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Human cytochrome P-450 (CYP)2J2 is abundant in heart and active in biosynthesis of epoxyeicosatrienoic acids (EEls). Recently, we demonstrated that these eicosanoid products protect myocardium from ischemia-reperfusion injury. The present study utilized trans- genic (Tr) mice with cardiomyocyte-specific overexpression of human CYP2J2 to investigate protection toward toxicity resulting from acute (0, 5, or 15 mg/kg daily for 3 days, followed by 24-h recovery) or chronic (0, 1.5, or 3.0 mg/kg biweekly for 5 wk, followed by 2-wk recovery) doxorubicin (Dox) administration. Acute treatment resulted in marked elevations of serum lactate dehydrogenase and creatine kinase levels that were significantly greater in wild-type (WT) than CYP2J2 Tr mice. Acute treatment also resulted in less activation of stress response enzymes in CYP2J2 Tr mice (catalase 750% vs. 300% of baseline, caspase-3 235% vs. 165% of baseline in WT vs. CYP2J2 Tr mice). Moreover, CYP2J2 Tr hearts exhibited less Dox-induced cardiomyocytes apoptosis (measured by TUNEL) compared with WT hearts. After chronic treatment, comparable decreases in body weight were observed in WT and CYP2J2 Tr mice. However, cardiac function, assessed by measurement of fractional shortening with M-mode transthoracic echocardiography, was significantly higher in CYP2J2 Tr than WT hearts after chronic Dox treatment (WT 37 ± 2%, CYP2J2 Tr 47 ± 1%). WT mice also had larger increases in β-myosin heavy chain and cardiac ankryin repeat protein compared with CYP2J2 Tr mice. CYP2J2 Tr hearts had a significantly higher rate of Dox metabolism than WT hearts (2.2 ± 0.25 vs. 1.6 ± 0.50 ng·min-1·100 μg protein-1). In vitro data from H9c2 cells demonstrated that EEls attenuated Dox-induced mitochondrial damage. Together, these data suggest that cardiac-specific overexpression of CYP2J2 limited Dox-induced toxicity. [ABSTRACT FROM AUTHOR]

Development of hypertension stems from both environmental and genetic factors wherein the kidney plays a central role. Spontaneously hypertensive rats (SHR) and the nonhypertensive Wistar-Kyoto (WKY) controls are widely used as a model for studying hypertension. The present study examined the renal gene expression profiles between SHR and WKY at a prehypertensive stage (3 wk of age) and hypertensive stage (9 wk of age). Additionally, age-related changes in gene expression patterns were examined from 3 to 9 wk in both WKY and SHR. Five to six individual kidney samples of the same experimental group were pooled together, and quadruplicate hybridizations were performed using the National Institute of Environmental Health Sciences Rat version 2.0 Chip, which contains ∼6,700 genes. Twenty two genes were found to be differentially expressed between SHR and WKY at 3 wk of age, and 104 genes were differentially expressed at 9 wk of age. Soluble epoxide hydrolase (Ephx2) was found to be significantly upregulated in SHR at both time points and was the predominant outlier. Conversely, elastase 1 (Ela1) was found to be the predominant gene downregulated in SHR at both time points. Analysis of profiles at 3 vs. 9 wk of age identified 508 differentially expressed genes in WKY rats. In contrast, only 211 genes were found to be differentially expressed during this time period in SHR. The altered gene expression patterns observed in the age-related analysis suggested significant differences in the vascular extracellular matrix system between SHR and WKY kidney. Together, our data highlight the complexity of hypertension and the numerous genes involved in and affected by this condition. [ABSTRACT FROM AUTHOR]

OBJECTIVE: Regulation of angiogenesis is critical for many diseases. Specifically, pathological retinal neovascularization, a major cause of blindness, is suppressed with dietary ω3-long-chain polyunsaturated fatty acids (ω3LCPUFAs) through antiangiogenic metabolites of cyclooxygenase and lipoxygenase. Cytochrome P450 epoxygenases (CYP2C8) also metabolize LCPUFAs, producing bioactive epoxides, which are inactivated by soluble epoxide hydrolase (sEH) to transdihydrodiols. The effect of these enzymes and their metabolites on neovascularization is unknown. APPROACH AND RESULTS: The mouse model of oxygen-induced retinopathy was used to investigate retinal neovascularization. We found that CYP2C (localized in wild-type monocytes/macrophages) is upregulated in oxygen-induced retinopathy, whereas sEH is suppressed, resulting in an increased retinal epoxide:diol ratio. With a ω3LCPUFA-enriched diet, retinal neovascularization increases in Tie2-driven human-CYP2C8-overexpressing mice (Tie2-CYP2C8-Tg), associated with increased plasma 19,20-epoxydocosapentaenoic acid and retinal epoxide:diol ratio. 19,20-Epoxydocosapentaenoic acids and the epoxide:diol ratio are decreased with overexpression of sEH (Tie2-sEH-Tg). Overexpression of CYP2C8 or sEH in mice does not change normal retinal vascular development compared with their wild-type littermate controls. The proangiogenic role in retina of CYP2C8 with both ω3LCPUFA and ω6LCPUFA and antiangiogenic role of sEH in ω3LCPUFA metabolism were corroborated in aortic ring assays. CONCLUSIONS: Our results suggest that CYP2C ω3LCPUFA metabolites promote retinal pathological angiogenesis. CYP2C8 is part of a novel lipid metabolic pathway influencing retinal neovascularization.

Enhanced cellular cAMP levels have been shown to increase apical membrane Cl- and HCO3- conductances in epithelia. We found that the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX) increases cAMP levels in Necturus gallbladder. We used conventional open-tip and double-barreled Cl- -selective microelectrodes to study the effects of IBMX on membrane conductances and intracellular Cl- activities in gallbladders mounted in a divided chamber and bathed with Ringer's solutions at 23 degrees C and pH 7.4. In HCO3- -free media, 0.1 mM IBMX added to the mucosal medium depolarized the apical membrane potential Va, decreased the fractional resistance FR, and significantly reduced intracellular Cl- activity (aCli). Under control conditions, aCli was above the value corresponding to passive distribution across the apical cell membrane. In media containing 25 mM HCO3-, IBMX caused a small transient hyperpolarization of Va followed by a depolarization not significantly different from that observed in HCO3- -free Ringer's. Removal of mucosal Cl-, Na+ or Ca2+ did not affect the IBMX-induced depolarization in Va. The basolateral membrane of Necturus gallbladder is highly K+ permeable. Increasing serosal K+ from 2.5 to 80 mM, depolarized Va. Mucosal IBMX significantly reduced this depolarization. Addition of 10 mM Ba2+, a K+ channel blocker, to the serosal medium depolarized Va and, essentially, blocked the depolarization induced by IBMX. These results indicate that mucosal IBMX increases apical HCO3- conductance and decreases basolateral K+ conductance in gallbladder epithelial cells via a cAMP-dependent mechanism. The latter effect, not previously reported in epithelial tissues, appears to be the major determinant of the IBMX-induced depolarization of Va. [ABSTRACT FROM AUTHOR]

Background The subcommissural organ (SCO) differentiates early in ontogeny and remains fully active during the entire life span. It secretes glycoproteins into the cerebrospinal fluid (CSF) flowing through the Sylvius aqueduct (SA); these proteins either assemble to form Reissner fiber (RF), or remain soluble in the CSF and circulate throughout the CSF compartments. Overholser et al. (1954) have demonstrated that offspring littered by rats maintained on a diet deficient in folic acid and/or Vitamin B12 lack a SCO and develop hydrocephalus. This led them to propose that a dysfunction of the SCO during development leads to stenosis of the SA and hydrocephalus. We have investigated several animal models in which the SCO would play a role in the pathogenesis of hydrocephalus. We have now performed a comparative analysis of the changes occurring in the SCO-RF complex of these animal models, with the aim to find a landmark common to all hydrocephalic animals that might help to unfold the mechanism of SCO-dependant hydrocephalus. Materials and methods A comparative study of the SCO-RF complex and SA of five animal models characterized by a dysfunction of the SCO and development of hydrocephalus was carried out. Conventional light and electron microscopy and immunocytochemistry were applied. Results Model 1. Immunological blockage of RF formation by maternal transfer of antibodies against RF-glycoproteins: undamaged and secretory active SCO, missing RF, stenosed SA and moderate hydrocephalus. Model 2. hyh mice with a point mutation of aSNAP gene: undamaged and secretory active SCO, missing RF, obliterated SA and severe hydrocephalus. Model 3. HTx rat: subcommissural portion (two distal thirds) of SCO missing, supracommissural portion (rostral third) of SCO secretory active, RF absent, obliterated SA and severe hydrocephalus. Model 4. Transgenic mice deficient in transcription factor RFX4- v3: subcommissural portion of SCO missing, supracommissural portion of SCO secretory active, RF absent, patent SA and moderate to severe hydrocephalus. Model 5. fyn knockout mice: subcommissural portion of SCO missing, supracommissural portion of SCO secretory active, RF absent, SA with an abnormal shape and severe hydrocephalus. Conclusion 1. The SCO is formed by two zones: the subcommissural and the supracommissural portions. 2. Differentiation of both zones would be controlled by different genes. 3. The subcommissural portion of the SCO is essential for RF formation. Mutant and transgenic animals lacking this portion, although still have a secretory active flow of CSF throughout SA. 6. Absence of RF could cause SA obliteration or a turbulent CSF flow through SA what, in turn, would lead to hydrocephalus. Supported by Fondecyt 1030265 (Chile), the Intramural Program of the NIH, NIEHS (USA), and MEXT (Japan). [ABSTRACT FROM AUTHOR]

Cytochrome P450 epoxygenase (CYP450)-derived epoxyeicosatrienoic acids (EETs) are important regulators of cardiac remodeling; but the underlying mechanism remains unclear. The present study aimed to elucidate how EETs regulated cardiac fibrosis in response to isoprenaline (Iso) or angiotensin (Ang) II. Cardiac-specific human CYP2J2 transgenic mice (Tr) and wild-type (WT) C57BL/6 littermates were infused with Iso- or Ang II. Two weeks after infusion, Tr mice showed more alleviative cardiac fibrosis and inflammation compared with WT mice. In vitro, we found Iso or Ang II induced nuclear transfer of NF-κB p65 and inflammatory cytokines expression in cardiomyocytes. Furthermore, inflammation response emerged in macrophages cultured in cardiomyocytes-conditioned medium. When pretreatment with 14,15-EET in cardiomyocytes, the inflammatory response was markedly suppressed and the transmission of inflammation from cardiomyocytes to macrophages was reduced. In conclusion, CYP2J2 and EETs prevent cardiac fibrosis and cardiac dysfunction by suppressing transmission of pro-inflammation from cardiomyocytes to macrophages in heart, suggesting that elevation of EETs level could be a potential strategy to prevent cardiac fibrosis., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Cyclooxygenases (COX)-1 and -2 are the key enzymes in the conversion of arachidonic acid to prostaglandins. COX-2 appears to play an emerging role in inflammation and carcinogenesis. Nonsteroidal anti-inflammatory drugs (NSAIDs) are used for the treatment of numerous diseases and reduce the risk of developing colorectal cancer. Polymorphisms in the COX-2 gene could alter enzyme expression, function, and/or the response to NSAIDs. Therefore, they could modify individual risks for developing cancer and other diseases or the occurrence of side effects or sensitivity toward selective or nonselective COX inhibitors. We sequenced the COX-2 gene of 72 individuals and identified rare polymorphisms in the promoter and the coding region. A COX-2 molecular model was used to locate the coding region polymorphisms relative to functional sites in the protein, and the COX-2 V511A polymorphism was very near to the active site. This variant protein was expressed, and function was evaluated, but no difference was detected in metabolism of the COX-2 substrates, arachidonic acid, linoleic acid, and 2-arachidonyl glycerol, compared with the wild type. The Km values for arachidonic acid showed no differences between the COX-2 wild type and V511A mutant. Inhibition with selective or nonselective COX inhibitors was essentially the same for the two enzymes. The absence of functionally important polymorphisms in the COX-2 gene may suggest that there has been selective pressure against those single nucleotide polymorphisms because of the critical role of this enzyme in maintenance of homeostasis.

Cytochrome P450 (CYP) 2C8 is the principal enzyme responsible for the metabolism of the anti-cancer drug paclitaxel (Taxol). It is also the predominant P450 responsible for the metabolism of arachidonic acid to biologically active epoxyeicosatrienoic acids (EETs) in human liver and kidney. In this study, we describe two new CYP2C8 alleles containing coding changes: CYP2C8*2 has an Ile269Phe substitution in exon 5 and CYP2C8*3 includes both Arg139Lys and Lys399Arg amino acid substitutions in exons 3 and 8. CYP2C8*2 was found only in African-Americans, while CYP2C8*3 occurred primarily in Caucasians. Neither occurred in Asians. The frequency of the CYP2C8*2 allele was 0.18 in African-Americans, and that of CYP2C8*3 was 0.13 in Caucasians. CYP2C8*1 (wild-type), CYP2C8*2 and CYP2C8*3 cDNAs were expressed in Escherichia coli, and the ability of these enzymes to metabolize both paclitaxel and arachidonic acid was assessed. Recombinant CYP2C8*3 was defective in the metabolism of both substrates. The turnover number of CYP2C8*3 for paclitaxel was 15% of CYP2C8*1. CYP2C8*2 had a two-fold higher Km and two-fold lower intrinsic clearance for paclitaxel than CYP2C8*1. CYP2C8*3 was also markedly defective in the metabolism of arachidonic acid to 11,12- and 14,15-EET (turnover numbers 35-40% that of CYP2C8*1). Thus, CYP2C8*3 is defective in the metabolism of two important CYP2C8 substrates: the anticancer drug paclitaxel and the physiologically important compound arachidonic acid. This polymorphism has important clinical and physiological implications in individuals homozygous for this allele.

Bacterial lipopolysaccharide (LPS) is a risk factor for exacerbation of asthma and causes airway inflammation. The aim of this study was to examine the effects of disruption of prostaglandin (PG) H synthase (PGHS)-1 and PGHS-2 genes on pulmonary responses to inhaled LPS. PGHS-1(-/-), PGHS-2(-/-), and wild-type (WT) mice were exposed to 4 to 6 microg/m(3) LPS via aerosol. Enhanced pause (PenH), a measure of bronchoconstriction, was assessed using a whole-body plethysmograph before and immediately after a 4-h LPS exposure. Bronchoalveolar lavage (BAL) was performed after LPS exposure to assess inflammatory cells, cytokines/chemokines (tumor necrosis factor-alpha, interleukin-6, and macrophage inflammatory protein-2), and PGE(2). The degree of lung inflammation was scored on hematoxylin-and-eosin-stained sections. PGHS-1 and PGHS-2 protein levels were determined by immunoblotting. All mice exhibited increased PenH and methacholine responsiveness after LPS exposure; however, these changes were much more pronounced in PGHS-1(-/-) and PGHS-2(-/-) mice relative to WT mice (P < 0.05). There were no significant differences in inflammation as assessed by BAL fluid (BALF) cells or lung histology between the genotypes despite reduced BALF cytokines/chemokines and PGE(2) in PGHS-1(-/-) and PGHS-2(-/-) mice relative to WT mice (P < 0.05). PGHS-2 was upregulated more in PGHS-1(-/-) mice compared with WT mice after LPS exposure. We conclude that: (1) airway inflammation and hyperresponsiveness are dissociated in PGHS-1(-/-) and PGHS-2(-/-) mice exposed to LPS; (2) the balance of PGHS-1 and PGHS-2 is important in regulating the functional respiratory responses to inhaled LPS; and (3) neither PGHS-1 nor PGHS-2 is important in regulating basal lung function or the inflammatory responses of the lung to inhaled LPS.

The CYP2C subfamily has been extensively studied in humans with respect to the metabolism of clinically important drugs, and polymorphisms have been identified in these enzymes. In the present study, a murine model was used to determine the possible physiological functions and extrahepatic distribution of CYP2Cs. Using the reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, and immununohistochemistry, this report demonstrates that the mouse CYP2Cs are extensively distributed in extrahepatic tissues and localized to heart muscle, lung Clara and ciliated cells, kidney collecting ducts, the X-zone of female adrenals, reproductive organs, white blood cells, and eyes (in the optic nerve, rods, and cones). RT-PCR, subcloning, and sequencing of the products indicate that each CYP2C has a unique tissue distribution. Four cDNA fragments representing potentially new CYP2Cs were identified, each with its own organ-specific pattern of expression. Using a bacterial cDNA expression system, we found that recombinant proteins for each of the five full-length murine CYP2Cs metabolize arachidonic acid to different regio- and stereospecific products, including epoxyeicosatrienoic acids and hydroxyeicosatetraenoic acids. Regio- and stereospecific metabolites of arachidonic acid have been reported to affect important physiological functions such as inflammation, neutrophil activation, ion transport, cellular proliferation, and vascular tone. Our results suggest that the presence of CYP2C enzymes in heart muscle, aorta, kidney, lung, adrenals, eyes, and reproductive organs could regulate important physiological and/or pathological processes in these tissues.

CYP2J2 is abundant in human heart and its arachidonic acid metabolites, the epoxyeicosatrienoic acids (EETs), have potent vasodilatory, antiinflammatory and cardioprotective properties. This study was designed to examine the role of CYP2J2 in hypoxia-reoxygenation-induced injury in cultured bovine aortic endothelial cells (BAECs). Early passage BAECs were exposed to 24-h hypoxia followed by 4-h reoxygenation (HR). HR resulted in cell injury, as indicated by significant increases in lactate dehydrogenase (LDH) release and trypan blue stained cells (p < 0.01) and was associated with a decrease in CYP2J2 protein expression. Transfection of BAECs with the CYP2J2 cDNA resulted in increased CYP2J2 expression and arachidonic acid epoxygenase activity, compared with cells transfected with an irrelevant green fluorescent protein (GFP) cDNA. HR induced significant injury in GFP-transfected BAECs, as indicated by increases in LDH release and trypan blue-stained cells (p < 0.01); however, the HR-induced injury was markedly attenuated in CYP2J2-transfected cells (p < 0.01). HR increased cellular 8-iso-prostaglandin F(2alpha) (p < 0.05), and decreased eNOS expression, L-arginine uptake and conversion, and nitrite production (p < 0.01) in GFP-transfected BAECs. CYP2J2 transfection attenuated the HR-induced increase in 8-iso-prostaglandin F(2alpha) (p < 0.05) and decreased the amount of extracellular superoxide detected by cytochrome c reduction under normoxic conditions (p < 0.05) but did not significantly affect HR-induced decreases in eNOS expression, L-arginine uptake and conversion, and nitrite production. Treatment of BAECs with synthetic EETs and/or epoxide hydrolase inhibitors also showed protective effects against HR injury (p < 0.05). These observations suggest: (1) HR results in endothelial injury and decreased CYP2J2 expression; (2) transfection with the CYP2J2 cDNA protects against HR injury; and (3) the cytoprotective effects of CYP2J2 may be mediated, at least in part, by antioxidant effects.

The epoxyeicosatrienoic acids (EETs) are products of cytochrome P450 (CYP) epoxygenases that have vasodilatory and anti-inflammatory properties. Here we report that EETs have additional fibrinolytic properties. In vascular endothelial cells, physiological concentrations of EETs, particularly 11,12-EET, or overexpression of the endothelial epoxygenase, CYP2J2, increased tissue plasminogen activator (t-PA) expression by 2.5-fold without affecting plasminogen activator inhibitor-1 expression. This increase in t-PA expression correlated with a 4-fold induction in t-PA gene transcription and a 3-fold increase in t-PA fibrinolytic activity and was blocked by the CYP inhibitor, SKF525A, but not by the calcium-activated potassium channel blocker, charybdotoxin, indicating a mechanism that does not involve endothelial cell hyperpolarization. The t-PA promoter is cAMP-responsive, and induction of t-PA gene transcription by EETs correlated with increases in intracellular cAMP levels and, functionally, with cAMP-driven promoter activity. To determine whether increases in intracellular cAMP levels were due to modulation of guanine nucleotide-binding proteins, we assessed the effects of EETs on Galpha(s) and Galpha(i2). Treatment with EETs increased Galpha(s), but not Galpha(i2), GTP-binding activity by 3.5-fold. These findings indicate that EETs possess fibrinolytic properties through the induction of t-PA and suggest that endothelial CYP2J2 may play an important role in regulating vascular hemostasis.

Arachidonic acid metabolites of the cyclooxygenase and lipoxygenase pathways have a variety of important lung functions. Recent observations indicate that cytochrome P-450 (P-450) monooxygenases are also expressed in the lung, localized to specific pulmonary cell types (e.g., epithelium, endothelium, and smooth muscle), and may modulate critical lung functions. This review summarizes recent data on the presence and biological activity of P-450-derived eicosanoids in the pulmonary vasculature and airways, including effects on pulmonary vascular and bronchial smooth muscle tone and airway epithelial ion transport. We hypothesize a number of potential functions of P-450-derived arachidonate metabolites in the lungs such as contribution to hypoxic pulmonary vasoconstriction, regulation of bronchomotor tone, control of the composition of airway lining fluid, and limitation of pulmonary inflammation. Finally, we describe a number of emerging technologies, including congenic and transgenic strains of experimental animals, P-450 isoform-specific inhibitors and inhibitory antibodies, eicosanoid analogs, and vectors for delivery of P-450 cDNAs and antisense oligonucleotides. These tools will facilitate further studies on the contribution of endogenously formed P-450 eicosanoid metabolites to lung function, under both normal and pathological conditions.

The cytochrome P450-derived epoxyeicosatrienoic acids (EETs) have potent effects on renal vascular reactivity and tubular sodium and water transport; however, the role of these eicosanoids in the pathogenesis of hypertension is controversial. The current study examined the hydrolysis of the EETs to the corresponding dihydroxyeicosatrienoic acids (DHETs) as a mechanism for regulation of EET activity and blood pressure. EET hydrolysis was increased 5- to 54-fold in renal cortical S9 fractions from the spontaneously hypertensive rat (SHR) relative to the normotensive Wistar-Kyoto (WKY) rat. This increase was most significant for the 14,15-EET regioisomer, and there was a clear preference for hydrolysis of 14, 15-EET over the 8,9- and 11,12-EETs. Increased EET hydrolysis was consistent with increased expression of soluble epoxide hydrolase (sEH) in the SHR renal microsomes and cytosol relative to the WKY samples. The urinary excretion of 14,15-DHET was 2.6-fold higher in the SHR than in the WKY rat, confirming increased EET hydrolysis in the SHR in vivo. Blood pressure was decreased 22+/-4 mm Hg (P:<0.01) 6 hours after treatment of SHRs with the selective sEH inhibitor N:, N:'-dicyclohexylurea; this treatment had no effect on blood pressure in the WKY rat. These studies identify sEH as a novel therapeutic target for control of blood pressure. The identification of a potent and selective inhibitor of EET hydrolysis will be invaluable in separating the vascular effects of the EET and DHET eicosanoids.