Cytochrome P450 Ω-hydroxylase Research Articles

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

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. We analysed human cantharidin-induced blister samples and inflammatory responses in TRPV1 transgenic mice. 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. 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.

The Functions of Cytochrome P450 ω-hydroxylases and the Associated Eicosanoids in Inflammation-Related Diseases.

The cytochrome P450 (CYP) ω-hydroxylases are a subfamily of CYP enzymes. While CYPs are the main metabolic enzymes that mediate the oxidation reactions of many endogenous and exogenous compounds in the human body, CYP ω-hydroxylases mediate the metabolism of multiple fatty acids and their metabolites via the addition of a hydroxyl group to the ω- or (ω-1)-C atom of the substrates. The substrates of CYP ω-hydroxylases include but not limited to arachidonic acid, docosahexaenoic acid, eicosapentaenoic acid, epoxyeicosatrienoic acids, leukotrienes, and prostaglandins. The CYP ω-hydroxylases-mediated metabolites, such as 20-hyroxyleicosatrienoic acid (20-HETE), 19-HETE, 20-hydroxyl leukotriene B4 (20-OH-LTB4), and many ω-hydroxylated prostaglandins, have pleiotropic effects in inflammation and many inflammation-associated diseases. Here we reviewed the classification, tissue distribution of CYP ω-hydroxylases and the role of their hydroxylated metabolites in inflammation-associated diseases. We described up-regulation of CYP ω-hydroxylases may be a pathogenic mechanism of many inflammation-associated diseases and thus CYP ω-hydroxylases may be a therapeutic target for these diseases. CYP ω-hydroxylases-mediated eicosanods play important roles in inflammation as pro-inflammatory or anti-inflammatory mediators, participating in the process stimulated by cytokines and/or the process stimulating the production of multiple cytokines. However, most previous studies focused on 20-HETE,and further studies are needed for the function and mechanisms of other CYP ω-hydroxylases-mediated eicosanoids. We believe that our studies of CYP ω-hydroxylases and their associated eicosanoids will advance the translational and clinal use of CYP ω-hydroxylases inhibitors and activators in many diseases.

β-adrenergic Receptor-stimulated Cardiac Myocyte Apoptosis: Role of Cytochrome P450 ω-hydroxylase.

Prolonged or excessive β-adrenergic activation leads to cardiac myocyte loss and heart dysfunction; however, the underlying cellular mechanisms are still unclear. Therefore, we first confirmed the effect of isoproterenol (ISO), a β-adrenergic receptor agonist, on cardiac toxicity using TUNEL and caspase activity assays in cultured rat cardiomyocytes. ISO treatment significantly increased cardiomyocyte apoptosis. Persistent ISO stimulation of cardiomyocytes also increased the expression of CYP4A3, a major CYP450 ω-hydroxylase that produces 20-hydroxyeicosatetraenoic acid (20-HETE) in a time-dependent manner. Next, we examined the effect of ISO and 20-HETE on cardiomyocyte apoptosis using annexin V and propidium iodide staining. Treatment with either 20-HETE or ISO significantly increased cardiomyocyte apoptosis, and inhibition of 20-HETE production using 17-ODYA, a CYP450 ω-hydroxylase inhibitor, dramatically attenuated ISO-induced cardiomyocyte apoptosis. To determine the apoptotic pathway involved, the mitochondrial membrane potential (ΔΨm) was measured by detecting the ratio of JC-1 green/red emission intensity. The results demonstrated that 17-ODYA significantly abolished ISO-induced disruption of ΔΨm and that 20-HETE alone induced a marked disruptive effect on ΔΨm in cardiomyocytes. In addition, 20-HETE-induced disruption of ΔΨm and apoptosis was significantly attenuated by KN93, a CaMKII inhibitor. Taken together, these results demonstrate that 20-HETE treatment induces significant apoptosis via mitochondrial-dependent pathways, and that inhibition of 20-HETE production using 17-ODYA attenuates ISO-induced cardiomyocyte apoptosis.

Cytochrome P450 eicosanoids in cerebrovascular function and disease.

Cytochrome P450 eicosanoids play important roles in brain function and disease through their complementary actions on cell-cell communications within the neurovascular unit (NVU) and mechanisms of brain injury. Epoxy- and hydroxyeicosanoids, respectively formed by cytochrome P450 epoxygenases and ω-hydroxylases, play opposing roles in cerebrovascular function and in pathological processes underlying neural injury, including ischemia, neuroinflammation and oxidative injury. P450 eicosanoids also contribute to cerebrovascular disease risk factors, including hypertension and diabetes. We summarize studies investigating the roles P450 eicosanoids in cerebrovascular physiology and disease to highlight the existing balance between these important lipid signaling molecules, as well as their roles in maintaining neurovascular homeostasis and in acute and chronic neurovascular and neurodegenerative disorders.

Abstract 5133: Targeting the eicosanoid 20-HETE suppresses pancreatic cancer growth and metastasis through regulation of inflammation

Abstract Pancreatic cancer incidence and death rates are on the rise. Most available therapies fail partly due to the uniquely inflammatory pancreatic tumor stromal compartment. Prolonged and unresolved inflammation is implicated in the pathogenesis of pancreatic cancer, yet the critical inflammatory mediators remain largely unknown. The role of lipid autacoids in pancreatic cancer, which are metabolites of arachidonic acid and other related fatty acids, has not been thoroughly studied. Among these, metabolites of the cytochrome P450 (CYP) epoxygenases and ω-hydroxylases, including EETs and 20-HETE, respectively, have been mostly neglected in cancer. We previously showed that epoxyeicosatrienoic acids (EETs) stimulate multi-organ metastasis and tumor dormancy escape. Here we show that elevated levels of 20-hydroxyeicosatetraenoic acid (20-HETE), a product of ω-hydroxylases of the CYP4A/F subfamilies, stimulated pancreatic primary tumor growth and metastasis. We measured 20-HETE and other lipid autacoids via LC/MS/MS in multiple cell lines and found that 20-HETE was produced primarily by pancreatic cancer cells, which express the 20-HETE generating enzyme, CYP4F2. Overexpression of CYP4F2 and exogenous 20-HETE induced Ras activity in HEK293 cells or pancreatic cancer and macrophage cell lines, respectively. 20-HETE attracted human macrophages in vitro, while macrophage migration was reduced towards pancreatic cancer cells treated with an inhibitor of 20-HETE synthesis, HET0016. Inhibition of 20-HETE synthesis in human macrophages and pancreatic cancer cells decreased HB-EGF and M-CSF production. Systemic administration of 20-HETE promoted primary human and murine pancreatic tumor growth in subcutaneous and orthotopic murine models. Transgenic mice overexpressing 20-HETE (Tie2-CYP4F2 Tr) exhibited spontaneous liver and mediastinal lymph node metastasis following resection of subcutaneous primary pancreatic tumors, in contrast to wild type control mice. Targeting 20-HETE by HET0016 or siRNA towards CYP4F2 suppressed primary pancreatic tumor growth. Systemic treatment of orthotopic pancreatic tumors with HET0016, 14,15-EEZE, and gemcitabine regressed established pancreatic tumors and prolonged survival without overt toxicity. Thus, 20-HETE critically regulates Ras activation, inflammation, and pancreatic tumor growth and metastasis. Our studies offer a mechanistic rationale for pharmacologically targeting 20-HETE as a novel approach to treat pancreatic cancer. Citation Format: Yael Gus-Brautbar, Julia Piwowarski, Jessica A. Casper, Diane R. Bielenberg, Jennifer Cheng, Birgitta Schmidt, Bruce D. Hammock, Darryl C. Zeldin, Mark W. Kieran, Dipak Panigrahy. Targeting the eicosanoid 20-HETE suppresses pancreatic cancer growth and metastasis through regulation of inflammation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5133.

N-3 fatty acids reduce plasma 20-hydroxyeicosatetraenoic acid and blood pressure in patients with chronic kidney disease.

Metabolism of arachidonic acid by cytochrome P450 ω-hydroxylase leads to the formation of 20-hydroxyeicosatetraenoic acid (20-HETE) that regulates vascular function, sodium homeostasis and blood pressure (BP). Supplementation with n-3 fatty acids is known to alter arachidonic acid metabolism and reduce the formation of the lipid peroxidation products F2-isoprostanes, but the effect of n-3 fatty acids on 20-HETE has not been studied. We previously reported a significant effect of n-3 fatty acids but not coenzyme Q10 (CoQ) to reduce BP in a double-blind, placebo-controlled intervention, wherein patients with chronic kidney disease (CKD) were randomized to n-3 fatty acids (4 g), CoQ (200 mg), both supplements or control (4 g olive oil), daily for 8 weeks. This study examined the effect of n-3 fatty acids on plasma and urinary 20-HETE in the same study, as well as plasma and urinary F2-isoprostanes, and relate these to changes in BP. Seventy-four patients completed the 8-week intervention. n-3 fatty acids but not CoQ significantly reduced plasma 20-HETE (P = 0.001) and F2-isoprostanes (P < 0.001). In regression models adjusted for BP at baseline, postintervention plasma 20-HETE was a significant predictor of the fall in SBP (P < 0.0001) and DBP (P < 0.0001) after n-3 fatty acids. This is the first report that n-3 fatty acid supplementation reduces plasma 20-HETE in humans and that this associates with reduced BP. These results provide a plausible mechanism for the reduction in BP observed in patients with CKD following n-3 fatty acid supplementation.

Abstract 015: Increased 20-HETE Levels Contribute to Impaired Glucose Metabolism and Type 2 Diabetes in Cyp4a14 Knockout Mice Fed on High Fat Diet.

20-HETE (20-Hydroxyeicosatetraenoic acid) is a cytochrome P450 ω-hydroxylase metabolite of arachidonic acid that promotes endothelial dysfunction, microvascular remodeling and hypertension. Previous studies have shown that urinary 20-HETE levels correlate with BMI and plasma insulin levels. However, there is no direct evidence for the role of 20-HETE in the regulation of glucose metabolism, obesity and type 2 diabetes mellitus. In this study we examined the effect of 20-SOLA (2,5,8,11,14,17-hexaoxanonadecan-19-yl-20-hydroxyeicosa-6(Z),15(Z)-dienoate), a water-soluble 20-HETE antagonist, on blood pressure, weight gain and blood glucose in Cyp4a14 knockout (Cyp4a14-/-) mice fed high-fat diet (HFD). The Cyp4a14-/- male mice exhibit high vascular 20-HETE levels and display 20-HETE-dependent hypertension. There was no difference in weight gain and fasting blood glucose between Cyp4a14-/- and wild type (WT) on regular chow. When subjected to HFD for 15 weeks, a significant increase in weight was observed in Cyp4a14-/- as compared to WT mice (56.5±3.45 vs. 30.2±0.7g, p&lt;0.05). Administration of 20-SOLA (10mg/kg/day in drinking water) significantly attenuated the weight gain (28.7±1.47g, p&lt;0.05) and normalized blood pressure in Cyp4a14-/- mice on HFD (116±0.3 vs. 172.7±4.6mmHg, p&lt;0.05). HFD fed Cyp4a14-/- mice exhibited hyperglycemia as opposed to normal glucose levels in WT on a HFD (154±1.9 vs. 96.3±3.0 mg/dL, p&lt;0.05). 20-SOLA prevented the HFD-induced hyperglycemia in Cyp4a14-/- mice (91±8mg/dL, p&lt;0.05). Plasma insulin levels were markedly high in Cyp4a14-/- mice vs. WT on HFD (2.66±0.7 vs. 0.58±0.18ng/mL, p&lt;0.05); corrected by the treatment with 20-SOLA (0.69±0.09 ng/mL, p&lt;0.05). Importantly, glucose and insulin tolerance tests showed impaired glucose homeostasis and insulin resistance in Cyp4a14-/- mice on HFD; ameliorated by treatment with 20-SOLA. This novel finding that blockade of 20-HETE actions by 20-SOLA prevents HFD-induced obesity and restores glucose homeostasis in Cyp4a14-/- mice suggests that 20-HETE contributes to obesity, hyperglycemia and insulin resistance in HFD induced metabolic disorder. The molecular mechanisms underlying 20-HETE mediated metabolic dysfunction are being currently explored.

Cytochrome P450 ω-Hydroxylases in Inflammation and Cancer.

Cytochrome P450-dependent ω-hydroxylation is a prototypic metabolic reaction of CYP4 family members that is important for the elimination and bioactivation of not only therapeutic drugs, but also endogenous compounds, principally fatty acids. Eicosanoids, derived from arachidonic acid, are key substrates in the latter category. Human CYP4 enzymes, mainly CYP4A11, CYP4F2, and CYP4F3B, hydroxylate arachidonic acid at the omega position to form 20-HETE, which has important effects in tumor progression and on angiogenesis and blood pressure regulation in the vasculature and kidney. CYP4F3A in myeloid tissue catalyzes the ω-hydroxylation of leukotriene B4 to 20-hydroxy leukotriene B4, an inactivation process that is critical for the regulation of the inflammatory response. Here, we review the enzymology, tissue distribution, and substrate selectivity of human CYP4 ω-hydroxylases and their roles as catalysts for the formation and termination of the biological effects of key eicosanoid metabolites in inflammation and cancer progression.

Human fetal ventricular cardiomyocyte, RL-14 cell line, is a promising model to study drug metabolizing enzymes and their associated arachidonic acid metabolites.

RL-14 cells, human fetal ventricular cardiomyocytes, are a commercially available cell line that has been established from non-proliferating primary cultures derived from human fetal heart tissue. However, the expression of different drug metabolizing enzymes (DMEs) in RL-14 cells has not been elucidated yet. Therefore, the main objectives of the current work were to investigate the capacity of RL-14 cells to express different cytochrome P450 (CYP) isoenzymes and correlate this expression to primary cardiomyocytes. The expression of CYP isoenzymes was determined at mRNA, protein and catalytic activity levels using real time-PCR, Western blot analysis and liquid chromatography-electron spray ionization-mass spectrometry (LC-ESI-MS), respectively. Our results showed that RL-14 cells constitutively express CYP ω-hydroxylases, CYP1A, 1B, 4A and 4F; CYP epoxygenases, CYP2B, 2C and 2J; in addition to soluble epoxide hydrolayse (EPHX2) at mRNA and protein levels. The basal expression of CYP ω-hydroxylases, epoxygenases and EPHX2 was supported by the ability of RL-14 cells to convert arachidonic acid to its biologically active metabolites, 20-hydroxyeicosatetraenoic acids (20-HETEs), 14,15-epoxyeicosatrienoic acids (14,15-EET), 11,12-EET, 8,9-EET, 5,6-EET, 14,15-dihydroxyeicosatrienoic acid (14,15-DHET), 11,12-DHET, 8,9-DHET and 5,6-DHET. Furthermore, RL-14 cells express CYP epoxygenases and ω-hydroxylase at comparable levels to those expressed in adult and fetal human primary cardiomyocytes cells implying the importance of RL-14 cells as a model for studying DMEs in vitro. Lastly, different CYP families were induced in RL-14 cells using 2,3,7,8-tetrachlorodibenzo-p-dioxin and fenofibrate at mRNA and protein levels. The current study provides the first evidence that RL-14 cells express CYP isoenzymes at comparable levels to those expressed in the primary cells and thus offers a unique in vitro model to study DMEs in the heart.

Abstract 4833: The eicosanoid 20-HETE triggers Ras activation, pancreatic cancer growth and metastasis through inflammatory macrophage signaling

Abstract Prolonged, unresolving inflammation is increasingly implicated in the pathogenesis of pancreatic cancer, but the critical inflammatory mediators remain largely unknown. Most efforts to elucidate these mediators have focused on protein cytokines rather than lipid autacoids. Lipid autacoids are metabolites of arachidonic acid and other related fatty acids that regulate inflammation and tumor growth. Among these, the metabolites of the cytochrome P450 (CYP) epoxygenases and ω-hydroxylases, including EETs and 20-HETE, respectively, have been largely neglected in cancer. Recently, we showed that epoxyeicosatrienoic acids (EETs) stimulate multi-organ metastasis and tumor dormancy escape. We report for the first time, that elevated levels of 20-hydroxyeicosatetraenoic acid (20-HETE), a product of the ω-hydroxylases CYP4A/F, stimulate primary pancreatic tumor growth and metastasis. Using genetic and pharmacological manipulation of endogenous 20-HETE, we demonstrate that systemic administration of 20-HETE promotes primary human and murine pancreatic cancer in subcutaneous and orthotopic models. Furthermore, a transgenic murine model of endogenously high 20-HETE level (Tie2-CYP4F2 Tr), exhibited spontaneous liver and mediastinal lymph node metastasis after resection of subcutaneous primary pancreatic tumors. In contrast, wild-type mice did not develop liver and mediastinal lymph node metastasis after primary tumor resection. We show that 20-HETE activated Ras in human pancreatic tumor cell lines and the 20-HETE antagonist, HET0016, decreased Ras activity. The p65 subunit of NF-κB also increased in a 20-HETE-dependent manner in these tumor cell lines. 20-HETE induced migration of human macrophages in vitro, and tumor-associated macrophages expressed the 20-HETE generating enzyme CYP4F2. Depletion of macrophages with clodronate liposomes impaired CYP-metabolite-stimulated tumor growth. In addition, we show that 14,15-EET promotes pancreatic tumor growth in vivo. Moreover, inhibitors of soluble epoxide hydrolase, the enzyme that metabolizes EETs, elevated EET levels and promoted pancreatic tumor growth. Either the 20-HETE antagonist HET0016, or the EET antagonist 14,15-EEZE alone significantly inhibited primary tumor growth. Remarkably, systemic administration of both antagonists together resulted in sustained regression of established pancreatic tumors. This combination of 20-HETE and EET antagonists also suppressed ascites formation and prolonged survival in an orthotopic pancreatic cancer model without toxic effects. Thus, 20-HETE and EET production by macrophages in the tumor microenvironment critically regulates Ras activation, pancreatic tumor growth and metastasis. Our studies offer a mechanistic rationale for using 20-HETE and EET antagonists as a novel approach and anti-cancer therapeutics for pancreatic cancer. Citation Format: Yael Gus-Brautbar, Jessica Casper, Diane R. Bielenberg, Jennifer Cheng, Birgitta Schmidt, Bruce D. Hammock, Darryl C. Zeldin, Mark W. Kieran, Dipak Panigrahy. The eicosanoid 20-HETE triggers Ras activation, pancreatic cancer growth and metastasis through inflammatory macrophage signaling. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4833. doi:10.1158/1538-7445.AM2014-4833

Acute arsenic treatment alters arachidonic acid and its associated metabolite levels in the brain of C57Bl/6 mice.

The toxic effects of arsenic on the whole brain, as well as the discrete regions, has been previously reported for mice. We investigated the effects of acute arsenite (As(III)) on brain levels of arachidonic acid (AA) and its associated metabolites generated through cytochrome P450 (CYP), cyclooxygenase (COX), and lipoxygenase (LOX) pathways. Our results demonstrated that acute As(III) treatment (12.5 mg·(kg body mass)(-1)) decreases cytosolic phospholipase A2 (cPLA2) with a subsequent decrease in its catalytic activity and brain AA levels. In addition, As(III) differentially altered CYP epoxygenases and CYP ω-hydroxylases, but it did not affect brain Ephx2 mRNA or sEH catalytic activity levels. As(III)-mediated effects on Cyps caused an increase in brain 5,6-epoxyeicosatrienoic acid (5,6-EET) and 16/17-hydroxyeicosatetreinoic acid (16/17-HETE) levels, and a decrease in 18- and 20-HETE levels. Furthermore, As(III) increased cyclooxygenase-2 (COX-2) mRNA while decreasing prostaglandins F2α (PGF2α) and PGJ2. As(III) also increased brain 5-lipoxygenase (5-LOX) and 15-LOX mRNA, but decreased 12-LOX mRNA. These changes in LOX mRNA were associated with a decrease in 8/12-HETE levels only. In conclusion, this is the first demonstration that As(III) decreases AA levels coinciding with alterations to EET, HETE, and PG levels, which affects brain development and neurochemistry.

Dysregulation of pressure‐induced Ca2+ signaling and myogenic constriction of cerebral arteries in aged hypertensive mice (1079.3)

Hypertension in the elderly substantially increases the risk of stroke and vascular cognitive impairment (VCI) in part due to an impaired functional adaptation of aged cerebral arteries to high blood pressure. To elucidate the mechanisms underlying impaired autoregulatory protection in aging, hypertension was induced in young (3 mo) and aged (24 mo) C57/BL6 mice by chronic infusion of angiotensin II and pressure‐induced changes in smooth muscle (SMC) [Ca2+]i and myogenic constriction of middle cerebral arteries (MCA) were assessed. In MCAs from young hypertensive mice pressure‐induced increases in VSMC [Ca2+]i and myogenic tone were increased and these adaptive responses were inhibited by the cytochrome P450 ω‐hydroxylase inhibitor HET0016 and the TRP channel blocker SKF96365. Administration of 20‐HETE increased SMC [Ca2+]i and constricted MCAs and these responses were inhibited by SKF96365. MCAs from aged hypertensive mice did not show adaptive increases in pressure‐induced calcium signal and myogenic tone and responses to HET0016 and SKF96365 were blunted. Inhibition of BK channels by iberiotoxin enhanced SMC [Ca2+]i and myogenic constriction in MCAs of young normotensive animals, whereas it was without effect in MCAs of young hypertensive mice. Iberiotoxin did not restore myogenic adaptation in MCAs of aged hypertensive mice. Thus, functional maladaptation of aged cerebral arteries to hypertension is due to the dysregulation of pressure‐induced, 20‐HETE and TRP channel‐mediated SMC calcium signaling, whereas overactivation of BK channels is unlikely to play a role in this phenomenon.Grant Funding Source: Supported by the American Heart Association and the Nemzeti Fejlesztési Ügynökség

Role of 20-HETE, TRPC channels, and BKCa in dysregulation of pressure-induced Ca2+ signaling and myogenic constriction of cerebral arteries in aged hypertensive mice

Hypertension in the elderly substantially increases the risk of stroke and vascular cognitive impairment in part due to an impaired functional adaptation of aged cerebral arteries to high blood pressure. To elucidate the mechanisms underlying impaired autoregulatory protection in aging, hypertension was induced in young (3 mo) and aged (24 mo) C57BL/6 mice by chronic infusion of angiotensin II and pressure-induced changes in smooth muscle cell (SMC) intracellular Ca(2+) concentration ([Ca(2+)]i) and myogenic constriction of middle cerebral arteries (MCA) were assessed. In MCAs from young hypertensive mice, pressure-induced increases in vascular SMC [Ca(2+)]i and myogenic tone were increased, and these adaptive responses were inhibited by the cytochrome P-450 ω-hydroxylase inhibitor HET0016 and the transient receptor potential (TRP) channel blocker SKF96365. Administration of 20- hydroxyeicosatetraenoic acid (HETE) increased SMC [Ca(2+)]i and constricted MCAs, and these responses were inhibited by SKF96365. MCAs from aged hypertensive mice did not show adaptive increases in pressure-induced calcium signal and myogenic tone and responses to HET0016 and SKF96365 were blunted. Inhibition of large-conductance Ca(2+)-activated K(+) (BK) channels by iberiotoxin enhanced SMC [Ca(2+)]i and myogenic constriction in MCAs of young normotensive animals, whereas it was without effect in MCAs of young hypertensive mice. Iberiotoxin did not restore myogenic adaptation in MCAs of aged hypertensive mice. Thus functional maladaptation of aged cerebral arteries to hypertension is due to the dysregulation of pressure-induced 20-HETE and TRP channel-mediated SMC calcium signaling, whereas overactivation of BK channels is unlikely to play a role in this phenomenon.

Differential Expression of Cytochrome P450 Omega-hydroxylase Isoforms and Their Association with Clinicopathological Features in Pancreatic Ductal Adenocarcinoma

The cytochrome P450 (CYP) superfamily consists of enzymes that catalyze the oxidation of lipids, steroids, and drugs. In particular, the CYP4 family plays an essential role in lipid metabolism by the ω-hydroxylation of terminal ends of fatty acids. Disturbance of this system has been associated with increased angiogenesis, proliferation, and metastasis of several cancers. This study aimed to detect the expression of CYP4 isoforms (CYP4A11, CYP4F2, CYP4F3) in pancreatic ductal adenocarcinoma (PDA) and their association with clinicopathological features. Pancreatic specimens were collected from 73 patients who underwent surgical resection at the Thomas Jefferson University Hospital. Quantitative polymerase chain reaction was used to examine the cytochrome P450 isoforms in PDA (n = 62), adjacent-normal (n = 30), and benign tissues (n = 11). Logistic regression models were used to analyze gene expression among tissue types. Spearman rank correlations were calculated for isoform expression and for age. Differences in expression by gender were assessed via t test. Other clinicopathological variables (diabetes, smoking, obesity, T stage, perineural invasion, nodal status) were analyzed by Wilcoxon rank sum. CYP4 expression for isoforms was significantly higher in PDA tissues versus matched-adjacent tissues (p < 0.01). PDA tumors expressed significantly higher levels of CYP4F2 and CYP4F3 when compared to benign lesions (p < 0.01). Significant associations were found between low levels of CYP4F2 and CYP4F3 and increased age of PDA patients. Interestingly, all isoforms were expressed at higher levels in male patients. Transcriptional upregulation of cytochrome P450 ω-hydroxylase suggests that these enzymes have the potential to be used as distinguishing markers in pancreatic pathology.

Abstract 4961: Control of tumor lymphangiogenesis by epoxyeicosanoids.

Abstract Background: Epoxyeicosatrienoic acids (EETs) are lipid autacoids biosynthesized by cytochrome P450 epoxygenases and metabolized by soluble epoxide hydrolase (sEH) to less active dihydroxyeicosatrienoic acids (DHETs). EETs are autocrine and paracrine mediators of arachidonic acid-induced vasorelaxation in the cardiovascular and renal systems. Thus, inhibitors of sEH, which raise endogenous EET levels, are being considered for long-term use in hypertension, diabetes, stroke and other medical conditions. 20-hydroxeicosatetraenoic acid (20-HETE) is a small molecule biosynthesized by cytochrome P450 ω-hydroxylases. We recently demonstrated that EETs stimulate multiorgan and lymph node metastasis, a major cause of disease dissemination and death. However, the mechanism of EETs in tumor lymphangiogenesis is unknown. Thus, we hypothesize that EETs stimulate lymph node metastasis via tumor lymphangiogenesis. Methods and Results: Using two murine tumor lines transfected with VEGFC, B16F10-VEGFC melanoma and T241-VEGFC fibrosarcoma tumors, we demonstrate that EETs stimulate primary tumor growth via tumor lymphangiogenesis. Systemic administration of 14,15-EET and/or 20-HETE by osmotic minipump accelerated primary B16F10-VEGFC melanoma and T241-VEGFC fibrosarcoma tumor growth. Inhibitors of soluble epoxide hydrolase (sEH), the enzyme that metabolizes EETs, elevated endogenous EET levels and also promoted tumor growth: sEH inhibitors (TUPS and AUCB) stimulated primary B16F10-VEGFC tumor growth. Conversely, systemic administration of a 20-HETE antagonist (HET0016) and/or EET antagonist (14,15-EEZE) regressed established primary B16F10-VEGFC tumors. 14,15-EET and 11,12-EET stimulated lymphatic endothelial cell proliferation, viability and migration. To determine whether tumor lymphangiogenesis contributes to the increase in B16F10-VEGFC tumor growth in 14,15-EET treated mice, we analyzed tumors for the specific lymphangiogenesis markers podoplanin and LYVE-1. Immunohistochemistry studies revealed an increase in podoplanin-positive and LYVE-1-positive vessels in B16F10-VEGFC tumors treated with 14,15-EET compared to vehicle treatment. 14,15-EET, 11,12-EET, and 20-HETE increased the production of VEGFC by B16F10-VEGFC and T241 fibrosarcoma tumor cells. Using a well-established model in which resection of a primary tumor (Lewis lung carcinoma) reproducibly stimulates the development of distant metastases 14-17 days after resection, we investigated whether EET antagonists inhibit spontaneous metastatic growth. The EET antagonist 14,15-EEZE and/or siRNA to VEGFR3 inhibited lung metastasis and the combination showed additive inhibition. Conclusion: EETs and 20-HETE stimulate tumor lymphangogenesis, offering a mechanistic rationale for using EET and 20-HETE antagonists as novel anti-cancer therapeutics to inhibit lymph node metastasis and tumor lymphangiogenesis. Citation Format: Dayna K. Mudge, Karolina Serhan, Diane R. Bielenberg, Sung Hee Hwang, Bruce D. Hammock, Mark W. Kieran, Dipak Panigrahy. Control of tumor lymphangiogenesis by epoxyeicosanoids. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4961. doi:10.1158/1538-7445.AM2013-4961

Abstract 3952: Cytochrome P450-derived eicosanoids regulate pancreatic cancer and metastasis.

Abstract Background: 20-hydroxeicosatetraenoic acid (20-HETE) and epoxyeicosatrenoic acids (EETs) are lipid autacoids biosynthesized by cytochrome P450 ω-hydroxylases (e.g. CYP4F2) and epoxygenases, respectively. These eicosanoids regulate inflammation, vascular tone and stimulate angiogenesis which is required for tumor growth and metastasis. However, despite their pleiotropic effects on cells, the role of these cytochrome P450-derived metabolites in pancreatic cancer has not been studied. We hypothesize that 20-HETE and EETs in the tumor microenvironment stimulate pancreatic cancer and metastasis. Methods and Results: Using genetic and pharmacological manipulation of endogenous 20-HETE and EET levels, we demonstrate that 20-HETE and EETs are critical for primary pancreatic tumor growth and metastasis. Native CYP4F2 protein was expressed in a series of human pancreatic tumor cell lines. Transgenic mice with endothelial-specific (tie2-promoter) overexpression of CYP4F2 were used to achieve increased endothelial levels of 20-HETE and stimulated pancreatic tumor growth. Systemic administration of 20-HETE and/or 14,15-EET by osmotic minipump stimulated primary human pancreatic cancer (Panc1) and murine pancreatic (PancOH7) tumor growth in both subcutaneous and orthotopic models. Inhibitors of soluble epoxide hydrolase, the enzyme that metabolizes EETs, elevated EET levels and promoted pancreatic tumor growth. Conversely, systemic administration of a 20-HETE antagonist (HET0016) and/or EET antagonist (14,15-EEZE) suppressed PancOH7 pancreatic tumor growth without toxic effects. Tie2-CYP4F2 transgenic mice exhibited spontaneous liver metastasis after resection of primary pancreatic (PancOH7) subcutaneous tumors. This is of interest because liver metastasis from the injection of pancreatic tumor cells is commonly only achieved by direct injection of tumor cells into the spleen or pancreas. Systemic administration of 20-HETE in WT mice stimulated a 4-fold increase in the number of surface lung metastases compared with control mice and led to an induction of liver and mediastinal lymph node metastasis 26-30 days after resection of the primary PancOH7 tumor. In contrast, the 20-HETE antagonist and/or EET antagonist prolonged survival in the PancOH7 resection metastasis model. Moreover, the 20-HETE antagonist and/or EET antagonist inhibited pancreatic tumor cell proliferation and viability induced by 20-HETE and 14,15-EET, respectively. Furthermore, the 20-HETE antagonist and/or EET antagonist suppressed tumor angiogenesis including a decrease in CD31-positive vessels and VEGF levels in PancOH7 pancreatic tumors. Conclusion: 20-HETE and EET production in the tumor microenvironment may be a critical regulator of pancreatic tumor growth and metastasis, offering a mechanistic rationale for using 20-HETE and EET antagonists as novel anti-cancer therapeutics for pancreatic cancer. Citation Format: Jessica A. Casper, Diane R. Bielenberg, Jennifer Cheng, Birgitta Schmidt, Bruce D. Hammock, Darryl C. Zeldin, Mark W. Kieran, Dipak Panigrahy. Cytochrome P450-derived eicosanoids regulate pancreatic cancer and metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3952. doi:10.1158/1538-7445.AM2013-3952

Treatment with the cytochrome P450 ω‐hydroxylase inhibitor HET0016 attenuates cerebrovascular inflammation, oxidative stress and improves vasomotor function in spontaneously hypertensive rats

Hypertension increases cerebrovascular oxidative stress and inflammation and impairs vasomotor function. These pathological alterations lead to dysregulation of cerebral blood flow and exacerbate atherogenesis, increasing the morbidity of ischaemic cerebrovascular diseases and promoting vascular cognitive impairment. We aimed to test the hypothesis that increased production of the arachidonic acid metabolite 20-hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE) contributes to hypertension-induced cerebrovascular alterations. We treated male spontaneously hypertensive rats (SHR) with HET0016 (N-hydroxy-N'-(4-butyl-2-methylphenyl)-formamidine), an inhibitor of 20-HETE synthesis. In middle cerebral arteries (MCAs) of SHRs, we focused on vasomotor responses and end points that are highly relevant for cellular reactive oxygen species (ROS) production, inflammatory cytokine expression and NF-κB activation. SHRs treated with HET0016 remained hypertensive (SHR + HET0016: 149 ± 8 mmHg, Wistar-Kyoto rat: 115 ± 4 mmHg; P < 0.05.), although their systolic blood pressure was decreased compared to untreated SHRs (191 ± 6 mmHg). In MCAs of SHRs, flow-induced constriction was increased, whereas ACh- and ATP-induced dilations were impaired. This functional impairment was reversed by treatment with HET0016. Treatment with HET0016 also significantly decreased oxidative stress in MCAs of SHRs (as shown by dihydroethidium staining and analysis of vascular 5-nitrotyrosine, 4-hydroxynonenal and carbonyl content) and inhibited cerebrovascular inflammation (shown by the reduced mRNA expression of TNFα, IL-1β and IL-6). Treatment of SHRs with HET0016 also attenuated vascular NF-κB activation. In vitro treatment with 20-HETE significantly increased vascular production of ROS and promoted NF-κB activation in cultured cerebromicrovascular endothelial cells. Taken together, treatment with HET0016 confers anti-oxidative and anti-inflammatory effects in the cerebral arteries of SHRs by disrupting 20-HETE-mediated autocrine/paracrine signalling pathways in the vascular wall. It is likely that HET0016-induced decreases in blood pressure also potentiate the cerebrovascular protective effects of the drug.

Cytochrome P450-derived eicosanoids and vascular dysfunction in coronary artery disease patients

ObjectiveAccumulating preclinical and epidemiologic evidence has emerged to suggest that modulation of cytochrome P450 (CYP)-mediated eicosanoid metabolism may be a viable vascular protective therapeutic strategy for the secondary prevention of coronary artery disease (CAD). The functional relationship between CYP-derived eicosanoid metabolite levels and vascular dysfunction in humans with established CAD, however, has not been evaluated. Therefore, we characterized the relationship between inter-individual variation in soluble epoxide hydrolase (sEH) and CYP ω-hydroxylase metabolism and established vascular function phenotypes predictive of prognosis in a cohort of patients with atherosclerotic cardiovascular disease. MethodsPlasma epoxyeicosatrienoic acid (EET), dihydroxyeicosatrienoic acid (DHET), and 20-hydroxyeicosatetraenoic acid (20-HETE) levels were quantified by HPLC-MS/MS in 106 patients with stable, angiographically-confirmed CAD. Relationships between biomarkers of CYP-mediated eicosanoid metabolism and vascular function phenotypes were evaluated by Pearson's correlation. ResultsA significant inverse association was observed between 20-HETE levels (a biomarker of CYP ω-hydroxylase metabolism) and brachial artery flow-mediated dilation (r = −0.255, p = 0.010). An inverse association was also observed between 14,15-EET:DHET ratios (a biomarker of sEH metabolism) and both monocyte chemoattractant protein-1 levels (r = −0.252, p = 0.009) and a consolidated cellular adhesion molecule ‘score’ reflecting the levels of E-selectin and P-selectin (r = −0.216, p = 0.027). No associations with C-reactive protein or epithelial neutrophil-activating protein-78 levels were observed. ConclusionsCollectively, these findings demonstrate that enhanced CYP ω-hydroxylase and sEH metabolic function are associated with more advanced endothelial dysfunction and vascular inflammation, respectively, in patients with established atherosclerotic cardiovascular disease. These findings lay the foundation for future clinical research in this area.

Introgression of Brown Norway CYP4A genes on to the Dahl salt-sensitive background restores vascular function in SS-5BN consomic rats

The present study tested the hypothesis that the Dahl SS (salt-sensitive) rat has vascular dysfunction due, in part, to the up-regulation of the CYP4A/20-HETE (cytochrome P450 ω-hydroxylase 4A)/20-hydroxyeicosatetraenoic acid) system. To assess the role of vascular 20-HETE, SS rats were compared with SS-5(BN) consomic rats, carrying CYP4A alleles on chromosome 5 from the normotensive BN (Brown Norway) introgressed on to the SS genetic background. Cerebral arteries from SS-5(BN) rats had less CYP4A protein than arteries from SS rats fed either NS (normal-salt, 0.4% NaCl) or HS (high-salt, 4.0% NaCl) diet. ACh (acetylcholine)-induced dilation of MCAs (middle cerebral arteries) from SS and SS-5(BN) rats was present in SS-5(BN) rats fed on either an NS or HS diet, but absent in SS rats. In SS rats fed on either diet, ACh-induced dilation was restored by acute treatment with the CYP4A inhibitor DDMS (N-methyl-sulfonyl-12,12-dibromododec-11-enamide) or the 20-HETE antagonist 20-HEDE [20-hydroxyeicosa-6(Z),15(Z)-dienoic acid]. The restored response to ACh in DDMS-treated SS rats was inhibited by L-NAME (N(G)nitro-L-arginine methyl ester) and unaffected by indomethacin or MS-PPOH [N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide]. Vascular relaxation responses to the NO donor C(5)FeN(6)Na(2)O were intact in both SS and SS-5(BN) rats and unaffected by the acute addition of DDMS, indicating that the vascular dysfunction of the SS rat is due to a reduced bioavailability of NO instead of failure of the VSMCs (vascular smooth muscle cells) to respond to the vasodilator. Superoxide levels in cerebral arteries of SS-5(BN) rats [evaluated semi-quantitatively by DHE (dihydroethidium) fluorescence] were lower than those in the arteries of SS rats. These findings indicate that SS rats have an up-regulation of the CYP4A/20-HETE pathway resulting in elevated ROS (reactive oxygen species) and reduced NO bioavailability causing vascular dysfunction.

Evaluation of cytochrome P450-derived eicosanoids in humans with stable atherosclerotic cardiovascular disease

ObjectivePreclinical and genetic epidemiologic studies suggest that modulating cytochrome P450 (CYP)-mediated arachidonic acid metabolism may have therapeutic utility in the management of coronary artery disease (CAD). However, predictors of inter-individual variation in CYP-derived eicosanoid metabolites in CAD patients have not been evaluated to date. Therefore, the primary objective was to identify clinical factors that influence CYP epoxygenase, soluble epoxide hydrolase (sEH), and CYP ω-hydroxylase metabolism in patients with established CAD. MethodsPlasma levels of epoxyeicosatrienoic acids (EETs), dihydroxyeicosatrienoic acids (DHETs), and 20-hydroxyeicosatetraenoic acid (20-HETE) were quantified by HPLC–MS/MS in a population of patients with stable, angiographically confirmed CAD (N=82) and healthy volunteers from the local community (N=36). Predictors of CYP epoxygenase, sEH, and CYP ω-hydroxylase metabolic function were evaluated by regression. ResultsObesity was significantly associated with low plasma EET levels and 14,15-EET:14,15-DHET ratios. Age, diabetes, and cigarette smoking also were significantly associated with CYP epoxygenase and sEH metabolic activity, while only renin-angiotensin system inhibitor use was associated with CYP ω-hydroxylase metabolic activity. Compared to healthy volunteers, both obese and non-obese CAD patients had significantly higher plasma EETs (P<0.01) and epoxide:diol ratios (P<0.01), whereas no difference in 20-HETE levels was observed (P=NS). ConclusionsCollectively, these findings suggest that CYP-mediated eicosanoid metabolism is dysregulated in certain subsets of CAD patients, and demonstrate that biomarkers of CYP epoxygenase and sEH, but not CYP ω-hydroxylase, metabolism are altered in stable CAD patients relative to healthy individuals. Future studies are necessary to determine the therapeutic utility of modulating these pathways in patients with CAD.