Altered Eicosanoid Production Research Articles

Trisomy 21 impairs PGE2 production in dermal fibroblasts

The triplication of human chromosome 21 results in Down syndrome (DS), the most common genetic form of intellectual disability. This aneuploid condition also results in an enhanced risk of a spectrum of comorbid conditions, such as leukemia, early onset Alzheimer’s disease, and diabetes. Individuals with DS also display an increased incidence of wound healing complications and resistance to solid tumor development. Due to this unique phenotype and the involvement of eicosanoids in key comorbidities like poor healing and tumor development, we hypothesized that cells from DS individuals would display altered eicosanoid production. Using age- and sex-matched dermal fibroblasts we interrogated this hypothesis. Briefly, assessment of over 90 metabolites derived from cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome p450 systems revealed a possible deficiency in the COX system. Basal gene expression and Western blotting experiments showed significantly decreased gene expression of COX1 and 2, and COX2 protein abundance in DS fibroblasts compared to euploid controls. Further, using two different stressors, scratch wound or LPS, we found that DS fibroblasts could not upregulate COX2 abundance and prostaglandin E2 production. Together, these findings show that dermal fibroblasts from DS individuals have a deficient COX2 response, which may contribute to wound healing complications and tumor resistance in DS.

Altered eicosanoid production and phospholipid remodeling during cell culture

The remodeling of PUFAs by the Lands cycle is responsible for the diversity of phospholipid molecular species found in cells. There have not been detailed studies of the alteration of phospholipid molecular species as a result of serum starvation or depletion of PUFAs that typically occurs during tissue culture. The time-dependent effect of cell culture on phospholipid molecular species in RAW 264.7 cells cultured for 24, 48, or 72 h was examined by lipidomic strategies. These cells were then stimulated to produce arachidonate metabolites derived from the cyclooxygenase pathway, thromboxane B2, PGE2, and PGD2, and the 5-lipoxygenase pathway, leukotriene (LT)B4, LTC4, and 5-HETE, which decreased with increasing time in culture. However, the 5-lipoxygenase metabolites of a 20:3 fatty acid, LTB3, all trans-LTB3, LTC3, and 5-hydroxyeicosatrienoic acid, time-dependently increased. Molecular species of arachidonate containing phospholipids were drastically remodeled during cell culture, with a new 20:3 acyl group being populated into phospholipids to replace increasingly scarce arachidonate. In addition, the amount of TNFα induced by lipopolysaccharide stimulation was significantly increased in the cells cultured for 72 h compared with 24 h, suggesting that the remodeling of PUFAs enhanced inflammatory response. These studies supported the rapid operation of the Lands cycle to maintain cell growth and viability by populating PUFA species; however, without sufficient n-6 fatty acids, 20:3 n-9 accumulated, resulting in altered lipid mediator biosynthesis and inflammatory response.

CLA and EPA inhibit LPS-induced prostaglandin release from bovine endometrial cells through an NF-κB-dependent signaling mechanism

Although their action in antagonizing arachidonic acid metabolism is a key antiinflammatory effect of conjugated linoleic acid (CLA) and omega-3 polyunsaturated fatty acids (n-3 PUFA), these molecules have other antiinflammatory effects that might occur upstream of altered eicosanoid production. We examined the effects of two CLA isomers (c9,t11 and t10,c12 isomers), n-3 PUFA (EPA and DHA), and κNF-κB inhibitor (ammonium pyrrolidine dithiocarbamate (PDTC)) on lipopolysaccharide (LPS)-induced secretion of prostaglandins (PGE 2 and PGF 2a ) by bovine endometrial (BEND) cells. LPS increased PGE 2 and PGF 2a concentrations in BEND cell- conditioned media in a concentration- and time-dependent manner. The c9,t11 CLA isomer and EPA decreased prostaglandin response to LPS. Addition of PDTC, alone or in combination with c9,t11 CLA or EPA, inhibited LPS-induced eicosanoid release into the culture medium. Results indicate that the c9,t11 CLA isomer and EPA inhibit the endometrial prostaglandin release in cattle and that these molecules may act through an NF-κB signaling mechanism.

Periodontal disease: modulation of the inflammatory cascade by dietary n‐3 polyunsaturated fatty acids

Periodontal disease, including gingivitis and periodontitis, is caused by the interaction between pathogenic bacteria and the host immune system. The ensuing oxidative stress and inflammatory cascade result in the destruction of gingival tissue, alveolar bone and periodontal ligament. This article reviews the underlying mechanisms and host-bacteria interactions responsible for periodontal disease and evidence that nutritional supplementation with fish oil may provide a protective effect. Historical investigations of diet and disease have highlighted an inverse relationship between ingestion of fish oil, which is high in n-3 polyunsaturated fatty acids, and the incidence of typical inflammatory diseases such as arthritis and coronary heart disease. Ingestion of n-3 polyunsaturated fatty acids, such as docosahexaenoic acid and eicosapentaenoic acid, results in their incorporation into membrane phospholipids, which can alter eicosanoid production after stimulation during the immune response. These eicosanoids promote a reduction in chronic inflammation, which has led to the proposal that fish oil is a possible modulator of inflammation and may reduce the severity of periodontal diseases. Tentative animal and human studies have provided an indication of this effect. Further human investigation is needed to establish the protective effects of fish oil in relation to periodontal disease.

The role of thromboxane A 2 in the pathogenesis of intrauterine growth restriction associated with maternal smoking in pregnancy

Background To examine the effect of maternal smoking in pregnancy on the production of two eicosanoids, thromboxane A 2 and prostacyclin I2, and their role in the pathogenesis of intrauterine growth restriction. Methods Prospective case control study enrolled smoking and non-smoking women at ≤14 weeks gestation. Maternal urine samples were obtained at ≤14, 28 and 36 weeks. High performance liquid chromatography tandem mass spectrometry (LC–MS–MS) was used to quantify 11-dehydrothromboxane B 2 (TX-M) and 2,3 dinor-6-ketoprostaglandin F1α (PG-M), stable urinary metabolites of thromboxane A 2 and prostacyclin I2. Confirmation of the smoking status was performed by quantitation of urinary nicotine metabolites. Data was analysed using SPSS and Stata ®. Results Thirty five were enrolled in the smoking group and 32 in the non-smoking group. Smoking resulted higher levels of TX-M at ≤14, 28 and 36 weeks gestation. There was no difference in PG-M at any gestational time point between the two groups. The median customised birthweight centile in the smoking group was 17.0 (0–78) compared to 55.5 (4–100) in the non-smoking group ( P < 0.001). A causal relationship between elevated TX-M and IUGR could not be established. Conclusions Maternal smoking in pregnancy is associated with altered eicosanoid production in favour of the vasoconstrictor thromboxane A 2 which occurs early in the first trimester.

Polyunsaturated fatty acids and inflammation

The n-6 polyunsaturated fatty acid, arachidonic acid, is a precursor of prostaglandins, leukotrienes and related compounds that have important roles as mediators and regulators of inflammation. Consuming increased amounts of long chain n-3 polyunsaturated fatty acids (found in oily fish and fish oils) results in a partial replacement of the arachidonic acid in cell membranes by eicosapentaenoic and docosahexaenoic acids. This leads to decreased production of arachidonic acid-derived mediators. This alone is a potentially beneficial anti-inflammatory effect of n-3 fatty acids. However, n-3 fatty acids have a number of other effects that might occur downstream of altered eicosanoid production or are independent of this. For example, they result in suppressed production of pro-inflammatory cytokines and can modulate adhesion molecule expression. These effects occur at the level of altered gene expression.

Enhanced 15-HPETE production during oxidant stress induces apoptosis of endothelial cells

Oxidant stress plays an important role in the etiology of vascular diseases by increasing rates of endothelial cell apoptosis, but few data exist on the mechanisms involved. Using a unique model of oxidative stress based on selenium deficiency (−Se), the effects of altered eicosanoid production on bovine aortic endothelial cells (BAEC) apoptosis was evaluated. Oxidant stress significantly increased the immediate oxygenation product of arachidonic acid metabolized by the 15-lipoxygenase pathway, 15-hydroxyperoxyeicosatetraenoic acid (15-HPETE). Treatment of −Se BAEC with TNFα/cyclohexamide (CHX) exhibited elevated levels of apoptosis, which was significantly reduced by the addition of a specific 15-lipoxygenase inhibitor PD146176. Furthermore, the addition of 15-HPETE to PD146176-treated BAEC, partially restored TNF/CHX-induced apoptosis. Increased exposure to 15-HPETE induced apoptosis, as determined by internucleosomal DNA fragmentation, chromatin condensation, caspase-3 activation, and caspase-9 activation, which suggests mitochondrial dysfunction. The expression of Bcl-2 protein also was decreased in −Se BAEC. Addition of a caspase-9 inhibitor (LEHD-fmk) completely blocked 15-HPETE-induced chromatin condensation in −Se BAEC, suggesting that 15-HPETE-induced apoptosis is caspase-9 dependent. Increased apoptosis of BAEC as a result of oxidant stress and subsequent production of 15-HPETE may play a critical role in a variety of inflammatory based diseases.

N-3 polyunsaturated fatty acids and inflammation: from molecular biology to the clinic.

The immune system is involved in host defense against infectious agents, tumor cells, and environmental insults. Inflammation is an important component of the early immunologic response. Inappropriate or dysfunctional immune responses underlie acute and chronic inflammatory diseases. The n−6 PUFA arachidonic acid (AA) is the precursor of prostaglandins, leukotrienes, and related compounds that have important roles in inflammation and in the regulation of immunity. Feeding fish oil results in partial replacement of AA in cell membranes by EPA. This leads to decreased production of AA-derived mediators, through several mechanisms, including decreased availability of AA, competition for cyclooxygenase (COX) and lipoxygenase (LOX) enzymes, and decreased expression of COX-2 and 5-LOX. This alone is a potentially beneficial anti-inflammatory effect of n−3 FA. However, n−3 FA have a number of other effects that might occur down-stream of altered eicosanoid production or might be independent of this effect. For example, dietary fish oil results in suppressed production of proinflammatory cytokines and can modulate adhesion molecule expression. These effects occur at the level of altered gene expression. Fish oil feeding has been shown to ameliorate the symptoms of some animal models of autoimmune disease and to protect against the effects of endotoxin. Clinical studies have reported that oral fish oil supplementation has beneficial effects in rheumatoid arthritis and among some asthmatics, supporting the idea that the n−3 FA in fish oil are anti-inflammatory. There are indications that the inclusion of fish oil in enteral and parenteral formulae is beneficial to patients.

Dietary modification of inflammation with lipids.

The n-3 polyunsaturated fatty acids (PUFA) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are found in high proportions in oily fish and fish oils. The n-3 PUFA are structurally and functionally distinct from the n-6 PUFA. Typically, human inflammatory cells contain high proportions of the n-6 PUFA arachidonic acid and low proportions of n-3 PUFA. The significance of this difference is that arachidonic acid is the precursor of 2-series prostaglandins and 4-series leukotrienes, which are highly-active mediators of inflammation. Feeding fish oil results in partial replacement of arachidonic acid in inflammatory cell membranes by EPA. This change leads to decreased production of arachidonic acid-derived mediators. This response alone is a potentially beneficial anti-inflammatory effect of n-3 PUFA. However, n-3 PUFA have a number of other effects which might occur downstream of altered eicosanoid production or might be independent of this activity. For example, animal and human studies have shown that dietary fish oil results in suppressed production of pro-inflammatory cytokines and can decrease adhesion molecule expression. These effects occur at the level of altered gene expression. This action might come about through antagonism of the effects of arachidonic acid-derived mediators or through more direct actions on the intracellular signalling pathways which lead to activation of transcription factors such as nuclear factor kappa B (NFB). Recent studies have shown that n-3 PUFA can down regulate the activity of the nuclear transcription factor NFB. Fish oil feeding has been shown to ameliorate the symptoms in some animal models of chronic inflammatory disease and to protect against the effects of endotoxin and similar inflammatory challenges. Clinical studies have reported that oral fish oil supplementation has beneficial effects in rheumatoid arthritis and among some patients with asthma, supporting the idea that the n-3 PUFA in fish oil are anti-inflammatory. There are indications that inclusion of n-3 PUFA in enteral and parenteral formulas might be beneficial to patients in intensive care or post-surgery.

Effect of shigatoxin-1 on arachidonic acid release by human glomerular epithelial cells

Altered arachidonic acid (AA) metabolism has been implicated in the pathogenesis of renal injury in the hemolytic uremic syndrome (HUS). However, there is very little information of the effect of shigatoxin (Stx; the putative mediator of renal damage in HUS) on AA release or metabolism by renal cells. Since recent studies have demonstrated that glomerular epithelial cells (GECs) may be important early targets of Stx, the current study was undertaken to examine the effects of Stx on AA release and metabolism by GECs. Cultured human GECs were exposed to Stx1 +/- lipopolysaccharide (LPS) for 4 to 48 hours followed by determination of (3)H-arachidonate release, thromboxane A(2) (TxA(2)) and prostacyclin (PGI(2)) production, cyclooxygenase (COX) activity, and Western and Northern analyses for phospholipase A(2) (PLA(2)) and COX protein and mRNA levels, respectively. Stx1 increased arachidonate release by GECs. LPS alone had no such effect, but increased arachidonate release in response to Stx1. Stx1-stimulated arachidonate release correlated with elevations in cPLA(2) and sPLA(2) protein and cPLA(2) mRNA levels. Stx1 also increased both TxA(2) and PGI(2) production by GECs; LPS alone did not alter eicosanoid production, but augmented Stx1 effects. Both Stx1 and LPS stimulated COX activity; however, these effects were not additive. Although there was an accompanying elevation of COX-1 and COX-2 mRNA, Stx1 decreased and LPS did not change COX1 and COX2 protein levels. Stx1 alone or in conjunction with LPS increases arachidonate release and eicosanoid production by human GECs; this effect correlates with increased PLA(2) protein and mRNA levels. To our knowledge, this is the first study identifying the mechanisms of Stx1-stimulated AA release. These results raise the possibility that arachidonate release and metabolism by GECs, and conceivably other renal cell types, are involved in renal injury in HUS.

Increased 15-HPETE production decreases prostacyclin synthase activity during oxidant stress in aortic endothelial cells

Selenium (Se) is an integral component of glutathione peroxidase and is able to detoxify peroxides that can affect arachidonic acid (AA) metabolism, thereby influencing eicosanoid biosynthesis. This study investigated the effects of oxidant stress, a consequence of Se deficiency, on eicosanoid formation and important key enzyme expression in bovine aortic endothelial cells (BAEC). Bovine aortic endothelial cells cultured in Se-deficient media and stimulated with tumor necrosis factor α or H 2O 2 produced significantly less prostacyclin (PGI 2) and more 15-hydroxyeicosatetraenoic acid, 15-hydroperoxyeicosatetraenoic acid (15-HPETE), and thromboxane than Se-supplemented BAEC. Additionally, reverse transcription polymerase chain reaction and immunoblotting determined that the mRNA and protein levels of the eicosanoid forming enzymes cyclooxygenase-1 (COX1), cyclooxygenase-2 (COX2), and PGI synthase were not significantly changed. The addition of 15-HPETE to Se-supplemented BAEC inhibited the production of PGI 2 suggesting that the accumulation of lipid hydroperoxides during Se-deficiency may be the underlying factor in the altered eicosanoid production during Se deficiency. Furthermore, inhibition of COX and addition of PGH 2 to Se-deficient or Se-supplemented BAEC still resulted in lower PGI 2 formation by Se-deficient cells. Together, these results suggest that Se deficiency modifies eicosanoid production by affecting the activity of key enzymes, particularly PGI synthase, rather than their transcription or translation.

Eicosanoid generation and responsiveness of human lymphatics in hyperlipoproteinemia

In this work, the oxidation injury in hyperlipoproteinemia (HLP) was determined by measuring the isoprostane 8-epi-prostaglandin (PG) F2αin human lymphatics, lymph fluid, plasma, serum and urine. Lymphatics from 6 patients with HLP generated less PGI2and contained more 8-epi-PGF2αas compared to 6 normolipemics without risk factors. Likewise, plasma (29.3 vs 19.7 pg/ml), lymph fluid (137.3 vs 65.3 pg/ml), serum (286.7 vs 204.1 pg/ml) and urinary (360.8 vs 241.0 pg/mg creatinine) values of 8-epi-PGF2αin HLP (as compared to normolipemics) were significantly elevated. Lymphatics from HLP showed an enhanced contractile response, less14C-arachidonic acid conversion to PGI2and less PGI2-formation upon various stimuli compared to normolipemics of comparable age. These findings indicate that HLP-induced oxidation injury, resulting in an altered (iso-)eicosanoid production and function, may also significantly affect (patho-) physiology of lympathics.

Umbilical vessels of preeclamptic women have low contents of both n−3 and n−6 long-chain polyunsaturated fatty acids

Preeclampsia is characterized by enhanced platelet aggregation and vasoconstriction and is related to an elevated ratio of thromboxane A2 to prostacyclin I2. We investigated whether altered eicosanoid production in preeclamptic women could be explained by the fatty acid composition of umbilical vessel walls and platelets. The fatty acid composition of maternal and umbilical platelets and of umbilical arteries and veins in 27 preeclamptic women and 24 normotensive women was determined. Between-group differences were analyzed with linear discriminant analysis, the Kruskal-Wallis test, or analysis of covariance with gestational age as the covariate. Platelets of preeclamptic women contained lower amounts of 20:5n-3 and a higher ratio of 20:4n-6 to 20:5n-3 than did platelets of normotensive women. Additionally, linear discriminant analysis revealed higher amounts of 20:4n-6 in platelets of preeclamptic women. Umbilical arteries and veins in preeclamptic women contained lower amounts of long-chain polyunsaturated fatty acids (PUFAs) of the n-3 series, n-6 long-chain PUFAs, and 20:3n-6 than did umbilical arteries and veins of normotensive women. Umbilical arteries also had lower amounts of 20:4n-6, higher amounts of 20:3n-9, and a higher ratio of 20:3n-9 to 20:4n-6. Low amounts of long-chain n-3 and n-6 PUFAs in umbilical vessels of preeclamptic women with adequate n-6 status may indicate insufficient transplacental transfer of long-chain PUFAs. The low amounts of 20:4n-6, high amounts of 20:3n-9, and high ratio of 20:3n-9 to 20:4n-6 in umbilical arteries may unfavorably affect local prostacyclin production. Low amounts of 20:3n-6 in umbilical arteries and veins and low amounts of 20:5n-3 in maternal platelets may contribute to the dominance of eicosanoids derived from 20:4n-6.

Acceleration of experimental diabetic retinopathy in the rat by omega-3 fatty acids

Omega-3 fatty acids exert several important biological effects on factors that may predispose to diabetic retinopathy. Potential pathogenetic mechanisms include platelet dysfunction, altered eicosanoid production, increased blood viscosity in association with impaired cell deformability and pathologic leucocyte/endothelium interaction. Therefore, we tested whether a 6-month administration of fish oil (750 mg Maxepa, 5 times per week), containing 14% eicosapentaenoic acid (EPA) and 10% docosahexaenic acid, could inhibit the development of experimental retinopathy of the streptozotocin-diabetic rat. The efficiency of fish oil supplementation was evaluated by measuring EPA concentrations in total, plasma and membrane fatty acids and by measuring the generation of lipid mediators (leukotrienes and thromboxanes). Retinal digest preparations were quantitatively analysed for pericyte loss, and the formation of acellular capillaries. Omega-3 fatty acid administration to diabetic rats resulted in a twofold increase of EPA 20:5 in total fatty acids, and a reduction of the thromboxane ratio from 600 (untreated diabetic rats) to 50 (treated diabetic rats). Despite these biochemical changes, diabetes-associated pericyte loss remained unaffected and the formation of acellular, occluded capillaries was increased by 75% in the fish oil treated diabetic group (115.1 +/- 26.8; untreated diabetic 65.2 +/- 15.0 acellular capillary segments/mm2 of retinal area). We conclude from this study that dietary fish oil supplementation may be harmful for the diabetic microvasculature in the retina.

Dietary n-3 Fatty Acids Reduce Antibody-Dependent Cell Cytotoxicity and Alter Eicosanoid Release by Chicken Immune Cells ,

The overall goal of the present study was to determine whether the incorporation of n-3 fatty acids into poultry rations would alter the immune response of broiler chickens. Female broiler chicks were fed a corn and soybean meal-based diet to which one of four dietary fats were added: lard (LA), corn oil (CO), flaxseed oil (SO), or menhaden fish oil (FO). The latter two fat sources are rich in n-3 polyunsaturated fatty acids (PUFA). Enriching the diet with n-3 PUFA did not alter the primary or secondary antibody response of broiler chickens to sheep red blood cells. Dietary fat source had no effect on antibody-dependent cell cytotoxicity (ADCC) by peripheral blood leukocytes, but ADCC by splenocytes was 50% lower in chickens fed SO and FO compared with LA and CO (P < .005). As expected, the fatty acid profile of the isolated immune cells reflected the fatty acid composition of the dietary fats fed. Basal release and calcium ionophore (A23187)-stimulated (10 μM) release of thromboxane B were significantly lower (P < .05) in the SO and FO groups compared with the LA and CO groups. Total leukotriene B release was not significantly altered by dietary fat source. In conclusion, feeding broiler chickens diets rich in n-3 PUFA reduced ADCC of splenocytes and altered eicosanoid production by isolated immune cells.

The efficacy of dexamethasone and flunixin meglumine in treating endotoxin-induced changes in calves.

Eicosanoids have been implicated in the pathophysiology of endotoxic shock. Drugs which alter eicosanoid production such as corticosteroids and non-steroidal anti-inflammatory drugs (NSAID) are beneficial in treating endotoxic shock. Experiments were conducted to investigate the efficacy of dexamethasone, a corticosteroid, and/or flunixin meglumine, a NSAID, in treating endotoxin-induced changes in calves. Fourteen male calves were assigned to one of four treatment groups: group 1, endotoxin-untreated; group 2, endotoxin-flunixin meglumine treated; group 3, endotoxin-dexamethasone-treated; group 4, endotoxin-flunixin meglumine and dexamethasone-treated. Each calf was given three intravenous and intraperitoneal injections of E. coli endotoxin. Hemodynamic, blood gas, blood chemical and eicosanoid level determinations were obtained. Thirty minutes after endotoxin injection, pulmonary artery pressure (PAP) increased and cardiac output (CO) decreased compared with baseline, corresponding to increased thromboxaneB2 levels in groups 1 and 3. These groups exhibited a decreased mean arterial pressure (MAP) at three and five hours corresponding to increased 6-keto-prostaglandinF1 alpha. The MAP, PAP and CO of group 4 remained near baseline for the entire six hours, except for a late drop in MAP. Lactic acid levels were significantly increased and arterial bicarbonate levels were reduced by six hours in all groups except for group 4. These results indicate that the combination treatment of flunixin meglumine and dexamethasone prevents many of the metabolic derangements observed during endotoxic shock in calves.