Soluble epoxide hydrolase regulates hematopoietic progenitor cell function via generation of fatty acid diols.

Fatty acid epoxides are important lipid signaling molecules involved in the regulation of vascular tone and homeostasis. Tissue and plasma levels of these mediators are determined by the activity of cytochrome P450 epoxygenases and the soluble epoxide hydrolase (sEH), and targeting the latter is an effective way of manipulating epoxide levels in vivo. We investigated the role of the sEH in regulating the mobilization and proliferation of progenitor cells with vasculogenic/reparative potential. Our studies revealed that sEH down-regulation/inhibition impaired the development of the caudal vein plexus in zebrafish, and decreased the numbers of lmo2/cmyb-positive progenitor cells therein. In mice sEH inactivation attenuated progenitor cell proliferation (spleen colony formation), but the sEH products 12,13-dihydroxyoctadecenoic acid (12,13-DiHOME) and 11,12- dihydroxyeicosatrienoic acid stimulated canonical Wnt signaling and rescued the effects of sEH inhibition. In murine bone marrow, the epoxide/diol content increased during G-CSF-induced progenitor cell expansion and mobilization, and both mobilization and spleen colony formation were reduced in sEH(-/-) mice. Similarly, sEH(-/-) mice showed impaired functional recovery following hindlimb ischemia, which was rescued following either the restoration of bone marrow sEH activity or treatment with 12,13-DiHOME. Thus, sEH activity is required for optimal progenitor cell proliferation, whereas long-term sEH inhibition is detrimental to progenitor cell proliferation, mobilization, and vascular repair.

Cytochrome P4502S1: a novel monocyte/macrophage fatty acid epoxygenase in human atherosclerotic plaques.

Cytochrome P450 (CYP) epoxygenases metabolize endogenous polyunsaturated fatty acids to their corresponding epoxides, generating bioactive lipid mediators. The latter play an important role in vascular homeostasis, angiogenesis, and inflammation. As little is known about the functional importance of extra-vascular sources of lipid epoxides, we focused on determining whether lipid epoxide-generating CYP isoforms are expressed in human monocytes/macrophages. Epoxides were generated by freshly isolated human monocytes and production increased markedly during differentiation to macrophages. Mass spectrometric analysis identified CYP2S1 as a novel macrophage CYP and CYP2S1-containing microsomes generated epoxides of arachidonic, linoleic and eicosapentaenoic acid. Macrophage CYP2S1 expression was increased by LPS and IFN-γ (classically activated), and oxidized LDL but not IL-4 and IL-13 (alternatively activated), and was colocalised with CD68 in inflamed human tonsils but not in breast cancer metastases. Prostaglandin (PG) E(2) is an immune modulator factor that promotes phagocytosis and CYP2S1 can metabolize its immediate precursors PGG(2) and PGH(2) to 12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT). We found that CYP inhibition and siRNA-mediated downregulation of CYP2S1 increased macrophage phagocytosis and that the latter effect correlated with decreased 12-HHT formation. Although no Cyp2s1 protein was detected in aortae from wild-type mice it was expressed in aortae and macrophage foam cells from ApoE(-/-) mice. Consistent with these observations CYP2S1 was colocalised with the monocyte marker CD68 in human atherosclerotic lesions. Thus, CYP2S1 generates 12-HHT and is a novel regulator of macrophage function that is expressed in classical inflammatory macrophages, and can be found in murine and human atherosclerotic plaques.

Soluble epoxide hydrolase deficiency attenuates neointima formation in the femoral cuff model of hyperlipidemic mice.

OBJECTIVE: Epoxyeicosatrienoic acids (EETs) have antiinflammatory effects and are required for normal endothelial function. The soluble epoxide hydrolase (sEH) metabolizes EETs to their less active diols. We hypothesized that knockout and inhibition of sEH prevents neointima formation in hyperlipidemic ApoE(-/-) mice. METHODS AND RESULTS: Inhibition of sEH by 12-(3-adamantan-1-yl-ureido) dodecanoic acid or knockout of the enzyme significantly increased plasma EET levels. sEH activity was detectable in femoral and carotid arteries. sEH knockout or inhibition resulted in a significant reduction of neointima formation in the femoral artery cuff model but not following carotid artery ligation. Although macrophage infiltration occurred abundantly at the site of cuff placement in both sEH(+/+) and sEH(-/-), the expression of proinflammatory genes was significantly reduced in femoral arteries from sEH(-/-) mice. Moreover, an in vivo 5-bromo-2'-deoxyuridine assay revealed that smooth muscle cell proliferation at the site of cuff placement was attenuated in sEH knockout and sEH inhibitor-treated animals. CONCLUSION: These observations suggest that inhibition of sEH prevents vascular remodeling in an inflammatory model but not in a blood flow-dependent model of neointima formation.

Inhibition of the soluble epoxide hydrolase by tyrosine nitration.

Inhibition of the soluble epoxide hydrolase (sEH) has beneficial effects on vascular inflammation and hypertension indicating that the enzyme may be a promising target for drug development. As the enzymatic core of the hydrolase domain of the human sEH contains two tyrosine residues (Tyr(383) and Tyr(466)) that are theoretically crucial for enzymatic activity, we addressed the hypothesis that the activity of the sEH may be affected by nitrosative stress. Epoxide hydrolase activity was detected in human and murine endothelial cells as well in HEK293 cells and could be inhibited by either authentic peroxynitrite (ONOO(-)) or the ONOO(-) generator 3-morpholino-sydnonimine (SIN-1). Protection of the enzymatic core with 1-adamantyl-3-cyclohexylurea in vitro decreased sensitivity to SIN-1. Both ONOO(-) and SIN-1 elicited the tyrosine nitration of the sEH protein and mass spectrometry analysis of tryptic fragments revealed nitration on several tyrosine residues including Tyr(383) and Tyr(466). Mutation of the latter residues to phenylalanine was sufficient to abrogate epoxide hydrolase activity. In vivo, streptozotocin-induced diabetes resulted in the tyrosine nitration of the sEH in murine lungs and a significant decrease in its activity. Taken together, these data indicate that the activity of the sEH can be regulated by the tyrosine nitration of the protein. Moreover, nitrosative stress would be expected to potentiate the physiological actions of arachidonic acid epoxides by preventing their metabolism to the corresponding diols.

Inhibition of the soluble epoxide hydrolase attenuates monocrotaline-induced pulmonary hypertension in rats.

OBJECTIVES: The soluble epoxide hydrolase (sEH) metabolizes epoxyeicosatrienoic acids (EETs) to their less active dihydroxy derivatives. Because EETs have antiinflammatory properties, we determined whether or not inhibition of sEH attenuates disease development in the monocrotaline model of pulmonary hypertension in rats. METHODS: sEH inhibition was achieved using 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (25 mg/l) and cis- 4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (5 mg/l) administered via drinking water starting 3 days prior to monocrotaline injection (60 mg/kg). RESULTS: Monocrotaline induced the development of progressive pulmonary hypertension. sEH inhibition increased the plasma ratio of EETs to DHETs and attenuated the monocrotaline-induced increase in pulmonary artery medial wall thickness as well as the degree of vascular muscularization. Moreover, right ventricular pressure was significantly lower in the group treated with sEH inhibitors. Pulmonary sEH protein expression and sEH activity, as well as pulmonary cytochrome P450 epoxygenase activity were all impaired in monocrotaline-treated rats as compared with control animals. sEH inhibitors, however, increased the plasma ratio of EETs to dihydroxy epoxyeicosatrienoic acids. Monocrotaline induced the proliferation of pulmonary endothelial and vascular smooth muscle cells in vivo as determined by 5-Bromo-2'-deoxy-Uridine incorporation, and this effect was significantly blunted in animals treated with sEH inhibitors. Proliferation of cultured pulmonary smooth muscle cell, however, was not affected by EETs or sEH inhibitors suggesting that the in-vivo effects are a consequence of a direct EET-mediated protection against the inflammation induced by monocrotaline. CONCLUSION: sEH inhibition reduces pulmonary vascular remodeling and the development of pulmonary hypertension in the monocrotaline model of primary pulmonary hypertension in rats.

Epoxyeicosatrienoic acids and the soluble epoxide hydrolase are determinants of pulmonary artery pressure and the acute hypoxic pulmonary vasoconstrictor response.

Recent findings have indicated a role for cytochrome P-450 (CYP) epoxygenase-derived epoxyeicosatrienoic acids (EETs) in acute hypoxic pulmonary vasoconstriction (HPV). Given that the intracellular concentration of EETs is determined by the soluble epoxide hydrolase (sEH), we assessed the influence of the sEH and 11,12-EET on pulmonary artery pressure and HPV in the isolated mouse lung. In lungs from wild-type mice, HPV was significantly increased by sEH inhibition, an effect abolished by pretreatment with CYP epoxygenase inhibitors and the EET antagonist 14,15-EEZE. HPV and EET production were greater in lungs from sEH(-/-) mice than from wild-type mice and sEH inhibition had no further effect on HPV, while MSPPOH and 14,15-EEZE decreased the response. 11,12-EET increased pulmonary artery pressure in a concentration-dependent manner and enhanced HPV via a Rho-dependent mechanism. Both 11,12-EET and hypoxia elicited the membrane translocation of a transient receptor potential (TRP) C6-V5 fusion protein, the latter effect was sensitive to 14,15-EEZE. Moreover, while acute hypoxia and 11,12-EET increased pulmonary pressure in lungs from TRPC6(+/-) mice, lungs from TRPC6(-/-) mice did not respond to either stimuli. These data demonstrate that CYP-derived EETs are involved in HPV and that EET-induced pulmonary contraction under normoxic and hypoxic conditions involves a TRPC6-dependent pathway.

Role of cytochrome P450 2C epoxygenases in hypoxia-induced cell migration and angiogenesis in retinal endothelial cells.

PURPOSE: Cytochrome P450 (CYP) epoxygenase-derived epoxyeicosatrienoic acids (EETs) elicit cell proliferation and promote angiogenesis. The aim of this study was to determine the expression of CYP epoxygenases in the bovine retina and the potential role of EETs in hypoxia-induced angiogenesis in bovine retinal endothelial cells. METHODS: Bovine retinal endothelial cells were cultured under normoxic (21% O(2)) or hypoxic (1% O(2)) conditions, and CYP2C expression was determined by Western blot analysis. The effect of hypoxia on EET levels was determined by LC-MS/MS. Cell migration (Transwell filter assays) and endothelial cell tube formation (on basement membrane matrix) were assessed in vitro in the absence and presence of pharmacologic inhibitors and CYP2C antisense oligonucleotides. RESULTS: Bovine retinal endothelial cells expressed CYP2C protein in culture and generated detectable levels of EETs under basal conditions. Hypoxia (6-48 hours) enhanced CYP2C protein expression (2-fold) and EET formation (1.5-fold). Moreover, endothelial cells preexposed to hypoxia demonstrated an increase in serum-induced cell migration that was sensitive to the CYP2C inhibitors sulfaphenazole and MS-PPOH and the EET antagonist 14,15-epoxyeicosa-5(Z)-enoic acid. Furthermore, preventing the hypoxia-induced expression of CYP2C (antisense oligonucleotides) suppressed hypoxia-induced cell migration. In an in vitro angiogenesis model, the preexposure of endothelial cells to hypoxia increased CYP2C expression and enhanced endothelial tube formation, which was blocked by the EET antagonist and by the CYP2C antisense oligonucleotides. CONCLUSIONS: Taken together, these data indicate that CYP2C-derived EETs are implicated in angiogenesis by retinal endothelial cells, especially under hypoxic conditions.

Mouse Cyp4a isoforms: enzymatic properties, gender- and strain-specific expression, and role in renal 20-hydroxyeicosatetraenoic acid formation.

AA (arachidonic acid) hydroxylation to 20-HETE (20-hydroxyeicosatetraenoic acid) influences renal vascular and tubular function. To identify the CYP (cytochrome P450) isoforms catalysing this reaction in the mouse kidney, we analysed the substrate specificity of Cyp4a10, 4a12a, 4a12b and 4a14 and determined sex- and strain-specific expressions. All recombinant enzymes showed high lauric acid hydroxylase activities. Cyp4a12a and Cyp4a12b efficiently hydroxylated AA to 20-HETE with V(max) values of approx. 10 nmol x nmol(-1) x min(-1) and K(m) values of 20-40 microM. 20-Carboxyeicosatetraenoic acid occurred as a secondary metabolite. AA hydroxylase activities were approx. 25-75-fold lower with Cyp4a10 and not detectable with Cyp4a14. Cyp4a12a and Cyp4a12b also efficiently converted EPA (eicosapentaenoic acid) into 19/20-OH- and 17,18-epoxy-EPA. In male mice, renal microsomal AA hydroxylase activities ranged between approx. 100 (NMRI), 45-55 (FVB/N, 129 Sv/J and Balb/c) and 25 pmol x min(-1) x mg(-1) (C57BL/6). The activities correlated with differences in Cyp4a12a protein and mRNA levels. Treatment with 5alpha-dihydrotestosterone induced both 20-HETE production and Cyp4a12a expression more than 4-fold in male C57BL/6 mice. All female mice showed low AA hydroxylase activities (15-25 pmol x min(-1) x mg(-1)) and very low Cyp4a12a mRNA and protein levels, but high Cyp4a10 and Cyp4a14 expression. Renal Cyp4a12b mRNA expression was almost undetectable in both sexes of all strains. Thus Cyp4a12a is the predominant 20-HETE synthase in the mouse kidney. Cyp4a12a expression determines the sex- and strain-specific differences in 20-HETE generation and may explain sex and strain differences in the susceptibility to hypertension and target organ damage.

Cognitive phenotype in ataxia-telangiectasia.

BACKGROUND: Pediatric cerebrocerebellar neurodegenerative disorders such as ataxia-telangiectasia (AT) have not been examined in detail for neuropsychologic changes. Such studies may contribute to the further understanding of ataxia-telangiectasia and to the role of the cerebrocerebellar system in the development of cognitive function in childhood. METHODS: Twenty-two patients with the classic phenotype of ataxia-telangiectasia were grouped into early stage cerebellar disease (group AT-I) versus late stage cerebrocerebellar disease (group AT-II) and examined for neurocognitive features. Results were compared with those of healthy control subjects and with standard norms. RESULTS: Patients in AT-I group scored low average compared with standard norms on all tests and were impaired compared with healthy control subjects for verbal intelligence quotient (P < 0.001), vocabulary and comprehension (P = 0.007), processing speed (P = 0.005), visuospatial processing (P = 0.020), and working memory (P = 0.046). Patients in AT-II group scored below average compared with standard norms on all tests and were impaired compared with control subjects for attention (P < 0.001), working memory (P < 0.001), and abstract reasoning (P < 0.001). Comprehension scores were lower for patients in AT-II than in AT-I group (P = 0.002), whereas vocabulary scores showed no difference between groups (P = 0.480). CONCLUSION: Cognitive impairments in ataxia-telangiectasia present early, coinciding with cerebellar pathology and are characteristic of the cerebellar cognitive affective syndrome. Widespread and deeper cognitive deficits manifest in later stages of ataxia-telangiectasia when additional noncerebellar pathology develops. These results are the first indications of distinct cerebellar and extracerebellar and/or subcortical contributions to the range of cognitive domains affected in ataxia-telangiectasia and need to be confirmed in future studies.

Hypoxic pulmonary vasoconstriction requires connexin 40-mediated endothelial signal conduction.

Hypoxic pulmonary vasoconstriction (HPV) is a physiological mechanism by which pulmonary arteries constrict in hypoxic lung areas in order to redirect blood flow to areas with greater oxygen supply. Both oxygen sensing and the contractile response are thought to be intrinsic to pulmonary arterial smooth muscle cells. Here we speculated that the ideal site for oxygen sensing might instead be at the alveolocapillary level, with subsequent retrograde propagation to upstream arterioles via connexin 40 (Cx40) endothelial gap junctions. HPV was largely attenuated by Cx40-specific and nonspecific gap junction uncouplers in the lungs of wild-type mice and in lungs from mice lacking Cx40 (Cx40-/-). In vivo, hypoxemia was more severe in Cx40-/- mice than in wild-type mice. Real-time fluorescence imaging revealed that hypoxia caused endothelial membrane depolarization in alveolar capillaries that propagated to upstream arterioles in wild-type, but not Cx40-/-, mice. Transformation of endothelial depolarization into vasoconstriction involved endothelial voltage-dependent α1G subtype Ca2+ channels, cytosolic phospholipase A2, and epoxyeicosatrienoic acids. Based on these data, we propose that HPV originates at the alveolocapillary level, from which the hypoxic signal is propagated as endothelial membrane depolarization to upstream arterioles in a Cx40-dependent manner.

Hypoxia-induced pulmonary hypertension: comparison of soluble epoxide hydrolase deletion vs. inhibition.

AIMS: The C-terminal domain of the soluble epoxide hydrolase (sEH) metabolizes epoxyeicosatrienoic acids (EETs) to their less active diols, while the N-terminal domain demonstrates lipid phosphatase activity. As EETs are potent vasoconstrictors in the pulmonary circulation, we assessed the development of pulmonary hypertension induced by exposure to hypoxia (10% O(2)) for 21 days in wild-type (WT) and sEH(-/-) mice and compared the effects with chronic (4 months) sEH inhibition. METHODS AND RESULTS: In isolated lungs from WT mice, acute hypoxic vasoconstriction (HPV) was potentiated by sEH inhibition and attenuated by an EET antagonist. After prolonged hypoxia, the acute HPV and sensitivity to the EET antagonist were increased, but potentiation of vasoconstriction following sEH inhibition was not evident. Chronic hypoxia also stimulated the muscularization of pulmonary arteries and decreased sEH expression in WT mice. In normoxic sEH(-/-) mice, acute HPV and small artery muscularization were greater than that in WT lungs and enhanced muscularization was accompanied with decreased voluntary exercise capacity. Acute HPV in sEH(-/-) mice was insensitive to sEH inhibition but inhibited by the EET antagonist and chronic hypoxia induced an exaggerated pulmonary vascular remodelling. In WT mice, chronic sEH inhibition increased serum EET levels but failed to affect acute HPV, right ventricle weight, pulmonary artery muscularization, or voluntary running distance. In human donor lungs, the sEH was expressed in the wall of pulmonary arteries, however, sEH expression was absent in samples from patients with pulmonary hypertension. CONCLUSION: These data suggest that a decrease in sEH expression is intimately linked to pathophysiology of hypoxia-induced pulmonary remodelling and hypertension. However, as sEH inhibitors do not promote the development of pulmonary hypertension it seems likely that the N-terminal lipid phosphatase may play a role in the development of this disease.

Increased cytochrome P4502E1 expression and altered hydroxyeicosatetraenoic acid formation mediate diabetic vascular dysfunction: rescue by guanylyl-cyclase activation.

OBJECTIVE: We investigated the mechanisms underlying vascular endothelial and contractile dysfunction in diabetes as well as the effect of HMR1766, a novel nitric oxide (NO)-independent activator of soluble guanylyl cyclase (sGC). RESEARCH DESIGN AND METHODS: Two weeks after induction of diabetes by streptozotocin, Wistar rats received either placebo or HMR1766 (10 mg/kg twice daily) for another 2 weeks; thereafter, vascular function was assessed. RESULTS: Endothelial function and contractile responses were significantly impaired, while vascular superoxide formation was increased in the aortae from diabetic versus healthy control rats. Using RNA microarrays, cytochrome P4502E1 (CYP2E1) was identified as the highest upregulated gene in diabetic aorta. CYP2E1 protein was significantly increased (16-fold) by diabetes, leading to a reduction in levels of the potent vasoconstrictor 20-hydroxy-eicosatetraenoic acid (20-HETE). Induction of CYP2E1 expression in healthy rats using isoniazide mimicked the diabetic noncontractile vascular response while preincubation of aortae from STZ-diabetic rats in vitro with 20-HETE rescued contractile function. Chronic treatment with the sGC activator HMR1766 improved NO sensitivity and endothelial function, reduced CYP2E1 expression and superoxide formation, enhanced 20-HETE levels, and reversed the contractile deficit observed in the diabetic rats that received placebo. CONCLUSIONS: Upregulation of CYP2E1 is essentially involved in diabetic vascular dysfunction. Chronic treatment with the sGC activator HMR1766 reduced oxidative stress, decreased CYP2E1 levels, and normalized vasomotor function in diabetic rats.

Inhibition of the soluble epoxide hydrolase promotes albuminuria in mice with progressive renal disease.

Epoxyeicotrienoic acids (EETs) are cytochrome P450-dependent anti-hypertensive and anti-inflammatory derivatives of arachidonic acid, which are highly abundant in the kidney and considered reno-protective. EETs are degraded by the enzyme soluble epoxide hydrolase (sEH) and sEH inhibitors are considered treatment for chronic renal failure (CRF). We determined whether sEH inhibition attenuates the progression of CRF in the 5/6-nephrectomy model (5/6-Nx) in mice. 5/6-Nx mice were treated with a placebo, an ACE-inhibitor (Ramipril, 40 mg/kg), the sEH-inhibitor cAUCB or the CYP-inhibitor fenbendazole for 8 weeks. 5/6-Nx induced hypertension, albuminuria, glomerulosclerosis and tubulo-interstitial damage and these effects were attenuated by Ramipril. In contrast, cAUCB failed to lower the blood pressure and albuminuria was more severe as compared to placebo. Plasma EET-levels were doubled in 5/6 Nx-mice as compared to sham mice receiving placebo. Renal sEH expression was attenuated in 5/6-Nx mice but cAUCB in these animals still further increased the EET-level. cAUCB also increased 5-HETE and 15-HETE, which derive from peroxidation or lipoxygenases. Similar to cAUCB, CYP450 inhibition increased HETEs and promoted albuminuria. Thus, sEH-inhibition failed to elicit protective effects in the 5/6-Nx model and showed a tendency to aggravate the disease. These effects might be consequence of a shift of arachidonic acid metabolism into the lipoxygenase pathway.

Apocynin is not an inhibitor of vascular NADPH oxidases but an antioxidant.

A large body of literature suggest that vascular reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidases are important sources of reactive oxygen species. Many studies, however, relied on data obtained with the inhibitor apocynin (4'-hydroxy-3'methoxyacetophenone). Because the mode of action of apocynin, however, is elusive, we determined its mechanism of inhibition on vascular NADPH oxidases. In HEK293 cells overexpressing NADPH oxidase isoforms (Nox1, Nox2, or Nox4), apocynin failed to inhibit superoxide anion generation detected by lucigenin chemiluminescence. In contrast, apocynin interfered with the detection of reactive oxygen species in assay systems selective for hydrogen peroxide or hydroxyl radicals. Importantly, apocynin interfered directly with the detection of peroxides but not superoxide, if generated by xanthine/xanthine oxidase or nonenzymatic systems. In leukocytes, apocynin is a prodrug that is activated by myeloperoxidase, a process that results in the formation of apocynin dimers. Endothelial cells and smooth muscle cells failed to form these dimers and, therefore, are not able to activate apocynin. Dimer formation was, however, observed in Nox-overexpressing HEK293 cells when myeloperoxidase was supplemented. As a consequence, apocynin should only inhibit NADPH oxidase in leukocytes, whereas in vascular cells, the compound could act as an antioxidant. Indeed, in vascular smooth muscle cells, the activation of the redox-sensitive kinases p38-mitogen-activate protein kinase, Akt, and extracellular signal-regulated kinase 1/2 by hydrogen peroxide and by the intracellular radical generator menadione was prevented in the presence of apocynin. These observations indicate that apocynin predominantly acts as an antioxidant in endothelial cells and vascular smooth muscle cells and should not be used as an NADPH oxidase inhibitor in vascular systems.

Epoxyeicosatrienoic acids are part of the VEGF-activated signaling cascade leading to angiogenesis.

Cytochrome P-450 (CYP) epoxygenases metabolize arachidonic acid to epoxyeicosatrienoic acid (EET) regioisomers, which activate several signaling pathways to promote endothelial cell proliferation, migration, and angiogenesis. Since vascular endothelial growth factor (VEGF) plays a key role in angiogenesis, we assessed a possible role of EETs in the VEGF-activated signal transduction cascade. Stimulation with VEGF increased CYP2C promoter activity in endothelial cells and enhanced CYP2C8 mRNA and protein expression resulting in increased intracellular EET levels. VEGF-induced endothelial cell tube formation was inhibited by the EET antagonist 14,15-epoxyeicosa-5(Z)-enoicacid (14,15-EEZE), which did not affect the VEGF-induced phosphorylation of its receptor or basic fibroblast growth factor (bFGF)-stimulated tube formation. Moreover, VEGF-stimulated endothelial cell sprouting in a modified spheroid assay was reduced by CYP2C antisense oligonucleotides. Mechanistically, VEGF stimulated the phosphorylation of the AMP-activated protein kinase (AMPK), which has also been linked to CYP induction, and the overexpression of a constitutively active AMPK mutant increased CYP2C expression. On the other hand, a dominant-negative AMPK mutant prevented the VEGF-induced increase in CYP2C RNA and protein expression in human endothelial cells. In vivo (Matrigel plug assay) in mice, endothelial cells were recruited into VEGF-impregnated plugs; an effect that was sensitive to 14,15-EEZE and the inclusion of small interfering RNA directed against the AMPK. The EET antagonist did not affect responses observed in plugs containing bFGF. Taken together, our data indicate that CYP2C-derived EETs participate as second messengers in the angiogenic response initiated by VEGF and that preventing the increase in CYP expression curtails the angiogenic response to VEGF.

Cytochrome P450 epoxygenase gene function in hypoxic pulmonary vasoconstriction and pulmonary vascular remodeling.

We assessed pulmonary cytochrome P450 (CYP) epoxygenase expression and activity during hypoxia and explored the effects of modulating epoxygenase activity on pulmonary hypertension. The acute hypoxic vasoconstrictor response was studied in Swiss Webster mice, who express CYP2C29 in their lungs. Animals were pretreated with vehicle, the epoxygenase inhibitor (N-methylsulfonyl-6-[2-propargyloxyphenyl] hexanamide) or an inhibitor of the soluble epoxide hydrolase. Whereas the epoxygenase inhibitor attenuated hypoxic pulmonary constriction (by 52%), the soluble epoxide hydrolase inhibitor enhanced the response (by 39%), indicating that CYP epoxygenase-derived epoxyeicosatrienoic acids elicit pulmonary vasoconstriction. Aerosol gene transfer of recombinant adenovirus containing the human CYP2C9 significantly elevated mean pulmonary artery pressure and total pulmonary resistance indices, both of which were sensitive to the inhibitor sulfaphenazole. The prolonged exposure of mice to hypoxia increased CYP2C29 expression, and transcript levels increased 5-fold after exposure to normobaric hypoxia (FIO2 0.07) for 2 hours. This was followed by a 2-fold increase in protein expression and by a significant increase in epoxyeicosatrienoic acid production after 24 hours. Chronic hypoxia (7 days) elicited pulmonary hypertension and pulmonary vascular remodeling, effects that were significantly attenuated in animals continually treated with N-methylsulfonyl-6-[2-propargyloxyphenyl] hexanamide (-46% and -55%, respectively). Our results indicate that endogenously generated epoxygenase products are associated with hypoxic pulmonary hypertension in mice and that selective epoxygenase inhibition significantly reduces acute hypoxic pulmonary vasoconstriction and chronic hypoxia-induced pulmonary vascular remodeling. These observations indicate potential novel targets for the treatment of pulmonary hypertension and highlight a pivotal role for CYP epoxygenases in pulmonary responses to hypoxia.

Cytochrome P450 epoxygenases 2C8 and 2C9 are implicated in hypoxia-induced endothelial cell migration and angiogenesis.

Recent studies suggest that cytochrome P450 (CYP) epoxygenase-derived epoxyeicosatrienoic acids (EETs) elicit cell proliferation and promote angiogenesis. The aim of this study was to determine the role of CYP 2C8/9-derived EETs in the process of angiogenesis under hypoxic conditions. In human endothelial cells, hypoxia enhanced the activity of the CYP 2C9 promoter, increased the expression of CYP 2C mRNA and protein and augmented 11,12-EET production. In Transwell assays, the migration of endothelial cells pre-exposed to hypoxia to increase CYP expression was abolished by CYP 2C antisense oligonucleotides as well as by the CYP inhibitor MS-PPOH and the EET antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (EEZE). Similar findings were obtained in porcine coronary artery endothelial cells. CYP 2C9 overexpression in endothelial cells increased the association of PAK-1 with Rac, a response also elicited by the CYP 2C9 product 11,12-EET. Matrix metalloprotease (MMP) activity was increased in CYP-2C9-overexpressing cells and correlated with increased invasion through Matrigel-coated Transwell chambers: an effect sensitive to the CYP 2C9 inhibitor sulfaphenazole as well as to EEZE and the MMP inhibitor GM6001. In in vitro angiogenesis models, the EET antagonist inhibited tube formation induced by CYP 2C9 overexpression as well as that in endothelial cells exposed to hypoxia to increase CYP 2C expression. Furthermore, in the chick chorioallantoic membrane assay, EEZE abolished hypoxia-induced angiogenesis. Taken together, these data indicate that CYP 2C-derived EETs significantly affect the sequence of angiogenic events under hypoxic conditions.

Eicosapentaenoic acid metabolism by cytochrome P450 enzymes of the CYP2C subfamily.

CYP2C enzymes epoxidize arachidonic acid (AA) to metabolites involved in the regulation of vascular and renal function. We tested the hypothesis that eicosapentaenoic acid (EPA), a n-3 polyunsaturated fatty acid, may serve as an alternative substrate. Human CYP2C8 and CYP2C9, as well as rat CYP2C11 and CYP2C23, were co-expressed with NADPH-CYP reductase in a baculovirus/insect cell system. The recombinant enzymes showed high EPA and AA epoxygenase activities and the catalytic efficiencies were almost equal comparing the two substrates. The 17,18-double bond was the preferred site of EPA epoxidation by CYPs 2C8, 2C11, and 2C23. 17(R),18(S)-Epoxyeicosatetraenoic acid was produced with an optical purity of about 70% by CYPs 2C9, 2C11, and 2C23 whereas CYP2C8 showed the opposite enantioselectivity. These results demonstrate that EPA is an efficient substrate of CYP2C enzymes and suggest that n-3 PUFA-rich diets may shift the CYP2C-dependent generation of physiologically active eicosanoids from AA- to EPA-derived metabolites.