Epoxyeicosatrienoic acids (EETs) are a class of cytochrome P450 (P450) arachidonic acid (AA) metabolites with diverse biological activities including anti-hypertensive, vasodilatory, angiogenic, and anti-inflammatory properties. While their functions as autocrine and paracrine mediators in cardiovascular and renal systems are well established, their mechanism of action and roles in hormonal functional responses are yet to be fully defined. In this review, we highlight extant evidence of their participation in hormonal transmembrane signal transduction leading to the activation of the ERK1/2 or Akt serine/threonine kinases. Based on studies with EGF (Epidermal Growth Factor), VEGF (Vascular Endothelial Growth Factor) and insulin binding to their membrane bound receptors, we propose to include EETs to the inventory of intracellular mediators associated with the functional responses elicited upon selected hormone/receptor interactions.

Glomerular diseases like focal and segmental glomerulosclerosis (FSGS) are the leading cause of chronic and end-stage kidney diseases. Although FSGS is associated with high morbidity and mortality, available treatments are limited, which emphasizes the need for novel therapy. We developed a dual inhibitor, PTUPB, that concurrently acts as a soluble epoxide hydrolase (sEH) inhibitor and a cyclooxygenase-2 (COX-2) inhibitor. In the current study, we investigated renal actions of PTUPB in a genetic renal hypoplasia FSGS model, ROP Os/+ mice. ROP Os/+ mice developed albuminuria and glomerular injury between 16 and 20 weeks of age with an 8-fold higher albumin/Cr ratio than wild-type ROP +/+ mice. Three groups were assessed: 20-week-old ROP +/+ mice, ROP Os/+ mice treated with vehicle, and ROP Os/+ mice treated with PTUPB (10 mg/kg/d, i.p.). After 4 weeks, urine, blood, and kidney tissue were collected in the mice groups for biochemical, molecular, and histological analysis. ROP Os/+ mice had 10-fold higher albuminuria than ROP +/+ mice, and PTUPB treatment markedly attenuated albuminuria in ROP Os/+ mice by 69 %. ROP Os/+ mice developed a glomerular injury with a 3-fold higher glomerular injury score and 10-fold higher glomerular expression of sclerotic marker fibronectin than ROP +/+ mice, and PTUPB treatment attenuated these by 40-50 %. We further demonstrated that there was significantly lower renal mRNA expression of nephrin, podocin, podoplanin, synaptopodin, and glomerular WT1 immunopositive cells in ROP Os/+ mice which was improved by PTUPB treatment in these mice. Glomerular permeability studies in isolated glomeruli determined that COX-2 metabolites of 8,9-epoxyeicosatrienoic acid (EET) increased glomerular permeability. PTUPB, 8,9-EET, or 8,9-EET analogs prevented angiotensin II-induced glomerular permeability and mesangial cell proliferation. These findings demonstrate that COX-2 metabolites harm the glomerulus, whereas 8,9-EET improves glomerular function. Lastly, kidney fibrosis and inflammation were increased in ROP Os/+ mice compared to ROP +/+ mice. PTUPB treatment to ROP Os/+ mice decreased kidney fibrosis and inflammation. These findings demonstrate that the dual inhibitor PTUPB that concurrently inhibits sEH and COX-2 is a potential treatment for FSGS and related glomerular diseases.

Abstract ID 92011Poster Board 280Background: Organ preservation solutions have been applied to diminish hypoxic injury during cold storage and subsequent re-warming (CS-RW), and this improve graft survival during kidney transplantation. Despite significant progress in the development of organ preservation solution and hypoxic injury mitigation, poor graft survival is still common. We studied 9 compounds that targets eicosanoid and nuclear receptor signaling on the adverse effects of CS-RW on normal rat renal epithelial cells (NRK-52E) and tested the hypothesis that these compounds have cytoprotective effects during CS-RW.Methods: Kidney epithelial NRK-52E cells were incubated in University of Wisconsin (UW) solution at 4°C for 18h contained vehicle (0.1% DMSO, vehicle-CS-RW) or the test compounds (1, 3, and 10μM) in triplicates (n = 6-9/group). Following18h cold exposure (CS), cells were washed off treatments and UW sooution and grown in cell culture medium at 37°C for 6h (RW). Two epoxyeicosatrienoic (EET) acid mimetics, three multi-target drugs, one soluble epoxide hydrolase inhibitor (sEHi), one sEH phosphatase inhibitor (sEH-Pi), and two hydroxyeicosatetraenoic acid (20-HETE) antagonists were tested at 1, 3, and 10mM. Cell counting kit-8 (CCK-8) and CellTiter-Glo® 2.0 Cell Viability (CellTiter-Glo assay) assays were conducted to determine cell viability, and the number of the viable cells is expressed as % of cells of that were incubated in normal cell growth conditions without cold storage (control).Results: Cell viability in control and vehicle-CS-RW was similar when analyzed with CCK-8 (89± 1 & 87 ± 1 %) or CellTiter-Glo(91± 2 & 91 ± 1 %) assays. Four compounds demonstrated cytoprotective effects comparable to SB202190, p38 MAPK inhibitor that is known to protect renal epithelial cells and kidneys during CS-RW. SB202190 maintained cell viability at 107 ± 3 and 113± 2% of control, in CCK-8 and CellTiter-Glo assays; respectively. Irrespective of the cell viability assay type, the EET mimetics demonstrated the strongest NRK-52E cytoprotective effects, followed by a sEH-Pi, and a dual sEHi / COX2 inhibitor. At 10μMconcentration, EET-A maintain maximum viable cell numbers at 100 ± 2 % (CCK-8) & 109 ± 1% (CellTiter-Glo assay) of control and SWE, a sEH-Pi maintain cell viability at 108 ± 2% (CellTiter-Glo assay) at 10μM concentration. Also, EET-C, an EET mimetic demonstrated strong cytoprotective effect and maintain cell viability at 96 ± 3 % (CCK-8; 3μM) & 104 ± 1% (CellTiter-Glo assay; 1μM) of control; respectively. Multi-target drug PTUPB that simultaneously acts as sEHi and cyclooxygenase-2 (COX-2) inhibitor, protected NRK-52E cells from CS-RW with lesser cytoprotective action. PTUPB maintain maximum cell viability at 97 ± 3 % (CCK-8; 3 μM) & 100 ± 1% (CellTiter-Glo assay; 1μM). Interestingly, ompounds that inhibited 20-HETE or included nuclear receptor agonism did not improve cell viability.Conclusion: Our findings demonstrate renal cytoprotective effects during cold storage in transplant solution of a series of compounds that acts via epoxyeicosanoid signaling. EET mimetics, sEH-Pi, and dual sEHi / COX-2 inhibitor demonstrate potential to better preserve kidneys in cold storage and improve graft survival.The National Institute of Diabetes and Digestive and Kidney Diseases grant DK103616 and the Arkansas Research Alliance provided support to John D. Imig.

Supplemental Digital Content is Available in the Text. Mounting evidence suggests that cytochrome P450 epoxygenase-derived metabolites of docosahexaenoic acid, called epoxydocosapentaenoic acids (EDPs), limit mitochondrial damage after cardiac injury. In particular, the 19,20-EDP regioisomer has demonstrated potent cardioprotective action. Thus, we investigated our novel synthetic 19,20-EDP analog SA-22 for protection against cardiac ischemia-reperfusion (IR) injury. Isolated C57BL/6J mouse hearts were perfused through Langendorff apparatus for 20 minutes to obtain baseline function, followed by 30 minutes of global ischemia. Hearts were then treated with vehicle, 19,20-EDP, SA-22, or SA-22 with the pan-sirtuin inhibitor nicotinamide or the SIRT3-selective inhibitor 3-(1H-1,2,3-triazol-4-yl) pyridine (3-TYP) at the start of 40 minutes reperfusion (N = 5-8). We assessed IR injury-induced changes in recovery of myocardial function, using left ventricular developed pressure and systolic and diastolic pressure change. Tissues were assessed for electron transport chain function, SIRT1 and SIRT3, optic atrophy type 1, and caspase-1. We also used H9c2 cells in an in vitro model of hypoxia/reoxygenation injury (N = 3-6). Hearts perfused with SA-22 had significantly improved postischemic left ventricular developed pressure, systolic and diastolic recovery (64% of baseline), compared with vehicle control (15% of baseline). In addition, treatment with SA-22 led to better catalytic function observed in electron transport chain and SIRT enzymes. The protective action of SA-22 resulted in reduced activation of pyroptosis in both hearts and cells after injury. Interestingly, although nicotinamide cotreatment worsened functional outcomes, cell survival, and attenuated sirtuin activity, it failed to completely attenuate SA-22-induced protection against pyroptosis, possibly indicating EDPs exert cytoprotection through pleiotropic mechanisms. In short, these data demonstrate the potential of our novel synthetic 19,20-EDP analog, SA-22, against IR/hypoxia-reoxygenation injury and justify further development of therapeutic agents based on 19,20-EDP.

Cytochrome P450 epoxygenase Cyp2c44, a murine epoxyeicosatrienoic acid (EET) producing enzyme, promotes insulin sensitivity and Cyp2c44(-/-) mice show hepatic insulin resistance. Because insulin resistance leads to hepatic lipid accumulation and hyperlipidemia, we hypothesized that Cyp2c44 regulates hepatic lipid metabolism. Standard chow diet (SD) fed male Cyp2c44(-/-) mice had significantly decreased EET levels and increased hepatic and plasma lipid levels compared to wild-type mice. We showed increased hepatic plasma membrane localization of the FA transporter 2 (FATP2) and total unsaturated fatty acids and diacylglycerol levels. Cyp2c44(-/-) mice had impaired glucose tolerance and increased hepatic plasma membrane-associated PKCδ and phosphorylated IRS-1, two negative regulators of insulin signaling. Surprisingly, SD and high fat diet fed (HFD) Cyp2c44(-/-) mice had similar glucose tolerance and hepatic plasma membrane PKCδ levels, suggesting that SD-fed Cyp2c44(-/-) mice have reached their maximal glucose intolerance. Inhibition of PKCδ resulted in decreased IRS-1 serine phosphorylation and improved insulin-mediated signaling in Cyp2c44(-/-) hepatocytes. Finally, Cyp2c44(-/-) HFD-fed mice treated with the analog EET-A showed decreased hepatic plasma membrane FATP2 and PCKDd levels with improved glucose tolerance and insulin signaling. In conclusion, loss of Cyp2c44 with concomitant decreased EET levels leads to increased hepatic FATP2 plasma membrane localization, diacylglycerol accumulation, and PKCδ-mediated attenuation of insulin signaling. Thus, Cyp2c44 acts as a regulator of lipid metabolism by linking it to insulin signaling.

Vascular function is dynamically regulated and dependent on a bevy of cell types and factors that work in concert across the vasculature. The vasoactive eicosanoid, 20-Hydroxyeicosatetraenoic acid (20-HETE) is a key player in this system influencing the sensitivity of the vasculature to constrictor stimuli, regulating endothelial function, and influencing the renin angiotensin system (RAS), as well as being a driver of vascular remodeling independent of blood pressure elevations. Several of these bioactions are accomplished through the ligand-receptor pairing between 20-HETE and its high-affinity receptor, GPR75. This 20-HETE axis is at the root of various vascular pathologies and processes including ischemia induced angiogenesis, arteriogenesis, septic shock, hypertension, atherosclerosis, myocardial infarction and cardiometabolic diseases including diabetes and insulin resistance. Pharmacologically, several preclinical tools have been developed to disrupt the 20-HETE axis including 20-HETE synthesis inhibitors (DDMS and HET0016), synthetic 20-HETE agonist analogues (20-5,14-HEDE and 20-5,14-HEDGE) and 20-HETE receptor blockers (AAA and 20-SOLA). Systemic or cell-specific therapeutic targeting of the 20-HETE-GPR75 axis continues to be an invaluable approach as studies examine the molecular underpinnings activated by 20-HETE under various physiological settings. In particular, the development and characterization of 20-HETE receptor blockers look to be a promising new class of compounds that can provide a considerable benefit to patients suffering from these cardiovascular pathologies.

This file contains the primer sequence of CYP2C9* variants (Supplementary Table S1); the SNPs genotyped in the BioVU NSCLC study (Supplementary Table S2); quality control measures of SNPs in the BioVU NSCLC study (Supplementary Table S3); clinical and demographic description of NSCLC cases (Supplementary Table S4); the association of clinical and demographic variables with survival (Supplementary Table S5); the genotypes in the NSCLC cases (Supplementary Table S6); the spectra of CYP2C9* variants and the effect of arachidonic acid on CYP2C9* variant (Supplementary Figure S1); the association between CYP2C9* variants and platinum chemotherapy (Supplementary Figure S2).

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-13(BN) consomic rat). Unlike SS rats, SS-13(BN) 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-13(BN) 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-13(BN) 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.

PDF file - 24K, Analysis of p60.5 and KrasLA2 tumor cells.

OBJECTIVE: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.METHODS: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.RESULTS: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.CONCLUSION: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.

Benzylic/allylic alcohols are converted via site-selective C(sp(2))-C(sp(3)) cleavage to value-added nitrogenous motifs, viz., anilines and/or nitriles as well as N-heterocycles, utilizing commercial hydroxylamine-O-sulfonic acid (HOSA) and Et3N in an operationally simple, one-pot process. Notably, cyclic benzylic/allylic alcohols undergo bis-functionalization with attendant increases in architectural complexity and step-economy.

20‐Hydroxyeicosatetraenoic acid (20‐HETE) and its receptor (20HR), GPR75 (Gq), exhibit diverse bioactions that promote the activation of pro‐hypertensive, ‐diabetic and ‐obesity signals. The pharmacological properties associated with analogues that target GPR75 remain unclear. The screening of 20‐HETE and synthetic 20‐HETE analogues using changes of intracellular calcium (iCa2+) in the endothelial cell line, EA.hy926, as a measure of 20HR activation, revealed that the compounds 20‐HETE (10 nM), sodium 20‐hydroxyeicosa‐5Z,14Z‐dienoate (20‐5,14‐HEDE) (10 nM) and sodium 14‐((6‐hydroxyhexyl)oxy)tetradec‐5(Z)‐enoate (5Z‐HOTE) (10 nM) promote significant and comparable elevations in iCa2+. In EA.hy926 cells, 20‐HETE elicited a half‐maximal effective concentrations (EC50) with respect to iCa2+ of 1.228 e‐9 M while 20‐5,14‐HEDE’s EC50 was 6.908 e‐10 M. The water‐soluble derivative of 20‐5,14‐HEDE, SOLAGO, exhibited a marked and leftward shift in the dose‐response with an EC50 of 2.702 e‐10 M. Interestingly, sodium 19(R)‐ and 19(S)‐hydroxyeicosa‐5(Z),14(Z)‐dienoate, analogues of 19(R)‐HETE, an endogenous 20HR blocker (20HRB), demonstrated partial agonist activity; elevating iCa2+to 1.6‐ and 2‐fold over baseline vehicle treatment, respectively. With respect to 20HR blockers, the water‐soluble 20HRBs 2,5,8,11,14,17‐hexaoxanonadecan‐19‐yl 20‐hydroxyeicosa‐6(Z),15(Z)‐dienoate (20‐SOLA) and N‐disodium succinate‐20‐hydroxyeicosa‐6(Z),15(Z)‐diencarboxamide (AAA) displayed potent half‐maximal inhibitory concentrations (IC50s) of 8.059 e‐10 and 5.356 e‐10 M, respectively. The 19(R)‐HETE analogue sodium (19(R)‐hydroxyeicosa‐5(Z),14(Z)‐dienoyl)glycinate (19(R)‐HEDGE) also demonstrated a strong yet less potent IC50 response of 6.715 e‐9 M. Further studies are necessary to better understand the structure‐function relationships between 20HR agonists, partial agonists, and receptor blockers. These data would allow for the development of novel 20HRBs for the treatment of various pathologies associated with elevations in 20‐HETE including hypertension, cancer, diabetes, and obesity.

While epoxyeicosatrienoic acids (EETs) have been implicated in breast cancer growth and progression, less is known about their effects on oncogene transcription. We have previously found that the oncogenic regioisomer (±)14,15-EET drives mitochondrial respiration, ATP synthesis, and proliferation of ER+/HER2- breast cancer cells [Cell Chem Biol. 2017 Oct 19;24(10):1259-1275]. RNAseq analysis was performed (5 replicates per condition) on serum starved (16 hours) MCF-7 cells which were then treated with (±)14,15-EET or vehicle (2 hours; serum and phenol red free medium without estradiol). Using gene set enrichment analysis (GSEA), we found that (±)14,15-EET activated an estrogen receptor alpha (ER) hallmark early response gene set and synchronously activated a MYC hallmark gene set. With activation of the MYC hallmark gene set, c-Myc gene expression was also induced at 2 hours (3.99-fold; P=2.3 x 10-17; FDR=2.8 x 10-14). These data suggest an alternative pathway for activation of estrogen and MYC regulated genes in the absence of estradiol. The 15 genes most transcriptionally activated by (±)14,15-EET at 2 hours were: VMP1, ZFP36, JUNB, FOS, IER3, EGR1, IER5L, ELF3, JUN, NR4A1, HES1, DUSP1, MYC, TOB1, and CITED2 [fold change range: 3.25 (CITED2) to 90.5 (FOS); all P< 2.86 x 10-17; all FDR < 3.03 x 10-14]. The ER regulated genes most transcriptionally activated (>1.5 fold) were: IER3, TOB1, AREG, CISH, KCNMB3, and PDK4 (fold change range: 1.77 to 4.35; P= 1.74 x 10-18 to 1.35 x 10-5; FDR=5.54 x 10-15 to 6.4 x 10-4). The MYC regulated genes most transcriptionally activated (> 1.5-fold change) were EIF4A1 (fold change= 2.37; P=1.0 x 10-7; FDR=1.33 x 10-5), IRF9 (fold change=1.58; P=0.0044; FDR= 0.026), and FOSL1 (fold change=1.51; P=0.008; FDR=0.04). Supporting the hypothesis of (±)14,15-EET activation of ER-regulated transcription, (±)14,15-EET promoted nuclear translocation of ER at 1 hour measured by DAPI normalized immunofluorescence [MCF-7 nuclear ER increase of 1.66-fold (P=0.031); ZR75-1 nuclear ER increase of 1.77-fold (P=0.015)]. Supporting the hypothesis of (±)14,15-EET activation of MYC-regulated transcription, (±)14,15-EET treatment promoted nuclear translocation of c-Myc with MCF-7 cells exhibiting a 1.22-fold increase at 2 hours (P=0.002). (±)14,15-EET also promoted nuclear translocation of FITC-70 kDa dextran with MCF-7 cells exhibiting an increase of 1.35-fold at 1 hour (P=0.029). These data suggest that (±)14,15-EET can induce an estradiol-like immediate early gene response in ER+/HER2- breast cancer cells correlating with c-Myc activation. In summary, while the effect of (±)14,15-EET on nuclear translocation may be partially cargo agnostic, (±)14,15-EET promotes ER and c-Myc nuclear translocation and associated transcription, mimicking a tandem hormonal and growth factor response. Citation Format: Jianxun Lei, Zhijun Guo, Julissa Molina-Vega, Paloma Cervantes, Swaathi Jayaraman, John R. Hawse, Carlos Perez, Juan Abrahante, Xiaojia Tang, Krishna Kalari, Jinhua Wang, John R. Falck, Carol Lange, Matthew P. Goetz, David Potter. (±) 14,15-epoxyeicosatrienoic acid induces hallmark ER and MYC gene expression and associated ER and c-Myc nuclear translocation in ER+/HER2- breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB564.

The androgen receptor (AR) and AR-driven genes are crucial in normal and neoplastic prostate tissue. Previous results showed a link between 20-hydroxyeicosatetraenoic acid (20-HETE) production and AR-driven prostate cancer (PCa) progression. This study aims to describe the contribution of GPR75, 20-HETE membrane receptor, in 20-HETE-mediated expression and transcriptional activity of AR in PCa. In LNCaP cells, 20-HETE increased AR expression, nuclear localization, and its transcriptional activity. Also, 20-HETE enhanced dihydrotestosterone (DHT) induced effects. All was abrogated by chemical antagonism of GPR75 (19-HEDE) or its transient knockdown. In human PCa, the expression of AR-driven genes correlated with GPR75. In LNCaP xenografts, tumors from castrated animals expressed higher levels of AR, this was impaired by inhibition of 20-HETE synthesis. These data suggest that 20-HETE, through the GPR75 receptor, regulates transcriptionally active AR in PCa cells, thus making 20-HETE/GRP75 potential targets to limit the expression of AR-driven phenotype in PCa cells.

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.

Iminodirhodium reactive intermediates generated in situ from O-tosyloximes using Rh2(esp)2 in CH2Cl2 at rt were exploited for an agile trichotomy of challenging transformations: (1) remote C-H functionalizations using an exceptionally broad diversity of inorganic and organic nucleophiles including several unconventional examples, for example, ethers and acyl silanes; (2) desaturative annulation, a biomimetic 1,3-methylene C-C ring-closure with an overall loss of two hydrogens; and (3) directed desaturation for the acceptor-less, regioselective creation of γ,δ- or γ,δ,ε,ζ-olefins. Compared with typical iminyl transition-metal-mediated and 1,5-hydrogen atom-transfer (1,5-HAT) processes, iminodirhodium intermediates are largely underexplored, especially with respect to C(sp3)-H centers and, yet, have the potential to be transformative by virtue of their substrate breadth, regiocontrol, and elusive reaction modality. A substrate scope includes benzylic, allylic, propargylic, tertiary, and α-alkyloxy centers.

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/G alpha(q/11) and GPR kinase interactor 1 (GIT1)/protein kinase C (PKC) alpha, the association of GPR75/GIT1, large conductance voltage and calcium-activated potassium subunit beta (MaxiK beta)/PKC alpha, MaxiK beta/proto-oncogene tyrosine-protein kinase (c-Src), and epidermal growth factor receptor (EGFR)/c-Src, MaxiK beta, 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 G alpha(q/11)/PKC alpha/MaxiK beta, GIT1/PKC alpha/MaxiK beta, GIT1/c-Src/MaxiK beta, 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.

We previously showed that global deletion of the cytochrome P450 epoxygenase Cyp2c44, a major epoxyeicosatrienoic acid (EET) producing enzyme in mice, leads to impaired hepatic insulin signaling resulting in insulin resistance. This finding led us to investigate whether administration of a water soluble EET analog restores insulin signaling in vivo in Cyp2c44(-/-) mice and investigated the underlying mechanisms by which this effect is exerted. Cyp2c44(-/-) mice treated with the analog EET-A for 4 weeks improved fasting glucose and glucose tolerance compared to Cyp2c44(-/-) mice treated with vehicle alone. This beneficial effect was accompanied by enhanced hepatic insulin signaling, decreased expression of gluconeogenic genes and increased expression of glycogenic genes. Mechanistically, we show that insulin-stimulated phosphorylation of insulin receptor β (IRβ) is impaired in primary Cyp2c44(-/-) hepatocytes and this can be restored by cotreatment with EET-A and insulin. Plasma membrane fractionations of livers indicated that EET-A enhances the retention of IRβ in membrane rich fractions, thus potentiating its activation. Altogether, EET analogs ameliorate insulin signaling in a genetic model of hepatic insulin resistance by stabilizing membrane-associated IRβ and potentiating insulin signaling.