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Lipoxygenase (ALOX) enzymes are implicated in both pro- and anti-atherogenic processes. The aim of this study was to investigate mRNA expression of 12- and 15-lipoxygenases (ALOX12, ALOX12B, ALOX15, ALOX15B) and the atypical ALOXE3 in human carotid atherosclerotic lesions, in relation to cerebrovascular symptoms and risk factors. The Biobank of Karolinska Endarterectomies (BiKE) collection of human carotid plaque tissue and associated clinical data was utilized (n=132). Lesion mRNA levels were analyzed by TaqMan qPCR (n=132) and microarray hybridization (n=77). Of the investigated mRNAs, only ALOX15B (15-LOX-2; epidermis-type 15-LOX) was readily detected in all plaque samples by qPCR, and thus suitable for quantitative statistical evaluation. ALOX12, ALOX12B, ALOX15 and ALOXE3 were detected with lower frequency and at lower levels, or virtually undetected. Microarray analysis confirmed ALOX15B as the most abundant 12- or 15-lipoxygenase mRNA in carotid lesions. Comparing plaques with or without attributable cerebrovascular symptoms (amaurosis fugax, transient ischemic attack, or stroke), ALOX15B mRNA levels were higher in symptomatic than asymptomatic plaques (1.31 [1.11-1.56], n=102; and 0.79 [0.55-1.15], n=30, respectively; p=0.008; mean [95% CI], arbitrary units). Multiple regression analysis confirmed symptomatic/asymptomatic status as a significant determinant of ALOX15B mRNA levels, independently of potentially confounding factors. Immunohistochemical analyses showed abundant ALOX15B expression in macrophage-rich areas of carotid lesions, and lipidomic analyses demonstrated the presence of typical ALOX15B products in plaque tissue. In summary, we observed associations between high ALOX15B expression in carotid lesions and a history of cerebrovascular symptoms. These findings suggest a link between ALOX15B and atherothrombotic events that merits further investigation.

Colorectal cancer (CRC) is the third most common cancer and the third leading cause of 'cancer related death' world-wide. In case of CRC patients who have large sized tumor, radiation therapy is essential to decrease tumor size without having to eliminate the anus. However, radiation effects vary according to patients. Also, the association between genes and radiation sensitivity is still rarely researched. In this study, we elucidated that the expression level of ALOX15 regulates radiation sensitivity by TNFα signaling pathway in CRC cells. ALOX15 is known as the main metabolic enzyme for linoleic acid and arachidonic acid. Interestingly, stable cells which express low ALOX15 inhibit radiation-induced apoptosis. Mechanistically, inhibition of ALOX15 increases NF-kB under radiation therapy condition. NF-kB that plays a key role in the cellular response to DNA damage is a reason for ALOX15 to act as a radiation sensitizer. Taken together, our results indicate that ALOX15 is suggested as a prediction marker for radiation therapy. In addition, screening natural compound for elevating ALOX15 would be used for the purpose of combination therapy in radiation-resistant CRC cells. Citation Format: Yoo Jin Na, Dae-Hee Lee, Bo Ram Kim, Jung Lim Kim, So Yeon Jeong, Seong Hye Park, Min Jee Jo, Yoon A Jeong, Hye Kyeong Yun, Sang Cheul Oh. The regulation of ALOX15 expression in colorectal cancer to overcome the resistance to radiation therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 669.

Aims. Genes of the 5-lipoxygenase pathway are compelling candidates for atherosclerosis. We hypothesize that polymorphisms inALOX12, ALOX15, ALOX5, andALOX5APgenes are associated with subclinical atherosclerosis in multiple vascular beds.Methods. Families with two or more siblings with type 2 diabetes and their nondiabetic siblings were studied as part of the Diabetes Heart Study (DHS). European American diabetic (n=828) and nondiabetic (n=170) siblings were genotyped for SNPs in theALOX12, ALOX15, ALOX5, andALOX5APgenes. Subclinical measures of atherosclerosis (IMT, coronary (CorCP), carotid (CarCP) and aortic (AorCP) calcified plaque) were obtained.Results. Associations were observed betweenALOX12with CorCP,ALOX5with CorCP, AorCP, and IMT, andALOX5APwith CorCP and CarCP, independent of known epidemiologic risk factors. Further, lipoxygenase pathway SNPs that were associated with measures of atherosclerosis were associated with markers of inflammation (CRP, ICAM-1) and calcification (MGP).Conclusions. Polymorphisms withinALOX12, ALOX5, andALOX5APare genetically associated with subclinical atherosclerosis and with biomarkers of disease in families with type 2 diabetes. These results suggest that variants in lipoxygenase pathway genes may have pleiotropic effects on multiple components that determine risk of cardiovascular disease.

To identify a novel target for the treatment of heart failure, we examined gene expression in the failing heart. Among the genes analyzed, Alox15 encoding the protein 12/15 lipoxygenase (LOX) was markedly up-regulated in heart failure. To determine whether increased expression of 12/15-LOX causes heart failure, we established transgenic mice that overexpressed 12/15-LOX in cardiomyocytes. Echocardiography showed that Alox15 transgenic mice developed systolic dysfunction. Cardiac fibrosis increased in Alox15 transgenic mice with advancing age and was associated with the infiltration of macrophages. Consistent with these observations, cardiac expression of monocyte chemoattractant protein 1 (MCP-1) was up-regulated in Alox15 transgenic mice compared with wild-type mice. Treatment with 12-hydroxy-eicosatetraenoic acid, a major metabolite of 12/15-LOX, increased MCP-1 expression in cardiac fibroblasts and endothelial cells but not in cardiomyocytes. Inhibition of MCP-1 reduced the infiltration of macrophages into the myocardium and prevented both systolic dysfunction and cardiac fibrosis in Alox15 transgenic mice. Likewise, disruption of 12/15-LOX significantly reduced cardiac MCP-1 expression and macrophage infiltration, thereby improving systolic dysfunction induced by chronic pressure overload. Our results suggest that cardiac 12/15-LOX is involved in the development of heart failure and that inhibition of 12/15-LOX could be a novel treatment for this condition.

Asthma is a chronic inflammatory disease of the lungs, characterized by airway hyperreactivity, mucus hypersecretion, and airflow obstruction. Despite recent advances, the genetic regulation of asthma pathogenesis is still largely unknown. Gene expression profiling techniques are well suited to study complex diseases and hold substantial promise for identifying novel genes and pathways in asthma; however, relatively few studies have been completed in human asthma. The few studies that have been done have identified many novel candidate genes and pathways in asthma pathogenesis, including ALOX15 and serine proteinase inhibitors cathepsin C and G. The interpretation of results of these studies should be cautious, as limitations include small sample sizes and heterogeneity of study populations and tissues sampled. In the future, the promise of gene expression studies would be enhanced by the use of larger sample sizes and attempts to standardize phenotype, sample collection techniques, and analysis. As the field of expression profiling in asthma advances, we hope it will improve our understanding of critical questions about mechanisms involved in susceptibility to the disease, as well as help to personalize care by improving appropriate selection of patients for prevention and treatment strategies.

Improved understanding of the role of inflammation in tendon disease is required to facilitate therapeutic target discovery. We studied supraspinatus tendons from patients experiencing pain before and after surgical subacromial decompression treatment. Tendons were classified as having early, intermediate, or advanced disease, and inflammation was characterized through activation of pathways mediated by interferon (IFN), nuclear factor κB (NF-κB), glucocorticoid receptor, and signal transducer and activator of transcription 6 (STAT-6). Inflammation signatures revealed expression of genes and proteins induced by IFN and NF-κB in early-stage disease and genes and proteins induced by STAT-6 and glucocorticoid receptor activation in advanced-stage disease. The proresolving proteins FPR2/ALX and ChemR23 were increased in early-stage disease compared to intermediate- to advanced-stage disease. Patients who were pain-free after treatment had tendons with increased expression of CD206 and ALOX15 mRNA compared to tendons from patients who continued to experience pain after treatment, suggesting that these genes and their pathways may moderate tendon pain. Stromal cells from diseased tendons cultured in vitro showed increased expression of NF-κB and IFN target genes after treatment with lipopolysaccharide or IFNγ compared to stromal cells derived from healthy tendons. We identified 15-epi lipoxin A4, a stable lipoxin isoform derived from aspirin treatment, as potentially beneficial in the resolution of tendon inflammation.

Lipoxygenases (LOX) form a family of lipid peroxidizing enzymes, which have been implicated in a number of physiological processes and in the pathogenesis of inflammatory, hyperproliferative and neurodegenerative diseases. They occur in two of the three domains of terrestrial life (bacteria, eucarya) and the human genome involves six functional LOX genes, which encode for six different LOX isoforms. One of these isoforms is ALOX15, which has first been described in rabbits in 1974 as enzyme capable of oxidizing membrane phospholipids during the maturational breakdown of mitochondria in immature red blood cells. During the following decades ALOX15 has extensively been characterized and its biological functions have been studied in a number of cellular in vitro systems as well as in various whole animal disease models. This review is aimed at summarizing the current knowledge on the protein–chemical, molecular biological and enzymatic properties of ALOX15 in various species (human, mouse, rabbit, rat) as well as its implication in cellular physiology and in the pathogenesis of various diseases.

Among lipoxygenases ALOX15 orthologs are somewhat peculiar because of their capability of oxygenating polyenoic fatty acids even if they are incorporated in complex lipid-protein assemblies. ALOX15 orthologs of different species have been characterized before, but little is known about the corresponding rat enzyme. Since rats are frequently employed as models in biomedical research we expressed rat Alox15 as recombinant protein in pro- and eukaryotic expression systems and characterized the enzyme with respect to its enzymatic properties. The enzyme oxygenated free arachidonic acid mainly to 12S-HpETE with 15S-HpETE only contributing 10% to the product mixture. Multiple directed mutagenesis studies indicated applicability of the triad concept with particular importance of Leu353 and Ile593 as specificity determinants. Ala404Gly exchange induced subtle alterations in enantioselectivity suggesting partial applicability of the Coffa/Brash concept. Wildtype rat Alox15 and its 15-lipoxygenating Leu353Phe mutant are capable of oxygenating ester lipids of biomembranes and high-density lipoproteins. For the wildtype enzyme 13S-HODE and 12S-HETE were identified as major oxygenation products but for the Leu353Phe mutant 13S-HODE and 15S-HETE prevailed. These data indicate for the first time that mutagenesis of triad determinants modifies the reaction specificity of ALOX15 orthologs with free fatty acids and complex ester lipids in a similar way.

ALOX12 and ALOX15 encode arachidonate lipoxygenases which produce lipid metabolites involved in inflammatory processes. Metabolites generated by ALOX12 and ALOX15 can activate the expression of the potent pro-inflammatory cytokine IL-6, and produce endogenous ligands for PPARG. In this study, polymorphisms in ALOX12, ALOX15, IL6 and PPARG were investigated for association with bone properties in young and elderly Swedish women.Three SNPs in ALOX12, five in ALOX15, one each in IL6 and PPARG were genotyped in the cohorts PEAK-25 (n=1061 women; all 25y) and OPRA (n=1044 women; all 75y). Bone mineral density (BMD) and quantitative ultrasound (QUS) were analyzed in both cohorts; trabecular bone score (TBS) in PEAK-25; bone loss, fracture incidence and serum C-reactive protein (CRP) were assessed in OPRA.In the elderly women ALOX15 (rs2619112) was associated with CRP levels (p=0.004) and incident fracture of any type (p=0.014), although not with BMD or ultrasound. In young women, carrying the common T allele (ALOX 15 rs748694) was associated with lower QUS values (p=0.002-0.006). The IL6 SNP was associated with lower BMD in PEAK-25 (femoral neck p=0.034; hip p=0.012). TBS was not associated with variation in any gene. Variants in the ALOX12 and PPARγ were not associated with BMD in either cohort.This study suggests that variation in inflammation related genes ALOX15 and IL6 was associated with bone microarchitecture and density in young adult women, but appears to be less important in the elderly, despite an observed association with CRP as a marker of inflammation and incident fracture.

The development of osteoporosis involves the interaction of multiple environmental and genetic factors. Through combined genetic and genomic approaches, we identified the lipoxygenase geneAlox15as a negative regulator of peak bone mineral density in mice. Crossbreeding experiments withAlox15knockout mice confirmed that 12/15-lipoxygenase plays a role in skeletal development. Pharmacologic inhibitors of this enzyme improved bone density and strength in two rodent models of osteoporosis. These results suggest that drugs targeting the 12/15-lipoxygenase pathway merit investigation as a therapy for osteoporosis.

Pro-inflammatory signaling has been shown to promote colorectal tumorigenesis and is a target for the development of effective chemopreventive approaches. The specialized pro-resolving lipid mediators (SPMs, e.g. resolvins), bioactive metabolites of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), actively terminate inflammation and have been proposed to possibly contribute to the anti-tumorigenic effects of DHA and EPA. The enzyme 15-lipoxygenase-1 (ALOX15) is a key biosynthetic enzyme in generation of resolvins. However, 15-LOX-1 expression is commonly lost during human CRC tumorigenesis starting in premalignant stages via transcriptional mechanisms independent of substrate availability. The impact of ALOX15 on DHA and EPA’s effects on tumorigenesis remain unknown. Mice with intestinal epithelium-specific expression of human ALOX15 (15-LOX-1-gut mice) and wild-type FVB controls were injected with azoxymethane (AOM, 7.5mg/kg) once weekly for 6w and followed for 20w. Mice were fed diet with 1% omega-3-acid ethyl esters (O3AEE, a pharmaceutical grade fish oil preparation of EPA and DHA ethyl esters) or control diet starting 3w before initiation with AOM. Colonic tumors developed in 10 of the 13 (77%) wild type (WT) mice fed control diet, 5 of the 10 (50%) WT mice fed O3AEE diet, 5 of the 12 (42%) 15-LOX-1-gut mice fed control diet, and 3 of the 10 (30%) 15-LOX-1-gut mice fed control diet. Lipid mediator levels were measured by liquid chromatography/ tandem mass spectrometry (LC-MS/MS). The SPMs resolvin E1 and D2 (RvE1, RvD2) as well as the pathway intermediates 18-HEPE and 17-HDHE were increased in ALOX15-gut mice on O3AEE (see Table; data are shown as ng/mg protein; mean ± SEM). Our results demonstrate that ALOX15 activity is important to DHA and EPA formation of resolvins and inhibition of colonic tumorigenesis. Supported by grants CPRIT RP150195 and NIH RO1 R01CA195686 SPMs in O3AEE or control diet fed miceWTWT15-LOX-1-gut15-LOX-1-gutControl dietO3AEE dietControl dietO3AEE diet18-HEPE00.46 + 0.500.82 + 0.3117-HDHE0.21 + 0.090.2 + 0.74.2 + 0.4113.35 + 3.1RvE10001.98 + 0.77RvD200.024 + 0.0240.17 + 0.0220.3 + 0.077 Citation Format: Xiangsheng Zuo, Jennifer K. Colby, Fuyao Liu, Shen Gao, Ling Wu, Jonathan C. Jaoude, Micheline J. Moussalli, Lin Tan, Peiying Yang, Imad Shureiqi. Increased 15-lipoxygenase-1 activity limits tumor development in the azoxymethane mouse model of colon cancer: impact of omega-3-acid ethyl esters [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2224. doi:10.1158/1538-7445.AM2017-2224

Background Gene expression patterns have not been extensively examined in the context of clinical features of eosinophilic esophagitis (EoE). Aims To assess whether gene expression is associated with clinically defined phenotypes in adults with EoE. Methods This was an analysis of prospectively collected esophageal biopsies in newly diagnosed EoE patients. We determined differential gene expression with a 94 gene panel in relation to clinical features and phenotypes. These included: endoscopic findings of esophageal rings, stricture, narrowing, linear furrows, exudates, edema, and dilation; an allergic phenotype; an inflammatory phenotype, and a fibrostenotic phenotype. Results In 89 EoE cases analyzed, patients with exudates on endoscopy had multiple differences in gene expression compared to patients without exudates, though patients with exudates also had higher eosinophil counts (172 vs 106 eos/hpf; p = .01). Genes associated with esophageal narrowing included CCL26 (q-value = 0.028), ALOX15 (q = 0.011), GRK5 (q = 0.029), CPA3 (q = 0.012), and TRIM2 (q = 0.0027). TRIM2 was also associated with the fibrostenotic phenotype (q = 0.0051). No genes were associated with the inflammatory or atopic phenotypes, or with dilation. Conclusions Multiple genes are associated with exudates, possibly related to higher eosinophil counts. However, a number of genes, including those related to both inflammation and remodelling, are associated with esophageal narrowing. In particular, TRIM2 is associated with clinical fibrotic phenotypes.

Lipoxygenases (LOXs) form a heterogeneous class of lipid peroxidizing enzymes, which have been implicated not only in cell proliferation and differentiation but also in the pathogenesis of various diseases with major public health relevance. As other fatty acid dioxygenases LOXs oxidize polyunsaturated fatty acids to their corresponding hydroperoxy derivatives, which are further transformed to bioactive lipid mediators (eicosanoids and related substances). On the other hand, lipoxygenases are key players in the regulation of the cellular redox homeostasis, which is an important element in gene expression regulation. Although the first mammalian lipoxygenases were discovered 40 years ago and although the enzymes have been well characterized with respect to their structural and functional properties the biological roles of the different lipoxygenase isoforms are not completely understood. This review is aimed at summarizing the current knowledge on the physiological roles of different mammalian LOX-isoforms and their patho-physiological function in inflammatory, metabolic, hyperproliferative, neurodegenerative and infectious disorders. This article is part of a Special Issue entitled "Oxygenated metabolism of PUFA: analysis and biological relevance".

BACKGROUND Pro- and anti-inflammatory metabolites of arachidonic acid - eicosanoids - participate in skin homeostasis, affecting the growth and differentiation of keratinocytes. Alterations of 12-lipoxygenase (LOX) and 15-LOX and their metabolites have been described in the epidermis of patients with psoriasis, but systemic production of 12-LOX and 15-LOX eicosanoids has not been studied in the disease. OBJECTIVES To ascertain the frequencies of the genetic variants ALOX12 rs1126667 and ALOX15 rs11568070 in cases and controls, and to compare urinary metabolites of 12(S)-hydroxyeicosatetraenoic acid (HETE) between patients with psoriasis and healthy controls. METHODS Patients with psoriasis (n = 200) were stratified depending on the severity of their dermal lesions. Genotyping was performed using a 5'-nuclease real-time assay. The concentrations of 12(S)-HETE, its metabolites and 15(S)-HETE were determined in urine samples using high-performance liquid chromatography-tandem mass spectrometry. RESULTS Tetranor-12(S)-HETE metabolite excretion was significantly higher in urine of patients with psoriasis, while excretion of 12(S)-HETE was decreased. Neither 12(S)-HETE nor tetranor-12(S)-HETE correlated with the type of disease or severity score. No difference in urinary 15(S)-HETE was found between the study groups. Genotype distribution of the ALOX12 rs1126667 or ALOX15 rs11568070 polymorphisms did not discriminate for the disease or its severity. CONCLUSIONS Systemic metabolism of 12(S)-HETE is accelerated in psoriasis because excretion of the tetranor-12(S)-HETE inactivation product is elevated. No correlation with the severity or extent of psoriasis is detectable. We propose that in patients with psoriasis, 12(S)-HETE to tetranor-12(S)-HETE conversion could be at least a marker for this disease, in which inflammation of the skin can induce microsomal beta-oxidation of this eicosanoid.

Aims: The reaction of nitric oxide and nitrite-derived species with polyunsaturated fatty acids yields electrophilic fatty acid nitroalkene derivatives (NO2-FA), which display anti-inflammatory properties. Given that the 5-lipoxygenase (5-LO, ALOX5) possesses critical nucleophilic amino acids, which are potentially sensitive to electrophilic modifications, we determined the consequences of NO2-FA on 5-LO activity in vitro and on 5-LO-mediated inflammation in vivo. Results: Stimulation of human polymorphonuclear leukocytes (PMNL) with nitro-oleic (NO2-OA) or nitro-linoleic acid (NO2-LA) (but not the parent lipids) resulted in the concentration-dependent and irreversible inhibition of 5-LO activity. Similar effects were observed in cell lysates and using the recombinant human protein, indicating a direct reaction with 5-LO. NO2-FAs did not affect the activity of the platelet-type 12-LO (ALOX12) or 15-LO-1 (ALOX15) in intact cells or the recombinant protein. The NO2-FA-induced inhibition of 5-LO was attributed to the alkylation of Cys418, and the exchange of Cys418 to serine rendered 5-LO insensitive to NO2-FA. In vivo, the systemic administration of NO2-OA to mice decreased neutrophil and monocyte mobilization in response to lipopolysaccharide (LPS), attenuated the formation of the 5-LO product 5-hydroxyeicosatetraenoic acid (5-HETE), and inhibited lung injury. The administration of NO2-OA to 5-LO knockout mice had no effect on LPS-induced neutrophil or monocyte mobilization as well as on lung injury. Innovation: Prophylactic administration of NO2-OA to septic mice inhibits inflammation and promotes its resolution by interfering in 5-LO-mediated inflammatory processes. Conclusion: NO2-FAs directly and irreversibly inhibit 5-LO and attenuate downstream acute inflammatory responses. Antioxid. Redox Signal. 20, 2667–2680.

5-Lipoxygenase (ALOX5) plays a key role in the biosynthesis of pro-inflammatory leukotrienes whereas 15-lipoxygenases (ALOX15) have been implicated in the formation of pro-resolving eicosanoids (lipoxins, resolvins). Recently, it has been suggested that a phosphorylation mimicking mutant (Ser663Asp) of a stabilized variant of human ALOX5 exhibits dominant arachidonic acid 15-lipoxygenase activity (> 95%). To test whether similar alterations in the reaction specificity can also be observed for ALOX5 orthologs of other species we expressed wildtype and phosphorylation mimicking mutants (Ser271Asp, Ser523Asp, Ser663Asp, Ser663Glu) of human, mouse and zebrafish ALOX5 in pro- and eukaryotic overexpression systems and characterized their reaction specificities. We found that neither of the phosphorylation mimicking mutants produced significant amounts of 15-hydroperoxyeicosatetraenoic acid and the 5-lipoxygenation/15-lipoxygenation ratio for all wildtype and mutant enzyme species was lower than 100:2. Taken together, this data suggest that phosphorylation of native ALOX5 orthologs of different vertebrates may not induce major alterations in the reaction specificity and thus may not inverse their biological activity.

Background Atherosclerosis is a multifactorial disease and the underlying cause of coronary artery disease (CAD), myocardial infarction and stroke. Two main features are involved in the progression of atherosclerosis, lipid retention and inflammation. 12/15-lipoxygenases are involved in inflammation and have been implicated in atherosclerosis. Genetic association studies of the 15-lipoxygenase 1 ( ALOX15 ) in humans revealed a neutral to atheroprotective role of the enzyme. Recently the epidermis-type 15-lipoxygenase 2 ( ALOX15B ) has been identified in human atherosclerotic plaques but its role in human atherosclerosis is still unclear. Methods We screened the ALOX15B gene for polymorphisms and investigated the association of 18 detected polymorphisms with angiographically documented CAD in a case–control study (n = 496). In addition, we measured in vitro the enzyme activity and Michaelis–Menten kinetics of the detected non-synonymous polymorphic variants p.Arg486His (c.1457G > A), p.Gln656Arg (c.1967A > G) and p.Ile676Val (c.2026A > G). Results We found that the linked polymorphisms at position c.1458-38G > C, c.1579 + 71C > T and c.1656G > A are associated with CAD (OR: 0.51 (0.27–0.94), p-value: 0.03). In addition, we show that the activity and the kinetics of the three non-synonymous ALOX15B enzyme variants (p.Arg486His, p.Gln656Arg and p.Ile676Val) are similar to the wild-type enzyme. Conclusions Our data indicate that the ALOX15B gene may be associated with coronary artery disease. However, larger studies would be necessary to confirm the association of these polymorphisms with CAD. In contrast, our study did not find frequent non-synonymous polymorphisms in ALOX15B altering enzyme activity in Europeans.

The lipoxygenase metabolism of arachidonic acid occurs in specific blood cell types and epithelial tissues and is activated in inflammation and tissue injury. In the course of studying lipoxygenase expression in human skin, we detected and characterized a previously unrecognized enzyme that at least partly accounts for the 15 S -lipoxygenase metabolism of arachidonic acid in certain epithelial tissues. The cDNA was cloned from human hair roots, and expression of the mRNA was detected also in prostate, lung, and cornea; an additional 16 human tissues, including peripheral blood leukocytes, were negative for the mRNA. The cDNA encodes a protein of 676 amino acids with a calculated molecular mass of 76 kDa. The amino acid sequence has approximately 40% identity to the known human 5 S -, 12 S -, and 15 S -lipoxygenases. When expressed in HEK 293 cells, the newly discovered enzyme converts arachidonic acid exclusively to 15 S -hydroperoxyeicosatetraenoic acid, while linoleic acid is less well metabolized. These features contrast with the previously reported 15 S -lipoxygenase, which oxygenates arachidonic acid mainly at C-15, but also partly at C-12, and for which linoleic acid is an excellent substrate. The different catalytic activities and tissue distribution suggest a distinct function for the new enzyme compared with the previously reported human 15 S -lipoxygenase.

The differential diagnosis between eosinophilic esophagitis (EoE) and gastroesophageal reflux disease (GERD) is often challenging. We recently showed that the ALOX15 protein is expressed in 95% of esophageal biopsies from patients with a definitive diagnosis of EoE. Here we correlated ALOX15 expression with the clinical classification of EoE or GERD in a cohort of consecutive pediatric patients (n = 62) with at least 1 esophageal biopsy containing at least 15 eosinophils per high-power field (eos/HPF). The patients were categorized into the following groups: (1) at least 15 eos/HPF in the distal esophagus only (n = 24), (2) at least 15 eos/HPF in the proximal esophagus only (n = 5), and (3) at least 15 eos/HPF in the distal and proximal biopsies (n = 33). Control groups included patients with GERD with biopsies containing 6 to 15 eos/HPF (n = 9), patients with GERD with 5 eos/HPF or less (n = 15), patients with candida esophagitis (n = 15), and patients with normal biopsies (n = 15). ALOX15 was positive in 90.5% of patients with EoE (13/16 in group 1, 4/4 in group 2, 31/33 in group 3) versus 44% of patients with GERD (4/8 in group 1, 0/1 in group 2, and 0/0 in group 3), 2 of 9 (22%) of patients with 6 to 15 eos/HPF, and was negative in all patients with GERD with biopsies containing 5 eos/HPF or less, all patients with candida esophagitis, and all normal controls. In conclusion, ALOX15 is a sensitive marker of EoE; however, subpopulations of patients with GERD with >5 eos/HPF also express ALOX15. Positive ALOX15 expression is more prevalent in EoE than in GERD and may prove to be a useful diagnostic marker in patients with discrepant biopsy findings between the proximal and distal esophagus.

Targeted disruption of the Alox15 gene makes mice resistant to angiotensin II-, DOCA/salt-, and Nω-nitro-l-arginine methyl ester (l-NAME)-induced experimental hypertension. Macrophages, a primary source of Alox15, are facilitating this resistance, but the underlying mechanism is not known. Because Alox15 metabolites are peroxisome proliferator-activated receptor (PPAR)γ agonists, we hypothesized that activation of macrophage PPARγ is the key step in Alox15 mediation of hypertension. Thioglycollate, used for macrophage elicitation, selectively upregulated PPARγ and its target gene CD36 in peritoneal macrophages of both wild-type (WT) and Alox15−/− mice. Moreover, thioglycollate-injected Alox15−/− mice became hypertensive upon l-NAME treatment. A similar hypertensive effect was observed with adoptive transfer of thioglycollate-elicited Alox15−/− macrophages into Alox15−/− recipient mice. The role of PPARγ was further specified by using the selective PPARγ antagonist GW9662. WT mice treated with 50 μg/kg daily dose of GW9662 for 12 days became resistant to l-NAME-induced hypertension. The PPARγ antagonist treatment also prevented l-NAME-induced hypertension in thioglycollate-injected Alox15−/− mice, indicating a PPARγ-mediated effect in macrophage elicitation and the resultant hypertension. These results indicate a regulatory role for macrophage-localized PPARγ in l-NAME-induced experimental hypertension.

Targeted disruption of the 12/15-lipoxygenase (Alox15) gene in mice prevents the development of angiotensin II-, DOCA-salt-, and L-NAME-induced experimental hypertension. Macrophages are the primary source of Alox15. Using Alox15-/- and wild type (WT) mice, we previously demonstrated that macrophage Alox15 is essential for experimental hypertension; however, the underlying mechanism is not known. Since Alox15-derived metabolites of arachidonic and linoleic acids are potent PPARγ agonists, we hypothesized that activation of macrophage PPARγ is the key step in Alox15 mediation of L-NAME-induced hypertension. Thioglycollate (TG) injection, used to elicit peritoneal macrophages, upregulated PPARγ protein expression and expression of its target gene CD36 in peritoneal macrophages, but not in aorta or kidney, of both WT (6.8±0.3 and 9.3±0.5 fold increase for PPARγ and CD36, respectively) and Alox15-/- mice (4.7±0.4 and 11.1±0.6 fold increase for PPARγ and CD36, respectively). Thus, TG bypassed Alox15 to activate PPARγ selectively in macrophages. Upregulation of PPARγ by TG restored the sensitivity to L-NAME hypertension (100 mg/kg/day L-NAME for 7 days) in Alox15-/- mice (129.4±7.3 mmHg for TG vs. 94.8±5.6 mmHg for non-injected controls, p

Foxa2 is a member of the Forkhead family of nuclear transcription factors that is highly expressed in respiratory epithelial cells of the developing and mature lung. Foxa2 is required for normal airway epithelial differentiation, and its deletion causes goblet-cell metaplasia and Th2-mediated pulmonary inflammation during postnatal development. Foxa2 expression is inhibited during aeroallergen sensitization and after stimulation with Th2 cytokines, when its loss is associated with goblet-cell metaplasia. Mechanisms by which Foxa2 controls airway epithelial differentiation and Th2 immunity are incompletely known. During the first 2 weeks after birth, the loss of Foxa2 increases the production of leukotrienes (LTs) and Th2 cytokines in the lungs of Foxa2 gene–targeted mice. Foxa2 expression inhibited 15-lipoxygenase (Alox15) and increased Alox5 transcription, each encoding key lipoxygenases associated with asthma. The inhibition of the cysteinyl LT (CysLT) signaling pathway by montelukast inhibited IL-4, IL-5, eotaxin-2, and regulated upon activation normal T cell expressed and presumably secreted expression in the developing lungs of Foxa2 gene–targeted mice. Montelukast inhibited the expression of genes regulating mucus metaplasia, including Spdef, Muc5ac, Foxa3, and Arg2. Foxa2 plays a cell-autonomous role in the respiratory epithelium, and is required for the suppression of Th2 immunity and mucus metaplasia in the developing lung in a process determined in part by its regulation of the CysLT pathway.

Background. Q fever is caused by Coxiella burnetii, a bacterium that persists in M2-polarized macrophages. We wondered whether the concept of M1/M2 polarization is applicable to Q fever patients. Methods. Monocytes from healthy controls were cultured with IFN-γ and IL-4, agonists of M1 and M2 macrophages, respectively, and their gene expression was assessed using whole-genome microarrays. Selected biomarkers were assessed in blood from Q fever patients by real-time reverse transcription polymerase chain reaction (RTPCR). Results. Monocytes exhibited early (6-hour) patterns of activation specific to IFN-γ or IL-4 and a late (18-hour) pattern of common activation. Because these responses were not reducible to M1/M2 polarization, we selected biomarkers and tested their relevance in Q fever patients. The early genes NLRC5, RTP4, and RHOH, which were modulated in response to IFN-γ, were up-regulated in patients with acute Q fever, and the expression levels of the late genes ALOX15, CLECSF1, CCL13, and CCL23 were specifically increased in patients with Q fever endocarditis. The RHOH and ALOX15 genes were associated with the activity of acute Q fever and Q fever endocarditis, respectively. Conclusions. Our results show that the kinetic model of monocyte activation enables a dynamic approach for the evaluation of Q fever patients.

Aims The aim of this study was to evaluate the combined contribution of 12 genetic variants to the risk of coronary heart disease (CHD). Methods Through a comprehensive literature search for genetic variants involved in the CHD association study, we harvested a total of 10 genes (12 variants) for the current meta-analyses. These genes consisted of GPX1 (rs1050450), PPARD (rs2016520), ALOX15 (rs34210653), SELPLG (rs2228315), FCGR2A (rs1801274), CCL5 (rs2107538), CYP1A1 (rs4646903), TP53 (rs1042522), CX37 (rs1764391), and PECAM1 (rs668, rs12953, and rs1131012). Results A total of 45 studies among 23,314 cases and 28,430 controls were retrieved for the meta-analyses of 12 genetic variants. The results showed a significant association between the GPX1 rs1050450 polymorphism and CHD (odd ratio (OR) = 1.61, 95% confidence interval (CI) = 1.25–2.07, P = 0.0002). Other meta-analyses of the rest 11 variants suggested a lack of association with the risk of CHD. Conclusion Our results confirmed that GPX1 rs1050450 was associated with susceptibility to CHD in Chinese and Indian populations.

Some asthmatic individuals undergoing allergen inhalation challenge develop an isolated early response whereas others develop a dual response (early plus late response). In the present study we have used transcriptomics (microarrays) and metabolomics (mass spectrometry) of peripheral blood to identify molecular patterns that can discriminate allergen-induced isolated early from dual asthmatic responses. Peripheral blood was obtained prior to (pre-) and 2 hours post allergen inhalation challenge from 33 study participants. In an initial cohort of 14 participants, complete blood counts indicated significant differences in neutrophil and lymphocyte counts at pre-challenge between early and dual responders. At post-challenge, significant genes (ALOX15, FADS2 and LPCAT2) and metabolites (lysolipids) were enriched in lipid metabolism pathways. Enzymes encoding for these genes are involved in membrane biogenesis and metabolism of fatty acids into pro-inflammatory and anti-inflammatory mediators. Correlation analysis indicated a strong negative correlation between ALOX15, FADS2, and IL5RA expression with 2-arachidonoylglycerophosphocholine levels in dual responders. However, measuring arachidonic acid and docosahexaenoic acid levels in a validation cohort of 19 participants indicated that the free form of DHA (nmoles/µg of protein) was significantly (p = 0.03) different between early and dual responders after allergen challenge. Collectively these results may suggest an imbalance in lipid metabolism which dictates pro- (anti-) inflammatory and pro-resolving mechanisms. Future studies with larger sample sizes may reveal novel mechanisms and therapeutic targets of the late phase asthmatic response.

Previously, we isolated the murine “leukocyte-type” 12-lipoxygenase (L-12LO) cDNA from RNA of peritoneal-elicited cells that consisted predominantly of leukocytes (Chen, X.-S., Kurre, U., Jenkins, N. A., Copeland, N. G., and Funk, C. D. (1994) J. Biol. Chem. 269, 13979-13987). By in situ hybridization we show that the L-12LO gene is expressed abundantly in a subset of peritoneal macrophages but not in elicited leukocytes, alveolar macrophages, or bone marrow-derived macrophages. L-12LO is highly related to human and rabbit 15-lipoxygenases, enzymes that have been implicated in the maturation process of red blood cells, and the oxidative modification of low density lipoproteins that is implicated in atherogenesis. Accordingly, these enzymes have been referred to as 12/15-lipoxygenases. We have inactivated the L-12LO gene in mice using homologous recombination in embryonic stem cells. Macrophage expression of L-12LO was abolished in homozygous deficient mice as was formation of 12-hydroxyeicosatetraenoic acid (12-HETE). In zymosan-stimulated cells, there was significant diversion of metabolism to the 5-lipoxygenase products leukotriene C4 and 5-HETE and in A23187-treated cells to 5-HETE only. The enhanced formation of 5-lipoxygenase metabolites was not due to compensatory changes of 5-lipoxygenase or 5-lipoxygenase activating protein but rather an apparent substrate diversion. L-12LO-deficient mice have no obvious abnormalities in reticulocyte or mature red blood cells, which suggest that in mice this pathway is not functionally important for erythrocytic development. Indices for oxidation of low density lipoprotein (measured as either thiobarbituric acid-reactive substances or the oxidant stress marker isoprostane 8-epi-prostaglandin F2α) were identical in incubations with unstimulated wild-type and L-12LO-deficient macrophages, but the zymosan-induced increase observed with wild-type macrophages was abolished in L-12LO-deficient cells. Thus, 12/15-lipoxygenase-deficient mice will be useful for the study of interaction between lipoxygenase pathways and determination of the in vivo role of 12/15-lipoxygenase-catalyzed oxidation of LDL in atherogenesis.

Background Sequence variation in the human 12/15 lipoxygenase (ALOX15) has been associated with atherosclerotic disease. We functionally characterized an ALOX15 promoter polymorphism, rs2255888, previously associated with carotid plaque burden. Methodology/Principal Findings We demonstrate specific in vitro and in vivo binding of the cytoskeletal protein, vimentin, to the ALOX15 promoter. We show that the two promoter haplotypes carrying alternate alleles at rs2255888 exhibit significant differences in promoter activity by luciferase reporter assay in two cell lines. Differences in in-vitro vimentin-binding to and formation of DNA secondary structures in the polymorphic promoter sequence are also detected by electrophoretic mobility shift assay and biophysical analysis, respectively. We show regulation of ALOX15 protein by vimentin. Conclusions/Significance This study suggests that vimentin binds the ALOX15 promoter and regulates its promoter activity and protein expression. Sequence variation that results in changes in DNA conformation and vimentin binding to the promoter may be relevant to ALOX15 gene regulation.

Dietary polyunsaturated fatty acids (PUFAs) can be converted to prostaglandins and leukotrienes. Oxygenation of omega-6 PUFAs generally results in the production of pro-inflammatory mediators, whereas oxygenated products of omega-3 (n-3) PUFAs generally have lower inflammatory activity. We hypothesize that elevated n-3 PUFA intakes from fish are associated with lower risk of colorectal cancer among those with genetic variants that result in higher levels of pro-inflammatory mediators. In population-based case–control studies of colon (case n = 1,574) and rectal cancer (case n = 791) and disease-free controls (n = 2,969), we investigated interactions between dietary fatty acid intake and 107 candidate polymorphisms and tagSNPs in PTGS1, PTGS2, ALOX12, ALOX5, ALOX15, and FLAP. The two studies used an identical genotyping protocol. We observed interactions and statistically significant increases in colon cancer risk for low docosahexaenoic acid intake among those with the PTGS1 rs10306110 (−1,053 A > G) variant genotypes (OR = 1.6, 95 % confidence interval = 1.1–2.3, adj. p = 0.06) and rectal cancer risk for low total fat intake among those with the variant PTGS1 rs10306122 (7,135 A > G) (ORvs.wt = 1.80, 1.02–2.99; adj. p = 0.08). The ALOX15 rs11568131 (10,339 C > T) wild type in combination with a high inflammation score (low EPA intake, high AA intake, no regular NSAID use, high BMI, smoking) was associated with increased colon cancer risk (OR = 2.28, 1.7–3.07). Rectal cancer risk was inversely associated with a low inflammation score among PTGS2 rs4648276 (3,934 T > C) variant allele carriers (OR = 0.49, 0.25–0.75). Overall, these data provide some modest evidence for interactions between dietary fat intake and genetic variation in genes involved in eicosanoid metabolism and colorectal cancer risk.

Objectives Atherosclerosis is a chronic disease characterized by two main features, lipid retention and inflammation. The 12/15-lipoxygenases play a two-faced role in atherosclerosis with pro-inflammatory effects through oxidation of LDL and anti-inflammatory effects through lipid mediator synthesis. In cells involved in atherosclerosis the 12-lipoxygenase ALOX12 and the two 15-lipoxygenases, ALOX15 and ALOX15B may be expressed but their expression has not yet been investigated in detail. Methods To investigate the regulation of ALOX12, ALOX15 and ALOX15B in human macrophages we measured basal mRNA and protein expression during differentiation of monocytes to macrophages and stimulated expression in macrophages. Results The results show an increase of ALOX15B during the differentiation of monocytes to macrophages, while the expression of ALOX12 and ALOX15 remains on the same low level. Stimulation of macrophages with a set of cytokines and with hypoxia revealed that IL-4, IL-13, LPS and hypoxia further increase the ALOX15B mRNA. Western blot analysis showed that IL-4, LPS and hypoxia increase the ALOX15B protein expression, whereas IL-13 has no effect on the protein levels. IL-4 and IL-13 also enhance ALOX15 mRNA and protein expression, whereas none of the stimuli has an impact on ALOX12 expression. Conclusion In summary, these data suggest that ALOX15B is the mainly expressed 12/15-lipoxygenase in human macrophages and that its expression is induced by IL-4, LPS and hypoxia. IL-4 and IL-13 also increase the expression of ALOX15, however, only IL-4 stimulation seems to drive ALOX15 expression to levels higher than the basal expression of ALOX15B. Hence, ALOX15B may play a major role in human atherosclerosis.

SummaryBackground To date, genetic studies of hypersensitivity reactions to non-steroidal anti-inflammatory drugs (NSAIDs) have been carried out mainly in aspirin-induced asthma and to a lesser extent in chronic urticaria, with no studies in patients with acute urticaria (AU), the most common entity induced by these drugs. Objective In this work, we analysed the association of common variants of 15 relevant genes encoding both enzymes and receptors from the arachidonic acid (AA) pathway with NSAID-induced AU. Methods Patients were recruited in several Allergy Services that are integrated into the Spanish network RIRAAF, and diagnosed of AU induced by cross-intolerance (CRI) to NSAIDs. Genotyping was carried out by TaqMan allelic discrimination assays. Results A total of 486 patients with AU induced by CRI to NSAIDs and 536 unrelated controls were included in this large Spanish case-control study. Seven variants from 31 tested in six genes were associated in a discovery study population from Malaga (0.0003 ≤ p-value ≤ 0.041). A follow-up analysis in an independent sample from Madrid replicated three of the SNPs from the ALOX15 (rs7220870), PTGDR (rs8004654) and CYSLTR1 (rs320095) genes (1.055x10-6≤meta-analysis p-value ≤ 0.003). Conclusions and Clinical Relevance Genetic variants of the AA pathway may play an important role in NSAID-induced AU. These data may help understand the mechanism underlying this disease.

In mouse arteries, Alox15 [leukocyte-type 12/15-lipoxygenase (LO)] is assumed to regulate vascular function by metabolizing arachidonic acid (AA) to dilator eicosanoids that mediate the endothelium-dependent relaxations to AA and acetylcholine (ACh). We used Alox15−/−mice, made by targeted disruption of the Alox15 gene, to characterize its role in the regulation of blood pressure and vascular tone. Systolic blood pressures did not differ between wild-type (WT) and Alox15−/−mice between 8–12 wk of age, but Alox15−/−mice exhibited resistance toward both NG-nitro-l-arginine-methyl ester (l-NAME)- and deoxycorticosterone acetate (DOCA)/high-salt-induced hypertension. ACh relaxed mesenteric arteries and abdominal aortas of WT and Alox15−/−mice to an identical extent. The LO inhibitor nordihydroguaiaretic acid attenuated the ACh relaxations by 35% in arteries from both WT and Alox15−/−mice. Reverse-phase HPLC analysis of [14C]AA metabolites in aorta and peritoneal macrophages (PM) revealed differences. Unlike PM, aorta tissue did not produce detectable amounts of 15-hydroxyeicosatetraenoic acid. Although Alox15 mRNA was detected in aorta, high-resolution gel electrophoresis with immunodetection revealed no Alox15 protein expression. Unlike aorta, Alox15 protein was detected in PM, intestine, fat, lung, spleen, and skin from WT, but not Alox15−/−, mice. Injection of WT PM, a primary source of Alox15 protein, into Alox15−/−mice abolished their resistance toward l-NAME-induced hypertension. On the other hand, WT mice acquired resistance to l-NAME-induced hypertension after depletion of macrophages by clodronate injection. These studies indicate that Alox15 is involved in development of experimental hypertension by altering macrophage functions but not via synthesis of the vasoactive LO metabolites in mouse arteries.

Background and Objectives. The purpose of the present study is to find the genes and SNP that influence BMD and postmenopausal Saudi women.Material and Methods. Two-hundred ethnic Saudi Arabian women with a diagnosis of postmenopausal osteoporosis were the subjects of this study. Baseline blood hematology, biochemistry, and bone panel were done. Blood was collected, and three TaqMan-MGB probes were used to analyze SNP variants in ALOX15 (rs7220870), LRP5 (C 25752205 10), and TNFRSF11B (C 11869235 10).Results. The variant of ALOX15 17p13 showed that the BMD of the spine was lower in the AA allele (Pvalue<0.002) and fractures were highest at 50% compared to CC allele. In the TNFRSF11B gene, BMD of the hip and spine was significantly higher in the GG allele and the history of fractures was significantly higher in GG group. With regard to the LRP5 (C 25752205 10) gene, there was no significant difference between allele groups.Conclusion(s). This study shows that the genetic influence of osteoporosis in the Caucasian and Saudi Arabians population is similar. We believe that the same genetic markers that influence osteoporosis in the Caucasian race could be used for further studies in the Saudi Arabian population.

Review on ALOX15 (arachidonate 15-lipoxygenase), with data on DNA, on the protein encoded, and where the gene is implicated.

Lipoxygenases (LOX) comprise a family of non-heme-iron-containing proteins that catalyze the dioxygenation of polyunsaturated fatty acids to form bioactive lipids. Metabolism of arachidonic acid by LOXs leads to the formation of regioisomeric cis/trans-conjugated hydroxyeicosatetraenoic acids (HETEs), leukotrienes, lipoxins, and hepoxilins. Dependent on the predominant position of the incorporation of hydroperoxy group, LOXs are classified as 3-, 5-, 8-, 12(S)-, 12(R)-, and 15-LOXs, whose main products are 3(S)-, 5(S)-, 8(S)-, 12(S), 12(R)-, and 15(S)-HETE, respectively. There are two 15-LOX: 15-LOX1 (gene: ALOX15) and 15-LOX2 (gene: ALOX15B). 15-LOX1, whose murine ortholog is leukocyte-type 12-LOX, prefers linoleic acid as the substrate to form 13(S)-HODE, although it can convert arachidonic acid to 15(S)-HETE and, to a lesser extent, 12(S)-HETE. On the other hand, 15-LOX2 mainly uses arachidonic acid to form 15(S)-HETE.1 The involvement of LOXs in carcinogenesis is complex, since both tumor-promoting and -suppressing activities are reported for the various members of LOX family. A number of studies suggest 15-LOX2 as a functional tumor suppressor, particularly in the prostate. Its expression or activity is frequently suppressed during carcinogenesis of prostate, lung, esophageal, and sebaceous gland. Restoration of 15-LOX2 expression in prostate cancer cells inhibited DNA replication, caused G0/G1 arrest, reduced tumor growth and even tumor development.2,3 Interestingly, when 15-LOX2 is expressed in mouse prostate, the transgenic mice presented indications of prostate hyperplasia,4 suggesting the complex function of 15-LOX2 in disrupting tissue homeostasis in the mouse prostate. Suraneni et al.5 in this issue presented experimental evidence unequivocally establishing the tumor-suppressing activities of 15-LOX2. By crossing 15-LOX2 transgenic mice with a Hi-Myc mouse model for prostate cancer, they found that the presence of 15-LOX2 inhibited Myc-induced tumor development in the prostate of double transgenic mice. Given the frequent amplification of Myc in human prostate cancer, it is reasonable to extrapolate that the loss of 15-LOX2 can contribute to Myc-induced prostate carcinogenesis. However, whether Myc suppresses 15-LOX2 expression remains to be elucidated. A common biological effect of 15-LOX2 is cell senescence. Suraneni et al. observed an increased cell senescence in the mouse prostate with expression of human 15-LOX2, possibly through induction of RB1CC1 expression.4 In this issue, Suraneni extended this observation.5 They found an upregulation of RB1CC1 in the prostates of double transgenic Myc;LOX mice when compared with the Hi-Myc prostates. Paradoxically, RB1CC1 is found upregulated, instead of being suppressed as 15-LOX2, in human prostate cancers, suggesting that the link between 15-LOX2 and RB1CC1 can be compounded by many other extraneous factors uniquely present in the human prostate. An important question is how to harness the tumor-suppressive activities of 15-LOX2 as possible prevention and treatment of prostate cancer. Reactivation of 15-LOX2 expression in human prostate cancer cells, while conceptually plausible, requires detailed understanding how 15-LOX2 is suppressed during carcinogenesis and identification of druggable targets. Another approach is to utilize the arachidonate product of 15-LOX2, 15(S)-HETE, to suppress prostate cancer. A limitation for this approach is the uncertainty whether 15(S)-HETE can recapitulate the tumor-suppressive activities of 15-LOX2. 15(S)-HETE treatment has been shown to suppress prostate cancer cell growth and induce RB1CC1, as 15-LOX2 did.3,5 However, the enzymatic activity of 15-LOX2 seems dispensable for its tumor-suppressing activities. PC-3 cells transfected with a splice variant of 15-LOX2, 15-LOX2sv-b, which did not have the capacity of synthesizing 15(S)-HETE, also had reduced tumor development.3 This evidence suggests that 15(S)-HETE may not fully recapitulate the tumor-suppressing activities of 15-LOX2. Further studies are needed to resolve the complex roles of lipoxygenases in cancers. From a gene evolution perspective, all ALOX genes come from one ancestral gene that has duplicated several times (Fig. 1). Comparisons of the orthologous genes between mouse and human ALOX5 or ALOX12R demonstrate that those genes have more than 85% amino acid identities. On the other hand, comparisons of paraologous ALOX genes, such as ALOX15 vs ALOX15B or ALOX12 vs ALOX15B, reveal only 40% amino acids identities. However, the amino acid identity between human ALOX15B and mouse ALOX8 is as high as 78%. In this sense, the murine ALOX8 is the ortholog of human ALOX15B, although their metabolic products can be different. Studies show that 8-LOX possesses anti-proliferation and anti-tumorigenic activities, as does 15-LOX2.6,7 Although 8-LOX expression is not detected in the prostate of 3- or 6-mo-old mice (human equivalent ages of 8 or 16 y), it is unknown whether 8-LOX is expressed in more senescent or diseased mouse prostate. Given the remarkably similar organization of murine ALOX8 and human ALOX15B in their respective genome (Fig. 1), further studies are needed to determine whether deletion of ALOX8 can lead to increased carcinogenesis, particularly in the prostate. Figure 1. Evolution of ALOX genes. Based on the arrangements and sequence homology, it is deduced all ALOX genes are derived from one ancient ALOX gene through duplications and evolutions. Note the remarkable similarity in genomic localization ...

Objective Nasal polyposis (NP) is a multifactorial disease manifesting in chronic inflammation of upper respiratory tract of unknown etiology. We studied mRNA gene expression profiles in NP compared with normal mucosa as well as pointed at genes characteristic of different expression in examined tissues. Material and methods Fifty-three patients with NP (36 eosinophilic and 17 neutrophilic NP) were included into the study. Transcriptional activity of genes was analyzed using oligonucleotide microarray in 17 NP and 8 cases of normal nasal mucosa. A study of mRNA expression of selected genes was performed using QRT-PCR. Results We identified 556 genes, which were differentially expressed between the studied and the control group. Among them 217 showed significantly higher expression, whereas 339 lower expression in NP than in controls. The microarray and QRT-PCR results were compatible for 7 of 8 evaluated genes. In NP strongly significant higher transcriptional activity of MMP10, NOS2A, ALOX15 and IL-8 genes was observed. In the control group, significantly higher expression of DMBT1, ALOX12 and LTF genes was detected. Conclusion The analysis of gene expression in inflammatory changed nasal polyp tissues may become a supplementary method in diagnostics and treatment. Molecular alterations may indicate changes during the clinical course of the disease.

Background— Many different genetic and clinical factors have been identified as causes or contributors to atherosclerosis. We present a model of preclinical atherosclerosis based on genetic and clinical data that predicts the presence of coronary artery calcification in healthy Americans of European descent 45 to 84 years of age in the Multi-Ethnic Study of Atherosclerosis (MESA). Methods and Results— We assessed 712 individuals for the presence or absence of coronary artery calcification and assessed their genotypes for 2882 single-nucleotide polymorphisms. With the use of these single-nucleotide polymorphisms and relevant clinical data, a Bayesian network that predicts the presence of coronary calcification was constructed. The model contained 13 single-nucleotide polymorphisms (from genes AGTR1, ALOX15, INSR, PRKAB1, IL1R2, ESR2, KCNK1, FBLN5, PPARA, VEGFA, PON1, TDRD6, PLA2G7, and 1 ancestry informative marker) and 5 clinical variables (sex, age, weight, smoking, and diabetes mellitus) and achieved 85% predictive accuracy, as measured by area under the receiver operating characteristic curve. This is a significant ( P Conclusions— We present an investigation of joint genetic and clinical factors associated with atherosclerosis that shows predictive results for both cases, as well as enhanced performance for their combination.

Background Elevated left ventricular mass (LVM) is a strong predictor of negative cardiovascular outcomes, including heart failure, stroke, and sudden cardiac death. A relationship between close (< 50 m compared with > 150 m) residential proximity to major roadways and higher LVM has previously been described, but the mechanistic pathways that are involved in this relationship are not known. Understanding genetic factors that influence susceptibility to these effects may provide insight into relevant mechanistic pathways. Objective We set out to determine whether genetic polymorphisms in genes affecting vascular and autonomic function, blood pressure, or inflammation influence the relationship between traffic proximity and LVM. Methods This was a cross-sectional study of 1,376 genotyped participants in the Multi-Ethnic Study of Atherosclerosis, with cardiac magnetic resonance imaging performed between 2000 and 2002. The impact of tagged single-nucleotide polymorphisms (tagSNPs) and inferred haplotypes in 12 candidate genes (ACE, ADRB2, AGT, AGTR1, ALOX15, EDN1, GRK4, PTGS1, PTGS2, TLR4, VEGFA, and VEGFB) on the relationship between residential proximity to major roadways and LVM was analyzed using multiple linear regression, adjusting for multiple potential confounders. Results After accounting for multiple testing and comparing homozygotes, tagSNPs in the type 1 angiotensin II receptor (AGTR1, rs6801836) and arachidonate 15-lipoxygenase (ALOX15, rs2664593) genes were each significantly (q < 0.2) associated with a 9–10% difference in the association between residential proximity to major roadways and LVM. Participants with suboptimal blood pressure control demonstrated stronger interactions between AGTR1 and traffic proximity. Conclusions Common polymorphisms in genes responsible for vascular function, inflammation, and oxidative stress appear to modify associations between proximity to major roadways and LVM. Further understanding of how genes modify effects of air pollution on CVD may help guide research efforts into specific mechanistic pathways.

The 12/15-lipoxygenase plays a janus-role in inflammation with pro-inflammatory and anti-inflammatory effects in cell systems and even opposite effects on atherosclerosis in two different animal species. Screening of the human 15-lipoxygenase (ALOX15) gene detected a polymorphic C to T substitution at position c.-292, which led to three times higher ALOX15 activity in macrophages and showed a trend to be atheroprotective in a small case-control study for coronary artery disease (CAD). A second polymorphism at position c.1693C>T leading to an T560M exchange and an inactive enzyme was recently associated with increased CAD. We now investigated whether these polymorphisms or a certain haplotype of ALOX15 are associated with myocardial infarction (MI) in a case-control subset from the population-based MONIKA/KORA cohort S3. Six polymorphisms in ALOX15 were analyzed in 2629 participants to cover all major haplotypes with a frequency higher than 1% in the Caucasian population. None of the polymorphism was associated with MI but a rare ALOX15 haplotype showed a significant protective effect on the risk for MI (p=0.03). However, none of the polymorphisms or haplotypes was associated with CRP levels. These data suggest that ALOX15 may play a less prominent role during later stages of atherosclerosis involving atherothrombotic mechanisms than eventually during early plaque development.

The aim of the present study, involving 463 women of reproductive age, was to evaluate for the first time the relationship between endometriosis, endometriosis-related infertility, and a recently described functional polymorphism in the ALOX15 gene, reported to be essential for implantation. In our study population, ALOX15 -292 C/T was not correlated either with the risk of developing an endometriosis or with the risk of infertility.

The major underlying cause of CHD is atherosclerosis, and oxidised LDL is known to play an important role in its development. We examined the role of three single nucleotide polymorphisms (SNPs) in the 15-lipoxygenase gene (ALOX15), in atherosclerosis. We genotyped three SNPs in the ALOX15 promoter in two Western Australian samples—1,111 community-based individuals and 556 with CHD. SNPs and haplotypes were tested for an association with carotid plaque, intima-media thickness and risk of CHD. The −611GG genotype was associated with increased likelihood of carotid plaque in CHD patients (OR = 4.01, 95%CI = 1.39–11.53, P = 0.005) and the C alleles of the G-220C and G-189C SNPs were associated with decreased likelihood of plaque among cases (OR = 0.66, 95%CI = 0.43–0.99, P = 0.05 and OR = 0.51, 95%CI = 0.34–0.78, P = 0.002 respectively). The GGG haplotype was associated with increased risk of carotid plaque in CHD patients (OR = 5.77, 95%CI = 1.82–18.29, P = 0.0007) and in community-based individuals under 53 years (OR = 4.15, 95%CI = 1.23–14.08, P = 0.02). No association was observed between ALOX15 SNPs or haplotypes and intima-media thickness. This study is novel as it is the first to examine the association between 15-lipoxygenase polymorphisms and atherosclerotic indicators. These findings suggest a possible role of ALOX15 polymorphisms in focal plaque formation.

Background Reticulocyte-type 15-lipoxygenase-1 (ALOX15) has anti-inflammatory and inflammatory effects and is implicated in the development of asthma, arthritis and atherosclerosis. Previously, we screened the human ALOX15 gene for variations because genetic variability in ALOX15 might influence these diseases. We found a C>T substitution at position c.-292 in the ALOX15 promoter that created a novel binding site for the transcription factor SPI1 and increased ALOX15 mRNA levels in monocytes from c.-292CT heterozygous volunteers. Methods To test whether the higher mRNA levels led to higher ALOX15 activity, we performed an activity assay and measured the arachidonic acid metabolite 15(S)-hydroxy-eicosatetraenoic acid [15(S)-HETE] by HPLC analysis. To test whether this polymorphism was associated with coronary artery disease (CAD), we investigated its association in a case-control study involving 498 Caucasians. Results The c.-292C>T polymorphism was associated with higher enzyme activity in heterozygous carriers. Intriguingly, this polymorphism also showed a tendency to be protective against atherosclerosis. Conclusions These results suggest that increased ALOX15 activity may attenuate inflammation, which could be caused by an increase in 15(S)-HETE and eventually by its metabolites, the lipoxins.

Aim: NSAIDs are the most frequent cause of hypersensitivity drug reactions. We have examined the association between NSAID-exacerbated respiratory disease (NERD) and genetic variants in arachidonic acid metabolism genes. Patients & methods: We included 250 NERD patients, 260 NSAID-tolerant asthmatic (NTA) subjects and 315 healthy controls. Results: Significant associations with NERD were identified for: ALOX15 rs3892408 C/C homozygous genotype (NERD vs NTA; p = 0.0001, pc = 0.0011; NERD vs controls; p = 0.0001, pc = 0.0011), PTGS-1 rs5789 A/A homozygous genotype (NERD vs NTA; p = 0.0001, pc = 0.0011; NERD vs controls; p = 0.0001, pc = 0.0011), PTGS-1 rs10306135 A/A homozygous genotype (NERD vs NTA; p = 0.0009, pc = 0.0091; NERD vs controls; p = 0.0064, pc = 0.045). Differences in ALOX5 copy number variations were also found (NERD vs NTA; p = 0.010; NERD vs controls; p = 0.0001). Conclusion: These results improve our understanding of the underlying mechanisms of NERD and may help develop a predictive test for this pathology. Original submitted 3 November 2014; Revision submitted 2 April 2015

The murine arachidonate 15-lipoxygenase gene (Alox15) has recently been identified as a negative regulator of peak bone mineral density (BMD). The human ALOX15 gene shares significant sequence homology with the murine Alox15 gene; however, the human arachidonate 12-lipoxygenase gene (ALOX12) is functionally more similar to the mouse gene. Multiple single-nucleotide polymorphisms (SNPs) in the human ALOX15 and ALOX12 genes have previously been reported to be significantly associated with BMD in humans. On the basis of these data, we carried out our own investigation of the human ALOX15 and ALOX12 genes and their relationship with hip and spine BMD parameters. The study population consisted of 779 postmenopausal women with a mean (+/- standard deviation) age of 62.5 +/- 5.9 years at BMD measurement and was recruited from a single large general practice in Chingford, northeast London. Three SNPs from ALOX15 and five from ALOX12 were analyzed. None of the SNPs that we analyzed in ALOX15 were significantly associated with any of the BMD parameters or fracture data. However, we found that three SNPs from ALOX12, all previously associated with spine BMD in women, were significantly associated with spine and various hip BMD parameters in our cohort (P = 0.029-0.049). In conclusion, we found no association between polymorphism in ALOX15 and BMD phenotypes but were able to replicate previous findings that genetic variation in ALOX12 seems to play a role in determining bone structure in Caucasian women.

The 12/15-lipoxygenase gene Alox15 has been identified as a susceptibility gene for bone mineral density (BMD) in mice through combined genetic and genomic analyses. Here we studied the association between bone mineral density and an ALOX15 gene single nucleotide polymorphism to assess the potential involvement of the human ALOX15 gene in postmenopausal osteoporosis. Specifically, we examined the association between a single nucleotide polymorphism at -5299G/A in the ALOX15 5'-flanking region with BMD in 319 postmenopausal Japanese women (66.7 +/- 8.9 years, mean +/- SD). We found that subjects bearing at least one variant A allele (GA + AA; n = 273) had significantly lower Z scores for lumbar spine and total body bone mineral density than did subjects with no A allele (GG; n = 46) (lumbar spine, -0.25 +/- 1.34 versus 0.48 +/- 1.70; P = 0.0014; total body, 0.25 +/- 1.01 vs 0.62 +/- 1.11; P = 0.048). These findings suggest that the ALOX15 gene is one of the genetic determinants of BMD in postmenopausal women. Accordingly, this polymorphism could be useful as a genetic marker for predicting the risk of osteoporosis.