Endothelin Receptor Subtypes Research Articles

Postoperative Management of Aneurysmal Subarachnoid Hemorrhage

Delayed cerebral ischemia(DCI) is one of the most significant complications of subarachnoid hemorrhage. Despite significant evolution in understanding DCI pathophysiology, vasospasm affecting cerebral vessels of large and moderate diameters remain the only clinically measurable component of DCI and is therefore the primary target for intervention in the postoperative management of subarachnoid hemorrhage. In Japan, fasudil hydrochloride and ozagrel sodium are widely used to prevent vasospasms; however, their effects are sometimes insufficient. Clazosentan, a selective endothelin receptor subtype A antagonist, reduces vasospasm-related morbidity and all-cause mortality following aneurysmal subarachnoid hemorrhage. This was demonstrated in a recent randomized phase 3 trial, leading to the approval of clazosentan by the Pharmaceuticals and Medical Devices Agency in Japan. Recent advances in our understanding of subarachnoid hemorrhage will facilitate improved management to reduce the incidence of DCI.

Prophylactic management of cerebral vasospasm with clazosentan in real clinical practice: a single-center retrospective cohort study.

Clazosentan, a selective endothelin receptor subtype A antagonist, reduces vasospasm-related morbidity and all-cause mortality following aneurysmal subarachnoid hemorrhage (SAH) in the Japanese population, as demonstrated by a recent randomized phase 3 trial. However, evidence to suggest clazosentan should be prioritized over the current standard of care to prevent cerebral vasospasm is still lacking. Therefore, we investigated the efficacy and safety of clazosentan in comparison with conventional postoperative management in real-world clinical practice. We conducted a single-center, retrospective, observational cohort study using prospectively collected data from consecutive patients with aneurysmal SAH. After clazosentan was approved for use in Japan, the conventional postoperative management protocol, composed of intravenous fasudil chloride and oral cilostazol (control group, April 2021 to March 2022), was changed to the clazosentan protocol (clazosentan group, April 2022 to March 2023). The primary endpoint was the incidence of vasospasm-related symptomatic infarction. The secondary endpoints were favorable functional outcomes (modified Rankin scale score < 3) at discharge, angiographic vasospasm, and the need for rescue therapy for delayed cerebral ischemia. The analysis included 100 and 81 patients in the control and clazosentan groups, respectively. The incidence of vasospasm-related symptomatic infarction was significantly lower in the clazosentan group than in the control group (6.2% vs. 16%, p = 0.032). Multiple logistic analyses demonstrated that the use of clazosentan was independently associated with fewer incidence of vasospasm-related symptomatic infarct (23.8% vs. 47.5%, odds ratio 0.34 [0.12-0.97], p = 0.032). Clazosentan was significantly associated with favorable outcomes at discharge (79% vs. 66%, p = 0.037). Moreover, both the incidence of angiographic vasospasm (25.9% vs. 44%, p = 0.013) and the need for rescue therapy (16.1% vs. 34%, p = 0.006) was lower in the clazosentan group. The occurrence of pulmonary edema was significantly higher with clazosentan use (19.8% vs. 5%, p = 0.002), which did not result in morbidity. A postoperative management protocol centering on clazosentan was associated with the reduced vasospasm-related symptomatic infarction and improved clinical outcomes compared to the conventional management protocol in Japanese clinical practice. Clazosentan might be a promising treatment option for counteracting cerebral vasospasm after aneurysmal SAH.

ENDOTHELIN-1 ROLE IN HUMAN EYE: A SYSTEMATIC REVIEW

Background: Endothelin-1 (ET-1) is a potent vasoactive factor implicated in development of diabetic retinopathy, which is commonly associated with retinal edema and hyperglycemia. Although the vasomotor activity of venules contributes to the regulation of tissue fluid homeostasis, responses of human retinal venules to ET-1 under euglycemia and hyperglycemia remain unknown and the ET-1 receptor subtype corresponding to vasomotor function has not been determined. The aim: This study aims to show endothelin-1 role in human eye. Methods: By comparing itself to the standards set by the Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) 2020, this study was able to show that it met all of the requirements. So, the experts were able to make sure that the study was as up-to-date as it was possible to be. For this search approach, publications that came out between 2013 and 2023 were taken into account. Several different online reference sources, like Pubmed and SagePub, were used to do this. It was decided not to take into account review pieces, works that had already been published, or works that were only half done. Result: In the PubMed database the results of our search brought up 44 articles, whereas the results of our search on SagePub brought up 95 articles. The results of the search conducted for the last year of 2013 yielded a total 15 articles for PubMed and 42 articles for SagePub. The result from title screening, a total 4 articles for PubMed and 12 articles for SagePub. In the end, we compiled a total of 8 papers. We included five research that met the criteria. Conclusion: ET-1 (vasoconstrictor, mitogenic, pro-oxidative, and proinflammatory) is one of the most potent vasoconstrictors in the body and its action has a dual effect: on the one hand, it acts on receptors of ECs and pericytes; on the other hand, it has a mitogenic effect on VSMCs.

The ring size of monocyclic ET-1 controls selectivity and signaling efficiency at both endothelin receptor subtypes.

Cardiovascular diseases (CVDs) like hypertension are a major cause for death worldwide. In the cardiovascular tissue, the endothelin system-consisting of the receptor subtypes A (ETA R) and B (ETB R) and the mixed agonist endothelin 1 (ET-1)-is a major key player in the regulation of vascular tone and blood pressure. Tight control of this system is required to maintain homeostasis; otherwise, the endothelin system can cause severe CVDs like pulmonary artery hypertension. The high sequence homology between both receptor subtypes limits the development of novel and selective ligands. Identification of small differences in receptor-ligand interactions and determination of selectivity constraints are crucial to fine-tune ligand properties and subsequent signaling events. Here, we report on novel ET-1 analogs and their detailed pharmacological characterization. We generated simplified ET-1-derived monocyclic peptides to provide an accessible synthesis route. By detailed in vitro characterization, we demonstrated that both G protein signaling and the subsequent arrestin recruitment of activated ETB R remain intact, whereas activation of the ETA R depends on the intramolecular ring size. Increasing of the intramolecular ring structure reduces activity at the ETA R and shifts the peptide toward ETB R selectivity. All ET-1 analogs displayed efficient ETB R-mediated signaling by G protein activation and arrestin 3 recruitment. Our study provides in-depth characterization of the ET-1/ETA R and ET-1/ETB R interactions, which has the potential for future development of endothelin-based drugs for CVD treatment. By identification of Lys9 for selective labeling, novel analogs for peptide-mediated shuttling by ET-1 are proposed.

Hyperglycemia Augments Endothelin-1-Induced Constriction of Human Retinal Venules.

PurposeEndothelin-1 (ET-1) is a potent vasoactive factor implicated in development of diabetic retinopathy, which is commonly associated with retinal edema and hyperglycemia. Although the vasomotor activity of venules contributes to the regulation of tissue fluid homeostasis, responses of human retinal venules to ET-1 under euglycemia and hyperglycemia remain unknown and the ET-1 receptor subtype corresponding to vasomotor function has not been determined. Herein, we addressed these issues by examining the reactivity of isolated human retinal venules to ET-1, and results from porcine retinal venules were compared.MethodsRetinal tissues were obtained from patients undergoing enucleation. Human and porcine retinal venules were isolated and pressurized to assess diameter changes in response to ET-1 after exposure to 5 mM control glucose or 25 mM high glucose for 2 hours.ResultsBoth human and porcine retinal venules exposed to control glucose developed similar basal tone and constricted comparably to ET-1 in a concentration-dependent manner. ET-1–induced constrictions of human and porcine retinal venules were abolished by ETA receptor antagonist BQ123. During high glucose exposure, basal tone of human and porcine retinal venules was unaltered but ET-1–induced vasoconstrictions were enhanced.ConclusionsET-1 elicits comparable constriction of human and porcine retinal venules by activation of ETA receptors. In vitro hyperglycemia augments human and porcine retinal venular responses to ET-1.Translational RelevanceSimilarities in vasoconstriction to ET-1 between human and porcine retinal venules support the latter as an effective model of the human retinal microcirculation to help identify vascular targets for the treatment of retinal complications in patients with diabetes.

Identification and Characterization of Endothelin Receptors in Rat Cerebellum

Endothelins (EIS), potent vasoconstrictors, are also neuropeptides in mammalian brain. The objectives of this study were to identify and characterize the receptors for ETs in rat cerebellum, using radioligand binding techniques and affinity cross-linking method. Iodinated ET-1 bound specifically to receptors in rat cerebellar membrane in a dose-dependent manner. Scatchard plot indicated a single class of high-affinity binding sites. Apparent Kd value was 110 pM, and the Bmax value was 4.85 pmol/mg of membrane protein. Through experiments of affinity cross-linking of ET receptors with 125I-ET-1, two forms of endothelin receptors with molecular masses of 47 KD and 32 KD were identified. Electrophoresis conducted in the presence of reducing reagents did not affect the mobilities of specifically labeled bands, suggesting that endothelin receptors exist as a single polypeptide chain. The order of potency for EIS and sarafotoxin 6b (S6b) in displacing the specifically bound iodinated ET-1 from cerebellar membrane was ET-1 = ET-2 = ET-3 >S6b. Result from receptor specificity indicated that endothelin receptors in rat cerebellum belong to endothelin receptor subtype B.

Endothelin-1 (ET-1) promotes a proinflammatory microglia phenotype in diabetic conditions.

Diabetes increases the risk and severity of cognitive impairment, especially after ischemic stroke. It is also known that the activation of the endothelin (ET) system is associated with cognitive impairment and microglia around the periinfarct area produce ET-1. However, little is known about the effect of ET-1 on microglial polarization, especially under diabetic conditions. We hypothesized that (i) ET-1 activates microglia to the proinflammatory M-1-like phenotype and (ii) hypoxia/ lipopolysaccharide (LPS) activates the microglial ET system and promotes microglial activation towards the M-1 phenotype in diabetic conditions. Microglial cells (C8B4) cultured under normal-glucose (25 mmol/L) conditions and diabetes-mimicking high-glucose (50 mmol/L) conditions for 48 h were stimulated with ET-1, cobalt chloride (200 μmol/L), or LPS (100 ng/mL) for 24 h. PPET-1, ET receptor subtypes, and M1/M2 marker gene mRNA expression were measured by RT-PCR. Secreted ET-1 was measured by ELISA. A high dose of ET-1 (1 μmol/L) increases the mRNA levels of ET receptors and activates the microglia towards the M1 phenotype. Hypoxia or LPS activates the ET system in microglial cells and shifts the microglia towards the M1 phenotype in diabetic conditions. These in vitro observations warrant further investigation into the role of ET-1-mediated activation of proinflammatory microglia in post-stroke cognitive impairment in diabetes.

Receptor- and cellular compartment-specific activation of the cAMP/PKA pathway by α1-adrenergic and ETA endothelin receptors

The signalling functions of many G protein-coupled receptors (GPCRs) expressed in the myocardium are incompletely understood. Among these are the endothelin receptor (ETR) family and α1-adrenergic receptor (α1-AR), which are thought to couple to the G protein Gαq. In this study, we used transcriptome analysis to compare the signalling networks downstream of these receptors in primary neonatal rat cardiomyocytes. This analysis indicated increased expression of target genes of cAMP responsive element modulator (CREM) after 24 h treatment with the α1-AR agonist phenylephrine, but not the ETR agonist endothelin-1, suggesting a specific role for the α1-AR in promoting cAMP production in cardiomyocytes. To validate the difference observed between these two GPCRs, we used heterologous expression of the receptors and genetically encoded biosensors in HEK 293 cell lines. We validated that both α1A- and α1B-AR subtypes were able to lead to the accumulation of cAMP in response to phenylephrine in both the nucleus and cytoplasm in a Gαs-dependent manner. However, the ETR subtype ETA did not affect cAMP levels in either compartment. All three receptors were coupled to Gαq signalling as expected. Further, we showed that activation of PKA in different compartments was α1-AR subtype specific, with α1B-AR able to activate PKA in the cytoplasm and nucleus and α1A-AR only able to in the nucleus. We provide evidence for a pathway downstream of the α1-AR, and show that distinct pools of a receptor lead to differential activation of downstream effector proteins dependent on their cellular compartment.

Microvascular endothelial function in lean versus obese women with polycystic ovary syndrome: role of the endothelin B receptor

Polycystic ovary syndrome (PCOS) affects 10% of women and is associated with myriad cardiovascular risk factors, including obesity and endothelial dysfunction. PCOS is also associated with elevated levels of plasma endothelin‐1 (ET‐1), one of several circulating indicators of endothelial injury and dysfunction. Women with PCOS have reduced vasodilatory responses to ET‐1 as mediated through the endothelin receptor subtype B (ETBR). We hypothesized that impairments in ETBR‐dependent and independent vasodilation are more severe in obese than in lean women with PCOS. We tested 3 lean (age 30 ± 6 y; BMI 23 ± 1 kg/m2) and 3 obese (33 ± 5 y; 36 ± 4 kg/m2) women diagnosed with PCOS according to the Rotterdam criteria. We assessed cutaneous vascular conductance (CVC) with laser Doppler flowmetry during intradermal microdialysis perfusions of ET‐1 (1, 3, 4, 5, and 7 pmol) with either lactated Ringer's solution, ETBR inhibition (BQ‐788), or nitric oxide inhibition (L‐NMMA). Dose‐response curves (%CVCmax) as a function of the log concentration of ET‐1 demonstrated reduced vasodilatory responses in lean PCOS (logEC50 0.61 ± 0.07) relative to obese PCOS (logEC50 0.52 ± 0.47). Inhibition of ETBR decreased the ET‐1‐induced vasodilation in both lean (i.e., logEC50 0.66 ± 0.11) and obese (logEC50 0.62 ± 0.21) groups. Contrary to our hypothesis, this inhibition was two‐fold greater in obese PCOS women, indicating a greater role for ETBR‐induced vasodilation under basal conditions relative to lean PCOS. Conversely, nitric oxide inhibition had little effect in either lean (logEC50 0.61 ± 0.32) or obese PCOS women (logEC50 0.54 ± 0.18) compared to ET‐1 alone. Our data suggest that impairments to endothelial function are more severe in lean than obese women with PCOS, and that ETBR plays an important role in this potentially adverse cardiovascular outcome.Support or Funding InformationSupported by The John B. Pierce Laboratory and the American Heart Association (15POST24990003).

Calcium‐handling of Endothelin A and B Receptors in Coronary Artery upon Post Ischemic Reperfusion Injury in Rats

BackgroundIschemia‐reperfusion has been demonstrated to increase coronary artery resistance and increase the sensitivity toward the potent vasoconstrictor peptide endothelin‐1 (ET‐1). The increased sensitivity to ET‐1 is associated with an up‐regulation of vasoconstrictive ETB receptors in the vascular smooth muscle cells. This project aims to investigate any causal link between the ETB receptor‐upregulation and a shift in calcium sensitivity/handling in the vascular smooth muscle cells after experimental myocardial ischemia‐reperfusion injury.MethodsMale SD rats (~320 gram) were either subjected to experimental left anterior descending (LAD) coronary artery occlusion for 30 min followed by 24 hours of reperfusion, or sham operation. Contractile properties of artery segments distal to the ligature were measured with wire‐myography or along with simultaneous measures of the intracellular Ca2+ concentration ([Ca2+]i) using Fura‐2. The Ca2+ sensitivity of the arteries was determined during stimulation of the ETB‐R agonist Sarafotoxin 6c (S6c) followed by cumulative addition of extracellular Ca2+ (up to 2.5 mM). The experiments were subsequent repeated with ET‐1 as stimulating agent to access dual ETB‐R and ETA‐R mediated Ca2+ sensitization. Thapsigargin, 2‐APB and Felodipine were in addition used in order to identify Ca2+ handling mechanisms and their relative contribution for the ETB‐R and ETA‐R mediated contraction following ischemia‐reperfusion injury.ResultsPost ischemic reperfusion injury significantly increased vascular tone of the LAD coronary artery in response to [Ca2+]o. Prior incubation with either 125 mM of K+ or the ETB‐R antagonist BQ788 showed, that this difference in contractility was neither due to changes in membrane depolarization or a constitutive activity of the ETB‐R after ischemia and reperfusion injury. The evoked agonist‐responses of the Endothelin receptor subtypes, confirmed the enhanced ETB‐R mediated tone after ischemia‐reperfusion injury, whereas dual ETA‐R and ETB‐R mediated tone seems unaffected between surgery groups. The enhanced ETB‐R contractility develops an equal [Ca2+]i –tone relationship mainly from extracellular Ca2+ influx, through activation of voltage‐gated Ca2+ channels.ConclusionThe ETA‐R mediated contraction was unaffected between surgery groups and show equal dependence on intracellular store‐release and extracellular Ca2+ influx. The ET‐1 evoked [Ca2+]i –tone balance was leftward‐shifted opposed to the ETB‐R and suggest the ETA‐Rs to additionally stimulate downstream Ca2+ sensitivity mechanisms.Support or Funding InformationFaculty of Health and Medical Sciences, University of Copenhagen, Denmark. Lundbeck Foundation, Grant of Excellence, Denmark. R59‐AR‐5404.

Endothelin ETA Receptor Blockade, by Activating ETB Receptors, Increases Vascular Permeability and Induces Exaggerated Fluid Retention.

Endothelin (ET) receptor antagonists have been associated with fluid retention. It has been suggested that, of the two endothelin receptor subtypes, ETB receptors should not be blocked, because of their involvement in natriuresis and diuresis. Surprisingly, clinical data suggest that ETA-selective antagonists pose a greater risk of fluid overload than dual antagonists. The purpose of this study was to evaluate the contribution of each endothelin receptor to fluid retention and vascular permeability in rats. Sitaxentan and ambrisentan as ETA-selective antagonists and bosentan and macitentan as dual antagonists were used as representatives of each class, respectively. ETA-selective antagonism caused a dose-dependent hematocrit/hemoglobin decrease that was prevented by ETB-selective receptor antagonism. ETA-selective antagonism led to a significant blood pressure reduction, plasma volume expansion, and a greater increase in vascular permeability than dual antagonism. Isolated vessel experiments showed that ETA-selective antagonism increased vascular permeability via ETB receptor overstimulation. Acutely, ETA-selective but not dual antagonism activated sympathetic activity and increased plasma arginine vasopressin and aldosterone concentrations. The hematocrit/hemoglobin decrease induced by ETA-selective antagonism was reduced in Brattleboro rats and in Wistar rats treated with an arginine vasopressin receptor antagonist. Finally, the decrease in hematocrit/hemoglobin was larger in the venous than in the arterial side, suggesting fluid redistribution. In conclusion, by activating ETB receptors, endothelin receptor antagonists (particularly ETA-selective antagonists) favor edema formation by causing: 1) fluid retention resulting from arginine vasopressin and aldosterone activation secondary to vasodilation, and 2) increased vascular permeability. Plasma volume redistribution may explain the clinical observation of a hematocrit/hemoglobin decrease even in the absence of signs of fluid retention.

Nrf2 Activation Attenuates Hemolysis but Promotes Pulmonary Vascular Dysfunction in Sickle Mice

Nuclear factor erythroid 2-related factor 2 (Nrf2) is the major transcription factor that coordinates the body’s antioxidant and cytoprotective defense against a variety of toxins. Several Nrf2 activators can reactivate gamma globin gene expression and augment fetal hemoglobin production. More recently, genetic and pharmacologic evidence have shown that Nrf2 activation can specifically mitigate the severity of hemolytic anemia, and systemic and local inflammation in transgenic sickle cell disease (SCD) mice. Based on these encouraging results Nrf2 activation has emerged as an attractive therapeutic strategy in SCD. However, the BEACON trial of the Nrf2 activator CDDO-Methyl Ester (CDDO-Me) showed that this therapeutic approach can cause adverse cardiovascular events in patients with chronic kidney disease with comorbid diabetes. Hitherto, the efficacy-toxicity profile generated by individual Nrf2 activating drugs has not been investigated in SCD. There are hundreds of synthetic and naturally occurring Nrf2 activating compounds, and each class of Nrf2 activating compound has a unique pharmacokinetic, pharmacodynamic, toxicokinetic and toxicodynamic profile. We have recently demonstrated that intravascular hemolysis deteriorates with aging in transgenic sickle (SS) mice in a process that can be mitigated by the Nrf2 activator 3H-1,2-dithiole-3-thione (D3T) (Ghosh et al., JCI Insight, 2016). In this study, an in vitro screen of five Nrf2 activating compounds revealed CDDO-Me to be the most potent inducer of cytoprotective enzymes in human pulmonary microvascular endothelial cells. Thus, we performed a long-term prophylactic CDDO-Me treatment of SS mice and examined the effect of the drug on intravascular hemolysis and vascular dysfunction. A cohort of newly weaned SS mice aged ~4 weeks were randomly assigned to receive CDDO-Me (20µmoles/kg/TIW, n=6) or Vehicle (DMSO/TIW, n=10) by oral gavage for 4 months. After the treatment, the total hemoglobin increased by 10% in the CDDO-Me group while it decreased by 5% in the vehicle-treated group (p<0.05). Plasma concentration of VCAM-1 increased in both groups compared to their respective baseline (vehicle p<0.001; CDDO-Me p<0.05). The lack of impact on VCAM-1 by CDDO-Me was contrary to our results with D3T (Ghosh et al., JCI Insight, 2016). To examine this further, we performed vascular reactivity studies on isolated pulmonary arteries. Second order pulmonary arteries were isolated, cut into 2mm rings, and mounted using 40 μm wire. Rings were set at a resting tension equivalent to 100 mmHg transmural pressure. Cumulative dose response curves of endothelin-1 (100 pM-10 nM) and acetylcholine (10nM-100 μM) were used to study vasoconstriction and vasodilation, respectively. Pulmonary arteries isolated from the CDDO-Me treated SS mice showed a 15% reduction in acetylcholine stimulated vasodilation compared to the arteries isolated from the vehicle treated littermates (vehicle n=5, CDDO-Me n=5; p<0.001). In vitro studies of rat smooth muscle cells revealed significant up-regulation of endothelin receptor subtype A (ETA) mRNA by CDDO-Me (p < 0.01, n=6). We obtained similar results of increased ETA expression in rat smooth muscle cells treated with another Nrf2 activator, Dimethyl Fumarate (DMF) (p < 0.01, n=6). Upregulation of endothelin or the ETA is frequently found in pulmonary hypertension. The hypertensive phenotype that we report here in SS mice treated with CDDO-Me is consistent with the adverse cardiovascular events found in the BEACON trial. Together our results show that while CDDO-Me can stabilize and potentially improve hemolytic anemia in SCD, this drug and potentially other Nrf2 activators may promote the development of pulmonary hypertension, which is a major adult complication of SCD. DisclosuresNo relevant conflicts of interest to declare.

Endothelin-1 down-regulates matrix metalloproteinase 14 and 15 expression in human first trimester trophoblasts via endothelin receptor type B.

STUDY QUESTIONDoes endothelin-1 (ET-1) regulate matrix metalloproteinase (MMP) 14 and 15 production and invasion of human first trimester trophoblasts?SUMMARY ANSWERET-1 in pathophysiological concentrations down-regulates MMP14 and MMP15 expression via endothelin receptor (ETR) type B and decreases trophoblast migration and invasion.WHAT IS KNOWN ALREADYMMP14 and MMP15 are involved in trophoblast invasion. Impairment of invasion has been linked to pregnancy complications such as pre-eclampsia (PE). ET-1 is up-regulated in PE.STUDY DESIGN, SIZE, DURATIONIn vitro study using primary human trophoblasts from 50 first trimester placentas (gestational week 7–12).PARTICIPANTS/MATERIALS, SETTING, METHODSTrophoblasts were cultured in the absence or presence of 10–100 nM ET-1. MMP14 and MMP15 mRNA and protein were quantified by RT-qPCR and Western blotting, respectively. Selective antagonists for ETRA (BQ-123) or ETRB (BQ-788) were used to identify ETR subtypes involved. Functional ET-1 effects were tested in first trimester chorionic villous explants and transwell invasion assays. The roles of tumor necrosis factor (TNF)-α (25 ng/ml) and oxygen (1%) in ET-1 regulation of MMP14 and 15 expression were assessed by Western blotting.MAIN RESULTS AND THE ROLE OF CHANCEET-1 down-regulated MMP14 and MMP15 mRNA (−21% and −26%, respectively, P < 0.05) and protein levels (–18% and –22%, respectively, P < 0.05). This effect was mediated via ETRB. ET-1 decreased trophoblast outgrowth in placental explants (−24%, P < 0.05) and trophoblast invasion (−26%, P ≤ 0.01). TNF-α enhanced ET-1 mediated MMP15 down-regulation (by 10%, P < 0.05), whereas hypoxia abolished the effect of ET-1 on both MMPs.LARGE SCALE DATAN/A.LIMITATIONS, REASONS FOR CAUTIONOnly primary trophoblasts were used in this study. Since trophoblast yield from first trimester placental material is limited, further aspects of MMP14 and 15 regulation could not be characterized. Other anti-invasive factors may be altered by ET-1 in trophoblasts and, thus, contribute to the reduced invasion, but have not been investigated. Oxygen levels similar to those found in the decidua (5–8% O2) were not analyzed in this study.WIDER IMPLICATIONS OF THE FINDINGSET-1 modifies placental function already during the first trimester of pregnancy, the time-window when the placental changes implicated in PE occur. Thus, our results improve the understanding of the placental mechanisms underlying trophoblast invasion and PE.STUDY FUNDING/COMPETING INTEREST(S)The study was funded by the Oesterreichische Nationalbank (Anniversary Fund, project number: 14796) and the Herzfelder'sche Familienstiftung (to J.P.; number: 00685). AMM received funding from the Austrian Science Fund FWF (W1241) and the Medical University Graz through the PhD Program Molecular Fundamentals of Inflammation (DK-MOLIN). The authors have no conflict of interest.

Pulmonary Inflammation Induced by Single Walled Carbon Nanotubes in Golden Syrian Hamsters

Single-walled carbon nanotubes (SWCNTs) have a broad range of applications; however, they may present a potential health hazard. The current study was undertaken to evaluate the effects of inhalation exposure of aerosolized SWCNTs on hamster lungs. Golden Syrian Hamsters were divided in to 2 groups: a control group that was exposed to an aerosol of distilled water, and a treated group exposed to aerosolized SWCNTs at concentrations of 1, 2 or 4mg/m3 for 2hrs/day for 4 consecutive days. Additional groups were used to investigate longer inhalation periods (8 and 14 days) with exposures of 4hrs/day to 2mg/m3 SWCNTs. One day post exposure, hamsters were euthanized. Tissue sections were evaluated by TUNEL assay to determine apoptosis and tissue homogenates were used to measure endothelin-1 (ET-1) levels. Bronchoalveolar lavage fluid (BALF) was used to determine total leukocyte cell numbers, tumor necrosis factor receptor type 1 (TNFR1) counts and endothelin receptor subtypes A (ETA) levels on alveolar macrophages. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were carried out on lung tissue sections. The current study demonstrates inhalation exposures to SWCNTs in hamster induce pulmonary inflammation. Results from histological analysis of tissue sections indicated increase in inflammatory cells predominantly macrophages in airways and hyperplasia of airway epithelial cells. SWCNTs treatment significantly increased total BAL leukocyte counts, TNFR1 and lung ET-1 levels. SWCNTs treatment caused an increase in ETA levels and evidence of lung toxicity was demonstrated by apoptosis. SWCNTs deposition in alveolar airspaces along with changes to alveolar epithelium was observed in tissue sections examined by TEM.

Endothelin receptor B protects granulocyte macrophage colony-stimulating factor mRNA from degradation.

Evidence is lacking on the differential effects of the two therapeutic concepts of endothelin receptor antagonists (ERAs): the blockade of only the endothelin receptor A (ETAR; selective antagonism) versus both ETAR and endothelin receptor B (ETBR; dual blockade). Ambrisentan, a selective ERA, and bosentan, a dual blocker, are both available for therapy. We hypothesized that there are differences in the potential of ERAs to ameliorate inflammatory processes in human airway smooth muscle cells (HASMCs) and aimed to unravel underlying mechanisms. We used HASMC culture, enzyme-linked immunosorbent assay, and quantitative reverse-transcription polymerase chain reaction. Tumor necrosis factor α (TNFα) induced transcription and expression of chemokine (C-X-C motif) ligand 2 (CXCL2), chemokine (C-X-C motif) ligand 3 (CXCL3), granulocyte macrophage colony-stimulating factor (GM-CSF), and matrix metalloproteinase 12 (MMP12) in HASMCs. In concentration-response experiments, bosentan led to a significantly greater reduction of GM-CSF and MMP12 protein release than ambrisentan, whereas there was no significant difference in their effect on GM-CSF and MMP12 mRNA. Both ERAs reduced CXCL3 protein and mRNA equally but had no effect on CXCL2. Blocking mitogen-activated protein kinases revealed that both ETAR and ETBR signal through p38 mitogen-activated protein kinase, but ETBR also signals through extracellular signal-regulated kinase (ERK) 1/2 to induce GM-CSF expression. In the presence of the transcription inhibitor actinomycin D, bosentan, but not ambrisentan, reduced GM-CSF but not MMP12 or CXCL3 mRNA. In conclusion, blockade of each endothelin receptor subtype reduces GM-CSF transcription, but blocking ETBR additionally protects GM-CSF mRNA from degradation via ERK-1/2. Accordingly, blocking both ETAR and ETBR leads to a stronger reduction of TNFα-induced GM-CSF protein expression. This mechanism might be specific to GM-CSF. Our data stress the anti-inflammatory potential of ERA and warrant further investigation of their utility in chronic inflammatory airway diseases.

Effects of age and caloric restriction on the cardiac and coronary response to endothelin-1 in rats

Background and aimsAging is associated with alterations in the cardiovascular system such as increased vasoconstriction and decreased vasodilatation. Some of these changes are partially reversed by caloric restriction. Endothelin-1 is a potent vasoconstrictor which levels increased with age. The aim of this study is to analyze the role of endothelin-1 in the cardiac and coronary changes induced by age and whether these changes may be attenuated by a three-month caloric restriction. Methods and resultsHearts from young (3months old), aged (24months old) and aged rats after 3months of caloric restriction were perfused according to the Langendorff technique. Coronary vasoconstriction to endothelin-1 was reduced in old rats, and endothelin-1 increased myocardial contractility (dP/dt) and heart rate in old but not in young rats. These changes observed in old rats were partly reversed by caloric restriction. Also, in the myocardial tissue of old rats the gene expression of endothelin-1, inducible nitric oxide synthase (iNOS) and tumor necrosis factor alpha (TNF-a) was increased, and the gene expression of endothelin ETB receptors and endothelial nitric oxide syntase (eNOS) was reduced, compared with young rats. Aging induced changes in the expression of ETB receptors and eNOS were reversed by caloric restriction. ConclusionsThese results suggest that aging produces alterations in myocardial and coronary responses to endothelin-1, that may be related to changes in expression of nitric oxide synthases and/or endothelin receptor subtypes, with some of these changes being prevented by caloric restriction.

Antiaging gene Klotho regulates endothelin-1 levels and endothelin receptor subtype B expression in kidneys of spontaneously hypertensive rats.

Klotho is an antiaging gene and is predominately expressed in kidneys. The endothelin system is critical in the regulation of kidney function. The objective of this study is to assess whether klotho affects the renal endothelin system in spontaneously hypertensive rats (SHRs). Four groups of male SHRs and one group of male Wistar-Kyoto (WKY) rats were used. In-vivo expression of klotho was achieved by AAV2 delivery of mouse klotho full-length cDNA (AAV.mKL). Four groups of SHRs were given (intravenously) AAV.mKL, AAV.LacZ, AAV.GFP, and phosphate-buffered saline, respectively. The WKY group was given phosphate-buffered saline and served as a control. At the end of week 12 after gene delivery, all animals were euthanized. Plasma endothelin-1 (ET-1) and renal ET-1 levels were increased in SHRs vs. WKY rats. In-vivo expression of klotho reversed the elevated ET-1 levels in SHRs. ETB receptor protein expression was decreased in both kidney cortex and medulla of SHRs. Interestingly, in-vivo expression of klotho abolished the downregulation of ETB protein expression in SHRs, suggesting that klotho regulates ETB receptor expression. Klotho gene delivery also eliminated the increase in the ratio of ETA/ETB in SHRs. Mitochondrial superoxide dismutase (Mn-SOD) protein expression was decreased in kidneys of SHRs, which was rescued by in-vivo expression of klotho. Klotho gene delivery abolished the upregulation of ET-1 levels and the downregulation of ETB and Mn-SOD expression in kidneys of SHRs. These findings revealed a previously unidentified role of klotho in the regulation of the renal ET system and Mn-SOD in SHRs.

Selective endothelin-A receptor blockade attenuates endotoxin-induced pulmonary hypertension and pulmonary vascular dysfunction.

Endothelin-1 is a potent mediator of sepsis-induced pulmonary hypertension (PH). The pulmonary vascular effects of selective blockade of endothelin receptor subtype A (ETAR) during endotoxemia remain unknown. We hypothesized that selective ETAR antagonism attenuates endotoxin-induced PH and improves pulmonary artery (PA) vasoreactivity. Adult male Sprague-Dawley rats (250-450 g) received lipopolysaccharide (LPS; Salmonella typhimurium; 20 mg/kg intraperitoneally) or vehicle 6 hours before hemodynamic assessment and tissue harvest. The selective ETAR antagonist sitaxsentan (10 or 20 mg/kg) or vehicle was injected intravenously 3 hours after receipt of LPS. Right ventricular systolic pressure, mean arterial pressure (MAP), cardiac output (CO), oxygenation (P/F ratio), and serum bicarbonate were measured. Bronchoalveolar lavage (BAL) cell differential and lung wet-to-dry ratios were obtained. Endothelium-dependent and endothelium-independent vasorelaxations were determined in isolated PA rings. PA interleukin (IL)-1β, IL-6, tumor necrosis factor α (TNF-α), and inducible nitric oxide synthase (iNOS) messenger RNA (mRNA) were measured. LPS caused PH, decreased MAP, CO, and serum bicarbonate, and increased PA IL-1β, IL-6, TNF-α, and iNOS mRNA. Sitaxsentan attenuated sepsis-induced PH and increased MAP. The P/F ratio, CO, serum bicarbonate, and BAL neutrophilia were not affected by sitaxsentan. In isolated PA rings, while not affecting phenylephrine-induced vasocontraction or endothelium-dependent relaxation, sitaxsentan dose-dependently attenuated LPS-induced alterations in endothelium-independent relaxation. PA cytokine mRNA levels were not significantly attenuated by ETAR blockade. We conclude that ETAR blockade attenuates endotoxin-induced alterations in systemic and PA pressures without negatively affecting oxygenation. This protective effect appears to be mediated not by attenuation of sepsis-induced cardiac dysfunction, acidosis, or alveolar inflammation but rather by improved endothelium-independent vasorelaxation.

Heart ischaemia–reperfusion induces local up‐regulation of vasoconstrictor endothelin ETB receptors in rat coronary arteries downstream of occlusion

Background and PurposeEndothelins act via two receptor subtypes, ETA and ETB. Under physiological conditions in coronary arteries, ETA receptors expressed in smooth muscle cells mediate vasoconstriction whereas ETB receptors mainly found in endothelial cells mediate vasorelaxation. However, under pathophysiological conditions, ETB receptors may also be expressed in vascular smooth muscle cells mediating vasoconstriction. Here, we have investigated whether vasoconstrictor ETB receptors are up‐regulated in coronary arteries after experimental myocardial ischaemia in rats.Experimental ApproachMale Sprague‐Dawley rats were subjected to either heart ischaemia–reperfusion (15 min ischaemia and 22 h reperfusion), permanent ischaemia (22 h) by ligation of the left anterior descending coronary artery, or sham operation. Using wire myography, the endothelin receptor subtypes mediating vasoconstriction were examined in isolated segments of the left anterior descending and the non‐ligated septal coronary arteries. Endothelin receptor‐mediated vasoconstriction was examined with cumulative administration of sarafotoxin 6c (ETB receptor agonist) and endothelin‐1 (with or without ETA or ETB receptor blockade). The distribution of ETB receptors was localized with immunohistochemistry and quantified by Western blot.Key ResultsEndothelin ETB receptor‐mediated vasoconstriction and receptor protein levels were significantly augmented in coronary arteries situated downstream of the occlusion after ischaemia–reperfusion compared with non‐ischaemic arteries. In contrast, the ETA receptor‐mediated vasoconstriction was unaltered in all groups.Conclusions and ImplicationsIschaemia–reperfusion induced local up‐regulation of ETB receptors in the smooth muscle cells of coronary arteries in the post‐ischaemic area. In contrast, in non‐ischaemic areas, ETB receptor function was unaltered.

Endothelial Krüppel-Like Factor 4 Modulates Pulmonary Arterial Hypertension

Krüppel-like factor 4 (KLF4) is a transcription factor expressed in the vascular endothelium, where it promotes anti-inflammatory and anticoagulant states, and increases endothelial nitric oxide synthase expression. We examined the role of endothelial KLF4 in pulmonary arterial (PA) hypertension (PAH). Mice with endothelial KLF4 knockdown were exposed to hypoxia for 3 weeks, followed by measurement of right ventricular and PA pressures, pulmonary vascular muscularization, and right ventricular hypertrophy. The effect of KLF4 on target gene expression was assessed in lungs from these mice, verified in vitro by small interfering RNA (siRNA) knockdown of KLF4, and further studied at the promoter level with cotransfection experiments. KLF4 expression was measured in lung tissue from patients with PAH and normal control subjects. We found that, after hypoxia, right ventricular and PA pressures were significantly higher in KLF4 knockdown animals than controls. Knockdown animals also had more severe pulmonary vascular muscularization and right ventricular hypertrophy. KLF4 knockdown resulted in increased pulmonary expression of endothelin-1 and decreased expression of endothelial nitric oxide synthase, endothelin receptor subtype B, and prostacyclin synthase. Concordant findings were observed in vitro, both with siRNA knockdown of KLF4 and promoter activity assays. Finally, KLF4 expression was reduced in lungs from patients with PAH. In conclusion, endothelial KLF4 regulates the transcription of genes involved in key pathways implicated in PAH, and its loss exacerbates pulmonary hypertension in response to chronic hypoxia in mice. These results introduce a novel transcriptional modulator of PAH, with the potential of becoming a new therapeutic target.