Increased Cyclooxygenase Activity Research Articles

Environmental cocaine places mussels at risk by increasing neurotransmitters

When going to the beach for a swim, probably the last thing on your mind is whether you are swimming in a thin soup of drug traces. Whether prescribed over the counter or illegal, many drugs taken by people wind up in our waterways. These drugs cannot be removed by wastewater treatment plants and thus get released back into the environment. A famous coastal region in Brazil, the Bay of All Saints (Baía de Todos os Santos in Portuguese), has been the subject of multiple environmental contaminant studies and researchers have found measurable quantities of cocaine in the seawater, sediment and mussels (Perna perna) of the bay. Although the environmental concentrations are too low to affect humans, whether cocaine at these levels has an effect on the nervous system of aquatic species that reside in the bay was unknown. So, Mayana Fontes and a team of researchers from Universidade Estadual Paulista Júlio de Mesquita Filho, Brazil, and other universities in Brazil investigated how levels of cocaine in the seawater affect the nervous system of P. perna mussels.Fontes and colleagues placed adult mussels in tanks containing seawater with two different concentrations of cocaine that were realistic for the Bay of All Saints (0.2 and 2 μg l−1) for 2, 4 or 7 days. Then, they collected the adductor muscles, which open and close the shell, and gonads of these mussels. In the adductor muscle, they measured the activity of acetylcholine esterase, an enzyme essential for a functioning nervous system. In the gonads, they measured the levels of two major neurotransmitters, serotonin and dopamine, the activity of the enzyme that breaks down these neurotransmitters, monoamine oxidase, and the activity of cyclooxygenase, an enzyme that is a sign of inflammation and is important for egg release in mussels. In addition, the authors also measured how much energy the mussels spent during the exposure to cocaine by measuring mitochondrial electron transport activity, and how much energy they set aside by measuring the level of total lipids in the gonads.The team found that mussels exposed to cocaine had a decreased acetylcholine esterase activity in the adductor muscle, which shows that cocaine disrupted the normal activity of the nervous system. They also found that mussels exposed to cocaine had raised levels of dopamine and serotonin. These neurotransmitters are essential for reproduction, and changes could lead to an unsustainable population in the wild. The mussels also increased cyclooxygenase activity in the gonads, which shows an inflammatory response to low doses of cocaine. In addition, the scientists found changes in the mitochondrial electron transport chain – which converts the energy from oxygen into ATP in the mitochondria – directly increasing the amount of energy the mussels spent after exposure to the drug, which decreases the amount of energy they can access for reproduction as energy is limited. Finally, they also found that there was an increase in levels of total lipid, which act as a convenient and easy to access energy source for the animals.Fontes and colleagues have shown that the current concentration of cocaine in the seawater around the Bay of All Saints can negatively impact the area's mussels in complex ways and this may put them at a greater risk from other chemicals that we dump into the environment.

Intrinsic exercise capacity induces divergent vascular plasticity via arachidonic acid-mediated inflammatory pathways in female rats

Metabolic syndrome prevalence has increased among US adults, particularly among non-hispanic white and black women. Sedentary behavior often leads to chronic inflammation, a triggering factor of metabolic syndrome. Given that intrinsic exercise capacity is genetically inherited, we questioned if low-grade chronic inflammation would be present in a female rat model of low intrinsic exercise capacity-induced metabolic syndrome, while beneficial increase of resolution of inflammation would be present in a female rat model of high intrinsic exercise capacity. In the vascular system, two primary markers for inflammation and resolution of inflammation are cyclooxygenase (COX) and lipoxygenase (LOX), respectively. Our study focused on the novel hypothesis that untrained, inherited exercise capacity induces divergent vascular plasticity via changes in the delicate balance between COX and LOX inflammatory mediators.We used divergent rat strains with low (LCR) and high (HCR) aerobic running capacity. By using animals with contrasting intrinsic exercise capacities, it is possible to determine the exact triggers that lead to inherited vascular plasticity in female rats. We observed that female LCR displayed increased periovarian fat pad and body weight, which is congruent with their obesity-presenting phenotype. Furthermore, LCR presented with vascular hypocontractility and increased COX and LOX-derived pro-inflammatory factors. On the other hand, HCR presented with a “shutdown” of COX-induced vasoconstriction and enhanced resolution of inflammation to maintain vascular tone and homeostasis. In conclusion, LCR display low-grade chronic inflammation via increased COX activity. These results provide mechanistic clues as to why lower intrinsic aerobic capacity correlates with a predisposition to risk of vascular disease. Conversely, being born with higher intrinsic aerobic capacity is a significant factor for improved vascular physiology in female rats.

Abstract P058: Female Rats Artificially Selected For Low Vs. High Intrinsic Aerobic Capacity Display Divergent Mechanisms In Vascular Inflammation

Hypertension is an important clinical symptom of metabolic syndrome (MetS). Rats selectively bred for low intrinsic aerobic capacity (LCR) are animal models for MetS, and present with increased blood pressure and vascular dysfunction. In contrast, rats selected for high intrinsic aerobic capacity (HCR) display reduced vascular inflammation and no metabolic abnormalities. Two important enzymes for vascular inflammation and the resolution of inflammation are cyclooxygenase (COX) and lipoxygenase (LOX), respectively; however, it is unknown whether COX and LOX play a role in the vascular function of LCR and HCR. We hypothesized that mesenteric resistance arteries (MRA) from untrained LCR present increased COX activity, while arteries from HCR show decreased COX and increased LOX activity. Female (18-38 weeks old) LCR, HCR, and high response trained (HRT) rats, control, were used. HRT rats present higher intrinsic aerobic capacity than LCR, but lower than HCR. MRA were mounted onto a wire myograph. One-way ANOVA: p<0.05: *vs. control (HRT); # vs. HCR; & vs. absence of indomethacin (INDO), a COX inhibitor. LCR rats showed increased periovarian fat pad [HRT: 0.95±0.1 (n=7) vs. LCR: 1.80±0.1* # (n=7) vs. HCR: 1.18±0.1 (n=7) (g)]. No significant differences were observed in the KCl (120 mM), acetylcholine, and sodium-nitroprusside-induced responses. However, LCR presented a decrease in phenylephrine (PE)-induced contraction [PE: E max %: HRT: 103±3 (n=8); LCR: 74±9* # (n=11); HCR: 112±5 (n=9)]. Inhibiting COX [INDO, 10 μM] decreased contraction in HRT arteries, but had little effect on HCR arteries. Contrarily, INDO abolished contraction in MRA from LCR [PE+INDO: E max %: HRT: 31±18 & (n=7); LCR: 2±0.9 & (n=8); HCR: 77±9 (n=8)]. Lipoxin (LXA4), a LOX-derived mediator for resolution of inflammation, induced contraction in MRA from HCR, but relaxation in LCR and HRT arteries [LXA4: E max %: HRT: -69±19 (n=4); LCR: -18±9 (n=3); HCR: 11±5 (n=4)*]. Thus, HCR are unresponsive to COX inhibition, suggesting a change from a normal inflammatory state to a higher resolution state. LCR display low-grade chronic inflammation via increased COX activity. These data reveal novel, inherited mechanisms for vascular physiology in high vs. low intrinsic aerobic capacity.

Female Rats Artificially Selected for Low and High Intrinsic Aerobic Capacity Swap Inflammatory Cascade in Resistance Arteries: Mechanisms of Cyclooxygenase‐Derived Prostanoids

Low‐capacity runner (LCR) rats and high‐capacity runner (HCR) rats are translational models of human physiology as they present a genetic predisposition towards disease and the ultra‐healthy state, respectively. Selected for their low (LCR) and high (HCR) intrinsic aerobic capacity, LCR rats display increased risk of metabolic syndrome and vascular inflammation, whereas HCR rats have decreased risk factors. Cyclooxygenase (COX) is an enzyme that plays a key role in inflammation as well as normal vascular physiology; however, it is unknown if rats with low or high intrinsic capacity present differences in the COX‐induced inflammatory cascade. As obese females display an increased risk to cardiovascular disease compared to males, female LCR rats were used as a model for metabolic syndrome. Therefore, we hypothesized that resistance arteries from female LCR rats would show increased COX activity, while arteries from female HCR rats would show decreased COX activity.Our rat model (female, 18–25 weeks old) study used LCR, HCR, and high‐response trained (HRT) rats as control (n=4/group). HRT rats have a trained aerobic capacity that is higher than LCR but lower than HCR. We quantified cardiac hypertrophy by dividing the left ventricle weight by 26.73 + tibia length (TL)3. Statistics: t‐test: p<0.05: * vs control (HRT); # vs. HCR; & vs. absence of indomethacin (Indo). No differences in fat pad weight between HRT and HCR were observed. However, LCR rats showed increased fat pad weight when compared to HCR [HRT: 1.05±0.2 vs. LCR: 1.57±0.2#; HCR: 0.92±0.1 (g)]. As expected, HCR presented an increased left ventricle weight, indicating physiologic hypertrophy, when compared to LCR. No differences were observed between LCR and control [HRT: 6.84±0.9 vs. LCR: 6.55±0.1#; HCR: 7.73±0.3; 26.73+TL3]. Mesenteric resistance arteries (MRA) were extracted and mounted onto a wire myograph. No significant differences were observed in the acetylcholine and phenylephrine (PE) concentration‐response curves between groups. As expected, arteries from HRT and LCR rats incubated with a nonspecific COX inhibitor [indomethacin (Indo), 10 μM] presented a significant decrease in contraction. Interestingly, indomethacin did not alter or decrease phenylephrine‐induced contraction in arteries from HCR [PE: Emax%: HRT: control 93±3 vs. Indo 3±0.6&; LCR: control 62±17 vs. Indo 2±0.7&; HCR: control 94±6 vs. Indo 80±9] (Fig 1). Overall, these data reveal a new, dichotomous mechanism for vascular inflammation and contractility in LCR and HCR rats. Specifically, we observed that arteries from female rats born with high aerobic capacity are unresponsive to COX inhibition during phenylephrine contraction, perhaps to maintain the high vascular flow needed for their higher exercise metabolism. This may be caused by a swap mechanism from a normal inflammatory state to a higher inflammatory‐resolution state. Our study constitutes the basis for the discovery of new therapeutic targets for vascular inflammation in people with high aerobic capacity.Support or Funding InformationThis work was supported by National Institutes of Health (NIH) (NIH: R00GM118885)Concentration‐response curves to phenylephrine, with and without indomethacin (10 uM), in mesenteric resistance arteries (MRA) from (A) HRT, (B) LCR, and (C) HCR rats. (D) The average maximum response (Emax) of MRA to phenylephrine, with and without indomethacin (10 uM). T‐test: & vs. absence of Indo p<0.05.Figure 1

Chronic Femoral Artery Ligation Increases Skeletal Muscle Interstitial Prostaglandin E2 in the Rat: Implications for the Exercise Pressor Reflex

The stimulation of primarily mechanically‐sensitive group III and primarily metabolically‐sensitive group IV muscle afferents during skeletal muscle contraction evokes reflex increases in blood pressure (i.e., the exercise pressor reflex). The exercise pressor reflex is exaggerated in many forms of cardiovascular disease when compared to the reflex found in healthy counterparts. In rats in which a femoral artery is ligated for 72 hours (a model of simulated peripheral artery disease), the mechanically‐sensitive component of the reflex (i.e., the mechanoreflex) contributes importantly to the overall exercise pressor reflex exaggeration. Using a dynamic hindlimb muscle stretch protocol to study the mechanoreflex, we found recently that the increase in blood pressure was greater during stretch of a ligated versus a freely perfused hindlimb. This suggested that a chronic sensitization of mechanically‐activated channels contributed to the exaggerated mechanoreflex in ligated hindlimbs. The identity of the sensitizing chemical, however, was unknown. Experimental evidence has established that cyclooxygenase (COX) products, especially prostaglandins, sensitize mechanically‐activated channels. Accordingly, the purpose of this investigation was to determine whether 72 hour femoral artery ligation increased resting skeletal muscle interstitial prostaglandin E2 (PGE2) concentration in decerebrate, unanesthetized rats. The microdialysis technique was used to collect hindlimb skeletal muscle interstitial fluid in eight rats with a previously ligated femoral artery and a freely perfused femoral artery. We found that resting PGE2 concentration was greater in the ligated (224±44 pg/ml) versus the freely perfused (180±33 pg/ml, p<0.01) hindlimb. To investigate the potential underlying mechanisms of the PGE2 elevation, we quantified both COX expression and COX activity in the soleus muscles (highly oxidative) and white portion of the gastrocnemius muscles (highly glycolytic). Western blot analyses revealed that femoral artery ligation had no effect on COX‐1 or COX‐2 protein expression in the soleus or white gastrocnemius muscles (p‐value range: 0.44 to 0.78). Total COX activity, however, was higher in the white gastrocnemius muscle of ligated hindlimbs compared to freely perfused hindlimbs (p<0.04). There was no difference in total COX activity in the soleus muscle between hindlimbs (p=0.21). We conclude that 72 hours of femoral artery ligation increased resting PGE2 concentration in rat hindlimb skeletal muscles. The higher PGE2 concentration in ligated hindlimb muscles appears attributable to increased COX activity in white, glycolytic muscle portions. Our findings suggest that chronically elevated skeletal muscle interstitial PGE2 concentration may contribute to the exaggerated mechanically‐sensitive component of the exercise pressor reflex found in ligated hindlimb versus a freely perfused hindlimb.Support or Funding Information1R01HL142877 to SWCThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Distinct influence of COX-1 and COX-2 on neuroinflammatory response and associated cognitive deficits during high altitude hypoxia

High-altitude hypoxia (HH) causes a spectrum of pathophysiological effects, including headaches, gliovascular dysfunction, and cognitive slowing. Previous studies have shown arachidonic acid (AA) metabolism due to cyclooxygenase (COX) activity before clinical manifestations in many diseases. AA metabolites, including COXs and prostaglandin E2 (PGE2), are well known immunomodulators. However, the relative contribution of COX-2 and COX-1 isoforms in the downstream proinflammatory responses and cognitive deficit in HH remains unknown. In the present study, AA metabolism via the COX pathway was investigated in Sprague Dawley rats after 0, 1, 3, and 7 days of HH exposure. Furthermore, we investigated the inflammatory response and cell-type-specific induction of both COXs. Our data revealed that AA metabolites peaked on day 3 of HH exposure. Interestingly, we observed endothelial and microglial activation on day 1, accompanied by an increase in the levels of proinflammatory cytokines, followed by astrocyte activation on day 3. We showed that the increase in COX activity during HH culminated in a significant increase in hippocampal inflammation, concomitant with spatial memory impairment and neuronal injury at day 7 of HH. We showed HH induced distinct COX-1 expression in endothelial and microglial cells, whereas it induced COX-2 expression predominantly in neurons, endothelial cells, and astrocytes. Notably, our data showed that the inhibition of COX-1 using valeryl salicylate had a prominent role in containing hippocampal inflammation by reducing microglial activation. COX-2 inhibition using celecoxib, along with COX-1 inhibition, ameliorated spatial memory impairment, astrocyte activation, and neurodegeneration after HH exposure.

Fate of silver nanoparticles in wastewater and immunotoxic effects on rainbow trout

Silver nanoparticles (AgNPs) are currently used in technology, medicine and consumer products, even though the fate and the ecotoxicological risks on aquatic organisms of these new materials are not well known. The purpose of this study was to investigate the fate, bioavailability of AgNPs and their effects on fish in presence of municipal effluents. Juvenile rainbow trout were exposed for 96h to 40μg/L of AgNPs or 4μg/L of dissolved silver (AgNO3) in diluted (10%) municipal wastewater. Silver (Ag) concentrations were measured both on water samples and fish tissues (liver and gills). Toxicity was investigated by following immunological parameters in the pronephros (viability, phagocytosis) and biomarkers in liver and gills (cyclooxygenase activity, lipid peroxidation, glutathione-S-transferase, metallothioneins, DNA strand breaks and labile zinc). Results indicated that AgNPs appeared as small non-charged aggregates in wastewaters (11.7±1.4nm). In gills, the exposure to AgNPs induced morphological modifications without visible nanoparticle bioaccumulation. Dissolved Ag+ was bioavailable in diluted effluent and induced oxidative stress (lipid peroxidation), labile zinc and a marginal decrease in superoxide dismutase in fish gills. Ag+ also increased significantly metallothionein levels and inhibited the DNA repair activity in the liver. Finally, the two silver forms were found in liver and induced immunosuppression and inflammation (increase in cyclooxygenase activity). This study demonstrated that both forms of Ag produced harmful effects and AgNPs in wastewater were bioavailable to fish despite of their formation of aggregates.

Abstract 38: Chemoprevention with COX2 and EGFR inhibition in familial adenomatous polyposis patients: mRNA signatures of duodenal neoplasia

Abstract Background and Purpose: Nearly 80% of colon cancers arise after acquired mutations in the adenomatous polyposis coli (APC) gene. Familial adenomatous polyposis (FAP) is an autosomal dominant inherited disorder caused by a germline mutation in the APC gene, leading to a nearly 100% risk of colorectal cancer and a 12% lifetime risk of duodenal cancer. Since prophylactic colectomy is now the standard of care when colorectal polyposis increases beyond endoscopic management, duodenal cancer has now become the leading cause of cancer death in FAP. Efficacious chemoprevention of duodenal neoplasia is, therefore, an unmet clinical need. As part of a Phase II double blind, randomized placebo-controlled trial of cyclooxygenase (COX) and epidermal growth factor receptor (EGFR) inhibition therapy with sulindac and erlotinib respectively, in FAP patients, we showed a profound 40% decrease in duodenal polyp burden at 6 months (p = 3 X10-9). For most patients on drug, both duodenal polyp number and size were decreased. However, the mechanisms by which COX and EGFR inhibition protects against duodenal neoplasia remain incompletely understood. Methods: To better understand the molecular changes in duodenal tissue caused by sulindac and erlotinib, RNA sequencing was performed on normal and polyp (≥3mm) biopsies from the duodenum of FAP patients obtained 6-month endpoint endoscopy. Ten FAP patients on placebo and ten FAP patients on sulindac and erlotinib were selected for this preliminary analysis. Differential RNA expression in paired baseline and endpoint normal colon tissue and paired normal and polyp tissue was determined using negative binomial statistics (DESeq2). Variations in RNA expression patterns between biopsy samples was determined by principal component analysis and unsupervised hierarchical clustering. Biological pathways affected by sulindac and erlotinib treatment were determined using Ingenuity Pathway Analysis (IPA) software. Results: Comparing paired polyp and normal tissue from patients on placebo, we identified more than 1100 differentially expressed genes (Fold ≥ 1.5, FDR < 0.05). In contrast, there were no differentially expressed genes found between paired polyp and normal tissue from patients on drug. Our clustering analysis revealed that 3 of 16 duodenal polyps from patients on drug had similar expression profiles as polyps from patients on placebo. These three duodenal polyps are hypothesized to have developed resistance. Using IPA we identified several protein kinases and transcription factors predicted to be activated in polyps from patients on placebo but not from patients on erlotinib and sulindac. These include IKBKB, ERBB2, EGFR, SMAD4, CTNNB1, CCND1 and JUN. As expected, Wnt/β-catenin signaling was among the top canonical pathways activated in adenomatous polyps from patients on placebo. Our analysis also predicted activation of prostaglandin E2 suggestive of increased cyclooxygenase activity in polyps. Directly comparing polyps from patients on placebo to polyps from patients on drug we identified many immediate early genes including EGR1, EGR2, FOSB and JUN that were preferentially more highly expressed in polyps from placebo patients. Conclusions: Animal models and molecular studies have supported a link between Wnt and EGFR signaling pathways and COX-2 expression in intestinal neoplasia. A chemoprevention trial in patients with germline APC mutations leading to familial adenomatous polyposis tested the potential for combined COX and EGFR inhibition to hinder growth of duodenal neoplasia. Sequencing of mRNA from tissue obtained at endpoint of the trial demonstrated inhibition of COX2, EGFR and Wnt/β-catenin signaling in duodenal neoplasia of patients treated with sulindac and erlotinib. The successful observed regression of duodenal neoplasia in drug-treated FAP patients on the trial is supported by differential expression in the mRNA. Citation Format: Deborah Neklason, Don Delker, Jewel Samadder, Austin Wood, Randall Burt, Phil Bernard. Chemoprevention with COX2 and EGFR inhibition in familial adenomatous polyposis patients: mRNA signatures of duodenal neoplasia. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Integrating Clinical Genomics and Cancer Therapy; Jun 13-16, 2015; Salt Lake City, UT. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(1_Suppl):Abstract nr 38.

The influence of zinc chloride and zinc oxide nanoparticles on air-time survival in freshwater mussels

The purpose of this study was to determine the cumulative effects of exposure to either dissolved zinc or nanozinc oxide (nanoZnO) and air-time survival in freshwater mussels. Mussels were exposed to each forms of zinc for 96h then placed in air to determine survival time. A sub-group of mussels before and after 7days of exposure to air were kept aside for the determination of the following biomarkers: arachidonate-dependent cyclooxygenase (COX) and peroxidase (inflammation and oxidative stress), lipid metabolism (total lipids, esterases activity, HO-glycerol, acetyl CoA and phospholipase A2) and lipid damage (lipid peroxidation [LPO]). The results showed that air-time survival was decreased from a mean value of 18.5days to a mean value of 12days in mussels exposed to 2.5mg/L of nanoZnO although it was not lethal based on shell opening at concentrations below 50mg/L after 96h. In mussels exposed to zinc only, the median lethal concentration was estimated at 16mg/L (10–25 95% CI). The air-time survival did not significantly change in mussels exposed to the same concentration of dissolved Zn. Significant weight losses were observed at 0.5mg/L of nanoZnO and at 2.5mg/L for dissolved zinc chloride, and were also significantly correlated with air-time survival (r=0.53; p<0.01). Air exposure significantly increased COX activity in control mussels and in mussels exposed to 0.5mg/L of nanoZnO and zinc chloride. The data also suggested fatty acid breakdown and β-oxidation. Mussels exposed to contaminants are more susceptible to prolonged exposure to air during low water levels.

Frataxin deficiency increases cyclooxygenase 2 and prostaglandins in cell and animal models of Friedreich's ataxia.

An inherited deficiency of the mitochondrial protein frataxin causes Friedreich's ataxia (FRDA); the mechanism by which this deficiency triggers neuro- and cardio-degeneration is unclear. Microarrays of neural tissue of animal models of the disease showed decreases in antioxidant genes, and increases in inflammatory genes. Cyclooxygenase (COX)-derived oxylipins are important mediators of inflammation. We measured oxylipin levels using tandem mass spectrometry and ELISAs in multiple cell and animal models of FRDA. Mass spectrometry revealed increases in concentrations of prostaglandins, thromboxane B2, 15-HETE and 11-HETE in cerebellar samples of knockin knockout mice. One possible explanation for the elevated oxylipins is that frataxin deficiency results in increased COX activity. While constitutive COX1 was unchanged, inducible COX2 expression was elevated over 1.35-fold (P < 0.05) in two Friedreich's mouse models and Friedreich's lymphocytes. Consistent with higher COX2 expression, its activity was also increased by 58% over controls. COX2 expression is driven by multiple transcription factors, including activator protein 1 and cAMP response element-binding protein, both of which were elevated over 1.52-fold in cerebella. Taken together, the results support the hypothesis that reduced expression of frataxin leads to elevation of COX2-mediated oxylipin synthesis stimulated by increases in transcription factors that respond to increased reactive oxygen species. These findings support a neuroinflammatory mechanism in FRDA, which has both pathomechanistic and therapeutic implications.

Increase in cyclooxygenase activity in the brain paraventricular nucleus mediates development of angiotensin II‐salt hypertension in the rat

One in three adults in the US have chronic high blood pressure (hypertension; HTN) putting them at risk for cardiovascular diseases. Previous work from our laboratory suggests that inhibition of brain cyclooxygenases (COXs), which catalyze the rate limiting step in prostaglandin synthesis, prevents HTN development in rats treated with angiotensin II (AngII) and high salt (2% NaCl) diet. However, brain COX enzyme levels during HTN development were unaltered. So, we hypothesized that an early increase in brain COX activity in organum vasculosum lamina terminalis (OVLT), subfornical organ (SFO), paraventricular nucleus (PVN) and/or rostral ventrolateral medulla (RVLM) leads to AngII‐salt HTN development. Male Sprague‐ Dawley rats, fed a high‐salt diet were fitted with radio‐telemeters for continuous measurement of BP. After recording baseline BP, osmotic mini‐pumps were surgically implanted to deliver saline vehicle or AngII (150ng/kg/min, SC). Brains were collected after 4 days of infusion as rats became hypertensive. Above regions were punched and COX activity was measured using a commercially available kit. Hypertensive rats showed a significant increase in COX activity only in the PVN (p&lt;0.05 vs. control rats)and not in other brain regions. Therefore, inhibition of COX signaling in specific regions of the CNS like PVN may represent a potential therapeutic target for patients with salt‐sensitive HTN.

Transient cyclooxygenase inhibition blocks development of chronic angiotensin II‐salt hypertension

Hypertension (HT) is the major risk factor for cardiovascular diseases. Previously, cyclooxygenase inhibition with ketoprofen was found to prevent Angiotensin II (AngII)‐salt HT development and sympathetic over‐activity. But ketoprofen did not reverse established HT. Recently, Johnson and colleagues reported that transient, short‐term exposure to circulating AngII can “prime” (i.e. accentuate) subsequent AngII HT development. So we hypothesized that an early increase in cyclooxygenase activity accentuates AngII‐salt HT; additionally, we attempted to determine the time‐course of the effect. Radio‐telemetric recording of arterial pressure (AP) in a 22‐day protocol was made. After 3 days of control recordings, rats on 2% NaCl diet were pretreated with vehicle or ketoprofen (2mg/kg, sc) for 5 days and 11 days. From day 8, all rats received AngII (150 ng/kg/min, sc). In vehicle‐treated rats MAP rose to 147±2 mmHg. In rats pretreated with ketoprofen for 5 days and 11 days, MAP rose to 129±8 mmHg (p&gt;0.05) and 109±7 mmHg (p&lt;0.05) compared to vehicle treated rats. The fall in MAP using ganglionic blocker hexamethonium in 11‐day ketoprofen treated rats (35±7mmHg) was less than in vehicle treated rats (61±5mmHg) and 5‐day ketoprofen treated rats (70±2mmHg). We conclude that early AngII‐mediated cyclooxygenase pathway activation is critical for the later full development of AngII‐salt sympathoexcitation and HT.

Evidence of feminization in wild Elliptio complanata mussels in the receiving waters downstream of a municipal effluent outfall

The endocrine-disrupting activity of municipal effluents has the potential to alter the reproductive system and induce feminization to aquatic organisms. The purpose of this study was to examine the sex ratio, vitellogenin (Vtg)-like proteins, serotonin, arachidonate cyclooxygenase (COX) activity and dopamine status in wild mussels living at sites upstream and downstream of two municipal effluent outfalls in the Mille-Îles River (Quebec, Canada). Gonad integrity was also studied by monitoring the gonado-somatic index (GSI), the activity of the rate-limiting enzyme aspartate transcarbamoylase (ATC) for purine synthesis, and changes in lipid peroxidation (LPO). The results showed that the proportion of females was dramatically increased from 30% at the upstream sites to 80% at the downstream sites. The levels of Vtg-like proteins were significantly elevated in the male mussels only. Male mussels downstream of the municipal effluent plumes expressed female-specific protein bands (Vtg-like), as determined by high-resolution gel electrophoresis and silver staining. The serotonin/dopamine ratio was significantly decreased in the downstream mussels, indicating that the gonad was in a state of early vitellogenesis. However, this change was not accompanied by changes in ATC, suggesting no significant egg production was underway; this was confirmed by the observation that the downstream mussels displayed significantly low GSIs. GSIs were rather dependent on the serotonin/dopamine ratio (r = 0.44; p < 0.001), while Vtg-like proteins were dependent on dopamine levels (r = 0.50; p < 0.001). The increase in COX activity at the downstream sites and its close relationship with increased serotonin levels suggest a concomitant serotonergic signalling in addition to VTG production. The production of Vtg-like proteins combined with the serotonergic effects of the municipal effluents was associated with oxidative damage (LPO) in the gonad. This study provides the first evidence of feminization in wild mussel populations and the disruption in gonad physiology by exposure to municipal effluents.

Upregulation of fibronectin expression by COX-2 is mediated by interaction with ELMO1

Engulfment and cell motility 1 (ELMO1), a bipartite guanine nucleotide exchange factor (GEF) for the small GTPase Rac 1, was identified as a susceptibility gene for glomerular disease. Here, we reported that ELMO1 interacted with COX-2 in human mesangial cells. Furthermore, we identified ELMO1 as a posttranslational regulator of COX-2 activity. We demonstrated that COX-2 cyclooxygenase activity increased fibronectin promoter activity. The protein–protein interaction between ELMO1 and COX-2 increased the cyclooxygenase activity of COX-2 and, correspondingly, fibronectin expression. We also found that ET625, the dominant negative form of ELMO1 lacking Rac1 activity, interacted with COX-2, increased cyclooxygenase activity of COX-2 and enhanced COX-2-mediated fibronectin upregulation. To further rule out Rac1 as an ELMO1-mediated regulator of COX-2 activity, we employed the constitutive active Rac1, Rac1 Q63E, and demonstrated that Rac1 signaling has no effect on COX-2-mediated fibronectin promoter activity. These results suggest that ELMO1 contributes to the development of glomerular injury through serving as a regulator of COX-2 activity. The interaction of ELMO1 with COX-2 could play an important role in the development and progression of renal glomerular injury.

Chronic Inhibition of Nitric-Oxide Synthase Potentiates Endothelium-Dependent Contractions in the Rat Aorta by Augmenting the Expression of Cyclooxygenase-2

Acute inhibition of nitric-oxide synthase (NOS) unmasks the release of endothelium-derived contracting factors (EDCFs). The present study investigated whether chronic inhibition of NOS modulates endothelium-dependent contractions. Eighteen-week-old male Sprague-Dawley rats were treated by daily gavage for 6 weeks with the NOS inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) (60 mg/kg) or vehicle (distilled water; 1 ml/kg). Chronic treatment with L-NAME increased arterial blood pressure. Isometric tension was measured in aortic rings with or without endothelium. Endothelium-dependent relaxations to acetylcholine and the calcium ionophore 5-(methylamino)-2-[(2R,3R,6S,8S,9R,11R)-3,9,11-trimethyl-8-[(1S)-1-methyl-2-oxo-2-(1H-pyrrol-2-yl)-ethyl]-1,7-dioxaspiro[5.5]undec-2-yl]methyl]-4-benzoxazolecarboxylic acid (A23187) were reduced in preparations from L-NAME-treated rats. The reduction in relaxation to A23187 was partially reversed by L-arginine (1 mM). In quiescent aortic rings, A23187 caused contractions in the presence of L-NAME and intact endothelium. The A23187-induced contractions were greater in rings from the L-NAME-treated rats than in those from the control group. These contractions were abolished by the cyclooxygenase (COX)-2 inhibitor N-[2-cyclohexyloxy-4-nitrophenyl]methanesulfonamide (NS-398) and the thromboxane-prostanoid (TP) receptor antagonist 3-((6R)-6-{[(4-chlorophenyl)sulfonyl]amido}-2-methyl-5,6,7,8-tetrahydronaphthalen-1-yl)propanoate (S18886), but not by the COX-1 inhibitor 5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazole (SC-560). Chronic L-NAME treatment reduced the level of nitric oxide in the plasma but increased COX activity in the aortic rings. Western blotting and immunohistochemical staining showed that endothelial NOS expression was reduced in the aortae of the chronic L-NAME-treated group. COX-1 expression was augmented slightly, whereas COX-2 expression was up-regulated markedly. The TP receptor expression was comparable with control. These experiments demonstrate that chronic NOS inhibition increases endothelium-dependent contractions of the rat aorta by inducing COX-2 expression and augmenting the production of EDCF.

Increased cyclooxygenase activity impairs apoptosis of inflammatory neutrophils in mice lacking gelatinase B/matrix metalloproteinase‐9

Matrix metalloproteinase-9 (MMP-9)/gelatinase B plays an important role in neutrophil infiltration during inflammation and cyclooxygenases (COX-1 and COX-2) and their products are important regulators of inflammation. Recently, we reported that a genetic lack of MMP-9 impairs neutrophil infiltration during early zymosan-induced peritonitis but at later stages (> 24 hr) neutrophils persist in the peritoneal cavity. Here we show that this is the result of impaired apoptosis of MMP-9(-/-)-derived leucocytes. As enhanced COX-1 expression was reported in MMP-9(-/-) mice, we evaluated the hypothesis that altered COX expression induced the above phenomenon as COX-dependent prostaglandins can act either anti-apoptotically (PGE(2)) or pro-apoptotically (PGD(2)). The current data demonstrate that messenger RNA and protein expression of both COX isoforms and their activities are increased in MMP-9(-/-) mice during late peritonitis. Application of selective COX inhibitors revealed enhanced COX-1-dependent PGE(2) production and impaired COX-2-dependent PGD(2) synthesis in MMP-9(-/-) mice. Most importantly, inhibition of COX-1 abolished prolonged neutrophil accumulation in the peritoneal cavity of MMP-9(-/-) mice and increased apoptosis of inflammatory leucocytes. Similarly, weaker apoptosis of MMP-9(-/-) bone marrow neutrophils treated in vitro with zymosan was reversed by COX-1 inhibition. In conclusion, enhanced COX-1 expression is responsible for persistent neutrophil presence in the peritoneum of MMP-9(-/-) mice because of increased synthesis of anti-apoptotic PGE(2). In non-transgenic mice, however, inflammatory leucocytes die apoptotically in the late stages of peritonitis as a result of COX-2-dependent PGD(2) activity. Overall, we show a dependence of COX expression on the presence of MMP-9.

Nutrition and Age‐Associated Inflammation: Implications for Disease Prevention

Accumulating evidence suggests that aging is associated with dysregulated immune and inflammatory responses. Investigation into the cellular and molecular mechanisms underlying this phenomenon suggests that an up-regulated cyclooxygenase (COX)-2 expression, and resulting increase in production of prostaglandin E(2) (PGE(2)), is a critical factor. Macrophages from old mice have significantly higher levels of PGE(2) production compared with those from young mice, a result of increased COX-2 expression and protein levels leading to increased COX enzyme activity. Furthermore, studies suggest that the age-associated increase in macrophage PGE(2) production is due to ceramide-induced up-regulation of nuclear factor-kappa B activation. Such processes may also occur in cell types other than macrophages, lending further insight into potential mechanisms of age-related diseases. Moreover, the excess PGE(2) induces harmful effects in other cell types such as T cells and adipocytes through the negative crosstalk between macrophages with other cells, resulting in further increased susceptibility to diseases. Nutrient/dietary medications, such as antioxidants and certain lipids have suggested a promising route to reduce the age-related increase in COX activity and PGE(2) production that is associated with several disease states.

Age-associated Inflammatory Changes: Role of Nutritional Intervention

Accumulating evidence suggests that aging is associated with dysregulated immune and inflammatory responses. Investigation into the cellular and molecular mechanisms underlying this phenomenon suggests that an up-regulated cyclooxygenase (COX)-2 expression, and resulting increase in production of prostaglandin E2 (PGE2), is a critical factor. Macrophages from old mice have significantly higher levels of PGE2 production compared with those from young mice, a result of increased COX-2 expression and protein levels leading to increased COX enzyme activity. Further, it is possible that the age-associated increase in macrophage PGE2 production is due to ceramide-induced up-regulation of nuclear factor-kappa B activation. Such processes may also occur in cell types other than macrophages, lending further insight into potential mechanisms of age-related disease. More research is necessary to determine the efficacy of nutrient/dietary modifications, such as antioxidants and lipids, for reducing the age-related increase in COX activity and PGE2 production that are associated with several disease states.

Cyclooxygenase Inhibition in Ischemic Brain Injury

Neuroinflammation is one of the key pathological events involved in the progression of brain damage caused by cerebral ischemia. Metabolism of arachidonic acid through cyclooxygenase (COX) enzymes is known to be actively involved in the neuroinflammatory events leading to neuronal death after ischemia. Two isoforms of COX, termed COX-1 and COX-2, have been identified. Unlike COX-1, COX-2 expression is dramatically induced by ischemia and appears to be an effector of tissue damage. This review article will focus specifically on the involvement of COX isozymes in brain ischemia. We will discuss issues related to the biochemistry and selective pharmacological inhibition of COX enzymes, and further refer to their expression in the brain under normal conditions and following excitotoxicity and ischemic cerebral injury. We will review present knowledge of the relative contribution of each COX isoform to the brain ischemic pathology, based on data from investigations utilizing selective COX-1/COX-2 inhibitors and genetic knockout mouse models. The mechanisms of neurotoxicity associated with increased COX activity after ischemia will also be examined. Finally, we will provide a critical evaluation of the therapeutic potential of COX inhibitors in cerebral ischemia and discuss new targets downstream of COX with potential neuroprotective ability.

Deficient renal 20-HETE release in the diabetic rat is not the result of oxidative stress

We confirmed that release of 20-hydroxyeicosatetraenoic acid (20-HETE) from the isolated perfused kidney of diabetic rats is greatly reduced compared with age-matched control rats. The present studies were undertaken to examine potential mechanisms for the deficit in renal 20-HETE in rats with streptozotocin-induced diabetes of 3-4 wk duration. A role for oxidative stress was excluded, inasmuch as treatment of diabetic rats with tempol, an SOD mimetic, for 4 wk did not affect the renal release of 20-HETE. Similarly, chronic inhibition of nitric oxide formation with nitro-l-arginine methyl ester or aldose reductase with zopolrestat failed to alter the release of 20-HETE from the diabetic rat kidney. Inasmuch as 20-HETE may be metabolized by cyclooxygenase (COX), the expression/activity of which is increased in diabetes, we included indomethacin in the perfusate of the isolated kidney to inhibit COX but found no effect on 20-HETE release. Diabetic rats were treated for 3 wk with fenofibrate to increase expression of cytochrome P-450 (CYP4A) in an attempt to find an intervention that would restore release of 20-HETE from the diabetic rat kidney. However, fenofibrate reduced 20-HETE release in diabetic and control rat kidneys but increased expression of CYP4A. Only insulin treatment of diabetic rats for 2 wk to reverse the hyperglycemia and maintain blood glucose levels at <200 mg/dl reversed the renal deficit in 20-HETE. We conclude that oxidative stress, increased aldose reductase activity, or increased COX activity does not contribute to the renal deficit of 20-HETE in diabetes, which may be directly related to insulin deficiency.