Endothelin Receptor Blockade Research Articles

Abstract P335: Greater Reduction in Sympathetic Tone following ET B Receptor Blockade in Rats Lacking the Clock Gene Bmal1 during High Salt Diet

The absence of diurnal oscillations in blood pressure is associated with increased cardiovascular morbidity and mortality. The clock gene Bmal1 plays important roles in diurnal cardiovascular control as mice lacking Bmal1 have lower blood pressure and lack a diurnal rhythm. Our lab has previously reported a global Bmal1 knockout rat model that lacks a night-day difference in sodium excretion. Due to the importance of endothelin signaling in sodium homeostasis and autonomic tone, we sought to characterize the hemodynamic and autonomic responses of our Bmal1 knockout (KO) rat to high salt diet and endothelin receptor blockade. Male rats homozygous for the Bmal1 mutation (KO, n = 4) and wild type (WT, n = 7) littermate controls were implanted with telemetry transmitters to record blood pressure. After a recovery period of at least one week, the rats were placed on 7 days each of normal salt (0.49% NaCl) diet, high salt (4.0% NaCl) diet, followed by high salt diet containing the specific ET B receptor antagonist A192621 (10 mg/kg/day, p.o.). Rats were placed in metabolic cages for the last three days of each diet. Surprisingly, KO rats had a similar night-day difference in mean arterial pressure (MAP) as WT during normal salt diet (6.3 ± 0.4 vs. 6.9 ± 0.9 mmHg; respectively), high salt diet (7.1 ± 0.1 vs. 5.4 ± 0.9 mmHg; respectively), and high salt + A192621 (5.4 ± 0.4 vs. 4.8 ± 1.1 mmHg; respectively). KO and WT rats had similar 24-hr MAP during normal salt diet (104.1 ± 3.3 vs. 107.3 ± 1.2 mmHg; respectively), high salt diet (113.8 ± 4.1 vs. 114.0 ± 1.4 mmHg; respectively), and high salt + A192621 (136.3 ± 8.6 vs. 133.4 ± 3.1 mmHg; respectively). Despite these similar blood pressure responses to high salt diet and ET B antagonism, KO rats had a significantly greater reduction in vasomotor sympathetic to parasympathetic tone compared to WT rats as demonstrated by low frequency to high frequency (LF/HF) analysis of diastolic blood pressure variability (-0.9 ± 0.3 vs. 0.1 ± 0.2 ΔLF/HF relative to normal salt; respectively; p = 0.01). These results indicate that lack of Bmal1 may result in greater ET B receptor mediated vasomotor sympathetic tone in rats fed a high salt diet and that factors other than Bmal1 may be influential in circadian control of blood pressure in rats.

Abstract P186: Selective ET A Receptor Antagonism versus Dual ET A /ET B Receptor Blockade for Preventing Angiogenesis Inhibitor-induced Hypertension

Angiogenesis inhibitors are a mainstay treatment for cancer. While effective in preventing tumor growth, angiogenesis inhibitors cause off-target effects including cardiovascular toxicity and renal injury, most likely via endothelin-1 (ET-1) upregulation. ET-1 via stimulation of the ET A receptor causes pro-hypertensive effects whereas stimulation of the ET B receptor can elicit both pro- or anti-hypertensive effects. In the present study, we hypothesized that selective ET A receptor blockade versus dual ET A /ET B blockade provides better cardiovascular protection from angiogenesis inhibitor-induced hypertension. Male WKY rats were treated with vehicle, sunitinib (VEGF inhibitor; 14 mg/kg/day) alone or in combination with macitentan (ET A/B receptor antagonist; 30 mg/kg/day) or sitaxentan (ET A receptor antagonist; 30 or 100 mg/kg/day) for 8 days. Mean arterial pressure (MAP) was measured via radiotelemetry at baseline and days 1-6 of treatment. Vasoreactivity to acetylcholine (ACh) and ET-1 was assessed in iliac vessels. Compared to vehicle treatment, sunitinib treatment caused a rapid and sustained increase in MAP (1±1 versus 23±2 mmHg on day 6 of treatment, respectively, P<0.001). Co-treatment with macitentan blunted the pressor response to sunitinib, such that on day 6 of treatment the increase in MAP was 7±4 mmHg (P<0.01 versus sunitinib-treated). Similar results were observed for co-treatment with 30 mg/kg/day of sitaxentan. Conversely, co-treatment with 100 mg/kg/day sitaxentan, which has been shown to induce a depressor response in normotensive Wistar rats, completely abolished sunitinib-induced hypertension, with MAP actually decreasing before returning to near baseline level (-1±1 mmHg on day 6 of treatment, P<0.001 versus sunitinib-treated). Compared to vehicle treatment, ET B receptor stimulation yielded a constrictor response after 8 days of sunitinib treatment, while the response to ACh was unaltered. Both macitentan and sitaxentan reversed the constrictor ET B receptor response. Our results support a key role for the ET-1 system in the development of sunitinib-induced hypertension and suggest that selective ET A receptor blockade is sufficient to block this effect.

P643Effect of moderate altitude on biomarkers of cardiovascular inflammation and endothelial function and their differential modulation by dual endothelin receptor blockade

P643Effect of moderate altitude on biomarkers of cardiovascular inflammation and endothelial function and their differential modulation by dual endothelin receptor blockade

Regular aerobic exercise reduces endothelin-1-mediated vasoconstrictor tone in overweight and obese adults.

What is the central question of this study? Does aerobic exercise training reduce endothelin-1 (ET-1)-mediated vasoconstrictor tone in overweight/obese adults? And, if so, does lower ET-1 vasoconstriction underlie the exercise-related enhancement in endothelium-dependent vasodilatation in overweight/obese adults? What is the main finding and its importance? Regular aerobic exercise reduces ET-1-mediated vasoconstrictor tone in previously sedentary overweight/obese adults, independent of weight loss. Decreased ET-1 vasoconstriction is an important mechanism underlying the aerobic exercise-induced improvement in endothelium-dependent vasodilator function in overweight/obese adults. Endothelin-1 (ET-1)-mediated vasoconstrictor tone is elevated in overweight and obese adults, contributing to vasomotor dysfunction and increased cardiovascular disease risk. Although the effects of habitual aerobic exercise on endothelium-dependent vasodilatation in overweight/obese adults have been studied, little is known regarding ET-1-mediated vasoconstriction. Accordingly, the aims of the present study were to determine the following: (i) whether regular aerobic exercise training reduces ET-1-mediated vasoconstrictor tone in overweight and obese adults; and, if so, (ii) whether the reduction in ET-1-mediated vasoconstriction contributes to exercise-induced improvement in endothelium-dependent vasodilatation in this population. Forearm blood flow (FBF) in response to intra-arterial infusion of selective ETA receptor blockade (BQ-123, 100nmolmin-1 for 60min), acetylcholine [4.0, 8.0 and 16.0μg(100mltissue)-1 min-1 ] in the absence and presence of ETA receptor blockade and sodium nitroprusside [1.0, 2.0 and 4.0μg(100mltissue)-1 min-1 ] were determined before and after a 3month aerobic exercise training intervention in 25 (16 men and nine women) overweight/obese (body mass index 30.1±0.5kgm-2 ) adults. The vasodilator response to BQ-123 was significantly lower (∼25%) and the FBF responses to acetylcholine were ∼35% higher after exercise training. Before the exercise intervention, the co-infusion of acetylcholine plus BQ-123 resulted in a greater vasodilator response than acetylcholine alone; however, after the exercise intervention the FBF response to acetylcholine was not significantly increased by ETA receptor blockade. These results demonstrate that regular aerobic exercise reduces ET-1-mediated vasoconstrictor tone in previously sedentary overweight and obese adults. Moreover, decreased ET-1-mediated vasoconstriction is an important mechanism underlying the aerobic exercise-induced improvement in endothelium-dependent vasodilator function in overweight/obese adults.

KL 30 Vascular mediators leading to hypertension in pregnancy

Preeclampsia is characterized by generalized endothelial dysfunction. This endothelial dysfunction underlies the hypertension and proteinuria as well as other complications in preeclampsia and is associated with an increased production of the extremely potent, endothelium-dependent vasoconstrictor endothelin-1 (ET-1) and a decrease in the production of the endothelium-dependent vasodilators nitric oxide and prostacyclin. Angiogenic imbalance, due to increased placental production of soluble fms-tyrosine kinase −1 (sFlt-1) leading to decreases in free plasma concentrations of the proangiogenic placenta growth factor and vascular endothelial growth factor (VEGF), is considered to be the main mediator of the endothelial dysfunction in preeclampsia. Recent studies in pregnant women with and without preeclampsia have shown that the degree of activation of the endothelin system is directly related to the increased levels of soluble fms-tyrosine kinase-1 (sFlt-1). In turn this activation of the endothelin has shown to underlie the suppression of plasma renin and aldosterone in preeclamptic compared to normal gestations. That the angiogenic imbalance is a mediator of the increase ET-1 levels in preeclampsia is supported by experimental studies and by studies in patients with cancer treated with VEGF-inhibitors aimed to impair angiogenesis and tumor growth. In these patients anti-angiogenic treatment is associated with increased ET-1 levels. In addition, in animals exposed to VEGF-inhibition the rise in blood pressure and proteinuria can almost completely be prevented by endothelin receptor blockade, proving that an activated endothelin system is instrumental for the rise in blood pressure and proteinuria. These observations may imply that preeclampsia is treatable with agents that interfere with placental sFlt-1 production, resulting in lower plasma ET-1 levels, as has recently been reported for proton pump inhibitors or alternatively with endothelin receptor blockers, providing that the latter agents are safe in the second and third trimester of pregnancy when organogenesis had been completed.

Abstract 1197: Endothelin-1 receptor/β-arrestin1 is an actionable node that regulates YAP/TAZ signaling and chemoresistance in high-grade ovarian cancer

Abstract The widespread activation of YAP and TAZ transcriptional regulators has been shown to promote tumor progression and resistance to anti-cancer therapies. Extensive studies have identified different upstream regulators of YAP/TAZ, including G-protein coupled receptor (GPCR). However how GPCR signals regulate YAP/TAZ puzzles have remained elusive. Among the GPCR, we reveal that the endothelin-1 (ET-1) receptor activates YAP/TAZ pathway in high-grade serous ovarian cancer (HG-SOC) sensitive and resistant cell lines and primary cultures. In HG-SOC cells, the aberrant activation of ET-1 receptor (ET-1R), promotes an enhanced nuclear shuttling of dephosphorylated YAP and TAZ in a time-dependent manner, through the scaffold protein β-arrestin1 (β-arr1). Interestingly, ET-1-induced YAP/TAZ nuclear accumulation is further improved in platinum-resistant cells and is prevented by expressing a mutant β-arr1 incapable of nuclear distribution. Moreover, the ET-1/ETAR axis activation induces the co-localization of β-arr1, YAP and TAZ in the nucleus. Mechanistically, in sensitive and to a greater extent in resistant cells, ET-1 induces the formation of a complex β-arr1/Trio, a Rho-Guanine nucleotide exchange factor family member, which dephosphorylates YAP, inducing its nuclear accumulation and the transcription of YAP/TAZ target genes. β-arr1, RhoA or Trio depletion, as well as the treatment with the dual ETAR/ETBR antagonist macitentan, significantly reduces RhoA GTPase activity and YAP nuclear translocation, suggesting that ET-1/ETAR axis regulates YAP/TAZ activity by driving β-arr1/Trio/RhoA pathway. According with these results, the ET-1-induced upregulation of YAP/TEAD target genes, such as CYR61, CTGF, ANKRD1 and EDN1, as well as TEAD transcriptional activity and cell invasion, are inhibited upon β-arr1, Trio, or YAP silencing, or macitentan treatment, demonstrating that β-arr1-mediated intersections are required for ET-1-induced YAP signaling. At the chromatin level, the activation of ET-1 axis promotes the recruitment of YAP, TEAD and β-arr1 on TEAD binding sites of CTGF, ANKRD1 and EDN1 promoters, an effect inhibited by macitentan treatment. These results indicate that β-arr1 can act as a transcription co-activator that bind TEAD thereby activating transcription of EDN1, which in turn, can sustain persistent YAP/TAZ activity through an autocrine loop. In murine orthotopic model of patient-derived xenograft (PDX) of HG-SOC, ET-1R blockade by macitentan, inhibits tumor growth, enhances the sensitivity to chemotherapy and reduces YAP and TEAD transcriptional activity. Altogether our results establish for the first time YAP/TAZ as critical downstream effectors of ET-1R/β-arr1 signaling, providing mechanistic insights that targeting ET-1R signaling overcomes YAP/TAZ driven platinum-based therapy failure in HG-SOC. Citation Format: Piera Tocci, Roberta Cianfrocca, Laura Rosanò, Rosanna Sestito, Valeriana Di Castro, Giovanni Blandino, Anna Bagnato. Endothelin-1 receptor/β-arrestin1 is an actionable node that regulates YAP/TAZ signaling and chemoresistance in high-grade ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1197. doi:10.1158/1538-7445.AM2017-1197

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.

ETB receptor contribution to vascular dysfunction in postmenopausal women.

Endothelin-1 (ET-1) contributes to age-related endothelial dysfunction in men via the ETA receptor. However, there are sex differences in the ET-1 system, and ETB receptors are modulated by sex hormones. The purpose of this study was to test the hypothesis that ETB receptors contribute to impaired vasodilatory function in postmenopausal women (PMW). We measured flow-mediated dilation (FMD) using ultrasound, and cutaneous nitric oxide-mediated vasodilation during local heating (42°C) via laser Doppler flowmetry in 18 young women (YW; 22 ± 1 yr) and 16 PMW (56 ± 1 yr). Cutaneous microdialysis perfusions of lactated Ringer (control), an ETB receptor antagonist (BQ-788, 300 nM), and an ETA receptor antagonist (BQ-123, 500 nM), were done through separate fibers, followed by perfusions of sodium nitroprusside (28 mM) and local heating to 43°C (max). Cutaneous vascular conductance (CVC) was calculated as cutaneous blood flow/mean arterial pressure and expressed as a percent of maximal dilation. FMD (YW: 7.5 ± 0.5 vs. PMW: 5.6 ± 0.6%) and cutaneous vasodilation (YW: 93 ± 2 vs. PMW: 83 ± 4%CVCmax) were lower in PMW (both P < 0.05). Blockade of ETB receptors decreased cutaneous vasodilation in YW (87 ± 2%CVCmax; P < 0.05 vs. control) but increased vasodilation in PMW (93 ± 1%CVCmax; P < 0.05 vs. control). ETA receptor blockade had minimal effect in YW (92 ± 1%CVCmax) but increased cutaneous vasodilation in PMW (91 ± 2%CVCmax; P < 0.05 vs. control). In conclusion, ETB receptors mediate vasodilation in YW, but this effect is lost after menopause. Impaired vasodilatory function in PMW is due in part to a loss of ETB-mediated dilation.

The endothelin B receptor plays a crucial role in the adhesion of neutrophils to the endothelium in sickle cell disease

Although the primary origin of sickle cell disease is a hemoglobin disorder, many types of cells contribute considerably to the pathophysiology of the disease. The adhesion of neutrophils to activated endothelium is critical in the pathophysiology of sickle cell disease and targeting neutrophils and their interactions with endothelium represents an important opportunity for the development of new therapeutics. We focused on endothelin-1, a mediator involved in neutrophil activation and recruitment in tissues, and investigated the involvement of the endothelin receptors in the interaction of neutrophils with endothelial cells. We used fluorescence intravital microscopy analyses of the microcirculation in sickle mice and quantitative microfluidic fluorescence microscopy of human blood. Both experiments on the mouse model and patients indicate that blocking endothelin receptors, particularly ETB receptor, strongly influences neutrophil recruitment under inflammatory conditions in sickle cell disease. We show that human neutrophils have functional ETB receptors with calcium signaling capability, leading to increased adhesion to the endothelium through effects on both endothelial cells and neutrophils. Intact ETB function was found to be required for tumor necrosis factor α-dependent upregulation of CD11b on neutrophils. Furthermore, we confirmed that human neutrophils synthesize endothelin-1, which may be involved in autocrine and paracrine pathophysiological actions. Thus, the endothelin-ETB axis should be considered as a cytokine-like potent pro-inflammatory pathway in sickle cell disease. Blockade of endothelin receptors, including ETB, may provide major benefits for preventing or treating vaso-occlusive crises in sickle cell patients.

Combined hydroxyurea and ETA receptor blockade on renal dysfunction in the humanized sickle mouse

Sickle cell disease (SCD) is associated with an increasing incidence of kidney disease, and the underlying causes have not been elucidated. Recent data has from our lab and others have shown that the vaso‐active peptide endothelin‐1 (ET‐1) plays a critical role in the pathophysiology of sickle cell nephropathy (SCN) and the blockade of its ETA receptor improves renal function and reduces renal injury. As hydroxyurea (HU) is a commonly prescribed drug for the treatment of SCD due to its ability to increase fetal hemoglobin, investigating how these two complex systems interact in the pathophysiology of SCN could provide insight into pathophysiologic mechanisms. Therefore, we hypothesized that combined ambrisentan (ETA selective antagonist) and HU treatment has a synergistic effect on renal injury in SCN when compared to HU treatment alone. Male 12 week old humanized sickle mice (Towne's mice; HbSS) and their genetic controls (HbAA) were treated with either vehicle (drinking water), HU (50mg/kg/day), or HU plus ambrisentan (10mg/kg/day). After 2 weeks of treatment, mice were placed in metabolic cages to assess renal function and then euthanized for blood and tissue collection. Vehicle treated HbSS mice exhibited significant proteinuria (4.38 ± 0.89 mg/day, p=0.0082), elevated plasma ET‐1 concentration (2.68 ± 0.44 pg/ml, p=0.01), and urinary ET‐1 excretion (1.43 ± 0.17pg/day, p=0.0042) as well as decreased urine osmolarity (1205 ± 63.3 mOsmol/kg, p&lt;0.0001) compared to HbAA controls. This renal injury phenotype was significantly attenuated in the HU treated animals (2.09 ± 0.28 mg/day, 0.881± 0.100 pg/ml, 0.336 ± 0.05 pg/day, 1686 ± 83.1mOsmol/kg, respectively; p&lt;0.05 compared to HbSS vehicle treated for all analyses); however, there was no additional improvement in HbSS mice treated with combined ambrisentan and HU. The absence of further attenuation of renal injury with combined treatment suggests that the mechanism of action for both treatments may converge on the same mechanistic pathway.

Beneficial Effects of Apelin on Vascular Function in Patients With Central Obesity.

Patients with central obesity have impaired insulin-stimulated vasodilation and increased ET-1 (endothelin 1) vasoconstriction, which may contribute to insulin resistance and vascular damage. Apelin enhances insulin sensitivity and glucose disposal but also acts as a nitric oxide (NO)-dependent vasodilator and a counter-regulator of AT1 (angiotensin [Ang] II type 1) receptor-induced vasoconstriction. We, therefore, examined the effects of exogenous (Pyr1)apelin on NO-mediated vasodilation and Ang II- or ET-1-dependent vasoconstrictor tone in obese patients. In the absence of hyperinsulinemia, forearm blood flow responses to graded doses of acetylcholine and sodium nitroprusside were not different during saline or apelin administration (both P>0.05). During intra-arterial infusion of regular insulin, however, apelin enhanced the vasodilation induced by both acetylcholine and nitroprusside (both P<0.05). Interestingly, the vasodilator effect of concurrent blockade of AT1 (telmisartan) and AT2 (PD 123,319) receptors was blunted by apelin (3±5% versus 32±9%; P<0.05). Similarly, during apelin administration, blockade of ETA receptors (BQ-123) resulted in lower vasodilator response than during saline (23±10% versus 65±12%; P<0.05). NO synthase inhibition by L-NMMA (l-N-monometylarginine) during the concurrent blockade of either Ang II or ETA receptors resulted in similar vasoconstriction in the absence or presence of apelin (P>0.05). In conclusion, in patients with central obesity, apelin has favorable effects not only to improve insulin-stimulated endothelium-dependent and endothelium-independent vasodilator responses but also to blunt Ang II- and ET-1-dependent vasoconstriction by a mechanism not involving NO. Taken together, our results suggest that targeting the apelin system might favorably impact some hemodynamic abnormalities of insulin-resistant states like obesity.

Endothelin-1 Regulates Molecules of the Major Histocompatibility Complex: Role in Sickle Cell Disease

Molecules of the Major Histocompatibility Complex (MHC), and in particular specific human leukocyte antigen (HLA) alleles, have been proposed in the pathophysiology of immune and vascular alterations leading to vasoocclusive crises (VOC) and stroke in Sickle Cell Disease (SCD). Endothelial cells express MHC molecules following exposure to cytokines. SCD is characterized, in part, by vascular endothelial cell activation, increased oxidative stress, sickle cell adhesion and excess levels of the potent mitogen, endothelin-1 (ET-1). ET-1 activates endothelial cells, induces oxidative stress and inflammation in the vascular wall and regulates erythrocyte homeostasis. However, the role of ET-1 on MHC regulation in SCD is not clear. We first characterized the effect of ET-1 on MHC expression in the human endothelial cell line, EA.hy926. We observed dose-dependent increases in the expression of MHC class I (HLA-A2 4.8 ± 2.1 folds p<0.01 n=4), MHC class II (HLA-DR 4.4 ± 1.7 folds p<0.01 n=4) and MHC transcription factor (CIITA 3.5 ± 1.8 folds p<0.05 n=4) in EA.hy926 cells. ET-1-stimulated expression of HLA-A2, HLA-DR and CIITA were significantly blocked by pre-incubation of cells with 10 µM BQ788, a selective blocker of ET-1 type B receptors (p<0.01, n=4). Chromatin immunoprecipitation (ChIP) studies in EA.hy926 cells showed that ET-1 increased Histone H3 acetylation of the promoter region within MHC molecules (HLA-A2 62% ± 5%, HLA-DRB 33% ± 18%, p<0.01, n=4); an event that was likewise blocked by BQ788. We then studied two sickle transgenic knockout mouse models of moderate to severe disease phenotype, βSAntilles and Berkeley (BERK) mice, respectively. We observed significant increases in MHC molecule, H2-Aa mRNA levels (n=7; p<0.01) in spleens from sickle transgenic mice when compared to transgenic knockout mice expressing human hemoglobin A (HbA). We then treated BERK, βSAntilles and HbA mice for 14 days with ET-1 receptor antagonists and observed significant reductions in H2-Aa mRNA levels in spleen tissue from sickle transgenic mice but not in HbA mice (n=7; p<0.05). These results implicate ET-1 as a novel regulator of MHC molecules and suggest that ET-1 receptor blockade represents a promising therapeutic approach to regulate both immune and vascular responses in SCD. DisclosuresNo relevant conflicts of interest to declare.

Lack of an apparent role for endothelin-1 in the prolonged reduction in renal perfusion following severe unilateral ischemia-reperfusion injury in the mouse.

Therapeutic approaches to block the progression from acute kidney injury to chronic kidney disease are currently lacking. Endothelin‐1 (ET‐1) is a powerful vasoconstrictor, induced by hypoxia, and previously implicated in renal ischemia‐reperfusion (IR) injury. This study tested the hypothesis that blunting the vascular influence of ET‐1, either through endothelin ETA receptor blockade (ABT‐627) or vascular endothelial cell deletion of ET‐1 (VEET KO), would improve recovery of renal perfusion and repair of injury following a severe ischemic insult in mice (45 min unilateral renal ischemia). Male C57Bl/6 mice receiving vehicle or ABT‐627 commencing 2 days prior to surgery, and VEET KO mice and wild‐type littermates (WT) underwent 45 min unilateral renal IR surgery followed by 28 days recovery. Renal blood velocity was measured by pulsed‐wave Doppler ultrasound before and after surgery. Renal blood velocity was not significantly different between pairs of groups before surgery. Unilateral IR induced a marked reduction in renal blood velocity of the IR kidney at 24 h postsurgery in all groups, which partially recovered but remained below baseline at 28 days post‐IR. Despite the lack of effect on renal blood velocity, ETA receptor blockade significantly attenuated the atrophy of the post‐IR kidney, whereas this was not significantly affected by lack of endothelial ET‐1 expression. These data suggest that although blockade of the ETA receptor is mildly beneficial in preserving renal mass following a severe ischemic insult, this protective effect does not appear to involve improved recovery of renal perfusion.

Pilot Study of Endothelin Receptor Blockade in Heart Failure with Diastolic Dysfunction and Pulmonary Hypertension (BADDHY-Trial).

In this multi-centre, randomised, placebo-controlled pilot trial, we investigated the clinical and haemodynamic effects of the endothelin-receptor blocker Bosentan in patients with heart failure, preserved ejection fraction and pulmonary hypertension (PH-HFpEF). Eligible patients received either 12 weeks of Bosentan therapy, or a placebo drug. Patients were thereafter followed for a further period of 12 weeks without the study medication. At three points during the study (study Commencement, Week 12 and Week 24), a six-minute walk test (6MWT), echocardiographic and laboratory assessments were performed, as well as a quality of life survey. Right heart catheterisation (RHC) was undertaken at commencement only. The study was aborted early, after an interim analysis favoured the placebo. Six-minute walk distance (6MWD) did not change in the Bosentan group (309.7±96.3m (Commencement), 317.0±126.1m (Week 12), 307.0±84.4m (Week 24); p=0.86), but almost reached statistical significance in the placebo group from 328.8±79.6m, to 361.6±98.2m and 384.0±74.9m (Week 24); p=0.075. In the placebo group, estimated systolic pulmonary artery pressure (measured via echocardiography) significantly decreased (from 62.3±16.7mmHg [Commencement], 45.3±13.9mmHg [Week 12], to 44.6±14.5mmHg [Week 24]; p=0.014) as did right atrial pressure (13.1±5.3 [Commencement], 10.0±3.8 [Week 12], to 9.4±3.2 [Week 24]; p=0.046). Despite this study's limited sample size and premature cessation, it nevertheless suggests that endothelin receptor blockade in patients with PH-HFpEF may have no beneficial effects and could even be detrimental in comparison to a placebo.

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.

Abstract 095: Ovariectomy Restores Purinergic Receptor Activation of Endothelin-dependent Natriuresis

We have recently reported interplay between renal medullary endothelin-1 (ET-1) and purinergic (P2) systems, which play central roles in controlling Na + homeostasis in male rats. Evidence suggests that sex hormones regulate ET-1 and P2 systems. To test whether activation of the renal medullary P2 receptors promotes ET-dependent natriuresis in females and whether ovariectomy (OVX) modulates this potential interaction, we studied the effect of medullary NaCl loading on Na + excretion in adult intact female and OVX SD rats in the presence and absence of P2 or ET receptor antagonism. Isosmotic saline (284 mOsmol/kg H 2 O) was infused into the renal medullary interstitium during a baseline urine collection period, followed by isosmotic or hyperosmotic saline (1800 mOsmol/kg H 2 O) infusion. Blood pressure, renal blood flow, urine Na + , K + and osmolality were measured. Medullary NaCl loading significantly enhanced Na + excretion in intact females and OVX (from 0.8±0.2 to 6.2±1.6 and from 0.7±0.1 to 5.6±0.8 μmol/min, respectively, n=6-8, p&lt;0.05). The natriuretic effect of NaCl loading in intact females was not attenuated by P2 or ET receptor blockade. Whereas, intramedullary infusion of the P2 receptor antagonist, suramin, inhibited the natriuresis induced by medullary NaCl loading in OVX (from 0.4±0.2 to 0.9±0.4 μmol/min, n=6). Additionally, combined ET A/B receptor blockade (ABT-627 + A-192621) abolished the natriuretic response to medullary NaCl load in OVX rats (from 0.2±0.1 to 1.4±0.7 μmol/min, n=4). Activation of medullary purinergic (P2Y 2/4 ) receptors by UTP infusion had no significant effect in intact females, but enhanced Na + excretion in OVX rats (from 0.5±0.1 to 2.3±0.8 μmol/min, n=5, p&lt;0.05). Combined ET A/B receptor blockade significantly inhibited the natriuretic response to UTP observed in OVX rats. These data suggest that increased medullary NaCl loading induces ET-independent and P2-independent natriuresis in intact females. In OVX, activation of medullary P2 receptors promotes ET-dependent natriuresis, similar to our previous findings in male rats and suggests that OVX restores the interplay between the renal ET-1 and purinergic (P2) signaling systems to facilitate Na + excretion. Funded by AHA 15POST25090329 to EYG and P01 HL95499 to DMP

Evaluation of the Effect of Bosentan-Mediated Endothelin Receptor Blockade on Flap Survival in Rats: An Experimental Study.

Local skin flaps are important tools for performing plastic surgery. Skin flaps are used for closure of defects after tumor excision or in tissue losses after trauma. However, problems associated with these flaps are commonly encountered, particularly in areas of marginal necrosis. Bosentan is a vasodilator that exerts its effect through endothelin receptor blockade, and has been shown to prevent ischemic tissue damage. However, no reports have addressed the effect of bosentan on skin flaps. The aim of the study was to investigate the effects of bosentan, which may be applied clinically to promote survival of ischemic skin flaps. A modified McFarlane flap was elevated in the dorsum of 20 Albino Wistar rats with a width-to-length ratio of 3 to 10 cm, respectively, with the caudal base. Perioperatively, 0.9% of physiologic NaCl and injectable distilled water of identical volume were injected into rats in Group 1 (n = 10), and 5 mg/kg bosentan was injected intraperitoneally into rats in Group 2 (n = 10). All of the rats were followed up for 7 days postoperatively. The surviving parts of the flaps were measured at the end of day 7. Acute and chronic inflammation, amount of granulation tissue, fibroblast maturation, amount of collagen, and amounts of reepithelialization and neovascularization present in the ischemic zones of the distal parts of the flaps were evaluated histopathologically, and results were compared statistically. The mean flap survivals were 61.1% in Group 1 and 91.1% in Group 2; the percentage of the surviving flap area in Group 2 was higher than that in Group 1 (p < 0.005). In both groups, there was significantly less acute inflammation in the ischemic zones in Group 2 than in Group 1 (p < 0.005). No significant difference was found in the amounts of chronic inflammation and granulation tissue between the two groups (p > 0.005). Fibroblast maturation, amount of collagen, and amounts of reepithelialization and neovascularization investigated in Group 2 were statistically significantly higher than those in Group 1 (p < 0.005). We believe that bosentan may be used prophylactically to increase survival in risky skin flaps because it decreases ischemic necrosis distal to skin flaps, thus exerting favorable effects on flap survival.

Effect of endothelin-1 and endothelin receptor blockade on the release of microparticles.

Increased levels of endothelial cell microparticles (EMP) are known to reflect endothelial dysfunction (ED). In diabetes mellitus type 2 (T2DM), the expression of endothelin (ET)-1 is increased. As treatment with an ET-1 antagonist significantly inhibited atherosclerosis in animal models, we sought to investigate whether treatment with ET-1 antagonists affects EMP levels in vitro and in vivo in patients with T2DM. In vitro study: Human umbilical vein endothelial cells (HUVEC) were stimulated with ET-1 alone and ET-1 in combination with a dual ET-A and ET-B endothelin receptor blocker. In vivo study: Patients with T2DM were randomized to treatment with the ET receptor antagonist bosentan or placebo. After 4 weeks, the patients were re-examined and blood samples were obtained. EMP counts in supernatants and plasma samples were determined using flow cytometry. In vitro study: In supernatants of ET-1-stimulated HUVECs, the increased release of EMP was reduced significantly by co-incubation with an ET-1 receptor antagonist (e.g. CD31+/CD42b-EMP decreased from 37·1% ± 2·8 to 31·5% ± 2·8 SEM, P = 0·0078). In vivo study: No changes in EMP levels in blood samples of patients with T2DM were found after 4 weeks of bosentan treatment (n = 36, P = ns). Our in vitro results suggest that ET-1 stimulates the release of EMP from HUVECs via a receptor-dependent mechanism. Co-incubation with an endothelin receptor blocker abolished ET-1-dependent EMP release. However, treatment with bosentan for 4 weeks failed to alter EMP levels in patients with T2DM. Other factors seem to have influenced EMP release in patients with T2DM independent of ET-1 receptor-mediated mechanisms.

Role of endothelin-1 for the regulation of renal pelvic function.

Endothelin-1 (ET-1) stimulates contractions in isolated rat renal pelves. The signal transduction mechanisms that mediate ET-1-induced renal pelvic contractions and the role of ET-1 for the in vivo regulation of renal pelvic function are not well characterized. We tested if ET-1 stimulates contractions in murine and human renal pelves, if ET-1 activates the renal pelvic RhoA/ROCK pathway, and if low renal ET-1 formation or ET receptor blockade reduce renal pelvic contractile activity. ET-1 increased contraction frequency and force in murine renal pelves. The majority of human renal pelvic tissue samples showed tonic contractions in response to ET-1. Seven out of 20 human tissue samples showed phasic contractions. In four samples, they were elicited by ET-1 at 10-33nmol/l. ET-1 increased renal pelvic RhoA-GTP content and myosin phosphatase target subunit 1 phosphorylation in isolated rat renal pelves. Renal pelvic contraction frequency (29 ± 2 vs. 29 ± 3min(-1)) and renal pelvic pressure (7.1 ± 0.9 vs. 5.9 ± 1.7mmHg) were similar in collecting duct-specific ET-1 knockout mice and in ET-1 floxed controls in vivo. ET-1 sensitivity of isolated renal pelves was similar in both groups. ET receptor blockade did not significantly affect pelvic contraction frequency and pressure in rats. We conclude that ET-1 stimulates phasic contractions in murine, rat, and, to a lesser extent, in human renal pelves. ET-1 activates the RhoA/ROCK pathway in the renal pelvic wall. Endogenous, kidney-derived ET-1 does not play a major role for the regulation of renal pelvic contractions in vivo.

Activation of purinergic receptors (P2) in the renal medulla promotes endothelin-dependent natriuresis in male rats.

Renal endothelin-1 (ET-1) and purinergic signaling systems regulate Na(+) reabsorption in the renal medulla. A link between the renal ET-1 and purinergic systems was demonstrated in vitro, however, the in vivo interaction between these systems has not been defined. To test whether renal medullary activation of purinergic (P2) receptors promotes ET-dependent natriuresis, we determined the effect of increased medullary NaCl loading on Na(+) excretion and inner medullary ET-1 mRNA expression in anesthetized adult male Sprague-Dawley rats in the presence and absence of purinergic receptor antagonism. Isosmotic saline (NaCl; 284 mosmol/kgH2O) was infused into the medullary interstitium (500 μl/h) during a 30-min baseline urine collection period, followed by isosmotic or hyperosmotic saline (1,800 mosmol/kgH2O) for two further 30-min urine collection periods. Na(+) excretion was significantly increased during intramedullary infusion of hyperosmotic saline. Compared with isosmotic saline, hyperosmotic saline infused into the renal medulla caused significant increases in inner medullary ET-1 mRNA expression. Renal intramedullary infusion of the P2 receptor antagonist suramin inhibited the increase in Na(+) excretion and inner medullary ET-1 mRNA expression induced by NaCl loading in the renal medulla. Activation of medullary P2Y2/4 receptors by infusion of UTP increased urinary Na(+) excretion. Combined ETA and ETB receptor blockade abolished the natriuretic response to intramedullary infusion of UTP. These data demonstrate that activation of medullary P2 receptors promotes ET-dependent natriuresis in male rats, suggesting that the renal ET-1 and purinergic signaling systems interact to efficiently facilitate excretion of a NaCl load.