20-HETE Synthase Research Articles

20-HETE mediated TRPV1 activation drives allokinesis via MrgprA3+ neurons in chronic dermatitis.

Rationale: Noxious stimuli are often perceived as itchy in patients with chronic dermatitis (CD); however, itch and pain mechanisms of CD are not known. Methods: TRPV1 involvement in CD was analyzed using a SADBE induced CD-like mouse model, and several loss- and gain-of-function mouse models. Trigeminal TRPV1 channel and MrgprA3+ neuron functions were analyzed by calcium imaging and whole-cell patch-clamp recordings. Lesional CD-like skin from mice were analyzed by unbiased metabolomic analysis. 20-HETE availability in human and mouse skin were determined by LC/MS and ELISA. And finally, HET0016, a selective 20-HETE synthase inhibitor, was used to evaluate if blocking skin TRPV1 activation alleviates CD-associated chronic itch or pain. Results: While normally a pain inducing chemical, capsaicin induced both itch and pain in mice with CD condition. DREADD silencing of MrgprA3+ primary sensory neurons in these mice selectively decreased capsaicin induced scratching, but not pain-related wiping behavior. In the mice with CD condition, MrgprA3+ neurons showed elevated ERK phosphorylation. Further experiments showed that MrgprA3+ neurons from MrgprA3;Braf mice, which have constitutively active BRAF in MrgprA3+ neurons, were significantly more excitable and responded more strongly to capsaicin. Importantly, capsaicin induced both itch and pain in MrgprA3;Braf mice in an MrgprA3+ neuron dependent manner. Finally, the arachidonic acid metabolite 20-HETE, which can activate TRPV1, was significantly elevated in the lesional skin of mice and patients with CD. Treatment with the selective 20-HETE synthase inhibitor HET0016 alleviated itch in mice with CD condition. Conclusion: Our results demonstrate that 20-HETE activates TRPV1 channels on sensitized MrgprA3+ neurons, and induces allokinesis in lesional CD skin. Blockade of 20-HETE synthesis or silencing of TRPV1-MrgprA3+ neuron signaling offers promising therapeutic strategies for alleviating CD-associated chronic itch.

Introduction of a carboxylic acid group into pyrazolylpyridine derivatives increased selectivity for inhibition of the 20-HETE synthase CYP4A11/4F2

20-Hydroxyeicosatetraenoic acid (20-HETE) is a lipid mediator and one of the major arachidonic acid metabolites whose formation is mainly catalyzed by the enzymes cytochrome P450 (CYP) 4F2 and CYP4A11. Several studies have suggested that 20-HETE is involved in the pathogenesis of renal diseases, including diabetic nephropathy and autosomal dominant polycystic kidney disease, and we previously reported compound 1 as a dual inhibitor of CYP4A11/4F2 with therapeutic potential against renal fibrosis. Subsequent studies revealed that compound 1, the dual CYP4A11/4F2 inhibitor, however, exhibited low selectivity over another CYP4F subtype, CYP4F22, which catalyzes ω-hydroxylation of ultra-long-chain fatty acids (ULCFAs); ULCFAs are important for the formation of acylceramides, which play a role in skin barrier formation. Therefore, we sought to develop a CYP4A11/4F2 inhibitor that would show greater CYP4A11/4F2 selectivity against CYP4F22, to avoid potential dermatological side effects. We re-evaluated a series of compounds from our 20-HETE program and identified pyrazolylpyridine derivatives containing a carboxylic acid group showing only weak CYP4F22 inhibition. Subsequent optimization studies from these derivatives led to identification of compound 15, which showed CYP4A11/4F2 inhibition with improved selectivity against CYP4F22. Compound 15 inhibited 20-HETE production in both human and rat renal microsomes and did not inhibit ω-hydroxylation of ULCFAs in human keratinocytes. Compound 15 also significantly inhibited renal 20-HETE production after oral administration.

The interaction between STING and NCOA4 exacerbates lethal sepsis by orchestrating ferroptosis and inflammatory responses in macrophages

The discovery of STING-related innate immunity has recently provided a deep mechanistic understanding of immunopathy. While the detrimental effects of STING during sepsis had been well documented, the exact mechanism by which STING causes lethal sepsis remains obscure. Through single-cell RNA sequence, genetic approaches, and mass spectrometry, we demonstrate that STING promotes sepsis-induced multiple organ injury by inducing macrophage ferroptosis in a cGAS- and interferon-independent manner. Mechanistically, Q237, E316, and S322 in the CBD domain of STING are critical binding sites for the interaction with the coiled-coil domain of NCOA4. Their interaction not only triggers ferritinophagy-mediated ferroptosis, but also maintains the stability of STING dimers leading to enhanced inflammatory response, and reduces the nuclear localization of NCOA4, which impairs the transcription factor coregulator function of NCOA4. Meanwhile, we identified HET0016 by high throughput screening, a selective 20-HETE synthase inhibitor, decreased STING-induced ferroptosis in peripheral blood mononuclear cells from patients with sepsis and mortality in septic mice model. Our findings uncover a novel mechanism by which the interaction between STING and NCOA4 regulates innate immune response and ferroptosis, which can be reversed by HET0016, providing mechanistic and promising targets insights into sepsis.

Activation of 20-HETE Synthase Triggers Oxidative Injury and Peripheral Nerve Damage in Type 2 Diabetic Mice

Diabetic Peripheral Neuropathy (DPN), highly prevalent among patients with diabetes, is characterized by peripheral nerve dysfunction. Reactive Oxygen Species (ROS) overproduction has been suggested to orchestrate diabetic complications including DPN. Untargeted antioxidant therapy has exhibited limited efficacy, highlighting a critical need to explore ROS sources altered in a cell-specific manner in DPN. Cytochromes P450 (CYP) enzymes are prominent sources of ROS. Particularly, the 20-HETE synthase, CYP4A, is reported to mediate diabetes-induced renal, retinal, and cardiovascular injuries. This work investigates the role of CYP4A/20-HETE in DPN and their mechanisms of action. Non-obese type 2 Diabetic mice (MKR) were used and treated with a CYP4A-inhibitor (HET0016) or AMPK-activator (Metformin). Peripheral nerves of MKR mice reflect increased CYP4A and 20-HETE levels, concurrent with altered myelin proteins and sensorimotor deficits. This was associated with increased ROS production and altered Beclin-1 and LC3 protein levels, indicative of disrupted autophagic responses in tandem with AMPK inactivation. AMPK activation via Metformin restored nerve integrity, reduced ROS production, and regulated autophagy. Interestingly, similar outcomes were revealed upon HET0016 treatment whereby ROS production, autophagic responses, and AMPK signaling were normalized in diabetic mice. Altogether, the results highlight hyperglycemia-mediated oxidative injury in DPN through a novel CYP4A/20-HETE/AMPK pathological axis. PerspectiveTo our knowledge, this is the first study to highlight the role of CYPs/20-HETE-induced oxidative injury in the pathogenesis of diabetic peripheral neuropathy. Targeting the identified pathological axis CYP4A/20-HETE/AMPK may be of clinical potential in predicting and alleviating peripheral nerve injury in patients with Type 2 Diabetes Mellitus.

Abstract 15873: Novel Contributions of Neutrophil-derived Myeloperoxidase and Hypochlorous Acid to 20-hydroxyeicosatetraenoic Acid Production That Drives Post-ischemic Angiogenesis

Introduction: Compensatory angiogenic response to ischemia is often insufficient in maintaining adequate tissue perfusion resulting in critical limb ischemia and amputation. Identifying a novel mechanism by which angiogenesis occurs in these conditions is clinically relevant. We recently uncovered that an increase in 20-HETE, an arachidonic acid metabolite of CYP4A/F ω-hydroxylases, regulates post-ischemic angiogenesis. However, the underlying mechanism resulting in this increase is unknown. Hypothesis: Neutrophil-derived myeloperoxidase (MPO) and hypochlorous acid (HOCl) critically contribute to post-ischemic 20-HETE increases that drive angiogenesis. Methods: Hindlimb ischemia was established in mice depleted of neutrophils, macrophages, and MPO (MPO -/- ). Angiogenesis was assessed by laser doppler perfusion imaging and micro-vessel density quantitation in the hindlimb gracilis muscles. MPO and HOCl were detected in these tissues using immunohistochemistry and a HOCl-specific fluorophore. We also determined the effects of MPO and HOCl on 20-HETE production, the expression of 20-HETE synthase CYP4A11, and hypoxia inducible factor-1α in cultured endothelial cells (EC) using LC/MS/MS, real time-PCR and western blot analysis, respectively. Results: We found that ischemia failed to increase 20-HETE production in mice depleted of neutrophils and MPO (13 ± 1.5 vs 35 ± 5 and ~2 ± .25 vs 35 ± 5 pg/mg of protein, respectively), accompanied with a decreased post-ischemic angiogenic phenotype. We also detected the formation of MPO and HOCl in post-ischemic gracilis muscles. MPO and HOCl also significantly stimulate CYP4A11 expression and 20-HETE production (40±12 vs 8±5 pg/mg of protein) in EC. Furthermore, HOCl quickly induces CYP4A11 mRNA/protein expression (2-fold,) and the protein expression of HIF-1α (2-fold) in as little as 15 min. Conclusion: Our studies establish for the first time that neutrophil-derived MPO and HOCl are responsible for promoting 20-HETE increases that critically drive angiogenesis post ischemia. Thus, identifying these novel mediators can further future therapeutic strategies to balance angiogenic responses during ischemia as well as treating diseases that are associated with abnormal angiogenesis.

20-HETE interferes with insulin signaling and contributes to obesity-driven insulin resistance

20-HETE, a metabolite of arachidonic acid produced by Cytochrome P450 (CYP) 4A/4 F, has been implicated in the development of obesity-associated complications such as diabetes and insulin resistance. In this study, we examined whether the acute elevation of 20-HETE levels contributes to the development of diet-driven hyperglycemia and insulin resistance. We employed a conditional transgenic mouse model to overexpress Cyp4a12 (Cyp4a12tg), a murine 20-HETE synthase, together with high fat diet (HFD) feeding. Mice in which Cyp4a12 was induced by doxycycline (DOX) at the onset of HFD feeding gained weight at a greater rate and extent than corresponding DOX-untreated Cyp4a12 mice. Cyp4a12tg mice fed HFD + DOX displayed hyperglycemia and impaired glucose metabolism while corresponding HFD-fed Cyp4a12tg mice (no DOX) did not. Importantly, administration of a 20-HETE antagonist, 20-SOLA, to Cyp4a12tg mice fed HFD + DOX significantly attenuated weight gain and prevented the development of hyperglycemia and impaired glucose metabolism. Levels of insulin receptor (IR) phosphorylation at Tyrosine 972 and insulin receptor substrate-1 (IRS1) phosphorylation at serine 307 were markedly decreased and increased, respectively, in liver, skeletal muscle and adipose tissues from Cyp4a12tg mice fed HFD + DOX; 20-SOLA prevented the IR and IRS1 inactivation, suggesting that 20-HETE interferes with insulin signaling. Additional studies in 3T3−1 differentiated adipocytes confirmed that 20-HETE impairs insulin signaling and that its effect may require activation of its receptor GPR75. Taken together, these results provide strong evidence that 20-HETE interferes with insulin function and contributed to diet-driven insulin resistance

Vascular dysfunction and remodeling associated with 20‐HETE‐dependent hypertension is prevented in 20‐HETE receptor (GPR75) null mice

The potent vasoactive lipid 20‐Hydroxyeicosatetraenoic acid (20‐HETE) exhibits a range of bioactions across the vasculature including stimulation of endothelial dysfunction (ED) and increases in smooth muscle contractility; all of which promote hypertension. We have recently identified the G‐protein coupled receptor 75 (GPR75) as the receptor to which 20‐HETE binds and triggers signaling pathways culminating with the aforementioned effects. Previous studies have clearly shown the Cyp4a12tg mouse to be an animal model for the study of 20‐HETE and phenotypes associated with 20‐HETE‐dependent hypertension. In this model, expression of the 20‐HETE synthase, Cyp4a12, is under the control of a tetracycline (doxycycline, DOX) promoter and treatment of Cyp4a12tg (Gpr75+/+4a12tg) mice with DOX elicits increases circulating 20‐HETE levels followed by the promotion of pro‐hypertensive signaling including decreases in nitric oxide (NO) production and increases in vascular angiotensin converting enzyme (ACE) resulting in elevations in blood pressure (BP) (136 ± 4 vs. 103 ± 4 mmHg; P < 0.05). In this study, we examined whether the deletion of GPR75 affects the onset and progression of hypertension and vascular remodeling in the Cyp4a12tg mice by crossing Gpr75−/− with Cyp4a12tg mice. Gpr75−/− 4a12tg treated with DOX chow for 42 days did not exhibit any changes in BP (103 ± 4 vs. 100 ± 6 mmHg). 20‐HETE‐mediated increases in BP in DOX‐treated Cyp4a12tg mice are associated with ED as indicated by reduced relaxation response to acetylcholine (ACh). Wire myography of renal interlobar arteries from DOX‐treated Gpr75+/+4a12tg mice pre‐constricted with phenylephrine exhibited a diminished relaxation response to ACh compared to mice receiving regular chow (44 ± 4% vs. 91 ± 2% relaxation; P < 0.05). In contrast, ACh‐induced relaxations in arteries from DOX‐treated Gpr75−/− 4a12tg mice were not impaired and responded similarly to ACh as vessels from vehicle‐treated Gpr75−/− 4a12tg mice (89 ± 1% vs. 89 ± 2% relaxation; P < 0.05). Another hallmark of persistent elevations in BP over time and 20‐HETE‐dependent hypertension is the development of dynamic vascular remodeling with increases in media thickness (MT), media‐to‐lumen ratio (M:L) and cross sectional area (CSA). Gpr75+/+4a12tg mice receiving DOX for 42 days exhibit an increase in remodeling of renal resistance arteries as indicated by elevations in MT (24 ± 1 vs. 12 ± 1 μm), M:L (0.29 ± 0.02 vs. 0.12 ± 0.01) and CSA (15 ± 1 vs. 8 ± 1 μm2 × 103) compared to Gpr75+/+4a12tg mice receiving regular chow. In contrast, DOX treated Gpr75−/− 4a12tg mice, as opposed to vehicle‐treated Gpr75−/− 4a12tg mice, did not display any changes to MT (12 ± 1 vs. 11 ± 1 μm), M:L (0.13 ± 0.01 vs. 0.11 ± 0.01 ) or CSA (8± 1 vs. 7 ± 0.3 μm2 × 103). Taken together, this data support the hypothesis that 20‐HETE required its receptor, GPR75, in order to drive pro‐hypertensive signaling that promotes endothelial dysfunction, vascular remodeling and hypertension.Support or Funding InformationSupported by NIH grants HL39793 (MLS), HL39793‐01S1 (VG) and by the Intramural Research Program of the NIH (Z01 ES025034 (DCZ)).

Activation of 20-HETE/PPARs involved in reno-therapeutic effect of naringenin on diabetic nephropathy

Naringenin is a flavanone compound found in citrus fruits. Recent researches showed that naringenin has many potentially pharmacological effects. However, the therapeutic effect and the potential mechanism of naringenin on diabetic nephropathy (DN) remain to be elucidated. DN model was established by a high-fat diet combined with streptozotocin (STZ), which was confirmed by the levels of fasting blood glucose (FBG, more than 11.1 mmol/L) and urinary albumin (10 times higher than the normal mice). After 5 weeks of STZ injection, the DN was developed in model mice. Then naringenin (25 or 75 mg/kg·d) were supplemented for 4 weeks. At the end of the experiment, the injury of the renal function and structure was deteriorated. Concomitantly, peroxisome proliferators-activated receptors (PPARs) protein expression was down-regulated, and CYP4A expression and 20-hydroxyeicosatetraenoic acid (20-HETE) level were reduced in DN mice. Naringenin administration improved the renal damage of DN mice, and up-regulated PPARs expression, increased CYP4A-20-HETE level. Consistent with the results of in vivo, glucose at 30 mmol/L (high glucose, HG) significantly induced cell proliferation and hypertrophy in NRK-52E cells, following the reductive PPARs protein expression and the downward CYP4A-20-HETE level. Naringenin (0.01, 0.1, 1 μmol/L) reversed these changes induced by HG in a dose-dependent manner. HET0016, a selective inhibitor of 20-HETE synthase, partially blocked the effects of naringenin. In conclusion, naringenin has a therapeutic effect on DN, which may be, at least partly, related to the activation of CYP4A-20-HETE and the up-regulation of PPARs.

Novel Contribution of Myeloperoxidase‐derived Hypochlorous Acid to 20‐HETE Production Post‐Ischemia

Ischemia‐induced angiogenesis occurs as a mechanism to minimize the tissue damage caused by atherosclerotic diseases but is often insufficient to maintain adequate tissue perfusion and results in critical limb ischemia and amputation. The discovery of novel factors that increase angiogenesis in response to ischemia is therefore of clinical relevance.Recently, we discovered that 20‐hydroxy‐eicosatetraenoic acid (20‐HETE), an arachidonic acid metabolite produced by a cytochrome P450 (CYP) 4A/F ω‐hydroxylases, is a novel contributor of ischemia‐induced angiogenesis based on the following two major findings: 1) pharmacological interference of the CYP4A/20‐HETE axis attenuates ischemic‐induced angiogenesis; and 2) the expression of cyp4a12a and its product, 20‐HETE are both elevated in ischemic tissues of mice models. The resulting increase in 20‐HETE is endothelial in origin. However, the factors that cause endothelial cells (EC) to produce 20‐HETE following ischemia are largely unknown. The aim of these studies was to investigate the mechanism of post‐ischemic endothelial 20‐HETE increases. Inflammatory cells such as macrophages and neutrophils are the first responders to ischemia injury. Thus, we hypothesized that inflammatory cells contribute to post‐ischemic endothelial increases of 20‐HETE.We first examined the potential contribution of inflammatory response to hindlimb ischemia‐induced angiogenesis using immune‐deficient SCID mice. Following a femoral artery ligation and comparing SCID mice to Balb/C control mice, SCID mice exhibited impaired blood perfusion (55 ± 9% vs 89 ± 10%), decreased micro‐vessel counts (capillary:fiber ratio of 1.5 ± .25 vs 2.5 ± 3.75), as well as an inability to produce 20‐HETE (7.5 ± 2.5 vs 29 ± 5 pg/mg of protein).Furthermore, immune‐competent Balb/c mice depleted of macrophages or neutrophils showed that ischemia failed to induce 20‐HETE production in Balb/c depleted of neutrophils (13 ± 1.5 vs 35 ± 5 pg/mg of protein), accompanied with compromised blood perfusion (58 ± 10% vs 82 ± 9%), but not those depleted of macrophages. Neutrophils are known to release Myeloperoxidase (MPO) at sites of inflammation. The possibility that MPO contributes to ischemia‐induced 20‐HETE production was investigated using MPO−/− mice. Remarkably, the ischemic hind limb tissues of MPO−/− mice produce negligible amount of 20‐HETE (~2 ± .25 vs 35 ± 5 pg/mg of protein). MPO is known to produce hypochlorous acid (HOCl). Therefore, we further studied the role of MPO‐derived HOCl in 20‐HETE production in cultured human EC. The addition of physiological amounts of either MPO or HOCl markedly stimulated the expression of 20‐HETE synthase CYP4A11 and production of 20‐HETE (40 ± 12 vs 8 ± 5 pg/mg of protein); potentially via a HIF1‐a dependent mechanism.These observations suggest for the first time that neutrophil‐derived MPO critically contributes to the 20‐HETE increases post‐ischemia via a mechanism involving the generation of HOCl and activation of the HIF pathway in endothelium.Support or Funding InformationAHA [11SDG6870004(AMG), 17GRNT33430003(AMG)]NIH [HL34300(MLS), DK38226(JRF)]RA Welch Foundation [GL625910(JRF)]This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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.

Abstract P221: Vascular Smooth Muscle-Specific Overexpression of Cyp4a12-20-HETE Synthase Causes Hypertension and Vascular Remodeling

20-Hydroxyeicosatetraenoic acid (20-HETE) is a potent vasoactive eicosanoid of the microcirculation exhibiting effects on vascular smooth muscle (VSM) function that include stimulation of contractility, migration and growth. We have previously demonstrated in mice which globally overexpress 20-HETE that hypertension as well as vascular remodeling is not fully prevented by pharmacological normalization of blood pressure. However, both hypertension and vascular remodeling are prevented by antagonism of 20-HETE, suggesting that 20-HETE exerts vascular effects independent of hypertension. To examine the contribution of VSM-derived 20-HETE to hypertension and vascular remodeling, we have developed a transgenic mouse model that overexpresses the primary mouse 20-HETE synthase, Cyp4a12, specifically in VSM by crossbreeding floxed Cyp4a12 with Myh11-Cre mice. All mice carry the floxed Cyp4a12 gene and express a GFP protein under control of a modified CMV promoter. Cre-recombination excises the GFP cDNA and inserts the Cyp4a12 cDNA. Three to four month old male Cyp4a12-floxed mice carrying the Myh11-Cre gene (Cyp4a12 fl/fl Myh11 Cre+/- ) exhibited higher systolic blood pressure than control mice Cyp4a12 fl/fl Myh11 Cr-/- (WT) mice (135 vs. 115 mmHg, n=4, *p<0.05). 20-HETE production in Cyp4a12 fl/fl Myh11 Cre+/- mice were higher than in WT in mesenteric (3333±891 vs. 545± 196 ng/mg/h) and renal interlobar arteries (RIA; 1859±376 vs. 242±62 ng/mg/h). Plasma levels of 20-HETE were also elevated (324±61 vs. 185±34 pg/mL) while urinary levels were not significantly different (146±26 vs. 117±29 pg/mL). We also observed increased medial thickness and decreased lumen area of blood vessels by Myh11 immunofluorescence in heart and kidney sections. RIA from male Myh11-Cyp4a12 mice displayed higher constrictor sensitivity to phenylephrine and impaired relaxation to acetylcholine compared to WT. Taken together, these data suggest that this model displays hypertension and pathological hypertrophic vascular remodeling. We therefore conclude that the Cyp4a12 fl/fl Myh11 Cre/- is a promising and unique transgenic mouse model to examine the contribution of smooth muscle-derived 20-HETE to hypertension and hypertrophic vascular remodeling.

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.

Female-favorable attenuation of coronary myogenic constriction via reciprocal activations of epoxyeicosatrienoic acids and nitric oxide.

Epoxyeicosatrienoic acids (EETs) are metabolites of arachidonic acid via CYP/epoxygenases, which are catabolized by soluble epoxide hydrolase (sEH) and known to possess cardioprotective properties. To date, the role of sEH in the modulation of pressure-induced myogenic response/constriction in coronary arteries, an important regulatory mechanism in the coronary circulation, and the issue as to whether the disruption of the sEH gene affects the myogenic response sex differentially have never been addressed. To this end, experiments were conducted on male (M) and female (F) wild-type (WT) and sEH-knockout (KO) mice. Pressure-diameter relationships were assessed in isolated and cannulated coronary arteries. All vessels constricted in response to increases in intraluminal pressure from 60 to 120 mmHg. Myogenic vasoconstriction was significantly attenuated, expressed as an upward shift in the pressure-diameter curve of vessels, associated with higher cardiac EETs in M-KO, F-WT, and F-KO mice compared with M-WT controls. Blockade of EETs via exposure of vessels to 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE) prevented the attenuated myogenic constriction in sEH-KO mice. In the presence of 14,15-EEZE, pressure-diameter curves of females presented an upward shift from those of males, exhibiting a sex-different phenotype. Additional administration of N(ω)-nitro-l-arginine methyl ester eliminated the sex difference in myogenic responses, leading to four overlapped pressure-diameter curves. Cardiac sEH was downregulated in F-WT compared with M-WT mice, whereas expression of endothelial nitric oxide synthase and CYP4A (20-HETE synthase) was comparable among all groups. In summary, in combination with NO, the increased EET bioavailability as a function of genetic deletion and/or downregulation of sEH accounts for the female-favorable attenuation of pressure-induced vasoconstriction.

Abstract 438: Regulation of Ischemia-induced Neovascularization by 20-HETE Involves Bone Marrow-derived EPC.

Objective: Compensatory neovascularization is an important adaptation for recovery from critical ischemia. The CYP4A-derived 20-HETE were recently identified as a novel regulator of angiogenesis. Here, we assessed the contribution of 20-HETE to ischemia-induced neovascularization and its underlying molecular and cellular mechanisms. Methods: The mouse ischemia hindlimb angiogenesis assay was established to study the role of CYP4A-20-HETE in compensatory angiogenesis using the 20-HETE synthase inhibitor (DDMS, 10mg/kg/d) and the 20-HETE antagonist (6, 15-20-HEDGE, 10mg/kg/d). Hindlimb blood flow and microvessels density in ischemic hindlimb muscle were measured by Laser Doppler Perfusion Imaging and Immunofluorescent staining, respectively. 20-HETE production in ischemic muscles was measured by LC-MS-MS. Western blot was performed to explore the underlying signaling pathways. Flow cytometry analysis was used to assess the contribution of 20-HETE to mobilization and homing of bone marrow (BM)-derived endothelial progenitor cells (EPC) to ischemic neovascularization. Results: 20-HETE inhibition by DDMS and 6, 15-20-HEDGE reduced the blood flow perfusion and microvessel formation in response to ischemia by 46±2.3% and 34±1.2%, respectively. Importantly, ischemic hindlimb muscles showed markedly elevated 20-HETE synthesis compared to non-ischemic controls (96 ± 17 pg/mg protein vs. non-detectable). HIF-1α, VEGF, and VEGFR2 expression were markedly induced in ischemic hindlimb muscles, but were negated by DDMS and 6, 15-20-HEDGE. Furthermore, ischemia induced BM-derived EPC mobilization and subsequent EPC homing to the ischemic hindlimb muscle by 5.5±0.7 and 20±2.8 folds, respectively, whereas DDMS and 6, 15-20-HEDGE significantly attenuated these processes. Conclusions: An increase in 20-HETE production in ischemic muscles regulates ischemic compensatory neovascularization via the induction of the HIF-1α/VEGF pathways that support the mobilization and homing of BM-derived EPC. These results strengthen the notion that 20-HETE is a key regulator of ischemic compensatory angiogenesis via regulation of EPC function and may represent a novel therapeutic target.

Abstract 338: 20-HETE Contributes To Ischemia-induced Compensatory Neovascularization

Objective: Compensatory neovascularizaiton is an important adaptation for recovery from critical ischemia. Recent studies identified the CYP4A-20-HETE system as a novel regulator of angiogenic processes. Here, we assessed the contribution of the 20-HETE system to ischemia-induced neovascularization and further explored its underlying molecular and cellular mechanisms. Methods: The mouse ischemia hindlimb angiogenesis assay was performed to evaluate the effect of systemic or local inhibition of the CYP4A-20-HETE system with the 20-HETE synthase inhibitor, DDMS or the 20-HETE antagonist, 20-HEDGE on the compensatory angiogenic responses. Laser Doppler Perfusion Imaging was conducted to assess hindlimb blood flow. Blood pressure was monitored with the non-invasive blood pressure monitoring system. 20-HETE production in ischemic muscles was also measured by LC-MS-MS analysis. IHC staining were carried out to quantify microvessel density and to assess the localization of 20-HETE system in hindlimb gracilis muscle. Finally, western blot was performed to explore the potential underlying signaling pathways. Results: Inhibition of 20-HETE synthesis or antagonizing its action either locally or systematically reduced the blood flow perfusion and microvessel formation in response to ischemia without affecting blood pressures. Importantly, ischemic hindlimb muscles showed markedly elevated 20-HETE synthesis compared to non-ischemic controls (96 ± 17 pg/mg of protein versus non-detectable amount). IHC staining showed that an upregulation of 20-HETE synthase ( cyp4a12a ) is co-localized with endothelial cells in the ischemic gracillis muscle, suggesting ischemia may induce the increased production of 20-HETE in the vasculature. Furthermore, the protein expression of HIF-1α, VEGF, VEGFR2, and p44/42 MAPK in ischemic hindlimb muscles were significantly induced. These increases were negated by DDMS or 20-HEDGE. Conclusions: Early increases in 20-HETE production in ischemic muscles may regulate ischemic angiogenesis via the induction of the HIF-1α/VEGF and MAPK pathways. These results strengthen the notion that 20-HETE may be a key regulator of ischemia-induced compensatory neovascularization processes.

Abstract 047: 20-SOLA, A Novel Water-soluble 20-HETE Antagonist, Elicits Natriuresis And Abrogates Hypertension In Cyp4a14 Knockout Mice.

20-HETE (20-hydroxyeicosatetraenoic acid) is a cytochrome P450 (CYP) 4A-derived arachidonate metabolite. In the vasculature, 20-HETE increases smooth muscle contraction, endothelial dysfunction/activation and sensitivity to constrictor stimuli; all of which contribute to hypertension. In renal tubules, 20-HETE inhibits ion transport and has pro-natriuretic functions in salt-sensitive models of hypertension. The pro-natriuretic effect of 20-HETE has not been reported in mouse models of 20-HETE-dependent hypertension. The CYP4a14 knockout (KO) male mice exhibit androgen-dependent upregulation of Cyp4a12, the sole 20-HETE synthase in mice, and display 20-HETE-dependent hypertension; female KO mice show no increase in Cyp412-20-HETE and are normotensive. We examined the effect of 20-SOLA (2,5,8,11,14,17-hexaoxanonadecan-19-yl-20-hydroxyeicosa-6(Z),15(Z)-dienoate), a water-soluble 20-HETE antagonist, and determined its effect on blood pressure (BP) and natriuresis in the KO mice. Administration of 20-SOLA (10mg/kg/day in drinking water) normalized BP in male KO mice; a significant reduction was observed on day 4 (146±1 vs153±2 mmHg in untreated littermates) reaching BP of the normotensive female KO mice (124±1 and 126±1 mmHg for treated and untreated, respectively) at day 10 of treatment (124±1 vs. 153±2 mmHg in untreated male KO; p<0.05). 20-SOLA decreased vascular reactivity to phenylephrine in male KO renal microvessels by 7-fold (EC50: 1.58±0.23 vs 0.22±0.03 μM). Moreover, treatment with 20-SOLA increased urine output in KO male mice as compared to untreated littermates (1.25±0.04 vs. 1.06±0.04 mL; p<0.05). The urine output to water intake ratio was significantly elevated in 20-SOLA-treated KO vs. untreated (0.25±0.01 vs. 0.19±0.01, p<0.05). Importantly, treatment with 20-SOLA increased urinary sodium excretion in the KO male mice (12.33±0.44 vs. 8.33±0.63 μmol/gr BW/24Hrs; p<0.05). These results suggest that 20-HETE has an anti-natriuretic effect in the kidneys of KO mice and that antagonism of 20-HETE’s action using 20-SOLA elicited natriuresis concurrent with reduction in blood pressure. The potential vascular and tubular mechanisms underlying 20-HETE anti-natriuretic action are being currently explored.

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.

Abstract 202: Activation of Cyp4a12-20-HETE Synthase Increases Blood Pressure and Promotes Vascular Hypertrophy and Cardiac Dysfunction

20-Hydroxyeicosatetraenoic acid (20-HETE) is a microcirculatory cytochrome P450-derived eicosanoid shown to increase smooth muscle contractions and proliferation, stimulate endothelial dysfunction and activation, and promotes hypertension. We developed a mouse model (Cyp4a12tg) in which the expression of Cyp4a12, the sole 20-HETE synthase in mouse, is under the control of the doxycycline (DOX) promoter. Administration of DOX to Cyp4a12tg mice increased blood pressure (131±3 vs 100±2 mmHg, p<0.05), which was prevented by co-treatment with the 20-HETE antagonist, 20-HEDGE (96±3 mmHg, p<0.05). Media-to-lumen ratio and medial cross sectional area of renal microvessels from DOX-treated Cyp4a12tg mice significantly increased compared to untreated (M/L, 0.15±0.01 vs 0.07±0.01; mCSAx103 10.8±0.92 vs. 6.4±0.48 mm2); these increases were abolished by co-treatment with 20-HEDGE. Cardiac output and heart rate were unchanged, whereas %EF and %FS were reduced and LV volume and diameter at systole increased in DOX-treated Cyp4a12tg. Total peripheral resistance (TPR) was significantly increased in DOX-treated Cyp4a12tg mice (8.00±0.39 vs 6.77±0.36 mmHg/ml/min, p<0.05); co-treatment with 20-HEDGE decreased (p<0.0%) DOX-induced TPR in Cyp4a12tg mice (7.45±0.22 mmHg/ml/min). These results indicate that activation of Cyp4a12-20-HETE synthase causes hypertension, microvascular remodeling, and cardiac dysfunction. The results also suggest that 20-HETE promotes hypertension by increasing TPR. However, the mechanisms underlying Cyp4a12-20-HETE-driven microvascular remodeling and cardiac dysfunction are yet to be explored.

20-HETE induces remodeling of renal resistance arteries independent of blood pressure elevation in hypertension

20-Hydroxyeicosatetraenoic acid (20-HETE) is a cytochrome P-450 (Cyp)-derived arachidonic acid metabolite that has been shown to increase smooth muscle contractions and proliferation, stimulate endothelial dysfunction and activation, and promote hypertension. We examined if 20-HETE contributes to microvascular remodeling in hypertension. In Sprague-Dawley rats, administration of the 20-HETE biosynthesis inhibitor HET0016 or the 20-HETE antagonist N-20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (20-HEDE) prevented 5α-dihydrotestosterone (DHT)-induced increases in blood pressure as well as abrogated DHT-induced increases in the media-to-lumen ratio (M/L), media thickness, and collagen IV deposition in renal interlobar arteries. Reserpine prevented blood pressure elevation in DHT-treated rats but did not affect microvascular remodeling (M/L, media thickness, and collagen deposition); under these conditions, treatment with the 20-HETE antagonist attenuated microvascular remodeling, suggesting that 20-HETE contributes to DHT-induced vascular remodeling independent of blood pressure elevation. In Cyp4a14(-/-) mice, which display androgen-driven and 20-HETE-dependent hypertension, treatment with the 20-HETE antagonist abolished remodeling of renal resistance arteries measured as media thickness (24 ± 1 vs. 15 ± 1 μm) and M/L (0.29 ± 0.03 vs. 0.17 ± 0.01). Moreover, in Cyp4a12 transgenic mice in which the expression of Cyp4a12-20-HETE synthase is driven by a tetracycline-sensitive promoter, treatment with doxycycline resulted in blood pressure elevation (140 ± 4 vs. 92 ± 5 mmHg) and a significant increase in remodeling of renal resistance arteries (media thickness: 23 ± 1 vs. 16 ± 1 μm; M/L: 0.39 ± 0.04 vs. 0.23 ± 0.02); these increases were abrogated by cotreatment with 20-HEDE. This study demonstrated that 20-HETE is a key regulator of microvascular remodeling in hypertension; its effect is independent of blood pressure elevation and androgen levels.

CYP4 enzymes as potential drug targets: focus on enzyme multiplicity, inducers and inhibitors, and therapeutic modulation of 20-hydroxyeicosatetraenoic acid (20-HETE) synthase and fatty acid ω-hydroxylase activities.

The Cytochrome P450 4 (CYP4) family of enzymes in humans is comprised of thirteen isozymes that typically catalyze the ω-oxidation of endogenous fatty acids and eicosanoids. Several CYP4 enzymes can biosynthesize 20- hydroxyeicosatetraenoic acid, or 20-HETE, an important signaling eicosanoid involved in regulation of vascular tone and kidney reabsorption. Additionally, accumulation of certain fatty acids is a hallmark of the rare genetic disorders, Refsum disease and X-ALD. Therefore, modulation of CYP4 enzyme activity, either by inhibition or induction, is a potential strategy for drug discovery. Here we review the substrate specificities, sites of expression, genetic regulation, and inhibition by exogenous chemicals of the human CYP4 enzymes, and discuss the targeting of CYP4 enzymes in the development of new treatments for hypertension, stroke, certain cancers and the fatty acid-linked orphan diseases.