Elevated 20-HETE in metabolic syndrome regulates arterial stiffness and systolic hypertension via MMP12 activation.

Arterial stiffness plays a causal role in development of systolic hypertension. 20-hydroxyeicosatetraeonic acid (20-HETE), a cytochrome P450 (CYP450)-derived arachidonic acid metabolite, is known to be elevated in resistance arteries in hypertensive animal models and loosely associated with obesity in humans. However, the role of 20-HETE in the regulation of large artery remodeling in metabolic syndrome has not been investigated. We hypothesized that elevated 20-HETE in metabolic syndrome increases matrix metalloproteinase 12 (MMP12) activation leading to increased degradation of elastin, increased large artery stiffness and increased systolic blood pressure. 20-HETE production was increased ~7 fold in large, conduit arteries of metabolic syndrome (JCR:LA-cp, JCR) vs. normal Sprague-Dawley (SD) rats. This correlated with increased elastin degradation (~7 fold) and decreased arterial compliance (~75% JCR vs. SD). 20-HETE antagonists blocked elastin degradation in JCR rats concomitant with blocking MMP12 activation. 20-HETE antagonists normalized, and MMP12 inhibition (pharmacological and MMP12-shRNA-Lnv) significantly improved (~50% vs. untreated JCR) large artery compliance in JCR rats. 20-HETE antagonists also decreased systolic (182 ±â€¯3 mmHg JCR, 145 ±â€¯3 mmHg JCR + 20-HETE antagonists) but not diastolic blood pressure in JCR rats. Whereas diastolic pressure was fully angiotensin II (Ang II)-dependent, systolic pressure was only partially Ang II-dependent, and large artery stiffness was Ang II-independent. Thus, 20-HETE-dependent regulation of systolic blood pressure may be a unique feature of metabolic syndrome related to high 20-HETE production in large, conduit arteries, which results in increased large artery stiffness and systolic blood pressure. These findings may have implications for management of systolic hypertension in patients with metabolic syndrome.

Harmonizing lipidomics: NIST interlaboratory comparison exercise for lipidomics using SRM 1950-Metabolites in Frozen Human Plasma.

As the lipidomics field continues to advance, self-evaluation within the community is critical. Here, we performed an interlaboratory comparison exercise for lipidomics using Standard Reference Material (SRM) 1950-Metabolites in Frozen Human Plasma, a commercially available reference material. The interlaboratory study comprised 31 diverse laboratories, with each laboratory using a different lipidomics workflow. A total of 1,527 unique lipids were measured across all laboratories and consensus location estimates and associated uncertainties were determined for 339 of these lipids measured at the sum composition level by five or more participating laboratories. These evaluated lipids detected in SRM 1950 serve as community-wide benchmarks for intra- and interlaboratory quality control and method validation. These analyses were performed using nonstandardized laboratory-independent workflows. The consensus locations were also compared with a previous examination of SRM 1950 by the LIPID MAPS consortium. While the central theme of the interlaboratory study was to provide values to help harmonize lipids, lipid mediators, and precursor measurements across the community, it was also initiated to stimulate a discussion regarding areas in need of improvement.

Elevated 20-HETE impairs coronary collateral growth in metabolic syndrome via endothelial dysfunction.

Coronary collateral growth (CCG) is impaired in metabolic syndrome (MetS). microRNA-145 (miR-145-Adv) delivery to our rat model of MetS (JCR) completely restored and neutrophil depletion significantly improved CCG. We determined whether low endogenous levels of miR-145 in MetS allowed for elevated production of 20-hydroxyeicosatetraenoic acid (20-HETE), which, in turn, resulted in excessive neutrophil accumulation and endothelial dysfunction leading to impaired CCG. Rats underwent 0-9 days of repetitive ischemia (RI). RI-induced cardiac CYP4F (neutrophil-specific 20-HETE synthase) expression and 20-HETE levels were increased (4-fold) in JCR vs. normal rats. miR-145-Adv and 20-HETE antagonists abolished and neutrophil depletion (blocking antibodies) reduced (~60%) RI-induced increases in CYP4F expression and 20-HETE production in JCR rats. Impaired CCG in JCR rats (collateral-dependent blood flow using microspheres) was completely restored by 20-HETE antagonists [collateral-dependent zone (CZ)/normal zone (NZ) flow ratio was 0.76 ± 0.07 in JCR + 20-SOLA, 0.84 ± 0.05 in JCR + 20-HEDGE vs. 0.11 ± 0.02 in JCR vs. 0.84 ± 0.03 in normal rats]. In JCR rats, elevated 20-HETE was associated with excessive expression of endothelial adhesion molecules and neutrophil infiltration, which were reversed by miR-145-Adv. Endothelium-dependent vasodilation of coronary arteries, endothelial nitric oxide synthase (eNOS) Ser1179 phosphorylation, eNOS-dependent NO·- production and endothelial cell survival were compromised in JCR rats. These parameters of endothelial dysfunction were completely reversed by 20-HETE antagonism or miR-145-Adv delivery, whereas neutrophil depletion resulted in partial reversal (~70%). We conclude that low miR-145 in MetS allows for increased 20-HETE, mainly from neutrophils, which compromises endothelial cell survival and function leading to impaired CCG. 20-HETE antagonists could provide viable therapy for restoration of CCG in MetS.NEW & NOTEWORTHY Elevated 20-hydroxyeicosatetraenoic acid (20-HETE) impairs coronary collateral growth (CCG) in metabolic syndrome by eliciting endothelial dysfunction and apoptosis via excessive neutrophil infiltration. 20-HETE antagonists completely restore coronary collateral growth in metabolic syndrome. microRNA-145 (miR-145) is an upstream regulator of 20-HETE production in metabolic syndrome; low expression of miR-145 in metabolic syndrome promotes elevated production of 20-HETE.

Cyp2c44 gene disruption exacerbated pulmonary hypertension and heart failure in female but not male mice.

Epoxyeicosatrienoicacids (EETs), synthesized from arachidonic acid by epoxygenases of the CYP2C and CYP2J gene subfamilies, contribute to hypoxic pulmonary vasoconstriction (HPV) in mice. Despite their roles in HPV, it is controversial whether EETs mediate or ameliorate pulmonary hypertension (PH). A recent study showed that deficiency of Cyp2j did not protect male and female mice from hypoxia-induced PH. Since CYP2C44 is a functionally important epoxygenase, we hypothesized that knockout of the Cyp2c44 gene would protect both sexes of mice from hypoxia-induced PH. We tested this hypothesis in wild-type (WT) and Cyp2c44 knockout (Cyp2c44 (-/-)) mice exposed to normoxia (room air) and hypoxia (10% O2) for 5 weeks. Exposure of WT and Cyp2c44 (-/-) mice to hypoxia resulted in pulmonary vascular remodeling, increased pulmonary artery resistance, and decreased cardiac function in both sexes. However, in female Cyp2c44 (-/-) mice, compared with WT mice, (1) pulmonary artery resistance and right ventricular hypertrophy were greater, (2) cardiac index was lower, (3) left ventricular and arterial stiffness were higher, and (4) plasma aldosterone levels were higher, but (5) there was no difference in levels of EET in lungs and heart. Paradoxically and unexpectedly, we found that Cyp2c44 disruption exacerbated hypoxia-induced PH in female but not male mice. We attribute exacerbated PH in female Cyp2c44 (-/-) mice to elevated aldosterone and as-yet-unknown systemic factors. Therefore, we suggest a role for the human CYP2C genes in protecting women from severe PH and that this could be one of the underlying causes for a better 5-year survival rate in women than in men.

Oxidized HDL is a potent inducer of adipogenesis and causes activation of the Ang-II and 20-HETE systems in human obese females.

BACKGROUND: Oxidized-HDL (OX-HDL) has been reported to increase coronary events in obese patients; however, OX-HDL has not been studied in subjects with the metabolic syndrome. A high body mass index (BMI) correlates positively with higher levels of metabolic syndrome biomarkers including vasoconstrictors and adipokines. We hypothesize that a subject with a high BMI would present with higher levels of OX-HDL, 20-HETE and Angiotensin II (Ang II) with a reciprocal reduction in serum adiponectin. METHODS: Female subjects with a BMI of 17-25 and a BMI of 30-40, without overt cardiovascular disease, were enrolled in the study. All patients had a history and physical exam documenting the absence of signs and symptoms of cardiovascular disease. Appropriate screening was done and documented. Blood pressure was taken at two discrete points. The BP data are presented as the average. Changes in the relationship between BMI, OX-HDL, 20-HETE, Ang II, TNFα, isoprostane and adiponectin were examined. In addition, the effects of OX-HDL, 20-HETE and Ang II on adipogenesis were examined in human MSC derived adipocytes. RESULTS: Subjects with a high BMI>30 displayed an increase in OX-HDL and isoprostane (P<0.05) compared to those with the lower BMI<25 which was associated with an increase in Ang II and 20-HETE (p<0.05). Serum TNFα levels increased in subjects with a high BMI, compared to subjects with the lower BMI (p<0.05). In contrast, adiponectin levels were increased in subjects with a low BMI compared to obese subjects (p<0.05). In MSC derived adipocytes OX-HDL increased adipogenesis 6 fold at a concentration of 50ng compared to untreated adipocytes. Adipocytes treated with Ang II and 20-HETE also displayed increased adipogenesis (p<0.05), which was attenuated by endogenous increases of the anti-oxidant heme oxygenase-1. Our study demonstrates that OX-HDL presents a unique inflammatory biomarker profile in obese females with the metabolic syndrome at risk for developing cardiovascular disease. CONCLUSIONS: Females with increased BMI (30-40) exhibit a marked increase in OX-HDL and isoprostane levels, which was associated with an increase in 20-HETE, TNF α and Ang II and decreased levels of adiponectin when compared to a group with a low BMI. OX-HDL had a more powerful adipogenic effect when compared to 20-HETE and Ang II. Our study demonstrates that OX-HDL presents a unique inflammatory biomarker profile in obese females with the metabolic syndrome at risk for developing cardiovascular disease. This represents a novel mechanism by which females with a high BMI and controlled blood pressure remain "at risk" for the development of the metabolic syndrome as a result of increased adipogenesis by OX-HDL and activation of the 20-HETE and Ang II systems.

20-HETE contributes to ischemia-induced angiogenesis.

Angiogenesis is an important adaptation for recovery from peripheral ischemia. Here, we determined whether 20-hydroxyeicosatetraenoic acid (20-HETE) contributes to ischemia-induced angiogenesis and assessed its underlying molecular and cellular mechanisms using a mouse hindlimb-ischemia angiogenesis model. Hindlimb blood flow was measured by Laser Doppler Perfusion Imaging and microvessel density was determined by CD31 and tomato lectin staining. We found that systemic and local administration of a 20-HETE synthesis inhibitor, DDMS, or a 20-HETE antagonist, 6,15-20-HEDGE significantly reduced blood flow recovery and microvessel formation in response to ischemia. 20-HETE production, measured by LC/MS/MS, was markedly increased in ischemic muscles (91±11 vs. 8±2pg/mg in controls), which was associated with prominent upregulation of the 20-HETE synthase, CYP4A12. Immunofluorescence co-localized increased CYP4A12 expression in response to ischemia to CD31-positive EC in the ischemic hindlimb microvessels. We further showed that ischemia increased HIF-1α, VEGF, and VEGFR2 expression in gracilis muscles and that these increases were negated by DDMS and 6,15-20-HEDGE. Lastly, we showed that ERK1/2 of MAPK is a component of 20-HETE regulated ischemic angiogenesis. Taken together, these data indicate that 20-HETE is a critical contributor of ischemia-induced angiogenesis in vivo.

20-HETE-induced mitochondrial superoxide production and inflammatory phenotype in vascular smooth muscle is prevented by glucose-6-phosphate dehydrogenase inhibition.

20-Hydroxyeicosatetraeonic acid (20-HETE) produced by cytochrome P-450 monooxygenases in NADPH-dependent manner is proinflammatory, and it contributes to the pathogenesis of systemic and pulmonary hypertension. In this study, we tested the hypothesis that inhibition of glucose-6-phosphate dehydrogenase (G6PD), a major source of NADPH in the cell, prevents 20-HETE synthesis and 20-HETE-induced proinflammatory signaling that promotes secretory phenotype of vascular smooth muscle cells. Lipidomic analysis indicated that G6PD inhibition and knockdown decreased 20-HETE levels in pulmonary arteries as well as 20-HETE-induced 1) mitochondrial superoxide production, 2) activation of mitogen-activated protein kinase 1 and 3, 3) phosphorylation of ETS domain-containing protein Elk-1 that activate transcription of tumor necrosis factor-α gene (Tnfa), and 4) expression of tumor necrosis factor-α (TNF-α). Moreover, inhibition of G6PD increased protein kinase G1α activity, which, at least partially, mitigated superoxide production and Elk-1 and TNF-α expression. Additionally, we report here for the first time that 20-HETE repressed miR-143, which suppresses Elk-1 expression, and miR-133a, which is known to suppress synthetic/secretory phenotype of vascular smooth muscle cells. In summary, our findings indicate that 20-HETE elicited mitochondrial superoxide production and promoted secretory phenotype of vascular smooth muscle cells by activating MAPK1-Elk-1, all of which are blocked by inhibition of G6PD.

Angiotensin II receptor blockade or deletion of vascular endothelial ACE does not prevent vascular dysfunction and remodeling in 20-HETE-dependent hypertension.

Increased vascular 20-HETE is associated with hypertension and activation of the renin-angiotensin system (RAS) through induction of vascular angiotensin-converting enzyme (ACE) expression. Cyp4a12tg mice, whose Cyp4a12-20-HETE synthase expression is under the control of a tetracycline (doxycycline, DOX) promoter, were used to assess the contribution of ACE/RAS to microvascular remodeling in 20-HETE-dependent hypertension. Treatment of Cyp4a12tg mice with DOX increased systolic blood pressure (SBP; 136 ± 2 vs. 102 ± 1 mmHg; P < 0.05), and this increase was prevented by administration of 20-HEDGE, lisinopril, or losartan. DOX-induced hypertension was associated with microvascular dysfunction and remodeling of preglomerular microvessels, which was prevented by 20-HEDGE, a 20-HETE antagonist, yet only lessened, but not prevented, by lisinopril or losartan. In ACE 3/3 mice, which lack vascular endothelial ACE, administration of 5α-dihydrotestosterone (DHT), a known inducer of 20-HETE production, increased SBP; however, the increase was about 50% of that in wild-type (WT) mice (151 ± 1 vs. 126 ± 1 mmHg). Losartan and 20-HEDGE prevented the DHT-induced increase in SBP in WT and ACE 3/3 mice. DHT treatment increased 20-HETE production and microvascular remodeling in WT and ACE 3/3 mice; however, remodeling was attenuated in the ACE 3/3 mice as opposed to WT mice (15.83 ± 1.11 vs. 22.17 ± 0.92 µm; P < 0.05). 20-HEDGE prevented microvascular remodeling in WT and ACE 3/3 mice, while losartan had no effect on microvascular remodeling in ACE 3/3. Taken together, these results suggest that RAS contributes to 20-HETE-mediated microvascular remodeling in hypertension and that 20-HETE-driven microvascular remodeling independent of blood pressure elevation does not fully rely on ACE activity in the vascular endothelium.

Androgen-induced hypertension in angiotensinogen deficient mice: role of 20-HETE and EETS.

20-HETE is a potent inducer of endothelial ACE in vitro and administration of lisinopril or losartan attenuates blood pressure in models of 20-HETE-dependent hypertension. The present study was undertaken to further define the relationship between 20-HETE and the renin-angiotensin system in hypertension using an angiotensinogen-deficient mouse (Agt+/-). Treatment of male AGT+/- with 5α-dihydrotestosterone (DHT) increased systolic BP from 102±2 to 125±3mmHg; in comparison, the same treatment raised BP in wild type (WT) from 110±2 to 138±2mmHg. DHT increased vascular 20-HETE levels in AGT+/- and WT from 1.5±0.7 and 2.1±0.6 to 13.0±2.0 and 15.8±4.0ng/mg, respectively. Concurrent treatment with the 20-HETE antagonist, 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (20-HEDE) prevented the increases in BP in both AGT+/- and WT mice. Administration of 20-HEDE at the peak of the DHT-induced BP increase (12 days) reduced BP to basal levels after 48h. Interestingly, basal levels of renal microvascular EETs were higher in AGT+/- compared to WT (55.2±9.7 vs 20.0±4.1ng/mg) and treatment of AGT+/- with DHT decreased the levels of EETs (28.4±5.1ng/mg). DHT-mediated changes in vascular EET level were not observed in WT mice. Vascular Cyp4a12 and ACE protein levels were increased in both AGT+/- and WT by 30-40% and decreased with concomitant administration of 20-HEDE. Lisinopril was as effective as 20-HEDE in preventing DHT-mediated increases in BP in both AGT+/- and WT mice. This study substantiates our previous findings that the RAS plays an important role in 20-HETE-mediated hypertension. It also proposes a novel interaction between 20-HETE and EETs.

Soluble epoxide hydrolase-dependent regulation of myogenic response and blood pressure.

Epoxyeicosatrienoic acids (EETs) are metabolites of arachidonic acid via cytochrome P450 (CYP)/epoxygenases. EETs possess cardioprotective properties and are catalyzed by soluble epoxide hydrolase (sEH) to dihydroxyeicosatrienoic acids (DHETs) that lack vasoactive property. To date, the role of sEH in the regulation of myogenic response of resistant arteries, a key player in the control of blood pressure, remains unknown. To this end, experiments were conducted on sEH-knockout (KO) mice, wild-type (WT) mice, and endothelial nitric oxide synthase (eNOS)-KO mice treated with t-TUCB, a sEH inhibitor, for 4 wk. sEH-KO and t-TUCB-treated mice displayed significantly lower blood pressure, associated with significantly increased vascular EETs and ratio of EETs/DHETs. Pressure-diameter relationships were assessed in isolated and cannulated gracilis muscle arterioles. All arterioles constricted in response to increases in transmural pressure from 60 to 140 mmHg. The myogenic constriction was significantly reduced, expressed as an upward shift of pressure-diameter curve, in arterioles of sEH-KO and t-TUCB-treated eNOS-KO mice compared with their controls. Removal of the endothelium, or treatment of the vessels with PPOH, an inhibitor of EET synthase, restored the attenuated pressure-induced constriction to the levels similar to those observed in their controls but had no effects on control vessels. No difference was observed in the myogenic index, or in the vascular expression of eNOS, CYP2C29 (EET synthase), and CYP4A (20-HETE synthase) among these groups of mice. In conclusion, the increased EET bioavailability, as a function of deficiency/inhibition of sEH, potentiates vasodilator responses that counteract pressure-induced vasoconstriction to lower blood pressure.

20-HETE regulates the angiogenic functions of human endothelial progenitor cells and contributes to angiogenesis in vivo.

Circulating endothelial progenitor cells (EPC) contribute to postnatal neovascularization. We identified the cytochrome P450 4A/F-20-hydroxyeicosatetraenoic acid (CYP4A/F-20-HETE) system as a novel regulator of EPC functions associated with angiogenesis in vitro. Here, we explored cellular mechanisms by which 20-HETE regulates EPC angiogenic functions and assessed its contribution to EPC-mediated angiogenesis in vivo. Results showed that both hypoxia and vascular endothelial growth factor (VEGF) induce CYP4A11 gene and protein expression (the predominant 20-HETE synthases in human EPC), and this is accompanied by an increase in 20-HETE production by ~1.4- and 1.8-fold, respectively, compared with the control levels. Additional studies demonstrated that 20-HETE and VEGF have a synergistic effect on EPC proliferation, whereas 20-HETE antagonist 20-HEDGE or VEGF-neutralizing antibody negated 20-HETE- or VEGF-induced proliferation, respectively. These findings are consistent with the presence of a positive feedback regulation on EPC proliferation between the 20-HETE and the VEGF pathways. Furthermore, we found that 20-HETE induced EPC adhesion to fibronectin and endothelial cell monolayer by 40 ± 5.6 and 67 ± 10%, respectively, which was accompanied by a rapid induction of very late antigen-4 and chemokine receptor type 4 mRNA and protein expression. Basal and 20-HETE-stimulated increases in adhesion were negated by the inhibition of the CYP4A-20-HETE system. Lastly, EPC increased angiogenesis in vivo by 3.6 ± 0.2-fold using the Matrigel plug angiogenesis assay, and these increases were markedly reduced by the local inhibition of 20-HETE system. These results strengthened the notion that 20-HETE regulates the angiogenic functions of EPC in vitro and EPC-mediated angiogenesis in vivo.

Dysregulated heme oxygenase-ferritin system in pterygium pathogenesis.

PURPOSE: Cyclooxygenase (COX)-, lipoxygenase (LOX)-, and cytochrome P450 monooxygenase (CYP)-derived eicosanoids have been implicated in ocular surface inflammation and neovascularization. These eicosanoids are subjected to regulation by enzymes, such as heme oxygenases (HOs) and ferritin. METHODS: Quantitative polymerase chain reaction and lipidomics based on liquid chromatography-tandem mass spectrometry were performed on pterygia from patients undergoing surgical pterygium excision. Control tissues consisted of donor corneas. In addition, lipidomics based on liquid chromatography-tandem mass spectrometry was performed on tears collected from patients before the surgery. RESULTS: Messenger RNA (mRNA) expression of HO-2, the constitutive HO isoform, was upregulated by 40% in pterygia compared with control tissue, whereas the mRNA level of the inducible form, HO-1, was downregulated by more than 50%. Levels of CYP4B1 mRNA showed an approximate 2-fold increase in pterygia compared with control. Lipidomic analysis of tissues indicated a moderate elevation in Prostaglandin E2 and thromboxane B2 levels in pterygia compared with control. Among the LOX-derived metabolites, the antiinflammatory-hydroxyeicosatetraenoic acid (15-HETE) levels were significantly reduced in pterygia (79.3 ± 48.11 pg/mg protein) compared with control (586.2 ± 213.5 pg/mg protein), whereas the proinflammatory LOX- and CYP4B1-derived 12-HETE levels were 10-fold higher in pterygia (2768 ± 832.3 pg/mg protein) compared with control (231.4 ± 87.35 pg/mg protein). Prostaglandin E2 and HETEs were also present in tears from patients with pterygium but were not detected in tears from healthy volunteers. The mRNA expression levels of both light and heavy chain ferritin were 60% and 30% lower, respectively, in pterygia compared with control. CONCLUSIONS: We believe that a dysfunctional HO-ferritin system leads to increased levels of proinflammatory mediators, thus contributing to the inflammation characteristic of pterygia.

Androgen-sensitive hypertension associates with upregulated vascular CYP4A12-20-HETE synthase.

Although the mechanism underlying the effect of androgen on BP and cardiovascular disease is not well understood, recent studies suggest that 20-hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE), a primary cytochrome P450 4 (Cyp4)-derived eicosanoid, may mediate androgen-induced hypertension. Here, treatment of normotensive mice with 5α-dihydrotestosterone increased BP and induced both Cyp4a12 expression and 20-HETE levels in preglomerular microvessels. Administration of a 20-HETE antagonist prevented and reversed the effects of dihydrotestosterone on BP. Cyp4a14(-/-) mice, which exhibit androgen-sensitive hypertension in the male mice, produced increased levels of vascular 20-HETE; furthermore, administration of a 20-HETE antagonist normalized BP. To examine whether androgen-independent increases in 20-HETE are sufficient to cause hypertension, we studied Cyp4a12-transgenic mice, which express the CYP4A12-20-HETE synthase under the control of a doxycycline-sensitive promoter. Administration of doxycycline increased BP by 40%, and administration of a 20-HETE antagonist prevented this increase. Levels of CYP4A12 and 20-HETE in preglomerular microvessels of doxycycline-treated transgenic mice approximately doubled, correlating with increased 20-HETE-dependent sensitivity to phenylephrine-mediated vasoconstriction and with decreased acetylcholine-mediated vasodilation in the renal microvasculature. We observed a similar contribution of 20-HETE to myogenic tone in the mesenteric microvasculature. Taken together, these results suggest that 20-HETE both mediates androgen-induced hypertension and can cause hypertension independent of androgen.

Elevated level of pro-inflammatory eicosanoids and EPC dysfunction in diabetic patients with cardiac ischemia.

BACKGROUND: Circulating endothelial progenitor cells (EPCs) are recruited from the blood system to sites of ischemia and endothelial damage, where they contribute to the repair and development of blood vessels. Since numerous eicosanoids including leukotrienes (LTs) and hydroxyeicosatetraenoic acids (HETEs) have been shown to exert potent pro-inflammatory activities, we examined their levels in chronic diabetic patients with severe cardiac ischemia in conjunction with the level and function of EPCs. RESULTS: Lipidomic analysis revealed a diabetes-specific increase (p<0.05) in inflammatory and angiogenic eicosanoids including the 5-lipoxygenase-derived LTB (4.11±1.17 vs. 0.96±0.27 ng/ml), the lipoxygenase/CYP-derived 12-HETE (117.08±35.05 vs. 24.34±10.03 ng/ml), 12-HETrE (17.56±4.43 vs. 4.15±2.07 ng/ml), and the CYP-derived 20-HETE (0.32±0.04 vs. 0.06±0.05 ng/ml) the level of which correlated with BMI (p=0.0027). In contrast, levels of the CYP-derived EETs were not significantly (p=0.36) different between these two groups. EPC levels and their colony-forming units were lower (p<0.05) with a reduced viability in diabetic patients compared with non-diabetics. EPC function (colony-forming units (CFUs) and MTT assay) also negatively correlated with the circulating levels of HgA1C. CONCLUSION: This study demonstrates a close association between elevated levels of highly pro-inflammatory eicosonoids, diabetes and EPC dysfunction in patients with cardiac ischemia, indicating that chronic inflammation impact negatively on EPC function and angiogenic capacity in diabetes.

Heme oxygenase (HO-1) rescue of adipocyte dysfunction in HO-2 deficient mice via recruitment of epoxyeicosatrienoic acids (EETs) and adiponectin.

BACKGROUND/AIMS: HO-1 and EETs are functionally linked and their interactions influence body weight, insulin sensitivity, and serum levels of inflammatory cytokines in metabolic syndrome phenotype of HO-2 null mice. The HO-2 isozyme is essential for regulating physiological levels of ROS. Recent studies have suggested a potential role of EET in modifying adipocyte differentiation through up-regulation of HO-1-adiponectin-AkT signaling in human mesenchymal stem cells (MSCs). Our aim was to examine the consequences of HO deficiency on MSC-derived adipogenesis in vitro using MSC derived from HO-2 null and WT mice in vivo. METHODS: Four-month-old HO-2 null (HO-2(-/-)) and B6/129SF2/J (WT) mice were divided into three groups (four mice/group): WT, HO-2(-/-), and HO-2(-/-) +CoPP. Adipogenesis was performed on purified MSC-derived adipocytes cultured in adipogenic differentiation media and an EET-agonist was added every 3 days. RESULTS: HO-2 depletion of MSC adipocytes resulted in increased adipogenesis (p<0.01) and increased levels of inflammatory cytokines including (TNF)-alpha (p<0.05), (MCP)-1 (p<0.05), and (IL-1)-beta (p<0.05). These results were accompanied by decreases in HO-1 (p<0.05) and subsequently EET and HO activity (p<0.05). Up-regulation of HO-1 resulted in decreased MSC-derived adipocyte differentiation, decreased production of TNF-alpha and MCP-1 and increased levels of adiponectin (p<0.05). Cyp2J5 (p<0.05), HO-1 (p<0.05), and adiponectin mRNA levels (p<0.05) were also decreased in visceral adipose tissue isolated from HO-2 null compared to WT mice. EET agonist stimulation of MSC adipocytes derived from HO-2 null mice yielded similar results. CONCLUSION: Increased levels of EET and HO-1 are essential for protection against the adverse effects of adipocyte hypertrophy and the ensuing metabolic syndrome. These results offer a portal into therapeutic approaches for the prevention of the metabolic syndrome.

Arachidonate 5 lipoxygenase expression in papillary thyroid carcinoma promotes invasion via MMP-9 induction.

Arachidonate 5-lipoxygenase (ALOX5) expression and activity has been implicated in tumor pathogenesis, yet its role in papillary thyroid carcinoma (PTC) has not been characterized. ALOX5 protein and mRNA were upregulated in PTC compared to matched, normal thyroid tissue, and ALOX5 expression correlated with invasive tumor histopathology. Evidence suggests that PTC invasion is mediated through the induction of matrix metalloproteinases (MMPs) that can degrade and remodel the extracellular matrix (ECM). A correlation between MMP-9 and ALOX5 protein expression was established by immunohistochemical analysis of PTC and normal thyroid tissues using a tissue array. Transfection of ALOX5 into a PTC cell line (BCPAP) increased MMP-9 secretion and cell invasion across an ECM barrier. The ALOX5 product, 5(S)-hydroxyeicosatetraenoic acid also increased MMP-9 protein expression by BCPAP in a dose-dependent manner. Inhibitors of MMP-9 and ALOX5 reversed ALOX5-enhanced invasion. Here we describe a new role for ALOX5 as a mediator of invasion via MMP-9 induction; this ALOX5/MMP9 pathway represents a new avenue in the search for functional biomarkers and/or potential therapeutic targets for aggressive PTC.

Cyp2c44 epoxygenase is essential for preventing the renal sodium absorption during increasing dietary potassium intake.

The aim of this study is to test whether the Cyp2c44 epoxygenase-dependent metabolism of arachidonic acid prevents the hypertensive effect of a high K (HK) intake by inhibiting the epithelial sodium channel (ENaC) activity. A HK intake elevated Cyp2c44 mRNA expression and 11,12-epoxyeicosatrienoic acid levels in the cortical collecting duct in Cyp2c44(+/+) mice (wild-type [wt]). However, an HK intake failed to increase 11,12-epoxyeicosatrienoic acid formation in the cortical collecting ducts of Cyp2c44(-/-) mice. Moreover, increasing K intake enhanced arachidonic acid-induced inhibition of ENaC in the wt but not in Cyp2c44(-/-) mice. In contrast, 11,12-epoxyeicosatrienoic acid, a Cyp2c44 metabolite, inhibited ENaC in the wt and Cyp2c44(-/-) mice. The notion that Cyp2c44 is the epoxygenase responsible for mediating the inhibitory effects of arachidonic acid on ENaC is further suggested by the observation that inhibiting Cyp-epoxygenase increased the whole-cell Na currents in principal cells of wt but not in Cyp2c44(-/-) mice. Feeding mice with an HK diet raised the systemic blood pressures of Cyp2c44(-/-) mice but was without an effect on wt mice. Moreover, application of amiloride abolished the HK-induced hypertension in Cyp2c44(-/-) mice. The HK-induced hypertension of Cyp2c44(-/-) mice was accompanied by decreasing 24-hour urinary Na excretion and increasing the plasma Na concentration, and the effects were absent in wt mice. In contrast, disruption of the Cyp2c44 gene did not alter K excretion. We conclude that Cyp2c44 epoxygenase mediates the inhibitory effect of arachidonic acid on ENaC and that Cyp2c44 functions as an HK-inducible antihypertensive enzyme responsible for inhibiting ENaC activity and Na absorption in the aldosterone-sensitive distal nephron.

Androgen-dependent hypertension is mediated by 20-hydroxy-5,8,11,14-eicosatetraenoic acid-induced vascular dysfunction: role of inhibitor of kappaB Kinase.

Increased vascular synthesis of 20-hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE) is associated with increased vascular contraction, endothelial dysfunction, and endothelial activation; all are believed to account for 20-HETE prohypertensive properties. We demonstrated previously that the 20-HETE-dependent inhibition of NO production is mediated through inhibitor of κB kinase (IKK), suggesting a cross-talk between 20-HETE-mediated endothelial dysfunction and activation. In this study, we examined the temporal relationship among blood pressure, endothelial dysfunction, and endothelial activation and the role of IKK in the rat model of androgen-driven 20-HETE-mediated hypertension. In Sprague-Dawley rats treated with 5α-dihydrotestosterone, renal vascular 20-HETE levels increased by day 2 of treatment from 17.7±2.4 to 57.7±9.7 ng/mg, whereas blood pressure elevation reached significance by day 3 (132.7±1.7 versus 117.2±0.8 mm Hg). In renal interlobar arteries, when compared with vehicle, 5α-dihydrotestosterone treatment increased the sensitivity to phenylephrine-induced vasoconstriction by 3.5-fold, decreased acetylcholine-induced vasorelaxation, and increased nuclear factor κB activity, all of which were attenuated by treatment with the 20-HETE antagonist, 20 hydroxyeicosa-6(Z),15(Z)-dienoic acid, (20-6,15-HEDE). Cotreatment with parthenolide, an IKK inhibitor, attenuated the androgen-dependent 20-HETE-mediated elevation in blood pressure (from 133.7±3.1 to 109.8±3.0 mm Hg). In addition, parthenolide treatment negated 20-HETE-mediated inhibition of the relaxing response to acetylcholine and 20-HETE-mediated increase in vascular nuclear factor κB activity. These findings suggest that inhibition of IKK attenuates the androgen-dependent 20-HETE-mediated increase in blood pressure by inhibiting both 20-HETE-dependent endothelial activation and dysfunction.

CYP4A2-induced hypertension is 20-hydroxyeicosatetraenoic acid- and angiotensin II-dependent.

We have shown previously that increased vascular endothelial expression of CYP4A2 leads to 20-hydroxyeicosatetraenoic (20-HETE)-dependent hypertension. The renin-angiotensin system is a key regulator of blood pressure. In this study, we examined possible interactions between 20-HETE and the renin-angiotensin system. In normotensive (110±3 mm Hg) Sprague-Dawley rats transduced with a lentivirus expressing the CYP4A2 cDNA under the control of an endothelial-specific promoter (VECAD-4A2), systolic blood pressure increased rapidly, reaching 139±1, 145±3, and 150±2 mm Hg at 3, 5, and 10 days after transduction; blood pressure remained elevated, thereafter, with maximum levels of 163±3 mm Hg. Treatment with lisinopril, losartan, or the 20-HETE antagonist 20-hydroxyeicosa-6(Z), 15(Z)-dienoic acid decreased blood pressure to control values, but blood pressure returned to its high levels after cessation of treatment. Endothelial-specific overexpression of CYP4A2 resulted in increased expression of vascular angiotensin-converting enzyme (ACE) and angiotensin II type 1 receptor and increased levels of plasma and tissue angiotensin II; all were attenuated by treatment with HET0016, an inhibitor of 20-HETE synthesis, or with 20-hydroxyeicosa-6(Z), 15(Z)-dienoic acid. In cultured endothelial cells, 20-HETE specifically and potently induced ACE expression without altering the expression of ACE2, angiotensinogen, or angiotensin II receptors. This is the first study to demonstrate that 20-HETE, a key constrictor eicosanoid in the microcirculation, induces ACE and angiotensin II type 1 receptor expression and increases angiotensin II levels, suggesting that the mechanisms by which 20-HETE promotes hypertension include activation of the renin-angiotensin system that is likely initiated at the level of ACE induction.