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Prostaglandin endoperoxide synthase: regulation of enzyme expression
... Cyclooxygenase-1 (COX-1) is constitutively expressed by many cell types. Its synthesis can be induced by several cytokines, albeit often to a modest degree [17]. In contrast, COX-2 belongs to the family of early response genes and is primarily inducible [18,19]. ...
... The effect of dexamethasone on ET-1-stimulated PGE2 production and cyclooxygenase subtype mRNA levels was determined since dexamethasone has been shown to inhibit prostaglandin synthesis in several cell types [17]. Dexamethasone had no effect on total cell protein or cell number. ...
... As discussed earlier, two isoforms of cyclooxygenase have been identified, COX-1 and COX-2. Until recently, only COX-1 was characterized; this enzyme was known to be constitutively expressed by mesangial cells and to be only modestly induced by a variety of cytokines [17]. The discovery of COX-2 generated a great deal of interest since this enzyme was shown to be an early response gene and to be capable of being induced to very high levels of expression [18,19]. ...
Prostaglandin E2 (PGE2) may be an important negative feedback modulator of endothelin-1 (ET-1)-stimulated mesangial cell proliferation and contraction. Recent studies suggest that ET-1 may induce prolonged mesangial cell PGE2 production, however the mechanism of this effect is unknown. The current study was undertaken, therefore, to examine the long-term effect of ET-1 on mesangial cell PGE2 synthesis. ET-1 markedly increased PGE2 release by rat mesangial cells for at least six hours. Cyclooxygenase (COX) activity was increased by one hour and persisted for at least six hours. ET-1 increased COX-2, but not COX-1, protein and mRNA levels. Actinomycin D reduced ET-1-stimulated PGE2 synthesis and COX-2 mRNA expression, while cycloheximide superinduced COX-2 mRNA. Dexamethasone decreased ET-1-stimulated PGE2 release and COX-2 protein and mRNA levels. ET-1-stimulated PGE2 release was prevented by BQ-123, an endothelin receptor A antagonist. We conclude that ET-1, via activation of the endothelin A receptor, causes a prolonged increase in mesangial cell PGE2 production that is partially dependent on induction of dexamethasone-inhibitable COX-2.
... COX is a key enzyme and a rate-limiting element in the biosynthetic pathway for the formation of PG. A correlation between the acquired capacity for synthesis of PG and enhanced COX expression has been observed in various cells (21,22). In the present study, we assessed the effect of PAF on the expression of COX-2, the inducible form of cyclooxygenase, in rat AM. ...
Platelet-activating factor (PAF) can stimulate alveolar macrophages (AM) to produce IL-6 through a PG-dependent process. In this study, the modulation by PAF of the expression of COX-2, the inducible isoform of PG synthase, was investigated. Expression of COX-2 mRNA was increased in rat AM within 2 h after treatment with either of the bacterial products LPS or muramyl dipeptide (MDP) alone. Although PAF had no effect by itself, stimulation of AM with a combination of PAF (10(-10) to 10(-8) M) and LPS or MDP resulted in a synergistic, three- to fivefold increase in levels of COX-2 mRNA. No significant change was observed in the mRNA expression of the constitutive isoform, COX-1. The antagonist WEB 2170 blocked the action of PAF, whereas lyso-PAF was inactive on the COX-2 gene expression. The effect of PAF was rapid, being evident by 30 to 60 min of stimulation and was accompanied by enhanced production of PGE2. Two relatively selective inhibitors of COX-2 abolished the PAF-dependent increase in PGE2 production. Moreover, inhibition of transcription with actinomycin D completely abrogated the effect of PAF on both COX-2 mRNA and PGE2 production. These results suggest that COX-2 expression can be regulated at the transcriptional level by PAF, in synergy with bacterial products. PAF-dependent up-regulation of COX-2 expression may constitute a novel element in the interrelationship between PAF and prostanoids in the context of allergic, inflammatory, and immune processes.
... COX-2 was shown to be induced by proinflammatory mediators and its inhibition by NSAIDs was thought to be responsible for the antiinflammatory effects of these compounds [6]. In contrast, inhibition of COX-1 was thought to interfere with the house keeping functions of this enzyme, particularly in the gastrointestinal tract, thereby accounting for the gastric side effects of NSAIDs [7]. Despite a high sequence homology between the two isoforms differences in the active sites of the enzymes were exploited by pharmaceutical companies to develop selective COX-2 inhibitors. ...
Acute inflammatory reactions, in contrast to chronic inflammatory reactions, are usually self-limiting and resolve. We have investigated the resolving phase of a number of immune and non-immune inflammatory reactions induced in the pleural cavity of rats. COX-2 is expressed during resolution of these models. Using carrageenan pleurisy, we showed that this enzyme has a proinflammatory role as the reaction develops but an antiinflammatory role as the lesion resolves. This antiinflammatory role is associated with production of cyclopentenone prostaglandins and the absence of PGE2. Dual COX-1/COX-2 inhibitors or COX-2 inhibitors when given at the peak of the inflammatory response delay resolution, an effect reversed by replacing CyPGs into the pleural space. PGF2alpha like the CyPGs appears to have a role in resolving this reaction. Stress proteins are also induced in a variety of acute inflammatory models during resolution. Heme oxygenase-1 (HO-1) is one such protein so too are members of the hsp70 family. An inducer of HO-1 promotes resolution whereas an inhibitor is proinflammatory. In most cases it appears to be the macrophage that is the source of proteins necessary for resolution to occur. Understanding how proinflammatory pathways switch to the antiinflammatory pathways necessary for resolution to take place may eventually allow the exploitation of endogenous antiinflammatory pathways in the treatment of chronic inflammation.
... An in vitro study in mice demonstrated a stimulatory effect of PGE 2 on cumulus expansion by isolated cumulusoocyte complexes (COCs) (Eppig, 1981). Prostanoids, which group prostaglandins and thromboxanes, are products of prostaglandin H synthase (PGHS) also known as cyclooxygenase (cox) pathway (De Witt, 1991;Smith, 1992;Funk, 1993;Vane et al., 1998). The two isoforms of cyclooxygenase, cox-1 and cox-2, mainly differ in their pattern of expression and their regulation in mammalian cells (DuBois et al., 1994;Inoue et al., 1995;Williams and DuBois, 1996). ...
Prostaglandins could be involved in various aspects of final differentiation of ovarian follicles. Prostaglandins are generated by the cyclooxygenase (cox) pathway. Until now, the expression pattern of isoforms cox-1 and cox-2 of cyclooxygenase in bovine cumulus-oocyte complexes (COCs) was unknown. Using immunodetection procedure, we demonstrated in the present study that cox-2 was expressed by cumulus cells during in vivo and in vitro maturation. Time course induction of cox-2 expression was investigated during in vitro maturation using Western blot analysis. Specific signal of cox-2 was markedly evidenced from 6 hr of culture and increased to reach a maximal level at 24 hr of culture. In vitro, cox-2 expression in COCs was associated with increased concentrations of PGE(2) and PGF(2alpha) in the maturation medium. In addition, the effects of culture conditions on cox-2 expression was considered using RT-PCR and Western-blot analysis. We demonstrated that the addition of 10 ng/ml of EGF to TCM199 clearly increased the expression level of cox-2 mRNA and protein. Higher levels of in vitro cox-2 expression was associated with greater rates of cumulus expansion and oocytes at metaphase II at 24 hr of culture. In conclusion, our present results suggest that cox-2 expression in cumulus cells may be involved in differentiation of COCs that occurs during oocyte maturation.
... Two distinct PGHS enzymes have been characterized, PGHS-1 and PGHS-2, both catalyzing the synthesis of prostaglandin H 2 , the prostanoid precursor of prostaglandin D 2 , E 2 , and F 2α , prostacyclin and thromboxanes. The genes encoding the two PGHSs have originally been described as constitutive for PGHS-1 and inducible for PGHS-2 [2]. However, examples where PGHS-1 gene is induced and where PGHS-2 gene is constitutively expressed are accumulating. ...
Prostaglandin endoperoxide H synthase-1 gene expression is described as inducible in a few contexts such as differentiation of megakaryoblastic MEG-01 cells into platelet-like structures. In the MEG-01 cells model of PGHS-1 gene induction, we previously reported a delay in protein synthesis and identified the translational step of gene expression as being regulated. In the current study, we mapped PGHS-1 mRNA sequences regulating translational efficiency and identified an RNA binding protein. The 5'UTR and first two exons of the PGHS-1 5' mRNA decreased the synthesis of Luciferase protein by approximately 80% without significant changes in mRNA levels when compared to controls. Both the PGHS-1 5'-UTR and the first two exons were required for activity. Sucrose density gradient fractionations of cytoplasmic extracts from MEG-01 cells infected with reporter constructs, either controls or containing PGHS-1 sequence, presented a similar profile of distribution of reporter transcripts between polysomal and non-polysomal fractions. RNA/protein interaction studies revealed nucleolin binding to the 135 nt PGHS-1 sequence. Mutation of the two NRE elements located in the 5'end of PGHS-1 mRNA sequence partially reduced the negative activity of the 135 nt sequence. Stable secondary structures predicted at the 5' end of the transcript are potentially involved in translational regulation. We propose that the 5'end of PGHS-1 mRNA represses translation and could delay the synthesis of PGHS-1 enzyme.
... Products of arachidonic acid metabolism are thus critical participants in the development of inflammatory responses after infection or tissue injury. Prostaglandin E 2 (PGE 2 ) is one of the most thoroughly studied mediators of this process (13). ...
The in vitro biological response to fluoro-edenite (FE) fibres, an asbestos-like amphibole, was evaluated in lung alveolar epithelial A549, mesothelial MeT-5A and monocyte-macrophage J774 cell lines. The mineral has been found in the vicinity of the town of Biancavilla (Catania, Sicily), where an abnormal incidence of mesothelioma has been documented. Cell motility, distribution of polymerized actin, and synthesis of vascular endothelial growth factor (VEGF) and of beta-catenin, critical parameters for tumour development, progression and survival, were investigated in A549 and MeT-5A cells exposed to 50 microg/ml FE fibres for 24 hr and 48 hr. The levels of cyclooxygenase (COX-2) and prostaglandin (PGE2), two molecules involved in cancer pathogenesis by affecting mitogenesis, cell adhesion, immune surveillance and apoptosis, were measured in J774 cells treated with FE fibres under the same experimental conditions. Finally, FE fibres were studied by SEM and EDS analysis to investigate their chemical composition. Exposure of A549 and MeT-5A cells to FE fibres affected differentially phalloidin-stained cytoplasmic F-actin networks, cell motility and VEGF and beta-catenin expression according to the different sensitivity of the two cell lines. In J774 cells it induced a significant increase in COX-2 expression, as assessed by Western blot analysis, and in the concentration of PGE2, measured in culture media by ELISA. SEM-EDS investigations demonstrated two types of FE fibres, edenite and fluoro-edenite, differing in chemical composition and both recognizable as calcic amphiboles. Fibre width ranged from less than 1 microm (prevalently 0.5 microm) to 2-3 microm (edenite) up to several microm (fluoro-edenite); length ranged from about 6 to 80 microm (edenite) up to some hundred microm (fluoro-edenite). Results provide convincing evidence that FE fibres are capable of inducing in vitro functional modifications in a number of parameters with crucial roles in cancer development and progression. Inhaled FE fibres have the potential to induce mesothelioma, even though their ability to penetrate lung alveoli depends on their aerodynamic diameter.
Fluoro-edenite (FE), asbestiform fiber found in Biancavilla (Sicily, Italy), presents various characteristics similar to the asbestos group, in particular two fibrous phases tremolite and actinolite. Indeed, epidemiological studies have shown that FE fibers have similar effects to those of asbestos fibers. Such studies have reported a high incidence of malignant mesothelioma (MM), an aggressive neoplasm of the serosal membranes lining the pleural cavity, in individuals residing there due to FE exposure in Biancavilla related to environmental contamination. Evidence has led to the classification of FE as a Group 1 human carcinogen by the International Agency for Research on Cancer (IARC). The aim of this systematic review is to compare the results achieved in in vitro, in vivo and ex vivo experimental studies involving FE in order to update the current knowledge on the pathogenesis and molecular mechanisms responsible for FE-mediated MM development as well as the availability of effective biomarkers for MM prevention and diagnosis. This review is focused on the pathophysiological mechanisms mediated by inflammation induced by FE fiber exposure and which are responsible for MM development. This review also discusses the discovery of new diagnostic and prognostic biomarkers for the management of this pathology. It is known that the risk of cancer development increases with chronic inflammation, arising from enhanced reactive oxygen species (ROS) and NO• production stimulated by the body to remove exogenous agents, causing DNA damage and enhanced signal transduction that may lead to activation of oncogenes. Studies concerning MM biomarker discovery indicate that several biomarkers have been proposed for MM, but mesothelin is the only Food and Drug Administration (FDA)-approved biomarker for MM, with limitations. In recent studies, in silico analysis to identify selected miRNAs highly deregulated in cancer samples when compared with normal control have been developed. This in silico approach could represent an effort in the field of biomarker discovery for MM.
Aberrant expression of COX-2 occurs in many types of malignancies including colon and lung cancers, and is implicated in development and progression of cancer. The molecular mechanisms associated with aberrant expression of COX-2 in lung cancer cells remain to be fully elucidated. In this study, we found that non-small cell lung cancer (NSCLC) NCI-H520 and NCI-H460 cells constitutively expressed COX-2 and produced prostaglandin E 2 (PGE 2 ) as measured by Western blotting and enzyme-linked immunosorbent assay (ELISA), respectively. Reporter assays showed that transcriptional regulation of COX-2 was blunted when either the NF-IL6 (C/EBPB) or nuclear factor-KB (NF-KB) binding site in the COX-2 promoter was mutated, suggesting that C/EBPB and NF-KB transcription factors have an important role in aberrant expression of COX-2 in these lung cancer cells. In addition, the eight herbal mixture PC-SPES (Lot. 5431219) caused growth arrest and apoptosis of NCI-H520 and NCI-H460 cells in association with blockade of NF-KB and down-regulation of C/EBPB, resulting in down-regulation of COX-2 and PGE 2 in these cells. On the other hand, PC-SPES up-regulated the level of C/EBPB in these cells. Taken together, C/EBPB and NF-KB may be promising molecular targets for COX-2 inhibition in lung cancer cells. PC-SPES might be useful in the adjuvant setting for the treatment of individuals with resected NSCLC as well as other types of cancer in which COX-2 is activated.
The probable existence of a biochemical cycle, involving histamine, interleukins and prostaglandins for regulating sleep and wakefulness in higher vertebrates is proposed in this paper. For convenience, it is given an acronym, H-l-P cycle. The proposed biochemical cycle consists of 13 essential steps, with four regulatory points. How this cycle differs from the previously described biochemical models for sleep and wakefulness, such as Jouvet's monaminergic cycle (5) and the multiple sleep factor model of Krueger (9,lO) is also mentioned. The regional distribution of mast cells in the mammalian brain, the fluctuation of their numbers under different light regimen and a progressive decrease of their numbers with aging suggest that they may be the candidate 'black box' for the anatomical seat of sleep.
Myocardial ischemia/reperfusion (MI/R) is a major cause for the events of cardiovascular disease. Oxidative stress plays a critical role in the development of ischemia/reperfusion (IR) injury. As a potent antioxidant, alpha-lipoic acid (LA) has been shown to provide a benefit for the inhibition of IR injury and inhibit reactive oxygen species (ROS) generation during MI/R in rats. However, the mechanism on the protective effect of LA is still to be clarified. The present study was aimed to investigate the protective effect of LA against MI/R injury and its mechanisms. We found that 2h of myocardial ischemia followed by different time periods of reperfusion resulted in significant increase of creatine kinase (CK) activity. MI/R also significantly promoted oxidative stress and decreased the activities of antioxidant enzymes. In addition, apoptosis and inflammatory response were activated and aggravated in a time-dependent manner by MI/R. All these alterations induced by MI/R were attenuated by the administration of LA 30 min before reperfusion. These results suggested that LA played a protective effect against MI/R injury via antioxidant, anti-apoptotic and anti-inflammatory effects. These findings may significantly better the understanding of the pharmacological actions of LA and advance therapeutic approaches to MI/R injury and cardiovascular diseases.
Cyclooxygenase-2 (COX-2) influences carcinogenesis through immune response suppression, apoptosis inhibition, regulation of angiogenesis and tumor cell invasion, and metastasis. It is now well established that COX-2 is overexpressed in many premalignant, malignant, and metastatic cancers, including hepatocellular carcinoma (HCC). DNA sequence variations in the COX-2 gene may lead to altered COX-2 production and/or activity, and so they cause inter-individual differences in the susceptibility to HCC. Functional coding region polymorphisms −1195A>G (rs689466), −765G>C (rs20417), and +8473T>C (rs5275) in the COX-2 gene have recently been shown to be associated with several human cancers but their association with HCC has yet to be investigated. We used hospital-based case–control study to assess the hypothesis that the functional COX-2 variation may affect individual susceptibility to the HCC. COX-2 polymorphisms were investigated in 129 confirmed subjects with HCC and 129 cancer-free control subjects matched on age, gender, smoking, and alcohol consumption using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. The distribution of the COX-2 −1195A>G and +8473T>C genotypes were not significantly different between HCC cases and control. However, proportion of the COX-2 −765CC genotype which leads to a 30% reduction of the COX-2 promoter activity was significantly lower in patients with HCC (3.1%) when compared to control subjects (11.6%) (P COX-2 −765G>C variant genotype (−765CC) was associated with a significantly decreased risk of HCC compared with the −765GG wild-type homozygotes [P COX-2 −765G>C polymorphism, causing lower COX-2 gen expression, is a genetic protective factor for HCC. However, because this is the first report concerning the COX-2 −1195A>G, −765G>C, and +8473T>C polymorphisms and the risk of HCC, independent studies are needed to validate our findings.
We have evaluated the selectivity of ketoprofen and two novel nonsteroidal anti‐inflammatory drugs, N‐[2‐(cyclohexyloxy)‐4‐nitrophenyl]methanesulphonamide (NS‐398) and 5‐methanesulphonamido‐6‐(2, 4‐difluorothiophenyl)‐1‐indanone (L‐745, 337), in inhibiting the cyclo‐oxygenase activity of prostaglandin endoperoxide synthase‐2 (PGHS‐2) vs PGHS‐1 in human blood monocytes and platelets, respectively.
Heparinized whole blood samples were drawn from healthy volunteers pretreated with aspirin, 300 mg 48 h before sampling, to suppress the activity of platelet PGHS‐1 and incubated at 37°C for 24 h with increasing concentrations of the test compounds in the presence of lipopolysaccharide (LPS, 10 μg ml ⁻¹ ). Immunoreactive PGE 2 levels were measured in plasma by a specific radioimmunoassay as an index of the cyclo‐oxygenase activity of LPS‐induced monocyte PGHS‐2.
The effects of the same inhibitors on platelet PGHS‐1 activity were assessed by allowing whole blood samples, drawn from the same subjects in aspirin‐free periods, to clot at 37°C for 1 h in the presence of the compounds and measuring immunoreactive thromboxane B 2 (TXB 2 ) levels in serum by a specific radioimmunoassay.
Under these experimental conditions, ketoprofen enantioselectively inhibited the cyclo‐oxygenase activity of both PGHS‐1 and PGHS‐2 with equal potency (IC 50 ratio: approx. 0.5 for both enantiomers), while L‐745, 337 and NS‐398 achieved selective inhibition of monocyte PGHS‐2 (IC 50 ratio: > 150). L‐745, 337 and NS‐398 did not affect LPS‐induced monocyte PGHS‐2 biosynthesis to any detectable extent.
We conclude that L‐745, 337 and NS‐398 are selective inhibitors of the cyclo‐oxygenase activity of human monocyte PGHS‐2. These compounds may provide adequate tools to test the contribution of this novel pathway of arachidonate metabolism to human inflammatory disease.
Ischemic stress causes damage to cerebrovascular endothelium and alters arteriolar responses to prostanoid-dependent stimuli. However, effects of ischemic stress on cyclooxygenase (COX) levels in endothelium are unclear. We examined the effect of ischemia and reperfusion and asphyxia and reventilation on production of COX isoforms in cerebral vascular endothelium.
Neonatal pigs were exposed to global ischemia (n = 4) or asphyxia (n = 3) for 5-10 min. Following 2-6 h of recovery, the animals were killed, and the cerebral arteries and arterioles were removed. Cerebral arteries and arterioles were also removed from untreated control animals (n = 1) and from time control animals (n = 3). Cerebral vessels were fixed in 4% formalin and paraffin embedded, and constitutive and inducible COX (COX-1 and COX-2, respectively) levels were assessed using indirect immunofluorescence.
Hemotoxylin and eosin staining indicated that anoxic stress leads to enlargement of endothelial cells. Immunofluorescence for COX-1 in endothelium was minimal in cerebral arteries and arterioles from control animals and did not show an increase in animals exposed to anoxic stress. Similarly, cerebral vessels from control animals showed little immunostaining for COX-2. In contrast, immunofluorescence for COX-2 was greatly increased in cerebral arteries and arterioles from animals exposed to asphyxia or ischemia.
We concluded that anoxic stress increases COX-2 but not COX-1 levels in cerebral endothelium.
Cyclo-oxygenase (COX), the enzyme responsible for the conversion of arachidonic acid (AA) to prostaglandin H2 (PGH2), exists in two forms, termed COX-1 and COX-2 which are encoded by different genes. COX-1 is expressed constitutively and is known to be the site of action of aspirin and other non-steroidal anti-inflammatory drugs. COX-2 may be induced by a series of pro-inflammatory stimuli and its role in the development of inflammation has been claimed.
Endothelial cells are an important physiological source of prostanoids and the selective induction of COX-2 activity has been described for finite cultures of endothelial cells, but not for permanent endothelial cell lines.
The HUV-EC-C line is a permanent endothelial cell line of human origin. We have determined the COX activity of these cells under basal conditions and after its exposure to two different stimuli, phorbol 12-myristate 13-acetate (PMA) and interleukin-1β (IL-1β).
Both PMA and IL-1β produced dose- and time-dependent increases of the synthesis of the COX-derived eicosanoids. These increases were maximal after the treatment with 10 nM PMA for 6 to 9 h. Under these conditions, the main eicosanoid produced by the cells was PGE2.
The increase of COX activity by PMA or IL-1β correlated with an increase of the enzyme's apparent Vmax, whilst the affinity for the substrate, measured as apparent Km, remained unaffected.
Treatment of the cells with PMA induced a time-dependent increase in the expression of both COX-1 and COX-2 mRNAs. Nevertheless, this increase was reflected only as an increase of the COX-2 isoenzyme at the protein level.
The enzymatic activity of the PMA-induced COX was measured in the presence of a panel of enzyme inhibitors, and the IC50 values obtained were compared with those obtained for the inhibition of human platelet COX activity, a COX-1 selective assay. Classical non-steroidal anti-inflammatory drugs (NSAIDs) inhibited both enzymes with varying potencies but only the three compounds previously shown to be selective COX-2 inhibitors (SC-58125, NS-398 and nimesulide) showed higher potency towards the COX of PMA-treated HUV-EC-C.
Overall, it appears that the stimulation of the HUV-EC-C line with PMA selectively induces the COX-2 isoenzyme. This appears to be a suitable model for the study of the physiology and pharmacology of this important isoenzyme, with a permanent endothelial cell line of human origin.
British Journal of Pharmacology (1997) 121, 171–180; doi:10.1038/sj.bjp.0701112
Cyclooxygenase (COX) is the pivotal enzyme in prostaglandin biosynthesis. It exists in two isoforms, constitutive COX-1 (responsible for physiological functions) and inducible COX-2 (involved in inflammation). Inhibition of COX explains both the therapeutic effects (inhibition of COX-2) and side effects (inhibition of COX-1) of non-steroidal anti-inflammatory drugs (NSAIDs). A NSAID which selectively inhibits COX-2 is likely to retain maximal anti-inflammatory efficacy combined with less toxicity. The activity of a number of NSAIDs has been investigated in several test systems, showing that most of those marketed have higher activities against COX-1 or are equipotent against both isoforms. Adverse event data of marketed NSAIDs show a relationship between a poor safety profile and more potent inhibition of COX-1 relative to COX-2. There are several new non-steroidal COX-2 inhibitors in development. The most clinically advanced is meloxicam, which consistently demonstrates higher activity against COX-2 than COX-1 in several test systems.
Neuropeptide and cyclooxygenase (Cox) gene expression was examined in the brains of catheterized pigs killed 30 or 120 min after intravenous injection of a low (20 microg) dose of lipopolysaccharide endotoxin (LPS), previously demonstrated to induce fever in this species. In the paraventricular hypothalamic nucleus (PVN), corticotrophin releasing hormone (CRH) mRNA was shown to be present in the pars parvocellularis but was not upregulated 30 or 120 min after 20 microg LPS, or 90 min after 60 microg LPS; there was also no change in proopiomelanocortin (POMC) message in the anterior pituitary (AP). Similarly, expression of mRNAs for lysine vasopressin (LVP) or oxytocin (OT) did not change in the PVN after LPS (20 microg), although LVP message was increased (p<0.05) at 30 min in the hypothalamic supraoptic nucleus (SON). Expression of Cox-1 and Cox-2 genes was quantified in the organum vasculosum lamina terminalis (OVLT) and choroid plexus (CP) in an attempt to determine whether altered expression of prostaglandin (PG) synthetic enzymes in brain vasculature is involved in LPS fever. Although vascular endothelial cells in both structures expressed Cox-1 and Cox-2 mRNAs, neither increased in the OVLT following LPS. However, in the CP, Cox-1 mRNA was enhanced (p<0.05) at 30 and 120 min after LPS injection and Cox-2 showed a similar (NS) change. These results provide the first description of CRH and Cox gene expression in the porcine brain. They also suggest that LPS may influence the activity of genes controlling LVP synthesis in the hypothalamus and PG production by the brain vasculature.
Atherogenesis is a multifactorial chronic inflammatory disease in which low plasma levels of HDLs are a strong predictor of the condition. Although the mechanism of protection by HDLs is not precisely known, HDLs have been shown to influence many of the events involved in the development of atherosclerosis. Previously we have shown that HDLs inhibited the cytokine-induced expression of adhesion molecules (E-selectin, VCAM-1, and ICAM-1) by endothelial cells (ECs). As the complete transcriptional regulation of all 3 genes requires the NF-kappaB family of transcription factors, we examined the effect of HDLs on activation of NF-kappaB. We also investigated the effect of HDLs on 2 other cytokine-induced genes, granulocyte-macrophage colony-stimulating factor (GM-CSF) and cyclooxygenase (Cox-2; prostaglandin H2 synthase, EC 0.1.14.99.1). E-selectin expression in response to tumor necrosis factor-alpha (TNFalpha) was, as expected, inhibited in ECs that had been preincubated with HDLs. However, the level of secretion of GM-CSF in the same cultures was no different from control. In a similar manner, although HDLs had no effect on steady-state mRNA levels of GM-CSF, the levels of E-selectin were significantly inhibited by HDLs. In transient cotransfection experiments we found that HDLs inhibited the cytokine-induced expression of a reporter gene driven by the E-selectin proximal promoter (-383 to 80) but had no effect on the expression of a reporter gene driven under the control of the proximal promoter of GM-CSF (-627 to 28). As would be predicted from this differential response, HDLs did not influence the nuclear translocation or DNA binding of NF-kappaB, or alter the kinetics of degradation and resynthesis of the inhibitory protein IkappaBalpha. We found that HDLs synergized with cytokine to enhance the expression of Cox-2 and induce the synthesis of its main EC product, prostacyclin (PGI2), a potent inhibitor of platelet and leukocyte functions. In conclusion, HDL induces an antiinflammatory phenotype in cytokine-induced ECs, synergizing with cytokine to induce elevation of Cox-2 in addition to inhibiting adhesion molecule expression. Our studies show that these differential effects are mediated in a manner that is likely to be independent of NF-kappaB per se.
The aim of the study was to determine PGE2 levels in venous blood plasma (VBP), gingival blood plasma (GBP) and gingival crevicular fluid (GCF) in advanced periodontal patients before and after 6 months surgical treatment.
There were 12 patients aged 28-45 years and seven healthy subjects as controls included into the study. Clinical parameters such as PLI, GI, BI, PD, AL and bone height % from radiographs were recorded before and after treatment. PGE2 levels were determined by radioimmunoassay (125 I RIA Kit, NEN(R)).
Before treatment the mean VBP, GBP and GCF PGE2 levels in periodontal patients were higher than healthy controls (P < 0.001, Student's t-test). Six months after treatment the mean levels of PGE2 in VBP, GBP and GCF were significantly reduced in patients with improvement of clinical and roentgenological parameters (P < 0.001, Student's t-test).
It can be concluded that PGE2 is involved in the pathogenesis of periodontal disease. The inflamed periodontal tissues may produce significant amount of PGE2 and the degree of inflammation might be determined by the ratios of PGE2. The lower levels of PGE2 in blood plasma and in gingival crevicular fluid after treatment are signs of improvement of periodontal disease.
Nitric oxide ((.)NO) signaling pathways and lipid oxidation reactions are of central importance in both the maintenance of vascular homeostasis and the progression of vascular disease. Because both of these pathways involve free radical species that can also react together at extremely fast rates, convergent interactions between these pathways are expected. Biochemical and cell biology studies have defined multiple interactions of (.)NO with oxidizing lipids that could lead to either vascular protection or potentiation of inflammatory vascular injury. For example, low levels of (.)NO generated by endothelial nitric oxide synthase can terminate propagating lipid radicals and inhibit lipoxygenases, reactions that would be protective. Alternatively, if generated at elevated levels, for example, after inducible nitric oxide synthase expression in inflammation, (.)NO can be converted to prooxidant species, such as peroxynitrite (ONOO(-)) and nitrogen dioxide ((.)NO(2)), that can potentiate inflammatory injury to vascular cells. Finally, both enzymatic and nonenzymatic lipid oxidation reactions can influence (.)NO bioactivity by directly scavenging (.)NO or altering the induction and catalytic activity of nitric oxide synthase enzymes. In this review, we summarize the biochemical interactions between (.)NO and lipid oxidation reactions and discuss the recognized and potential roles of these reactions in the vasculature.
Ischemia-reperfusion injury (IRI) is a common event in organ transplantation, being implicated as a potential contributor for the development of chronic allograft nephropathy. There are new evidences showing a tissue inflammatory response following renal IRI. Cyclooxygenases (COX) 1 and 2 can be detected in tissue submitted to IRI and may have impact on organ function outcome. We evaluated the role of COX inhibition on the renal tissue damage that follows IRI. Mice were submitted to 45 min of renal pedicle ligature and allowed to reperfuse for 24, 48, 72 and 120 h. Blood and kidney samples were collected at reperfusion times. mRNA was extracted from the kidney samples to amplify COX-1, COX-2 and beta-actin genes. Animals were pretreated with indomethacin or rofecoxib before the surgery. Indomethacin treatment induced a better renal function (serum urea) when compared to control animals at 24, 48 and 72 h (219+/-54.5 vs. 338+/-51 mg/dl; 106+/-51 vs. 326+/-86 mg/dl; 94+/-14 vs. 138+/-38 mg/dl, respectively). Surprisingly, rofecoxib use was associated with even better renal improvement following IR. Animals treated with the later drug showed lower urea values at 24 h post reperfusion compared to indomethacin-treated animals (128+/-33 vs. 219+/-54.5 mg/dl, P<0.05). Blockade of COX-1 and -2 resulted in a decrease of tubular necrosis. mRNA COX-2 was up-regulated post IRI and considerable inhibited after indomethacin or rofecoxib treatment. Our data show COX-1/-2 participates in the inflammatory tissue response to IR injury and its inhibition is associated with an improvement in renal function.
It is known that cyclooxygenase (COX)-2 expression is increased in Barrett's esophagus and esophageal adenocarcinomas. We studied COX-2 expression and the effect sulindac has on the genesis of Barrett's esophagus and adenocarcinoma in rats undergoing esophagogastroduodenal anastomosis (EGDA).
Fifty-one rats were divided into a control group (n=27), a 500 ppm sulindac-treated group (n=15) and 1000 ppm sulindac-treated group (n=9). Randomly selected rats were killed by diethyl ether inhalation at 20 and 40 weeks after surgery.
At 40 weeks, rats treated with 1000 ppm sulindac showed narrower esophageal diameter and milder inflammation than the control rats. At 40 weeks, the incidence of Barrett's esophagus was similar between control and sulindac-treated groups, but the incidence of adenocarcinoma was significantly lower in the 1000 ppm sulindac-treated group than either the control or 500 ppm sulindac-treated groups. COX-2 was significantly increased in the lower esophagus of control rats killed at 40 weeks. Cyclin D1 expression was negligible in the sulindac- treated group compared with the control group.
We suggest that the chemopreventive effect of sulindac is related to decreased COX-2 and cyclin D1 expression, which may be influenced by reduced inflammation.
We previously demonstrated that angiotensin II (Ang II) receptor signaling is involved in azoxymethane-induced mouse colon tumorigenesis. In order to clarify the role of Ang II in COX-2 expression in the intestinal epithelium, the receptor subtype-specific effect on COX-2 expression in a rat intestinal epithelial cell line (RIE-1) has been investigated. Ang II dose- and time-dependently increased the expression of COX-2, but not COX-1 mRNA and protein. This stimulation was completely blocked by the AT(1) receptor antagonist but not the AT(2) receptor antagonist. Ang II and lipopolysaccharide (LPS) additively induced COX-2 protein in RIE-1 cells, whereas the LPS-induced COX-2 expression was significantly attenuated by low concentrations of Ang II or the AT(2) agonistic peptide CGP-42112A only in AT(2) over-expressed cells. These data indicate that Ang II bi-directionally regulates COX-2 expression via both AT(1) and AT(2) receptors. Control of COX-2 expression through Ang II signaling may have significance in cytokine-induced COX-2 induction and colon tumorigenesis.
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