Distinctive Lipid Characteristics of Colorectal Cancer Revealed through Non-targeted Lipidomics Analysis of Tongue Coating.

The metabolites and microbiota in tongue coating display distinct characteristics in certain digestive disorders, yet their relationship with colorectal cancer (CRC) remains unexplored. Here, we employed liquid chromatography coupled with tandem mass spectrometry to analyze the lipid composition of tongue coating using a nontargeted approach in 30 individuals with colorectal adenomas (CRA), 32 with CRC, and 30 healthy controls (HC). We identified 21 tongue coating lipids that effectively distinguished CRC from HC (AUC = 0.89), and 9 lipids that differentiated CRC from CRA (AUC = 0.9). Furthermore, we observed significant alterations in the tongue coating lipid composition in the CRC group compared to HC/CRA groups. As the adenoma-cancer sequence progressed, there was an increase in long-chain unsaturated triglycerides (TG) levels and a decrease in phosphatidylethanolamine plasmalogen (PE-P) levels. Furthermore, we noted a positive correlation between N-acyl ornithine (NAOrn), sphingomyelin (SM), and ceramide phosphoethanolamine (PE-Cer), potentially produced by members of the Bacteroidetes phylum. The levels of inflammatory lipid metabolite 12-HETE showed a decreasing trend with colorectal tumor progression, indicating the potential involvement of tongue coating microbiota and tumor immune regulation in early CRC development. Our findings highlight the potential utility of tongue coating lipid analysis as a noninvasive tool for CRC diagnosis.

12-HETE activates Müller glial cells: The potential role of GPR31 and miR-29.

Diabetic retinopathy (DR) is a neurovascular complication of diabetes, driven by an intricate network of cellular and molecular mechanisms. This study sought to explore the mechanisms by investigating the role of 12-hydroxyeicosatetraenoic acid (12-HETE), its receptor GPR31, and microRNA (miR-29) in the context of DR, specifically focusing on their impact on Müller glial cells. We found that 12-HETE activates Müller cells (MCs), elevates glutamate production, and induces inflammatory and oxidative responses, all of which are instrumental in DR progression. The expression of GPR31, the receptor for 12-HETE, was prominently found in the retina, especially in MCs and retinal ganglion cells, and was upregulated in diabetes. Interestingly, miR29 showed potential as a protective agent, mitigating the harmful effects of 12-HETE by attenuating inflammation and oxidative stress, and restoring the expression of pigment epithelium-derived factor (PEDF). Our results underline the central role of 12-HETE in DR progression through activation of a neurovascular toxic pathway in MCs and illuminate the protective capabilities of miR-29, highlighting both as promising therapeutic targets for the management of DR.

Sustained activation of 12/15 lipoxygenase (12/15 LOX) contributes to impaired renal recovery post ischemic injury in male SHR compared to females.

BACKGROUND: Acute kidney injury (AKI) due to ischemia-reperfusion (IR) is a serious and frequent complication in clinical settings, and mortality rates remain high. There are well established sex differences in renal IR, with males exhibiting greater injury following an ischemic insult compared to females. We recently reported that males have impaired renal recovery from ischemic injury vs. females. However, the mechanisms mediating sex differences in renal recovery from IR injury remain poorly understood. Elevated 12/15 lipoxygenase (LOX) activity has been reported to contribute to the progression of numerous kidney diseases. The goal of the current study was to test the hypothesis that enhanced activation of 12/15 LOX contributes to impaired recovery post-IR in males vs. females. METHODS: 13-week-old male and female spontaneously hypertensive rats (SHR) were randomized to sham or 30-minute warm bilateral IR surgery. Additional male and female SHR were randomized to treatment with vehicle or the specific 12/15 LOX inhibitor ML355 1 h prior to sham/IR surgery, and every other day following up to 7-days post-IR. Blood was collected from all rats 1-and 7-days post-IR. Kidneys were harvested 7-days post-IR and processed for biochemical, histological, and Western blot analysis. 12/15 LOX metabolites 12 and 15 HETE were measured in kidney samples by liquid chromatography-mass spectrometry (LC/MS). RESULTS: Male SHR exhibited delayed recovery of renal function post-IR vs. male sham and female IR rats. Delayed recovery in males was associated with activation of renal 12/15 LOX, increased renal 12-HETE, enhanced endoplasmic reticulum (ER) stress, lipid peroxidation, renal cell death and inflammation compared to females 7-days post-IR. Treatment of male SHR with ML355 lowered levels of 12-HETE and resulted in reduced renal lipid peroxidation, ER stress, tubular cell death and inflammation 7-days post-IR with enhanced recovery of renal function compared to vehicle-treated IR male rats. ML355 treatment did not alter IR-induced increases in plasma creatinine in females, however, tubular injury and cell death were attenuated in ML355 treated females compared to vehicle-treated rats 7 days post-IR. CONCLUSION: Our data demonstrate that sustained activation 12/15 LOX contributes to impaired renal recovery post ischemic injury in male and female SHR, although males are more susceptible on this mechanism than females.

Neonatal imprinting of alveolar macrophages via neutrophil-derived 12-HETE.

Resident-tissue macrophages (RTMs) arise from embryonic precursors1,2, yet the developmental signals that shape their longevity remain largely unknown. Here we demonstrate in mice genetically deficient in 12-lipoxygenase and 15-lipoxygenase (Alox15-/- mice) that neonatal neutrophil-derived 12-HETE is required for self-renewal and maintenance of alveolar macrophages (AMs) during lung development. Although the seeding and differentiation of AM progenitors remained intact, the absence of 12-HETE led to a significant reduction in AMs in adult lungs and enhanced senescence owing to increased prostaglandin E2 production. A compromised AM compartment resulted in increased susceptibility to acute lung injury induced by lipopolysaccharide and to pulmonary infections with influenza A virus or SARS-CoV-2. Our results highlight the complexity of prenatal RTM programming and reveal their dependency on in trans eicosanoid production by neutrophils for lifelong self-renewal.

The Heat Is On: 20-HETE Instructs an Immunosuppressive Phenotype in Cancer-Associated Fibroblasts.

Immunotherapy of cancer is a burgeoning field of research since the realization that our immune system intrinsically has the capacity to restrict tumor occurrence and progression. Though strategies to maximize antitumor T-cell activation are well established, the efficacy of these therapies is limited by an insufficient knowledge of the intricate tumor microenvironment and its capacity to thwart antitumor immunity. Chen and colleagues now uncover a novel immunosuppressive pathway in non-small cell lung carcinoma. Overexpression of cytochrome P450F2 in cancer cells increases production of 20-hydroxyeicosatetraenoic acid, which instructs the expression of immunosuppressive molecules in cancer-associated fibroblasts by binding the GPR75 receptor and activating STAT3/c-Jun signaling. This work proposes several innovative therapeutic anchor points that may improve the efficacy of existing immunotherapies. See related article by Chen et al., p. 4016.

Targeted metabolomics combined with network pharmacology to reveal the protective role of luteolin in pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a progressive disease that significantly endangers human health, where metabolism may drive pathogenesis: a shift from mitochondrial oxidation to glycolysis occurs in diseased pulmonary vessels and the right ventricle. An increase in pulmonary vascular resistance in patients with heart failure with a preserved ejection fraction portends a poor prognosis. Luteolin exists in numerous foods and is marketed as a dietary supplement assisting in many disease treatments. However, little is known about the protective effect of luteolin on metabolism disorders in diseased pulmonary vessels. In this study, we found that luteolin apparently reversed the pulmonary vascular remodeling of PAH rats by inhibiting the abnormal proliferation of pulmonary artery smooth muscle cells (PASMCs). Moreover, network pharmacology and metabolomics results revealed that the arachidonic acid pathway, amino acid pathway and TCA cycle were dysregulated in PAH. A total of 14 differential metabolites were significantly changed during the PAH, including DHA, PGE2, PGD2, LTB4, 12-HETE, 15-HETE, PGF2α, and 8-iso-PGF2α metabolites in the arachidonic acid pathway, and L-asparagine, oxaloacetate, N-acetyl-L-ornithine, butane diacid, ornithine, glutamic acid metabolites in amino acid and TCA pathways. However, treatment with luteolin recovered the LTB4, PGE2, PGD2, 12-HETE, 15-HETE, PGF2α and 8-iso-PGF2α levels close to normal. Meanwhile, we showed that luteolin also downregulated the gene and protein levels of COX 1, 5-LOX, 12-LOX, and 15-LOX in the arachidonic acid pathway. Collectively, this work highlighted the metabolic mechanism of luteolin-protected PAH and showed that luteolin would hold great potential in PAH prevention.

Intestinal metabolomics of juvenile lenok (Brachymystax lenok) in response to heat stress.

Changes in the metabolic profile within the intestine of lenok (Brachymystax lenok) when challenged to acute and lethal heat stress (HS) are studied using no-target HPLC-MS/MS metabonomic analysis. A total of 51 differentially expressed metabolites (VIP > 1, P < 0.05) were identified in response to HS, and 34 occurred in the positive ion mode and 17 in negative ion mode, respectively. After heat stress, changes in metabolites related to glycolysis (i.e., alpha-D-glucose, stachyose, and L-lactate) were identified. The metabolites (acetyl carnitine, palmitoylcarnitine, carnitine, and erucic acid) related to fatty acid ß-oxidation accumulated significantly, and many amino acids (L-tryptophan, D-proline, L-leucine, L-phenylalanine, L-aspartate, L-tyrosine, L-methionine, L-histidine, and L-glutamine) were significantly decreased in HS-treated lenok. The mitochondrial ß-oxidation pathway might be inhibited, while severe heat stress might activate the anaerobic glycolysis and catabolism of amino acid for energy expenditure. Oxidative damage in HS-treated lenok was indicated by the decreased glycerophospholipid metabolites (i.e., glycerophosphocholine, 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine, 1-palmitoyl-sn-glycero-3-phosphocholine, 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine, and 1, 2-dioleoyl-sn-glycero-3-phosphatidylcholine) and the increased oxylipin production (12-HETE and 9R, 10S-EpOME). The minor oxidative pathways (omega-oxidation and peroxisomal beta-oxidation) were likely to be induced in HS-treated lenok.

INTERCEPT Pathogen Reduction in Platelet Concentrates, in Contrast to Gamma Irradiation, Induces the Formation of trans-Arachidonic Acids and Affects Eicosanoid Release during Storage.

Pathogen inactivation techniques for blood products have been implemented to optimize clinically safe blood components supply. The INTERCEPT system uses amotosalen together with ultraviolet light wavelength A (UVA) irradiation. Irradiation-induced inactivation of nucleic acids may actually be accompanied by modifications of chemically reactive polyunsaturated fatty acids known to be important mediators of platelet functions. Thus, here, we investigated eicosanoids and the related fatty acids released upon treatment and during storage of platelet concentrates for 7 days, complemented by the analysis of functional and metabolic consequences of these treatments. Metabolic and functional issues like glucose consumption, lactate formation, platelet aggregation, and clot firmness hardly differed between the two treatment groups. In contrast to gamma irradiation, here, we demonstrated that INTERCEPT treatment immediately caused new formation of trans-arachidonic acid isoforms, while 11-hydroxyeicosatetraenoic acid (11-HETE) and 15-HETE were increased and two hydroperoxyoctadecadienoic acid (HpODE) isoforms decreased. During further storage, these alterations remained stable, while the release of 12-lipoxygenase (12-LOX) products such as 12-HETE and 12-hydroxyeicosapentaenoic acid (12-HEPE) was further attenuated. In vitro synthesis of trans-arachidonic acid isoforms suggested that thiol radicals formed by UVA treatment may be responsible for the INTERCEPT-specific effects observed in platelet concentrates. It is reasonable to assume that UVA-induced molecules may have specific biological effects which need to be further investigated.

Chronic social defeat stress increases the amounts of 12-lipoxygenase lipid metabolites in the nucleus accumbens of stress-resilient mice.

Severe and prolonged social stress induces mood and cognitive dysfunctions and precipitates major depression. Neuroinflammation has been associated with chronic stress and depression. Rodent studies showed crucial roles of a few inflammation-related lipid mediators for chronic stress-induced depressive-like behaviors. Despite an increasing number of lipid mediators identified, systematic analyses of synthetic pathways of lipid mediators in chronic stress models have not been performed. Using LC-MS/MS, here we examined the effects of chronic social defeat stress on multiple synthetic pathways of lipid mediators in brain regions associated with stress susceptibility in mice. Chronic social defeat stress increased the amounts of 12-lipoxygenase (LOX) metabolites, 12-HETE and 12-HEPE, specifically in the nucleus accumbens 1 week, but not immediately, after the last stress exposure. The increase was larger in stress-resilient mice than stress-susceptible mice. The S isomer of 12-HETE was selectively increased in amount, indicating the role of 12S-LOX activity. Among the enzymes known to have 12S-LOX activity, only Alox12 mRNA was reliably detected in the brain and enriched in brain endothelial cells. These findings suggest that chronic social stress induces a late increase in the amounts of 12S-LOX metabolites derived from the brain vasculature in the nucleus accumbens in a manner associated with stress resilience.

Key Role of 12-Lipoxygenase and Its Metabolite 12-Hydroxyeicosatetraenoic Acid (12-HETE) in Diabetic Retinopathy.

PURPOSE: Abnormal lipid metabolism has been proved to be implicated in the complex pathogenesis of diabetic retinopathy (DR). 12-lipoxygenase (12-LOX) is a member of lipoxygenase family responsible for the oxygenation of cellular polyunsaturated fatty acids to produce lipid mediators which modulate cell inflammation. This review explores the role of 12-lipoxygenase and its products in the pathogenesis of DR. METHODS: A comprehensive medical literature search was conducted on PubMed till September 2021. RESULTS: Emerging evidence has demonstrated that 12-LOX and its main product 12- hydroxyeicosatetraenoic acid (12-HETE) activate retinal cells, especially retinal vascular endothelial cells, through the activation of NADPH oxidase and the subsequent generation of reactive oxygen species (ROS), mediating multiple pathological changes during DR. Genetic deletion or pharmacological inhibition models of 12-LOX in mice show protection from DR. CONCLUSION: 12-LOX and its product 12-HETE take important part in DR pathogenesis and show their potential as future therapeutic targets for DR. Further studies are needed on the specific mechanism including 12-LOX pathway related molecules, 12-HETE receptors and downstream signaling pathways.

Oxylipin metabolism is controlled by mitochondrial ß-oxidation during bacterial inflammation.

Oxylipins are potent biological mediators requiring strict control, but how they are removed en masse during infection and inflammation is unknown. Here we show that lipopolysaccharide (LPS) dynamically enhances oxylipin removal via mitochondrial ß-oxidation. Specifically, genetic or pharmacological targeting of carnitine palmitoyl transferase 1 (CPT1), a mitochondrial importer of fatty acids, reveal that many oxylipins are removed by this protein during inflammation in vitro and in vivo. Using stable isotope-tracing lipidomics, we find secretion-reuptake recycling for 12-HETE and its intermediate metabolites. Meanwhile, oxylipin ß-oxidation is uncoupled from oxidative phosphorylation, thus not contributing to energy generation. Testing for genetic control checkpoints, transcriptional interrogation of human neonatal sepsis finds upregulation of many genes involved in mitochondrial removal of long-chain fatty acyls, such as ACSL1,3,4, ACADVL, CPT1B, CPT2 and HADHB. Also, ACSL1/Acsl1 upregulation is consistently observed following the treatment of human/murine macrophages with LPS and IFN-γ. Last, dampening oxylipin levels by ß-oxidation is suggested to impact on their regulation of leukocyte functions. In summary, we propose mitochondrial ß-oxidation as a regulatory metabolic checkpoint for oxylipins during inflammation.

Development of heart failure with preserved ejection fraction in type 2 diabetic mice is ameliorated by preserving vascular function.

AIMS: Heart failure with preserved ejection fraction (HFpEF) is associated with endothelial dysfunction and is frequent in people with type 2 diabetes mellitus. In diabetic patients, increased levels of the eicosanoid 12-hydroxyeicosatetraenoic acid (12-HETE) are linked to vascular dysfunction. Here, we aimed to identify the importance of 12-HETE in type 2 diabetic patients exhibiting diastolic dysfunction, and mice exhibiting HFpEF and whether targeting 12-HETE is a means to ameliorate HFpEF progression by improving vascular function in diabetes. MATERIAL AND METHODS: Subjects with diagnosed type 2 diabetes mellitus and reported diastolic dysfunction or healthy controls were recruited and 12(S)-HETE levels determined by ELISA. 12(S)-HETE levels were determined in type 2 diabetic, leptin receptor deficient mice (LepRdb/db) and HFpEF verified by echocardiography. Mitochondrial function, endothelial function and capillary density were assessed using Seahorse technique, pressure myography and immunohistochemistry in LepRdb/db or non-diabetic littermate controls. 12/15Lo generation was inhibited using ML351 and 12(S)-HETE action by using the V1-cal peptide. KEY FINDINGS: Endothelium-dependent vasodilation and mitochondrial functional capacity both improved in response to either application of ML351 or the V1-cal peptide. Correlating to improved vascular function, mice treated with either pharmacological agent exhibited improved diastolic filling and left ventricular relaxation that correlated with increased myocardial capillary density. SIGNIFICANCE: Our results suggest that 12-HETE may serve as a biomarker indicating endothelial dysfunction and the resulting cardiovascular consequences such as HFpEF in type 2 diabetic patients. Antagonizing 12-HETE is a potent means to causally control HFpEF development and progression in type 2 diabetes by preserving vascular function.

Unraveling the Role of 12- and 20- HETE in Cardiac Pathophysiology: G-Protein-Coupled Receptors, Pharmacological Inhibitors, and Transgenic Approaches.

ABSTRACT: Arachidonic acid-derived lipid mediators play crucial roles in the development and progression of cardiovascular diseases. Eicosanoid metabolites generated by lipoxygenases and cytochrome P450 enzymes produce several classes of molecules, including the epoxyeicosatrienoic acid (EET) and hydroxyeicosatetraenoic acids (HETE) family of bioactive lipids. In general, the cardioprotective effects of EETs have been documented across a number of cardiac diseases. In contrast, members of the HETE family have been shown to contribute to the pathogenesis of ischemic cardiac disease, maladaptive cardiac hypertrophy, and heart failure. The net effect of 12(S)- and 20-HETE depends upon the relative amounts generated, ratio of HETEs:EETs produced, timing of synthesis, as well as cellular and subcellular mechanisms activated by each respective metabolite. HETEs are synthesized by and affect multiple cell types within the myocardium. Moreover, cytochrome P450-derived and lipoxygenase- derived metabolites have been shown to directly influence cardiac myocyte growth and the regulation of cardiac fibroblasts. The mechanistic data uncovered thus far have employed the use of enzyme inhibitors, HETE antagonists, and the genetic manipulation of lipid-producing enzymes and their respective receptors, all of which influence a complex network of outcomes that complicate data interpretation. This review will summarize and integrate recent findings on the role of 12(S)-/20-HETE in cardiac diseases.

Evidence that ABC transporter-mediated autocrine export of an eicosanoid signaling molecule enhances germ cell chemotaxis in the colonial tunicate Botryllus schlosseri.

The colonial ascidian Botryllus schlosseri regenerates the germline during repeated cycles of asexual reproduction. Germline stem cells (GSCs) circulate in the blood and migrate to new germline niches as they develop and this homing process is directed by a Sphigosine-1-Phosphate (S1P) gradient. Here, we find that inhibition of ABC transporter activity reduces migration of GSCs towards low concentrations of S1P in vitro In addition, inhibiting phospholipase A2 (PLA2) or lipoxygenase (Lox) blocks chemotaxis towards low concentrations of S1P. These effects can be rescued by addition of the 12-Lox product 12-S-HETE. Blocking ABC transporter, PLA2 or 12-Lox activity also inhibits homing of germ cells in vivo Using a live-imaging chemotaxis assay in a 3D matrix, we show that a shallow gradient of 12-S-HETE enhances chemotaxis towards low concentrations of S1P and stimulates motility. A potential homolog of the human receptor for 12-S-HETE, gpr31, is expressed on GSCs and differentiating vasa+ germ cells. These results suggest that 12-S-HETE might be an autocrine signaling molecule exported by ABC transporters that enhances chemotaxis in GSCs migrating towards low concentrations of S1P.

Cytochrome P450 1A1 enhances inflammatory responses and impedes phagocytosis of bacteria in macrophages during sepsis.

The hydroxylase cytochrome P450 1A1 (CYP1A1) is regulated by the inflammation-limiting aryl hydrocarbon receptor (AhR), but CYP1A1 immune functions remain unclear. We observed CYP1A1 overexpression in peritoneal macrophages (PMs) isolated from mice following LPS or heat-killed Escherichia. coli (E. coli) challenge. CYP1A1 overexpression augmented TNF-α and IL-6 production in RAW264.7 cells (RAW) by enhancing JNK/AP-1 signalling. CYP1A1 overexpression also promoted 12S-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (12(S)-HETE) production in activated RAW, while a 12(S)-HETE antibody attenuated and 12(S)-HETE alone induced inflammatory responses. Macrophages harbouring hydroxylase-deficient CYP1A1 demonstrated reduced 12(S)-HETE generation and LPS-induced TNF-α/IL-6 secretion. CYP1A1 overexpression also impaired phagocytosis of bacteria via decreasing the expression of scavenger receptor A (SR-A) in PMs. Mice injected with CYP1A1-overexpressing PMs were more susceptible to CLP- or E. coli-induced mortality and bacteria invading, while Rhapontigenin, a selective CYP1A1 inhibitor, improved survival and bacteria clearance of mice in sepsis. CYP1A1 and 12(S)-HETE were also elevated in monocytes and plasma of septic patients and positively correlated with SOFA scores. Macrophage CYP1A1 disruption could be a promising strategy for treating sepsis. Video abstract.

12-Lipoxygenase as a key pharmacological target in the pathogenesis of diabetic nephropathy.

The characteristic features of diabetic nephropathy include thickening of the glomerular basement membranes, expansion of mesangium, and appearance of albumin in the urine (microalbuminuria and macroalbuminuria). Experimental studies have documented that 12-lipoxygenase (12-LOX) and its metabolite 12(S)-hydroxyeicosatetraenoic acid (HETE) play an important role in the pathogenesis of diabetic nephropathy. 12(S)-HETE may work in association with angiotensin II and transforming growth factor- ß (TGF-ß) reciprocally to induce fibrotic changes in the diabetic kidneys. The fibrotic actions of angiotensin II on the kidneys are mediated indirectly through an increase in the synthesis of 12(S)-HETE. Conversely, 12(S)-HETE may also enhance the actions of angiotensin II by upregulating the expression of AT1 receptors on the glomerulus, mesangium, and podocytes. 12(S)-HETE may also cross-talk with TGF-ß in a reciprocal manner to induce the fibrotic changes in the diabetic kidney. 12(S)-HETE-triggered signaling pathways may involve activation of p38 mitogen-activated protein (p38MAP) kinase, increase in cAMP-responsive element-binding protein (CREB) transcriptional activity, epigenetic changes involving histone methylation through an increase in histone methyltransferase activity along with an upregulation of cyclin-kinase inhibitors including, p16, p21, and p27. The present review discusses the role of 12-LOX in the pathogenesis of diabetic nephropathy along with the possible mechanisms.

12-LOX catalyzes the oxidation of 2-arachidonoyl-lysolipids in platelets generating eicosanoid-lysolipids that are attenuated by iPLA2γ knockout.

The canonical pathway of eicosanoid production in most mammalian cells is initiated by phospholipase A2-mediated release of arachidonic acid, followed by its enzymatic oxidation resulting in a vast array of eicosanoid products. However, recent work has demonstrated that the major phospholipase in mitochondria, iPLA2γ (patatin-like phospholipase domain containing 8 (PNPLA8)), possesses sn-1 specificity, with polyunsaturated fatty acids at the sn-2 position generating polyunsaturated sn-2-acyl lysophospholipids. Through strategic chemical derivatization, chiral chromatographic separation, and multistage tandem MS, here we first demonstrate that human platelet-type 12-lipoxygenase (12-LOX) can directly catalyze the regioselective and stereospecific oxidation of 2-arachidonoyl-lysophosphatidylcholine (2-AA-LPC) and 2-arachidonoyl-lysophosphatidylethanolamine (2-AA-LPE). Next, we identified these two eicosanoid-lysophospholipids in murine myocardium and in isolated platelets. Moreover, we observed robust increases in 2-AA-LPC, 2-AA-LPE, and their downstream 12-LOX oxidation products, 12(S)-HETE-LPC and 12(S)-HETE-LPE, in calcium ionophore (A23187)-stimulated murine platelets. Mechanistically, genetic ablation of iPLA2γ markedly decreased the calcium-stimulated production of 2-AA-LPC, 2-AA-LPE, and 12-HETE-lysophospholipids in mouse platelets. Importantly, a potent and selective 12-LOX inhibitor, ML355, significantly inhibited the production of 12-HETE-LPC and 12-HETE-LPE in activated platelets. Furthermore, we found that aging is accompanied by significant changes in 12-HETE-LPC in murine serum that were also markedly attenuated by iPLA2γ genetic ablation. Collectively, these results identify previously unknown iPLA2γ-initiated signaling pathways mediated by direct 12-LOX oxidation of 2-AA-LPC and 2-AA-LPE. This oxidation generates previously unrecognized eicosanoid-lysophospholipids that may serve as biomarkers for age-related diseases and could potentially be used as targets in therapeutic interventions.

12-HETE is a regulator of PGE2 production via COX-2 expression induced by a snake venom group IIA phospholipase A2 in isolated peritoneal macrophages.

The snake venom miotoxin (MT)-III is a group IIA secreted phospholipase A2 (sPLA2) with pro-inflammatory activities. Previous studies have demonstrated that MT-III has the ability to stimulate macrophages to release inflammatory lipid mediators derived from arachidonic acid metabolism. Among them, we highlight prostaglandin (PG)E2 produced by the cyclooxygenase (COX)-2 pathway, through activation of nuclear factor (NF)-κB. However, the mechanisms coordinating this process are not fully understood. This study investigates the regulatory mechanisms exerted by other groups of bioactive eicosanoids derived from 12-lipoxygenase (12-LO), in particular 12-hydroxyeicosatetraenoic (12-HETE), on group IIA sPLA2-induced (i) PGE2 release, (ii) COX-2 expression, and (iii) activation of signaling pathways p38 mitogen-activated protein kinases(p38MAPK), protein C kinase (PKC), extracellular signal-regulated kinase 1/2 (ERK1/2), and NF-κB. Stimulation of macrophages with group IIA sPLA2 resulted in release of 12-HETE without modification of 12-LO protein levels. Pre-treatment of these cells with baicalein, a 12-LO inhibitor, decreased the sPLA2-induced PGE2 production, significantly reduced COX-2 expression, and inhibited sPLA2-induced ERK; however, it did not affect p38MAPK or PKC phosphorylation. In turn, sPLA2-induced PGE2 release and COX-2 expression, but not NF-κB activation, was attenuated by pre-treating macrophages with PD98059 an inhibitor of ERK1/2. These results suggest that, in macrophages, group IIA sPLA2-induced PGE2 release and COX-2 protein expression are distinctly mediated through 12-HETE followed by ERK1/2 pathway activation, independently of NF-κB activation. These findings highlight an as yet undescribed mechanism by which 12-HETE regulates one of the distinct signaling pathways for snake venom group IIA sPLA2-induced PGE2 release and COX-2 expression in macrophages.

Ischemia reperfusion injury promotes recurrence of hepatocellular carcinoma in fatty liver via ALOX12-12HETE-GPR31 signaling axis.

BACKGROUND: Ischemia reperfusion injury (IRI) has been shown to increase the risk of tumor recurrence after liver surgery. Also, nonalcoholic fatty liver disease (NAFLD) is associated with increased HCC recurrence. ALOX12-12-HETE pathway is activated both in liver IRI and NASH. Also, ALOX12-12-HETE has been shown to mediate tumorigenesis and progression. Therefore, our study aims to investigate whether the ALOX12-12-HETE-GPR31 pathway involved in IRI induced HCC recurrence in NAFLD. METHODS: HCC mouse model was used to mimic the HCC recurrence in NAFLD. Western Blot, qPCR, Elisa and Immunofluorescence analysis were conducted to evaluate the changes of multiple signaling pathways during HCC recurrence, including ALOX12-12-HETE axis, EMT, MMPs and PI3K/AKT/NF-κB signaling pathway. We also measured the expression and functional changes of GPR31 by siRNA. RESULTS: ALOX12-12-HETE pathway was activated in liver IRI and its activation was further enhanced in NAFLD, which induced more severe HCC recurrence in fatty livers than normal livers. Inhibition of ALOX12-12-HETE by ML355 reduced the HCC recurrence in fatty livers. In vitro studies showed that 12-HETE increased the expression of GPR31 and induced epithelial-mesenchymal transition (EMT) and matrix metalloprotein (MMPs) by activating PI3K/AKT/NF-κB pathway. Furthermore, knockdown of GPR31 in cancer cells inhibited the HCC recurrence in NAFLD. CONCLUSIONS: ALOX12-12-HETE-GPR31 played an important role in HCC recurrence and might be a potential therapeutic target to reduce HCC recurrence after surgery in fatty livers.

Preferential Incorporation of Administered Eicosapentaenoic Acid Into Thin-Cap Atherosclerotic Plaques.

OBJECTIVE: n-3 polyunsaturated fatty acids, especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), have beneficial effects on atherosclerosis. Although specific salutary actions have been reported, the detailed distribution of n-3 polyunsaturated fatty acids in plaque and their relevance in disease progression are unclear. Our aim was to assess the pharmacodynamics of EPA and DHA and their metabolites in atherosclerotic plaques. Approach and Results: Apolipoprotein E-deficient (Apoe-/-) mice were fed a Western diet supplemented with EPA (1%, w/w) or DHA (1%, w/w) for 3 weeks. Imaging mass spectrometry analyses were performed in the aortic root and arch of the Apoe-/- mice to evaluate the distribution of EPA, DHA, their metabolites and the lipids containing EPA or DHA in the plaques. Liquid chromatography-mass spectrometry and histological analysis were also performed. The intima-media thickness of atherosclerotic plaque decreased in plaques containing free EPA and EPAs attached with several lipids. EPA was distributed more densely in the thin-cap plaques than in the thick-cap plaques, while DHA was more evenly distributed. In the aortic root, the distribution of total EPA level and cholesteryl esters containing EPA followed a concentration gradient from the vascular endothelium to the media. In the aortic arch, free EPA and 12-hydroxy-EPA colocalized with M2 macrophage. CONCLUSIONS: Administered EPA tends to be incorporated from the vascular lumen side and preferentially taken into the thin-cap plaque.