The Effects of 16-HETE Enantiomers on Hypertrophic Markers in Human Fetal Ventricular Cardiomyocytes, RL-14 Cells.

BACKGROUND: Cytochrome P450 (CYP) metabolizes arachidonic acid to produce bioactive metabolites such as EETs and HETEs: mid-chain, subterminal, and terminal HETEs. Recent studies have revealed the role of CYP1B1 and its associated cardiotoxic mid-chain HETE metabolites in developing cardiac hypertrophy and heart failure. Subterminal HETEs have also been involved in various physiological and pathophysiological processes; however, their role in cardiac hypertrophy has not been fully defined. OBJECTIVE: The objective of the current study is to determine the possible effect of subterminal HETEs, R and S enantiomers of 16-HETE, on CYP1B1 expression in vitro using human cardiomyocytes RL-14 cells. METHODS: In the study, RL14 cell line was treated with vehicle and either of the 16-HETE enantiomers for 24 h. Subsequently, the following markers were assessed: cell viability, cellular size, hypertrophic markers, CYP1B1 gene expression (at mRNA, protein, and activity levels), luciferase activity, and CYP1B1 mRNA and protein half-lives. RESULTS: The results of the study showed that 16-HETE enantiomers significantly increased hypertrophic markers and upregulated CYP1B1 mRNA and protein expressions in RL-14 cell line. The upregulation of CYP1B1 by 16-HETE enantiomers occurs via a transcriptional mechanism as evidenced by transcriptional induction and luciferase reporter assay. Furthermore, neither post-transcriptional nor post-translational modification was involved in such modulation since there was no change in CYP1B1 mRNA and protein stabilities upon treatment with 16-HETE enantiomers. CONCLUSION: The current study provides the first evidence that 16R-HETE and 16S-HETE increase CYP1B1 gene expression through a transcriptional mechanism.

17-(R/S)-hydroxyeicosatetraenoic acid (HETE) induces cardiac hypertrophy through the CYP1B1 in enantioselective manners.

Cardiac cellular hypertrophy is the increase in the size of individual cardiac cells. Cytochrome P450 1B1 (CYP1B1) is an extrahepatic inducible enzyme that is associated with toxicity, including cardiotoxicity. We previously reported that 19-hydroxyeicosatetraenoic acid (19-HETE) inhibited CYP1B1 and prevented cardiac hypertrophy in enantioselective manner. Therefore, our aim is to investigate the effect of 17-HETE enantiomers on cardiac hypertrophy and CYP1B1. Human adult cardiomyocyte (AC16) cells were treated with 17-HETE enantiomers (20 µM); cellular hypertrophy was evaluated by cell surface area and cardiac hypertrophy markers. In addition, CYP1B1 gene, protein and activity were assessed. Human recombinant CYP1B1 and heart microsomes of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-treated rats were incubated with 17-HETE enantiomers (10-80 nM). Our results demonstrated that 17-HETE induced cellular hypertrophy, which is manifested by increase in cell surface area and cardiac hypertrophy markers. 17-HETE enantiomers allosterically activated CYP1B1 and selectively upregulated CYP1B1 gene and protein expression in AC16 cells at uM range. In addition, CYP1B1 was allosterically activated by 17-HETE enantiomers at nM range in recombinant CYP1B1 and heart microsomes. In conclusion, 17-HETE acts as an autocrine mediator, leading to the cardiac hypertrophy through induction of CYP1B1 activity in the heart.

20-Hydroxyeicosatetraenoic acid (20-HETE): Bioactions, receptors, vascular function, cardiometabolic disease and beyond.

Vascular function is dynamically regulated and dependent on a bevy of cell types and factors that work in concert across the vasculature. The vasoactive eicosanoid, 20-Hydroxyeicosatetraenoic acid (20-HETE) is a key player in this system influencing the sensitivity of the vasculature to constrictor stimuli, regulating endothelial function, and influencing the renin angiotensin system (RAS), as well as being a driver of vascular remodeling independent of blood pressure elevations. Several of these bioactions are accomplished through the ligand-receptor pairing between 20-HETE and its high-affinity receptor, GPR75. This 20-HETE axis is at the root of various vascular pathologies and processes including ischemia induced angiogenesis, arteriogenesis, septic shock, hypertension, atherosclerosis, myocardial infarction and cardiometabolic diseases including diabetes and insulin resistance. Pharmacologically, several preclinical tools have been developed to disrupt the 20-HETE axis including 20-HETE synthesis inhibitors (DDMS and HET0016), synthetic 20-HETE agonist analogues (20-5,14-HEDE and 20-5,14-HEDGE) and 20-HETE receptor blockers (AAA and 20-SOLA). Systemic or cell-specific therapeutic targeting of the 20-HETE-GPR75 axis continues to be an invaluable approach as studies examine the molecular underpinnings activated by 20-HETE under various physiological settings. In particular, the development and characterization of 20-HETE receptor blockers look to be a promising new class of compounds that can provide a considerable benefit to patients suffering from these cardiovascular pathologies.

Unsaturated oxidated fatty acid 12(S)-HETE attenuates TNF-α expression in TNF-α/IFN-γ-stimulated human keratinocytes.

Chronic skin inflammatory diseases are associated with abnormal immune responses characterized by skin barrier dysfunction. Keratinocytes participate in immune homeostasis regulated by immune cells. Immune homeostasis dysfunction contributes to the pathogenesis of skin diseases mediated by pro-inflammatory cytokines and chemokines, such as tumor necrosis factor (TNF)-α, which are produced by activated keratinocytes. 12(S)-Hydroxy eicosatetraenoic acid [12(S)-HETE], an arachidonic acid metabolite, has anti-inflammatory properties. However, the role of 12(S)-HETE in chronic skin inflammatory diseases has not been elucidated yet. In this study, we investigated the effect of 12(S)-HETE on TNF-α/interferon (IFN)-γ-induced pro-inflammatory cytokine and chemokine expression. Our data showed that 12(S)-HETE modulates TNF-α mRNA and protein expression in TNF-α-/IFN-γ-treated human keratinocytes. Molecular docking analyses demonstrated that 12(S)-HETE bound to extracellular signal-regulated kinase (ERK)1/2, thus preventing ERK activation and downregulating phosphorylated ERK expression. We also demonstrated that 12(S)-HETE treatment inhibited IκB and ERK phosphorylation and nuclear factor (NF)-κB, p65/p50, and CCAAT/enhancerbindingproteinß (C/EBPß) translocation. Overall, our results showed that 12(S)-HETE attenuated TNF-α expression and secretion by inhibiting the mitogen-activated protein kinase ERK/NF-κB and C/EBPß signaling pathways. Overall, these results suggest that 12(S)-HETE effectively resolved TNF-α-induced inflammation.

Fiber-like Action of d-Fagomine on the Gut Microbiota and Body Weight of Healthy Rats.

The goal of this work is to explore if the changes induced by d-fagomine in the gut microbiota are compatible with its effect on body weight and inflammation markers in rats. Methods: Sprague Dawley rats were fed a standard diet supplemented with d-fagomine (or not, for comparison) for 6 months. The variables measured were body weight, plasma mediators of inflammation (hydroxyeicosatetraenoic acids, leukotriene B4, and IL-6), and the concentration of acetic acid in feces and plasma. The composition and diversities of microbiota in cecal content and feces were estimated using 16S rRNA metabarcoding and high-throughput sequencing. We found that after just 6 weeks of intake d-fagomine significantly reduced body weight gain, increased the plasma acetate concentration, and reduced the plasma concentration of the pro-inflammatory biomarkers' leukotriene B4, interleukin 6 and 12 hydroxyeicosatetraenoic acids. These changes were associated with a significantly increased prevalence of Bacteroides and Prevotella feces and increased Bacteroides, Prevotella, Clostridium, and Dysgonomonas while reducing Anaerofilum, Blautia, and Oribacterium in cecal content. In conclusion, d-fagomine induced changes in the composition and diversity of gut microbiota similar to those elicited by dietary fiber and compatible with its anti-inflammatory and body-weight-reducing effects.

Identification of a Feed-Forward Loop Between 15(S)-HETE and PGE2 in Human Amnion at Parturition.

Human parturition is associated with massive arachidonic acid (AA) mobilization in the amnion, indicating that large amounts of AA-derived eicosanoids are required for parturition. Prostaglandin E2 (PGE2) synthesized from the cyclooxygenase (COX) pathway is the best characterized AA-derived eicosanoid in the amnion which plays a pivotal role in parturition. The existence of any other pivotal AA-derived eicosanoids involved in parturition remains elusive. Here, we screened such eicosanoids in human amnion tissue with AA-targeted metabolomics and studied their role and synthesis in parturition by using human amnion fibroblasts and a mouse model. We found that lipoxygenase (ALOX) pathway-derived 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE) and its synthetic enzymes ALOX15 and ALOX15B were significantly increased in human amnion at parturition. Although 15(S)-HETE is ineffective on its own, it potently potentiated the activation of NF-κB by inflammatory mediators including lipopolysaccharide, interleukin-1ß, and serum amyloid A1, resulting in the amplification of COX-2 expression and PGE2 production in amnion fibroblasts. In turn, we determined that PGE2 induced ALOX15/15B expression and 15(S)-HETE production through its EP2 receptor-coupled PKA pathway, thereby forming a feed-forward loop between 15(S)-HETE and PGE2 production in the amnion at parturition. Our studies in pregnant mice showed that 15(S)-HETE injection induced preterm birth with increased COX-2 and PGE2 abundance in the fetal membranes and placenta. Conclusively, 15(S)-HETE is identified as another crucial parturition-pertinent AA-derived eicosanoid in the amnion, which may form a feed-forward loop with PGE2 in parturition. Interruption of this feed-forward loop may be of therapeutic value for the treatment of preterm birth.

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.

Caloric restriction reduces the pro-inflammatory eicosanoid 20-hydroxyeicosatetraenoic acid to protect from acute kidney injury.

Acute kidney injury is a frequent complication in the clinical setting and associated with significant morbidity and mortality. Preconditioning with short-term caloric restriction is highly protective against kidney injury in rodent ischemia reperfusion injury models. However, the underlying mechanisms are unknown hampering clinical translation. Here, we examined the molecular basis of caloric restriction-mediated protection to elucidate the principles of kidney stress resistance. Analysis of an RNAseq dataset after caloric restriction identified Cyp4a12a, a cytochrome exclusively expressed in male mice, to be strongly downregulated after caloric restriction. Kidney ischemia reperfusion injury robustly induced acute kidney injury in male mice and this damage could be markedly attenuated by pretreatment with caloric restriction. In females, damage was significantly less pronounced and preconditioning with caloric restriction had only little effect. Tissue concentrations of the metabolic product of Cyp4a12a, 20-hydroxyeicosatetraenoic acid (20-HETE), were found to be significantly reduced by caloric restriction. Conversely, intraperitoneal supplementation of 20-HETE in preconditioned males partly abrogated the protective potential of caloric restriction. Interestingly, this effect was accompanied by a partial reversal of caloric restriction--induced changes in protein but not RNA expression pointing towards inflammation, endoplasmic reticulum stress and lipid metabolism. Thus, our findings provide an insight into the mechanisms underlying kidney protection by caloric restriction. Hence, understanding the mediators of preconditioning is an important prerequisite for moving towards translation to the clinical setting.

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.

20-hydroxyeicosatetraenoic acid (20-HETE) is a pivotal endogenous ligand for TRPV1-mediated neurogenic inflammation in the skin.

BACKGROUND AND PURPOSE: Transient receptor potential cation channel subfamily V member 1 (TRPV1) is localized to sensory C-fibres and its opening leads to membrane depolarization, resulting in neuropeptide release and neurogenic inflammation. However, the identity of the endogenous activator of TRPV1 in this setting is unknown. The arachidonic acid metabolites 12-hydroperoxyeicosatetraenoyl acid (12-HpETE) and 20-hydroxyeicosatetraenoic acid (20-HETE) have emerged as potential endogenous activators of TRPV1. However, whether these lipids underlie TRPV1-mediated neurogenic inflammation remains unknown. EXPERIMENTAL APPROACH: We analysed human cantharidin-induced blister samples and inflammatory responses in TRPV1 transgenic mice. KEY RESULTS: In a human cantharidin-blister model, the potent TRPV1 activators 20-HETE but not 12-HETE (stable metabolite of 12-HpETE) correlated with arachidonic acid levels. Similarly, in mice, levels of 20-HETE (but not 12-HETE) and arachidonic acid were strongly positively correlated within the inflammatory milieu. Furthermore, LPS-induced oedema formation and neutrophil recruitment were substantially and significantly attenuated by pharmacological block or genetic deletion of TRPV1 channels, inhibition of 20-HETE formation or SP receptor neurokinin 1 (NK1 ) blockade. LPS treatment also increased cytochrome P450 ω-hydroxylase gene expression, the enzyme responsible for 20-HETE production. CONCLUSION AND IMPLICATIONS: Taken together, our findings suggest that endogenously generated 20-HETE activates TRPV1 causing C-fibre activation and consequent oedema formation. These findings identify a novel pathway that may be useful in the therapeutics of diseases/conditions characterized by a prominent neurogenic inflammation, as in several skin diseases.

Disruption of pulmonary resolution mediators contribute to exacerbated silver nanoparticle-induced acute inflammation in a metabolic syndrome mouse model.

Pre-existing conditions modulate sensitivity to numerous xenobiotic exposures such as air pollution. Specifically, individuals suffering from metabolic syndrome (MetS) demonstrate enhanced acute inflammatory responses following particulate matter inhalation. The mechanisms associated with these exacerbated inflammatory responses are unknown, impairing interventional strategies and our understanding of susceptible populations. We hypothesize MetS-associated lipid dysregulation influences mediators of inflammatory resolution signaling contributing to increased acute pulmonary toxicity. To evaluate this hypothesis, healthy and MetS mouse models were treated with either 18-hydroxy eicosapentaenoic acid (18-HEPE), 14-hydroxy docosahexaenoic acid (14-HDHA), 17-hydroxy docosahexaenoic acid (17-HDHA), or saline (control) via intraperitoneal injection prior to oropharyngeal aspiration of silver nanoparticles (AgNP). In mice receiving saline treatment, AgNP exposure resulted in an acute pulmonary inflammatory response that was exacerbated in MetS mice. A targeted lipid assessment demonstrated 18-HEPE, 14-HDHA, and 17-HDHA treatments altered lung levels of specialized pro-resolving lipid mediators (SPMs). 14-HDHA and 17-HDHA treatments more efficiently reduced the exacerbated acute inflammatory response in AgNP exposed MetS mice as compared to 18-HEPE. This included decreased neutrophilic influx, diminished induction of inflammatory cytokines/chemokines, and reduced alterations in SPMs. Examination of SPM receptors determined baseline reductions in MetS mice compared to healthy as well as decreases due to AgNP exposure. Overall, these results demonstrate AgNP exposure disrupts inflammatory resolution, specifically 14-HDHA and 17-HDHA derived SPMs, in MetS contributing to exacerbated acute inflammatory responses. Our findings identify a potential mechanism responsible for enhanced susceptibility in MetS that can be targeted for interventional therapeutic approaches.

Novel Synthetic Analogues of 19(S/R)-Hydroxyeicosatetraenoic Acid Exhibit Noncompetitive Inhibitory Effect on the Activity of Cytochrome P450 1A1 and 1B1.

BACKGROUND AND OBJECTIVES: Cytochrome P450 (CYP) 1A1 and CYP1B1 enzymes play a significant role in the pathogenesis of cancer and cardiovascular diseases (CVD) such as cardiac hypertrophy and heart failure. Previously, we have demonstrated that R- and S-enantiomers of 19-hydroxyeicosatetraenoic acid (19-HETE), an arachidonic acid endogenous metabolite, enantioselectively inhibit CYP1B1. The current study was conducted to test the possible inhibitory effect of novel synthetic analogues of R- and S-enantiomers of 19-HETE on the activity of CYP1A1, CYP1A2, and CYP1B1. METHODS: The O-dealkylation rate of 7-ethoxyresorufin (EROD) by recombinant human CYP1A1 and CYP1B1, in addition to the O-dealkylation rate of 7-methoxyresorufin (MROD) by recombinant human CYP1A2, were measured in the absence and presence of varying concentrations (0-40 nM) of the synthetic analogues of 19(R)- and 19(S)-HETE. Also, the possible inhibitory effect of both analogues on the catalytic activity of EROD and MROD, using RL-14 cells and human liver microsomes, was assessed. RESULTS: The results showed that both synthetic analogues of 19(R)- and 19(S)-HETE exhibited direct inhibitory effects on the activity of CYP1A1 and CYP1B1, while they had no significant effect on CYP1A2 activity. Nonlinear regression analysis and comparisons showed that the mode of inhibition for both analogues is noncompetitive inhibition of CYP1A1 and CYP1B1 enzymes. Also, nonlinear regression analysis and Dixon plots showed that the R- and S-analogues have KI values of 15.7 ± 4.4 and 6.1 ± 1.5 nM for CYP1A1 and 26.1 ± 2.9 and 9.1 ± 1.8 nM for CYP1B1, respectively. Moreover, both analogues were able to inhibit EROD and MROD activities in a cell-based assay and human liver microsomes. CONCLUSIONS: Therefore, the synthetic analogues of 19-HETE could be considered as a novel therapeutic approach in the treatment of cancer and CVD.

The anti-inflammatory effects of 15-HETE on osteoarthritis during treadmill exercise.

AIMS: Investigate the involvement of 15-hydroxyeicosatetraenoic acid (15-HETE), an anti-inflammatory molecule, on the beneficial effects of exercise therapy for osteoarthritis (OA). MAIN METHODS: 15-HETE (10 µM, twice a week) and monosodium iodoacetate (MIA) (1 mg) were injected into rat knee joints. Treadmill exercise was applied on OA rat. Primary rat chondrocytes were treated with 15-HETE, LY294002 and interleukin (IL)-1ß. Rats undergo a 1 hour single session treadmill exercise once. 15-HETE levels in the knee joint were evaluated using ELISA after a single session of treadmill exercise on healthy and OA rats. Matrix metalloproteinase (MMP)3, MMP-13, a disintegrin and metalloproteinase with thrombospondin motif (ADAMTS)-5, p-Akt, Akt, and collagen type 2 (COL2) expression were evaluated using RT-PCR and western blotting. OA degree was evaluated using X-ray, scored by Osteoarthritis Research Society International (OARSI) and Mankin scores. COL2 and MMP-13 expression in articular was evaluated using immunohistochemistry. KEY FINDINGS: Medium intensity exercise alleviated OA. 15-HETE levels after exercise was increased. 15-HETE inhibited IL-1ß-induced inflammation in primary chondrocytes and increased p-Akt levels. LY294002 blocked the effect of 15-HETE in vitro. Finally, 15-HETE alleviated cartilage damage, inhibited MMP-13 expression, and increased COL2 expression in joint cartilage tissue. SIGNIFICANCE: Treadmill exercise alleviates OA and increases 15-HETE levels in the knee joint, which suppresses inflammation in chondrocytes via PI3k-Akt signalling in vitro and in vivo.

20-HETE synthesis inhibition attenuates traumatic brain injury-induced mitochondrial dysfunction and neuronal apoptosis via the SIRT1/PGC-1α pathway: A translational study.

OBJECTIVES: 20-hydroxyeicosatetraenoic acid (20-HETE) is a metabolite of arachidonic acid catalysed by cytochrome P450 enzymes and plays an important role in cell death and proliferation. We hypothesized that 20-HETE synthesis inhibition may have protective effects in traumatic brain injury (TBI) and investigated possible underlying molecular mechanisms. MATERIALS AND METHODS: Neurologic deficits, and lesion volume, reactive oxygen species (ROS) levels and cell death as assessed using immunofluorescence staining, transmission electron microscopy and Western blotting were used to determine post-TBI effects of HET0016, an inhibitor of 20-HETE synthesis, and their underlying mechanisms. RESULTS: The level of 20-HETE was found to be increased significantly after TBI in mice. 20-HETE synthesis inhibition reduced neuronal apoptosis, ROS production and damage to mitochondrial structures after TBI. Mechanistically, HET0016 decreased the Drp1 level and increased the expression of Mfn1 and Mfn2 after TBI, indicating a reversal of the abnormal post-TBI mitochondrial dynamics. HET0016 also promoted the restoration of SIRT1 and PGC-1α in vivo, and a SIRT1 activator (SRT1720) reversed the downregulation of SIRT1 and PGC-1α and the abnormal mitochondrial dynamics induced by 20-HETE in vitro. Furthermore, plasma 20-HETE levels were found to be higher in TBI patients with unfavourable neurological outcomes and were correlated with the GOS score. CONCLUSIONS: The inhibition of 20-HETE synthesis represents a novel strategy to mitigate TBI-induced mitochondrial dysfunction and neuronal apoptosis by regulating the SIRT1/PGC-1α pathway.

A paracrine effect of 15 (S)-hydroxyeicosatetraenoic acid revealed in prostaglandin production by human follicular dendritic cell-like cells.

Lipid mediators play active roles in each stage of inflammation under physiological and pathologic conditions. We have investigated the cellular source and functions of several prostanoids in the immune inflammatory responses using follicular dendritic cell (FDC)-like cells. In this study, we report a novel finding on the role of 15(S)- hydroxyeicosatetraenoic acid (HETE). Our observation of 15(S)-HETE uptake by FDC-like cells prompted to hypothesize that 15(S)-HETE might have a regulatory role in the other branch of eicosanoid production. The effects of 15(S)-HETE on COX-2 expression and prostaglandin (PG) production were analyzed by immunoblotting and specific enzyme immunoassays. The addition of 15(S)-HETE resulted in elevated levels of COX-2 expression and PG production. The enhanced PG production was not due to growth stimulation of FDC-like cells since 15(S)-HETE did not modulate FDC-like cell proliferation by the culture period of PG measurement. Peroxisome proliferator-activated receptor gamma (PPARγ) seems to mediate the augmenting activity as the antagonist GW9662 dose- dependently prevented 15(S)-HETE from increasing PG production. In addition, PPARγ protein expression was readily detected in FDC-like cells. These effects of 15(S)-HETE were displayed in the combined addition with IL-1ß. Based on these results, we suggest that 15(S)-HETE is an inflammatory costimulator of FDC acting in a paracrine fashion.

Vascular Lipidomic Profiling of Potential Endogenous Fatty Acid PPAR Ligands Reveals the Coronary Artery as Major Producer of CYP450-Derived Epoxy Fatty Acids.

A number of oxylipins have been described as endogenous PPAR ligands. The very short biological half-lives of oxylipins suggest roles as autocrine or paracrine signaling molecules. While coronary arterial atherosclerosis is the root of myocardial infarction, aortic atherosclerotic plaque formation is a common readout of in vivo atherosclerosis studies in mice. Improved understanding of the compartmentalized sources of oxylipin PPAR ligands will increase our knowledge of the roles of PPAR signaling in diverse vascular tissues. Here, we performed a targeted lipidomic analysis of ex vivo-generated oxylipins from porcine aorta, coronary artery, pulmonary artery and perivascular adipose. Cyclooxygenase (COX)-derived prostanoids were the most abundant detectable oxylipin from all tissues. By contrast, the coronary artery produced significantly higher levels of oxylipins from CYP450 pathways than other tissues. The TLR4 ligand LPS induced prostanoid formation in all vascular tissue tested. The 11-HETE, 15-HETE, and 9-HODE were also induced by LPS from the aorta and pulmonary artery but not coronary artery. Epoxy fatty acid (EpFA) formation was largely unaffected by LPS. The pig CYP2J homologue CYP2J34 was expressed in porcine vascular tissue and primary coronary artery smooth muscle cells (pCASMCs) in culture. Treatment of pCASMCs with LPS induced a robust profile of pro-inflammatory target genes: TNFα, ICAM-1, VCAM-1, MCP-1 and CD40L. The soluble epoxide hydrolase inhibitor TPPU, which prevents the breakdown of endogenous CYP-derived EpFAs, significantly suppressed LPS-induced inflammatory target genes. In conclusion, PPAR-activating oxylipins are produced and regulated in a vascular site-specific manner. The CYP450 pathway is highly active in the coronary artery and capable of providing anti-inflammatory oxylipins that prevent processes of inflammatory vascular disease progression.

Feed­back loops integrating RELA, SOX18 and FAK mediate the break­down of the lymph­endothelial barrier that is triggered by 12(S)­HETE.

Metastatic cancer cells cross endothelial barriers and travel through the blood or lymphatic fluid to pre­metastatic niches, leading to their colonisation. 'S' stereoisomer 12S­hydroxy­5Z,8Z,10E,14Z­eicosatetraenoic acid [12(S)­HETE] is secreted by a variety of cancer cell types and has been indicated to open up these barriers. In the present study, another aspect of the endothelial unlocking mechanism was elucidated. This was achieved by investigating 12(S)­HETE­treated lymph endothelial cells (LECs) with regard to their expression and mutual interaction with v­rel avian reticuloendotheliosis viral oncogene homolog A (RELA), intercellular adhesion molecule 1, SRY­box transcription factor 18 (SOX18), prospero homeobox 1 (PROX1) and focal adhesion kinase (FAK). These key players of LEC retraction, which is a prerequisite for cancer cell transit into vasculature, were analysed using western blot analysis, reverse transcription­quantitative PCR and transfection with small interfering (si)RNA. The silencing of a combination of these signalling and executing molecules using siRNA, or pharmacological inhibition with defactinib and Bay11­7082, extended the mono­culture experiments to co­culture settings using HCT116 colon cancer cell spheroids that were placed on top of LEC monolayers to measure their retraction using the validated 'circular chemorepellent­induced defect' assay. 12(S)­HETE was indicated to induce the upregulation of the RELA/SOX18 feedback loop causing the subsequent phosphorylation of FAK, which fed back to RELA/SOX18. Therefore, 12(S)­HETE was demonstrated to be associated with circuits involving RELA, SOX18 and FAK, which transduced signals causing the retraction of LECs. The FAK­inhibitor defactinib and the NF­κB inhibitor Bay11­7082 attenuated LEC retraction additively, which was similar to the suppression of FAK and PROX1 (the target of SOX18) by the transfection of respective siRNAs. FAK is an effector molecule at the distal end of a pro­metastatic signalling cascade. Therefore, targeting the endothelial­specific activity of FAK through the pathway demonstrated herein may provide a potential therapeutic method to combat cancer dissemination via vascular routes.

Cytochrome P450 1A1 enhances Arginase-1 expression, which reduces LPS-induced mouse peritonitis by targeting JAK1/STAT6.

The polarization of macrophages is critical to inflammation and tissue repair, with unbalanced macrophage polarization associated with critical dysfunctions of the immune system. Cytochrome P450 1A1 (CYP1A1) is a hydroxylase mainly controlled by the inflammation-limiting aryl hydrocarbon receptor (AhR), which plays a critical role in mycoplasma infection, oxidative stress injury, and cancer. Arginase-1 (Arg-1) is a surrogate for polarized alternative macrophages and is important to the production of nitric oxide (NO) by the modulation of arginine. In the present study, we found CYP1A1 to be upregulated in IL-4-stimulated mouse peritoneal macrophages (PMs) and human peripheral blood monocytes. Using CYP1A1-overexpressing RAW264.7 cells (CYP1A1/RAW) we found that CYP1A1 augmented Arg-1 expression by strengthening the activation of the JAK1/STAT6 signaling pathway in macrophages treated with IL-4. 15(S)-HETE, a metabolite of CYP1A1 hydroxylase, was elevated in IL-4-induced CYP1A1/RAW cells. Further, in macrophages, the loss-of-CYP1A1-hydroxylase activity was associated with reduced IL-4-induced Arg-1 expression due to impaired 15(S)-HETE generation. Of importance, CYP1A1 overexpressing macrophages reduced the inflammation associated with LPS-induced peritonitis. Taken together, these findings identified a novel signaling axis, CYP1A1-15(S)-HETE-JAK1-STAT6, that may be a promising target for the proper maintenance of macrophage polarization and may also be a means by which to treat immune-related disease due to macrophage dysfunction.

20-hydroxyeicosatetraenoic acid alters endothelial cell barrier integrity independent of oxidative stress and cell death.

Unregulated inflammation during bovine mastitis is characterized by severe mammary tissue damage with systemic involvement. Vascular dysfunction underlies tissue pathology because of concurrent oxidative stress mediated by several inflammatory mediators. We recently demonstrated increased production of 20-hydroxyeicosatetraenoic acid (20-HETE), a cytochrome P450-derived (CYP) oxylipid that correlated with oxidative stress during severe bovine coliform mastitis. The hypothesis for this study was that 20-HETE-induced oxidative stress disrupts barrier function of endothelial cells. Primary endothelial cells from the bovine aorta were utilized to investigate the effects of 20-HETE on barrier integrity in an in-vitro model of oxidative stress. The effects of various antioxidants on modulating the 20-HETE barrier integrity effects also were investigated. Our results showed that 20-HETE decreased endothelial barrier integrity, which was associated with increased reactive metabolite production and decreased total glutathione. The antioxidant, vitamin E, partially delayed the loss of endothelial resistance upon exposure to 20-HETE but did not prevent complete loss of barrier integrity. The decrease in barrier resistance due to 20-HETE was neither associated with oxidative stress as assessed by oxidative protein or lipid damage nor endothelial cell apoptosis; however, selenium supplementation conferred resistance to loss of barrier integrity suggesting a role for shifts in redox status. Specific mechanisms by which 20-HETE alters vascular barrier integrity require further investigation to identify targets for therapy during inflammatory conditions with enhanced CYP450 activity.

Cytochrome 450 metabolites of arachidonic acid (20-HETE, 11,12-EET and 14,15-EET) promote pheochromocytoma cell growth and tumor associated angiogenesis.

The importance of cytochrome P450 (CYP)-derived arachidonic acid (AA) metabolites, 20-hydroxyeicosatetraenoic acid (20-HETE) and epoxyeicosatrienoic acids (EETs) as tumor growth promotors has already been described in several cancer types. The aim of this study was to evaluate the role of these compounds in the biology of pheochromocytoma/paraganglioma. These tumors originate from chromaffin cells derived from adrenal medulla (pheochromocytomas) or extra-adrenal autonomic paraganglia (paragangliomas), and they represent the most common hereditary endocrine neoplasia. According to mutations in the driver genes, these tumors are divided in two clusters: pseudo-hypoxic and kinase-signaling EETs, but not 20-HETE, exhibited a potent ability to sustain growth in a murine pheochromocytoma cell line (MPC) in vitro, EETs promoted an increase in cell proliferation and a decrease in cell apoptosis. In a mouse model of pheochromocytoma, the inhibition of CYP-mediated AA metabolism using 1-aminobenzotriazol resulted in slower tumor growth, a decreased vascularization, and a lower final volume. Also, the expression of AA-metabolizing CYP monooxygenases was detected in tumor samples from human origin, being their apparent abundance and the production of both metabolites higher in tumors from the kinase-signaling cluster. This is the first evidence of the importance of CYP- derived AA metabolites in the biology and development of pheochromocytoma/paraganglioma tumors.