Pharmacology of celecoxib in rat brain after kainate administration

Prostaglandin E(2) (PGE(2)) is the major prostaglandin produced both centrally and in the periphery in models of acute and chronic inflammation, and its formation in both locations is blocked by cyclooxygenase-2 (COX-2) inhibitors such as celecoxib. In animal models of inflammation, PGE(2) inhibition in the brain may occur secondarily to a peripheral action by inhibiting local PG formation that elicits increased firing of pain fibers and consequent activation of PG synthesis in the central nervous system (CNS). Celecoxib was studied in the kainate-induced seizure model in the rat, a model of direct central prostaglandin induction, to determine whether it can act directly in the CNS. In the kainate-treated rat brain there was increased PGE(2), PGF(2alpha), and PGD(2) production, with COX activity and PGE(2) formation increased about 7-fold over normal. We quantitated mRNA levels for enzymes involved in the prostaglandin biosynthetic pathways and found that both COX-2 and PGE synthase (PGEs) mRNA levels were increased in the brain; no changes were found for expression of COX-1 or PGD synthase mRNA. By Western blot analysis, COX-2 and PGEs were induced in total brain, hippocampus, and cortex, but not in the spinal cord. Immunohistological studies showed that COX-2 protein expression was enhanced in neurons. Dexamethasone treatment reduced the expression of both COX-2 and PGEs in kainate-treated animals. Celecoxib reduced the elevated PGE(2) levels in brain of kainate-treated rats and inhibited induced COX activity, demonstrating the ability of this compound to act on COX-2 in CNS. Doses of celecoxib that inhibited brain COX-2 were lower than those needed for anti-inflammatory activity in adjuvant arthritis, demonstrating a potent direct central action of the compound.     

Effects of selective and unselective cyclooxygenase inhibitors on prostanoid release from various rat organs

It has been assumed that cyclooxygenase-2 (COX-2) is solely responsible for inflammatory processes. Recently, this view has been challenged because COX-2-selective agents caused a delay of gastric ulcer healing and exacerbation of inflammation in rats. To further characterize organ-specific toxic effects of selective and nonselective COX inhibitors, we assessed the eicosanoid release from different rat organs ex vivo after oral administration of the COX-2-selective inhibitor NS-398 and the unselective COX inhibitors diclofenac, meloxicam, and ketorolac. Prostanoid and leukotriene release from tissue fragments of the stomach, kidney, lung, and brain were determined after ex vivo incubation of tissue fragments in Tyrode's solution for 10 min at 37 degrees C. Ketorolac (0.1, 0.3, and 0.9 mg/kg) inhibited prostanoid release from all organs most potently and led to a significant increase of leukotriene release from the lung. Effects of diclofenac and meloxicam (1, 3, and 9 mg/kg each) were similar for all organs tested. At 9 mg/kg, 6keto-prostaglandin F (PGF)(1alpha) release from gastric mucosa was reduced by 79.1 +/- 11.4 and 87.6 +/- 7.7% and PGE(2) release from rat kidney was inhibited by 60.4 +/- 6.8 and 78.6 +/- 16.6% by diclofenac and meloxicam, respectively. NS-398 did not reduce prostanoid release from the lung. Consistent with the reported constitutive expression of COX-2, prostanoid release from kidney and brain was reduced by 20 to 30%. The release of 6keto-PGF(1alpha) from gastric mucosa was reduced by 34.7 +/- 22.2% at 3 mg/kg and by 86.9 +/- 12.7% at 9 mg/kg. At these doses, NS-398 has been previously shown to be COX-2 selective. Because PGF(1alpha) is the stable breakdown product of PGI(2), these results suggest that COX-2 contributes to PGI(2) synthesis in the rat stomach.     

Urinary prostaglandin F2 alpha excretion is not pH-dependent in the conscious rat: implications for the urinary prostaglandin E2/prostaglandin F2 alpha ratio

1. The influence of urine pH on the urinary excretion of prostaglandin (PG) F2 alpha and the PGE2/PGF2 alpha ratio has been examined in the conscious rat. 2. The basal urinary PGF2 alpha excretion rate of 3.9 pmol/h (n = 23) did not vary with urine pH. In marked contrast, PGE2 excretion increased as the urine became more alkaline. The PGE2/PGF2 alpha ratio therefore progressively increased from 1.5 to 22 as the pH of the urine changed from pH 5.8 to pH 7.8. 3. The independence of PGF2 alpha excretion from urine pH: (a) excludes cyclo-oxygenase as a potential site of action for the pH-dependence of urinary PGE2 excretion; (b) suggests that the urinary PGE2/PGF2 alpha ratio measured in alkaline urine may be a more accurate reflection of the kidneys, ability to synthesize these two prostaglandins in vivo; (c) suggests that control of urine pH is required before the urinary PGE2/PGF2 alpha ratio can be employed as an index of PGE2 9-ketoreductase (EC 1.1.1.189) activity in vivo.     

Isomer-specific effects of conjugated linoleic acid on lipid peroxidation in humans: regulation by alpha-tocopherol and cyclo-oxygenase-2 inhibitor

We have found previously that supplementation with conjugated linoleic acid (CLA) induces lipid peroxidation and inflammation in humans as indicated by an increase of 8-iso-prostaglandin F2alpha (PGF2alpha) and 15-keto-dihydro-PGF2alpha respectively. The present study was undertaken firstly to study the regulatory mechanisms behind these effects, and secondly to see if these effects are specific to different isomers of CLA. Sixty healthy men and women, divided into six groups, were given a cyclo-oxygenase (COX)-2 inhibitor (rofecoxib; 12 mg/day), alpha-tocopherol (200 mg/day) or neither treatment (control group) over a period of 6 weeks. During the last 4 weeks, three groups were given a CLA preparation (3.5 g/day) mainly containing the isomers cis-9, trans-11 and trans-10, cis-12 (CLA mix), and the three other groups a CLA preparation mainly containing the isomer trans-10, cis-12 (CLA 1012; 4.0 g/day). Treatment with alpha-tocopherol or COX-2 inhibitor did not alter the basal urinary levels of either 8-iso-PGF2alpha or 15-keto-dihydro-PGF2alpha. Both CLA preparations induced an increase in 8-iso-PGF2alpha and 15-keto-dihydro-PGF2alpha in the urine, with a larger increase being found in the CLA 1012 group. Treatment with the COX-2 inhibitor suppressed the increase in urinary 15-keto-dihydro-PGF2alpha in the CLA 1012 group, but not in the CLA mix group. Neither the COX-2 inhibitor nor alpha-tocopherol had any effect on 8-iso-PGF2alpha levels after supplementation with CLA. The CLA-induced production of PGF2alpha metabolites is probably partially mediated by COX-2. Levels of the induced lipid peroxidation may be dependent on the isomeric property of CLA.     

Effect of regulated expression of human cyclooxygenase isoforms on eicosanoid and isoeicosanoid production in inflammation

To examine the role of cyclooxygenase (COX) isozymes in prostaglandin formation and oxidant stress in inflammation, we administered to volunteer subjects placebo or bolus injections of lipopolysaccharide (LPS), which caused a dose-dependent increase in temperature, heart rate, and plasma cortisol. LPS caused also dose-dependent elevations in urinary excretion of 2,3-dinor 6-keto PGF(1alpha) (PGI-M) and 11-dehydro thromboxane B(2) (Tx-M). Platelet COX-1 inhibition by chronic administration of low-dose aspirin before LPS did not alter the symptomatic and febrile responses to LPS, but the increment in urinary PGI-M and Tx-M were both partially depressed. Pretreatment with ibuprofen, a nonspecific COX inhibitor, attenuated the febrile and systemic response to LPS and inhibited prostanoid biosynthesis. Both celecoxib, a selective COX-2 inhibitor, and ibuprofen attenuated the pyrexial, but not the chronotropic, response to LPS. Experimental endotoxemia caused differential expression of the COX isozymes in monocytes and polymorphonuclear leucocytes ex vivo. LPS also increased urinary iPF(2alpha)-III, iPF(2alpha)-VI, and 8,12-iso-iPF(2alpha)-VI, isoprostane (iP) indices of lipid peroxidation, and none of the drugs blunted this response. These studies indicate that (a) although COX-2 predominates, both COX isozymes are induced and contribute to the prostaglandin response to LPS in humans; (b) COX activation contributes undetectably to lipid peroxidation induced by LPS; and (c) COX-2, but not COX-1, contributes to the constitutional response to LPS in humans.     

Effect of furosemide on the renal prostaglandin system in liver cirrhosis with ascites

Daily urine levels of PGE2 and PGF2 alpha as well as water-electrolyte exchange were measured in 72 patients with chronic active hepatitis (CAH) and hepatic cirrhosis (HC) in comparison with healthy controls. Furosemide treatment was performed in 26 HC patients. It was established that in CAH and compensated HC daily excretion of PGE2 and PGF2 alpha as well as 22Na elimination were within the range registered in the control. In association of HC with nonrefractory ascites the above excretion was elevated, with refractory one daily excretion of PGE2 was low, the urine ratio PGF2 alpha/PGE2 rose. Daily urine excretion of prostaglandins and PGF2 alpha/PGE2 value may be of prognostic significance in evaluating efficacy of diuretic therapy in HC patients with ascites.     

Type 1 diabetes is associated with increased cyclooxygenase- and cytokine-mediated inflammation

OBJECTIVE: The extent of involvement of cyclooxygenase (COX)-mediated inflammation in type 1 diabetes is unknown, and the association between the COX- and cytokine-mediated inflammatory responses in type 1 diabetes is not fully understood. RESEARCH DESIGN AND METHODS: Plasma high-sensitivity C-reactive protein (CRP), 24-h urinary and plasma 15-keto-dihydro-prostaglandin F(2alpha) (a metabolite of prostaglandin F(2alpha) [PGF(2alpha)] and an indicator of COX-mediated inflammation), serum amyloid protein A (SAA), and interleukin (IL)-6 (indicators of inflammation) were measured in 38 subjects with type 1 diabetes and 41 healthy age- and sex-matched control subjects. RESULTS: The inflammatory indicators (urinary 15-keto-dihydro-PGF(2alpha), P < 0.01; IL-6, P < 0.04) were increased in men with diabetes. CRP and SAA did not show any significant difference between the diabetic and the control subjects. Urinary levels of 15-keto-dihydro-PGF(2alpha) correlated with the degree of glycemic control, HbA(1c) (r = 0.42, P < 0.0005). No correlation was found between the duration of diabetes and the inflammatory biomarkers or metabolic measurements. CONCLUSIONS: These results suggest that an early low-grade inflammatory process reflected by elevated levels of PGF(2alpha) and IL-6 is involved in type 1 diabetes. Thus, both COX- and cytokine-mediated inflammatory pathways are significantly related to type 1 diabetes.     

Prostacyclin antagonism reduces pain and inflammation in rodent models of hyperalgesia and chronic arthritis

The inhibition of prostaglandin (PG) synthesis is at the center of current anti-inflammatory therapies. Because cyclooxygenase-2 (COX-2) inhibitors and nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit the formation of multiple PGs, there is currently a strong focus on characterizing the role of the different PGs in the inflammation process and development of arthritis. Evidence to date suggests that both PGE(2) and PGI(2) act as mediators of pain and inflammation. Most of the data indicating a role for PGI(2) in this context have been generated in animal models of acute pain. Herein, we describe the role of PGI(2) in models of osteoarthritis (OA) and rheumatoid arthritis using a highly selective PGI(2) receptor (IP, Ptgir) antagonist and IP receptor-deficient mice. In the rat OA model using monoiodoacetate injection into the knee joint, the IP antagonist reduced pain with an efficacy approaching that of the NSAID diclofenac. In a chronic model of inflammatory arthritis, collagen-antibody induced arthritis model in mice, IP receptor-deficient mice displayed a 91% reduction in arthritis score. Interestingly, pretreatment with the IP [N-[4-(imidazolidin-2-ylideneamino)-benzyl]-4-methoxy-benzamide] antagonist in this model also caused a significant reduction of the symptoms, whereas administration of the compound after the initiation of arthritis had no detectable effect. Our data indicate that, in addition to its role in acute inflammation, PGI(2) is involved in the development of chronic inflammation. The results also suggest that the inhibition of PGI(2) synthesis by NSAIDs and COX-2 inhibitors, in addition to that of PGE(2), contributes to their efficacy in treating the signs of arthritis.     

Differential impact of prostaglandin H synthase 1 knockdown on platelets and parturition

Platelet activation is a hallmark of severe preeclampsia, and platelet PGH synthase 1-derived (PGHS1-derived) thromboxane A(2) (TxA(2)) has been implicated in its pathogenesis. However, genetic disruption of PGHS1 delays parturition. We created hypomorphic PGHS1 (PGHS1(Neo/Neo)) mice, in which the substantial but tissue-dependent variability in the inhibition of PGHS1-derived eicosanoids achieved by low-dose aspirin treatment is mimicked, to assess the relative impact of this strategy on hemostatic and reproductive function. Depression of platelet TxA(2) by 98% in PGHS1(Neo/Neo) mice decreased platelet aggregation and prevented thrombosis. Similarly, depression of macrophage PGE(2) by 75% was associated with selectively impaired inflammatory responses. PGF(2alpha) at 8% WT levels was sufficient to induce coordinated temporal oxytocin receptor (OTR) expression in uterus and normal ovarian luteolysis in PGHS1(Neo/Neo) mice at late gestation, while absence of PGHS1 expression in null mice delayed OTR induction and the programmed decrease of serum progesterone during parturition. Thus, extensive but tissue-dependent variability in PG suppression, as occurs with low-dose aspirin treatment, prevents thrombosis and impairs the inflammatory response but sustains parturition. PGHS1(Neo/Neo) mice provide a model of low-dose aspirin therapy that elucidates how prevention or delay of preeclampsia might be achieved without compromising reproductive function.     

Cyclooxygenase expression in splanchnic hyposensitivity to glypressin of bleeding portal hypertensive rats

BACKGROUND: Prostacyclin mediates, at least partly, the splanchnic vascular hyporesponsiveness to glypressin in bleeding portal hypertensive rats. This study investigated the relative contribution of cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) in the splanchnic hyposensitivity to glypressin in rats with portal hypertension induced by partial portal vein ligation (PVL). METHODS: Fourteen days after the operation, the rats were divided into without- and with-bleeding groups. Three series of PVL rats were used to investigate (i). the haemodynamic effects of glypressin (0.07 mg x kg(-1) intravenously), (ii). COX-1/COX-2 mRNA expression over abdominal aorta and superior mesenteric artery and (iii). plasma levels of 6-keto-prostaglandin-F1alpha. In rats with a hypotensive haemorrhage, 4.5 mL of blood was withdrawn and 50% of the withdrawn blood was re-infused before blood and vessel sampling or the administration of glypressin. RESULTS: Splanchnic hyposensitivity to glypressin was demonstrated in the haemorrhage-transfused PVL rats with enhanced COX-1 expression of superior mesenteric artery and increased plasma levels of 6-keto-prostaglandin-F1alpha. There were no differences in the COX-2 expression of superior mesenteric artery and COX-1 and COX-2 expressions of abdominal aorta between without- and with-bleeding groups. CONCLUSION: In portal hypertensive rats with acute haemorrhage, COX-1 over-expression in the superior mesenteric artery plays a role in mediating the splanchnic hyposensitivity to glypressin.     

Cytochrome P450 metabolites of arachidonic acid but not cyclooxygenase-2 metabolites contribute to the pulmonary vascular hyporeactivity in rats with acute Pseudomonas pneumonia

We have previously demonstrated depressed vascular contractility in intralobar pulmonary artery (PA) rings isolated from rats with acute Pseudomonas pneumonia. Here we describe the role of arachidonic acid (AA) metabolites in the regulation of pulmonary vascular tone in inflammation. Pneumonia was induced by intratracheal injection of P. aeruginosa organisms. Rats were sacrificed 44 h later. EETs and 20-HETE were formed at significantly lower rates in pneumonia compared with control lung microsomes. Vasoactive effects of CYP metabolites (5,6-EET, 8,9-EET, 11,12-EET, 14,15-EET, and 20-HETE) on small PA rings from control or pneumonia rats were assessed in vitro. All four EETs and 20-HETE were more potent PA vasoconstrictors than KCl or phenylephrine (PE). However, this potency was attenuated in PA rings from pneumonia lungs compared with control. In contrast, pneumonia had no effect on COX activity [total pulmonary prostaglandin (PG), PGE(2), and 6-keto-PGF(1 alpha)]. In vitro vascular contractility to KCl, PE, or PGF(2 alpha) was assessed in small PA rings from control and pneumonia rats in the presence and absence of the COX-2 inhibitor NS-398 (10 microM). NS-398 did not reverse the attenuated contractile responses to KCl, PE, or PGF(2 alpha) in pneumonia rats. Nitrite/nitrate levels, inducible nitric-oxide synthase and heme oxygenase activities were all significantly elevated in pneumonia lungs. In conclusion, vasodilator PGs produced by COX-2 do not contribute to the depressed PA contractility in this model of pneumonia. Depressed pulmonary production and vasoconstrictor effects of CYP metabolites of AA (possibly due to increased NO and/or carbon monoxide) indicate a potential role for these vasoactive metabolites in this model of acute pneumonia.     

Repetitive, noninvasive imaging of cyclooxygenase-2 gene expression in living mice.

The cyclooxygenase-2 (COX-2) gene plays a role in a wide variety of normal physiologic pathways and is a major target of pharmacologic intervention in a large number of pathophysiologic contexts, including pain, fever, inflammation, and cancer. Expression of the COX-2 gene is induced in a wide range of cells, in response to an ever-increasing number of stimuli. The regulation of the COX-2 gene has been the subject of extensive study, using traditional transfection techniques with reporter gene constructs. Regulation of the COX-2 gene in living animals, however, requires sacrifice of the animal and in situ hybridization and/or immunohistochemical studies. We have utilized in vivo optical imaging technology with a cooled charged coupled device camera to image the expression of the firefly luciferase gene in tumor xenografts that are stably transfected with a chimeric gene containing the first kilobase of the murine COX-2 promoter. Induction of luciferase gene expression following systemic lipopolysaccharide/endotoxin administration can be robustly demonstrated; both a dose-response relationship and a time course for luciferase expression from the COX-2 promoter can be noninvasively analyzed in the tumor xenografts. These data suggest expression from the COX-2 promoter will be easily analyzed in transgenic mice, in knock-in mice, and in somatic cell and gene transfer experiments.     

Mechanism of action of paracetamol

Paracetamol (acetaminophen) is generally considered to be a weak inhibitor of the synthesis of prostaglandins (PGs). However, the in vivo effects of paracetamol are similar to those of the selective cyclooxygenase-2 (COX-2) inhibitors. Paracetamol also decreases PG concentrations in vivo, but, unlike the selective COX-2 inhibitors, paracetamol does not suppress the inflammation of rheumatoid arthritis. It does, however, decrease swelling after oral surgery in humans and suppresses inflammation in rats and mice. Paracetamol is a weak inhibitor of PG synthesis of COX-1 and COX-2 in broken cell systems, but, by contrast, therapeutic concentrations of paracetamol inhibit PG synthesis in intact cells in vitro when the levels of the substrate arachidonic acid are low (less than about 5 mumol/L). When the levels of arachidonic acid are low, PGs are synthesized largely by COX-2 in cells that contain both COX-1 and COX-2. Thus, the apparent selectivity of paracetamol may be due to inhibition of COX-2-dependent pathways that are proceeding at low rates. This hypothesis is consistent with the similar pharmacological effects of paracetamol and the selective COX-2 inhibitors. COX-3, a splice variant of COX-1, has been suggested to be the site of action of paracetamol, but genomic and kinetic analysis indicates that this selective interaction is unlikely to be clinically relevant. There is considerable evidence that the analgesic effect of paracetamol is central and is due to activation of descending serotonergic pathways, but its primary site of action may still be inhibition of PG synthesis. The action of paracetamol at a molecular level is unclear but could be related to the production of reactive metabolites by the peroxidase function of COX-2, which could deplete glutathione, a cofactor of enzymes such as PGE synthase.     

环氧合酶-2在胃癌细胞中的表达及意义

目的　通过研究环氧合酶 2 (COX 2 )在胃癌、癌前病变及慢性炎症中的表达情况 ,探讨COX 2与胃癌发生、发展的关系及意义。方法　采用免疫组化和原位分子杂交法 ,对 15例正常胃黏膜组织及 12 3例不同病变胃黏膜组织标本中COX 2进行检测。结果　COX 2蛋白在胃癌组织中的表达明显增强 ,其阳性率 (6 2 .2 6 % )显著高于正常胃黏膜、慢性浅表性胃炎、慢性萎缩性胃炎伴肠化及不典型增生胃黏膜组织 (6 .6 7% ,17.86 % ,30 .4 3% ,4 2 .11% ;P均 <0 .0 5 ) ;COX 2蛋白的阳性表达与胃癌组织学分级、浸润深度及有无淋巴结转移有关 ;高、中分化胃癌组中COX 2的表达明显高于低分化组 ;同时 ,在COX 2蛋白阳性的相关胃黏膜组织细胞中亦可见到COX 2mRNA的阳性表达。结论　COX 2的过度表达可能参与了胃癌形成的全过程 ,不仅在胃癌形成的早期 ,且与胃癌的侵袭、转移及预后亦有一定关系。     

Combination treatment using the COX-2 inhibitor meloxicam and an anti-allergic in pollinosis

The value of combination therapies that aim to affect both immediate and delayed inflammatory reactions is well known in the treatment of pollinosis. Use of a selective cyclooxygenase-2 (COX-2) inhibitor such as meloxicam is a new approach, however. COX-2 inhibitors target the delayed reaction, which plays a major role in chronic nature of this allergic reaction through activation of the arachidonate cascade and the production of prostaglandins. In this study, four treatment regimens were compared for effectiveness in relieving the symptoms of pollinosis: (i) an anti-allergic alone; (ii) an anti-allergic plus a conventional non-steroidal anti-inflammatory drug; (iii) an anti-allergic plus a selective COX-2 inhibitor; and (iv) an anti-allergic plus an oral steroid. Use of a COX-2 inhibitor plus an anti-allergic produced significantly more relief for most symptoms. The significance of this is as yet unclear, but it is hoped that these results will further our understanding of chronic inflammation.     

Importance of renal prostaglandins in control of renal function after chronic ligation of the common bile duct in dogs

To explore the possible vasoregulatory role of renal prostaglandins during liver disease, excretory rates of PGE2, PGF2 alpha, and a metabolite of PGI2, 6k-PGF1 alpha, were determined before and after chronic ligation of the common bile duct in 23 dogs. Bile duct ligation for 50 +/- 3.7 days (mean +/- SEM) significantly increased serum bilirubin and alkaline phosphatase. PGE2, PGF2 alpha, and 6k-PGF1 alpha excretion rates were significantly (p less than 0.01) increased following chronic bile duct ligation, by approximately 100%, 80%, and 500%, respectively, with similar increments in both ascitic and nonascitic animals. In 10 sham-ligated animals, PGE2, PGF2 alpha, and 6k-PGF1 alpha excretion rates were unchanged. In 6 dogs sequential measurements of urine prostaglandins indicated that PGE2 and 6k-PGF1 alpha excretion were significantly increased at 2, 4, and 6 weeks after ligation, whereas the increase in PGF2 alpha excretion was not significant until 6 weeks. Indomethacin (2 mg/kg) reduced prostaglandin excretion by 65% to 90% and significantly increased arterial pressure, decreased glomerular filtration rate and renal blood flow, and increased renal vascular resistance from 0.53 +/- 0.09 to 0.90 +/- 0.13 mm Hg/ml/min. Fractional renal blood flow, assessed by microspheres, was disproportionately reduced in the inner cortex after prostaglandin inhibition in the chronic bile duct ligation group. Indomethacin did not significantly alter renal function in sham animals, despite comparable reductions in prostaglandin excretion. These data demonstrate that, in dogs with experimental liver disease produced by chronic bile duct ligation, renal prostaglandin synthesis is increased, and the enhanced synthesis of vasodilatory prostaglandins serves to maintain renal blood flow and glomerular filtration rate.     

Protective effect of tyrphostin AG-556 on shock induced by endotoxin or gram positive bacteria.

The effects of tyrphostin AG-556 (TYR), a tyrosine kinase inhibitor, were evaluated on shock induced by lipopolysaccharide (LPS) or group B streptococcus (GBS) in rats. Mortality and mean survival time were monitored. Plasma 6-keto prostaglandin F1alpha (6-keto PGF1alpha) was also measured at four hours after LPS injection. The effects of TYR on the production of 6-keto PGF1alpha thromboxane B2(TXB2) and nitrite (NO) from LPS or GBS stimulated in vitro peritoneal rat macrophage were also examined. Salmonella enteritidis LPS (12 mg/kg, i.v. ) (n=6) produced severe shock (100% mortality). Simultaneous treatment with TYR (n=6) significantly (p < 0.01) extended mean survival time and 33% of rats survived. Plasma 6-keto PGF1alpha concentrations were increased in LPS controls, whereas TYR (5 mg/kg) significantly (p < 0.05) decreased the production. Animals treated with GBS/D-galactosamine (n=9) also exhibited shock with 100% lethality and TYR again prolonged survival time (p < 0.05) with 55% of the animals surviving. To evaluate direct effects of TYR on mediator production induced by LPS or GBS, rat macrophages were stimulated with heat-killed GBS or LPS with or without TYR. Supernatants were collected at 24 h for determination of TXB2, 6-keto PGF1alpha and NO. All mediators measured were significantly increased (p < 0.05) with LPS or GBS. TYR inhibited (p < 0.05) the production of all mediators from macrophages induced by LPS or GBS. The decrease in eicosanoids was associated with a reduction of the content of cyclooxygenase-2 (COX-2) as determined by western blotting. Collectively, these results suggest that TYR ameliorates toxic shock induced by LPS or gram positive bacteria. This protection is associated with suppression of macrophage mediator production.     

Recruited bone marrow cells expressing the EP3 prostaglandin E receptor subtype enhance angiogenesis during chronic inflammation

Chronic inflammation, which is characterized by the proliferation of granulation tissues, is known to be regulated by angiogenesis. Recent results suggest that bone marrow-derived (BM-derived) hematopoietic cells regulate angiogenesis in vivo. We previously reported that the angiogenesis occurring during chronic inflammation is enhanced in response to the endogenous prostaglandins (PGs) derived from an inducible cyclooxygenase-2 (COX-2). In the present study, we examined the role of BM-derived cells expressing an E-type PG receptor subtype, EP3, in sponge-induced angiogenesis. The replacement of wild-type (WT) BM with BM cells (BMCs) from green fluorescent protein (GFP) transgenic mice revealed that the formation of granulation tissue around the sponge implants developed via the recruitment of BMCs. This recruitment was enhanced by topical injections of vascular endothelial growth factor (VEGF)-A, and a VEGF-dependent increase in the recruitment of BMCs was inhibited by a COX-2 inhibitor, celecoxib. FACS analysis of the granulation tissues after treatment with collagenase revealed that the Mac-1-positive macrophage fraction was enhanced by topical injections of VEGF-A, and that this increased recruitment of Mac-1-positive BMCs was inhibited by celecoxib. Selective knockdown of EP3 performed by BM transplantation with BMCs isolated from EP3 knockout (EP3) mice reduced sponge-induced angiogenesis, as estimated by mean vascular number and CD31 expression in the granulation tissues. This reduction in angiogenesis in EP3^−/− BM chimeric mice was accompanied by reductions in the recruitment of BMCs, especially of Mac-1-positive cells and Gr-1-positive cells. These results indicate that the recruited bone marrow cells that express the EP3 receptor have a significant role in enhancing angiogenesis during chronic proliferative inflammation.     

Failure of chronic aspirin treatment to inhibit urinary prostaglandin excretion in spontaneously hypertensive rats: comparison with indomethacin and flurbiprofen

The inability of chronic treatment with aspirin to cause sustained inhibition of urinary prostaglandin (PG) excretion observed previously prompted us to compare the effects of 9-day treatment of spontaneously hypertensive rats with aspirin, 200 mg/kg/day s.c., flurbiprofen, 2.5 mg/kg/b.i.d. s.c. and indomethacin, 2.5 mg/kg/b.i.d. s.c. on the excretion rate of radioimmunoassayable PGE2 and PGF2 alpha. Conversion of 1-[14C]arachidonic acid and the release of PGs from endogenous substrate by the renal papilla were also examined. In vehicle-treated control rats, PGF2 alpha excretion ranged from 32.2 +/- 6.2 (mean +/- S.E.M.) to 41.6 +/- 7.3 ng/6 h, and was 2- to 4-fold higher than that of PGE2. Within 6 h of administration all three drugs reduced excretion of PGF2 alpha and PGE2 to less than 20% and 35% of control rats, respectively. Thereafter, PGF2 alpha and PGE2 excretion in aspirin-treated rats returned to values similar to the vehicle-treated group, whereas inhibition of PG excretion in indomethacin and flurbiprofen groups was sustained. Urine volume was doubled by aspirin throughout the study. In contrast, urine volume in flurbiprofen- and indomethacin-treated rats was unaffected. Paradoxically, metabolism of 1-[14C]arachidonic acid to PGs by renal papilla dissected on day 10, 2 to 4 h after the last drug dose, was reduced markedly by aspirin as was the release of immunoreactive PGs but was unaffected by flurbiprofen or indomethacin. The failure of long-term aspirin treatment to inhibit urinary PG excretion and the disparity between in vivo and ex vivo indices of PG release emphasize the need to verify their intended action by measuring PGs in biological fluids.     

Rofecoxib modulates multiple gene expression pathways in a clinical model of acute inflammatory pain

New insights into the biological properties of cyclooxygenase-2 (COX-2) and its response pathway challenge the hypothesis that COX-2 is simply pro-inflammatory and inhibition of COX-2 solely prevents the development of inflammation and ameliorates inflammatory pain. The present study performed a comprehensive analysis of gene/protein expression induced by a selective inhibitor of COX-2, rofecoxib, compared with a non-selective COX inhibitor, ibuprofen, and placebo in a clinical model of acute inflammatory pain (the surgical extraction of impacted third molars) using microarray analysis followed by quantitative RT-PCR verification and Western blotting. Inhibition of COX-2 modulated gene expression related to inflammation and pain, the arachidonic acid pathway, apoptosis/angiogenesis, cell adhesion and signal transduction. Compared to placebo, rofecoxib treatment increased the gene expression of ANXA3 (annexin 3), SOD2 (superoxide dismutase 2), SOCS3 (suppressor of cytokine signaling 3) and IL1RN (IL1 receptor antagonist) which are associated with inhibition of phospholipase A(2) and suppression of cytokine signaling cascades, respectively. Both rofecoxib and ibuprofen treatment increased the gene expression of the pro-inflammatory mediators, IL6 and CCL2 (chemokine C-C motif ligand 2), following tissue injury compared to the placebo treatment. These results indicate a complex role for COX-2 in the inflammatory cascade in addition to the well-characterized COX-dependent pathway, as multiple pathways are also involved in rofecoxib-induced anti-inflammatory and analgesic effects at the gene expression level. These findings may also suggest an alternative hypothesis for the adverse effects attributed to selective inhibition of COX-2.