ET Antagonist Research Articles

Endothelin Inhibitors in Chronic Kidney Disease: New Treatment Prospects.

The endothelin system is reported to play a significant role in glomerular and tubulointerstitial kidney disease. In the kidney, endothelins are produced in mesangial cells and the glomerular basement membrane by the endothelium and podocytes. The endothelin system regulates glomerular function by inducing proliferation, increasing permeability and in effect proteinuria, and stimulating inflammation, tubular fibrosis, and glomerular scarring. Endothelin A receptor antagonists have been proven to delay the progression of chronic kidney disease and play a protective role in immunoglobulin A nephropathy, focal segmental glomerulosclerosis, and diabetic nephropathy. There are several ongoing research studies with ETAR antagonists in nondiabetic nephropathy, Alport disease, vasculitis and scleroderma nephropathy, which results are promising. Some reports suggest that the endothelin system might contribute to ischemia-reperfusion injury, acute graft rejection and deterioration of graft function. Endothelin inhibition in renal transplantation and its influence on graft survival is the future direction needing further research. The most frequent side effects associated with ETAR antagonists is fluid retention. Additionally, it should be considered if selective ETAR antagonists therapy needs to be co-administered with sodium-glucose co-transporter 2 inhibitors, renin-angiotensin-aldosterone inhibitors or diuretics and which patients should be recruited to such treatment to minimize the risk of adverse outcomes.

Engineered SPIONs functionalized with endothelin a receptor antagonist ameliorate liver fibrosis by inhibiting hepatic stellate cell activation

Endothelin-1/endothelin A receptor (ET-1/ETAR) pathway plays an important role in the progression of liver fibrosis by activating hepatic stellate cells (HSCs) - a key cell type involved in the pathogenesis of liver fibrosis. Inactivating HSCs by blocking the ET-1/ETAR pathway using a selective ETAR antagonist (ERA) represents a promising therapeutic approach for liver fibrosis. Unfortunately, small-molecule ERAs possess limited clinical potential due to poor bioavailability, short half-life, and rapid renal clearance. To improve the clinical applicability, we conjugated ERA to superparamagnetic iron-oxide nanoparticles (SPIONs) and investigated the therapeutic efficacy of ERA and ERA-SPIONs in vitro and in vivo and analyzed liver uptake by in vivo and ex vivo magnetic resonance imaging (MRI), HSCs-specific localization, and ET-1/ETAR-pathway antagonism in vivo. In murine and human liver fibrosis/cirrhosis, we observed overexpression of ET-1 and ETAR that correlated with HSC activation, and HSC-specific localization of ETAR. ERA and successfully synthesized ERA-SPIONs demonstrated significant attenuation in TGFβ-induced HSC activation, ECM production, migration, and contractility. In an acute CCl4-induced liver fibrosis mouse model, ERA-SPIONs exhibited higher liver uptake, HSC-specific localization, and ET-1/ETAR pathway antagonism. This resulted in significantly reduced liver-to-body weight ratio, plasma ALT levels, and α-SMA and collagen-I expression, indicating attenuation of liver fibrosis. In conclusion, our study demonstrates that the delivery of ERA using SPIONs enhances the therapeutic efficacy of ERA in vivo. This approach holds promise as a theranostic strategy for the MRI-based diagnosis and treatment of liver fibrosis.

Low expression of miR-1929-3p mediates murine cytomegalovirus-induced fibrosis in cardiac fibroblasts via targeting endothelin a receptor/NLRP3 inflammasome pathway.

MicroRNAs are crucial in the development of myocardial remodeling in hypertension. Low miR-1929-3p expression induced by murine cytomegalovirus (MCMV) infection is closely related to hypertensive myocardial remodeling. This study investigated the molecular mechanism of miR-1929-3p-induced myocardial remodeling after MCMV infection. We modeled MCMV-infected mouse cardiac fibroblasts (MMCFs) as the primary cell model. First, MCMV infection reduced the expression of miR-1929-3p and increased the mRNA and protein expression of its target gene endothelin receptor type A (ETAR) in mouse cardiac fibroblasts (MCFs), which demonstrated an internal relationship with myocardial fibrosis (MF) based on high proliferation, phenotypic transformation (α-SMA), and collagen expression in MMCFs. The transfection of the miR-1929-3p mimic downregulated the high expression of ETAR and alleviated these adverse effects in MMCFs. Inversely, these effects were exacerbated by the miR-1929-3p inhibitor. Second, the transfection of endothelin receptor type A over-expressed adenovirus (adETAR) reversed these positive effects of the miR-1929-3p mimic on MF improvement. Third, the transfection of adETAR exhibited a strong inflammatory response in MMCFs with increased expression of NOD-like receptors pyrin domain containing 3 (NLRP3) and increased secretion of interleukin-18. However, we found that the ETAR antagonist BQ123 and the selected NLRP3 inflammasome inhibitor MCC950 effectively eliminated the inflammatory response induced by both MCMV infection and miR-1929-3p inhibitor. Moreover, the MCF supernatant was related to cardiomyocyte hypertrophy. Our findings suggest that MCMV infection promotes MF by inducing the downregulation of miR-1929-3p and the high expression of ETAR, which activates NLRP3 inflammasomes in MCFs.

Evidence of Sex Differences in the Endothelin Pathway in Diabetes

BackgroundThe endothelin system has been identified as a key regulator of vascular function and is comprised of two receptors; a constricting A‐receptor (ETAR) and dilating B‐receptor (ETBR). Sex differences in the ET‐1 pathway have been established. Specifically, when compared to men, activation of the ETBR in women results in a favorable dilatory response. In the presence of diabetes; however, the endothelin system in general, is negatively altered. Whether sex differences in the endothelin pathway are observed in type 1 diabetes (T1D), is unknown. Thus, the present investigation sought to test the hypothesis that ETB receptor function is reduced in women with T1D compared to men with T1D.MethodsPremenopausal women (n=2, HbA1c=10.4±0.8%, age=29±13 years, BMI=29.9±2.8 kg/m2) and men (n=4, HbA1c=8.5±1.0%, age=22±3 years, BMI=21.5±2.8 kg/m2) with a clinical diagnosis of T1D were recruited to participate. Three microdialysis fibers were inserted into the dermal layer of the right forearm where cutaneous blood flow was measured using laser speckle contrast imaging. Microvascular function was determined by a ten dose incremental perfusion of acetylcholine (Ach; 10‐2‐2*103 M). Endothelin receptor function was determined two ways: 1) by a seven dose incremental perfusion of ET‐1 (10‐20‐10‐8 M), and 2) by a seven dose incremental perfusion of ET‐1 (10‐20‐10‐8 M) combined with the selective ETAR antagonist, BQ‐123 (750 nM). The dose response area under the curve (AUC) for cutaneous vascular conductance of each fiber was calculated for each individual.ResultsWomen with T1D had a significantly greater BMI (p=0.026) and HbA1C(p=0.089) compared to men with T1D. The Ach dose response AUC was greater (p=0.135) in women with T1D (753.5±12.7 AU) when compared to men with T1D (691.7±61.1 AU). The ET‐1 + BQ‐123 dose response AUC tended (p=0.298) to be lower in women with T1D (295.5±14.1 AU) when compared to men with T1D (357.8±98.6 AU). A similar lower response in women was observed for the ET‐1 AUC (p=0.102; men: 1411.9±508.9 AU vs women: 821.7±38.5 AU).ConclusionThese data suggest that, compared to men with T1D, microvascular function is greater in women, albeit weighing more and having a higher HbA1c. In contrast, preliminary findings indicate that ETBR function may be impaired in women with T1D compared to men with T1D.

The acute pressure natriuresis response is suppressed by selective ETA receptor blockade.

Hypertension is a major risk factor for cardiovascular disease. In a significant minority of people, it develops when salt intake is increased (salt-sensitivity). It is not clear whether this represents impaired vascular function or disruption to the relationship between blood pressure (BP) and renal salt-handling (pressure natriuresis, PN). Endothelin-1 (ET-1) regulates BP via ETA and ETB receptor subtypes. Blockade of ETA receptors reduces BP but promotes sodium retention by an unknown mechanism. ETB blockade increases both BP and sodium retention. We hypothesized that ETA blockade promotes sodium and water retention by suppressing PN. We also investigated whether suppression of PN might reflect off-target ETB blockade. Acute PN was induced by sequential arterial ligation in male Sprague Dawley rats. Intravenous atrasentan (ETA antagonist, 5 mg/kg) halved the normal increase in medullary perfusion and reduced sodium and water excretion by >60%. This was not due to off-target ETB blockade because intravenous A-192621 (ETB antagonist, 10 mg/kg) increased natriuresis by 50% without modifying medullary perfusion. In a separate experiment in salt-loaded rats monitored by radiotelemetry, oral atrasentan reduced systolic and diastolic BP by ∼10 mmHg, but additional oral A-192621 reversed these effects. Endogenous ETA stimulation has natriuretic effects mediated by renal vascular dilation while endogenous ETB stimulation in the kidney has antinatriuretic effects via renal tubular mechanisms. Pharmacological manipulation of vascular function with ET antagonists modifies the BP set-point, but even highly selective ETA antagonists attenuate PN, which may be associated with salt and water retention.

Investigation of the Molecular Mechanisms by Which Endothelin-3 Stimulates Preadipocyte Growth.

Endothelins induce many biological responses, and they are composed of three peptides: ET-1, ET-2, and ET-3. Reports have indicated that ET-1 regulates cell proliferation, adipogenesis, and other cell responses and that ET-3 stimulates the growth of gastrointestinal epithelial cells and melanocytes. However, the signalling pathways of ET3 that mediate the growth of fat cells are still unclear. Using 3T3-L1 white preadipocytes, we found that ET-3 induced increases in both cell number and BrdU incorporation. Pretreatment with an ETAR antagonist (but not an ETBR antagonist) blocked the ET-3-induced increases in both cell number and BrdU incorporation. Additionally, BQ610 suppressed the ET-3-induced increases in phosphorylation of AMPK, c-JUN, and STAT3 proteins, and pretreatment with specific inhibitors of AMPK, JNK/c-JUN, or JAK/STAT3 prevented the ET-3-induced increases in phosphorylation of AMPK, c-JUN, and STAT3, respectively. Neither p38 MAPK inhibitor nor PKC inhibitor altered the effects of ET-3 on cell growth. These data suggest that ET-3 stimulates preadipocyte growth through the ETAR, AMPK, JNK/c-JUN, and STAT3 pathways. Moreover, ET-3 did not alter HIB1B brown preadipocyte and D12 beige preadipocyte growth, suggesting a preadipocyte type-dependent effect. The results of this study may help explain how endothelin mediates fat cell activity and fat cell-associated diseases.

Sex‐Specific Role of Endothelin‐A Receptors in the Cardiac Susceptibility to Ischemia/Reperfusion Insult in Adult Offspring Exposed to Prenatal Hypoxia

Introduction Fetal hypoxia, a common consequence of complicated pregnancies, leads to the development of cardiac dysfunction in adult offspring by impairing cardiac tolerance to an ischemia/reperfusion (I/R) insult. Extensive evidence demonstrates that cardiac I/R upregulates the endothelin-1 (ET-1) system leading to elevated levels of ET-1 (a peptide with potent physiological and pathophysiological effects on the cardiovascular system). Active ET-1 exerts its effects via the endothelin-A receptors (ETAR; of which enhanced activation has been associated with impaired post-I/R cardiac function) and endothelin-B receptors (ETBR; which are essential for post-I/R cardiac recovery). However, whether activation of ETAR contributes to cardiac dysfunction in adult offspring exposed to prenatal hypoxia is unknown. We hypothesize that, in adult offspring exposed to prenatal hypoxia, cardiac ETAR expression is increased, and its inhibition improves post-I/R cardiac recovery. Methods Pregnant Sprague-Dawley rats (n=4-6 dams/group) were exposed to normoxia (21% O2; normoxia) or hypoxia (11% O2; p-hypoxia) from gestational day 15-21 (term=22 days). Cardiac function was assessed ex vivo in 4-month old male and female offspring (n=1-2 offspring/dam) by subjecting isolated hearts to I/R (25 min. of ischemia with 40 min. of reperfusion). The contribution of ETAR to cardiac dysfunction was assessed by pre-infusion with an ETAR antagonist (ABT-627) before I/R (ABT-627 was recirculated during reperfusion). ETAR and ETBR levels were assessed in non-ischemic left ventricle tissues by Western blotting. Data (mean±SEM) were compared by two-way ANOVA (Sidak's post hoc test), or by unpaired t-test. Results : ABT-627 induced a dramatic reduction in cardiac recovery after I/R in prenatally hypoxic males compared to their respective controls (control: 60.5±16.3% vs ABT-627: 4.4±2.6%, p=0.009), without effect in normoxic males. In contrast, ABT-627 improved I/R recovery in prenatal hypoxia-exposed females (control: 53.5±10.6% vs ABT-627: 87±5.6%, p=0.05) with no effect in normoxic females. In males, exposure to prenatal hypoxia did not alter cardiac ETAR levels, but decreased ETBR levels compared to the normoxic group (normoxia: 100±3.2% vs p-hypoxia: 70.6±4%, p=0.0003), without changes in females. Conclusion Prenatal hypoxia alters the cardiac ET-1 system in a sex-specific manner. Inhibition of ETAR in prenatally hypoxic males impaired cardiac recovery post-I/R, potentially due to lower ETBR expression; while cardiac recovery was improved by ETAR inhibition in females. Activation of ETAR may thus be essential for cardiac tolerance to I/R in prenatally hypoxic males, while it may contribute to the development of cardiac dysfunction in females. Our data suggests that developmental influences and sex differences need to be considered during the implementation of therapeutic strategies to prevent the long-term effects of pregnancy complications on offspring cardiac health.

Targeting the endothelin axis as a therapeutic strategy for oral cancer metastasis and pain

Metastasis reduces survival in oral cancer patients and pain is their greatest complaint. We have shown previously that oral cancer metastasis and pain are controlled by the endothelin axis, which is a pathway comprised of the endothelin A and B receptors (ETAR and ETBR). In this study we focus on individual genes of the pathway, demonstrating that the endothelin axis genes are methylated and dysregulated in cancer tissue. Based on these findings in patients, we hypothesize that ETAR and ETBR play dichotomous roles in oral carcinogenesis and pain, such that ETAR activation and silenced ETBR expression result in increased carcinogenesis and pain. We test a treatment strategy that targets the dichotomous functions of the two receptors by inhibiting ETAR with macitentan, an ETAR antagonist approved for treatment of pulmonary hypertension, and re-expressing the ETBR gene with adenovirus transduction, and determine the treatment effect on cancer invasion (i.e., metastasis), proliferation and pain in vitro and in vivo. We demonstrate that combination treatment of macitentan and ETBR gene therapy inhibits invasion, but not proliferation, in cell culture and in a mouse model of tongue cancer. Furthermore, the treatment combination produces an antinociceptive effect through inhibition of endothelin-1 mediated neuronal activation, revealing the analgesic potential of macitentan. Our treatment approach targets a pathway shown to be dysregulated in oral cancer patients, using gene therapy and repurposing an available drug to effectively treat both oral cancer metastasis and pain in a preclinical model.

Endothelin-1 stimulates preadipocyte growth via the PKC, STAT3, AMPK, c-JUN, ERK, sphingosine kinase, and sphingomyelinase pathways.

Endothelin (ET)-1 regulates adipogenesis and the endocrine activity of fat cells. However, relatively little is known about the ET-1 signaling pathway in preadipocyte growth. We used 3T3-L1 preadipocytes to investigate the signaling pathways involved in ET-1 modulation of preadipocyte proliferation. As indicated by an increased number of cells and greater incorporation of bromodeoxyuridine (BrdU), the stimulation of preadipocyte growth by ET-1 depends on concentration and timing. The concentration of ET-1 that increased preadipocyte number by 51-67% was ~100 nM for ~24-48 h of treatment. ET-1 signaling time dependently stimulated phosphorylation of ERK, c-JUN, STAT3, AMPK, and PKCα/βII proteins but not AKT, JNK, or p38 MAPK. Treatment with an ETAR antagonist, such as BQ610, but not ETBR antagonist BQ788, blocked the ET-1-induced increase in cell proliferation and phosphorylated levels of ERK, c-JUN, STAT3, AMPK, and PKCα/βII proteins. In addition, pretreatment with specific inhibitors of ERK1/2 (U0126), JNK (SP600125), JAK2/STAT3 (AG490), AMPK (compound C), or PKC (Ro318220) prevented the ET-1-induced increase in cell proliferation and reduced the ET-1-stimulated phosphorylation of ERK1/2, c-JUN, STAT3, AMPK, and PKCα/β. Moreover, the SphK antagonist suppressed ET-1-induced cell proliferation and ERK, c-JUN, STAT3, AMPK, and PKC phosphorylation, and the SMase2 antagonist suppressed ET-1-induced cell proliferation. However, neither the p38 MAPK antagonist nor the CerS inhibitor altered the effect of ET-1. The results indicate that ETAR, JAK2/STAT3, ERK1/2, JNK/c-JUN, AMPK, PKC, SphK, and SMase2, but not ETBR, p38 MAPK, or CerS, are necessary for the ET-1 stimulation of preadipocyte proliferation.

Endothelin receptor antagonists alleviate blood-brain barrier disruption and cerebral edema in a mouse model of traumatic brain injury: A comparison between bosentan and ambrisentan

Traumatic brain injury (TBI) is induced by the immediate physical disruption of brain tissue. TBI causes disruption of the blood–brain barrier (BBB) and brain edema. In the cerebrospinal fluid (CSF) of TBI patients, endothelin-1 (ET-1) is increased, suggesting that ET-1 aggravates TBI-induced brain damage. In this study, the effect of bosentan (ETA/ETB antagonist) and ambrisentan (ETA antagonist) on BBB dysfunction and brain edema were examined in a mouse model of TBI using lateral fluid percussion injury (FPI). FPI to the mouse cerebrum increased the expression levels of ET-1 and ETB receptors. Administration of bosentan (3 or 15 mg/kg/day) and ambrisentan (0.1 or 0.5 mg/kg/day) at 6 and 24 h after FPI ameliorated BBB disruption and cerebral brain edema. Delayed administration of bosentan from 2 days after FPI also reduced BBB disruption and brain edema, while ambrisentan had no significant effects. FPI-induced expression levels of ET-1 and ETB receptors were reduced by bosentan, but not by ambrisentan. In cultured mouse astrocytes and brain microvessel endothelial cells, ET-1 (100 nM) increased prepro--ET-1 mRNA, which was inhibited by bosentan, but not by ambrisentan. FPI-induced alterations of the expression levels of matrix metalloproteinase-9, vascular endothelial growth factor-A, and angiopoietin-1 in the mouse cerebrum were reduced by delayed administration of bosentan, while ambrisentan had no significant effects. These results suggest that ET antagonists are effective in improving BBB disruption and cerebral edema in TBI patients and that an ETA/ETB non-selective type of antagonists is more effective.

Blockade of endothelin receptor A enhances the therapeutic efficacy of gemcitabine in pancreatic cancer cells

Pancreatic adenocarcinoma is currently one of the leading causes of cancer-related death worldwide. The high rate of mortality in pancreatic cancer patients is due to the inability to detect early-stage disease and the disease being highly refractory to therapy. Gemcitabine has been the standard chemotherapy for advanced pancreatic cancer patients for the last two decades. However, gemcitabine resistance develops within a few weeks of treatment, and the associated mechanism remains poorly understood. Therefore, a novel therapeutic strategy is needed to overcome the limited clinical efficacy of gemcitabine in pancreatic adenocarcinoma.In this study, we demonstrated that ET-1/ETAR axis gene expression was upregulated in pancreatic cancer cells after treatment with gemcitabine. Additionally, ETAR expression was significantly higher in tumor tissues than in normal tissues, and patients with high ETAR expression had a notably worse overall survival rate than those with low ETAR expression. Furthermore, our results revealed that bosentan, an ETAR antagonist, enhanced the growth-inhibiting and proapoptotic effects of gemcitabine on pancreatic cancer cells.Thus, our findings indicate that blockade of the ET-1/ETAR axis signaling pathway promotes the antiproliferative effect of gemcitabine on pancreatic cancer. Therefore, combination of ETAR blockade and gemcitabine serves as an effective therapeutic approach to achieve clinical benefits in pancreatic adenocarcinoma patients.

Evidence of Endothelin‐B Receptor Dysfunction in Obesity

BackgroundThe endothelin system has been identified as a key regulator of vascular function and is comprised of two opposing receptors; a constricting A‐receptor (ETAR) and dilating B‐receptor (ETBR). Dysregulation of the ETBR and obesity both independently contribute to vascular endothelial dysfunction. However, whether obese individuals exhibit ETBR dysfunction has yet to be determined. Thus, this study sought to test the hypothesis that microvascular ETBR dysfunction is present in obesity compared to normal weight individuals.MethodsIntradermal microdialysis assessments were completed on 13 individuals (9 men; 4 women; age range: 19–38 years old). Participants were recruited according to BMI: lean (BMI ≤ 24.9 kg/m2; n = 6) or overweight/obese (BMI ≥ 25.0 kg/m2; n = 7). Two microdiaylsis fibers were inserted into the dermal layer of the right forearm where cutaneous blood flow was measured using laser speckle contrast imaging. ETBR function was determined two ways: 1) by a seven dose incremental perfusion of ET‐1 (10−20–10−8 M) combined with the selective ETAR antagonist, BQ‐123 (750 nM), and 2) by a seven dose incremental perfusion of ET‐3 (10−17–10−11 M), the isoform that has ETBR selectivity at low doses. The dose response area under the curve (AUC) for cutaneous vascular conductance was calculated for each individual.ResultsThe AUC for the ET‐1 + BQ‐123 dose response was significantly (p=0.02 reduced in the overweight/obese group (AUC = 157.0 ± 11.0 AU) when compared to the lean group (AUC = 221.8 ± 41.1 AU). The AUC for the ET‐3 dose response tended (p=0.21) to be lower in the overweight/obese group (AUC = 155.2 ± 8.5 AU) compared to the lean group (AUC = 191.3 ± 46.4 AU).ConclusionThese data suggest that ETBR function is impaired in obesity compared to normal weight individuals. The reduced ETBR function may contribute to the observed prevalence of vascular endothelial dysfunction in obesity; however, this hypothesis warrants further investigation.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Ovarian hormones modulate endothelin-1 receptor responses in young women.

We recently demonstrated ETBR mediate vasodilation in young but not postmenopausal women; it is unclear if this is related to age or a decline in ovarian hormones. The purpose of this study was to test the hypothesis that ETBR responses are modulated by ovarian hormones. We measured cutaneous vasodilatory responses in 12 young women (22±1years, 23±1kg/m2 ) during the ML (days 20-25) and EF (days 2-5) phases of the menstrual cycle. Cutaneous microdialysis perfusions of lactated Ringer (control), ETBR antagonist (BQ-788, 300nmol/L), and ETAR antagonist (BQ-123, 500nmol/L) were performed, followed by local heating to 42°C. Serum estradiol (ML: 118±16 vs EF: 44±9pg/mL, P<0.05) and progesterone (ML: 8.3±1.0 vs EF: 0.7±0.2ng/mL, P<0.05) were higher during ML vs EF phase. ETBR blockade decreased vasodilation during ML (control: 91±2 vs BQ-788: 83±2%CVCmax, P<0.05) but not EF (control: 89±2 vs BQ-788: 89±1%CVCmax). ETAR blockade also decreased vasodilation during ML (control: 91±2 vs BQ-123: 87±2%CVCmax, P<0.05) but not EF (control: 89±2 vs BQ-123: 92±2%CVCmax). These data suggest that fluctuations in ovarian hormones modulate ETBR and ETAR responses in young women.

Renoprotective effects of ET(A) receptor antagonists therapy in experimental non-diabetic chronic kidney disease: Is there still hope for the future?

Chronic kidney disease (CKD) is a life-threatening disease arising as a frequent complication of diabetes, obesity and hypertension. Since it is typically undetected for long periods, it often progresses to end-stage renal disease. CKD is characterized by the development of progressive glomerulosclerosis, interstitial fibrosis and tubular atrophy along with a decreased glomerular filtration rate. This is associated with podocyte injury and a progressive rise in proteinuria. As endothelin-1 (ET-1) through the activation of endothelin receptor type A (ET(A)) promotes renal cell injury, inflammation, and fibrosis which finally lead to proteinuria, it is not surprising that ET(A) receptors antagonists have been proven to have beneficial renoprotective effects in both experimental and clinical studies in diabetic and non-diabetic CKD. Unfortunately, fluid retention encountered in large clinical trials in diabetic CKD led to the termination of these studies. Therefore, several advances, including the synthesis of new antagonists with enhanced pharmacological activity, the use of lower doses of ET antagonists, the addition of diuretics, plus simply searching for distinct pathological states to be treated, are promising targets for future experimental studies. In support of these approaches, our group demonstrated in adult subtotally nephrectomized Ren-2 transgenic rats that the addition of a diuretic on top of renin-angiotensin and ET(A) blockade led to a further decrease of proteinuria. This effect was independent of blood pressure which was normalized in all treated groups. Recent data in non-diabetic CKD, therefore, indicate a new potential for ET(A) antagonists, at least under certain pathological conditions.

Delayed Administration of BQ788, an ETB Antagonist, after Experimental Traumatic Brain Injury Promotes Recovery of Blood-Brain Barrier Function and a Reduction of Cerebral Edema in Mice.

Traumatic brain injury (TBI) is induced by immediate physical disruption of brain tissue, and causes death and disability. Studies on experimental TBI animal models show that disruption of the blood-brain barrier (BBB) underlies brain edema and neuroinflammation during the delayed phase of TBI. In neurological disorders, endothelin-1 (ET-1) is involved in BBB dysfunction and brain edema. In this study, the effect of ET antagonists on BBB dysfunction and brain edema were examined in a mouse focal TBI model using lateral fluid percussion injury (FPI). ET-1 and ETB receptors were increased at 2-7 days after FPI, which was accompanied by extravasation of Evans blue (EB) and brain edema. Repeated intracerebroventricular administration of BQ788 (15 nmol/day), an ETB antagonist, from 2 days after FPI promoted recovery of EB extravasation and brain edema, while FR 139317, an ETA antagonist, had no effect. Delayed intravenous administration of BQ788 also promoted recovery from FPI-induced EB extravasation and brain edema. While FPI caused decreases in claudin-5, occludin, and zonula occludens-1 proteins, BQ788 reversed FPI-induced reductions of them. Immunohistochemical observation of the cerebrum after FPI showed that ETB receptors are predominantly expressed in glial fibrillary acidic protein (GFAP)-positive astrocytes. BQ788 reduced FPI-induced increases in GFAP-positive astrocytes. GFAP-positive astrocytes produced vascular endothelial growth factor-A (VEGF-A) and matrix metalloproteinase-9 (MMP9). FPI-induced increases in VEGF-A and MMP-9 production were reversed by BQ788. These results suggest that ETB receptor antagonism during the delayed phase of focal TBI promotes recovery of BBB function and reduction of brain edema.

Blocking endothelin-1-receptor/\u03b2-catenin circuit sensitizes to chemotherapy in colorectal cancer

The limited clinical response to conventional chemotherapeutics observed in colorectal cancer (CRC) may be related to the connections between the hyperactivated β-catenin signaling and other pathways in CRC stem-like cells (CRC-SC). Here, we show the mechanistic link between the endothelin-1 (ET-1)/ET-1 receptor (ET-1R) signaling and β-catenin pathway through the specific interaction with the signal transducer β-arrestin1 (β-arr1), which initiates signaling cascades as part of the signaling complex. Using a panel of patient-derived CRC-SC, we show that these cells secrete ET-1 and express ETAR and β-arr1, and that the activation of ETAR/β-arr1 axis promotes the cross-talk with β-catenin signaling to sustain stemness, epithelial-to-mesenchymal transition (EMT) phenotype and response to chemotherapy. Upon ETAR activation, β-arr1 acts as a transcription co-activator that binds β-catenin, thereby promoting nuclear complex with β-catenin/TFC4 and p300 and histone acetylation, inducing chromatin reorganization on target genes, such as ET-1. The enhanced transcription of ET-1 increases the self-sustained ET-1/β-catenin network. All these findings provide a strong rationale for targeting ET-1R to hamper downstream β-catenin/ET-1 autocrine circuit. Interestingly, treatment with macitentan, a dual ETAR and ETBR antagonist, able to interfere with tumor and microenvironment, disrupts the ET-1R/β-arr1-β-catenin interaction impairing pathways involved in cell survival, EMT, invasion, and enhancing sensitivity to oxaliplatin (OX) and 5-fluorouracil (5-FU). In CRC-SC xenografts, the combination of macitentan and OX or 5-FU enhances the therapeutic effects of cytotoxic drugs. Together, these results provide mechanistic insight into how ET-1R coopts β-catenin signaling and offer a novel therapeutic strategy to manage CRC based on the combination of macitentan and chemotherapy that might benefit patients whose tumors show high ETAR and β-catenin expression.

The Endothelin Type A Receptor as a Potential Therapeutic Target in Preeclampsia.

Preeclampsia (PE) is a disorder of pregnancy typically characterized by new onset hypertension after gestational week 20 and proteinuria. Although PE is one of the leading causes of maternal and perinatal morbidity and death worldwide, the mechanisms of the pathogenesis of the disease remain unclear and treatment options are limited. However, there is increasing evidence to suggest that endothelin-1 (ET-1) plays a critical role in the pathophysiology of PE. Multiple studies report that ET-1 is increased in PE and some studies report a positive correlation between ET-1 and the severity of symptoms. A number of experimental models of PE are also associated with elevated tissue levels of prepro ET-1 mRNA. Moreover, experimental models of PE (placental ischemia, sFlt-1 infusion, Tumor necrosis factor (TNF) -α infusion, and Angiotensin II type 1 receptor autoantibody (AT1-AA) infusion) have proven to be susceptible to Endothelin Type A (ETA) receptor antagonism. While the results are promising, further work is needed to determine whether ET antagonists could provide an effective therapy for the management of preeclampsia.

Endothelin causes transactivation of the EGFR and HER2 in non-small cell lung cancer cells

Endothelin (ET)-1 is an important peptide in cancer progression stimulating cellular proliferation, tumor angiogenesis and metastasis. ET-1 binds with high affinity to the ETA receptor (R) and ETBR on cancer cells. High levels of tumor ET-1 and ETAR are associated with poor survival of lung cancer patients. Here the effects of ET-1 on epidermal growth factor (EGF)R and HER2 transactivation were investigated using non-small cell lung cancer (NSCLC) cells. ETAR mRNA was present in all 10 NSCLC cell lines examined. Addition of ET-1 to NCI-H838 or H1975 cells increased EGFR, HER2 and ERK tyrosine phosphorylation within 2min. The increase in EGFR and HER2 transactivation caused by ET-1 addition to NSCLC cells was inhibited by lapatinib (EGFR and HER2 tyrosine kinase inhibitor (TKI)), gefitinib (EGFR TKI), ZD4054 or BQ-123 (ETAR antagonist), GM6001 (matrix metalloprotease inhibitor), PP2 (Src inhibitor) or Tiron (superoxide scavenger). ET-1 addition to NSCLC cells increased cytosolic Ca2+ and reactive oxygen species. ET-1 increased NSCLC clonal growth, whereas BQ123, ZD4054, lapatinib or gefitinib inhibited proliferation. The results indicate that ET-1 may regulate NSCLC cellular proliferation in an EGFR- and HER2-dependent manner.

Endothelin.

The endothelins comprise three structurally similar 21-amino acid peptides. Endothelin-1 and -2 activate two G-protein coupled receptors, ETA and ETB, with equal affinity, whereas endothelin-3 has a lower affinity for the ETA subtype. Genes encoding the peptides are present only among vertebrates. The ligand-receptor signaling pathway is a vertebrate innovation and may reflect the evolution of endothelin-1 as the most potent vasoconstrictor in the human cardiovascular system with remarkably long lasting action. Highly selective peptide ETA and ETB antagonists and ETB agonists together with radiolabeled analogs have accurately delineated endothelin pharmacology in humans and animal models, although surprisingly no ETA agonist has been discovered. ET antagonists (bosentan, ambrisentan) have revolutionized the treatment of pulmonary arterial hypertension, with the next generation of antagonists exhibiting improved efficacy (macitentan). Clinical trials continue to explore new applications, particularly in renal failure and for reducing proteinuria in diabetic nephropathy. Translational studies suggest a potential benefit of ETB agonists in chemotherapy and neuroprotection. However, demonstrating clinical efficacy of combined inhibitors of the endothelin converting enzyme and neutral endopeptidase has proved elusive. Over 28 genetic modifications have been made to the ET system in mice through global or cell-specific knockouts, knock ins, or alterations in gene expression of endothelin ligands or their target receptors. These studies have identified key roles for the endothelin isoforms and new therapeutic targets in development, fluid-electrolyte homeostasis, and cardiovascular and neuronal function. For the future, novel pharmacological strategies are emerging via small molecule epigenetic modulators, biologicals such as ETB monoclonal antibodies and the potential of signaling pathway biased agonists and antagonists.

Endothelin therapeutics in cancer: Where are we?

In human cancers, the autocrine and paracrine loop mediated by the aberrantly activation of endothelin-1 (ET-1) receptor (ET-1R) elicits pleiotropic effects, preferentially mediated by the scaffold protein β-arrestin 1 (β-arr1), on tumor cells and on the host microenvironment, providing a strong rationale for targeting ET-1 receptors. This review describes the most up-to-date preclinical and clinical results obtained by using ET-1 therapeutics. The previous negative clinical results of ET-1 therapeutics should not prevent us from setting the standard of this class of drugs for future well-designed clinical trials. The preclinical data obtained with the dual ETAR and ETBR antagonist macitentan indicate that this molecule, which targets cancer cells and tumor-associated microenvironmental elements, could be a cancer therapeutic option. The field of ET-1 therapeutics will be improved in the next decade, facilitated by the new knowledge on the genomic landscape of the human stroma and tumor, and by the low invasive approaches based on liquid biopsies for the discovery of predictive biomarkers. The information obtained from preclinical studies in patient-derived models and from the Cancer Genome Atlas will set the scene of precision medicine for cancer. Results from these studies are expected to open the possibility that ET-1R antagonists might be more efficacious as molecular cancer therapeutics, able to hamper the functional β-arr1-dependent signaling complexes, either alone or coupled with new targeted approaches.