20-HETE Levels Research Articles

Study on the mechanism of 20-hydroxyeicosatetraenoic acid in retinal ischemia-reperfusion injury.

To explore the effect of 20-hydroxyeicosatetraenoic acid (20-HETE) on retinal ischemia-reperfusion injury (RIRI) and the protective effect of N-hydroxy-N'-(4-n-butyl-2-methylphenyl)formamidine (HET0016) on RIRI. Male Sprague-Dawley rats were randomly divided into the normal control group, experimental model group (RIRI group), experimental solvent group (RIRI + solvent group), and experimental treatment group (RIRI + HET0016 group). The levels of 20-HETE, tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) in the retina of rats at 24 h after reperfusion were measured by enzyme-linked immunosorbent assay. Hematoxylin-eosin staining was used to observe the retinal morphological and thickness changes at 24 h, 48 h, and 7 days after reperfusion. The number and localized expression of matrix metalloproteinase-9-positive cells in the retina of the rats at 24 h after reperfusion and the activation and localized expression of retinal microglia at 48 h after reperfusion were measured using an immunohistochemical method. The nuclear metastasis of nuclear factor kappa-B (NF-κB, p65) cells at 24 h after reperfusion was observed using an immunofluorescence method. Overall, 20-HETE might activate microglia to aggravate RIRI by the NF-κB pathway, but HET0016 has significant protective effects for the retina.

20-HETE As an Emerging Target to Improve Renal Health in Sickle Cell Disease

The kidney is one of the most severely affected organs in sickle cell disease (SCD), with glomerular hyperfiltration and microalbuminuria beginning in childhood, and progressing to chronic kidney disease (CKD) in majority of adults. Sickle individuals also suffer from acute kidney injury (AKI) often during characteristic vaso-occlusive crisis. Renal pathology in sickle cell nephropathy includes extensive microvascular congestions and thrombotic microangiopathy. The hypercoagulable and vasoconstrictive environment of SCD, in conjunction with medullary hypoperfusion and cortical hyperperfusion contributes to vaso-occlusive injury and deregulates intrarenal hemostasis leading to CKD development. Ischemic injury increases arachidonic acid hydroxylation producing 20-hydroxyeicosatetraenoic acid (20-HETE), which in turn triggers thrombosis and promotes vasoconstriction in the kidney. However, the role of 20-HETE in regulating renal hemostasis in SCD nephropathy has never been reported. We found that plasma 20-HETE is significantly elevated in SCD patients (n=9-11; p<0.01) compared to normal individuals. Similarly, transgenic SCD mice (SS), homozygous for human hemoglobin S, have increased plasma 20-HETE compared to control mice (AA), homozygous for human hemoglobin A (n=6; p<0.01). We have previously shown that elevated circulating heme mimicking hemolytic crisis triggers clinically relevant acute kidney injury (AKI) in SS mice (Blood (2020) 135(13): 1044-1048). We postulated that hemolytic stress may exacerbate 20-HETE production which in turn aggravates renal microvascular occlusion and vasoconstriction, leading to renal damage in SCD. Immunohistochemistry and biochemical experiments showed that the expression of CYP4A12a, a cytochrome 450-dependent enzyme responsible 20-HETE production in mice, is substantially elevated in the kidneys of SS mice compared to AA mice, associated with increased vascular congestion and renal thrombogenesis. Renal CYP4A12a and plasma 20-HETE were increased substantially following heme-induced AKI in SS mice, associated with a heightened expression of renal angiotensin II receptor 1 (ATIIR1), thus indicating induced vasoconstrictive activity. In vitro, we found that 20-HETE induced ATIIR1 expression in human renal glomerular endothelial cells (hRGEC), which was further exacerbated by low-concentration of heme challenge (5 mM for 24 hour). Nitro-oleic acid (NO 2-OA) is a nitrated fatty acid that binds ATIIR1 and inhibits angiotensin II induced vasoconstriction. Pretreatment with NO 2-OA reduced 20-HETE-dependent ATIIR1 expression in hRGEC, while NO 2-OA prophylaxis during hemin-induced AKI inhibited albuminuria, normalized plasma creatinine levels, and lowered the expression of ATIIR1 in SS mice kidneys. Overall, our data show that elevated 20-HETE levels during hemolytic stress may aggravate renal injury in SCD which can be prevented by NO 2-OA prophylaxis. Ongoing experiments will determine the direct effect of elevated 20-HETE in renal hemostasis and CKD development in SCD.

Renin-Angiotensin System Components and Arachidonic Acid Metabolites as Biomarkers of COVID-19.

Through the ACE2, a main enzyme of the renin-angiotensin system (RAS), SARS-CoV-2 gains access into the cell, resulting in different complications which may extend beyond the RAS and impact the Arachidonic Acid (ArA) pathway. The contribution of the RAS through ArA pathways metabolites in the pathogenesis of COVID-19 is unknown. We investigated whether RAS components and ArA metabolites can be considered biomarkers of COVID-19. We measured the plasma levels of RAS and ArA metabolites using an LC-MS/MS. Results indicate that Ang 1-7 levels were significantly lower, whereas Ang II levels were higher in the COVID-19 patients than in healthy control individuals. The ratio of Ang 1-7/Ang II as an indicator of the RAS classical and protective arms balance was dramatically lower in COVID-19 patients. There was no significant increase in inflammatory 19-HETE and 20-HETE levels. The concentration of EETs was significantly increased in COVID-19 patients, whereas the DHETs concentration was repressed. Their plasma levels were correlated with Ang II concentration in COVID-19 patients. In conclusion, evaluating the RAS and ArA pathway biomarkers could provide helpful information for the early detection of high-risk groups, avoid delayed medical attention, facilitate resource allocation, and improve patient clinical outcomes to prevent long COVID incidence.

Bioactive lipid mediators in plasma are predictors of preeclampsia irrespective of aspirin therapy

There are few early biomarkers to identify pregnancies at risk of preeclampsia (PE) and abnormal placental function. In this cross-sectional study, we utilized targeted ultra-performance liquid chromatography-ESI MS/MS and a linear regression model to identify specific bioactive lipids that serve as early predictors of PE. Plasma samples were collected from 57 pregnant women prior to 24-weeks of gestation with outcomes of either PE (n = 26) or uncomplicated term pregnancies (n = 31), and the profiles of eicosanoids and sphingolipids were evaluated. Significant differences were revealed in the eicosanoid, (±)11,12 DHET, as well as multiple classes of sphingolipids; ceramides, ceramide-1-phosphate, sphingomyelin, and monohexosylceramides; all of which were associated with the subsequent development of PE regardless of aspirin therapy. Profiles of these bioactive lipids were found to vary based on self-designated race. Additional analyses demonstrated that PE patients can be stratified based on the lipid profile as to PE with a preterm birth linked to significant differences in the levels of 12-HETE, 15-HETE, and resolvin D1. Furthermore, subjects referred to a high-risk OB/GYN clinic had higher levels of 20-HETE, arachidonic acid, and Resolvin D1 versus subjects recruited from a routine, general OB/GYN clinic. Overall, this study shows that quantitative changes in plasma bioactive lipids detected by ultra-performance liquid chromatography-ESI-MS/MS can serve as an early predictor of PE and stratify pregnant people for PE type and risk.

Effect of Chronically Suppressed Plasma Angiotensin II on Regulation of the CYP4A/20-HETE Pathway in the Dahl Salt-Sensitive Rat.

In Dahl salt-sensitive (SS) rats, impaired vascular relaxation can be restored by: (1) minipump infusion of a low (sub-pressor) dose of angiotensin II (ANG II) to restore physiological levels of plasma ANG II, (2) inhibition of 20-HETE production, and (3) introgression of a normally functioning renin allele from the Brown Norway rat (SS-13BN consomic rat). Unlike SS rats, SS-13BN rats have normal levels of ANG II on a normal-salt diet and suppressed ANG II on a high-salt (HS) diet. This study tested whether chronically low ANG II levels in SS rats upregulate cytochrome P450-4A (CYP4A) increasing the production of the vasoconstrictor 20-HETE. Although salt-induced suppression of ANG II levels increased reactive oxygen species (ROS) in basilar arteries from SS-13BN rats in previous studies, this study showed no change in vascular 20-HETE levels in response to ANGII suppression. CYP4A inhibition significantly reduced vascular ROS levels and restored endothelium-dependent relaxation in response to acetylcholine in the middle cerebral artery (MCA) of SS rats and HS-fed SS-13BN rats. These data demonstrate that both the renin-angiotensin system and the CYP4A/20-HETE pathway play a direct role in the vascular dysfunction of the Dahl SS rat but are independent of each other, even though they may both contribute to vascular dysfunction through ROS production.

Mediators of inflammation resolution and vasoactive eicosanoids in gestational diabetes and preeclampsia.

Women with gestational diabetes (GDM) have an increased risk of preeclampsia and postpartum diabetes. Inflammation associates with both GDM and preeclampsia. This study examined specialized proresolving mediators (SPM) that direct inflammation resolution and eicosanoids that are involved in inflammation, in relation to the development of preeclampsia and ongoing postpartum glucose intolerance in GDM. Participants were selected from a prospective study examining the development of preeclampsia in women with GDM. Four groups of age-matched women were studied: GDM ( n = 20), GDM who developed preeclampsia (GDM+PE, n = 21), GDM who remained glucose-intolerant postpartum (GDM+PPIGT, n = 20), or pregnancies with glucose tolerance within the normal range (NGT, n = 21). Measurement of SPM (E-series resolvins and D-series resolvins), SPM pathway intermediates (14-HDHA, 18-HEPE and 17-HDHA), 20-hydroxyeicosatetraenoic acid (20-HETE), and the urinary metabolite of the vasodilator prostacyclin 2,3-dinor-6-Keto-PGF 1α , were made at 28, 32 and 36 weeks gestation and at 6 months postpartum. Compared with GDM, GDM+PE had elevated levels of 20-HETE and the SPM pathway intermediates 14-HDHA, 18-HEPE, 17-HDHA, at 32 weeks, and the SPM RvE1 at 32 and 36 weeks gestation. Compared with NGT and regardless of whether they developed preeclampsia or PPIGT, GDM had lower levels of 2,3-dinor-6-Keto-PGF 1α during pregnancy. Reduced levels of the prostacyclin metabolite 2,3-dinor-6-Keto-PGF 1α may contribute to the increased risk of preeclampsia in women with GDM. The increase in 20-HETE, a vasoconstrictor and mediator of inflammation, and SPM that contribute to inflammation resolution, prior to the onset of preeclampsia require further investigation to clarify their clinical significance.

Bioactive lipid screening during respiratory tract infections with bacterial and viral pathogens in mice

IntroductionRespiratory tract infections are a worldwide health problem for humans and animals. Different cell types produce lipid mediators in response to infections, which consist of eicosanoids like hydroxyeicosatetraenoic acids (HETEs) or oxylipins like hydroxydocosahexaenoic acids (HDHAs). Both substance classes possess immunomodulatory functions. However, little is known about their role in respiratory infections.ObjectivesHere, we aimed to analyze the lipid mediator imprint of different organs of C57BL/6J mice after intranasal mono-infections with Streptococcus pneumoniae (pneumococcus), Staphylococcus aureus or Influenza A virus (IAV) as wells as pneumococcal-IAV co-infection.MethodsC57BL/6J mice were infected with different pathogens and lungs, spleen, and plasma were collected. Lipid mediators were analyzed using HPLC-MS/MS. In addition, spatial-distribution of sphingosine 1-phosphate (S1P) and ceramide 1-phosphates (C1P) in tissue samples was examined using MALDI-MS-Imaging. The presence of bacterial pathogens in the lung was confirmed via immunofluorescence staining.ResultsWe found IAV specific changes for different HDHAs and HETEs in mouse lungs as well as enhanced levels of 20-HETE in severe S. aureus infection. Moreover, MALDI-MS-Imaging analysis showed an accumulation of C1P and a decrease of S1P during co-infection in lung and spleen. Long chain C1P was enriched in the red and not in the white pulp of the spleen.ConclusionsLipid mediator analysis showed that host synthesis of bioactive lipids is in part specific for a certain pathogen, in particular for IAV infection. Furthermore, MS-Imaging displayed great potential to study infections and revealed changes of S1P and C1P in lungs and spleen of co-infected animals, which was not described before.

Sexual dimorphism in renal heme oxygenase-1 and arachidonic acid metabolizing enzymes in spontaneously hypertensive rats versus normotensive Wistar Kyoto rats

Numerous studies have demonstrated a sexual dimorphism in blood pressure (BP) control in spontaneously hypertensive rats (SHR), however the mechanisms remain to be further elucidated. Based on the established role of arachidonic acid metabolites and heme oxygenase (HO) in BP control, we hypothesize that higher BP in male SHR is associated with differential expression in renal HO and arachidonic acid metabolizing enzymes vs. female SHR. Higher BP in male SHR coincided with significant increases in renal cortical superoxide production and thiobarbituric acid reactive substances (TBARs) levels as measures of oxidative stress compared to normotensive female WKY and female SHR. The elevations in BP and oxidative stress in male SHR were also associated with a decrease in cortical heme oxygenase-1 (HO-1) expression when compared to normotensive female WKY. Although there was no sex or strain differences in cortical expression of the epoxyeicosatrienoic acids (EETs) producing enzyme, cytochrome P450 epoxygenase (CYP2C23), in male and female SHR and WKY, SHR had greater expression of the EETs metabolizing enzyme, soluble epoxide hydrolase (sEH) vs. WKY. Cortical expression of the 20–hydroxyeicosatetraenoic acid (20-HETE) producing enzyme, cytochrome P450 hydroxylase (CYP4A), was less in female WKY and SHR compared to strain-matched males and cortical 20-HETE levels were also less in female SHR vs. male SHR. Cortical cyclooxygenase-2 (COX-2) expression was significantly greater in female SHR and WKY vs. males and cortical prostaglandin E2 (PGE2) levels in female SHR was significantly greater than male WKY. In conclusion, our data suggest that sex differences in renal oxidative stress, HO-1 and arachidonic acid metabolizing enzymes could contribute to sexual dimorphism in hypertension in young SHR.

Anti‐inflammatory effects of Angiotensin‐(1‐7) through Renin‐Angiotensin System and Arachidonic Acid Pathway

ObjectiveRheumatoid arthritis (RA) is a prevalent chronic inflammatory disease that affects 1‐2% of the population worldwide. Renin‐angiotensin system (RAS) and Arachidonic acid (ArA) pathway are associated with the pathogenesis of inflammatory diseases (Fig. 1). Inflammatory conditions such as RA disrupt the balance between pro‐ and anti‐inflammatory components of these two pathways. The RAS is composed of two opposing axes. Ang II is one of its components that is implicated in the up‐regulation of pro‐inflammatory mediators and contributes to the pathogenesis of RA. On the other hand, Ang‐(1‐7), counteracts the inflammatory effects of Ang II. Several studies have shown a cross‐talk between the RAS and ArA metabolites, especially eicosanoids such as 20‐hydroxyeicosatetraenoic acid (20‐HETE) and epoxyeicosatrienoic acids (EETs). Ang II stimulates 20‐HETE synthesis in microvessels and decreases EET levels by increasing their degradation. In this study, using Ang‐(1‐7) and its long‐acting bone‐targeting conjugate, we studied the impact of Ang‐(1‐7) anti‐inflammatory effects on the RAS, ArA pathways and their interrelation.MethodsTwenty‐four male Sprague‐Dawley rats were randomly divided into healthy control and adjuvant arthritic (AA) rats. The rats in the AA group were injected with Mycobacterium butyrium in squalene to induce AA. AA rats were further divided into and treated with four groups INF, Ang‐(1‐7), and Ang. Conj. After 21 days of treatment, animals’ plasma samples were collected for analysis of Ang II, Ang‐(1‐7), 20‐HETE, and different EETs. The quantification was done using an LC‐MS/MS system in positive ESI mode for Ang peptides and negative for HETE and EETs. The one‐way ANOVA with Tukey posthoc test was used to test the significance difference (p‐value < 0.05).ResultsThe Ang peptide analysis shows that the level of Ang‐(1‐7) in control (0.75 ± 0.08) and INF (0.19 ± 0.05) rats was significantly different. However, treating with Ang‐(1‐7) peptide (0.90 ± 0.17) or Ang. Conj. peptide conjugate (1.45 ± 0.22) could increase the Ang‐(1‐7) peptide level in rats. In contrast to Ang‐(1‐7), Ang II levels in the INF group were significantly different from control, Ang‐(1‐7), and Ang. Conj. (Fig. 2A). Analyzing the level of total EETs and 20‐HETE in plasma reveals that the total EETs/20‐HETE ratio decreases in INF rats (0.10 ± 0.02) significantly, whereas this ratio increased in Ang‐(1‐7) (3.48 ± 0.65), and Ang. Conj. (7.73 ± 1.21) in comparison to the control (6.90 ± 1.03) group (Fig. 2B). Our finding reveals a positive correlation between the level of Ang II and 20‐HETE, Ang‐(1‐7), and total‐EETs. This trend was reversed when Ang II was correlated with total EETs or the ratio of total‐EETs/20‐HETE (Fig. 3B). Concentrations are reported as ng/mL.ConclusionThe physiological balance of CYP‐mediated ArA metabolites and the RAS components are disturbed by the inflammation, resulting in the augmentation of pro‐inflammatory arms of both pathways. Our approach of using Ang‐(1‐7) and its long‐acting bone targeting conjugate could reverse such an effect and promote anti‐inflammatory effect in the AA animal model.

Urinary 20-HETE: A prospective Non-Invasive prognostic and diagnostic marker for diabetic kidney disease

IntroductionThe identification and validation of a non-invasive prognostic marker for early detection of diabetic kidney disease (DKD) can lead to substantial improvement in therapeutic decision-making. ObjectivesThe main objective of this study is to assess the potential role of the arachidonic acid (AA) metabolite 20-hydroxyeicosatetraenoic (20-HETE) in predicting the incidence and progression of DKD. MethodsHealthy patients and patients with diabetes were recruited from the Hamad General Hospital in Qatar, and urinary 20-HETE levels were measured. Data analysis was done using the Statistical Package for Social Sciences (SPSS). ResultsOur results show that urinary 20-HETE-to-creatinine (20-HETE/Cr) ratios were significantly elevated in patients with DKD when compared to patients with diabetes who did not exhibit clinical signs of kidney injury (p < 0.001). This correlation was preserved in the multivariate linear regression accounting for age, diabetes, family history of kidney disease, hypertension, dyslipidemia, stroke and metabolic syndrome. Urinary 20-HETE/Cr ratios were also positively correlated with the severity of kidney injury as indicated by albuminuria levels (p < 0.001). A urinary 20-HETE/Cr ratio of 4.6 pmol/mg discriminated between the presence and absence of kidney disease with a sensitivity of 82.2 % and a specificity of 67.1%. More importantly, a 10-unit increase in urinary 20-HETE/Cr ratio was tied to a 10-fold increase in the risk of developing DKD, suggesting a 20-HETE prognostic efficiency. ConclusionTaken together, our results suggest that urinary 20-HETE levels can potentially be used as non-invasive diagnostic and prognostic markers for DKD.

Gut Microbiome and Metabolome Changes in Mice With Acute Vestibular Deficit.

Vestibular deficit is a very common disorder in clinical practice and is characterized by vertigo, spontaneous nystagmus, and autonomic nervous symptoms, including nausea, vomiting, and sweating. In addition, the comorbidity of vestibular deficit and anxiety has long been an integral component of the medical literature. Previous studies have suggested that the mechanisms underlying this comorbidity involved overlap of vestibular and cerebellar networks. Emerging evidence has shown that the microbiota–gut–brain axis plays a key role in the regulation of affective disorders. Thus, we hypothesized that the gut microbiota may be involved in the comorbidity of vestibular deficit and anxiety. To verify this, we constructed a unilateral labyrinthectomy mouse model to simulate vestibular deficit. Then, 16S rRNA gene sequencing and liquid chromatography–mass spectrometry (LC-MS) were used to analyze the microbiome and metabolome of the cecal samples collected from mice in the unilateral labyrinthectomy, sham surgery, and control groups. Notably, unilateral labyrinthectomy shaped the composition of the mouse gut microbiome, resulting in increased abundance of Lachnospiraceae NK4A136 group, Odoribacter and Roseburia and decreased abundance of Prevotella and Parasutterella at the genus level. Tax4Fun functional prediction indicated a decrease in tryptophan metabolism in mice in the unilateral labyrinthectomy group. Moreover, functional correlation of changes in gut microbes and metabolites between different groups showed that the oleamide level was negatively correlated with Odoribacter abundance (r = -0.89, p = 0.0002). The butyric acid level was positively correlated with Parasutterella abundance (r = 0.85, p = 0.0010). The propanoate level was negatively correlated with Prevotella abundance (r = -0.81, p = 0.0020). The 20-HETE level was positively correlated with Parasutterella abundance (r = 0.84, p = 0.0013). The altered microbes and metabolites were closely related to the pathogenesis of affective disorders. Our results not only offer novel insights into the vestibular deficit comorbid with anxiety but also build an important basis for future research on this etiology.

IDF21-0159 Asymmetric Dimethylarginine and 20-Hydroxyeicosatetraenoic Acid – A lethal combo in diabetic kidney disease

Background: 20-Hydroxyeicosatetraenoic Acid (20-HETE), an important metabolite of arachidonic acid, is known to be involved in causing renal cell injury. Cytochrome P450 is the enzyme responsible for the synthesis of 20-HETE. Cytochrome P450 is negatively related to nitric oxide (NO) levels. We previously reported that Asymmetric Dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide, is significantly elevated in diabetes patients with albuminuria. Hence, we hypothesize that the inhibition of NO by ADMA in turn may augment the synthesis of 20-HETE. Further, we hypothesis that 20-HETE may also play a vital role in the ADMA induced kidney fibrosis. Hence, in this study we plan to measure the HETE levels in patients with Type 2 DM and study its association with ADMA and NO levels. Aim: Our aim is to study the urinary level of 20-HETE in patients with type 2 DM with and without nephropathy and to analyse the association of 20-HETE with ADMA, NO and other biochemical parameters. We also aimed to evaluate the efficiency of 20-HETE as a potential non-invasive biomarker for predicting diabetic nephropathy. Method: We recruited 120 patients from a centre for diabetes South India. Patients were divided into four groups namely normal glucose tolerance (NGT) (n=30), type 2 DM without nephropathy (T2DM) (n=30), type 2DM with microalbuminuria (MIC) (n=30) and type 2 DM with macroalbuminuria (MAC) (n=30). The urinary 20-HETE levels and circulatory ADMA levels were assessed using ELISA kit. Plasma total nitrite was quantified as a measure of nitric oxide formation by using Griess method as per the manufacturer's instructions. Urinary albumin was measured in fasting urine sample using immunoturbiometric assay and serum creatinine measured using Jaffe's method. Statistical analysis was performed using SPSS 20. Results: Urinary 20-HETE levels and the circulatory ADMA levels were significantly increased in concomitant with the significant decrease in nitric oxide levels in MIC and MAC groups when compared with NGT and T2DM groups. 20-HETE showed a significant positive correlation with the ADMA (r=0.362, p<0.001). As expected, 20-HETE showed a significant negative correlation with total nitric oxide (r=-0.252, p<0.05). Also with FBS (r=0.537, p<0.001), HbA1c (r=0.502, p<0.001) and urinary albumin to creatinine ratio (r=0.576, p<0.001) 20-HETE showed significant positive correlation confirming its association with diabetes and kidney damage. We found that the urinary 20-HETE level of 252.79 pg/ml was significant to detect the diabetic nephropathy in T2DM. The predicted cut-off value of 20-HETE had a specificity of 75% and sensitivity of 81%. Discussion: From this clinical study, we found that the exaggerated synthesis of ADMA under diabetic conditions contributes to the synthesis of 20-HETE. This is due to the inhibitory action of ADMA on the nitric oxide synthesis and the reduction in NO level promotes the synthesis of 20-HETE via CYP4A enzyme. Thus under hyperglycemic conditions, ADMA and 20-HETE together contribute to the diabetic kidney injury. Further, we suggest that 20-HETE can be used as a non-invasive biomarker for detecting diabetic kidney disease at an early stage.

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.

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

Transient receptor potential cation channel subfamily V member 1 (TRPV1) is localized to sensory C-fibres and its opening leads to membrane depolarization, resulting in neuropeptide release and neurogenic inflammation. However, the identity of the endogenous activator of TRPV1 in this setting is unknown. The arachidonic acid metabolites 12-hydroperoxyeicosatetraenoyl acid (12-HpETE) and 20-hydroxyeicosatetraenoic acid (20-HETE) have emerged as potential endogenous activators of TRPV1. However, whether these lipids underlie TRPV1-mediated neurogenic inflammation remains unknown. We analysed human cantharidin-induced blister samples and inflammatory responses in TRPV1 transgenic mice. In a human cantharidin-blister model, the potent TRPV1 activators 20-HETE but not 12-HETE (stable metabolite of 12-HpETE) correlated with arachidonic acid levels. Similarly, in mice, levels of 20-HETE (but not 12-HETE) and arachidonic acid were strongly positively correlated within the inflammatory milieu. Furthermore, LPS-induced oedema formation and neutrophil recruitment were substantially and significantly attenuated by pharmacological block or genetic deletion of TRPV1 channels, inhibition of 20-HETE formation or SP receptor neurokinin 1 (NK1 ) blockade. LPS treatment also increased cytochrome P450 ω-hydroxylase gene expression, the enzyme responsible for 20-HETE production. Taken together, our findings suggest that endogenously generated 20-HETE activates TRPV1 causing C-fibre activation and consequent oedema formation. These findings identify a novel pathway that may be useful in the therapeutics of diseases/conditions characterized by a prominent neurogenic inflammation, as in several skin diseases.

Blockade of 20-hydroxyeicosatetraenoic acid receptor lowers blood pressure and alters vascular function in mice with smooth muscle-specific overexpression of CYP4A12-20-HETE synthase.

20-Hydroxyeicosatetraenoic acid (20-HETE) is a vasoactive eicosanoid exhibiting effects on vascular smooth muscle cell (VSMC) via G-protein coupled receptor 75 (GPR75) and include stimulation of contractility, migration, and growth. We examined whether VSMC-targeted overexpression of CYP4A12, the primary 20-HETE-producing enzyme in mice, is sufficient to promote hypertension. Mice with VSM-specific Cyp4a12 overexpression (Myh11-4a12) and their littermate controls (WT) were generated by crossbreeding Cyp4a12-floxed with Myh11-Cre mice. The 20-HETE receptor blocker, N-disodium succinate-20-hydroxyeicosa-6(Z),15(Z)-diencarboxamide (AAA), was administered in the drinking water. Experiments were carried out for 12 days. SBP was measured by tail cuff. Renal interlobar and mesenteric arteries were harvested for assessment of gene expression, 20-HETE levels, vascular contractility, vasodilation, and remodeling. Vascular and circulatory levels of 20-HETE were several folds higher in Myh11-4a12 mice compared with WT. The Myh11-4a12 mice compared with WT were hypertensive (145 ± 2 vs. 127 ± 2 mmHg; P < 0.05) and their vasculature displayed a contractile phenotype exemplified by increased contractility, reduced vasodilatory capacity, and increased media to lumen ratio. All these features were reversed by the administration of AAA. The mechanism of increased contractility includes, at least in part, Rho-kinase activation followed by increased myosin light chain phosphorylation and activation of the contractile apparatus. VSM-specific Cyp4a12 overexpression is sufficient to alter VSM cell phenotype through changes in contractile markers and enhancement in contractility that promote hypertension and vascular dysfunction in a 20-HETE-dependent manner. The 20-HETE receptor GPR75 may represent a novel target for the treatment of hypertension and associated vascular conditions.

Effect of rosiglitazone on 20-hydroxyeicosatetraenoic acid levels and CYP4F2 expression in HepG2 cells

&#x0D; &#x0D; &#x0D; &#x0D; Purpose: To determine the effect of rosiglitazone on the levels of the cardiotoxic arachidonic acid metabolite, 20-hydroxyeicosatetraenoic acid (20-HETE), in the human liver hepatocellular carcinoma cell line, HepG2.&#x0D; Methods: HepG2 cells were treated with thiazolidinedione rosiglitazone and the mRNA and protein expressions of cytochrome P450 4F2 (CYP4F2) responsible for synthesizing 20-HETE were measured using quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting. The levels of 20-HETE were evaluated using liquid chromatography/mass spectrometry (LC-MS).&#x0D; Results: Rosiglitazone significantly increased the levels of CYP4F2 mRNA and protein when compared with the control group (p &lt; 0.05). This was correlated with significantly increased 20-HETE levels in the culture medium of rosiglitazone-treated cells in a dose-dependent manner (p &lt; 0.05). The PPARγ antagonist, GW9662, significantly repressed the increased production of 20-HETE and CYP4F2 mRNA protein (p &lt; 0.05).&#x0D; Conclusion: Rosiglitazone increases the synthesis of 20-HETE via activation of PPARγ receptor and upregulation of CYP4F2. These findings may provide an additional explanation, at least in part, for the unwanted side effects of rosiglitazone on the cardiovascular system.&#x0D; &#x0D; &#x0D; &#x0D;

20-hydroxyeiscosatetraenoic acid may be as a predictor of malignant middle cerebral artery infarction in patients with massive middle cerebral artery infarction

BackgroundEarly identification of massive middle cerebral artery infarction (MCAI) at risk for malignant MCAI (m-MCAI) may be useful in selecting patients for aggressive therapies. The aim of this study was to determine whether CYP metabolites may help to predict impending m-MCAI.MethodsThis is a prospective, two-center observational study in 256 patients with acute massive MCAI. Plasma levels of 20-hydroxyeicosatetraenoic acid (20-HETE), epoxyeicosatrienoic acids, and dihydroxyeicosatrienoic acids were measured at admission. Brain computed tomography (CT) was performed at admission and repeated between day 3 and 7, or earlier if there was neurological deterioration. The primary outcome was m-MCAI. The m-MCAI was diagnosed when follow-up brain CT detected a more than two-thirds space-occupying MCAI with midline shift, compression of the basal cisterns, and neurological worsening.ResultsIn total of 256 enrolled patients, 77 (30.1%) patients developed m-MCAI. Among the 77 patients with m-MCAI, 60 (77.9%) patients died during 3 months of stroke onset. 20-HETE level on admission was significantly higher in patients with m-MCAI than those without m-MCAI. There was an increase in the risk of m-MCAI with increase of 20-HETE levels. The third and fourth quartiles of 20-HETE levels were independent predictors of m-MCAI (OR: 2.86; 95% CI: 1.16 – 6.68; P = 0.025, and OR: 4.23; 95% CI: 1.35 – 8.26; P = 0.002, respectively).ConclusionsIncidence of m-MCAI was high in patients with massive MCAI and the prognosis of m-MCAI is very poor. Elevated plasma 20-HETE may be as a predictor for m-MCAI in acute massive MCAI, and it might useful in clinical practice in therapeutic decision making.

Increased 20-HETE Signaling Suppresses Capillary Neurovascular Coupling After Ischemic Stroke in Regions Beyond the Infarct.

Neurovascular coupling, the process by which neuronal activity elicits increases in the local blood supply, is impaired in stroke patients in brain regions outside the infarct. Such impairment may contribute to neurological deterioration over time, but its mechanism is unknown. Using the middle cerebral artery occlusion (MCAO) model of stroke, we show that neuronal activity-evoked capillary dilation is reduced by ∼75% in the intact cortical tissue outside the infarct border. This decrease in capillary responsiveness was not explained by a decrease in local neuronal activity or a loss of vascular contractility. Inhibiting synthesis of the vasoconstrictive molecule 20-hydroxyeicosatetraenoic acid (20-HETE), either by inhibiting its synthetic enzyme CYP450 ω-hydroxylases or by increasing nitric oxide (NO), which is a natural inhibitor of ω-hydroxylases, rescued activity-evoked capillary dilation. The capillary dilation unmasked by inhibiting 20-HETE was dependent on PGE2 activation of endoperoxide 4 (EP4) receptors, a vasodilatory pathway previously identified in healthy animals. Cortical 20-HETE levels were increased following MCAO, in agreement with data from stroke patients. Inhibition of ω-hydroxylases normalized 20-HETE levels in vivo and increased cerebral blood flow in the peri-infarct cortex. These data identify 20-HETE-dependent vasoconstriction as a mechanism underlying capillary neurovascular coupling impairment after stroke. Our results suggest that the brain’s energy supply may be significantly reduced after stroke in regions previously believed to be asymptomatic and that ω-hydroxylase inhibition may restore healthy neurovascular coupling post-stroke.

Genetics Variants in the Epoxygenase Pathway of Arachidonic Metabolism Are Associated with Eicosanoids Levels and the Risk of Diabetic Nephropathy.

Genes in the epoxygenase pathway of arachidonic acid metabolism leading to vasoactive eicosanoids, mainly 20-hydroxyeicosatetraenoic (20-HETE) and epoxyeicosatrienoic (EETs) acids, have been related to glucose-induced renal damage in preclinical reports. We genotyped 1088 diabetic kidney disease (DKD) patients and controls for seven polymorphisms in five genes (CYP2C8, CYP2J2, CYP4F2, CYP4A11, and EPHX2) along this metabolic route and evaluated their effect on DKD risk, clinical outcomes, and the plasma/urine levels of eicosanoids measured by LC/MS/MS and immunoenzymatic assays. The CYP4F2 433M variant allele was associated with lower incidence of DKD (OR = 0.65 (0.48–0.90), p = 0.008), whilst the CYP2C8*3/*3 genotype was related to increased risk (OR = 3.21 (1.05–9.87), p = 0.036). Patients carrying the 433M allele also showed lower eGFR [median and interquartile range vs. wildtype carriers: 30.8 (19.8) and 33.0 (23.2) mL/min/1.73 m2, p = 0.037). Finally, the 433VM/MM variant genotypes were associated with lower urinary levels of 20-HETE compared with 433VV (3.14 (0.86) vs. 8.45 (3.69) ng/mg Creatinine, p = 0.024). Our results indicate that the CYP4F2 V433M polymorphism, by decreasing 20-HETE levels, may play an important role in DKD.

Early type 2 diabetes and obesity does not affect eicosanoids level and renal morphology in a rat model/ Diabetes tipo 2 no estágio inicial e obesidade não afetam o nível de eicosanóides e a morfologia renal em um modelo de rato

This study evaluated the effects of the early development of Diabetes Mellitus 2 (T2D) and diet-induced Obesity in the eicosanoid pathways and its effects on renal tissue. Thirty male Wistar rats were fed with a high-fat or standard diet and were divided into 3 groups: The Control group received a standard diet, the T2D group received a high-fat diet and a single dose of streptozotocin (25mg/Kg) and the Obesity group received high-fat diet. Caloric intake, feed efficiency, body weight gain, visceral fat, blood glucose, plasma levels of 14,15 EET/DHET, 20-HETE, and kidneys’ morphology were analyzed. Total caloric intake and feed efficiency were higher in the animals of the Obesity group than in Control. Body weight gain, visceral fat, and blood glucose were higher in Obesity and T2D induced groups than in Control. Body weight gain, visceral fat, and feed efficiency associated positively with blood glucose. However, there was no difference in 14,15 EET/DHET, 20-HETE levels, or kidney injury between groups. In conclusion, we were unable to assess whether changes in eicosanoids are due to obesity or diabetes induction. So, this study suggests that longer periods of homeostatic disturbance caused by these protocols seem to be necessary to induce complications related to the disruption of the eicosanoid’s pathway and its effects on renal tissue.