Endothelin receptor antagonism improves glucose handling, dyslipidemia, and adipose tissue inflammation in obese mice

Endothelin-1 (ET-1) is elevated in patients with obesity; however, its contribution to the pathophysiology related to obesity is not fully understood. We hypothesized that high ET-1 levels cause dyslipidemia, inflammation, and insulin resistance within the adipose tissue of obese mice. To test this hypothesis, male C57BL/6J mice were fed either normal diet (NMD) or high fat diet (HFD) for 8 weeks followed by 2 weeks of treatment with either vehicle, atrasentan (ETA receptor antagonist, 10mg/kg/day), or bosentan (ETA/ETB receptor antagonist, 100mg/kg/day). Atrasentan and bosentan lowered circulating non-esterified free fatty acids and triglycerides seen in HFD mice, while atrasentan-treated mice had significantly lower liver triglycerides compared to non-treated HFD mice. ET-1 receptor blockade significantly improved insulin tolerance compared to insulin resistant HFD mice and lowered expression of genes in epididymal white adipose tissue (eWAT) associated with insulin resistance and inflammation. Flow cytometric analyses of eWAT indicated that HFD mice had significantly higher percentages of both CD4+ and CD8+ T cells compared to NMD mice, which was attenuated by treatment with atrasentan or bosentan. Atrasentan treatment also abolished the decrease in eosinophils seen in HFD mice. Taken together, these data indicate that ETA and ETA/ETB receptor blockade improves peripheral glucose homeostasis, dyslipidemia, and liver triglycerides, and also attenuates the proinflammatory immune profile in eWAT of mice fed a HFD. These data suggest a potential use for ETA and ETA/ETB receptor blockers in the treatment of obesity-associated dyslipidemia and insulin resistance.

Addition of Endothelin A-Receptor Blockade Spoils the Beneficial Effect of Combined Renin-Angiotensin and Soluble Epoxide Hydrolase Inhibition: Studies on the Course of Chronic Kidney Disease in 5/6 Nephrectomized Ren-2 Transgenic Hypertensive Rats

Introduction: Previous studies in Ren-2 transgenic hypertensive rats (TGR) after 5/6 renal ablation (5/6 NX) have shown that besides pharmacological blockade of the renin-angiotensin system (RAS) also increasing kidney tissue epoxyeicosatrienoic acids (EET) levels by blocking soluble epoxide hydrolase (sEH), an enzyme responsible for degradation of EETs, and endothelin type A (ETA) receptor blockade retards chronic kidney disease (CKD) progression. This prompted us to evaluate if this progression will be alleviated by the addition of sEH inhibitor and ETA receptor antagonist to the standard complex blockade of RAS (angiotensin-converting enzyme inhibitor plus angiotensin II type 1 receptor blocker) in rats with established CKD. Methods: The treatment regimens were initiated 6 weeks after 5/6 NX in TGR, and the follow-up period was 60 weeks. Results: The addition of sEH inhibition to RAS blockade improved survival rate, further reduced albuminuria and renal glomerular and kidney tubulointerstitial injury, and attenuated the decline in creatinine clearance – all this as compared with 5/6 NX TGR treated with RAS blockade alone. Addition of ETA receptor antagonist to the combined RAS and sEH blockade not only offered no additional renoprotection but, surprisingly, also abolished the beneficial effects of adding sEH inhibitor to the RAS blockade. Conclusion: These data indicate that pharmacological strategies that combine the blockade of RAS and sEH could be a novel tool to combat the progression of CKD. Any attempts to further extend this therapeutic regimen should be made with extreme caution.

Novel treatment avenues for uterine leiomyoma: a new implication for endothelin?

Cysteine-rich angiogenic inducer 61 (CYR61), an angiogenic factor whose expression is decreased in fibroids. The aim of the present study was to determine if CYR61 secretion in smooth muscle cells (SMCs) is regulated by hypoxia and through the endothelin A (ETA) receptor. SMCs from fibroids (fSMC) and the adjacent myometrium smooth muscle cells (mSMCs) were extracted from ten women undergoing hysterectomy for uterine fibroids and cultured with or without 1.0 µM of an ETA receptor antagonist for 24 h under either normal or hypoxic oxygen conditions. Cellular secretion of endothelin-1 (ET-1) and CYR61 were measured via enzyme linked immunosorbent assay in the cell culture media. SMCs were collected to determine cell proliferation and CYR61 protein expression via Western blot. ET-1 secretion was significantly increased in fSMC and was decreased with blockade of the ETA receptor under both normoxia (P=0.0004) and hypoxia (P=0.008). CYR61 expression was decreased in fSMCs and significantly increased with blockade of the ETA receptor under hypoxia (P=0.04). Cell proliferation decreased with ETA blockade under normoxia (P=0.0001) and hypoxia (P=0.001). These results suggest that suppression of CYR61 secretion in fSMC is regulated by the ET-1 and that blockade with ETA could be considered for a future treatment option.

Endothelin A Receptor Blockade Improves Endothelium-Dependent Relaxation in Obese Woman

Hypertension in obesity is associated with increased insulin resistance, vascular mass and body mass index (BMI). The purpose of the study was to visualize endothelin-1 (ET-1) mediated constriction in arteries isolated from subcutaneous adipose tissue from obese hypertensive women previously operated by gastric bypass. Functional studies were conducted in a microvascular myograph. Expressed as percentage of contraction elicited by 124 mM KCl concentration-response curves for ET-1 were shifted leftward in arteries from obese hypertensive patients compared to healthy normotensive subjects. The vasodilator response to the ET-1 antagonist BQ123 (1 µM) was significantly higher in arteries from obese hypertensive patients (p<0.001). BQ123 induced relaxation was inhibited by NO synthase inhibitor L-NAME (0.1 nM). Preincubation with BQ123 enhanced the relaxation induced by acetylcholine (ACh; 0.1 nM – 0.1 mM) (p<0.001), but not that induced by NO donor sodium nitroprusside (SNP; 0.1 nM – 0.1 mM), in arteries from obese hypertensive patients. The present study show that hypertension yet prevail after gastric bypass surgery and the ETA receptor antagonist BQ123 may be a useful tool in reducing blood pressure in obese hypertensive patients.

Pharmacological Modulation of Mucosa-Related Impairment of β-Adrenoceptor-Mediated Relaxation in Human Detrusor

Objectives: The mucosa of human detrusor strips impairs catecholamine-induced relaxation. In order to elucidate which signal transduction pathways are involved in this cross talk between the mucosa and detrusor, we have studied the effects of several pharmacological agonists and antagonists on noradrenaline-mediated relaxation in intact and mucosa-denuded detrusor strips. Patients and Methods: Strips of detrusor tissue were obtained from patients who had undergone cystectomy for bladder cancer and were set up for force measurement. KCl- or carbachol-precontracted strips were relaxed with increasing concentrations of noradrenaline in the absence and in the presence of nitric oxide synthase inhibitor, L-NAME; P2X-receptor antagonist, PPADS; ETA-receptor antagonist, BQ-123; ETB-receptor antagonist, BQ-788; cyclooxygenase inhibitor, diclofenac; AT1-receptor antagonist, candesartan; and NK1-receptor antagonist, L-703,606. Results: In intact strips, KCl-stimulated force was enhanced by all blockers; carbachol-stimulated force increased with L-703,606. In denuded strips, only L-NAME augmented the KCl-stimulated contraction. Noradrenaline relaxed the precontracted detrusor strips to a significantly larger extent and at lower concentrations in denuded than in intact strips. L-NAME, PPADS and BQ-123/BQ-788 had little effect on noradrenaline-induced relaxation, whereas diclofenac, candesartan and L-703,606 sensitized intact carbachol-stimulated detrusor strips to noradrenaline-induced relaxation. Conclusion: Inhibition of the noradrenaline-induced relaxation of precontracted human detrusor strips by the mucosa is attenuated by diclofenac, candesartan and L-703,606 suggesting the involvement of prostanoids, angiotensin and neurokinin pathways. Further experiments are required to unravel the exact mechanisms.

Abstract 386: Circulating Endothelial Microparticles, Elevated Blood Pressure and Endothelin-1 Mediated Vasoconstrictor Tone

Clinical interest in circulating microparticles originating from both endothelial cells and platelets has increased due to their putative role in inflammation, endovascular function, angiogenesis and thrombosis. Elevated blood pressure is associated with profound endothelial dysfunction, particularly enhanced endothelin (ET)-1-mediated vasoconstrictor tone. There is some evidence to suggest that circulating microparticles are influenced by blood pressure and may contribute to associated vascular abnormalities. As part of an ongoing study, we are determining: 1) whether circulating endothelial (EMP) and platelet (PMP) microparticles are higher in adults with elevated blood pressure (SBP >130 mmHg) and if so; 2) if these microparticles are associated with ET-1 mediated vasoconstriction. To date, 22 sedentary, non-obese middle-aged adults have been studied: 11 normotensive (age: 55+2 yr; 7 M/4 F; BP: 118/74+3/2 mm Hg) and 11 prehypertensive/hypertensive (age: 55+2 yr; 7 M/4 F; BP: 140/85+2/3 mm Hg). All subjects were free of overt cardiometabolic disease. EMPs and PMPs were measured in platelet-poor plasma by flow cytometry. EMPs were defined as CD31+/CD42b- events and PMPs were defined as CD31+/CD42+ events. Forearm blood flow (FBF: plethysmography) responses to intra-arterial infusion of BQ-123 (100 nmol/min; for 60 min), a selective ETA receptor antagonist. EMPs were ~70% higher (p<0.01) in the prehypertensive/hypertensive (39072+3951 MP/μL) compared with normotensive (22726+2552 MP/μL). There was no difference in EMPs with the elevated blood pressure group between the prehypertensive (n=5) and hypertensive (n=6) adults. PMPs were not significantly different between the groups (503+132 vs 431+80 MP/μL). Resting FBF increased ~40% (p<0.01) in response to BQ-123 in the prehypertensive/hypertensive group only. EMPs were significantly correlated with systolic blood pressure (r=0.68) and peak FBF response to ETA receptor blockade (r=0.61). These initial results indicate that circulating EMPs, but not PMPs, are elevated in prehypertensive/hypertensive adults. Moreover, circulating EMPs are associated with systolic blood pressure and enhanced ET-1 mediated vasoconstrictor tone.