20-HETE Antagonist Research Articles

20-Hydroxytetraenoic acid induces hepatic fibrosis via the TGF-β1/Smad3 signaling pathway

Hepatic fibrosis is caused by excessive accumulation of extracellular matrix (ECM) due to repeated liver injury. Hepatic stellate cells (HSCs) play a key role in the pathogenesis and progression of hepatic fibrosis. A study showed that CYP4A14 gene defect can inhibit hepatic fibrosis, but the specific mechanism was not clear. In this experiment, patients with hepatic fibrosis, LX-2 cells (a human HSCs line), and mice with liver fibrosis induced by carbon tetrachloride (CCl4) were used to study the effect of 20-Hydroxytetraenoic acid (20-HETE), one of the main metabolites of arachidonic acid (AA) catalyzed by CYP4A enzyme, on hepatic fibrosis and its mechanism. Our experimental results showed that the 20-HETE of patients with hepatic fibrosis is significantly higher than that of normal people and is closely related to the degree of fibrosis. 20-HETE could induce activation of LX-2 cells and 20-HETE antagonist could inhibit the induction of 20-HETE. 20-HETE was significantly increased in CCl4-induced liver fibrosis mice and inhibition of 20-HETE production could attenuate hepatic fibrosis. 20-HETE induced hepatic fibrosis mainly via the TGF- β1/Smad3 signal pathway. In conclusion, the results suggest that 20-HETE plays an important role in hepatic fibrosis and may be a possible target for the clinical treatment of hepatic fibrosis.

Activation of GPR75 Signaling Pathway Contributes to the Effect of a 20-HETE Mimetic, 5,14-HEDGE, to Prevent Hypotensive and Tachycardic Responses to Lipopolysaccharide in a Rat Model of Septic Shock.

The orphan receptor, G protein-coupled receptor (GPR) 75, which has been shown to mediate various effects of 20-hydroxyeicosatetraenoic acid (20-HETE), is considered as a therapeutic target in the treatment of cardiovascular diseases in which changes in the production of 20-HETE play a key role in their pathogenesis. Our previous studies showed that 20-HETE mimetic, N -(20-hydroxyeicosa-5[Z],14[Z]-dienoyl)glycine (5,14-HEDGE), protects against vascular hyporeactivity, hypotension, tachycardia, and arterial inflammation induced by lipopolysaccharide (LPS) in rats. This study tested the hypothesis that the GPR75 signaling pathway mediates these effects of 5,14-HEDGE in response to systemic exposure to LPS. Mean arterial pressure reduced by 33 mm Hg, and heart rate increased by 102 beats/min at 4 hours following LPS injection. Coimmunoprecipitation studies demonstrated that (1) the dissociation of GPR75/Gα q/11 and GPR kinase interactor 1 (GIT1)/protein kinase C (PKC) α, the association of GPR75/GIT1, large conductance voltage and calcium-activated potassium subunit β (MaxiKβ)/PKCα, MaxiKβ/proto-oncogene tyrosine-protein kinase (c-Src), and epidermal growth factor receptor (EGFR)/c-Src, MaxiKβ, and EGFR tyrosine phosphorylation were decreased, and (2) the association of GIT1/c-Src was increased in the arterial tissues of rats treated with LPS. The LPS-induced changes were prevented by 5,14-HEDGE. N -[20-Hydroxyeicosa-6( Z ),15( Z )-dienoyl]glycine, a 20-HETE antagonist, reversed the effects of 5,14-HEDGE in the arterial tissues of LPS-treated rats. Thus, similar to 20-HETE, by binding to GPR75 and activating the Gα q/11 /PKCα/MaxiKβ, GIT1/PKCα/MaxiKβ, GIT1/c-Src/MaxiKβ, and GIT1/c-Src/EGFR signaling pathways, 5,14-HEDGE may exert its protective effects against LPS-induced hypotension and tachycardia associated with vascular hyporeactivity and arterial inflammation.

Epoxyeicosatrienoic Acid Analog and 20-HETE Antagonist Combination Prevent Hypertension Development in Spontaneously Hypertensive Rats.

Numerous studies indicate a significant role for cytochrome P-450-dependent arachidonic acid metabolites in blood pressure regulation, vascular tone, and control of renal function. Epoxyeicosatrienoic acids (EETs) exhibit a spectrum of beneficial effects, such as vasodilatory activity and anti-inflammatory, anti-fibrotic, and anti-apoptotic properties. 20-Hydroxyeicosatetraenoic acid (20-HETE) is a potent vasoconstrictor that inhibits sodium reabsorption in the kidney. In the present study, the efficiency of EET-A (a stable analog of 14,15-EET) alone and combined with AAA, a novel receptor antagonist of 20-HETE, was tested in spontaneously hypertensive rats (SHR). Adult SHR (16 weeks old) were treated with two doses of EET-A (10 or 40 mg/kg/day). In the following experiments, we also tested selected substances in the prevention of hypertension development in young SHR (6 weeks old). Young rats were treated with EET-A or the combination of EET-A and AAA (both at 10 mg/kg/day). The substances were administered in drinking water for 4 weeks. Blood pressure was measured by telemetry. Once-a-week observation in metabolic cages was performed; urine, blood, and tissue samples were collected for further analysis. The combined treatment with AAA + EET-A exhibited antihypertensive efficiency in young SHR, which remained normotensive until the end of the observation in comparison to a control group (systolic blood pressure, 134 ± 2 versus 156 ± 5 mmHg, respectively; p < 0.05). Moreover the combined treatment also increased the nitric oxide metabolite excretion. Considering the beneficial impact of the combined treatment with EET-A and AAA in young rats and our previous positive results in adult SHR, we suggest that it is a promising therapeutic strategy not only for the treatment but also for the prevention of hypertension.

20-HETE Participates in Intracerebral Hemorrhage-Induced Acute Injury by Promoting Cell Ferroptosis.

Intracerebral hemorrhage (ICH) is a highly fatal type of stroke that leads to various types of neuronal death. Recently, ferroptosis, a form of cell death resulting from iron-dependent lipid peroxide accumulation, was observed in a mouse ICH model. N-hydroxy-N′-(4-n-butyl-2-methylphenyl)-formamidine (HET0016), which inhibits synthesis of the arachidonic acid metabolite 20-hydroxyeicosatetraenoic acid (20-HETE), has shown a protective effect after ICH. However, the underlying mechanisms of the neuroprotective effect need further investigation. We explored whether 20-HETE participates in ICH-induced ferroptosis ex vivo by using hemoglobin-treated organotypic hippocampal slice cultures (OHSCs) and in vivo by using a collagenase-induced ICH mouse model. Ex vivo, we found that the 20-HETE synthesis inhibitor HET0016 and antagonist 20-6,15-HEDGE reduced hemoglobin-induced cell death, iron deposition, and lipid reactive oxygen species levels in OHSCs. Furthermore, 20-HETE inhibition in OHSCs increased the expression of glutathione peroxidase (GPX) 4, an antioxidant enzyme that serves as a main regulator of ferroptosis. In contrast, exposure of OHSCs to the 20-HETE stable mimetic 20-5,14-HEDGE induced cell death that was significantly inhibited by the ferroptosis inhibitor ferrostatin-1. In vivo, HET0016 treatment ameliorated focal deficits, reduced lesion volume, and decreased iron accumulation around the lesion at day 3 and 7 after ICH. In addition, lipid peroxidation was decreased and expression of GPX4 was increased in the HET0016-treated ICH group. The mitogen-activated protein kinase pathway also was inhibited by HET0016 in vivo. These results indicate that 20-HETE contributes to ICH-induced acute brain injury in part by activating ferroptosis pathways, thereby providing an upstream target for inhibiting ferroptosis.

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

The Effects of 20-HETE Antagonism on Myocardial Infarction in Metabolic Syndrome Rats

20-hydroxyeicosatetraenoic acid (20-HETE) is an eicosanoid metabolite of that has a wide range of effects on the vascular system such as Collateral cell growth, vascular rebuilding of the heart. Metabolic Syndrome and 20-HETE have been shown to be correlated together.There is a higher concentration of 20-HETE in Metabolic Syndrome patients. With a higher concentration of 20-HETE patients with Metabolic syndrome have symptoms that are more severe. The effect of elevated 20-HETE is negative and can influence the cell growth after a Myocardial Infarction. Myocardial Infarction (MI) is another term for a heart attack. In previous studies, it shows an MI size increases with an elevated level of 20-HETE. During the study, the Metabolic Syndrome rats and control group of rats will be induced with an MI for about 30-minutes. After rats from both groups are given an MI and 20-HETE antagonist named 20-SOLA which counteracts 20-HETE levels will be given. 20-SOLA treatment was given to the rats at 48 hours, 1 week and 8 weeks. The results indicated that the AMPK antibody for both total and phosphorylated showed a significant decrease in 48-hour samples. 20-SOLA was found to create an equilibrium in 20-HETE levels in all tissue samples and significantly more in JCR MI rats. 20-SOLA aided the decrease in ischemia for both rats, but again results indicate a more reliable significance in JCR MI rats. These findings are relevant to the epidemic of cardiovascular diseases plaguing populations globally.

CYP4A/20-HETE regulates ischemia-induced neovascularization via its actions on endothelial progenitor and preexisting endothelial cells.

20-Hydroxyeicosatetraenoic acid (20-HETE) was recently identified as a novel contributor of ischemia-induced neovascularization based on the key observation that pharmacological interferences of CYP4A/20-HETE decrease ischemic neovascularization. The objective of the present study is to examine whether the underlying cellular mechanisms involve endothelial progenitor cells (EPCs) and preexisting endothelial cells (ECs). We found that ischemia leads to a time-dependent increase of cyp4a12 expression and 20-HETE production, which are endothelial in origin, using immunofluorescent microscopy, Western blot analysis, and LC-MS/MS. This is accompanied by increases in the tissue stromal cell-derived factor-1α (SDF-1α) expressions as well as SDF-1α plasma levels, EPC mobilization from bone marrow, and subsequent homing to ischemic tissues. Pharmacological interferences of CYP4A/20-HETE with a 20-HETE synthesis inhibitor, dibromo-dodecenyl-methylsulfimide (DDMS), or a 20-HETE antagonist, N-(20-hydroxyeicosa-6(Z), 15(Z)-dienoyl) glycine (6, 15-20-HEDGE), significantly attenuated these increases. Importantly, we also determined that 20-HETE plays a novel role in maintaining EPC functions and increasing the expression of Oct4, Sox2, and Nanog, which are indicative of increased progenitor cell stemness. Flow cytometric analysis revealed that pharmacological interferences of CYP4A/20-HETE decrease the EPC population in culture, whereas 20-HETE increases the cultured EPC population. Furthermore, ischemia also markedly increased the proliferation, oxidative stress, and ICAM-1 expression in the preexisting EC in the hindlimb gracilis muscles. We found that these increases were markedly negated by DDMS and 6, 15-20-HEDGE. Taken together, CYP4A/20-HETE regulates ischemia-induced compensatory neovascularization via its combined actions on promoting EPC and local preexisting EC responses that are associated with increased neovascularization. NEW & NOTEWORTHY CYP4A/20-hydroxyeicosatetraenoic acid (20-HETE) was recently discovered as a novel contributor of ischemia-induced neovascularization. However, the underlying molecular and cellular mechanisms are completely unknown. Here, we show that CYP4A/20-HETE regulates the ischemic neovascularization process via its combined actions on both endothelial progenitor cells (EPCs) and preexisting endothelial cells. Moreover, this is the first study, to the best of our knowledge, that associates CYP4A/20-HETE with EPC differentiation and stemness.

The CYP4A/20‐HETE Axis Regulates Ischemia‐induced Neovascularization via Its Actions on Endothelial Progenitor and Preexisting Endothelial Cells

20‐hydroxyeicosatetraenoic acid (20‐HETE) was recently identified as a novel contributor of ischemia‐induced neovascularization based on the key observation that pharmacological interferences of the CYP4A/20‐HETE axis decrease ischemic neovascularization. The objective of the present study is to determine whether the underlying cellular mechanisms involve endothelial progenitor cells (EPC) and pre‐existing endothelial cells (EC).We found that ischemia leads to a time‐dependent increase in 20‐HETE that is endothelial in origin using LC/MS/MS analysis and immunofluorescent (IF) microscopy. This is accompanied by increases in SDF‐1a expressions, EPC mobilization from bone marrow, and subsequent homing to ischemic tissues. Pharmacological interferences of the CYP4A/20‐HETE axis with a 20‐HETE synthesis inhibitor Dibromo‐dodecenyl‐methylsulfimide(DDMS) or a 20‐HETE antagonist N‐(20‐hydroxyeicosa‐6(Z), 15(Z)‐dienoyl) glycine(6, 15‐20‐HEDGE) significantly attenuated these increases. Importantly, we also determined that 20‐HETE plays a novel role in maintaining EPC functions and increases in the expression of Oct4, Sox2, and Nanog expression that are indicative of increased progenitor cell stemness. Flow cytometric analysis revealed that pharmacological interferences of the CYP4A/20‐HETE axis decrease the EPC population in culture, whereas 20‐HETE increases the cultured EPC population. Furthermore, ischemia also markedly increased the proliferation, oxidative stress, and ICAM‐1 expression in preexisting EC in the hindlimb gracilis muscles. We found that these increases were markedly negated by DDMS and 6, 15‐20‐HEDGE.Taken together, the CYP4A/20‐HETE axis regulates ischemia‐induced compensatory neovascularization via its combined actions on promoting EPC and local preexisting EC responses that are associated with increased neovascularization.Support or Funding InformationAmerican Heart Association [Grant 11SDG6870004]; National Institutes of Health [Grants HL34300], [Grant DK38226]; Robert A. Welch Foundation [GL625910]This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

20‐HETE Antagonism Reduces Left Ventricular Remodeling Post‐Myocardial Infarction

20‐Hydroxyeicosatetraenoic acid (20‐HETE) is a cytochrome p450‐derived eicosanoid that stimulates endothelial dysfunction and inflammation via binding to its receptor, GPR75 (GPCR‐Gαq/11). Increased 20‐HETE in animals and humans is associated with hypertension, stroke, myocardial infarction (MI), metabolic syndrome (MetS), and increased reactive oxygen species (ROS). However, investigated effectiveness of 20‐HETE antagonists on ROS generation and MI size has been limited by short‐term follow up and administration of inhibitors prior to onset of MI only in healthy animals. Here, we evaluated the effect of a 20‐HETE antagonist, 20‐SOLA, administered at onset of reperfusion on post MI remodeling 48 hours and 8 weeks after reperfusion in normal (SD) and MetS (JCR:LA‐cp) rats. 20‐HETE was elevated in response to ischemia and more so during reperfusion; this elevation was greater in MetS (ischemia: 2‐fold SD and JCR; reperfusion: 3‐fold SD, 4‐fold JCR). MI size was markedly greater in JCR vs. SD rate (50% vs. 25% of LV). Treatment with 20‐SOLA significantly decreased MI size in both SD (~50%) and JCR (~65%) rats. Equivalent results were obtained in animals treated with GPR75‐shRNA‐Lnv at onset of reperfusions. 20‐SOLA improved coronary blood flow (1.00 ± 0.01 to 1.84 ± 0.03 mg/ml/g in SD to 0.99 ± 0.03 to 1.75±0.01 mg/ml/g in JCR). Mechanistically, smaller MI size in 20‐SOLA‐treated animals correlated with decreased ROS production in JCR rats (90%). Furthermore, survival and left ventricular (LV) function were preserved 8 weeks post MI in 20‐SOLA‐treated animals (ejection fraction (EF) = 80.5% (SD, 100% survival) and 82.5% (JCR, 100% survival)). This correlated with decreased ROS, preserved myocyte morphology, and preserved intact collagen. Differential activation of MMPs in SD vs. JCR rats vs. 20‐SOLA‐treated animals underlie the observed morphological, structural and functional changes. ROS production was decreased in JCR rats treated with a NOX inhibitor (~60%); NOX inhibition also markedly reduced MI size at 48h (60%) and preserved LV function at 8 weeks (EF = 74%, 85% survival) vs. non‐treated rats demonstrating an important role for NOX‐derived ROS in determining MI size and long‐term post‐MI remodeling. In the aggregate, these results indicated that targeting 20‐HETE actions may be an important consideration in prevention of detrimental LV remodeling and mortality post MI. Funding: HL093052.Support or Funding InformationFunding: HL093052This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

20-Hydroxyeicosatetraenoic acid antagonist attenuates the development of malignant hypertension and reverses it once established: a study in Cyp1a1-Ren-2 transgenic rats.

We hypothesized that vascular actions of 20-hydroxyeicosatetraenoic acid (20-HETE), the product of cytochrome P450 (CYP450)-dependent ω-hydroxylase, potentiate prohypertensive actions of angiotensin II (ANG II) in Cyp1a1-Ren-2 transgenic rats, a model of ANG II-dependent malignant hypertension. Therefore, we evaluated the antihypertensive effectiveness of 20-HETE receptor antagonist (AAA) in this model. Malignant hypertension was induced in Cyp1a1-Ren-2 transgenic rats by activation of the renin gene using indole-3-carbinol (I3C), a natural xenobiotic. Treatment with AAA was started either simultaneously with induction of hypertension or 10 days later, during established hypertension. Systolic blood pressure (SBP) was monitored by radiotelemetry, indices of renal and cardiac injury, and kidney ANG II levels were determined. In I3C-induced hypertensive rats, early AAA treatment reduced SBP elevation (to 161 ± 3 compared with 199 ± 3 mmHg in untreated I3C-induced rats), reduced albuminuria, glomerulosclerosis index, and cardiac hypertrophy (P<0.05 in all cases). Untreated I3C-induced rats showed augmented kidney ANG II (405 ± 14 compared with 52 ± 3 fmol/g in non-induced rats, P<0.05) which was markedly lowered by AAA treatment (72 ± 6 fmol/g). Remarkably, in TGR with established hypertension, AAA also decreased SBP (from 187 ± 4 to 158 ± 4 mmHg, P<0.05) and exhibited organoprotective effects in addition to marked suppression of kidney ANG II levels. In conclusion, 20-HETE antagonist attenuated the development and largely reversed the established ANG II-dependent malignant hypertension, likely via suppression of intrarenal ANG II levels. This suggests that intrarenal ANG II activation by 20-HETE is important in the pathophysiology of this hypertension form.

High-fat diet-induced obesity and insulin resistance in CYP4a14-/- mice is mediated by 20-HETE.

20-Hydroxyeicosatetraenoic acid (20-HETE) has been shown to positively correlate with body mass index, hyperglycemia, and plasma insulin levels. This study seeks to identify a causal relationship between 20-HETE and obesity-driven insulin resistance. Cyp4a14-/- male mice, a model of 20-HETE overproduction, were fed a regular or high-fat diet (HFD) for 15 wk. 20-SOLA [2,5,8,11,14,17-hexaoxanonadecan-19-yl 20-hydroxyeicosa-6( Z),15( Z)-dienoate], a 20-HETE antagonist, was administered from week 0 or week 7 of HFD. HFD-fed mice gained significant weight (16.7 ± 3.2 vs. 3.8 ± 0.35 g, P < 0.05) and developed hyperglycemia (157 ± 3 vs. 121 ± 7 mg/dl, P < 0.05) and hyperinsulinemia (2.3 ± 0.4 vs. 0.5 ± 0.1 ng/ml, P < 0.05) compared with regular diet-fed mice. 20-SOLA attenuated HFD-induced weight gain (9.4 ± 1 vs. 16.7 ± 3 g, P < 0.05) and normalized the hyperglycemia (157 ± 7 vs. 102 ± 5 mg/dl, P < 0.05) and hyperinsulinemia (1.1 ± 0.1 vs. 2.3 ± 0.4 ng/ml, P < 0.05). The impaired glucose homeostasis and insulin resistance in HFD-fed mice evidenced by reduced insulin and glucose tolerance were also ameliorated by 20-SOLA. Circulatory and adipose tissue 20-HETE levels significantly increased in HFD-fed mice correlating with impaired insulin signaling, including reduction in insulin receptor tyrosine (Y972) phosphorylation and increased serine (S307) phosphorylation of the insulin receptor substrate-1 (IRS-1). 20-SOLA treatments prevented changes in insulin signaling. These findings indicate that 20-HETE contributes to HFD-induced obesity, insulin resistance, and impaired insulin signaling.

Intra‐Abdominal Lipectomy Reduces Large Arterial Stiffness and Blood Pressure in Metabolic Syndrome

Increased intra‐abdominal (visceral) adipose tissue is a key feature of the metabolic syndrome. Expansion of visceral adipose tissue and the associated altered adipokine profile are linked to the development of hypertension. While reduction in visceral adipose tissue volume offers cardioprotective effects, the mechanisms underlying these effects remain understudied and unclear. In this study, we removed ~90% of visceral adipose tissue (=~5% body weight) by intra‐abdominal lipectomy and assessed large arterial stiffness, large artery structural matrix components, and blood pressure in a metabolic syndrome rat model (JCR:LA‐cp, JCR). Large artery compliance was significantly decreased in JCR vs. normal (Sprague Dawley, SD) rats (75±2% JCR vs. SD (carotid)) with a concomitant significant increase in MMP12‐dependent elastin degradation (3–6 fold vs. SD). Intra‐abdominal lipectomy normalized large artery stiffness, blocked MMP12 activation and reduced elastin degradation in JCR animals (~75% (carotid) vs. untreated JCR). Likewise, hypertension in JCR animals was significantly attenuated by intra‐abdominal lipectomy (MABP=156±3 mmHg JCR vs. 90±6 mmHg SD vs. 132±4 mmHg JCR+lipectomy). 20‐hydroxyeicosatetraeonic acid (20‐HETE), an arachidonic acid metabolite known to be a potent vasoconstrictor in resistance arteries, was significantly elevated in the visceral adipose tissue of JCR rats (~6 fold vs. SD). Like intra‐abdominal lipectomy, 20‐HETE antagonists restored large artery elasticity, blocked MMP12 activation and elastin degradation and significantly decreased blood pressure (125±3 mmHg JCR+20‐HETE antagonists) in JCR rats. Thus, 20‐HETE may be an important adipokine that mediates, in part, the adverse effects of expanded visceral fat volume in the metabolic syndrome. Its removal and inhibition may provide therapeutic and pharmacological approaches, respectively, for the management of central obesity‐driven large artery stiffness and hypertension in the metabolic syndrome.Support or Funding InformationNational Heart, Lung and Blood Institute (NHLBI): 1F31HL137356‐01This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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 ± 3 mmHg JCR, 145 ± 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.

The Blood Pressure-Lowering Effect of 20-HETE Blockade in Cyp4a14(-/-) Mice Is Associated with Natriuresis.

20-Hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE) has been linked to pro-hypertensive and anti-hypertensive actions through its ability to promote vasoconstriction and inhibit Na transport in the ascending limb of the loop of Henle, respectively. In this study, we assessed the effects of 20-HETE blockade on blood pressure, renal hemodynamics, and urinary sodium excretion in Cyp4a14(-/-) male mice, which display androgen-driven 20-HETE-dependent hypertension. Administration of 2,5,8,11,14,17-hexaoxanonadecan-19-yl 20-hydroxyicosa-6(Z),15(Z)-dienoate (20-SOLA), a water-soluble 20-HETE antagonist, in the drinking water normalized the blood pressure of male Cyp4a14(-/-) hypertensive mice (±124 vs. ±153 mmHg) while having no effect on age-matched normotensive wild-type (WT) male mice. Hypertension in Cyp4a14(-/-) male mice was accompanied by decreased renal perfusion and reduced glomerular filtration rates, which were corrected by treatment with 20-SOLA. Interestingly, Cyp4a14(-/-) male mice treated with 20-SOLA displayed increased urinary sodium excretion that was paralleled by the reduction of blood pressure suggestive of an antinatriuretic activity of endogenous 20-HETE in the hypertensive mice. This interpretation is in line with the observation that the natriuretic response to acute isotonic saline loading in hypertensive Cyp4a14(-/-) male mice was significantly impaired relative to that in WT mice; this impairment was corrected by 20-SOLA treatment. Hence, endogenous 20-HETE appears to promote sodium conservation in hypertensive Cyp4a14(-/-) male mice, presumably, as a result of associated changes in renal hemodynamics and/or direct stimulatory action on tubular sodium reabsorption.

Abstract 122: 20-HETE Antagonists Normalize Large Artery Stiffness and Systolic Blood Pressure in Metabolic Syndrome

Large artery stiffness is a causal factor in development of systolic hypertension. 20-hydroxyeicosatetraeonic acid (20-HETE), a cytochrome CYP450-derived arachidonic acid metabolite, is known to be elevated in resistance arteries in hypertensive animal models and in obesity in humans, but the role of 20-HETE in regulation of large artery remodeling in metabolic syndrome has not been investigated. Unlike normal (Sprague-Dawley (SD)) rats, large arteries (aorta, carotid and &gt;100μM mesenteric arteries) of metabolic syndrome rats (JCR:LA-cp, JCR) express CYP4A and 4F, CYP450s which make 20-HETE in rats (2-fold increase vs. SD). Consequently, 20-HETE production is elevated in large arteries of JCR rats. We hypothesized that this elevated 20-HETE increases matrix metalloproteinase 12 (MMP12, an elastase) activation leading to increased degradation of elastin, increased large artery stiffness and increased systolic blood pressure. A 3-4 fold increase in 20-HETE production in large arteries of JCR vs. SD rats correlated with increased elastin degradation (3-6 fold) and increased arterial stiffness (~75%). 20-HETE antagonists blocked elastin degradation in JCR rats concomitant with blocking MMP12 activation. Importantly, 20-HETE antagonists and MMP12 inhibition (pharmacological and MMP12-shRNA-Lnv) significantly decreased (~60% vs. untreated JCR) large artery stiffness in JCR rats. 20-HETE antagonists also decreased systolic (182±3 mmHg JCR, 145±3 mmHg JCR+20-HETE antagonists) but not diastolic (125±4 mmHg JCR, 124±4 mmHg JCR+20-HETE antagonists) 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 in JCR rats was Ang II-independent. These results suggest that 20-HETE-dependent regulation of systolic blood pressure may be a unique feature of metabolic syndrome related to high CYP4A/4F expression and resultant high 20-HETE production in large conduit arterial stiffness, which is a primary determinant of systolic blood pressure. These findings may have implications for management of systolic hypertension in patients with metabolic syndrome.

Abstract P211: Intra-abdominal Lipectomy Normalizes Arterial Stiffness and Blood Pressure via Reduction in 20-HETE

Increased intra-abdominal (visceral) adipose tissue is a key feature of the metabolic syndrome affecting over 30% of the U.S. population. Expansion of visceral adipose tissue is linked to the development of hypertension and is a risk factor for cardiovascular disease that can ultimately lead to end-organ damage. While reduction in visceral adipose tissue volume offers cardioprotective effects, the cardiovascular mechanisms behind these beneficial effects remain unclear. In this study, we removed ~90% of visceral adipose tissue (=~5% body weight) by intra-abdominal lipectomy and assessed large arterial stiffness, large artery structural matrix components, and blood pressure in a metabolic syndrome rat model (JCR:LA-cp, JCR). Large artery stiffness was significantly elevated in JCR vs. normal (Sprague Dawley, SD) rats (75±2% JCR vs. SD (carotid)) with a concomitant significant increase in MMP12-dependent elastin degradation (3-6 fold vs. SD). Intra-abdominal lipectomy normalized large artery stiffness, blocked MMP12 activation and reduced elastin degradation in JCR animals (~75% (carotid) vs. untreated JCR). Likewise, hypertension in JCR animals was significantly attenuated by intra-abdominal lipectomy (MABP=156±3 mmHg JCR vs. 90±6 mmHg SD vs. 132±4 mmHg JCR+lipectomy). 20-hydroxyeicosatetraeonic acid (20-HETE), an arachidonic acid metabolite known to be a potent vasoconstrictor in resistance arteries, was significantly elevated in the visceral adipose tissue of JCR rats (~6 fold vs. SD). Intra-abdominal lipectomy normalized 20-HETE levels in JCR rats. Like intra-abdominal lipectomy, 20-HETE antagonists restored large artery elasticity, blocked MMP12 activation and elastin degradation, and significantly decreased blood pressure (125±3 mmHg JCR+20-HETE antagonists) in JCR rats. Thus, 20-HETE may be an important adipokine that mediates the adverse effects of expanded visceral fat volume in the metabolic syndrome and its inhibition may provide a pharmacological approach for the management of central obesity-driven large artery stiffness and hypertension.

Abstract P426: Suppression of Intrarenal Ras With a 20-hydroxyeicosatetraenoic Acid Antagonist Attenuates Ang II-dependent Malignant Hypertension and Reverses Established Ang-II-dependent Hypertension in Cyp1a1-ren-2 Transgenic Rats

Rationale and Objective: Interplay of 20-HETE and ANG II might be a factor in pathophysiology of many forms of experimental hypertension. We hypothesized that intrarenal 20-HETE potentiates prohypertensive actions of ANG II in Cyp1a1-Ren-2 transgenic rats (TGR), a model of ANG II-dependent malignant hypertension. Therefore, we evaluated the antihypertensive effectiveness of a new, orally active 20-HETE antagonist (SOLA) in this model. Methods: Treatment with SOLA (10 mg.kg -1 .day - 1 in drinking water) was started either simultaneously with induction of hypertension (early treatment) by indol-3-carbinol or 10 days later, during established hypertension (late treatment). Systolic blood pressure (SBP, measured by radiotelemetry) was monitored continuously and indices of renal and cardiac injury, and kidney 20-HETE and ANG II levels were determined at the end of experiments. Results: In TGR with induced hypertension, early SOLA treatment reduced SBP elevation (to 161±3 vs. 199±3 mmHg in induced TGR, p&lt;0.001), reduced albuminuria (16±2 vs. 35±3 mg/24 h, p&lt;0.002), glomerulosclerosis index (0.12±0.01 vs. 0.32±0.02, p&lt;0.001) and cardiac hypertrophy (left ventricle weight (mg)/tibial length ratio: 16.9±0.4 vs 20.6±0.5, p&lt;0.02). TGR with induced hypertension exhibited elevated intrarenal 20-HETE levels (15.2±0.03 vs. 9.2±0.02 μg/g in noninduced rats, p&lt;0.01); however, the elevation was not altered by SOLA treatment. Hypertensive TGR showed also augmented kidney ANG II levels (405±30 vs. 51±2 fmol/g in noninduced rats, p&lt;0.001). SOLA treatment significantly lowered kidney ANG II (95±19). Remarkably, in TGR with established hypertension, late SOLA treatment also decreased SBP (from 187±4 to 158±4 mmHg, p&lt;0.002) and exhibited cardio- and renoprotective effects in addition to a marked suppression of kidney ANG II levels (72±12 fmol/g). Conclusion: In hypertensive TGR the new orally active 20-HETE antagonist attenuated the development and largely reversed the established ANG II-dependent experimental malignant hypertension, likely via suppression of intrarenal ANG II levels. This suggests that intrarenal RAS activation by 20-HETE is important in pathophysiology of this hypertension form

Clinical Implications of 20-Hydroxyeicosatetraenoic Acid in the Kidney, Liver, Lung and Brain: An Emerging Therapeutic Target

Cytochrome P450-mediated metabolism of arachidonic acid (AA) is an important pathway for the formation of eicosanoids. The ω-hydroxylation of AA generates significant levels of 20-hydroxyeicosatetraenoic acid (20-HETE) in various tissues. In the current review, we discussed the role of 20-HETE in the kidney, liver, lung, and brain during physiological and pathophysiological states. Moreover, we discussed the role of 20-HETE in tumor formation, metabolic syndrome and diabetes. In the kidney, 20-HETE is involved in modulation of preglomerular vascular tone and tubular ion transport. Furthermore, 20-HETE is involved in renal ischemia/reperfusion (I/R) injury and polycystic kidney diseases. The role of 20-HETE in the liver is not clearly understood although it represents 50%–75% of liver CYP-dependent AA metabolism, and it is associated with liver cirrhotic ascites. In the respiratory system, 20-HETE plays a role in pulmonary cell survival, pulmonary vascular tone and tone of the airways. As for the brain, 20-HETE is involved in cerebral I/R injury. Moreover, 20-HETE has angiogenic and mitogenic properties and thus helps in tumor promotion. Several inhibitors and inducers of the synthesis of 20-HETE as well as 20-HETE analogues and antagonists are recently available and could be promising therapeutic options for the treatment of many disease states in the future.

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 P168: 20-HETE-mediated Neutrophil Adhesion Impairs Coronary Collateral Growth in Metabolic Syndrome

Transient, repetitive myocardial ischemia (RI)-induced coronary collateral growth (CCG) is impaired in metabolic syndrome patients and animal models. Endothelial cell (EC) dysfunction and chronic inflammation are hallmarks of metabolic syndrome. We showed that while in normal animals (SD), RI induces transient infiltration of monocytes, associated with successful CCG, in metabolic syndrome rats (JCR), RI induces sustained accumulation of neutrophils, which contributes to compromised CCG. 20-hydroxyeicosatetraeonic acid (20-HETE) is a pro-inflammatory metabolite of arachidonic acid. Its role in the regulation of CCG is unknown. We hypothesized that enhanced 20-HETE-mediated neutrophil adhesion to ECs and consequent EC dysfunction and apoptosis result in impaired CCG in metabolic syndrome. P-selectin and ICAM-1 expression was increased ~40% in JCR vs. SD rats. This increase was prevented by 20-HETE antagonists, 20-SOLA or 20-HEDGE. 20-HETE antagonists also prevented neutrophil accumulation observed in JCR rats. Coronary arteries from JCR rats exhibited reduced endothelium (Ach)-dependent vasodilation (20% JCR vs. 50% of max. SD). RI-induced eNOS activation and NO production were likewise decreased (~60% and~70%, respectively) in JCR vs. SD rats. EC apoptosis (TUNEL) was severely increased in response to RI in JCR rats (~75% vs. SD). Neutrophil adhesion-blocking antibodies partially attenuated EC apoptosis (~70%) and EC dysfunction (~75% eNOS activation and NO production, 75% Ach-dependent vasodilation). 20-HETE antagonists fully reversed impaired endothelium-dependent vasodilation, eNOS activation, NO production and prevented EC apoptosis. Finally, impaired CCG in JCR rats (collateral-dependent blood flow, microspheres) was completely restored by 20-HETE antagonists (CZ/NZ flow 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 ml/min/g in SD rats) and partially restored by neutrophil-blocking antibodies (0.49±0.05 ml/min/g). Taken together, these results indicate that 20-HETE-dependent neutrophil adhesion and accumulation compromises EC survival and function leading to impaired CCG. 20-HETE antagonists could provide therapy for restoration of CCG in metabolic syndrome.