-TNF therapies are in current use for the treatment of RA, including certolizumab pegol (CZP), (Cimzia ®; UCB, Belgium). Anti-TNF therapy has been associated with reduced clinical cardiovascular disease risk and ameliorated vascular function in RA patients. However, the specific effects of TNF inhibitors on endothelial cell function are largely unknown. Our aim was to investigate the mechanisms underpinning CZP effects on TNF-activated human endothelial cells. Methods: Human aortic endothelial cells (HAoECs) were cultured in vitro and exposed to a) TNF alone, b) TNF plus CZP, or c) neither agent. Microarray analysis was used to examine the transcriptional profile of cells treated for 6 hrs and quantitative polymerase chain reaction (qPCR) analysed gene expression at 1, 3, 6 and 24 hrs. NF-κB localization and IκB degradation were investigated using immunocytochemistry, high content analysis and western blotting. Flow cytometry was conducted to detect microparticle release from HAoECs. Results: Transcriptional profiling revealed that while TNF alone had strong effects on endothelial gene expression, TNF and CZP in combination produced a global gene expression pattern similar to untreated control. The two most highly up-regulated genes in response to TNF treatment were adhesion molecules E-selectin and VCAM-1 (q < 0.00005). This was supported by qPCR analysis at 6 hrs (E-selectin and VCAM-1; 208.5 fold and 40.5, respectively above control) and also at 1, 3 and 24 hrs (E-selectin; 25.6, 93.5, 12.7 fold, respectively) (VCAM-1; 4.7, 47.2, 17.6 fold) (n = 3; p < 0.05). In contrast, HAoECs treated with TNF in combination with CZP exhibited control levels of E-selectin and VCAM-1 transcript (p > 0.2 compared to control; p > 0.05 compared to TNF alone). The NF-κB pathway was confirmed as a downstream target of TNF-induced HAoEC activation, via nuclear translocation of NF-κB and degradation of IκB, effects which were abolished by treatment with CZP. In addition, flow cytometry detected an increased production of endothelial microparticles in TNF-activated HAoECs, which was prevented by treatment with CZP. Conclusions: We have found at a cellular level that a clinically available TNF inhibitor, CZP reduces the expression of adhesion molecule expression, and prevents TNF-induced activation of the NF-κB pathway. Furthermore, CZP prevents the production of microparticles by activated endothelial cells. This could be central to the prevention of inflammatory environments underlying these conditions and measurement of microparticles has potential as a novel prognostic marker for future cardiovascular events in this patient group. Disclosure statement: Y.A. received a research grant from UCB. I.B. received a research grant from UCB. S.H. received a research grant from UCB. All other authors have declared no conflicts of interest

This thesis investigates the role of the urothelium in bladder mechanosensation with focus on the cholinergic signalling pathway. The urothelium can be seen as a first responder to physiological changes in the bladder. It releases a host of mediators including ACh, ATP and NO that play a coordinated role in the stimulation of signalling cascades, which then lead to the onset of afferent nerve activity and detrusor muscle contraction, and finally trigger the micturition reflex. However, the underlying signalling pathways remain elusive. Understanding the signalling pathways involved in the mechanosensation of the urinary bladder will improve the understanding of bladder function, both in health and pathology and ultimately lead to novel treatment options available to patients of lower urinary tract conditions such as overactive bladder syndrome.Mediator release experiments in the whole, isolated murine bladder depicted that ACh is released in a mechanosensitive manner from the urothelium. Distension of as little as 5 mm Hg stimulated a significantly greater release of ACh compared to resting level. However implementation of rising pressure levels showed that the amount of ACh decreased in a negative correlation to the applied pressure. A stretch dependent choline uptake appears to be a convincing explanation for these results. Spontaneous contractions were also measured in the whole, isolated murine bladder and showed a positive correlation to increasing distension levels. Blocking different components of ACh release and choline uptake in the urothelium showed that interfering at any point in the cholinergic pathway does trigger the same response in the muscle, leading to an extremely contracted detrusor muscle, even at low pressure levels. It has been hypothesized in previous studies, that the balance of inhibitory and excitatory mediators released from the urothelium modulates afferent nerve activity and therefore bladder contractility. This balance might be altered when blocking components of the cholinergic pathway. In the present thesis it was also shown, that application of BoNT/A, a novel treatment option for lower urinary tract symptoms, alters this balance by decreasing the excitatory mediator ATP and by increasing the inhibitory mediator NO. How exactly these mediators are balanced and which imbalances occur in the onset of lower urinary tract symptoms will be the topic of future research.New insights on the physiological changes occurring in the naturally aged murine bladder have been shown in this thesis. A significantly higher sensitivity of the aged detrusor in the contractile response to purinergic and muscarinic stimulation was observed. Furthermore, urothelial release of ATP was increased while release of ACh was decreased in the aged bladder. Urothelial NO release was not affected by age and Substance P could not be shown to be released by the urothelium of adult or aged murine bladders. Moreover an increased purinergic receptor sensitivity of aged urothelial cells was shown, which is probably facilitated via the purinergic P2X3 receptor. Further characterisations of the studied pathways are now required to fully validate the data, most suitably in human tissue, as it is not clear if the same pathways are affected by age in the human bladder.Spontaneous contractions and carbachol-induced contractions were measured in male and female porcine bladder strips isolated from the dome, body and trigone region. The amplitude and frequency of these contractions were analysed. After cholinergic stimulation the amplitude of denuded tissue strips was higher compared to the intact counterparts in all bladder regions and in both genders. The existence of an urothelial derived inhibitory factor had been proposed before and would explain these results. Functional differences were also observed between the three bladder regions, particularly the female trigone seems to act differently compared to the bladder dome and body. Gender differences were not observed when comparing spontaneous activity of male and female bladder strips. However, after cholinergic stimulation, female tissue strips from the trigone region showed significantly higher amplitude and frequency in the contractile response. Gender differences and regional disparities should be considered when comparing the findings of detrusor contractility studies. In further experiments it should be examined if mediator release and receptor distribution of the urothelium differs between the different regions of the bladder and between the genders.In summary it can be stated that the urothelium plays an important role in signalling processes and in detrusor contractility of the porcine and murine urinary bladder. Further research is required to fully understand the signalling pathways in the different bladder cells and their interactions.

Multiple Sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) causing neurological disability in young adults, characterised by discrete acute and chronic lesions that are predominantly located in the CNS white matter. The role the extracellular matrix (ECM) has been widely studied as it is thought to be implicated in the pathogenesis of MS. Components such as chondroitin sulphate proteoglycans (CSPGs) are up-regulated in response to injury and inflammation and participate in the formation of sclerotic lesions. Research into the role of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) in CNS injury and inflammation has demonstrated that this protease may aid the recovery process by cleaving CSPGs, which are inhibitory to neurite outgrowth and axonal regeneration.In vivo studies were carried out investigating the distribution and expression of aggrecan and versican by dual label immunohistochemistry (IHC) and western blotting (WB) using antibodies which specially recognise cleavage-derived aggrecan and versican neoepitopes and intact protein. ADAMTS-9 expression was assessed by real time PCR (qRT-PCR), IHC and western blotting. In active lesions, both IHC and WB indicated that intact versican and aggrecan as well as their neoepitopes and ADAMTS-9 expression were all increased in areas of myelin thinning with ongoing demyelination and macrophage activation, compared to control and normal appearing white matter (NAWM) brain tissue. Immunostaining for CSPG neoepitope was particularly strong at the plaque border in chronic active lesions (CAL) compared to chronic inactive lesions (CL), NAWM and control brain tissue, suggesting active enzymatic cleavage of intact protein. IHC studies demonstrated that ADAMTS-9 was expressed predominantly by astrocytes, endothelium, macrophages and neurons with increased expression within MS active lesions. qRT-PCR studies confirmed ADAMTS-9 expression in MS tissue. In summary this study provides evidence that ADAMTS-9 plays an important role in cleavage of the ECM CSPGs, aggrecan and versican, in active lesions in MS.The role of CSPGs in neurite outgrowth from human neurons has been largely untested but is critical for understanding of regeneration following CNS injury. This thesis aimed to assess the effect of the ECM components, aggrecan, on neurite outgrowth of neuroblastoma cell line (SHSY-5Y) and expression of ADAMTS-9. This was achieved by several methods including qRT-PCR, ICC and WB. These data provided evidence that neurite outgrowth from SHSY-5Y cells is inhibited by aggrecan, this inhibition was associated with high level expression of ADAMTS-9.This thesis also aimed to investigate the expression of ADAMTS-9 by cells of the CNS to gain a better understanding of how these enzymes might be regulated and their possible role in the pathogenesis of MS. Cells were treated with pro-inflammatory cytokines in vitro, to mimic in vivo inflammatory conditions, and following this, ICC and WB data demonstrated that ADAMTS-9 was constitutively expressed by primary human astrocytes, microglia (CHME3) and neuronal (SHSY-5Y) cell lines in vitro, under basal condition. In primary astrocytes, ADAMTS-9 expression was increased following treatment with 10ng/mL interferon-y compared to control untreated cells. In contrast, dual treatment with interleukin-1 and tumour necrosis factor resulted in down regulation of ADAMTS-9. Thus illustrating the external inflammatory mileu influences the expression of ADAMTS-9 in lesions in MS, which in turn influences the degradation of the ECM components aggrecan and versican, which are also upregulated in lesions. Further work to understand how these changes in the ECM influence CNS repair is needed.

Degeneration of the intervertebral disc (IVD) is thought to account for 40% of all cases of chronic low back pain. Alterations in the behaviour of the IVDs’ native cell\ud population mediate the processes that lead to structural failure, as seen in IVD degeneration.\ud Cytokines are implicated in this process, several studies have identified that Interleukin-1 (IL-1) and Tumour Necrosis Factor-α (TNF-α) expression is increased\ud in degenerate IVDs compared to their normal counterparts. Furthermore, it has been shown in vitro that these cytokines stimulate alterations in the behaviour of\ud the IVDs’ native cells in a similar manner to those observed in IVD degeneration.\ud However, IL-1 and TNF-α are only two of a large group of intercellular signalling molecules known as cytokines, and studies investigating the expression of other\ud cytokines in the human IVD are limited.\ud This thesis demonstrates the production of numerous cytokines and chemokines (chemoattractant cytokines) by the human IVDs’ native cell population. Detailed gene and protein expression studies identified several novel cytokines and chemokines that are differentially expressed in cells isolated from degenerate or prolapsed IVDs compared to those isolated from the normal counterpart. Coexpression of receptors for these molecules was also identified, indicating a capacity of these cells to respond to cytokine intercellular signalling.\ud The response of IVD cells to cytokine and chemokine stimulation in vitro was investigated. The data presented indicates that inter-regulatory relationships exist\ud between the cytokines and chemokines of the intervertebral disc. Particularly, IL-1 exerts modulatory potential over the expression of other cytokines and chemokines\ud by IVD cells. Effects of stimulation were also observed in relation to reduced anabolic metabolism and increased catabolic metabolism, both of which are characteristic features of IVD degeneration.\ud Together, the findings presented in this thesis indicate that cytokines and chemokines are integral to the pathogenesis of IVD degeneration and prolapse that\ud may lead to low back pain.

ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs)-1, -4 and -5 are secreted enzymes which are members of the glutamyl endopeptidases (GEPs) group of ADAMTSs. These enzymes break down chondroitin sulphate proteoglycans (CSPGs) which are key components of brain extracellular matrix (ECM). In multiple sclerosis (MS), CSPG breakdown by ADAMTSs may enable axonal regeneration or conversely it may lead to alterations of the ECM, allowing influx of inflammatory cells promoting tissue damage.ADAMTS-1, -4 and -5 mRNA expression was studied by quantitative real-time PCR (qRT-PCR) using the Taqman method in SHSY-5Y and SK-N-DZ human neuroblastoma cells, undifferentiated or differentiated to a more neuronal phenotype using retinoic acid (RetA). Modulation by pro-inflammatory cytokines ((interleukin-1 IL-1) or tumour necrosis factor (TNF)), which are involved in the pathogenesis of MS, was also studied. As ADAMTS-1 was the most abundant ADAMTS in the neuronal cell lines, it was investigated at its protein level in both cell lines by Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) with western blotting. Furthermore, the presence of ADAMTS-1 at its mRNA and protein levels was confirmed by the small interfering RNA (siRNA) technique in SHSY-5Y cells. Cryostat sections of normal and MS central nervous system (CNS) tissue white matter, obtained from the UK Multiple Sclerosis Tissue Bank, were used to determine the localisation of V0/V2 neoepitopes of versican, derived by ADAMTS cleavage, using immunohistochemistry.SHSY-5Y and SK-N-DZ cells expressed mRNA for ADAMTS-1, -4 and -5. ADAMTS-1 expression was significantly increased on cellular differentiation with Ret A in SHSY-5Y cells. Its expression was confirmed at the mRNA and protein level. IL-1 B and TNF had no effect on ADAMTS mRNA expression in SHSY-5 Y cells. However, ADAMTS-1 mRNA expression was upregulated by IL-1 B in differentiated SK-N-DZ and there was also a significant increase in ADAMTS-4 mRNA expression with TNF treatment. ADAMTS-mediated versican breakdown, as determined immuohistochemically by versican (V0/V2) neoepitopes expression, was increased in MS brain tissue compared to normal brain tissue.In conclusion, ADAMTS-1, -4 and -5 were constitutively expressed in SHSY-5Y and SK-N-DZ neuronal cells. Modulation by the cytokines tested was seen in the SK-N-DZ cells. From these in vitro studies, neuronal ADAMTSs in the CNS may have a potential role in MS pathogenesis. However further investigation is needed on primary neuronal cells and CNS to elucidate the role of neuronal ADAMTSs and their contribution in MS.ADAMTSs do appear to be involved in increased proteolysis of versican at the known cleavage site in human brain tissue as indicated by (V0/V2) versican neoepitopes expression. Upregulation of versican (V0/V2) neoepitopes was observed in lesional MS sections on immunohistochemistry. These enzymes require further investigation, by immunohistochemical methods for co-localisation with versican (V0/V2) neoepitopes in MS, to determine which of the ADAMTSs generates these neoepitopes.