Quercetin attenuates reduced uterine perfusion pressure -induced hypertension in pregnant rats through regulation of endothelin-1 and endothelin-1 type A receptor.

BACKGROUND: Quercetin was reported to be crucial for a broad range of activities, including attenuating inflammation, platelet aggregation, capillary permeability, and lipid peroxidation. However, the effect of quercetin in hypertension during pregnancy, was not fully understood. METHODS: The model of hypertension in pregnancy was established in rats by reduced uterine perfusion pressure (RUPP). Quercetin was administrated by gavage. Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured using the CODA 6 BP system. Plasma concentrations of Endothelin-1 (ET-1), soluble fms-like tyrosine kinase-1 (sFlt-1), and vascular endothelial growth factor (VEGF) were detected using enzyme-linked immunosorbent assay kits. The mRNA and protein levels of ET-1 and endothelin-1 type A receptor (ETAR) were determined by RT-PCR and Western blotting. The ETAR antagonist BQ-123 was performed by osmotic minipumps. RESULTS: In RUPP induced rats, quercetin treatment decreased SBP and DBP, fetal resorptions percentage, plasma ET-1 and sFlt-1 concentrations, ET-1 and ETAR levels, but increased fetal body weight and VEGF expression. BQ-123 administration attenuated SBP and DBP, suppressed fatal resorptions percentage, and increased fetal body weight of RUPP rats. CONCLUSION: Quercetin attenuates RUPP induced hypertension in pregnant rats through the regulation of ET-1 and ETAR.

Autoantibodies Directed Against the Endothelin A Receptor in Patients With Benign Prostatic Hyperplasia.

BACKGROUND: Over-stimulation of G-protein coupled receptors (GPCRs) such as α1-adrenergic, muscarinic, endothelin, and AT1 receptors is considered to drive benign prostatic hyperplasia (BHP) which is often associated with lower urinary tract syndrome (LUTS). However, in addition to physiologic GPCR ligands, there is a new class of autoantibodies called functional autoantibodies that target the same GPCRs (GPCR-AABs) for over-stimulation, thus, presenting pathogenic potency. We hypothesize that patients with BPH/LUTS could carry GPCR-AABs representing potential targets for treatment. METHODS: GPCR-AABs were identified, quantified, and characterized in the serum from 20 patients (aged 55-82 years, median 71 years) with BPH using the bioassay of spontaneously beating cultured neonatal rat cardiomyocytes. RESULTS: A sum of 60% of the patients were positive for agonistic autoantibodies directed against the endothelin A receptor (ETA-AABs). ETA-AABs were associated with the IgG 1 subclass, targeted an epitope located on the second extracellular receptor loop and their agonistic activity could be neutralized by the aptamer BC007. CONCLUSIONS: Agonistic ETA-AABs could-via uncontrolled over-boarding endothelin A receptor stimulation-contribute to the pathogenesis of BPH/LUTS. The in vitro demonstrated ETA-AAB neutralization by the aptamer BC007 could open the door for a new treatment strategy in patients with BPH/LUTS. Prostate 77:458-465, 2017. © 2016 Wiley Periodicals, Inc.

Endothelin-1 receptor blockade as new possible therapeutic approach in multiple myeloma.

New effective treatments are needed to improve outcomes for multiple myeloma (MM) patients. Receptors with restricted expression on plasma cells (PCs) represent attractive new therapeutic targets. The endothelin-1 (EDN1) axis, consisting of EDN1 acting through EDN-receptor A (EDNRA) and B (EDNRB), was previously shown to be overexpressed in several tumours, including MM. However, there is incomplete understanding of how EDN1 axis regulates MM growth and response to therapy. Besides EDNRA, the majority of MM cell lines and primary malignant PCs express high levels of EDNRB and release EDN1. Similarly, bone-marrow microenvironment cells also secrete EDN1. Investigating the extent of epigenetic dysregulation of EDNRB gene in MM, we found that hypermethylation of EDNRB promoter and subsequent down-regulation of EDNRB gene was observed in PCs or B lymphocytes from healthy donors compared to EDNRB-expressing malignant PCs. Pharmacological blockade with the dual EDN1 receptor antagonist bosentan decreased cell viability and MAPK activation of U266 and RPMI-8226 cells. Interestingly, the combination of bosentan and the proteasome inhibitor bortezomib, currently approved for MM treatment, resulted in synergistic cytotoxic effects. Overall, our data has uncovered EDN1-mediated autocrine and paracrine mechanisms that regulate malignant PCs growth and drug response, and support EDN1 receptors as new therapeutic targets in MM.

A Mediterranean diet supplemented with extra virgin olive oil or nuts improves endothelial markers involved in blood pressure control in hypertensive women.

PURPOSE: Serum nitric oxide (NO) reduction and increased endothelin-1 (ET-1) play a pivotal role in endothelial dysfunction and hypertension. Considering that traditional Mediterranean diet (TMD) reduces blood pressure (BP), the aim of this study was to analyze whether TMD induced changes on endothelial physiology elements such as NO, ET-1 and ET-1 receptors which are involved in BP control. METHODS: Non-smoking women with moderate hypertension were submitted for 1 year to interventions promoting adherence to the TMD, one supplemented with extra virgin olive oil (EVOO) and the other with nuts versus a control low-fat diet (30 participants/group). BP, NO, ET-1 and related gene expression as well as oxidative stress biomarkers were measured. RESULTS: Serum NO and systolic BP (SBP) or diastolic BP (DBP) were negatively associated at baseline, as well as between NO and ET-1. Our findings also showed a DBP reduction with both interventions. A negative correlation was observed between changes in NO metabolites concentration and SBP or DBP after the intervention with TMD + EVOO (p = 0.033 and p = 0.044, respectively). SBP reduction was related to an impairment of serum ET-1 concentrations after the intervention with TMD + nuts (p = 0.008). We also observed changes in eNOS, caveolin 2 and ET-1 receptors gene expression which are related to NO metabolites levels and BP. CONCLUSIONS: The changes in NO and ET-1 as well as ET-1 receptors gene expression explain, at least partially, the effect of EVOO or nuts on lowering BP among hypertensive women.

Bosentan improves exercise capacity in adolescents and adults after Fontan operation: the TEMPO (Treatment With Endothelin Receptor Antagonist in Fontan Patients, a Randomized, Placebo-Controlled, Double-Blind Study Measuring Peak Oxygen Consumption) study.

BACKGROUND: The Fontan procedure has improved survival in children with functionally univentricular hearts. With time, however, complications such as reduced exercise capacity are seen more frequently. Exercise intolerance is multifactorial, but pulmonary vascular resistance probably plays a crucial role. Elevated pulmonary vascular resistance has been associated with raised levels of endothelin-1, which are common both before and after Fontan operations. Treatment with endothelin-1 receptor antagonists could theoretically improve cardiopulmonary hemodynamics and exercise capacity. The aim of this study was therefore to examine the efficacy and safety of bosentan in Fontan patients. METHODS AND RESULTS: Seventy-five adolescents and adults were randomized 1:1 to 14 weeks of treatment with bosentan or placebo. Cardiopulmonary exercise test, functional class, blood samples, and quality-of-life questionnaires were evaluated at baseline and at the end of treatment. Sixty-nine patients (92%) completed the study. Peak oxygen consumption increased 2.0 mL·kg(-1)·min(-1) (from 28.7 to 30.7 mL·kg(-1)·min(-1)) in the bosentan group compared with 0.6 mL·kg(-1)·min(-1) (from 28.4 to 29.0 mL·kg(-1)·min(-1)) in the placebo group (P=0.02). Cardiopulmonary exercise test time increased by 0.48 minute (from 6.79 to 7.27 minutes) versus 0.08 minute (from 6.94 to 7.02 minutes; P=0.04). Nine bosentan-treated patients improved 1 functional class, whereas none improved in the placebo group (P=0.0085). Side effects were mild and occurred equally in both groups. No serious adverse effects were seen, and no patients had liver enzyme levels above the 3-fold upper limit. CONCLUSIONS: Bosentan improves exercise capacity, exercise time, and functional class in Fontan patients without serious adverse events or hepatotoxicity. CLINICAL TRIAL REGISTRATION URL: http://www.clinicaltrials.gov. Unique identifier: NCT01292551.

Anti-ETAR and suPAR as markers of disease activity in renal ANCA-associated vasculitis.

PURPOSE: In anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), there is a lack of reliable biomarkers of disease activity. The aim of the study was to evaluate soluble urokinase plasminogen activator receptor (suPAR) and anti-endothelin-1 type A receptor (anti-ETAR) antibodies levels in active phase and remission of AAV. PATIENTS AND METHODS: We enrolled 60 patients (median age 63.0 years) with renal AAV into this study. Plasma suPAR, urine suPAR (expressed as urine suPAR/creatinine ratio) and serum anti-ETAR antibodies were assayed by ELISA. Disease activity was assessed using Birmingham Vasculitis Activity Score (BVAS) and patients were divided into 2 subgroups based on their BVAS scores, namely: active AAV subgroup (BVAS≥1) and remission subgroup (BVAS â€‹= â€‹0). Median follow-up was 12 months. RESULTS: Patients with active AAV had higher levels of all candidate biomarkers in comparison to those in remission (p â€‹< â€‹0.05). C-statistics for plasma suPAR, urine suPAR/creatinine ratio and serum anti-ETAR were 0.807, 0.713 and 0.783, respectively. In multivariable analysis, no clear associations were found between serum anti-ETAR and BVAS, while both plasma suPAR and serum anti-ETAR were independently influenced by estimated glomerular filtration rate (eGFR). CONCLUSIONS: Plasma suPAR better discriminated between active AAV and remission in comparison to urine suPAR/creatinine ratio and serum anti-ETAR antibodies.

Antibodies Against Angiotensin II Receptor Type 1 and Endothelin A Receptor Are Associated With an Unfavorable COVID19 Disease Course.

Background: Lung histopathology demonstrates vasculopathy in a subset of deceased COVID19 patients, which resembles histopathology observed in antibody-mediated lung transplant rejection. Autoantibodies against angiotensin II type 1 receptor (AT1R) and Endothelin receptor Type A (ETAR) have been demonstrated in antibody-mediated rejection and may also be associated with severe COVID19 infection. Objective To assess AT1R and ETAR auto-antibodies in COVID19 patients and controls, and explore their association with disease course. Methods: 65 hospitalized patients with COVID19 infection were included. Clinical and laboratory findings were retrospectively assessed. Patients with unfavorable disease course, admitted at the intensive care unit and/or deceased during hospital admission (n=33) were compared to admitted COVID19 patients with favorable disease course (n=32). The presence of antinuclear antibodies (ANA) and auto-antibodies against AT1R or ETAR in peripheral blood were compared between COVID19 with unfavorable and favorable disease course and age matched controls (n=20). Results: The presence of ANA was not significantly different between COVID19 patients with unfavorable (n=7/33; 21%) and favorable disease course (n=6/32; 19%) (p= 0.804) and controls (n=3/20; 15%). Auto-antibodies against AT1R were significantly increased in unfavorable disease course (median 14.59 U/mL, IQR 11.28 - 19.89) compared to favorable disease course (median 10.67 U/mL, IQR 8.55 - 13.0, p< 0.01). ETAR antibody titers were also significantly increased in unfavorable disease course (median 7.21, IQR 5.0 - 10.45) as compared to favorable disease course (median 4.0, IQR 3.0 - 6.0, p <0.05). Conclusion: Auto-antibodies against AT1R and ETAR are significantly increased in COVID19 patients with an unfavorable disease course.

Endothelin A and B Receptors: Potential Targets for Microcirculatory-Mitochondrial Therapy in Experimental Sepsis.

The hypoxia-sensitive endothelin (ET) system plays an important role in circulatory regulation through vasoconstrictor ETA and ETB2 and vasodilator ETB1 receptors. Sepsis progression is associated with microcirculatory and mitochondrial disturbances along with tissue hypoxia. Our aim was to investigate the consequences of treatments with the ETA receptor (ETA-R) antagonist, ETB1 receptor (ETB1-R) agonist, or their combination on oxygen dynamics, mesenteric microcirculation, and mitochondrial respiration in a rodent model of sepsis. Sprague Dawley rats were subjected to fecal peritonitis (0.6 g kg i.p.) or a sham operation. Septic animals were treated with saline or the ETA-R antagonist ETR-p1/fl peptide (100 nmol kg i.v.), the ETB1-R agonist IRL-1620 (0.55 nmol kg i.v.), or a combination therapy 22 h after induction. Invasive hemodynamic monitoring and blood gas analysis were performed during a 90-min observation, plasma ET-1 levels were determined, and intestinal capillary perfusion (CPR) was detected by intravital videomicroscopy. Mitochondrial Complex I (CI)- and CII-linked oxidative phosphorylation (OXPHOS) was evaluated by high-resolution respirometry in liver biopsies. Septic animals were hypotensive with elevated plasma ET-1. The ileal CPR, oxygen extraction (ExO2), and CI-CII-linked OXPHOS capacities decreased. ETR-p1/fl treatment increased ExO2 (by >45%), CPR, and CII-linked OXPHOS capacity. The administration of IRL-1620 countervailed the sepsis-induced hypotension (by >30%), normalized ExO2, and increased CPR. The combined ETA-R antagonist-ETB1-R agonist therapy reduced the plasma ET-1 level, significantly improved the intestinal microcirculation (by >41%), and reversed mitochondrial dysfunction. The additive effects of a combined ETA-R-ETB1-R-targeted therapy may offer a tool for a novel microcirculatory and mitochondrial resuscitation strategy in experimental sepsis.

Association of endothelin receptor type A rs5333 gene polymorphism with steroid response in Egyptian children with idiopathic nephrotic syndrome.

AIM: To investigate ENDRA rs5333 gene polymorphism distribution in idiopathic nephrotic syndrome (INS) and to analyze their association with response to steroid therapy, and biochemical markers of INS. SUBJECTS & METHODS: The PCR-restriction fragment length polymorphism was used to analyze ENDRA rs5333 polymorphism in 100 children with idiopathic nephrotic syndrom (INS) and 100 healthy children. Plasma endothelin-1 were measured by ELISA. RESULTS: The ENDRA rs5333 gene polymorphism was not associated with risk of INS. The frequency of minor allele (C) was significantly higher in the steroid resistant nephrotic syndrome group than the steroid sensitive group. The CC and TC mutant variants were associated with higher plasma levels of cholesterol, albumin, urea and 24-h urinary protein, but were not associated with risk of hypertension. The endothelin-1 plasma level was higher in INS than control and in steroid resistant nephrotic syndrome group when compared with steroid sensitive group cases. CONCLUSION: The ENDRA rs5333 gene polymorphism may be associated with genetic predisposition to steroid resistance in INS Egyptian children.

Common genetic variation in the type A endothelin-1 receptor is associated with ambulatory blood pressure: a family study.

The endothelins are among the most potent vasoconstrictors known. Pharmacological inhibition of endothelin receptors lowers blood pressure (BP). It is unknown whether naturally occurring genetic variation in the endothelin receptors influences BP. We have evaluated the type A endothelin receptor (EDNRA) as a candidate gene for hypertension in a large family study. A total of 1425 members of 248 families selected via a proband with hypertension were studied. Ambulatory BP monitoring was conducted using the A&D TM2421 device. Four haplotype-tagging single nucleotide polymorphisms (SNPs) spanning the EDNRA gene were typed. There was evidence of association between genotype at the rs5335 (C+70G) SNP and night systolic blood pressure (+1.24% (s.e. 0.64) per G allele; P=0.05); night diastolic blood pressure (+1.64% (s.e. 0.71) per G allele; P=0.021) and night mean BP (+1.51% (s.e. 0.64) per G allele; P=0.017). Borderline significant trends in the same direction were seen for daytime BPs. Proportions of hypertensives in each of the three genotype groups were C/C 34.7%, C/G 37.9%, G/G 42.4% yielding an odds ratio for hypertension per G allele of 1.19 (95% confidence interval 1.00-1.41; P=0.05). In conclusion, the rs5335 (C+70G) polymorphism of the EDNRA gene has small effects on the risk of hypertension. Natural variation in other genes in the endothelin-signalling pathway should be explored to identify additional influences on BP regulation.

Involvement of Central Endothelin ETA and Cannabinoid CB1 Receptors and Arginine Vasopressin Release in Sepsis Induced by Cecal Ligation and Puncture in Rats.

We previously reported that endothelin-1 (ET-1) reduced the frequency of spontaneous excitatory currents in vasopressinergic magnocellular cells through the activation of endothelin ETA receptors in rat brain slices. This effect was abolished by a cannabinoid CB1 receptor antagonist, suggesting the involvement of endocannabinoids. The present study investigated whether the blockade of ETA or CB1 receptors during the phase of increased levels of ET-1 after severe sepsis increases the survival rate of animals concomitantly with an increase in plasma arginine vasopressin (AVP) levels. Sepsis was induced in male Wistar rats by cecal ligation and puncture (CLP). Treatment with the CB1 receptor antagonist rimonabant (Rim; 10 and 20 mg/kg, orally) 4 h after CLP (three punctures) significantly increased the survival rate compared with the CLP per vehicle group. Intracerebroventricular treatment with the ETA receptor antagonist BQ123 (100 pmol) or with Rim (2 µg) 4 and 8 h after CLP but not the ETB receptor antagonist BQ788 (100 pmol), also significantly improved the survival rate. Sham-operated and CLP animals that were treated with Rim had significantly lower core temperature than CLP animals. However, oral treatment with Rim did not change bacterial count in the peritoneal exudate, neutrophil migration to the peritoneal cavity, leucopenia or increased plasma interleukin-6 levels induced by CLP. Both Rim and BQ123 also increased AVP levels 12 h after CLP. The blockade of central CB1 and ETA receptors in the late phase of sepsis increased the survival rate, reduced body temperature and increased the circulating AVP levels.

Tezosentan reduces the microvascular filtration coefficient in isolated lungs from rats subjected to cecum ligation and puncture.

INTRODUCTION: We recently demonstrated that the non-selective endothelin-1 (ET-1) receptor blocker tezosentan antagonizes ovine acute lung injury (ALI) following infusion of endotoxin or ET-1 by reducing the enhanced lung microvascular pressure, although we could not exclude the possibility of a simultaneous decline in microvascular permeability. In the present study, our aim was to find out if tezosentan reverses the rise in microvascular filtration coefficient (Kfc) in rat lungs that have been isolated and perfused 12 h after cecum ligation and puncture (CLP) or infusion of ET-1. METHODS: Wistar rats (n = 42) were subjected to CLP. Postoperatively, rats were randomized to a CLP group (n = 7) and a CLP + tezosentan group (n = 7); the latter received tezosentan 30 mg/kg. A sham-operated group (n = 5) underwent laparotomy without CLP. Twelve hours postoperatively, the lungs were isolated and perfused with blood from similarly treated rats that also were used to assess plasma concentration of ET-1 and protein kinase Calpha (PKCalpha) in lung tissue. Additionally, isolated blood perfused lungs from healthy rats were randomized to a control group (n = 8), an ET-1 group (n = 7) subjected to pulmonary arterial injection of ET-1 10 nM, and an ET-1 + tezosentan group (n = 7) that received tezosentan 30 mg/kg. All lung preparations received papaverine 0.1 microg/kg added to the perfusate for vasoplegia. Pulmonary hemodynamic variables, Kfc and lung compliance (CL) were assessed. RESULTS: After CLP, the plasma concentration of ET-1 increased. Papaverine abolished the vasoconstrictor response to ET-1 and the pulmonary vascular pressures remained close to baseline throughout the experiments. Both CLP and injection of ET-1 caused significant changes in Kfc and CL that were prevented in tezosentan-treated rats. Compared to sham-operated animals, CLP increased the content of PKCalpha by 50% and 70% in the cytosolic and the membrane fractions of lung tissue homogenates, respectively. Tezosentan prevented the upregulation of PKCalpha in the membrane fraction. CONCLUSION: In rat lungs isolated and perfused after CLP, tezosentan precludes both the increase in Kfc and the upregulation of PKCalpha in the membrane fraction of lung tissue.

Anti-ETAR and anti-AT1R autoantibodies are elevated in patients with endstage cystic fibrosis.

Autoantibodies against endothelin-1 type A receptor (ETAR) are present in systemic sclerosis complicated by lung fibrosis and pulmonary hypertension. As increased serum levels and local overproduction of endothelin-1 in the airways are reported in cystic fibrosis (CF) patients, we reasoned that anti-ETAR antibodies could be prevalent in endstage CF patients prior to lung transplantation (LTx). Also, ETAR autoantibodies are frequently associated with autoantibodies against the angiotensin II type 1 receptor (AT1R). We analyzed the presence of anti-ETAR and anti-AT1R autoantibodies in 43 LTx patients (chronic obstructive pulmonary disease (COPD), n=20; CF, n=13; interstitial lung disease (ILD), n=1). We observed overall higher anti-ETAR and anti-AT1R autoantibody titers in sera taken prior to LTx in the CF patient group as compared to COPD. No difference was found in autoantibody levels between patients with CF versus ILD. In sera taken post-LTx we found the same difference in anti-ETAR and anti-AT1R autoantibody titers between patients with CF versus COPD. No difference was found in antibody titers between sera taken prior to or 6 months after LTx. There was no association between autoantibody levels and other relevant demographic parameters, and we found no association between autoantibody titers and the development of the bronchiolitis obliterans syndrome. Both autoantibody titers were strongly correlated. We hypothesize that due to prolonged exposure to bacterial infection, increased levels of AT1R and ETAR result in a deregulated immune response causing autoantibody formation. Further research is expedient to elucidate the occurrence of autoantibodies against ETAR and AT1R and their role in disease progression.

Elevation of serum endothelins and cardiotoxicity induced by particulate matter (PM2.5) in rats with acute myocardial infarction.

Epidemiological studies have defined a significant positive association of acute exposure to ambient concentrations of particulate matter (PM) with increased daily mortality and hospital admission for cardiovascular diseases. Experimental studies have shown that animals with pre-existing cardiovascular diseases are more susceptible to the cardiac effect of PM exposure. The present study was undertaken to investigate possible involvement of upregulation of the endothelin system in PM exposure-induced cardiotoxicity in rats with acute myocardial infarction (MI). Adult male Sprague Dawley rats were subjected to occlusion of the left coronary artery and displayed myocardial infarction 12 h after the surgery. The heart rate significantly decreased and premature ventricular complexes of the electrocardiogram occurred in the myocardial infarct animals. Exposure to PM(2.5) via intratracheal instillation with 2.0 mg in 0.3 mL normal saline significantly worsened the ventricular arrhythmia along with a further decrease in heart rate. The same PM exposure only caused slight cardiac changes in the sham-operated animals. Serum total endothelin concentrations were significantly elevated in both myocardial infarct rats and shamoperated controls in response to PM exposure. However, increased numbers of the endothelin receptor type A on the cardiomyocytes were observed only in the infarct myocardium. This study thus suggests that upregulation of the endothelin system in rats with MI is likely involved in the PM exposure-induced cardiotoxicity.

Pillar cell and erythrocyte localization of fugu ET(A) receptor and its implication.

Endothelin, a vasoconstrictor peptide, plays important roles not only in the mammalian circulatory system but also in non-mammalian systems, such as the gill lamellar vascular network with complex structural characteristics. Here, we show that (i) the contraction of pillar cells that delimit the lamellar vasculature is controlled by endothelin through the type A endothelin receptor (ET(A)) linked to the intracellular calcium signaling system and (ii) ET(A) receptor is also highly expressed on fugu erythrocytes, a hitherto unexpected finding. Database mining revealed the presence of five endothelin receptor (ETR) sequences in the fugu genome. By Northern blotting, cDNA cloning, and fura-2 monitoring, the branchial ETR subtype was shown to be ET(A) able to induce a Ca(2+) transit. Immunohistochemistry revealed its pillar cell and erythrocyte localization. These results suggest an endothelin/ET(A)-mediated coordinated regulation of the pillar cell shape and erythrocyte membrane flexibility.