Steven M. Weldon

Soluble epoxide hydrolase is a susceptibility factor for heart failure in a rat model of human disease

We aimed to identify genetic variants associated with heart failure by using a rat model of the human disease. We performed invasive cardiac hemodynamic measurements in F2 crosses between spontaneously hypertensive heart failure (SHHF) rats and reference strains. We combined linkage analyses with genome-wide expression profiling and identified Ephx2 as a heart failure susceptibility gene in SHHF rats. Specifically, we found that cis variation at Ephx2 segregated with heart failure and with increased transcript expression, protein expression and enzyme activity, leading to a more rapid hydrolysis of cardioprotective epoxyeicosatrienoic acids. To confirm our results, we tested the role of Ephx2 in heart failure using knockout mice. Ephx2 gene ablation protected from pressure overload–induced heart failure and cardiac arrhythmias. We further demonstrated differential regulation of EPHX2 in human heart failure, suggesting a cross-species role for Ephx2 in this complex disease.

Compensatory Mechanism for Homeostatic Blood Pressure Regulation in Ephx2 Gene-disrupted Mice

Arachidonic acid-derived epoxides, epoxyeicosatrienoic acids, are important regulators of vascular homeostasis and inflammation, and therefore manipulation of their levels is a potentially useful pharmacological strategy. Soluble epoxide hydrolase converts epoxyeicosatrienoic acids to their corresponding diols, dihydroxyeicosatrienoic acids, modifying or eliminating the function of these oxylipins. To better understand the phenotypic impact of Ephx2 disruption, two independently derived colonies of soluble epoxide hydrolase-null mice were compared. We examined this genotype evaluating protein expression, biofluid oxylipin profile, tissue oxylipin production capacity, and blood pressure. Ephx2 gene disruption eliminated soluble epoxide hydrolase protein expression and activity in liver, kidney, and heart from each colony. Plasma levels of epoxy fatty acids were increased, and fatty acid diols levels were decreased, while measured levels of lipoxygenase- and cyclooxygenase-dependent oxylipins were unchanged. Liver and kidney homogenates also show elevated epoxide fatty acids. However, in whole kidney homogenate a 4-fold increase in the formation of 20-hydroxyeicosatetraenoic acid was measured along with a 3-fold increase in lipoxygenase-derived hydroxylation and prostanoid production. Unlike previous reports, however, neither Ephx2-null colony showed alterations in basal blood pressure. Finally, the soluble epoxide hydrolase-null mice show a survival advantage following acute systemic inflammation. The data suggest that blood pressure homeostasis may be achieved by increasing production of the vasoconstrictor, 20-hydroxyeicosatetraenoic acid in the kidney of the Ephx2-null mice. This shift in renal metabolism is likely a metabolic compensation for the loss of the soluble epoxide hydrolase gene.

Interaction Between P450 Eicosanoids and Nitric Oxide in the Control of Arterial Tone in Mice

Objective—Epoxyeicosatrienoic acids (EETs) serve as endothelial-derived hyperpolarizing factors (EDHF), but may also affect vascular function by other mechanisms. We identified a novel interaction between EETs and endothelial NO release using soluble epoxide hydrolase (sEH) −/− and +/+ mice. Methods and Results—EDHF responses to acetylcholine in pressurized isolated mesenteric arteries were neither affected by the sEH inhibitor, N-adamantyl-N′-dodecylurea (ADU), nor by sEH gene deletion. However, the EDHF responses were abolished by catalase and by apamin/charybdotoxin (ChTx), but not by iberiotoxin, nor by the cytochrome P450 inhibitor PPOH. All four EETs (order of potency: 8,9-EET >14,15-EET≈5,6-EET >11,12-EET) and all 4 dihydroxy derivatives (14,15-DHET≈8,9-DHET≈11,12-DHET >5,6-DHET) produced dose-dependent vasodilation. Endothelial removal or L-NAME blocked 8,9-EET and 14,15-DHET-dependent dilations. The effects of apamin/ChTx were minimal. 8,9-EET and 14,15-DHET induced NO production in endothelial cells. ADU (100 &mgr;g/mL in drinking water) lowered blood pressure in angiotensin II–infused hypertension, but not in L-NAME–induced hypertension. Blood pressure and EDHF responses were similar in L-NAME–treated sEH +/+ and −/− mice. Conclusions—Our data indicate that the EDHF response in mice is caused by hydrogen peroxide, but not by P450 eicosanoids. Moreover, P450 eicosanoids are vasodilatory, largely through their ability to activate endothelial NO synthase (eNOS) and NO release.

GDF11 Does Not Rescue Aging-Related Pathological Hypertrophy

RATIONALE Growth differentiation factor 11 (GDF11) is a member of the transforming growth factor-β super family of secreted factors. A recent study showed that reduced GDF11 blood levels with aging was associated with pathological cardiac hypertrophy (PCH) and restoring GDF11 to normal levels in old mice rescued PCH. OBJECTIVE To determine whether and by what mechanism GDF11 rescues aging dependent PCH. METHODS AND RESULTS Twenty-four-month-old C57BL/6 mice were given a daily injection of either recombinant (r) GDF11 at 0.1 mg/kg or vehicle for 28 days. rGDF11 bioactivity was confirmed in vitro. After treatment, rGDF11 levels were significantly increased, but there was no significant effect on either heart weight or body weight. Heart weight/body weight ratios of old mice were not different from 8- or 12-week-old animals, and the PCH marker atrial natriuretic peptide was not different in young versus old mice. Ejection fraction, internal ventricular dimension, and septal wall thickness were not significantly different between rGDF11 and vehicle-treated animals at baseline and remained unchanged at 1, 2, and 4 weeks of treatment. There was no difference in myocyte cross-sectional area rGDF11 versus vehicle-treated old animals. In vitro studies using phenylephrine-treated neonatal rat ventricular myocytes, to explore the putative antihypertrophic effects of GDF11, showed that GDF11 did not reduce neonatal rat ventricular myocytes hypertrophy, but instead induced hypertrophy. CONCLUSIONS Our studies show that there is no age-related PCH in disease-free 24-month-old C57BL/6 mice and that restoring GDF11 in old mice has no effect on cardiac structure or function.

Redox Regulation of Soluble Epoxide Hydrolase by 15-Deoxy-Δ-Prostaglandin J2 Controls Coronary Hypoxic Vasodilation

Rationale: 15-Deoxy-&Dgr;-prostaglandin (15d-PG)J2 is an electrophilic oxidant that dilates the coronary vasculature. This lipid can adduct to redox active protein thiols to induce oxidative posttranslational modifications that modulate protein and tissue function. Objective: To investigate the role of oxidative protein modifications in 15d-PGJ2–mediated coronary vasodilation and define the distal signaling pathways leading to enhanced perfusion. Methods and Results: Proteomic screening with biotinylated 15d-PGJ2 identified novel vascular targets to which it adducts, most notably soluble epoxide hydrolase (sEH). 15d-PGJ2 inhibited sEH by specifically adducting to a highly conserved thiol (Cys521) adjacent to the catalytic center of the hydrolase. Indeed a Cys521Ser sEH “redox-dead” mutant was resistant to 15d-PGJ2–induced hydrolase inhibition. 15d-PGJ2 dilated coronary vessels and a role for hydrolase inhibition was supported by 2 structurally different sEH antagonists each independently inducing vasorelaxation. Furthermore, 15d-PGJ2 and sEH antagonists also increased coronary effluent epoxyeicosatrienoic acids consistent with their vasodilatory actions. Indeed 14,15-EET alone induced relaxation and 15d-PGJ2–mediated vasodilation was blocked by the EET receptor antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE). Additionally, the coronary vasculature of sEH-null mice was basally dilated compared to wild-type controls and failed to vasodilate in response to 15d-PGJ2. Coronary vasodilation to hypoxia in wild-types was accompanied by 15d-PGJ2 adduction to and inhibition of sEH. Consistent with the importance of hydrolase inhibition, sEH-null mice failed to vasodilate during hypoxia. Conclusion: This represents a new paradigm for the regulation of sEH by an endogenous lipid, which is integral to the fundamental physiological response of coronary hypoxic vasodilation.

Renal Ischemia/Reperfusion Injury in Soluble Epoxide Hydrolase-Deficient Mice

Aim 20-hydroxyeicosatetraenoic acid (20-HETE) and epoxyeicosatrienoic acids (EETs) are cytochrome P450 (CYP)-dependent eicosanoids that play opposite roles in the regulation of vascular tone, inflammation, and apoptosis. 20-HETE aggravates, whereas EETs ameliorate ischemia/reperfusion (I/R)-induced organ damage. EETs are rapidly metabolized to dihydroxyeicosatrienoic acids (DHETs) by the soluble epoxide hydrolase (sEH). We hypothesized that sEH gene (EPHX2) deletion would increase endogenous EET levels and thereby protect against I/R-induced acute kidney injury (AKI). Methods Kidney damage was evaluated in male wildtype (WT) and sEH-knockout (KO)-mice that underwent 22-min renal ischemia followed by two days of reperfusion. CYP-eicosanoids were analyzed by liquid chromatography tandem mass spectrometry. Results Contrary to our initial hypothesis, renal function declined more severely in sEH-KO mice as indicated by higher serum creatinine and urea levels. The sEH-KO-mice also featured stronger tubular lesion scores, tubular apoptosis, and inflammatory cell infiltration. Plasma and renal EET/DHET-ratios were higher in sEH-KO than WT mice, thus confirming the expected metabolic consequences of sEH deficiency. However, CYP-eicosanoid profiling also revealed that renal, but not plasma and hepatic, 20-HETE levels were significantly increased in sEH-KO compared to WT mice. In line with this finding, renal expression of Cyp4a12a, the murine 20-HETE-generating CYP-enzyme, was up-regulated both at the mRNA and protein level, and Cyp4a12a immunostaining was more intense in the renal arterioles of sEH-KO compared with WT mice. Conclusion These results indicate that the potential beneficial effects of reducing EET degradation were obliterated by a thus far unknown mechanism leading to kidney-specific up-regulation of 20-HETE formation in sEH-KO-mice.

Selectivity of BI 689648, a Novel, Highly Selective Aldosterone Synthase Inhibitor: Comparison with FAD286 and LCI699 in Nonhuman Primates

The mineralocorticoid aldosterone is an important regulator of blood pressure, volume, and electrolyte balance. However, excess aldosterone can be deleterious as a driver of vascular remodeling and tissue fibrosis associated with cardiometabolic diseases. Aldosterone synthase (AS) inhibitors (ASI) attenuate the production of aldosterone directly and have been proposed as an alternative to mineralocorticoid receptor antagonists for blocking the pathologic effects of excess aldosterone. Discovery of selective ASIs has been challenging because of the high sequence identity (93%) AS shares with cortisol synthase (CS), and the low identity of rodent AS compared with human (63%). Using cynomolgus (cyno) monkey-based models, we identified BI 689648 [6-(5-methoxymethyl-pyridin-3-yl)-3,4-dihydro-2H-[1,8]naphthyridine-1-carboxylic acid amide], a novel, highly selective ASI that exhibits an in vitro IC50 of 2 nM against AS and 300 nm against CS (150-fold selectivity) compared with the recently described ASIs FAD286 [4-(5,6,7,8-tetrahydroimidazo[1,5-a]pyridin-5-yl)benzonitrile] (3 nM AS; 90 nM CS; 40-fold) and LCI699 (4-[(5R)-6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-5-yl]-3-fluorobenzonitrile) (10 nM AS; 80 nM CS; 8-fold). After oral administration in cyno monkeys, BI 689648 (5 mg/kg) exhibits a peak plasma concentration of ∼500 nM. For in vivo profiling we used an adrenocorticotropin-challenge model in which BI 689648 was >20-fold more selective compared with FAD286 and LCI699. Because both FAD286 and LCI699 failed to provide adequate selectivity for CS when tested in patients, the desire for more selective molecules to test the ASI hypothesis remains high. Therefore, highly selective aldosterone synthase inhibitors such as BI 689648 represent an important step forward toward developing ASIs with greater potential for clinical success in cardiometabolic diseases.

Elevated Steroid Hormone Production in the db/db Mouse Model of Obesity and Type 2 Diabetes

Obesity and type 2 diabetes have become a major public health problem worldwide. Steroid hormone dysfunction appears to be linked to development of obesity and type 2 diabetes and correction of steroid abnormalities may offer new approaches to therapy. We therefore analyzed plasma steroids in 15-16 week old obese and diabetic db/db mice using liquid chromatography-tandem mass spectrometry. Lean db/+ served as controls. Db/db mice developed obesity, hyperglycemia, hyperleptinemia, and hyperlipidemia. Hepatic triglyceride storage was increased and adiponectin and pancreatic insulin were lowered. Aldosterone, corticosterone, 11-deoxycorticosterone, and progesterone were respectively increased by 3.6-, 2.9-, 3.4, and 1.7-fold in db/db mice compared to controls. Ratios of aldosterone-to-progesterone and corticosterone-to-progesterone were respectively 2.0- and 1.5-fold higher in db/db mice. Genes associated with steroidogenesis were quantified in the adrenal glands and gonadal adipose tissues. In adrenals, Cyp11b2, Cyp11b1, Cyp21a1, Hsd3b1, Cyp11a1, and StAR were all significantly increased in db/db mice compared with db/+ controls. In adipose tissue, no Cyp11b2 or Cyp11b1 transcripts were detected and no differences in Cyp21a1, Hsd3b1, Cyp11a1, or StAR expression were found between db/+ and db/db mice. In conclusion, the present study showed an elevated steroid hormone production and adrenal steroidogenesis in the db/db model of obesity and type 2 diabetes.

The Aldosterone Synthase Inhibitor FAD286 is Suitable for Lowering Aldosterone Levels in ZDF Rats but not in db/db Mice

Inhibition of aldosterone synthase is an alternative treatment option to mineralocorticoid receptor antagonism to prevent harmful aldosterone actions. FAD286 is one of the best characterized aldosterone synthase inhibitors to date. FAD286 improves glucose tolerance and increases glucose-stimulated insulin secretion in obese and diabetic ZDF rats. However, there is limited knowledge about the dose-dependent effects of FAD286 on plasma aldosterone, corticosterone, and 11-deoxycorticosterone in ZDF rats and in db/db mice, a second important rodent model of obesity and type 2 diabetes. In addition, effects of FAD286 on plasma steroids in mice and rats are controversial. Therefore, obese Zucker diabetic fatty (ZDF) rats and db/db mice were treated with FAD286 for up to 15 weeks and plasma steroids were evaluated using highly sensitive liquid chromatography-tandem mass spectrometry. In ZDF rats, FAD286 (10 mg/kg/d) treatment resulted in nearly complete disappearance of plasma aldosterone while corticosterone levels remained unaffected and those of 11-deoxycorticosterone were increased ~4-fold compared to vehicle control. A lower dose of FAD286 (3 mg/kg/d) showed no effect on plasma aldosterone or corticosterone, but 11-deoxycorticosterone was again increased ~4-fold compared to control. In contrast to ZDF rats, a high dose of FAD286 (40 mg/kg/d) did not affect plasma aldosterone levels in db/db mice although 11-deoxycorticosterone increased ~2.5-fold. A low dose of FAD286 (10 mg/kg/d) increased plasma aldosterone without affecting corticosterone or 11-deoxycorticosterone. In conclusion, the aldosterone synthase inhibitor, FAD286, lowers plasma aldosterone in obese ZDF rats, but not in obese db/db mice.

Impact of Aldosterone Synthase Inhibitor FAD286 on Steroid Hormone Profile in Human Adrenocortical Cells

Inhibition of aldosterone synthase (CYP11B2) is an alternative treatment option to mineralocorticoid receptor antagonism to prevent harmful aldosterone effects. FAD286 is the best characterized aldosterone synthase inhibitor. However, to date, no study has used sensitive liquid chromatography-tandem mass spectrometry to characterize in detail the effect of FAD286 on the secreted steroid hormone profile of adrenocortical cells. Basal aldosterone production in NCI-H295R cells was detectable and 9-fold elevated after stimulation with angiotensin II. FAD286 inhibited this increase, showing a maximal effect at 10 nmol/l. Higher concentrations of FAD286 did not further reduce aldosterone concentrations, but showed a parallel reduction in corticosterone, cortisol and cortisone levels, reflecting additional inhibition of steroid-11β-hydroxylase (CYP11B1). Pregnenolone, progesterone and 17-OH-progesterone levels remained unaffected. In conclusion, the aldosterone synthase inhibitor FAD286 lowers angiotensin II-induced aldosterone concentrations in adrenocortical cells but the relative lack of selectivity over CYP11B1 is evident at higher FAD286 concentrations.