Prostaglandin E synthases in periodontitis-affected gingival tissue and in gingival fibroblasts

Periodontitis is a chronic inflammatory disease resulting in the destruction of the tissue and alveolar bone supporting the teeth and leading ultimately to tooth loss. Prostaglandin E2 (PGE2) is an important inflammatory mediator in the pathogenesis of periodontitis. The biosynthesis of PGE2 is catalysed by three groups of enzymes acting sequentially: phospholipase A2 (PLA2), cyclooxygenases (COX-1 and COX-2) and prostaglandin E (PGE) synthases, which catalyse the final step of PGE2 synthesis. Three PGE synthase isoforms have been identified: i) the inducible microsomal membrane-associated and glutathione-dependent PGE synthase, mPGES-1, ii) the constitutively expressed cytosolic PGE synthase, cPGES, and iii) the glutathione-independent, membrane-associated mPGES-2. The aim of this thesis was to investigate the expression of PGE synthases in gingival tissue from periodontitis patients, as well as to study their expression and regulation in relation to PGE2 production in gingival fibroblasts. In periodontitis-affected gingival tissue, we demonstrated in vivo protein expression of mPGES-1, mPGES-2 and cPGES, as well as COX-2 in fibroblasts, endothelial cells, smooth muscle cells, epithelial cells and immune cells. We further showed that, in cell cultures of gingival fibroblasts and smooth muscle cells, the inflammatory cytokines tumour necrosis factor α (TNFα) and interleukin-1β (IL-1β), or co-culture with lymphocytes, markedly induced mPGES-1 and COX-2 expression, accompanied by an increase in PGE2 production. In cultured endothelial cells, only TNFα was found to increase PGE2 production, via enhanced COX-2 expression. In mast cell cultures, basal levels of PGE2 were detected, but no increase was observed in response to TNFα or IL-1β. To elucidate the impact of mPGES-1 inhibition on mPGES-2 and cPGES expression, as well as on PGE2 production we used knock-down of mPGES-1 expression by small interfering RNA (siRNA). The cytokine-induced protein expression of mPGES-1 was reduced by up to 79% by siRNA silencing, without affecting mPGES-2 or cPGES expression. Moreover, mPGES-1 siRNA did not affect the cytokine-stimulated PGE2 production, whereas levels of the downstream prostaglandin F2α (PGF2α) were enhanced. Using inhibitors and activators of various signalling pathways, we demonstrated that cytokine-induced mPGES-1 expression in gingival fibroblasts did not involve protein kinase C, p38 mitogen-activated protein kinase or tyrosine kinase pathways, in contrast to COX-2 expression. We further observed a possible positive feedback loop in which PGE2 and PGF2α increased the expression of mPGES-1. Furthermore, cytokine-induced mPGES-1 expression and PGE2 production were reduced after the inhibition of the upstream enzyme PLA2 and increased after the addition of arachidonic acid, the product of PLA2. The proposed anti-inflammatory prostaglandin 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), reduced mPGES-1 expression but not COX-2 expression or PGE2 production. To further explore the pathways involved in increased PGE2 synthesis in TNFα- stimulated gingival fibroblasts, a global gene expression profile was established using a microarray platform. Enrichment analysis of the gene expression data led to further investigation of nuclear factor-κB (NF-κB) and c-Jun N-terminal kinase (JNK) signalling pathways, revealing that these pathways are involved in the signal transduction of TNFα- induced mPGES-1 and COX-2 expression. In conclusion, all three PGE synthases are expressed in gingival tissue from patients with periodontitis. The isoenzyme mPGES-1 is the main PGE synthase involved in cytokine-induced PGE2 production in gingival fibroblasts. The cytokine-increased expression of mPGES-1 involves the signal pathways JNK and NF-κB. Furthermore, the prostaglandins PGE2 and PGF2α increase mPGES-1 expression, which may create a positive feedback loop. Collectively, these results suggest that inflammation-induced production of PGE2 by gingival fibroblasts, mediated by the increased expression of mPGES-1 and COX-2, may contribute to chronic inflammation in periodontitis. The results provide new insights into the expression and regulation of mPGES-1 in gingival fibroblasts and gingival tissue

Envolvimento das endotelinas na nocicepção articular induzida por carragenina e lipopolissacarídeo de E. coli no rato /

Dissertação (Mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas

Inflammatory mechanisms in acute pancreatitis

Acute pancreatitis is an inflammatory condition. It is associated with a systemic inflammatory response, the degree of which appears to correlate with the severity of the illness. The role of circulating leucocytes and their production of cytokines in the development of severe acute pancreatitis is unknown. Monocytes are believed to be a major source of pro-inflammatory cytokines, but lymphocytes and endothelial cells also produce such cytokines. These cell types, in particular lymphocytes, also produce a variety of down-regulatory signals so that monocytes, lymphocytes and endothelial cells interact to produce a net systemic inflammatory signal, influenced further by the varying degree of lymphocyte sub-populations to undergo blastogenesis in response to inflammation. The focus of this thesis is on pro-inflammatory cytokines and their release in vitro from peripheral blood mononuclear cells (PBMCs) isolated from patients with acute pancreatitis.On admission to hospital, patients with acute pancreatitis demonstrated increased interleukin-6 and interleukin-8 release but not tumour necrosis factor-a release from isolated PBMCs compared with healthy volunteers. The severity of the disease was not related to the level of cytokine release from a standard cell number. However, when allowance was made for the variation in PBMC numbers in the blood, the estimated IL-6 and IL-8 release per unit of blood was greater in those patients with severe disease compared with those with mild disease. Severe disease is also characterised by a more prolonged duration of increased pro-inflammatory cytokine release compared with patients with mild disease. Products of the cyclooxygenase pathway play a down-regulatory role in PBMCs in patients with acute pancreatitis as indomethacin (a cyclo-oxygenase inhibitor) had no significant effect on pro-inflammatory cytokine release by PBMCs isolated from healthy volunteers, but increased IL-6 and IL-8 release by PBMCs isolated from patients with both mild and severe disease. PBMC pro-inflammatory cytokine release remains sensitive to the down-regulatory action of the T-cell regulatory cytokines, interleukin-4 and interleukin-10. Lymphocyte proliferation (as measured by thymidine incorporation) is impaired in acute pancreatitis and correlates with the severity of the disease. Following the successful isolation and culture of human umbilical vein endothelial cells, IL-4 and IL-10 (in contrast to their inhibitory action on PBMCs), produce a dose dependent increase in endothelial cell IL-6 and IL-8 release. TNFa is often undetectable in patients with acute pancreatitis on admission, even in severe disease. However, elevation in the serum concentration of soluble TNFa receptors would suggest significant TNFa-induced inflammation early in the course of the disease. Glutamine is a conditionally essential amino acid in patients with severe acute pancreatitis and is important for immune function. A double blind, randomised controlled trial of glutamine supplemented versus conventional total parenteral nutrition in patients with severe acute pancreatitis demonstrated a trend towards improved lymphocyte proliferation in the glutamine supplemented group. Furthermore, PBMC IL-8 release but not TNFa and IL-6 release was significantly reduced over the study period.Severe acute pancreatitis is associated with prolonged PBMC pro¬ inflammatory cytokine release and impaired lymphocyte proliferation. However, these cells remain sensitive to the down-regulatory action of T-cell cytokines in vitro, but the exogenous administration of these cytokines may have an unpredictable clinical effect because of their different actions on various cell types. More general methods of immuno-modulation, such as the exogenous administration of glutamine may have therapeutic benefit in patients with severe acute pancreatitis

Investigating the role of endothelin receptor subtypes in the response to vascular injury

Neointimal hyperplasia, the proliferative growth of the innermost layer of the blood vessel wall, is a key process in the response to vascular injury, underlying conditions such as post-interventional restenosis and vein/arterial graft disease. One of the many mediators implicated in this process is endothelin-1 (ET-1), a potent vasoconstrictor with pro-inflammatory and pro-mitogenic actions, which acts through ETA and ETB receptor subtypes. It is well established that ET-1 increases, and ETA blockade reduces, neointima formation following vascular injury. The role of ETB is less clear because these receptors mediate potentially beneficial actions in endothelial cells (EC; such as nitric oxide production, and ET-1 clearance) but detrimental effects elsewhere (such as vascular smooth muscle) and it has been recently reported that non-cell-specific ETB deficiency is associated with increased neointimal lesion size following injury. The work described in this thesis addressed the hypothesis that endogenous ET-1 contributes to neointimal hyperplasia by activation of the ETA receptor, and that this action is moderated by concurrent activation of the ETB receptor expressed in EC. The role of ET receptors in neointimal lesion development was assessed using two models of femoral arterial injury in the mouse: (i) an established method of intraluminal wire-injury, and (ii) adaptation of a model of ligation injury that induces robust neointimal lesion formation without physical damage to the endothelium. Lesion development was assessed using standard histological techniques and this was augmented by development of quantitative optical projection tomography (OPT) to allow three-dimensional analysis of lesions. The role of ETA and ETB receptors in these models was addressed using suitable pharmacological ET receptor antagonists. Following wire-injury, selective ETB blockade (A192621; 30mg.kg-1.day-1; 35 days) increased lesion size and blood pressure without significant altering lesion composition. In contrast, selective ETA blockade (atrasentan; 10mg.kg-1.day-1; 35 days) reduced lesion size and blood pressure. Combined ETA+ETB antagonism had no effect on lesion size, despite reducing blood pressure, and reducing collagen content of the lesions. In the ligation model, neither ETA selective, ETB selective nor ETA+ETB blockade altered lesion size as assessed by standard histology but analysis by OPT indicated that ETA blockade, with or without concurrent ETB blockade, reduced lesion volume. The influence of ETB receptors expressed by ECs on lesion formation was addressed using EC-specific ETB knockout mice. Small vessel myography indicated that endothelium-dependent relaxation was unaltered in femoral arteries from these mice. In addition, no effect on lesion size or rate of development was observed in either wire- or ligation-injury models of neointima formation (although subtle effects on lesion and medial composition were apparent after intra-luminal injury). These results indicate that ETB receptor activation can moderate the detrimental actions of the ETA receptor on neointimal lesion progression, and that this role is dependent on the mode of vascular injury. Furthermore, in this setting, this beneficial action is not primarily mediated by ETB expressed by EC, suggesting that ETB in other cell types can reduce lesion development through another, unidentified mechanism. Therefore, while both ETA selective and non-selective ETA/B antagonists are currently in clinical use, in conditions where similar arterial remodelling processes occur, selective ETA receptor antagonists might be preferred