Arachidonic Metabolism Research Articles

Aberrant expression of 5-lipoxygenase-activating protein (5-LOXAP) has prognostic and survival significance in patients with breast cancer

5-lipoxygenase (5-LOX)-activating protein, 5-LOXAP also known as LOX5AP or FLAP, is a protein that works closely with 5-LOX in regulating the metabolism of arachidonate. Some of the eicosanoid products of 5-LOX/5-LOXAP are known to play active roles in the function of cancer cells, including breast cancer cells. The current study investigated the expression of 5-LOXAP in clinical breast cancer and the prognostic impact of 5-LOXAP and 5-LOX in patients with breast cancer. A cohort of breast tumour tissues ( n = 1 2 2 ) with normal background tissues ( n = 3 2 ) were investigated. 5-LOXAP and 5-LOX transcripts were determined using RT-PCR and quantitative RT-PCR. Levels of the transcripts were analysed against clinical and pathological information. Breast tumour tissues had significantly higher levels of 5-LOX transcript compared with normal tissues ( P = 0.0 1 5 ). The transcript was seen at significantly higher levels in node positive tumours than that in node negative tumours ( P = 0.0 2 ). The prognostic significance was assessed using both a prognostic index and clinical outcome. Value of 5-LOXAP was first demonstrated when using the Nottingham Prognostic Index (NPI) as an indicator, in that patients with predicted poor prognosis had significantly higher levels of 5-LOXAP than patients with good prognosis ( P = 0.0 4 0 7 ). Furthermore, patients who died of breast cancer-related causes had significant higher levels of 5-LOXAP than those patients who remained disease free, following a median 10-year followup. A survival analysis has shown that high levels of 5-LOXAP were significantly correlated with overall survival (mean survival 109.6 month vs. 139.4 months, in tumour from patients with high and low levels of 5-LOXAP, P = 0.0 5 ). The same disadvantage of high levels of 5-LOXAP was also seen with disease-free survival (105.2 months vs. 135.6 months, P = 0.0 1 7 ). Analysis of 5-LOXAP together with 5-LOX transcript did not enhance the significance of the survival. However, when 5-LOXAP was considered together with 12-LOX, it improved the predictive power for both overall and disease-free survival (109.0 month vs. 143.1 months, P = 0.0 1 5 6 for overall survival and 98.3 months vs. 141.3 months for disease-free survival, P = 0.0 0 2 2 ). In conclusion, 5-LOXAP expression was aberrant in human breast cancer, particularly in aggressive tumours. Furthermore, 5-LOXAP had a significant prognostic value in patients with breast cancer. This identifies 5-LOXAP as a potential therapeutic target in breast cancer.

Systemic inflammation induces COX-2 mediated prostaglandin D2 biosynthesis in mice spinal cord

Although prostaglandin (PG)D 2 is one of the main metabolites of the cyclooxygenase (COX) pathway of arachidonate metabolism in the brain, relatively little is known about the regulation of PGD 2 biosynthesis in the spinal cord during systemic inflammation. Therefore, the present study was aimed at investigating the effect of endotoxin treatment on spinal PGD 2 biosynthesis in BALB/c mice. Spinal inflammatory response to systemic endotoxin was verified by determination of spinal TNFα and IL-1β mRNA. COX-1, COX-2, membrane-bound prostaglandin E synthase-1 (mPGES-1), and lipocalin-type prostaglandin D synthase (L-PGDS) mRNA and protein were determined by RT-PCR and western blot, respectively. The concentrations of immunoreactive PGD 2 and PGE 2 were measured in superfusion media of spinal cord samples in-vitro. Endotoxin treatment (1 mg/kg; 24 h before) enhanced the expression of COX-2, mPGES-1, and L-PGDS mRNA and protein in spinal cord, while there was no significant effect on COX-1 mRNA and protein. In superfusion media of spinal cord samples obtained from endotoxin treated mice, the concentrations of immunoreactive PGE 2 and PGD 2 were higher than in the control group suggesting enhanced spinal PG biosynthesis after endotoxin treatment. Addition of the selective COX-2 inhibitor lumiracoxib (100 nM) to the superfusion medium did not significantly affect PGE 2 or PGD 2 release in spinal cord obtained from non-treated mice. In spinal cord of endotoxin-treated mice, lumiracoxib (100 nM) attenuated PGE 2 and PGD 2 release to values similar to those observed in tissue obtained from non-endotoxin-treated mice. These results show enhanced expression of spinal L-PGDS and increased spinal PGD 2 biosynthesis during systemic inflammation whereby enhanced biosynthesis seems to be dependent primarily on COX-2 activity.

Platelets and new antiplatelet drugs

Platelets play an important, life-saving role in hemostasis and blood clotting at sites of vascular injury. However, unwanted platelet activation and arterial thrombus formation are implicated in the onset of myocardial infarction, stroke and other cardiovascular diseases. Different mechanisms, such as vascular damage, the development of mural platelet thrombi as a response to injury and the biochemical effects of intraplatelet substances that are released in response to damage, may be involved. Thus, antiplatelet therapy has become a mainstay of treatment for these conditions and the benefit of antiplatelet drugs is documented across a wide spectrum of clinical conditions. Aspirin has been regarded as the prototype antiplatelet drug and is still the most widely used agent. Aspirin’s antiplatelet effect is directly due to irreversible inactivation of arachidonic metabolism and suppression of thromboxane A2 synthesis. However, platelet activation occurs via several pathways that do not rely on amplificatio...

Upregulation of Angiotensin II Type 1 Receptor, Inflammatory Mediators, and Enzymes of Arachidonate Metabolism in Obese Zucker Rat Kidney

Severe obesity can result in proteinuria and progressive glomerulosclerosis in humans and experimental animals. The associated renal disease is ameliorated by weight reduction and/or blockade of the renin-angiotensin system. Various growth factors, cytokines, and lipid mediators are implicated in the pathogenesis of renal disease. To explore the possible involvement of these mediators in obesity-induced renal disease, we examined the expression of key enzymes of arachidonate metabolism and inflammatory genes in untreated and losartan-treated obese Zucker rats, a model of obesity, insulin resistance, and renal injury. Seven-week-old male obese Zucker rats were randomized to losartan-treated (100 mg/L drinking H2O) and untreated groups, with lean Zucker rats as controls. After 4 months, RNA and protein were obtained from renal cortical tissue for relative reverse transcription-polymerase chain reaction, Western blots, and immunohistochemistry. Compared with the lean controls, obese Zucker rats showed significant glomerular matrix expansion and increased mRNA expression of the extracellular matrix protein fibronectin, inflammatory mediators interleukin-6 and monocyte chemoattractant protein-1, and 2 major enzymes of arachidonate metabolism, namely, 12/15-lipoxygenase and cyclooxygenase-2. This was associated with significant increases in p38 and extracellular signal-regulated kinase (ERK) 1/2 mitogen-activated protein kinase activities and marked upregulation of angiotensin II type 1 receptor (AT1R) mRNA and protein expression. These abnormalities and the associated glomerulopathy and proteinuria were prevented by administration of the AT1R blocker losartan. These findings indicate that obesity-induced glomerulopathy is associated with upregulation of key inflammatory mediators. These events are associated with and perhaps in part due to upregulation of AT1R, as evidenced by their reversal with AT1R blocker treatment.

The Plasma Levels of Prostanoids and Plasminogen Activator Inhibitor-1 in Primary and Secondary Thrombocytosis

An elevated platelet count is a common finding in both hospitalized and ambulatory patients. Thrombosis and bleeding complications are more frequently observed in patients with clonal thrombocytosis than secondary thrombocytosis. The aim of this study was to investigate the behaviors of plasminogen activator inhibitor type 1 (PAI-1), the inhibitor of fibrinolysis; and thromboxane A2 and 6-keto-PGF1 alpha, the products of endoperoxides, in 16 patients affected with clonal thrombocytemia as compared with 16 patients with reactive thrombocytosis and 15 normal controls. In the clonal thrombocytemia group, plasma levels of PAI-1 antigen and activity were significantly higher than both reactive thrombocytosis and control group. Plasma levels of 6-keto-PGF1alpha were significantly higher in the clonal thrombocytemia group than the other two groups and also higher in the reactive thrombocytosis group than the control group, which was also significant. This study confirms that arachidonate metabolism is frequently deranged in patients with thrombocytosis and hypofibrinolysis due to increased PAI-1 plasma levels as shown in the clonal thrombocytosis group. This may explain the thrombotic tendency in myeloproliferative disorders.

Arachidonic Acid Signaling in Pathogenesis of Allergy: Therapeutic Implications

In recent years, significant progress has been made in understanding the involvement of pro-inflammatory lipidic mediators in the pathogenesis of allergic diseases. The most relevant lipidic mediator is arachidonic acid and its metabolites. Arachidonic acid is the precursor for biosynthesis of eicosanoids, potent mediators of inflammation that have been implicated in the pathogenesis of diverse disease processes. Eicosanoids are mainly synthesized by the action of cyclo-oxygenase (prostaglandin endoperoxide synthase) that generates prostaglandins and thromboxane, and 5-lipoxygenase, which leads to the production of leukotrienes. In addition, 12- and 15-lipoxygenase are found in mammalian systems. The activity of these enzymes results in the formation of different hydroxyeicosatetraenoic acids, but their functions in vivo have not been clearly established in normal or pathological states. Since several arachidonic acid metabolites clearly play an important role in allergic response, a substantial effort has been directed to understanding the cellular and molecular aspects of these pathways and their pharmacological modulation. This review summarizes some of these aspects based on our current knowledge of the involvement of arachidonic metabolism in the pathogenesis of allergic diseases and outlines the potential therapeutic opportunities that can result from the modulation of these metabolites.

Expression of secretory phospholipase A2 enzymes in lungs of humans with pneumonia and their potential prostaglandin-synthetic function in human lung-derived cells

Although a number of sPLA2 (secretory phospholipase A2) enzymes have been identified in mammals, the localization and functions of individual enzymes in human pathologic tissues still remain obscure. In the present study, we have examined the expression and function of sPLA2s in human lung-derived cells and in human lungs with pneumonia. Group IID, V and X sPLA2s were expressed in cultured human bronchial epithelial cells (BEAS-2B) and normal human pulmonary fibroblasts with distinct requirement for cytokines (interleukin-1b, tumour necrosis factor a and interferon-g). Lentivirus- or adenovirus-mediated transfection of various sPLA2s into BEAS-2B or normal human pulmonary fibroblast cells revealed that group V and X sPLA2s increased arachidonate release and prostaglandin production in both cell types, whereas group IIA and IID sPLA2s failed to do so. Immunohistochemistry of human lungs with pneumonia demonstrated that group V and X sPLA2s were widely expressed in the airway epithelium, interstitium and alveolar macrophages, in which group IID sPLA2 was also positive, whereas group IIA sPLA2 was restricted to the pulmonary arterial smooth muscle layers and bronchial chondrocytes, and group IIE and IIF sPLA2s were minimally detected. These results suggest that group V and X sPLA2s affect lung pathogenesis by facilitating arachidonate metabolism or possibly through other functions.

Various secretory phospholipase A2 enzymes are expressed in rheumatoid arthritis and augment prostaglandin production in cultured synovial cells

Although group IIA secretory phospholipase A2 (sPLA2-IIA) is known to be abundantly present in the joints of patients with rheumatoid arthritis (RA), expression of other sPLA2s in this disease has remained unknown. In this study, we examined the expression and localization of six sPLA2s (groups IIA, IID, IIE, IIF, V and X) in human RA. Immunohistochemistry of RA sections revealed that sPLA2-IIA was generally located in synovial lining and sublining cells and cartilage chondrocytes, sPLA2-IID in lymph follicles and capillary endothelium, sPLA2-IIE in vascular smooth muscle cells, and sPLA2-V in interstitial fibroblasts. Expression levels of these group II subfamily sPLA2s appeared to be higher in severe RA than in inactive RA. sPLA2-X was detected in synovial lining cells and interstitial fibers in both active and inactive RA sections. Expression of sPLA2-IIF was partially positive, yet its correlation with disease states was unclear. Expression of sPLA2 transcripts was also evident in cultured normal human synoviocytes, in which sPLA2-IIA and -V were induced by interleukin-1 and sPLA2-X was expressed constitutively. Adenovirus-mediated expression of sPLA2s in cultured synoviocytes resulted in increased prostaglandin E2 production at low ng x mL(-1) concentrations. Thus, multiple sPLA2s are expressed in human RA, in which they may play a role in the augmentation of arachidonate metabolism or exhibit other cell type-specific functions.

Effects of SKI306X on Arachidonate Metabolism and Other Inflammatory Mediators

SKI306X was previously reported to have good anti-inflammatory and analgesic efficacy in various studies. To determine its mode of action, an investigation was carried out for some representative mediators. Arachidonic acid metabolism and its products are particularly important in inflammation and pain. The pro-inflammatory cytokines, especially interleukin-1 (IL-1) and tumor necrosis factor (TNF-alpha), and induced nitric oxide (NO) appear to be most involved in the inflammatory process such as osteoarthritis (OA). Thus SKI306X was tested to determine the effects on enzymes related to arachidonic acid metabolism and the release or synthesis of inflammatory mediators. SKI306X did inhibit the expression of cyclooxygenase-2 (COX-2) enzyme without affecting COX-1 and COX-2 activity. Leukotriene B4 (LTB4) production also was inhibited by SKI306X (IC50 = 98.7+/-4.26 microg/ml). SKI306X inhibited significantly TNF-alpha release (IC50 = 97.6+/-17.8 microg/ml) and NO production (IC50 = 280+/-17.8 microg/ml). But IL-1alpha release was not attenuated by SKI306X. This study suggests that inhibition of these mediators by SKI306X may be one of the mechanisms responsible for its anti-inflammatory effects.

Diagnostic Assessment of Patients with Inherited Mucocutaneous Hemorrhages (IMCH): Opening a Pandora Box?.

Diagnosis of patients with IMCH is plagued with difficulties: 1. Type 1 VWD (VWD-1) and platelet function disorders (PFD), the best characterized disorders of primary hemostasis, present inherent diagnostic difficulties. 2. Bleeding may be present in healthy individuals and may be absent in patients with diagnosed diseases. 3. An unknown proportion of bleeders do not have an identifiable haemostatic disorder. We studied prospectively 280 consecutive patients (age = 14.5±9.5, range 4–48 years) with family bleeding history who had a high probability of being pathological bleeders, based on an objective bleeding score. Only one physician interviewed the patients and the non-bleeder matched controls, (n=299, age 12.2±6.5, range 4–44 years). Exclusion criteria: age <4 and >50 years; platelet count <100.000/μL; other concurrent diseases, drug intake and C-reactive protein >1mg/dL. VWD was diagnosed when at least two tests (VWF:Ag, VWF:RCo, VWF:CB) had values <percentil 2.5 of the controls, without considering ABO type. PFD were diagnosed by PRP aggregation and 14C-5-HT secretion using pre-established criteria and reference values obtained in the 299 controls. Only 66 (23.6%) of the patients had prolonged bleeding time (BT). Frequency of diseases: 38 (13.6%) patients had VWD-1; 1 (0.35%) had type 2A VWD; 52 (18.6%) had PFD; 8 (2.9%) had concomitant VWD-1 + PFD; 7 patients (2.5%) had mild coagulation factor deficiencies (CFD, 4 with ↓FVIII:C; 2 with ↓ FIX:C; 1 with ↓ FXI:C); and 3 (1.1%) had concomitant VWD-1 + CFD; 171 bleeders (61.1%) had an unknown disorder (UD); in this UD group, 29 (17%) patients had prolonged BT, but all the other hemostatic tests were within the normal range. No distinctive bleeding pattern/severity was found among the above diagnostic categories. One patient with type 2B VWD was excluded because of thrombocytopenia and no patients with type 2N VWD were found. Patients with PFD had a secretion defect, but only 2 of them had typical storage pool disease (↓platelet 5-HT and ADP and ↓ADP/ATP ratio). No phenotypic patterns of Glanzmann or Bernard-Soulier diseases, arachidonate metabolism, or of absent ADP, collagen and TxA2 receptors were observed among the patients with PFD. Interestingly, the 170 patients with UD and the controls had almost the same mean values and distribution of VWF and platelet function variables; so we expect that very few patients with UD would qualify as VWD or PFD in repeated studies. Conclusions: 1. Prevalence of platelet secretion defects is higher than that of type 1 VWD-1 in our population. 2. Types 2 and 3 VWD, as well as paradigmatic platelet disorders (i.e., Glanzmann′s thrombasthenia, Bernard Soulier syndrome) were not detected in this study; they are of very low frequency and they are likely diagnosed at earlier age. 3. Bleeding time lacks sensitivity as a global test of primary haemostasis defects and should be omitted as a diagnostic tool. 4. After a first complete lab workup, 60% of the population with IMCH remains undiagnosed. The overwhelming majority of patients with UD probably constitute a heterogeneous group, independent of VWD or PFD, with a bleeding pattern similar to that of other disorders of primary hemostasis. Hypothetically, structural vascular defects or substances derived from the vascular wall which interfere with the normal platelet-vessel wall interaction could be involved in the pathogenesis of the hemorrhages.

Bidirectional regulation of renal cortical Na+,K+-ATPase by protein kinase C.

We examined the role of protein kinase C (PKC) in the regulation of Na+,K+- ATPase activity in the renal cortex. Male Wistar rats were anaesthetized and the investigated reagents were infused into the abdominal aorta proximally to the renal arteries. A PKC-activating phorbol ester, phorbol 12,13-dibutyrate (PDBu), had a dose-dependent effect on cortical Na+,K+-ATPase activity. Low dose of PDBu (10(-11) mol/kg per min) increased cortical Na+,K+-ATPase activity by 34.2%, whereas high doses (10(-9) and 10(-8) mol/kg per min) reduced this activity by 22.7% and 35.0%, respectively. PDBu administration caused changes in Na+,K+-ATPase Vmax without affecting K(0.5) for Na+, K+ and ATP as well as Ki for ouabain. The effects of PDBu were abolished by PKC inhibitors, staurosporine, GF109203X, and Gö 6976. The inhibitory effect of PDBu was reversed by pretreatment with inhibitors of cytochrome P450-dependent arachidonate metabolism, ethoxyresorufin and 17-octadecynoic acid, inhibitors of phosphatidylinositol 3-kinase (PI3K), wortmannin and LY294002, and by actin depolymerizing agents, cytochalasin D and latrunculin B. These results suggest that PKC may either stimulate or inhibit renal cortical Na+,K+-ATPase. The inhibitory effect is mediated by cytochrome P450-dependent arachidonate metabolites and PI3K, and is caused by redistribution of the sodium pump from the plasma membrane to the inactive intracellular pool.

Bidirectional regulation of renal cortical Na+,K+-ATPase by protein kinase C.

We examined the role of protein kinase C (PKC) in the regulation of Na+,K+- ATPase activity in the renal cortex. Male Wistar rats were anaesthetized and the investigated reagents were infused into the abdominal aorta proximally to the renal arteries. A PKC-activating phorbol ester, phorbol 12,13-dibutyrate (PDBu), had a dose-dependent effect on cortical Na+,K+-ATPase activity. Low dose of PDBu (10(-11) mol/kg per min) increased cortical Na+,K+-ATPase activity by 34.2%, whereas high doses (10(-9) and 10(-8) mol/kg per min) reduced this activity by 22.7% and 35.0%, respectively. PDBu administration caused changes in Na+,K+-ATPase Vmax without affecting K(0.5) for Na+, K+ and ATP as well as Ki for ouabain. The effects of PDBu were abolished by PKC inhibitors, staurosporine, GF109203X, and Gö 6976. The inhibitory effect of PDBu was reversed by pretreatment with inhibitors of cytochrome P450-dependent arachidonate metabolism, ethoxyresorufin and 17-octadecynoic acid, inhibitors of phosphatidylinositol 3-kinase (PI3K), wortmannin and LY294002, and by actin depolymerizing agents, cytochalasin D and latrunculin B. These results suggest that PKC may either stimulate or inhibit renal cortical Na+,K+-ATPase. The inhibitory effect is mediated by cytochrome P450-dependent arachidonate metabolites and PI3K, and is caused by redistribution of the sodium pump from the plasma membrane to the inactive intracellular pool.

Balance between PGD synthase and PGE synthase is a major determinant of atherosclerotic plaque instability in humans.

Inducible cyclooxygenase (COX-2) catalyzes the first step in prostanoid biosynthesis and is considered a proinflammatory enzyme. COX-2 and type 1 inducible PGE synthase (mPGES-1) have a role in metalloproteinase (MMP) release leading to plaque rupture. In contrast, lipocalin-type PGD synthase (L-PGDS) has been shown to exert antiinflammatory actions. Thus, in this study we investigated whether a shift from a PGDS-oriented to a PGES-oriented profile in arachidonate metabolism leads to inflammatory activation in rupture-prone plaque macrophages. Atherosclerotic plaques were obtained from 60 patients who underwent carotid endarterectomy, symptomatic (n=30) and asymptomatic (n=30) according to evidence of recent transient ischemic attack or stroke. Plaques were analyzed for COX-2, mPGES-1, L-PGDS, PPARgamma, IkappaBalpha, NF-kappaB, and MMP-9 by immunocytochemistry, Western blot, reverse-transcriptase polymerase chain reaction, enzyme immunoassay, and zymography. Prostaglandin E2 (PGE2) pathway was significantly prevalent in symptomatic plaques, whereas PGD2 pathway was overexpressed in asymptomatic ones, associated with NF-kappaB inactivation and MMP-9 suppression. In vitro COX-2 inhibition in monocytes was associated with reduced MMP-9 release only when PGD2 pathway overcame PGE2 pathway. These results suggest that COX-2 may have proinflammatory and antiinflammatory properties as a function of expression of downstream PGH2 isomerases, and that the switch from L-PGDS to mPGES-1 in plaque macrophages is associated with cerebral ischemic syndromes, possibly through MMP-induced plaque rupture.

The effect of chronic lithium on arachidonic acid release and metabolism in rat brain does not involve secretory phospholipase A2 or lipoxygenase/cytochrome P450 pathways

The effect of chronic lithium on arachidonic acid release and metabolism in rat brain does not involve secretory phospholipase A2 or lipoxygenase/cytochrome P450 pathways

Human Lung Cancer Chemoprevention Strategies: Parker B. Francis Lecture

Pharmacologic or nutritional prevention of lung cancers is needed, especially for 60 million Americans who are former smokers. A portfolio of large-scale trials of beta-carotene, beta-carotene with and without vitamin E, and beta-carotene plus vitamin A demonstrated no benefit whatsoever from beta-carotene. The alpha-Tocopherol/beta-Carotene Trial and the beta-Carotene and Retinol Efficacy Trial found significant increases in lung cancer risk and total mortality. Laboratory research soon identified multiple adverse molecular effects. Nevertheless, chemoprevention remains an active, promising strategy, with new hypotheses and new candidate agents, including many already approved as therapies. The most active area currently is focused on selective inhibition of arachidonic metabolism, both Cox-2 and Lox pathways.

5-lipoxygenase and cyclooxygenase regulate wound closure in NIH/3T3 fibroblast monolayers.

Wound healing involves multiple cell signaling pathways, including those regulating cell-extracellular matrix adhesion. Previous work demonstrated that arachidonate oxidation to leukotriene B(4) (LTB(4)) by 5-lipoxygenase (5-LOX) signals fibroblast spreading on fibronectin, whereas cyclooxygenase-2 (COX-2)-catalyzed prostaglandin E(2) (PGE(2)) formation facilitates subsequent cell migration. We investigated arachidonate metabolite signaling in wound closure of perturbed NIH/3T3 fibroblast monolayers. We found that during initial stages of wound closure (0-120 min), all wound margin cells spread into the wound gap perpendicularly to the wound long axis. At regular intervals, between 120 and 300 min, some cells elongated to project across the wound and meet cells from the opposite margin, forming distinct cell bridges spanning the wound that act as foci for later wound-directed cell migration and resulting closure. 5-LOX inhibition by AA861 demonstrated a required LTB(4) signal for initial marginal cell spreading and bridge formation, both of which must precede wound-directed cell migration. 5-LOX inhibition effects were reversible by exogenous LTB(4). Conversely, COX inhibition by indomethacin reduced directed migration into the wound but enhanced early cell spreading and bridge formation. Exogenous PGE(2) reversed this effect and increased cell migration into the wound. The differential effects of arachidonic acid metabolites produced by LOX and COX were further confirmed with NIH/3T3 fibroblast cell lines constitutively over- and underexpressing the 5-LOX and COX-2 enzymes. These data suggest that two competing oxidative enzymes in arachidonate metabolism, LOX and COX, differentially regulate sequential aspects of fibroblast wound closure in vitro.

Multiple roles of COX-2 in tumor angiogenesis: a target for antiangiogenic therapy

Angiogenesis is required for multistage carcinogenesis. The inducible enzyme cyclooxygenase-2 (COX-2) is an important mediator of angiogenesis and tumor growth. COX-2 expression occurs in a wide range of preneoplastic and malignant conditions; and the enzyme has been localized to the neoplastic cells, endothelial cells, immune cells, and stromal fibroblasts within tumors. The proangiogenic effects of COX-2 are mediated primarily by three products of arachidonic metabolism: thromboxane A 2 (TXA 2), prostaglandin E 2 (PGE 2), and prostaglandin I 2 (PGI 2). Downstream proangiogenic actions of these eicosanoid products include: (1) production of vascular endothelial growth factor; (2) promotion of vascular sprouting, migration, and tube formation; (3) enhanced endothelial cell survival via Bcl-2 expression and Akt signaling; (4) induction of matrix metalloproteinases; (5) activation of epidermal growth factor receptor-mediated angiogenesis; and (6) suppression of interleukin-12 production. Selective inhibition of COX-2 activity has been shown to suppress angiogenesis in vitro and in vivo. Because these agents are safe and well tolerated, selective COX-2 inhibitors could have clinical utility as antiangiogenic agents for cancer prevention, as well as for intervention in established disease alone or in combination with chemotherapy, radiation, and biological therapies.

Leukotriene-related gene polymorphisms in ASA-intolerant asthma: an association with a haplotype of 5-lipoxygenase.

A recent study has demonstrated the possible involvement of a leukotriene C4 synthase (LTC4S) gene polymorphism in ASA-intolerant asthma (AIA) in a Polish population, whereas no significant association was noted in other populations. To investigate the role of genetic polymorphism in AIA development, we screened single nucleotide polymorphisms (SNPs) of the key enzymes involved in arachidonate metabolism, and the cysteinyl leukotriene receptor 1 (CYSLTR1) in a large Korean population with AIA: 93 AIA and 181 ASA-tolerant asthma (ATA) patients, and 123 normal controls. The single-base extension method was used to genotype SNPs in 5-lipoxygenase (ALOX5, -1708G-->A, 21C-->T, 270G-->A, 1728G-->A), ALOX5-activating protein (ALOX5AP, 218A-->G), prostaglandin-endoperoxide synthase 2 (PTGS2, COX2, -162C-->G, 10T-->G, R228H, V511A), LTC4S (-444A-->C), and CYSLTR1 (927T-->C). Haplotype analyses were undertaken for the SNPs in ALOX5. No significant differences in allele and genotype frequencies of single SNPs were observed between the patient groups ( P>0.05). However, the frequency of the ALOX5-ht1[G-C-G-A] haplotype in the AIA group was significantly higher than its frequency in the ATA group with a probability ( P) of 0.01, odds ratio (OR) of 5.0, and 95% confidence interval (95%CI) of 1.54-17.9, and in the normal controls ( P=0.03, OR=4.5, 95%CI=1.1-18.4), by using a dominant model. These results suggest a lack of association between the ALOX5AP, PTGS2, LTC4S, and CYSLTR1 gene polymorphisms and the AIA phenotype in the Korean population. However, the possible involvement of ALOX5-ht1[G-C-G-A] in AIA development is suggested.

Non-prostaglandin eicosanoids in fever and anapyrexia.

Until recently, studies on the role of the metabolites of arachidonic acid (AA), eicosanoids in fever have primarily focused on prostaglandins, prostaglandin E2 (PGE2) in particular, derived from the pathway related to cyclooxygenases (COX). COX exists in two known isoforms; a constitutive COX-1, and COX-2, which is inducible upon the action of pyrogens. Data accumulated in our laboratories suggest a thermoregulatory role for two other pathways of arachidonate metabolism; 5-lipoxygenase (5-LOX) and cytochrome P-450 (epoxygenase). We have demonstrated that leukotrienes (LTs; 5-LOX-derived eicosanoids) and various isomers of epoxyeicosatrienoic acids (EETs; epoxygenase-derived eicosanoids) contribute to the process of endogenous antipyresis or cryogenesis, which limits the height of fever. In support of this are several lines of evidence based on both in vivo and in vitro experiments. 1) Intracerebroventricular (icv) injections of LTC4 at nanomolar concentrations cause a dose-dependent decrease of body temperature (Tb) in mice. 2) Lipopolysaccharide (LPS)-induced anapyrexia in mice is preceded and accompanied by elevation in hypothalamic cysteinyl-LT (CysLT) production. 3) The inhibitor of LT synthesis MK-886 suppresses both of these processes. 4) EETs as well as inducers of the epoxygenase attenuate, whereas inhibitors of epoxygenase enhance the LPS-induced fever in rats. 5) One of the isomers of EET, 11,12-EET, in in vitro studies inhibited both the generation of PGE2 and IL-6 in monocytes stimulated with LPS. These results, together with a well-established pyrogenic role of PGE2, indicate that AA cascade may be regarded as an endogenous system to regulate the temperature response upon disease. COX, 5-LOX, and epoxygenase products may act at the level of hypothalamus as proximal mediators of, respectively, fever (PGE2) or cryogenesis (CysLTs and EETs), or indirectly by influencing the other endogenous cryogens and pyrogens.

Measurement of platelet arachidonic acid metabolism.

AbstractPlatelets respond to low levels of agonists such as collagen or thrombin with primary signals that, although small, are functionally significant because they are amplified by autocrine and paracrine pathways such as the secretion of ADP and ATP from the dense granules. An important mediator of the action of low-dose collagen, especially, which is not dependent on granule secretion, is thromboxane A2, a prostanoid that is a potent platelet agonist in its own right, binding to G-protein-linked receptors on the cell surface to stimulate calcium signals via phospholipase Cβ. The precursor of the prostanoids is arachidonic acid (AA), liberated from platelet membrane lipids by the action of phospholipase A2. AA is processed in two stages by cyclooxygenase to yield prostaglandin (PG) H2. This process is aspirin-sensitive, as the drug irreversibly acetylates and inhibits cyclooxygenase, providing the basis for the old and effective antithrombotic use for aspirin, which itself underscores the involvement of collagen in arterial thrombosis. Thromboxane synthase then consumes a proportion of PGH2 to generate thromboxane (Tx) A2. Other products of platelet arachidonate metabolism include PGD2, which exercises an inhibitory role on platelet function as a consequence of its capacity to activate adenylate cyclase, like its homolog, PGI2 (prostacyclin). PGI2 in circulation, however, is largely produced by the vascular endothelial cells rather than the platelets.KeywordsArachidonic AcidLoad BufferMicrofuge TubePLA2 ActivityArachidonic Acid ReleaseThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.