Effects of sEH inhibition on the eicosanoid and cytokine storms in SARS-CoV-2-infected mice.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection involves an initial viral infection phase followed by a host-response phase that includes an eicosanoid and cytokine storm, lung inflammation and respiratory failure. While vaccination and early anti-viral therapies are effective in preventing or limiting the pathogenic host response, this latter phase is poorly understood with no highly effective treatment options. Inhibitors of soluble epoxide hydrolase (sEH) increase levels of anti-inflammatory molecules called epoxyeicosatrienoic acids (EETs). This study aimed to investigate the impact of sEH inhibition on the host response to SARS-CoV-2 infection in a mouse model with human angiotensin-converting enzyme 2 (ACE2) expression. Mice were infected with SARS-CoV-2 and treated with either vehicle or the sEH inhibitor 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU). At day 5 post-infection, SARS-CoV-2 induced weight loss, clinical signs, a cytokine storm, an eicosanoid storm, and severe lung inflammation with ~50% mortality on days 6-8 post-infection. SARS-CoV-2 infection induced lung expression of phospholipase A2 (PLA2), cyclooxygenase (COX) and lipoxygenase (LOX) pathway genes, while suppressing expression of most cytochrome P450 genes. Treatment with the sEH inhibitor TPPU delayed weight loss but did not alter clinical signs, lung cytokine expression or overall survival of infected mice. Interestingly, TPPU treatment significantly reversed the eicosanoid storm and attenuated viral-induced elevation of 39 fatty acids and oxylipins from COX, LOX and P450 pathways, which suggests the effects at the level of PLA2 activation. The suppression of the eicosanoid storm by TPPU without corresponding changes in lung cytokines, lung inflammation or mortality reveals a surprising dissociation between systemic oxylipin and cytokine signaling pathways during SARS-CoV-2 infection and suggests that the cytokine storm is primarily responsible for morbidity and mortality in this animal model.

Production and Release of Proinflammatory Mediators by the Cockroach Allergen Bla g 1 via a Shared Membrane-Destabilization Mechanism.

The cockroach allergen Bla g 1 encloses an exceptionally large hydrophobic cavity, which allows it to bind and deliver unsaturated fatty acid ligands. Bla g 1-mediated delivery of naturally occurring (nMix) ligands has been shown to destabilize lipid membranes, contributing to its digestive/antiviral functions within the source organism. However, the consequences of this activity on Bla g 1 allergenicity following human exposure remain unknown. In this work, we show that Bla g 1-mediated membrane disruption can induce a proinflammatory immune response in mammalian cells via two complementary pathways. At high concentrations, the cytotoxic activity of Bla g 1 induces the release of proinflammatory cytosolic contents including damage-associated molecular patterns (DAMPs) such as heat-shock Protein-70 (HSP70) and the cytokine interleukin-1 (IL-1ß). Sublytic concentrations of Bla g 1 enhanced the ability of phospholipase A2 (PLA2) to extract and hydrolyze phospholipid substrates from cellular membranes, stimulating the production of free polyunsaturated fatty acids (PUFAs) and various downstream inflammatory lipid mediators. Both of these effects are dependent on the presence of Bla g 1's natural fatty-acid (nMix) ligands with CC50 values corresponding to the concentrations required for membrane destabilization reported in previous studies. Taken together, these results suggest that mechanisms through which Bla g 1-mediated lipid delivery and membrane destabilization could directly contribute to cockroach allergic sensitization.

Disruption of Ephx2 in cardiomyocytes but not endothelial cells improves functional recovery after ischemia-reperfusion in isolated mouse hearts.

Cytochromes P450 metabolize arachidonic acid to epoxyeicosatrienoic acids (EETs) which have numerous effects. After cardiac ischemia, EET-induced coronary vasodilation increases delivery of oxygen/nutrients to the myocardium, and EET-induced signaling protects cardiomyocytes against postischemic mitochondrial damage. Soluble epoxide hydrolase 2 (EPHX2) diminishes the benefits of EETs through hydrolysis to less active dihydroxyeicosatrienoic acids. EPHX2 inhibition or genetic disruption improves recovery of cardiac function after ischemia. Immunohistochemical staining revealed EPHX2 expression in cardiomyocytes and some endothelial cells but little expression in cardiac smooth muscle cells or fibroblasts. To determine specific roles of EPHX2 in cardiac cell types, we generated mice with cell-specific disruption of Ephx2 in endothelial cells (Ephx2fx/fx/Tek-cre) or cardiomyocytes (Ephx2fx/fx/Myh6-cre) to compare to global Ephx2-deficient mice (global Ephx2-/-) and WT (Ephx2fx/fx) mice in expression, EET hydrolase activity, and heart function studies. Most cardiac EPHX2 expression and activity is in cardiomyocytes with substantially less activity in endothelial cells. Ephx2fx/fx/Tek-cre hearts have similar EPHX2 expression, hydrolase activity, and postischemic cardiac function as control Ephx2fx/fx hearts. However, Ephx2fx/fx/Myh6-cre hearts were similar to global Ephx2-/- hearts with significantly diminished EPHX2 expression, decreased hydrolase activity, and enhanced postischemic cardiac function compared to Ephx2fx/fx hearts. During reperfusion, Ephx2fx/fx/Myh6-cre hearts displayed increased ERK activation compared to Ephx2fx/fx hearts, which could be reversed by EEZE treatment. EPHX2 did not regulate coronary vasodilation in this model. We conclude that EPHX2 is primarily expressed in cardiomyocytes where it regulates EET hydrolysis and postischemic cardiac function, whereas endothelial EPHX2 does not play a significant role in these processes.

CYP eicosanoid pathway mediates colon cancer-promoting effects of dietary linoleic acid.

Human and animal studies support that consuming a high level of linoleic acid (LA, 18:2ω-6), an essential fatty acid and key component of the human diet, increases the risk of colon cancer. However, results from human studies have been inconsistent, making it challenging to establish dietary recommendations for optimal LA intake. Given the importance of LA in the human diet, it is crucial to better understand the molecular mechanisms underlying its potential colon cancer-promoting effects. Using LC-MS/MS-based targeted lipidomics, we find that the cytochrome P450 (CYP) monooxygenase pathway is a major pathway for LA metabolism in vivo. Furthermore, CYP monooxygenase is required for the colon cancer-promoting effects of LA, since the LA-rich diet fails to exacerbate colon cancer in CYP monooxygenase-deficient mice. Finally, CYP monooxygenase mediates the pro-cancer effects of LA by converting LA to epoxy octadecenoic acids (EpOMEs), which have potent effects on promoting colon tumorigenesis via gut microbiota-dependent mechanisms. Overall, these results support that CYP monooxygenase-mediated conversion of LA to EpOMEs plays a crucial role in the health effects of LA, establishing a unique mechanistic link between dietary fatty acid intake and cancer risk. These results could help in developing more effective dietary guidelines for optimal LA intake and identifying subpopulations that may be especially vulnerable to LA's negative effects.

Overexpression of Human Soluble Epoxide Hydrolase Exacerbates Coronary Reactive Hyperemia Reduction in Angiotensin-II-Treated Mouse Hearts.

ABSTRACT: Coronary reactive hyperemia (CRH) is impaired in cardiovascular diseases, and angiotensin-II (Ang-II) exacerbates it. However, it is unknown how Ang-II affects CRH in Tie2-sEH Tr (human-sEH-overexpressed) versus wild-type (WT) mice. sEH-overexpression resulted in CRH reduction in Tie2-sEH Tr versus WT. We hypothesized that Ang-II exacerbates CRH reduction in Tie2-sEH Tr versus WT. The Langendorff system measured coronary flow in Tie2-sEH Tr and WT. The hearts were exposed to 15-second ischemia, and CRH was assessed in 10 mice each. Repayment volume was reduced by 40.50% in WT treated with Ang-II versus WT (7.42 ± 0.8 to 4.49 ± 0.8 mL/g) and 48% in Tie2-sEH Tr treated with Ang-II versus Tie2-sEH Tr (5.18 ± 0.4 to 2.68 ± 0.3 mL/g). Ang-II decreased repayment duration by 50% in WT-treated with Ang-II versus WT (2.46 ± 0.5 to 1.24 ± 0.4 minutes) and 54% in Tie2-sEH Tr treated with Ang-II versus Tie2-sEH Tr (1.66 ± 0.4 to 0.76 ± 0.2 minutes). Peak repayment flow was reduced by 11.2% in WT treated with Ang-II versus WT (35.98 ± 0.7 to 32.11 ± 1.4 mL/g) and 4% in Tie2-sEH Tr treated with Ang-II versus Tie2-sEH Tr (32.18 ± 0.6 to 30.89 ± 1.5 mL/g). Furthermore, coronary flow was reduced by 43% in WT treated with Ang-II versus WT (14.2 ± 0.5 to 8.15 ± 0.8 mL/min/g) and 32% in Tie2-sEH Tr treated with Ang-II versus Tie2-sEH Tr (12.1 ± 0.8 to 8.3 ± 1.2 mL/min/g). Moreover, the Ang-II-AT 1 -receptor and CYP4A were increased in Tie2-sEHTr. Our results demonstrate that Ang-II exacerbates CRH reduction in Tie2-sEH Tr mice.

Adam19 Deficiency Impacts Pulmonary Function: Human GWAS Follow-up in a Mouse Knockout Model.

PURPOSE: Over 550 loci have been associated with human pulmonary function in genome-wide association studies (GWAS); however, the causal role of most remains uncertain. Single nucleotide polymorphisms in a disintegrin and metalloprotease domain 19 (ADAM19) are consistently related to pulmonary function in GWAS. Thus, we used a mouse model to investigate the causal link between Adam19 and pulmonary function. METHODS: We created an Adam19 knockout (KO) mouse model and validated the gene targeting using RNA-Seq and RT-qPCR. Mouse body composition was assessed using dual-energy X-ray absorptiometry. Mouse lung function was measured using flexiVent. RESULTS: Contrary to prior publications, the KO was not neonatal lethal. KO mice had lower body weight and shorter tibial length than wild-type (WT) mice. Their body composition revealed lower soft weight, fat weight, and bone mineral content. Adam19 KO had decreased baseline respiratory system elastance, minute work of breathing, tissue damping, tissue elastance, and forced expiratory flow at 50% forced vital capacity but higher FEV0.1 and FVC. Adam19 KO had attenuated tissue damping and tissue elastance in response to methacholine following LPS exposure. Adam19 KO also exhibited attenuated neutrophil extravasation into the airway after LPS administration compared to WT. RNA-Seq analysis of KO and WT lungs identified several differentially expressed genes (Cd300lg, Kpna2, and Pttg1) implicated in lung biology and pathogenesis. Gene set enrichment analysis identified negative enrichment for TNF pathways. CONCLUSION: Our murine findings support a causal role of ADAM19, implicated in human GWAS, in regulating pulmonary function.

Dynamic expression of mucins and the genes controlling mucin-type O-glycosylation within the mouse respiratory system.

The COVID-19 global pandemic has underscored the need to understand how viruses and other pathogens are able to infect and replicate within the respiratory system. Recent studies have highlighted the role of highly O-glycosylated mucins in the protection of the respiratory system as well as how mucin-type O-glycosylation may be able to modify viral infectivity. Therefore, we set out to identify the specific genes controlling mucin-type O-glycosylation throughout the mouse respiratory system as well as determine how their expression and the expression of respiratory mucins is influenced by infection or injury. Here, we show that certain mucins and members of the Galnt family are abundantly expressed in specific respiratory tissues/cells and demonstrate unique patterns of O-glycosylation across diverse respiratory tissues. Moreover, we find that the expression of certain Galnts and mucins is altered during lung infection and injury in experimental mice challenged with infectious agents, toxins, and allergens. Finally, we examine gene expression changes of Galnts and mucins in a mouse model of SARS-CoV-2 infection. Our work provides foundational knowledge regarding the specific expression of Galnt enzyme family members and mucins throughout the respiratory system, and how their expression is altered upon lung infection and injury.

Epoxide hydrolase 3 (Ephx3) gene disruption reduces ceramide linoleate epoxide hydrolysis and impairs skin barrier function.

The mammalian epoxide hydrolase (EPHX)3 is known from in vitro experiments to efficiently hydrolyze the linoleate epoxides 9,10-epoxyoctadecamonoenoic acid (EpOME) and epoxyalcohol 9R,10R-trans-epoxy-11E-13R-hydroxy-octadecenoate to corresponding diols and triols, respectively. Herein we examined the physiological relevance of EPHX3 to hydrolysis of both substrates in vivo. Ephx3-/- mice show no deficiency in EpOME-derived plasma diols, discounting a role for EPHX3 in their formation, whereas epoxyalcohol-derived triols esterified in acylceramides of the epidermal 12R-lipoxygenase pathway are reduced. Although the Ephx3-/- pups appear normal, measurements of transepidermal water loss detected a modest and statistically significant increase compared with the wild-type or heterozygote mice, reflecting a skin barrier impairment that was not evident in the knockouts of mouse microsomal (EPHX1/microsomal epoxide hydrolase) or soluble (EPHX2/sEH). This barrier phenotype in the Ephx3-/- pups was associated with a significant decrease in the covalently bound ceramides in the epidermis (40% reduction, p < 0.05), indicating a corresponding structural impairment in the integrity of the water barrier. Quantitative LC-MS analysis of the esterified linoleate-derived triols in the murine epidermis revealed a marked and isomer-specific reduction (∼85%) in the Ephx3-/- epidermis of the major trihydroxy isomer 9R,10S,13R-trihydroxy-11E-octadecenoate. We conclude that EPHX3 (and not EPHX1 or EPHX2) catalyzes hydrolysis of the 12R-LOX/eLOX3-derived epoxyalcohol esterified in acylceramide and may function to control flux through the alternative and crucial route of metabolism via the dehydrogenation pathway of SDR9C7. Importantly, our findings also identify a functional role for EPHX3 in transformation of a naturally esterified epoxide substrate, pointing to its potential contribution in other tissues.

Soluble epoxide hydrolase deficiency attenuates lipotoxic cardiomyopathy via upregulation of AMPK-mTORC mediated autophagy.

Obesity-driven cardiac lipid accumulation can progress to lipotoxic cardiomyopathy. Soluble epoxide hydrolase (sEH) is the major enzyme that metabolizes epoxyeicosatrienoic acids (EETs), which have biological activity of regulating lipid metabolism. The current study explores the unknown role of sEH deficiency in lipotoxic cardiomyopathy and its underlying mechanism. Wild-type and Ephx2 knock out (sEH KO) C57BL/6 J mice were fed with high-fat diet (HFD) for 24 weeks to induce lipotoxic cardiomyopathy animal models. Palmitic acid (PA) was utilized to induce lipotoxicity to cardiomyocytes for in vitro study. We found sEH KO, independent of plasma lipid and blood pressures, significantly attenuated HFD-induced myocardial lipid accumulation and cardiac dysfunction in vivo. HFD-induced lipotoxic cardiomyopathy and dysfunction of adenosine 5'-monophosphate-activated protein kinase-mammalian target of rapamycin complex (AMPK-mTORC) signaling mediated lipid autophagy in heart were restored by sEH KO. In primary neonatal mouse cardiomyocytes, both sEH KO and sEH substrate EETs plus sEH inhibitor AUDA treatments attenuated PA-induced lipid accumulation. These effects were blocked by inhibition of AMPK or autophagy. The outcomes were supported by the results that sEH KO and EETs plus AUDA rescued HFD- and PA-induced impairment of autophagy upstream signaling of AMPK-mTORC, respectively. These findings revealed that sEH deficiency played an important role in attenuating myocardial lipid accumulation and provided new insights into treating lipotoxic cardiomyopathy. Regulation of autophagy via AMPK-mTORC signaling pathway is one of the underlying mechanisms.

Identification of a homozygous recessive variant in PTGS1 resulting in a congenital aspirin-like defect in platelet function.

We have identified a rare missense variant on chromosome 9, position 125145990 (GRCh37), in exon 8 in PTGS1 (the gene encoding cyclo-oxygenase 1, COX-1, the target of anti-thrombotic aspirin therapy). We report that in the homozygous state within a large consanguineous family this variant is associated with a bleeding phenotype and alterations in platelet reactivity and eicosanoid production. Western blotting and confocal imaging demonstrated that COX-1 was absent in the platelets of three family members homozygous for the PTGS1 variant but present in their leukocytes. Platelet reactivity, as assessed by aggregometry, lumi-aggregometry and flow cytometry, was impaired in homozygous family members, as were platelet adhesion and spreading. The productions of COX-derived eicosanoids by stimulated platelets were greatly reduced but there were no changes in the levels of urinary metabolites of COX-derived eicosanoids. The proband exhibited additional defects in platelet aggregation and spreading which may explain why her bleeding phenotype was slightly more severe than those of other homozygous affected relatives. This is the first demonstration in humans of the specific loss of platelet COX-1 activity and provides insight into its consequences for platelet function and eicosanoid metabolism. Notably despite the absence of thromboxane A2 (TXA2) formation by platelets, urinary TXA2 metabolites were in the normal range indicating these cannot be assumed as markers of in vivo platelet function. Results from this study are important benchmarks for the effects of aspirin upon platelet COX-1, platelet function and eicosanoid production as they define selective platelet COX-1 ablation within humans.

Profiling the eicosanoid networks that underlie the anti- and pro-thrombotic effects of aspirin.

Aspirin prevents thrombosis by inhibiting platelet cyclooxygenase (COX)-1 activity and the production of thromboxane (Tx)A2 , a pro-thrombotic eicosanoid. However, the non-platelet actions of aspirin limit its antithrombotic effects. Here, we used platelet-COX-1-ko mice to define the platelet and non-platelet eicosanoids affected by aspirin. Mass-spectrometry analysis demonstrated blood from platelet-COX-1-ko and global-COX-1-ko mice produced similar eicosanoid profiles in vitro: for example, formation of TxA2 , prostaglandin (PG) F2α , 11-hydroxyeicosatraenoic acid (HETE), and 15-HETE was absent in both platelet- and global-COX-1-ko mice. Conversely, in vivo, platelet-COX-1-ko mice had a distinctly different profile from global-COX-1-ko or aspirin-treated control mice, notably significantly higher levels of PGI2 metabolite. Ingenuity Pathway Analysis (IPA) predicted that platelet-COX-1-ko mice would be protected from thrombosis, forming less pro-thrombotic TxA2 and PGE2 . Conversely, aspirin or lack of systemic COX-1 activity decreased the synthesis of anti-aggregatory PGI2 and PGD2 at non-platelet sites leading to predicted thrombosis increase. In vitro and in vivo thrombosis studies proved these predictions. Overall, we have established the eicosanoid profiles linked to inhibition of COX-1 in platelets and in the remainder of the cardiovascular system and linked them to anti- and pro-thrombotic effects of aspirin. These results explain why increasing aspirin dosage or aspirin addition to other drugs may lessen antithrombotic protection.

Soluble Epoxide Hydrolase in Aged Female Mice and Human Explanted Hearts Following Ischemic Injury.

Myocardial infarction (MI) accounts for a significant proportion of death and morbidity in aged individuals. The risk for MI in females increases as they enter the peri-menopausal period, generally occurring in middle-age. Cytochrome (CYP) 450 metabolizes N-3 and N-6 polyunsaturated fatty acids (PUFA) into numerous lipid mediators, oxylipids, which are further metabolised by soluble epoxide hydrolase (sEH), reducing their activity. The objective of this study was to characterize oxylipid metabolism in the left ventricle (LV) following ischemic injury in females. Human LV specimens were procured from female patients with ischemic cardiomyopathy (ICM) or non-failing controls (NFC). Female C57BL6 (WT) and sEH null mice averaging 13-16 months old underwent permanent occlusion of the left anterior descending coronary artery (LAD) to induce myocardial infarction. WT (wild type) mice received vehicle or sEH inhibitor, trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (tAUCB), in their drinking water ad libitum for 28 days. Cardiac function was assessed using echocardiography and electrocardiogram. Protein expression was determined using immunoblotting, mitochondrial activity by spectrophotometry, and cardiac fibre respiration was measured using a Clark-type electrode. A full metabolite profile was determined by LC-MS/MS. sEH was significantly elevated in ischemic LV specimens from patients, associated with fundamental changes in oxylipid metabolite formation and significant decreases in mitochondrial enzymatic function. In mice, pre-treatment with tAUCB or genetic deletion of sEH significantly improved survival, preserved cardiac function, and maintained mitochondrial quality following MI in female mice. These data indicate that sEH may be a relevant pharmacologic target for women with MI. Although future studies are needed to determine the mechanisms, in this pilot study we suggest targeting sEH may be an effective strategy for reducing ischemic injury and mortality in middle-aged females.

Expression of Cyp2c/Cyp2j subfamily members and oxylipin levels during LPS-induced inflammation and resolution in mice.

Inflammatory stimuli, such as bacterial LPS, alter the expression of many cytochromes P450. CYP2C and CYP2J subfamily members actively metabolize fatty acids to bioactive eicosanoids, which exhibit potent anti-inflammatory effects. Herein, we examined mRNA levels of the 15 mouse Cyp2c and 7 mouse Cyp2j isoforms in liver, kidney, duodenum, and brain over a 96-h time course of LPS-induced inflammation and resolution. Plasma and liver eicosanoid levels were also measured by liquid chromatography with tandem mass spectrometry. Expression changes in Cyp2c and Cyp2j isoforms were both isoform and tissue specific. Total liver Cyp2c and Cyp2j mRNA content was reduced by 80% 24 h after LPS but recovered to baseline levels by 96 h. Total Cyp2c and Cyp2j mRNA in kidney (-19%) and duodenum (-64%) were reduced 24 h after LPS but recovered above baseline by 72 h. Total Cyp2c and Cyp2j mRNA content in brain was elevated at all time points after LPS dosing. Plasma eicosanoids transiently increased 3-6 h after administration of LPS. In liver, esterified oxylipin levels decreased during acute inflammation and before recovering. The biphasic suppression and recovery of mouse Cyp2c and Cyp2j isoforms and associated changes in eicosanoid levels during LPS-induced inflammation and resolution may have important physiologic consequences.-Graves, J. P., Bradbury, J. A., Gruzdev, A., Li, H., Duval, C., Lih, F. B., Edin, M. L., Zeldin, D. C. Expression of Cyp2c/Cyp2j subfamily members and oxylipin levels during LPS-induced inflammation and resolution in mice.

Ephx2-gene deletion affects acetylcholine-induced relaxation in angiotensin-II infused mice: role of nitric oxide and CYP-epoxygenases.

Previously, we showed that adenosine A2A receptor induces relaxation independent of NO in soluble epoxide hydrolase-null mice (Nayeem et al. in Am J Physiol Regul Integr Comp Physiol 304:R23-R32, 2013). Currently, we hypothesize that Ephx2-gene deletion affects acetylcholine (Ach)-induced relaxation which is independent of A2AAR but dependent on NO and CYP-epoxygenases. Ephx2-/- aortas showed a lack of sEH (97.1%, P < 0.05) but an increase in microsomal epoxide hydrolase (mEH, 37%, P < 0.05) proteins compared to C57Bl/6 mice, and no change in CYP2C29 and CYP2J protein (P > 0.05). Ach-induced response was tested with nitro-L-arginine methyl ester (L-NAME) NO-inhibitor; 10-4 M), N-(methylsulfonyl)-2-(2-propynyloxy)-benzenehexanamide (MS-PPOH) (CYP-epoxygenase inhibitor; 10-5 M), 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE, an epoxyeicosatrienoic acid-antagonist; 10-5 M), SCH-58261 (A2AAR-antagonist; 10-6 M), and angiotensin-II (Ang-II, 10-6 M). In Ephx2-/- mice, Ach-induced relaxation was not different from C57Bl/6 mice except at 10-5 M (92.75 ± 2.41 vs. 76.12 ± 3.34, P < 0.05). However, Ach-induced relaxation was inhibited with L-NAME (Ephx2-/-: 23.74 ± 3.76% and C57Bl/6: 11.61 ± 2.82%), MS-PPOH (Ephx2-/-: 48.16 ± 6.53% and C57Bl/6: 52.27 ± 7.47%), and 14,15-EEZE (Ephx2-/-: 44.29 ± 8.33% and C57Bl/6: 39.27 ± 7.47%) vs. non-treated (P < 0.05). But, it did not block with SCH-58261 (Ephx2-/-: 68.75 ± 11.41% and C57Bl/6: 66.26 ± 9.43%, P > 0.05) vs. non-treated (P > 0.05). Interestingly, Ang-II attenuates less relaxation in Ehx2-/- vs. C57Bl/6 mice (58.80 ± 7.81% vs. 45.92 ± 7.76, P < 0.05). Our data suggest that Ach-induced relaxation in Ephx2-/- mice depends on NO and CYP-epoxygenases but not on A2A AR, and Ephx2-gene deletion attenuates less Ach-induced relaxation in Ang-II-infused mice.

Kidney Transplantation in a Patient Lacking Cytosolic Phospholipase A2 Proves Renal Origins of Urinary PGI-M and TX-M.

RATIONALE: The balance between vascular prostacyclin, which is antithrombotic, and platelet thromboxane A2, which is prothrombotic, is fundamental to cardiovascular health. Prostacyclin and thromboxane A2 are formed after the concerted actions of cPLA2α (cytosolic phospholipase A2) and COX (cyclooxygenase). Urinary 2,3-dinor-6-keto-PGF1α (PGI-M) and 11-dehydro-TXB2 (TX-M) have been taken as biomarkers of prostacyclin and thromboxane A2 formation within the circulation and used to explain COX biology and patient phenotypes, despite concerns that urinary PGI-M and TX-M originate in the kidney. OBJECTIVE: We report data from a remarkable patient carrying an extremely rare genetic mutation in cPLA2α, causing almost complete loss of prostacyclin and thromboxane A2, who was transplanted with a normal kidney resulting in an experimental scenario of whole-body cPLA2α knockout, kidney-specific knockin. By studying this patient, we can determine definitively the contribution of the kidney to the productions of PGI-M and TX-M and test their validity as markers of prostacyclin and thromboxane A2 in the circulation. METHODS AND RESULTS: Metabolites were measured using liquid chromatography-tandem mass spectrometry. Endothelial cells were grown from blood progenitors. Before kidney transplantation, the patient's endothelial cells and platelets released negligible levels of prostacyclin (measured as 6-keto-prostaglandin F1α) and thromboxane A2 (measured as TXB2), respectively. Likewise, the urinary levels of PGI-M and TX-M were very low. After transplantation and the establishment of normal renal function, the levels of PGI-M and TX-M in the patient's urine rose to within normal ranges, whereas endothelial production of prostacyclin and platelet production of thromboxane A2 remained negligible. CONCLUSIONS: These data show that PGI-M and TX-M can be derived exclusively from the kidney without contribution from prostacyclin made by endothelial cells or thromboxane A2 by platelets in the general circulation. Previous work relying on urinary metabolites of prostacyclin and thromboxane A2 as markers of whole-body endothelial and platelet function now requires reevaluation.

Cytochrome P450 monooxygenase lipid metabolites are significant second messengers in the resolution of choroidal neovascularization.

Age-related macular degeneration (AMD) is the most common cause of blindness for individuals age 50 and above in the developed world. Abnormal growth of choroidal blood vessels, or choroidal neovascularization (CNV), is a hallmark of the neovascular (wet) form of advanced AMD and leads to significant vision loss. A growing body of evidence supports a strong link between neovascular disease and inflammation. Metabolites of long-chain polyunsaturated fatty acids derived from the cytochrome P450 (CYP) monooxygenase pathway serve as vital second messengers that regulate a number of hormones and growth factors involved in inflammation and vascular function. Using transgenic mice with altered CYP lipid biosynthetic pathways in a mouse model of laser-induced CNV, we characterized the role of these lipid metabolites in regulating neovascular disease. We discovered that the CYP-derived lipid metabolites epoxydocosapentaenoic acids (EDPs) and epoxyeicosatetraenoic acids (EEQs) are vital in dampening CNV severity. Specifically, overexpression of the monooxygenase CYP2C8 or genetic ablation or inhibition of the soluble epoxide hydrolase (sEH) enzyme led to increased levels of EDP and EEQ with attenuated CNV development. In contrast, when we promoted the degradation of these CYP-derived metabolites by transgenic overexpression of sEH, the protective effect against CNV was lost. We found that these molecules work in part through their ability to regulate the expression of key leukocyte adhesion molecules, on both leukocytes and endothelial cells, thereby mediating leukocyte recruitment. These results suggest that CYP lipid signaling molecules and their regulators are potential therapeutic targets in neovascular diseases.

Epoxide hydrolase 1 (EPHX1) hydrolyzes epoxyeicosanoids and impairs cardiac recovery after ischemia.

Stimuli such as inflammation or hypoxia induce cytochrome P450 epoxygenase-mediated production of arachidonic acid-derived epoxyeicosatrienoic acids (EETs). EETs have cardioprotective, vasodilatory, angiogenic, anti-inflammatory, and analgesic effects, which are diminished by EET hydrolysis yielding biologically less active dihydroxyeicosatrienoic acids (DHETs). Previous in vitro assays have suggested that epoxide hydrolase 2 (EPHX2) is responsible for nearly all EET hydrolysis. EPHX1, which exhibits slow EET hydrolysis in vitro, is thought to contribute only marginally to EET hydrolysis. Using Ephx1-/-, Ephx2-/-, and Ephx1-/-Ephx2-/- mice, we show here that EPHX1 significantly contributes to EET hydrolysis in vivo Disruption of Ephx1 and/or Ephx2 genes did not induce compensatory changes in expression of other Ephx genes or CYP2 family epoxygenases. Plasma levels of 8,9-, 11,12-, and 14,15-DHET were reduced by 38, 44, and 67% in Ephx2-/- mice compared with wildtype (WT) mice, respectively; however, plasma from Ephx1-/-Ephx2-/- mice exhibited significantly greater reduction (100, 99, and 96%) of those respective DHETs. Kinetic assays and FRET experiments indicated that EPHX1 is a slow EET scavenger, but hydrolyzes EETs in a coupled reaction with cytochrome P450 to limit basal EET levels. Moreover, we also found that EPHX1 activities are biologically relevant, as Ephx1-/-Ephx2-/- hearts had significantly better postischemic functional recovery (71%) than both WT (31%) and Ephx2-/- (51%) hearts. These findings indicate that Ephx1-/-Ephx2-/- mice are a valuable model for assessing EET-mediated effects, uncover a new paradigm for EET metabolism, and suggest that dual EPHX1 and EPHX2 inhibition may represent a therapeutic approach to manage human pathologies such as myocardial infarction.

Lipopolysaccharide Potentiates Insulin-Driven Hypoglycemic Shock.

Critically ill patients typically present with hyperglycemia. Treatment with conventional insulin therapy (targeting 144-180 mg/dl) improves patient survival; however, intensive insulin therapy (IIT) targeting normal blood glucose levels (81-108 mg/dl) increases the incidence of moderate and severe hypoglycemia, and increases mortality. Septic patients are especially prone to IIT-induced hypoglycemia, but the mechanism remains unknown. Here, we show that codelivery of insulin with otherwise sublethal doses of LPS induced hypoglycemic shock in mice within 1-2 h. LPS impaired clearance of insulin, which amplified insulin receptor signaling. These effects were mediated by caspase-11, TLR4, and complement, each of which trigger eicosanoid production that potentiates insulin signaling. Finally, in an animal model of sepsis, we observed that Salmonella typhimurium-infected mice exhibited simultaneous impaired insulin clearance coexisting with insulin resistance. Our results raise the possibility that septic patients have impaired insulin clearance, which could increase their susceptibility to hypoglycemia during IIT, contraindicating its use.

CYP450-derived oxylipins mediate inflammatory resolution.

Resolution of inflammation has emerged as an active process in immunobiology, with cells of the mononuclear phagocyte system being critical in mediating efferocytosis and wound debridement and bridging the gap between innate and adaptive immunity. Here we investigated the roles of cytochrome P450 (CYP)-derived epoxy-oxylipins in a well-characterized model of sterile resolving peritonitis in the mouse. Epoxy-oxylipins were produced in a biphasic manner during the peaks of acute (4 h) and resolution phases (24-48 h) of the response. The epoxygenase inhibitor SKF525A (epoxI) given at 24 h selectively inhibited arachidonic acid- and linoleic acid-derived CYP450-epoxy-oxlipins and resulted in a dramatic influx in monocytes. The epoxI-recruited monocytes were strongly GR1(+), Ly6c(hi), CCR2(hi), CCL2(hi), and CX3CR1(lo) In addition, expression of F4/80 and the recruitment of T cells, B cells, and dendritic cells were suppressed. sEH (Ephx2)(-/-) mice, which have elevated epoxy-oxylipins, demonstrated opposing effects to epoxI-treated mice: reduced Ly6c(hi) monocytes and elevated F4/80(hi) macrophages and B, T, and dendritic cells. Ly6c(hi) and Ly6c(lo) monocytes, resident macrophages, and recruited dendritic cells all showed a dramatic change in their resolution signature following in vivo epoxI treatment. Markers of macrophage differentiation CD11b, MerTK, and CD103 were reduced, and monocyte-derived macrophages and resident macrophages ex vivo showed greatly impaired phagocytosis of zymosan and efferocytosis of apoptotic thymocytes following epoxI treatment. These findings demonstrate that epoxy-oxylipins have a critical role in monocyte lineage recruitment and activity to promote inflammatory resolution and represent a previously unidentified internal regulatory system governing the establishment of adaptive immunity.

Characterization of the Tissue Distribution of the Mouse Cyp2c Subfamily by Quantitative PCR Analysis.

The CYP2C subfamily of the cytochrome P450 gene superfamily encodes heme-thiolate proteins that have a myriad of biologic functions. CYP2C proteins detoxify xenobiotics and metabolize endogenous lipids such as arachidonic acid to bioactive eicosanoids. We report new methods and results for the quantitative polymerase reaction (qPCR) analysis for the 15 members of the mouse Cyp2c subfamily (Cyp2c29, Cyp2c37, Cyp2c38, Cyp2c39, Cyp2c40, Cyp2c44, Cyp2c50, Cyp2c54, Cyp2c55, Cyp2c65, Cyp2c66, Cyp2c67, Cyp2c68, Cyp2c69, and Cyp2c70). Commercially available TaqMan primer/probe assays were compared with developed SYBR Green primer sets for specificity toward the mouse Cyp2c cDNAs and analysis of their tissue distribution. TaqMan primer/probe assays for 10 of the mouse Cyp2c isoforms were shown to be specific for their intended mouse Cyp2c cDNA; however, there were no TaqMan primer/probe assays specific for the mouse Cyp2c29, Cyp2c40, Cyp2c67, Cyp2c68, or Cyp2c69 transcripts. Each of the SYBR Green primer sets was specific for its intended mouse Cyp2c cDNA. The two qPCR methods confirmed similar patterns of Cyp2c tissue expression: Cyp2c37, Cyp2c38, Cyp2c39, Cyp2c44, Cyp2c50, Cyp2c54, and Cyp2c70 were most highly expressed in liver; Cyp2c55 was highly expressed in large intestine; Cyp2c65 was highly expressed in stomach, duodenum, and large intestine; and Cyp2c66 was highly expressed in both duodenum and jejunum. For isoforms without specific TaqMan primer/probe assays, the SYBR Green primer sets detected high level expression of Cyp2c29, Cyp2c40, Cyp2c67, Cyp2c68, and Cyp2c69 in the liver. Lower expression levels of the mouse Cyp2cs were also detected in other tissues.