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1.
Horm Metab Res ; 53(10): 699-704, 2021 Oct.
Article in English | MEDLINE | ID: mdl-34607368

ABSTRACT

The impact of dietary sodium reduction on mouse models of type 2 diabetes is not well understood. Therefore, we analyzed the effect of a low-salt diet on obesity and parameters of type 2 diabetes in db/db mice. Five-week-old male db/db and lean db/m mice were fed a normal salt (0.19% Na+, NS) or a low-salt diet (<0.03% Na+, LS) for 5 weeks. Body and organ weight and parameters of glucose and insulin tolerance were analyzed. Plasma levels of steroids were determined by liquid chromatography tandem mass spectrometry. Body weight, glucose, and insulin tolerance were not affected by LS. The amount of gonadal adipose tissue showed a trend to be increased by LS whereas liver, pancreas, kidney, heart, and adrenal weight remained unaffected. LS reduced urinary sodium-to-creatinine ratio but did not affect plasma Na+ levels in both genotypes. Plasma and urinary potassium-to-creatinine ratio did not differ in all groups of mice. Aldosterone as a major determinant of changes in dietary sodium remained unaffected by LS in db/db mice as well as further investigated steroid hormones. The present study showed reduced sodium-to-creatinine ratio, but no additional effects of dietary sodium reduction on major metabolic parameters and steroid levels in obese and hyper-glycemic db/db mice.


Subject(s)
Diabetes Mellitus, Type 2/diet therapy , Diet, Sodium-Restricted , Obesity/diet therapy , Animals , Body Weight/drug effects , Diabetes Mellitus, Type 2/metabolism , Diabetes Mellitus, Type 2/pathology , Disease Models, Animal , Disease Progression , Down-Regulation , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Obesity/metabolism , Obesity/pathology , Organ Size/drug effects , Sodium Chloride, Dietary/administration & dosage , Sodium Chloride, Dietary/pharmacology , Sodium, Dietary/pharmacology
2.
Proc Natl Acad Sci U S A ; 116(20): 10156-10161, 2019 05 14.
Article in English | MEDLINE | ID: mdl-31028142

ABSTRACT

Transient receptor potential canonical type 6 (TRPC6) is a nonselective receptor-operated cation channel that regulates reactive fibrosis and growth signaling. Increased TRPC6 activity from enhanced gene expression or gain-of-function mutations contribute to cardiac and/or renal disease. Despite evidence supporting a pathophysiological role, no orally bioavailable selective TRPC6 inhibitor has yet been developed and tested in vivo in disease models. Here, we report an orally bioavailable TRPC6 antagonist (BI 749327; IC50 13 nM against mouse TRPC6, t1/2 8.5-13.5 hours) with 85- and 42-fold selectivity over the most closely related channels, TRPC3 and TRPC7. TRPC6 calcium conductance results in the stimulation of nuclear factor of activated T cells (NFAT) that triggers pathological cardiac and renal fibrosis and disease. BI 749327 suppresses NFAT activation in HEK293T cells expressing wild-type or gain-of-function TRPC6 mutants (P112Q, M132T, R175Q, R895C, and R895L) and blocks associated signaling and expression of prohypertrophic genes in isolated myocytes. In vivo, BI 749327 (30 mg/kg/day, yielding unbound trough plasma concentration ∼180 nM) improves left heart function, reduces volume/mass ratio, and blunts expression of profibrotic genes and interstitial fibrosis in mice subjected to sustained pressure overload. Additionally, BI 749327 dose dependently reduces renal fibrosis and associated gene expression in mice with unilateral ureteral obstruction. These results provide in vivo evidence of therapeutic efficacy for a selective pharmacological TRPC6 inhibitor with oral bioavailability and suitable pharmacokinetics to ameliorate cardiac and renal stress-induced disease with fibrosis.


Subject(s)
Cardiomegaly/drug therapy , Nephrosclerosis/drug therapy , TRPC6 Cation Channel/antagonists & inhibitors , Animals , Drug Evaluation, Preclinical , Fibrosis , HEK293 Cells , Heart/drug effects , Humans , Kidney/drug effects , Mice
3.
Vitam Horm ; 109: 211-239, 2019.
Article in English | MEDLINE | ID: mdl-30678857

ABSTRACT

The mineralocorticoid aldosterone is an important regulator of blood pressure and electrolyte balance. However, excess aldosterone can be deleterious as a driver of inflammation, vascular remodeling and tissue fibrosis associated with cardiometabolic diseases. Mineralocorticoid receptor antagonists (MRA) and renin-angiotensin-aldosterone system (RAAS) antagonists are current clinical therapies used to antagonize deleterious effects of aldosterone in patients. MRAs compete with aldosterone for binding at its cognate receptor thereby limiting its effect while RAS antagonists reduce aldosterone levels indirectly by blocking the stimulatory effect of angiotensin. Both MRAs and RAS antagonists can result in incomplete inhibition of the harmful effects of excess aldosterone. Aldosterone synthase (AS) inhibitors (ASI) attenuate the production of aldosterone directly and have been proposed as an alternative to MRAs and RAS blockers. Cortisol synthase (CS) is an enzyme closely related to AS and responsible for generating the important glucocorticoid cortisol, required for maintaining critical metabolic and immune responses. The importance of selectivity against CS is shown by early examples of ASIs that were only modestly selective and as such, attenuated cortisol responses when evaluated in patients. Recently, next-generation, highly selective ASIs have been described and are presently being evaluated in the clinic as an alternative to angiotensin and MR antagonists for cardiometabolic disease. Herein we provide a brief review of the challenges associated with discovery of selective ASIs and the transition from the early compounds that paved the way toward the next-generation of highly selective ASIs currently under development.


Subject(s)
Mineralocorticoid Receptor Antagonists/pharmacology , Aldosterone/metabolism , Animals , Blood Pressure/physiology , Humans , Renin-Angiotensin System/physiology
4.
Bioorg Med Chem Lett ; 28(5): 979-984, 2018 03 01.
Article in English | MEDLINE | ID: mdl-29254646

ABSTRACT

6,7-Dihydro-5H-2,1-benzisoxazol-4-one analogs are potent inhibitors of aldosterone synthase (CYP11B2) with selectivity over the highly homologous enzyme cortisol synthase (CYP11B1). These compounds are unique among inhibitors of CYP11B2 in their lack of a strong-heme binding group such as a pyridine or imidazole. Poor metabolic stability in hepatocyte incubations was found to proceed via a reduction of the isoxazole ring. While the enzyme responsible for the reductive metabolism remains unknown, the rate of metabolism could be attenuated by the addition of polar functionality. The in vitro CYP11B2 potency and selectivity were confirmed in vivo in a cynomolgus monkey model by the inhibition of ACTH stimulated aldosterone production without impacting plasma cortisol concentrations.


Subject(s)
Cytochrome P-450 CYP11B2/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Isoxazoles/pharmacology , Cytochrome P-450 CYP11B2/metabolism , Dose-Response Relationship, Drug , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Humans , Isoxazoles/chemical synthesis , Isoxazoles/chemistry , Molecular Structure , Structure-Activity Relationship
5.
Horm Metab Res ; 49(9): 701-706, 2017 Sep.
Article in English | MEDLINE | ID: mdl-28759940

ABSTRACT

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-11ß-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.


Subject(s)
Adrenal Cortex/cytology , Cytochrome P-450 CYP11B2/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Fadrozole/pharmacology , Hormones/metabolism , Steroids/metabolism , Aldosterone/metabolism , Angiotensin II/pharmacology , Angiotensin II Type 1 Receptor Blockers/pharmacology , Benzimidazoles/pharmacology , Biphenyl Compounds , Cell Line , Cytochrome P-450 CYP11B2/genetics , Cytochrome P-450 CYP11B2/metabolism , Gene Expression Regulation, Enzymologic/drug effects , Glucocorticoids/metabolism , Humans , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptor, Angiotensin, Type 1/metabolism , Tetrazoles/pharmacology
6.
Horm Metab Res ; 49(6): 466-471, 2017 Jun.
Article in English | MEDLINE | ID: mdl-28427090

ABSTRACT

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.


Subject(s)
Aldosterone/blood , Cytochrome P-450 CYP11B2/antagonists & inhibitors , Cytochrome P-450 Enzyme Inhibitors/pharmacology , Diabetes Mellitus, Experimental/blood , Diabetes Mellitus, Experimental/enzymology , Fadrozole/pharmacology , Adrenal Glands/metabolism , Animals , Corticosterone/biosynthesis , Cytochrome P-450 CYP11B2/metabolism , Diabetes Mellitus, Experimental/pathology , Male , Mice, Obese , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats, Zucker , Steroid 11-beta-Hydroxylase/metabolism
7.
Horm Metab Res ; 49(1): 43-49, 2017 Jan.
Article in English | MEDLINE | ID: mdl-27813053

ABSTRACT

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.


Subject(s)
Adrenal Cortex Hormones/metabolism , Diabetes Mellitus, Experimental/metabolism , Diabetes Mellitus, Type 2/metabolism , Gonadal Steroid Hormones/metabolism , Obesity/metabolism , Adrenal Cortex Hormones/blood , Aldosterone/blood , Aldosterone/metabolism , Animals , Corticosterone/blood , Corticosterone/metabolism , Desoxycorticosterone/blood , Desoxycorticosterone/metabolism , Diabetes Mellitus, Experimental/complications , Diabetes Mellitus, Type 2/pathology , Gonadal Steroid Hormones/blood , Humans , Male , Mice , Mice, Obese , Obesity/complications , Progesterone/blood , Progesterone/metabolism
8.
Am J Physiol Renal Physiol ; 311(6): F1198-F1210, 2016 12 01.
Article in English | MEDLINE | ID: mdl-27681558

ABSTRACT

Activation of the thick ascending limb (TAL) Na+-K+-2Cl- cotransporter (NKCC2) by the antidiuretic hormone arginine vasopressin (AVP) is an essential mechanism of renal urine concentration and contributes to extracellular fluid and electrolyte homeostasis. AVP effects in the kidney are modulated by locally and/or by systemically produced epoxyeicosatrienoic acid derivates (EET). The relation between AVP and EET metabolism has not been determined. Here, we show that chronic treatment of AVP-deficient Brattleboro rats with the AVP V2 receptor analog desmopressin (dDAVP; 5 ng/h, 3 days) significantly lowered renal EET levels (-56 ± 3% for 5,6-EET, -50 ± 3.4% for 11,12-EET, and -60 ± 3.7% for 14,15-EET). The abundance of the principal EET-degrading enzyme soluble epoxide hydrolase (sEH) was increased at the mRNA (+160 ± 37%) and protein levels (+120 ± 26%). Immunohistochemistry revealed dDAVP-mediated induction of sEH in connecting tubules and cortical and medullary collecting ducts, suggesting a role of these segments in the regulation of local interstitial EET signals. Incubation of murine kidney cell suspensions with 1 µM 14,15-EET for 30 min reduced phosphorylation of NKCC2 at the AVP-sensitive threonine residues T96 and T101 (-66 ± 5%; P < 0.05), while 14,15-DHET had no effect. Concomitantly, isolated perfused cortical thick ascending limb pretreated with 14,15-EET showed a 30% lower transport current under high and a 70% lower transport current under low symmetric chloride concentrations. In summary, we have shown that activation of AVP signaling stimulates renal sEH biosynthesis and enzyme activity. The resulting reduction of EET tissue levels may be instrumental for increased NKCC2 transport activity during AVP-induced antidiuresis.


Subject(s)
Deamino Arginine Vasopressin/pharmacology , Eicosanoids/metabolism , Epoxide Hydrolases/metabolism , Kidney/drug effects , Solute Carrier Family 12, Member 1/metabolism , Animals , Kidney/metabolism , Mice , Phosphorylation/drug effects , Rats , Rats, Brattleboro
9.
J Pharmacol Exp Ther ; 359(1): 142-50, 2016 10.
Article in English | MEDLINE | ID: mdl-27481775

ABSTRACT

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.


Subject(s)
Cytochrome P-450 CYP11B2/antagonists & inhibitors , Cytochrome P-450 Enzyme Inhibitors/pharmacology , Fadrozole/pharmacology , Imidazoles/pharmacology , Naphthyridines/pharmacology , Pyridines/pharmacology , Adrenocorticotropic Hormone/pharmacology , Animals , Humans , Macaca fascicularis , Male , Substrate Specificity
10.
Endocrinology ; 157(10): 3844-3855, 2016 Oct.
Article in English | MEDLINE | ID: mdl-27526033

ABSTRACT

Plasma aldosterone is elevated in type 2 diabetes and obesity in experimental and clinical studies and can act to inhibit both glucose-stimulated insulin secretion by the ß-cell and insulin signaling. Currently mineralocorticoid receptor antagonism is the best characterized treatment to ameliorate aldosterone-mediated effects. A second alternative is inhibition of aldosterone synthase, an approach with protective effects on end-organ damage in heart or kidney in animal models. The effect of aldosterone synthase inhibition on metabolic parameters in type 2 diabetes is not known. Therefore, male Zucker diabetic fatty (ZDF) rats were treated for 11 weeks with the aldosterone synthase inhibitor FAD286, beginning at 7 weeks of age. Results were compared with the mineralocorticoid receptor antagonist eplerenone. Plasma aldosterone was abolished by FAD286 and elevated more than 9-fold by eplerenone. The area under the curve calculated from an oral glucose tolerance test (OGTT) was lower and overall insulin response during OGTT was increased by FAD286. In contrast, eplerenone elevated blood glucose levels and blunted insulin secretion during the OGTT. Fasting glucose was lowered and fasting insulin was increased by FAD286 in the prediabetic state. Glycated hemoglobin was lowered by FAD286, whereas eplerenone showed no effect. We conclude that aldosterone synthase inhibition, in contrast to mineralocorticoid receptor antagonism, has the potential for beneficial effects on metabolic parameters in type 2 diabetes.


Subject(s)
Cytochrome P-450 CYP11B2/antagonists & inhibitors , Diabetes Mellitus, Type 2/prevention & control , Fadrozole/therapeutic use , Adrenal Glands/drug effects , Aldosterone/blood , Animals , Blood Glucose/drug effects , Body Weight/drug effects , Disease Models, Animal , Drug Evaluation, Preclinical , Eating/drug effects , Eplerenone , Fadrozole/pharmacology , Glucose Tolerance Test , Glycated Hemoglobin/metabolism , Insulin/metabolism , Insulin Resistance , Insulin Secretion , Liver/drug effects , Liver/metabolism , Male , Mineralocorticoid Receptor Antagonists/pharmacology , Mineralocorticoid Receptor Antagonists/therapeutic use , Organ Size/drug effects , Potassium/blood , Random Allocation , Rats, Zucker , Sodium/blood , Spironolactone/analogs & derivatives , Spironolactone/pharmacology , Spironolactone/therapeutic use , Triglycerides/metabolism
11.
PLoS One ; 11(1): e0145645, 2016.
Article in English | MEDLINE | ID: mdl-26727266

ABSTRACT

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.


Subject(s)
Epoxide Hydrolases/genetics , Kidney/blood supply , Reperfusion Injury/enzymology , Animals , Chromatography, Liquid , Cytochrome P-450 Enzyme System/metabolism , Cytochrome P450 Family 4 , Hydroxyeicosatetraenoic Acids/biosynthesis , Kidney/enzymology , Male , Mice , Mice, Knockout , Oxylipins/metabolism , Tandem Mass Spectrometry
12.
Circ Res ; 117(11): 926-32, 2015 Nov 06.
Article in English | MEDLINE | ID: mdl-26383970

ABSTRACT

RATIONALE: Growth differentiation factor 11 (GDF11) is a member of the transforming growth factor-ß 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.


Subject(s)
Aging/pathology , Bone Morphogenetic Proteins/pharmacology , Cardiomegaly/prevention & control , Growth Differentiation Factors/pharmacology , Myocytes, Cardiac/drug effects , Ventricular Remodeling/drug effects , Adrenergic alpha-1 Receptor Agonists/pharmacology , Age Factors , Aging/metabolism , Animals , Bone Morphogenetic Proteins/administration & dosage , Cardiomegaly/metabolism , Cardiomegaly/pathology , Cardiomegaly/physiopathology , Cells, Cultured , Drug Administration Schedule , Fibroblasts/drug effects , Fibroblasts/metabolism , Fibroblasts/pathology , Fibrosis , Growth Differentiation Factors/administration & dosage , Injections, Intraperitoneal , Male , Mice, Inbred C57BL , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/pathology , Recombinant Proteins/pharmacology , Time Factors , Ventricular Function, Left/drug effects , Ventricular Pressure/drug effects
13.
Can J Physiol Pharmacol ; 90(6): 811-23, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22624559

ABSTRACT

Epoxyeicosatrienoic acids (EETs) are active metabolites of arachidonic acid that are inactivated by soluble epoxide hydrolase enzyme (sEH) to dihydroxyeicosatrienoic acid. EETs are known to render cardioprotection against ischemia reperfusion (IR) injury by maintaining mitochondrial function. We investigated the effect of a novel sEH inhibitor (sEHi) in limiting IR injury. Mouse hearts were perfused in Langendorff mode for 40 min and subjected to 20 min of global no-flow ischemia followed by 40 min of reperfusion. Hearts were perfused with 0.0, 0.1, 1.0 and 10.0 µmol·L(-1) of the sEHi N-(2-chloro-4-methanesulfonyl-benzyl)-6-(2,2,2-trifluoro-ethoxy)-nicotinamide (BI00611953). Inhibition of sEH by BI00611953 significantly improved postischemic left-ventricular-developed pressure and reduced infarct size following IR compared with control hearts, and similar to hearts perfused with 11,12-EETs (1 µmol·L(-1)) and sEH(-/-) mice. Perfusion with the putative EET receptor antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE, 10 µmol·L(-1)), or the plasma membrane K(ATP) channels (pmK(ATP)) inhibitor (glibenclamide, 10 µmol·L(-1)) abolished the improved recovery by BI00611953 (1 µmol·L(-1)). Mechanistic studies in H9c2 cells demonstrated that BI0611953 decreased ROS generation, caspase-3 activity, proteasome activity, increased HIF-1∝ DNA binding, and delayed the loss of mitochondrial membrane potential (ΔΨ(m)) caused by anoxia-reoxygenation. Together, our data demonstrate that the novel sEHi BI00611953, a nicotinamide-based compound, provides significant cardioprotection against ischemia reperfusion injury.


Subject(s)
Enzyme Inhibitors/pharmacology , Epoxide Hydrolases/antagonists & inhibitors , Mitochondria, Heart/drug effects , Myocardial Reperfusion Injury/drug therapy , Reperfusion Injury/drug therapy , Animals , Arachidonic Acid/metabolism , Caspase 3/metabolism , Cells, Cultured , Epoxide Hydrolases/metabolism , Heart/drug effects , Hypoxia/drug therapy , Hypoxia/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Membrane Potential, Mitochondrial/drug effects , Mice , Mice, Inbred C57BL , Mitochondria, Heart/metabolism , Myocardial Infarction/drug therapy , Myocardial Infarction/metabolism , Myocardial Reperfusion Injury/metabolism , Myocardium/enzymology , Myocardium/metabolism , Proteasome Endopeptidase Complex/metabolism , Rats , Reactive Oxygen Species/metabolism , Reperfusion Injury/metabolism
14.
Circ Res ; 108(3): 324-34, 2011 Feb 04.
Article in English | MEDLINE | ID: mdl-21164107

ABSTRACT

RATIONALE: 15-Deoxy-Δ-prostaglandin (15d-PG)J(2) 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-PGJ(2)-mediated coronary vasodilation and define the distal signaling pathways leading to enhanced perfusion. METHODS AND RESULTS: Proteomic screening with biotinylated 15d-PGJ(2) identified novel vascular targets to which it adducts, most notably soluble epoxide hydrolase (sEH). 15d-PGJ(2) 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-PGJ(2)-induced hydrolase inhibition. 15d-PGJ(2) dilated coronary vessels and a role for hydrolase inhibition was supported by 2 structurally different sEH antagonists each independently inducing vasorelaxation. Furthermore, 15d-PGJ(2) and sEH antagonists also increased coronary effluent epoxyeicosatrienoic acids consistent with their vasodilatory actions. Indeed 14,15-EET alone induced relaxation and 15d-PGJ(2)-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-PGJ(2). Coronary vasodilation to hypoxia in wild-types was accompanied by 15d-PGJ(2) 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.


Subject(s)
Epoxide Hydrolases/metabolism , Hypoxia/metabolism , Myocardium/metabolism , Prostaglandin D2/analogs & derivatives , Vasodilation/physiology , Amino Acid Sequence , Animals , Epoxide Hydrolases/analysis , Epoxide Hydrolases/genetics , Hypoxia/physiopathology , Mice , Mice, Inbred C57BL , Mice, Knockout , Models, Animal , Molecular Sequence Data , Oxidation-Reduction , Prostaglandin D2/metabolism , Rats , Rats, Wistar , Signal Transduction
15.
Arterioscler Thromb Vasc Biol ; 29(1): 54-60, 2009 Jan.
Article in English | MEDLINE | ID: mdl-18927469

ABSTRACT

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 approximately 5,6-EET >11,12-EET) and all 4 dihydroxy derivatives (14,15-DHET approximately 8,9-DHET approximately 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 microg/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.


Subject(s)
Arteries/physiology , Cytochrome P-450 Enzyme System/metabolism , Eicosanoids/physiology , Muscle Tonus/physiology , Nitric Oxide/physiology , Vasodilation/physiology , Animals , Arteries/drug effects , Biological Factors/physiology , Blood Pressure/drug effects , Catalase/metabolism , Charybdotoxin/pharmacology , Cytochrome P-450 Enzyme Inhibitors , Eicosanoids/pharmacology , Endothelium, Vascular/drug effects , Endothelium, Vascular/physiology , Epoxide Hydrolases/deficiency , Epoxide Hydrolases/metabolism , Heart Rate/drug effects , Hydrogen Peroxide/pharmacology , Kidney/drug effects , Kidney/physiology , Male , Mesenteric Arteries/drug effects , Mesenteric Arteries/physiology , Mice , Mice, Inbred Strains , Mice, Knockout , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/physiology , NG-Nitroarginine Methyl Ester/pharmacology , Peptides/pharmacology , Vasodilation/drug effects
16.
Nat Genet ; 40(5): 529-37, 2008 May.
Article in English | MEDLINE | ID: mdl-18443590

ABSTRACT

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.


Subject(s)
Disease Models, Animal , Epoxide Hydrolases/genetics , Genetic Predisposition to Disease , Heart Failure/genetics , Rats/genetics , Animals , Chromosome Mapping , Epoxide Hydrolases/analysis , Epoxide Hydrolases/metabolism , Gene Expression Profiling , Genetic Linkage , Heart Failure/enzymology , Heart Failure/physiopathology , Humans , Hypertension/complications , Hypertension/genetics , Mice , Mice, Knockout , Myocardium/enzymology , Polymorphism, Genetic , Polymorphism, Single Nucleotide , Promoter Regions, Genetic , Quantitative Trait Loci , Rats, Mutant Strains , Sequence Analysis, DNA , Sequence Deletion , Transcription Factor AP-1/metabolism
17.
J Biol Chem ; 282(5): 2891-8, 2007 Feb 02.
Article in English | MEDLINE | ID: mdl-17135253

ABSTRACT

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.


Subject(s)
Blood Pressure/physiology , Epoxide Hydrolases/deficiency , Epoxide Hydrolases/genetics , Animals , Blood Pressure/genetics , Crosses, Genetic , Epoxy Compounds/metabolism , Exons , Fatty Acids, Nonesterified/metabolism , Female , Homeostasis , Humans , Kidney/physiology , Liver/physiology , Male , Mice , Mice, Inbred C57BL , Mice, Inbred Strains , Mice, Knockout
18.
Nature ; 426(6963): 186-9, 2003 Nov 13.
Article in English | MEDLINE | ID: mdl-14578911

ABSTRACT

Hepatitis C virus (HCV) infection is a serious cause of chronic liver disease worldwide with more than 170 million infected individuals at risk of developing significant morbidity and mortality. Current interferon-based therapies are suboptimal especially in patients infected with HCV genotype 1, and they are poorly tolerated, highlighting the unmet medical need for new therapeutics. The HCV-encoded NS3 protease is essential for viral replication and has long been considered an attractive target for therapeutic intervention in HCV-infected patients. Here we identify a class of specific and potent NS3 protease inhibitors and report the evaluation of BILN 2061, a small molecule inhibitor biologically available through oral ingestion and the first of its class in human trials. Administration of BILN 2061 to patients infected with HCV genotype 1 for 2 days resulted in an impressive reduction of HCV RNA plasma levels, and established proof-of-concept in humans for an HCV NS3 protease inhibitor. Our results further illustrate the potential of the viral-enzyme-targeted drug discovery approach for the development of new HCV therapeutics.


Subject(s)
Antiviral Agents/therapeutic use , Carbamates/pharmacology , Hepacivirus/drug effects , Hepacivirus/physiology , Hepatitis C/drug therapy , Macrocyclic Compounds , Quinolines , Serine Proteinase Inhibitors/therapeutic use , Thiazoles/pharmacology , Viral Nonstructural Proteins/antagonists & inhibitors , Administration, Oral , Antiviral Agents/administration & dosage , Antiviral Agents/pharmacokinetics , Antiviral Agents/pharmacology , Carbamates/administration & dosage , Carbamates/chemistry , Carbamates/pharmacokinetics , Double-Blind Method , Hepacivirus/enzymology , Hepacivirus/genetics , Hepatitis C/virology , Humans , Male , Polyproteins/metabolism , Protein Processing, Post-Translational/drug effects , Serine Proteinase Inhibitors/administration & dosage , Serine Proteinase Inhibitors/pharmacokinetics , Serine Proteinase Inhibitors/pharmacology , Thiazoles/administration & dosage , Thiazoles/chemistry , Thiazoles/pharmacokinetics , Viral Load , Viral Nonstructural Proteins/metabolism , Viral Proteins/metabolism
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