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Aims Elevated intraocular pressure is primarily induced by the increased resistance of conventional outflow of aqueous humor. Dysfunction of the juxtacanalicular region of trabecular meshwork (TM) and Schlemm's canal (SC) endothelium, as the main conventional outflow tissue, have been implicated as the major reasons for the increased resistance. Integrins are widespread in these tissues, especially alpha8 integrin (ITGA8). We aim to investigate the properties of cells expressing ITGA8 in the conventional outflow tissue. Main methods Fluorescence in situ hybridization (FISH) and immunofluorescence (IF) were performed to detect the mRNA and protein levels of ITGA8 in human conventional outflow tissue. ITGA8-positive cells were isolated from the cultured human TM cells through a magnetic bead-based approach. Flow Cytometry was used to determine the purification efficiency. The expressions of TM and SC biomarkers and dexamethasone-induced myocilin secretion capacity of ITGA8-positive cells was assessed by Real-time PCR, IF and Western blot. A gel contraction assay was performed to evaluate contractility of ITGA8-positive cells after endothelin 1 treatment. Key findings ITGA8 was found with robust expression near the inner wall of SC endothelium. After purification, the proportion of ITGA8-positive cells were increased by about 10%. ITGA8-positive cells were identified with the properties as SC endothelial cells, such as more robust expressions of SC biomarkers, less dexamethasone-inducible myocilin expression, and stronger contractility. Significance This study demonstrated that cells expressing ITGA8 in SC region possess more properties as SC endothelial cells. Our data implicate a crucial role of ITGA8 in aqueous humor (AH) outflow resistance regulation.

Kidney fibrosis is a common final pathway of chronic kidney diseases, which are characterized by renal architecture damage, inflammation, fibroblast expansion and myofibroblast formation. Endothelin converting enzyme-1 (ECE-1) contributes to activation of Endothelin-1 (ET-1), a potent vasoconstrictor and pro-fibrotic substance. This study elucidated the effect of ECE-1 knockout in kidney fibrosis model in mice in association of ET-1 downregulation. Kidney fibrosis was performed in ECE-1 knockout (ECE-1 KO) and vascular endothelial derived ET-1 KO (VEETKO) mice (2 months, 20–30 g, n = 30) and their wild type (WT) littermates using unilateral ureteral obstruction (UUO) procedure. Mice were euthanized on day-7 and day-14 after UUO. Histopathological analysis was conducted for fibrosis and tubular injury. Immunostainings were done to quantify macrophages (F4/80), fibroblasts (FSP-1) and myofibroblasts (α-SMA). Monocyte Chemoattractant Protein-1 (MCP-1), ECE-1 and preproET-1 (ppET-1) mRNA expression were quantified with qRT-PCR, while Transforming Growth Factor-β1 (TGF-β1) and α-SMA protein level were quantified with Western blot. ECE-1 KO mice demonstrated reduction of ECE-1 and ppET-1 mRNA expression, attenuation of kidney fibrosis, tubular injury, MCP-1 mRNA expression and macrophage number compared to WT. Double immunostaining revealed fibroblast to myofibroblast formation after UUO, while ECE-1 KO mice had significantly lower fibroblast number and myofibroblast formation compared to WT, which were associated with significantly lower TGF-β1 and α-SMA protein levels in day-14 of UUO. VEETKO mice also demonstrated attenuation of ET-1 protein level, fibrosis and myofibroblast formation. In conclusion, ECE-1 knockout and ET-1 downregulation attenuated kidney fibrosis.

Aims Hypercholesterolemia is a hazard for increasing susceptibility of the heart to myocardial infarction (MI) by inducing platelet hyperaggregability. Clopidogrel and prasugrel have documented cardioprotective effects in clinical studies. Herein, we investigated whether clopidogrel and prasugrel could protect against isoproterenol-induced acute MI (A-MI) under hypercholesterolemic conditions in rats. Main methods Dietary hypercholesterolemic rats were subjected to acute doses of isoproterenol. Serum lipids, inflammatory markers, aortic endothelin1 and endothelial nitric oxide synthase (eNOS) mRNAs expression and immunexpression of BCL2 were determined. Key findings Hypercholesterolemic rats showed infiltration of inflammatory cells and reduction in aortic wall thickness, deposition of fibrous tissue between cardiac muscle fibers. Protective doses of prasugrel or clopidogrel for 28 days before A-MI increased survival, amended the ECG parameters -including ST segment elevation- and improved the histopathological picture in hypercholesterolemic rats. This was coupled with reductions in platelet aggregation, creatine kinase-MB activity, endothelin 1, systemic inflammation and cardiac lipid peroxidation and increment in aortic eNOS expression. Clopidogrel and prasugrel groups showed enhanced BCL2 expression in cardiac fibers and aortic wall. Significance Prasugrel and clopidogrel protected against A-MI via anti-aggregatory and anti-inflammatory effects. These results add to the value of these drugs in correcting cardiovascular dysfunction in patients vulnerable to A-MI after confirmation by appropriate human studies.

Among the dysfunctions and pathologies associated with sepsis, the underlying molecular mechanisms of sepsis-induced acute lung injury (ALI) are poorly understood. Endothelin (ET)-1, a potent vasoconstrictor and pro-inflammatory peptide, is known to be involved in the pathogenesis of ALI in a rat model of sepsis. Here, we investigated whether landiolol hydrochloride, an ultra-short-acting β-blocker, plays a crucial role in ameliorating and attenuating LPS-induced ALI through modulation of the ET-1 system. Male Wistar rats at 8 weeks of age were administered with either saline or lipopolysaccharide (LPS) for three hours (3 h) and some of the LPS-administered rats were continuously treated with landiolol for 3 h. ALI was induced by LPS, including levels of both circulatory and pulmonary TNF-α and IL-6 but [PaO2] was significantly decreased. LPS also induced a significant increase in levels of pulmonary ET-1 and ET-A receptor, but levels of ET-B receptor, which has vasodilating effects, were remarkably diminished. Further, LPS administration upregulated the pulmonary expression of HIF-1α. Finally, the treatment of LPS-administered rats with landiolol for 3 h ameliorated and prevented ALI, normalized the altered levels of pulmonary ET-1 and ET-A receptors. Landiolol also induced significant down-regulation of ET-B receptor in lung tissues in the early hours (phase) of sepsis. However, Landiolol treatment had no effect on the up-regulated inflammatory mediators (TNF-α, IL-6) in both plasma and lung tissues during sepsis, and expression of pulmonary HIF-1α also remained unchanged after landiolol treatment. Collectively, these data led us to conclude that landiolol may ameliorate sepsis-induced ALI via the pulmonary ET system.

Hypertension is one of the most prevalent diseases worldwide. Increased synthesis of the vasoconstrictor peptide endothelin 1 (encoded by EDN1) might be responsible for high blood pressure. The present study further confirmed the abnormal EDN1 upregulation within adipose tissue-derived stromal cells (ADSCs) derived from morbidly obese subjects. The overexpression of EDN1 in ADSCs derived from non-obese subjects significantly promoted the proliferation and migration of HUVECs and tube formation by human umbilical vein endothelial cell (HUVEC). Transcription factor NR4A3 was positively correlated with EDN1, binding to EDN1 promoter region to upregulate EDN1 expression. Similarly, the overexpression of NR4A3 in ADSCs derived from non-obese subjects significantly promoted the proliferation and migration of HUVECs and tube formation by HUVECs, as well as EDN1 protein levels in ADSCs. However, the effects of NR4A3 overexpression on EDN1 protein levels in ADSCs and the proliferation and migration of HUVECs and tube formation by HUVECs were significantly reversed by EDN1 silencing in ADSCs. In conclusion, NR4A3 is abnormally upregulated in ADSCs derived from morbidly obese subjects; NR4A3 could promote HUVEC angiogenesis through binding to EDN1 promoter and upregulating EDN1 expression.

Aims The study aims to determine the modulatory roles of ovarian hormones, estrogen (E 2 ) and progesterone (P), on the expression of endothelin A (ET A ) and B (ET B ) receptors in lung, liver and kidney tissues. Main methods Female Sprague–Dawley rats were subjected to bilateral ovariectomy and divided into four groups ovariectomized (OVX), OVX + E 2 , OVX + P, and OVX + E 2 + P. A separate group of rats underwent sham surgery and served as a control. Three weeks after OVX or sham surgery, tissues from lungs, liver, renal cortex, and inner medulla were collected, snap-frozen, and kept at − 80 °C for assessment of ET A and ET B receptor expression using real-time PCR. Key findings E 2 -treated OVX animals had significantly lower expression of ET A receptors in the lungs, compared to OVX rats. Pulmonary ET B receptor mRNA was not measurably affected by any of the interventions. Hepatic ET A and ET B were significantly increased in OVX + E 2 + P rats, compared to sham rats. Renal inner medullary ET A and ET B receptor expressions were significantly elevated in OVX compared to sham, an effect that was prevented by co-supplementation of OVX with E 2 and P. Additionally, both ET A and ET B receptor expression in the renal cortex were significantly attenuated by ovariectomy, and this reduction was not evident in OVX + E 2 rats. Significance These data suggest that ovarian hormones regulate ET receptor expression and may contribute to sex differences in cardiovascular and renal health.

Aims Endothelin-1 (ET-1) is a potent vasoconstrictor which regulates the physiology of cardiorenal system. The aim of this study was to evaluate ET-1-mediated elevation of intracellular Ca 2 + in smooth muscle cells (SMC) of renal resistance arteries. Main methods In in vitro studies of primary SMC, which were isolated from rat renal microvessels, the levels of intracellular Ca 2 + were calculated from the ratio of emissions at 340 and 380 nm after loading cells with Fura 2-AM dye. In ex vivo studies we used two-photon imaging of renal resistance arteries excised from rat kidneys and loaded with fluorescent Ca 2 + indicator Fluo-4 AM. Key findings The two-photon imaging demonstrates that treatment of isolated rat renal resistance arteries with ET-1 causes a rapid increase of intracellular Ca 2 + concentration in smooth muscle vasculature of these vessels. These ex vivo observations are in accordance with in vitro findings indicating that ET-1 mediates activation of TRPC channels and increases the level of intracellular Ca 2 + in cultured SMC to 510 ± 83 nM. Significance ET-1-mediated elevation of intracellular Ca 2 + is strongly linked to renal microvascular contraction and is crucial for ET-1-induced contraction of SMC. The two-photon imaging of intracellular Ca 2 + in intact SMC of rat renal resistance arteries is a powerful technique which allows the detailed ex vivo analysis of intracellular Ca 2 + handling by ET-1, an important player in hypertension-related kidney diseases.

C-reactive protein (CRP) is an inflammatory cytokine that has been shown to be an independent predictor of future atherothrombotic events. Hyperactivity of endothelin-1 (ET-1), a potent vasoconstrictor peptide produced by the endothelium, is linked with cardiovascular disease development and progression. ET-1 is sensitive to inflammatory stimuli, though the influence of CRP on ET-1 system activity is unknown. We tested the hypothesis that ET-1-mediated vasoconstrictor tone is enhanced in adults with elevated plasma CRP concentrations.Sixty non-obese adults (43-70years) were studied: 20 with hsCRP1.0mg/L (low CRP; 0.5±0.1mg/L); 20 with hsCRP 1.0-3.0mg/L (moderate CRP; 2.0±0.1mg/L); and 20 with hsCRP3.0mg/L (high CRP; 6.3±0.5mg/L). Forearm blood flow (FBF; plethysmography) was determined in response to intra-arterial infusions of ET-1 (5pmol/min for 20min) and selective ETA receptor blockade (BQ-123, 100nmol/min for 60min).In response to ET-1, FBF decreased ~10% in the low (-10.0±2.3%), moderate (-10.7±4.0%), and high (-6.6±5.2%) CRP groups, with no significant differences between groups. Additionally, all groups demonstrated a marginal, though significant (~10%), vasodilator response to BQ-123; however, there were no differences in the FBF response to BQ-123 across CRP groups. There were no significant correlations between plasma CRP concentrations and peak FBF response to either ET-1 or BQ-123.These results indicate that ET-1 system activity is not influenced by elevations in CRP. Enhanced ET-1 system activity may not be involved in the increased cardiovascular disease risk associated with elevations in plasma CRP concentrations.

Sickle cell disease (SCD) is a genetic hematologic disorder that is characterized by a variety of potentially life threatening acute and chronic complications. Currently, hydroxyurea is the only clinically approved pharmacological therapy for the treatment of SCD, and the continued prevalence of severe disease complications underscores the desperate need for the development of new therapeutic agents. Central features of the sickle cell disease milieu, including hypoxia, oxidative stress, and thrombosis, are established enhancers of endothelin-1 (ET-1) synthesis. This conceptual connection between ET-1 and SCD was confirmed by multiple studies that demonstrated markedly elevated plasma and urinary levels of ET-1 in SCD patients. Direct evidence for the involvement of ET-1 signaling in the development of SCD pathologies has come from studies using endothelin receptor antagonists in SCD mice. This review summarizes recent studies that have implicated ET-1 signaling as a mechanistic contributor to renal, vascular, pulmonary, and nociceptive complications of sickle cell disease and discusses the potential for the use of ET receptor antagonists in the treatment of SCD.

Aims Hemangiosarcoma (HSA) that originates from vascular endothelial cells is the most common splenic malignant neoplasm in dogs, as it accounts for approximately 20% of all canine soft tissue sarcomas. In this study, inhibitory effects of endothelin receptor antagonists on the growth of HSA cells were examined using cell lines established from canine HSA. Main methods The preproendothelin-1 ( PPET-1 ), endothelin type A receptor ( ETA ) and endothelin type B receptor ( ETB ) mRNA expression levels in HSA cell lines (n = 5) were analyzed quantitatively by real-time RT-PCR. These levels were compared with those in HSA tissues (n = 11) and those in normal splenic tissues (n = 6). ETA and ETB protein expression was examined by western blot. The production and secretion of endothelin-1 (ET-1) and big ET-1 by cell lines were analyzed by measuring the levels in the culture medium by ELISA. The inhibitory effects of endothelin receptor antagonists (ambrisentan, BQ788 and bosentan) on cell growth were evaluated by WST-8 assay. Key findings The PPET1 and ETA mRNA expression levels were elevated in HSA tissues and HSA cell lines compared with normal tissues. In cell lines, the production of ET-1 and big ET-1 peptide as well as the expression of ETA protein were detected, but the levels of ETB were not measured. Ambrisentan and bosentan inhibited growth activity in cell lines. Ambrisentan was more effective than bosentan. Significance These findings demonstrate the importance of the ETA axis in canine HSA as well as the potential of ETA inhibitors in the treatment of canine HSA.

Aims Cerium oxide nanoparticles (CeO2NPs) have been recently introduced into the medical field for their antioxidant properties. The ability of CeO2NPs alone or in combination with spironolactone (SP) to attenuate monocrotaline (MCT)-induced pulmonary hypertension and associated right ventricular hypertrophy was studied in rats. A special emphasis was given to endothelin-1 pathway. Materials and methods Pulmonary hypertension was induced in albino rats by a single subcutaneous injection of MCT (60 mg/kg). Rats received either single CeO2NPs therapy or combined therapy with SP for 2 weeks. Key findings CeO2NPs improved pulmonary function tests with concomitant decrease in serum endothelin-1 and pulmonary expression of endothelin-1 and its receptor ETAR. Besides, CeO2NPs diminished MCT-induced right ventricular hypertrophy and reduced cardiac oxidative stress and apoptosis. Significance CeO2NPs could improve pulmonary hypertension and associated right ventricular hypertrophy with no additive value for SP. Besides being an antioxidant, CeO2NPs work through endothelin-1 pathway to improve pulmonary hypertension.

AimsAging, a physiological process and main risk factor for cardiovascular and renal diseases, is associated with endothelial cell dysfunction partly resulting from NADPH oxidase-dependent oxidative stress. Because increased formation of endothelium-derived endothelin-1 (ET-1) may contribute to vascular aging, we studied the role of NADPH oxidase function in age-dependent contractions to ET-1.Main methodsRenal arteries and abdominal aortas from young and old C57BL6 mice (4 and 24months of age) were prepared for isometric force measurements. Contractions to ET-1 (0.1–100nmol/L) were determined in the presence and absence of the NADPH oxidase-selective inhibitor gp91ds-tat (3μmol/L). To exclude age-dependent differential effects of NO bioactivity between vascular beds, all experiments were conducted in the presence of the NO synthase inhibitor L-NAME (300μmol/L).Key findingsIn young animals, ET-1-induced contractions were 6-fold stronger in the renal artery than in the aorta (p

AimsCalcitonin gene-related peptide (CGRP) is a potent vasodilator neuropeptide. We investigated the ameliorating effect of CGRP in myocardial ischemia induced by endothelin-1 (ET-1), with special emphasis on myocardial microvascular hemodynamics and levels of energy-related metabolites.Main methodsThe Langendorff preparations of rat isolated heart were perfused at a constant flow rate. Microvascular blood flow was also visualized in the anterior epicardium of the left ventricle by means of an intravital fluorescence microscope system. Energy-related metabolite contents in the myocardium were measured by means of 31P-magnetic resonance spectroscopy (31P-MRS).Key findingsIntracoronary bolus injections of CGRP caused dose-dependent decreases in coronary perfusion pressure (CPP) in the hearts exposed to ET-1 (30pmol). The vasodilator potency of CGRP was about 10,000-fold greater than that of nitroglycerin and 1,000-fold greater than that of isobutylmethylxanthine. Vasodilation of the small-sized arterioles (10–40μm in diameter) in response to CGRP (100pmol) was confirmed by direct microscopic observation. After ET-1 (30pmol) plus vehicle administration, high energy phosphates (phosphocreatine (PCr), ATP) were markedly reduced (p

Aims: Endothelin-1 (ET-1) is a mediator of various physiological and pathological processes, including vascular inflammation, cell proliferation and vasoconstriction. Attenuation of ET action using ET-1 antagonists reduces pulmonary vascular leakage and inflammation in several models of lung injuries and experimental acute respiratory distress syndrome (ARDS). Based on these earlier reports, the current study investigates the patterns of ET-1 levels in circulation and pulmonary tissues in an experimental model of lavage-induced surfactant-depleted lung injury. Additionally, we also test the effects of open endotracheal suctioning (OES) and hyperinflation (HI) as recruitment maneuver following OES on ET-1 levels. Main methods: Briefly, 24 Japanese white rabbits were anesthetized and intubated. Normal saline was instilled into the lung and washed mildly. After instillation, rabbits were ventilated at definite settings at a total duration of 3 hours. OES and HI were performed every 15 minutes from the beginning of the protocol. Key findings: Here, we show that both circulatory and pulmonary ET-1 levels increased in models with lung injury induced by saline lavage compared to healthy control group. No further aggravation in expression of pulmonary ET-1 was seen after OES and HI, although OES and HI worsened arterial hypoxygenation and severity of lung injury. In contrast, circulatory ET-1 levels significantly decreased after OES and HI but were not associated with blood pressure changes. Significance: We conclude that in a saline lavage-induced lung injury model, both circulatory and pulmonary ET-1 levels increased. Further, OES and HI exerted differential effects on ET-1 expression at both circulatory and pulmonary levels.

Aims: The nitric oxide and endothelin systems are key components of a local paracrine hormone network in the heart. We previously reported that diastolic dysfunction observed in mice lacking the endothelial nitric oxide synthase (eNOS-/-) can be prevented by a genetic overexpression of ET-1. Sexual dimorphisms have been reported in both ET-1 and NO systems. Particularly, eNOS-/- mice present sex related phenotypic differences. Main methods: We used the ET-1 transgenic (ET+/+), eNOS-/-, and crossbred ET+/+ eNOS-/- mice, and wild type controls. We measured cardiac function by heart catheterization. Cardiac ventricles were collected for histological and molecular profiling. Key findings: We report here that (i) the level of ET-1 expression in eNOS-/- mice was elevated in males but not in females. (ii) Left ventricular end-diastolic blood pressure was higher in male eNOS-/- mice than in females. (ii) eNOS-/- males but not females developed cardiomyocyte hypertrophy. (iv) Perivascular fibrosis of intra-cardiac arteries developed in female ET+/+ and eNOS-/- mice but not in males. Additionally, (v) the cardiac expression of metalloprotease-9 was higher in eNOS-/- males compared to females. Finally, (vi) cardiac proteome analysis revealed that the protein abundance of the oxidative stress related enzyme superoxide dismutase presented with sexual dimorphism in eNOS-/- and ET+/+ mice. Significance: These results indicate that the cardiac phenotypes of ET-1 transgenic mice and eNOS knockout mice are sex specific. Since both systems are key players in the pathogenesis of cardiovascular diseases, our findings might be important in the context of gender differences in patients with such diseases. (C) 2013 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

article i nfo Aims: Functional and structural changes in pulmonary vasculature characterize pulmonary arterial hypertension (PAH) and the prognosis of advanced PAH remains poor despite progress in pharmacotherapy. Adipose-derived regenerative cells (ADRCs) promote cell regeneration at pathological sites and comprise a novel therapy for ail- ments of various organs. We investigated the effects of ADRC therapy in rat models of monocrotaline (MCT)-in- duced pulmonary hypertension (PH) and the underlying mechanisms. Mainmethods:RatswereassignedtoControl andMCT groupswithoutandwith (M/A)intravenoustransfusionof seven million ADRCs on day 7. We echocardiographically evaluated pulmonary hypertension as pulmonary ar- tery flow acceleration time (PAAT) and deceleration (PADc). Right ventricular (RV) systolic pressure was mea- sured by catheterization on day 28 and then pathological changes in pulmonary vessels were assessed. We analyzed PAH-associated gene expression on day 14 using real-time RT-PCR. Key findings: Echocardiography and RV catheterization showed that ADRC therapy inhibited PH development (assessedasPAAT,PADc,andRVsystolicpressure)atday28(MCTvs.M/A,Pb 0.05).Pulmonaryvascularremod- eling was also inhibited (vessel wall thickness: MCT vs. M/A, P b 0.01). Messenger RNA levels of endothelin (ET) A and B receptors, ET-1 and transforming growth factor (TGF)-β increased in the lungs by MCT were suppressed by ADRCs (MCT vs. M/A, P b 0.05). Significance: The development of PH was inhibited by ADRCs through suppressing changes in the expression of genes associated with ET and TGF-β systems. We believe that ADRC therapy could serve as a novel strategy for treating PH.

When endothelin-1(ET-1) was discovered it was hailed as the prototypical endothelium-derived contracting factor (EDCF). However, over the years little evidence emerged convincingly demonstrating that the peptide actually contributes to moment-to-moment changes in vascular tone elicited by endothelial cells. This has been attributed to the profound inhibitory effect of nitric oxide (NO) on both the production (by the endothelium) and the action (on vascular smooth muscle) of ET-1. Hence, the peptide is likely to initiate acute changes in vascular diameter only under extreme conditions of endothelial dysfunction when the NO bioavailability is considerably reduced if not absent. The present essay discusses whether or not this concept should be revised, in particular in view of the potent inhibitory effect exerted by calcitonin gene related peptide (CGRP) released from sensorimotor nerves on vasoconstrictor responses to ET-1.

Recently, microRNAs (miRNAs) have been implicated in control of Edn1 mRNA in several tissues. Here we examined the role of miRNA action on Edn1 mRNA expression in renal distal collecting duct cells.A microarray study was conducted to provide a comprehensive assessment of miRNAs present in a murine inner medullary collecting duct (mIMCD-3) cell line. The experiment was designed as a comparison between mIMCD-3 cells grown in the presence and absence of aldosterone. Argonaute (Ago) immunoprecipitation experiments were used to investigate binding of the RNA induced silencing complex (RISC) to Edn1 mRNA.Thirty-four miRNAs were detected in very high abundance in mIMCD-3 cells, and a large number of others were present at lower levels. The microarray experiments were validated by quantitative PCR analysis of selected miRNAs. The microarray data, in combination with in silico examination of the Edn1 3' UTR provided a panel of candidate miRNAs that could act upon the Edn1 expression. Edn1 mRNA was co-immunoprecipitated with an Argonaute protein antibody, and this interaction was blocked by anti-miR-709 oligonucleotides.These results define the miRNA landscape of the mIMCD-3 cell line. Moreover, Edn1 was shown to interact with Argonaute protein suggesting that it is a target of the RNA induced silencing complex (RISC).

Aims The effects of hormone replacement therapy with estrogen on cardiovascular disease in postmenopausal women are still controversial. In the present study, we examined the effects of an endothelin (ET) receptor antagonist (ERA) and/or angiotensin receptor blocker (ARB) on neointimal formation following vascular injury in ovariectomized (OVX) female rats. Main methods Female rats were divided into intact female and OVX groups. The right carotid artery was subjected to balloon injury, and harvested 2 weeks later. Key findings In the intact female groups, treatment with ARB (L-158809; 1 mg/kg/day) for two weeks after the injury significantly decreased neointimal formation, whereas treatment with the ERA (J-104132; 10 mg/kg/day) did not affect neointimal formation. On the other hand, the ERA markedly decreased neointimal formation after the injury in the OVX groups; however, neointimal formation was not significantly improved by the ARB treatment. In addition, the combined treatment with 17β-estradiol (20 μg/kg/day) or the ERA and ARB markedly suppressed neointimal formation after the balloon injury in the OVX groups, whereas no combinational effects were observed due to the combined treatment with 17β-estradiol and the ERA. Significance These results suggest that ERAs have estrogen-like vasoprotective effects on neointimal formation following balloon injury in OVX rats. ERAs may be useful as an alternative therapy to prevent vascular disease in postmenopausal women.

AimsThe present study is designed to consider a role for the circadian clock protein Per1 in the regulation of the endothelin axis in mouse kidney, lung, liver and heart. Renal endothelin-1 (ET-1) is a regulator of the epithelial sodium channel (ENaC) and blood pressure (BP), via activation of both endothelin receptors, ETA and ETB. However, ET-1 mediates many complex events in other tissues.Main methodsTissues were collected in the middle of murine rest and active phases, at noon and midnight, respectively. ET-1, ETA and ETB mRNA expressions were measured in the lung, heart, liver, renal inner medulla and renal cortex of wild type and Per1 heterozygous mice using real-time quantitative RT-PCR.Key findingsThe effect of reduced Per1 expression on levels of mRNAs and the time-dependent regulation of expression of the endothelin axis genes appeared to be tissue-specific. In the renal inner medulla and the liver, ETA and ETB exhibited peaks of expression in opposite circadian phases. In contrast, expressions of ET-1, ETA and ETB in the lung did not appear to vary with time, but ET-1 expression was dramatically decreased in this tissue in Per1 heterozygous mice. Interestingly, ET-1 and ETA, but not ETB, were expressed in a time-dependent manner in the heart.SignificancePer1 appears to regulate expression of the endothelin axis genes in a tissue-specific and time-dependent manner. These observations have important implications for our understanding of the best time of day to deliver endothelin receptor antagonists.

Aims Endothelin-1 (ET-1) and endothelin-2 (ET-2; Trp 6 Leu 7 ET-1) are expressed by different cell types, but are considered to display identical pharmacological properties on endothelin receptors. We studied agonist-dependent aspects of endothelin A (ET A )-receptor function and the importance of amino acids 6 and 7 of ET-1 and ET-2 in this respect. Main methods We used isolated rat mesenteric resistance arteries in wire myographs, in a setting that minimizes influences of endothelium and sensorimotor nerves, to study arterial smooth muscle ET A -receptor-mediated vasomotor responses, to ET-1, ET-2 and chimeras thereof (Trp 6 ET-1 and Leu 7 ET-1). Key findings ET-1 and ET-2 cause arterial contractions with comparable sensitivities and maximal responses. BQ123 (ET A -antagonist) reduces sensitivity to ET-1 more potently than that to ET-2 (pK B : 7.1 ± 0.2 versus 5.6 ± 0.4). However, 1 μM BQ123 relaxes maximal contractile responses to ET-2 more markedly than those to ET-1. Leu 7 ET-1 is a contractile agonist with lower potency and similar maximal effect compared to ET-1 and greater sensitivity to BQ123 than ET-2. Up to 256 nM Trp 6 ET-1 did not cause contraction and did not antagonize arterial responses to ET-1. Significance Arterial smooth muscle ET A -receptor function displays agonist-dependent aspects. This involves roles of amino acids on position 6 and 7 of the endothelin sequence. Agonist-dependent pathologies may benefit from the design of specific, agonist-selective ET-receptor antagonists.

Aims MicroRNA-1 (miR-1) has been demonstrated as a tumor-suppressive miRNA, which shows a down-regulated pattern in several human malignancies including hepatocellular carcinoma (HCC). However, the pathophysiologic roles of miR-1 and their mechanisms in HCC tumorigenesis are still not totally elucidated. Main methods Pre-miR-1 was cloned into pSuper plasmid to overexpress the miR-1 in hepatoma cells. Real-time PCR and Western blot were applied to detect miR-1, ET-1 mRNA and protein levels respectively. Dual luciferase reporter assay was conducted to investigate the binding site of miR-1 on 3′UTR of ET-1 mRNA. Proliferation of hepatoma cells was evaluated by MTT assay. Key findings We observed that over-expression of miR-1 by miRNA-expressing plasmid transfection in HepG2 and Hep3B cells significantly reduced the proliferation of these cells. To explore the mechanism, we examined the potential target genes of miR-1 by bioinformatics. A potent mitogen, Endothelin-1 (ET-1), attracted our attention. Elevated expression of ET-1 but reduced miR-1 level was detected both in human liver cancer tissues and in hepatoma cell lines using Western Blot and miRNA real-time PCR respectively. By the over-expression and inhibition of miR-1 in HepG2 and Hep3B, we confirmed that miR-1 negatively regulated ET-1 expression in hepatoma cells. A luciferase reporter assay showed that miR-1 regulation was established by pairing to a complementary binding site within the ET-1 3′UTR. Finally, attenuated proliferation of hepatoma cells by over-expression of miR-1 could be partially restored by exogenous ET-1 treatment. Significance Our findings demonstrate that miR-1 could inhibit ET-1 expression to attenuate the proliferation of hepatoma cells.

AimsEndothelin (ET) receptor A antagonism decreases neuronal damage in experimental models of stroke. Since large arteries like basilar artery contribute significantly to total cerebrovascular resistance and are major determinants of microvascular pressure, dysregulation of basilar artery function may worsen stroke injury. ET-1 is involved in the regulation of basilar constriction. However, whether stroke influences vasoreactivity of basilar artery and to what extent ET-1 contributes to basilar vascular dysfunction after stroke remained unknown. The goal of this study was to test the hypothesis that ET-1 impairs basilar artery vasorelaxation after ischemia/reperfusion (I/R) injury via activation of ETA receptor.Main methodsMale Wistar rats were subjected to 3h middle cerebral artery occlusion (MCAO) and 21h reperfusion. One group received ETA receptor antagonist atrasentan (5mg/kg, i.p.) at reperfusion. At 24h, basilar arteries were isolated from control non-stroked, stroked and stroked+atrasentan-treated animals for vascular reactivity measurements using pressurized arteriograph.Key findingsAcetylcholine (Ach)-induced maximum relaxation (Rmax) was decreased in stroked animals as compared to non-stroked group and ETA antagonism partially restored it. There was also a trend for decreased EC50 value for the antagonist treatment group indicating improved Ach sensitivity.SignificanceThese findings suggest that I/R not only affects vessels distal to the occlusion but also impairs relaxation of proximal large vessels. ET-1-mediated basilar artery dysfunction may contribute to neurovascular damage after stroke and early restoration of vascular function by ET receptor antagonism after I/R injury may offer a therapeutic strategy.

Aims The present study aimed to explore possible effects of endothelin-1 (ET-1) on s 2 -adrenoceptor gene transcription in human lung fibroblasts. Main methods MRC-5 human lung fibroblasts were cultured in absence or presence of test substances, followed by s 2 -adrenoceptor mRNA determination by quantitative real time PCR. Key findings ET-1 caused a marked and rapid in onset (1 hr) increase in β 2 -adrenoceptor mRNA, an effect additive to that of short time (1 hr) exposure to the β 2 -adrenoceptor agonist olodaterol. The stimulatory effect of ET-1 on β 2 -adrenoceptor mRNA was prevented by the non-selective ET-A/ET-B receptor antagonist bosentan, indicating that it was mediated via specific ET receptors. In the presence of actinomycin D the effect of ET-1 was prevented indicating that ET-1 acts via increased transcription of the β 2 -adrenoceptor gene. ET-1-induced up-regulation of β 2 -adrenoceptor mRNA was also seen in the presence of cycloheximide excluding indirect effects via up-regulation of other regulatory proteins. Conclusions ET-1 can up-regulate β-adrenoceptor gene transcription in human lung fibroblasts.

Aims Hypertensive patients develop cardiac hypertrophy and fibrosis with increased stiffness, contractile deficit and altered perfusion. Angiotensin II (AngII) is an important factor in the promotion of this pathology. The effects of AngII are partly mediated by endothelin-1 (ET-1) and transforming growth factor-β. The exact feature of these pathways and the intercellular communications involved remain unclear. In this study, we explored the role of endothelial cell-derived ET-1 in the development of AngII-induced cardiac fibrosis and hypertrophy. Main methods We used mice with vascular endothelial cell specific ET-1 deficiency (VEETKO) and their wild type littermates (WT). Mice were infused for one week with AngII (3.2 mg/kg/day, n = 12) or vehicle (0.15 mol/L NaCl and 1 mmol/L acetic acid, n = 5), using subcutaneous mini-pumps. Hearts were stained with hematoxylin–eosin and masson's trichrome for histology. Cardiac gene expression and protein abundance were measured by Northern Blot, real time PCR and Western Blot. Key findings AngII-induced cardiac hypertrophy, interstitial and perivascular fibrosis were less pronounced in VEETKO mice compared to WT. Blood pressure increased similarly in both genotypes. Expression of connective tissue growth factor, tumor growth factor-β, collagen I and III in response to AngII required endothelial ET-1. Endothelial ET-1 was also necessary to the elevation in protein kinase C δ abundance and ERK1/2 activation. AngII-induced elevation in PKCe abundance was however ET-1 independent. Significance This study underscores the significance of ET-1 from the vasculature in the process of AngII-induced cardiac hypertrophy and fibrosis, independently from blood pressure. Endothelial ET-1 represents therefore a possible pharmacological target.

Aims The aims of this study were to examine the role of endothelin-1 (ET-1), a pleiotropic peptide found at elevated levels in a number of malignancies and which has been shown to influence oral cancer cell behaviour via paracrine signalling pathways, on the phenotype of oral fibroblasts. Main methods The effect of ET-1 on proliferation and migration of human primary oral fibroblasts was assessed using MTS and scratch assays, respectively. The ability of ET-1 to affect fibroblast contractility was analysed using type-I collagen gels. Changes in gene expression in oral fibroblasts exposed to ET-1 were examined using quantitative PCR. The invasiveness of oral cancer cells in the presence of conditioned media collected from ET-1 treated fibroblasts was determined using 2D Matrigel assays. Key findings Here we provide evidence that ET-1 increases the migration of oral fibroblasts and induces a more contractile phenotype which is not associated with changes in gene expression indicative of myofibroblast transdifferentiation. In addition we provide evidence that conditioned medium of ET-1-stimulated oral fibroblasts promotes invasion of OSCC cells in vitro. Significance In oral squamous cell carcinoma, a frequently fatal and increasingly common epithelial malignancy of the oral cavity, ET-1 is known to contribute to pro-migratory paracrine signalling between stromal fibroblasts and cancer cells. The ability of ET-1 to modulate the phenotype of human oral stromal fibroblasts, however, has not previously been reported. The findings presented here suggest that targeting the stromal endothelin system may be a viable and novel therapeutic strategy for invasive oral cancer.

AimsTo determine if endothelin-1 (ET-1) stimulates the phosphorylation of ERK1/2 in the mouse inner medullary collecting duct (IMCD), and if this in turn upregulates nitric oxide (NO) production.Main methodsConfluent mouse IMCD segment-3 cells (mIMCD-3) were stimulated with 50nM ET-1 for 24h with and without various doses of ET receptor antagonists, BQ123 (ETA antagonist,) or BQ788 (ETB antagonist) and phosphorylation of ERK1/2 determined by immunoblots. As well, NOS isoform expression and nitrite production were assessed. Finally, increasing doses of the MEK inhibitors, PD98,059 or U0126, were incubated with mIMCD-3 cells and the ET-1 dependent nitrite production determined.Key findingsET-1 via the ETB receptor significantly increased ERK1/2 phosphorylation, and was prevented by MEK inhibition. ET-1 also stimulates nitrite production by mIMCD-3 cells (basal: 54.5±26pmol/mgpr/h vs ET-1: 221±28pmol/mg pr/h; N=4) via the ETB receptor (BQ788+ET-1: 83.7±27pmol/mgpr/h); however, ET-1 does not regulate NOS1 or NOS3 expression. MEK inhibition did not prevent the ET-1 stimulated nitrite production contrary to our initial hypothesis (vehicle+ET-1: 157±13pmol/mgpr/hr vs PD98,059+ET-1: 305.7±24pmol/mgpr/h, N=4, P>0.05).SignificanceAlthough the mouse IMCD-3 cells only express the NOS1β splice variant, ET-1 did regulate mouse IMCD nitrite production. ET-1 stimulates ERK1/2 phosphorylation in the mouse IMCD, but ERK1/2 signaling is not involved in the ET-1 dependent increase in NO production by IMCD cells. Thus, we propose that ET-1 regulates protein–protein interactions that are necessary for NO production, that are independent of MAPK signaling cascades.

Aims An increase in intracellular vascular smooth muscle cell calcium concentration (VSMC [Ca2 +]i) is essential for endothelin-1 (ET-1)-induced vasoconstriction. Based on previous findings that activation of the G protein-coupled estrogen receptor (GPER) inhibits vasoconstriction in response to ET-1 and regulates [Ca2 +]i in cultured VSMC, we investigated whether endogenous GPER regulates ET-1-induced changes in VSMC [Ca2 +]i and constriction of intact arteries. Main methods Pressurized carotid arteries of GPER-deficient (GPER0) and wildtype (WT) mice were loaded with the calcium indicator fura 2-AM. Arteries were stimulated with the GPER-selective agonist G-1 or solvent followed by exposure to ET-1. Changes in arterial diameter and VSMC [Ca2 +]i were recorded simultaneously. Vascular gene expression levels of ETA and ETB receptors were determined by qPCR. Key findings ET-1-dependent vasoconstriction was increased in arteries from GPER0 compared to arteries from WT mice. Despite the more potent vasoconstriction to ET-1, GPER deficiency was associated with a marked reduction in the ET-1-stimulated VSMC [Ca2 +]i increase, suggesting an increase in myofilament force sensitivity to [Ca2 +]i. Activation of GPER by G-1 had no effect on vasoconstriction or VSMC [Ca2 +]i responses to ET-1, and expression levels of ETA or ETB receptor were unaffected by GPER deficiency. Significance These results demonstrate that endogenous GPER inhibits ET-1-induced vasoconstriction, an effect that may be associated with reduced VSMC Ca2 + sensitivity. This represents a potential mechanism through which GPER could contribute to protective effects of endogenous estrogen in the cardiovascular system.

Aims Endothelin-1 (ET-1) has been suggested to be involved in different types of pain due to its neuromodulatory nature. However, its role in inflammatory pain processing, specifically the origin-specific effect, has not yet been clearly defined. Therefore, the aim of this study is to determine the role of cell-type specific ET-1 induction in the modulation of inflammatory pain processing. Main methods The current study assesses the effects of ET-1 over-expression specifically targeted to astrocytes (GET-1) or endothelial cells (TET-1) on the expression of pain-like behaviors induced by a model of inflammatory pain, consisting of a formalin injection into the hind paw. Key findings The baseline sensitivity thresholds of GET-1 and TET-1 mice to the response elicited by tactile and radiant heat stimulation were similar to those observed in age-matched non-transgenic (NTg) controls. Relative to the NTg controls, GET-1 mice displayed a marked decrease in pain-like behavioral responses during the second phase of formalin-induced pain (i.e., 15–20 min after injection), whereas the responses elicited in TET-1 mice were unaltered. The levels of mRNA encoding adrenomedullin, calcitonin gene-related peptide and calcitonin-like receptor were elevated in the spinal cord of saline-injected GET-1 mice compared to those of NTg mice. Significance The current results support a suppressor role for astrocyte-derived ET-1 in inflammatory pain and suggest that the study of GET-1 mice might provide mechanistic insights for improving the treatment of inflammatory pain.

Aims It has been reported that endothelin-1 (ET-1) overproduction and reduced nitric oxide (NO) production are closely related to the progression of renal diseases. In the present study, we examined the interrelation between ET-1 and NO system using rats treated with the combination of deoxycorticosterone acetate (DOCA)-salt and a non selective NO synthase inhibitor N ω -nitro- l -arginine (NOARG). Main methods Rats were treated with DOCA–salt (15 mg/kg, plus drinking water containing 1% NaCl) for two weeks, and then additional treatment of NOARG (0.6 mg/ml in the drinking water) was performed for three days. Key findings Combined treatment of DOCA–salt and NOARG drastically developed the severe renal dysfunction and tissue injury. This treatment additionally enhanced renal ET-1 production compared to the rats treated with DOCA–salt alone, whereas a selective ET A receptor antagonist ABT-627 completely prevented renal dysfunction and tissue injury. On the other hand, combined treatment of DOCA–salt and NOARG induced the phosphorylation of inhibitory protein kappa B (IκB), followed by the activation of nuclear factor-kappa B (NF-κB) in the kidney. In addition, pyrrolidine-dithiocarbamate completely suppressed not only NF-κB activation but also renal dysfunction and ET-1 overproduction. Significance These results suggest that NF-κB/ET-1/ET A receptor-mediated actions are responsible for the increased susceptibility to DOCA–salt induced renal injuries in the case of reduced NO production.

Article history:Received 21 November 2011Accepted 13 April 2012Keywords:FCP1CTD phosphataseCardiac hypertrophyEndothelin-1RNA polymerase II Aims: Cardiac hypertrophy is associated with the increase of total amount of RNA, which is in accordance withRNA polymerase II (RNAPII) activation via C-terminal domain (CTD) phosphorylation of the largest subunit ofRNAPII. It has been demonstrated that endothelin-1 (ET-1) phosphorylates CTD at the hypertrophic responseincardiomyocytes.However,itisunclearwhetherET-1-inducedhypertrophyisaffectedbytheCTDphosphatase,transcription factor IIF-interacting CTD phosphatase1 (FCP1).Mainmethods: We analyzed whether ET-1-induced cardiomyocyte hypertrophy was affected by overexpressionof FCP1 or dominant-negative form of FCP1 (dnFCP1) in neonatal rat cardiomyocytes.Key findings: The level of ET-1-induced RNAPII CTD phosphorylation was decreased by FCP1 overexpression,whereas it was sustained by dnFCP1. Global RNA synthesis evaluated by [ 3 H]-uridine incorporation showedthat the ET-1-induced increase in RNA synthesis was suppressed by FCP1 and was augmented by dnFCP1. ET-1-induced increase in cell surface area was suppressed by FCP1 and was preserved by dnFCP1. Furthermore, theET-1-induced increase in molecular markers of cardiac hypertrophy, expression of ANP and β-MHC gene, wassuppressed by FCP1 and was not inhibited by dnFCP1.Significance: ET-1-inducedcardiachypertrophyandCTDphosphorylationlevelarefunctionallyregulatedbyFCP1.These findings suggest that FCP1 plays an important role in ET-1-induced cardiac hypertrophy via controllingphosphorylation level of the RNAPII CTD.© 2012 Elsevier Inc. All rights reserved.

Aims To determine the impact of mixed endothelin receptor antagonist and mouse mast cell protease-4 (mMCP-4) in the development of atherosclerosis in the mouse model. Materials and methods Apolipoprotein E (ApoE) KO mice were crossed with mMCP-4 KO mice to generate ApoE/mMCP-4 double KO mice. Atherosclerosis was induced with a normal- or high-fat diet for 12, 27 or 52 weeks. Macitentan (30 mg/kg/day), a dual ET A /ET B receptor antagonist, was given orally for 6 weeks (27 week protocol). At sacrifice, aortas and brachiocephalic arteries (BCAs) were collected. En face Sudan IV staining was performed on aortas and BCA sections were subjected to Masson's trichrome stain and α-smooth muscle actin labeling. Key findings Under normal diet, both macitentan treatment and the absence of mMCP-4 reduced the development of aortic atherosclerotic lesions in 27-week old ApoE KO mice, but mMCP-4 deletion failed to maintain this effect on 52-week old mice. Under high-fat diet (WD), macitentan, but not the absence of mMCP-4, reduced aortic lesion development in ApoE KO mice. On BCA lesions of 27-week old WD mice, macitentan treatment had a small impact while mMCP-4 deletion showed improved features of plaque stability. Significance These results suggest that the inhibition of mMCP-4 reduces lesion spreading in the earlier phases of atherosclerosis development and can help stabilise the more advanced plaque. Macitentan treatment was more effective to prevent lesion spreading but did not improve plaque features to the same extent.

Endothelin-1 (ET-1), the most potent vasoconstrictor, has been shown to be mitogenic in many tumor cells as well as in vascular cells. It was previously reported that the mRNA of ET-1 and endothelin receptors (ETRs) are expressed in lung cancer cells. However, their biological role in lung cancer remains to be explored. The purpose of this study was to determine whether ET-1 stimulates proliferation of the human lung adenocarcinoma cell SPC-A1 and probe its cellular mechanism. Reverse-transcription polymerase chain reaction and Western blot analysis showed that both the mRNA and protein of ET-1, ET A R and ET B R are expressed in SPC-A1 cells. Application of ET-1 at 10 − 15 –10 − 8 M caused a dose-dependent cell proliferation and an increase in intracellular free Ca 2+ concentration ([Ca 2+ ] i ). This ET-1-induced cell proliferation and [Ca 2+ ] i increase were completely abolished by BQ123, a selective ET A R antagonist, but not by BQ788, a selective ET B R antagonist. Furthermore, it was significantly reduced by U73122, a specific inhibitor of phospholipase C (PLC), but not by U73433, the structural isomer of U73122. Chelating extracellular Ca 2+ or blocking voltage dependent calcium channels by nifedipine also significantly reduced the mitogenic effect of ET-1 and [Ca 2+ ] i increase in SPC-A1 cells. These results indicate that ET-1 acts as an autocrine growth factor and enhances proliferation of SPC-A1 cells via activation of ET A R. The phosphoinositol/Ca 2+ pathway and Ca 2+ influx through voltage dependent Ca 2+ channels activated by ET A R contribute to this process.

Aims Endothelin-1 (ET-1) is an autocrine inhibitor of collecting duct (CD) Na + and water reabsorption. CD ET-1 production is increased by a high salt diet and is important in promoting a natriuretic response. The mechanisms by which a high salt diet enhances CD ET-1 are being uncovered. In particular, elevated tubule fluid flow, as occurs in salt loading, enhances CD ET-1 synthesis. Tubule fluid solute content and interstitial osmolality can also be altered by a high salt diet, however their effect on CD ET-1 alone, or in combination with flow, is poorly understood. Main methods ET-1 mRNA production by a mouse inner medullary CD cell line (mIMCD3) in response to changing flow and/or osmolality was assessed. Key findings Flow or hyperosmolality (using NaCl, mannitol or urea) individually caused an ~ 2-fold increase in ET-1 mRNA, while flow and hyperosmolality together increased ET-1 mRNA by ~ 14 fold. The hyperosmolality effect alone and the synergistic effect of flow + hyperosmolality was inhibited by chelation of intracellular Ca 2 + , however were not altered by blockade of downstream Ca 2 + -signaling pathways (calcineurin or NFATc), inhibition of cellular Ca 2 + entry channels (purinergic receptors or polycystin-2), or blockade of the epithelial Na + channel. Inhibition of NFAT5 with rottlerin or NFAT5 siRNA greatly reduced the stimulatory effect of osmolality alone and osmolality + flow on mIMCD3 ET-1 mRNA levels. Significance Both flow and osmolality individually and synergistically stimulate mIMCD3 ET-1 mRNA content. These findings may be relevant to explaining high salt diet induction of CD ET-1 production.

Coronary effects of endothelin-1 and vasopressin during acute hypotension, and the role of NO and prostanoids in these effects were examined in anesthetized goats. Left circumflex coronary artery flow was measured electromagnetically, and hypotension was induced by constriction of the caudal vena cava in animals non-treated (7 goats) or treated with the inhibitor of NO synthesis N(w)-nitro-L-arginine methyl esther (L-NAME, 5 goats), the cyclooxygenase inhibitor meclofenamate (5 goats) or both drugs (5 goats). Under normotension (22 goats), mean arterial pressure averaged 93 +/- 3 mm Hg and coronary vascular conductance (CVC) 0.37 +/- 0.025 ml/min/mm Hg. Endothelin-1 (0.01-0.3 nmol) and vasopressin (0.03-1 nmol), intracoronarily injected, dose-dependently decreased CVC by up to 56% for endothelin-1 and 40% for vasopressin. During hypotension in every condition tested, mean arterial pressure decreased to approximately 60 mm Hg, and CVC only decreased during hypotension pretreated with L-NAME (23%) or L-NAME + meclofenamate (34%). Under non-treated hypotension, the decreases in CVC by endothelin-1 were augmented approximately 1.5 fold, and those by vasopressin were not modified. This increase in CVR by endothelin-1 was not affected by L-NAME and was reversed by meclofenamate or L-NAME + meclofenamate. The coronary effects of vasopressin were not modified by any of these treatments. Therefore, acute hypotension increases the coronary vasoconstriction in response to endothelin-1 but not to vasopressin. This increased response to endothelin-1 may be related to both inhibition of NO release and release of vasoconstrictor prostanoids.

Endothelin-1 secretion and sympathetic activation may play important role in cardiovascular pathophysiology. In vivo interactions between these systems are not defined. We aimed to study the electrophysiological and haemodynamic effects of simultaneous intracoronary endothelin-1 and intravenous isoproterenol infusions. 18 anaesthetised open chest dogs were studied after AV-ablation. Mean arterial blood pressure, coronary blood flow, left ventricular contractility, standard electrocardiograms, right and left ventricular epi- and endocardial monophasic action potential (MAP) signals were recorded. Intracoronary endothelin-1 (30 pmol/min) was given to Group ET (n=6), intravenous isoproterenol (0.2 microg/kg/min) to Group ISO (n=6), both endothelin-1 and isoproterenol to Group ET+ISO (n=6) for 30 min. MAP duration increased in all studied regions of Group ET, decreased in all studied regions of Group ISO and ET+ISO (control vs. maximal changes of left ventricular epicardial MAP 90% duration, Group ET: 296+/-22 vs 369+/-20 ms, p

Endothelin (ET)-1(1–31) is a novel vasoconstrictor peptide produced by human mast cell chymase, which selectively cleaves big ET-1 at the Try31-Gly32 bond. We investigated the localization of ET-1(1–31) in various hamster tissues by immunohistochemistry and compared it to the distribution of ET-1(1–21). We found that the localization and amount of ET-1(1–31) were different from those of ET-1(1–21) in each tissue. ET-1(1–31)-like immunoreactivities (IR) in the heart, lung, and adrenal gland were observed in the same areas as ET-1(1–21) but were significantly weaker, suggesting that ET-1(1–31) might play a role only in mast cell/chymase-related pathological conditions in these tissues. In the liver, ET-1(1–31)-like IR was strongly detected in Kupffer cells where ET-1(1–21)-like IR was seen more weakly. In the kidney, ET-1(1–31)-like IR was slightly higher than ET-1(1–21). These results suggest that ET-1(1–31) might have physiological roles distinct from those of ET-1(1–21) in some hamster tissues.

Urocortin (Ucn) is a member of the corticotropin-releasing hormone (CRH)-related peptides that has been reported to have cardiac inotropic and hypertrophic effects. In addition, Ucn mRNA was expressed in cardiac myocytes (MCs) and Ucn was suggested to have cardioprotective effects. Recently, it was reported that Ucn mRNA was expressed in cardiac non-myocytes (NMCs). Based on these facts, Ucn is assumed to affect not only MCs but also NMCs in an autocrine fashion. The present study was designed to elucidate a pathophysiological role of Ucn on NMCs. NMCs were prepared by the discontinuous Percoll gradient and adhesion method. Ucn increased [ 3 H]-thymidine uptake into NMCs. Ucn also enhanced endothelin-1-induced increase of [ 3 H]-thymidine uptake into NMCs. Effects of Ucn on [ 3 H]-thymidine uptake into NMCs were significantly abolished by the protein kinase A inhibitor, H89 (10 −5 M), but not by a competitive antagonist of CRH receptors, astressin (10 −5 M). Ucn also increased intracellular cAMP accumulation more potently than CRH on a molar basis. Finally, both MCs and NMCs also secreted Ucn. Together with the recent findings, at least in NMCs, these data suggest that Ucn could exert its own actions via the cAMP signaling pathway, but not through known CRH type 2 receptors, in an autocrine fashion. In conclusion, the present study indicated that Ucn was secreted not only from MCs but also from NMCs and that the primary source of Ucn acting on heart was the heart itself. On the other hand, Ucn could proliferate NMCs as well as MCs, suggesting that Ucn could be involved in cardiac hypertrophy and fibrosis, i.e., cardiac remodeling, in spite of its putative cardioprotective actions.

The effects of Cl- channel blockers on endothelin-1 (ET-1)-induced proliferation of rat aortic vascular smooth muscle cells (VSMC) were examined. We found ET-1 concentration-dependently increased cell count and [3H]-thymidine incorporation into VSMC, with EC50 values of 24.8 and 11.4 nM, respectively. Both nifedipine and SKF96365 inhibited 10 nM ET-1-induced [3H]-thymidine incorporation into VSMC with the maximal inhibitory concentrations of 1 and 10 microM, respectively. DIDS inhibited 10 nM ET-1-induced increase in cell count and [3H]-thymidine incorporation into VSMC in a concentration-dependent manner, whereas other Cl- channel blockers including IAA-94, NPPB, DPC, SITS and furosemide did not produce these effects. 3 microM DIDS reduced 10 nM ET-1-induced sustained increase in cytoplasmic Ca2+ concentration ([Ca2+]) by 52%. Pretreatment of VSMC with 1 microM nifedipine completely inhibited the DIDS effect on 10 nM ET-1-induced [3H]-thymidine incorporation into VSMC and sustained increase in [Ca2+]i, whereas pretreatment with 10 microM SKF96365 did not completely block these effects of DIDS. DIDS did not affect ET-1-induced Ca2+ release and 30 mM KCl-induced increase in [Ca2+]i. Our data suggest that DIDS-sensitive Cl- channels mediate VSMC proliferation induced by ET-1 by mechanisms related to membrane depolarization and Ca2+ influx through voltage-dependent Ca2+ channels.

Endothelins are potent regulators of vascular tone, which also have mitogenic, apoptotic, and immunomodulatory properties (Rubanyi and Polokoff, 1994; Kedzierski and Yanagisawa, 2001; Bagnato et al., 2011). Three isoforms of endothelin have been identified to date, with endothelin-1 (ET-1) being the best studied. ET-1 is classically considered a potent vasoconstrictor. However, in addition to the effects of ET-1 on vascular smooth muscle cells, the peptide is increasingly recognized as a pro-inflammatory cytokine (Teder and Noble, 2000; Sessa et al., 1991). ET-1 causes platelet aggregation and plays a role in the increased expression of leukocyte adhesion molecules, the synthesis of inflammatory mediators contributing to vascular dysfunction. High levels of ET-1 are found in alveolar macrophages, leukocytes (Sessa et al., 1991) and fibroblasts (Gu et al., 1991). Clinical and experimental data indicate that ET-1 is involved in the pathogenesis of sepsis (Tschaikowsky et al., 2000; Goto et al., 2012), viral and bacterial pneumonia (Schuetz et al., 2008; Samransamruajkit et al., 2002), Rickettsia conorii infections (Davi et al., 1995), Chagas disease (Petkova et al., 2000, 2001), and severe malaria (Dai et al., 2012; Machado et al., 2006; Wenisch et al., 1996a; Dietmann et al., 2008). In this minireview, we will discuss the role of endothelin in the pathogenesis of infectious processes.

To evaluate the effects of endothelin (ET)-converting enzyme (ECE) inhibitor on vascular remodeling in dogs with congestive heart failure (CHF), we chronically administered an ECE inhibitor, FR901533 (FR, iv. 0.3mg/kg/hr, n=6), to dogs with CHF induced by rapid ventricular pacing. Vehicle CHF dogs were given saline (n=7). In the vehicle CHF group after 3 weeks of pacing, the ET system was activated in the plasma and vasculature (3 and 5 times higher than normal, respectively). Inward remodeling occurred in the femoral artery; medial thickness (MT, 225±5 vs 193±4 μm, P

Aims Cardiac hypertrophy is elicited by endothelin (ET)-1 as well as other neurohumoral factors, hemodynamic overload, and oxidative stress; HMG-CoA reductase inhibitors (statins) were shown to inhibit cardiac hypertrophy partly via the anti-oxidative stress. One of their common intracellular pathways is the phosphorylation cascade of MEK signaling. Pin1 specifically isomerizes the phosphorylated protein with Ser/Thr-Pro bonds and regulates their activity through conformational changes. There is no report whether the Pin1 activation contributes to ET-1-induced cardiomyocyte hypertrophy and whether the Pin1 inactivation contributes to the inhibitory effect of statins. The aim of this study was to reveal these questions. Main methods We assessed neonatal rat cardiomyocyte hypertrophy using ET-1 and fluvastatin by the cell surface area, ANP mRNA expression, JNK and c-Jun phosphorylation, and [ 3 H]-leucine incorporation. Key findings Fluvastatin inhibited ET-1-induced increase in the cell surface area, ANP expression, and [ 3 H]-leucine incorporation; and it suppressed the signaling cascade from JNK to c-Jun. The phosphorylated Pin1 level, an inactive form, was decreased by ET-1; however, it reached basal level by fluvastatin. Furthermore, Pin1 overexpression clearly elicited cardiomyocyte hypertrophy, which was inhibited by fluvastatin. Significance This is the first report that ET-1-induced cardiomyocyte hypertrophy is mediated through the Pin1 activation and that the inhibitory effect of fluvastatin on cardiomyocyte hypertrophy would partly be attributed to the suppression of the Pin1 function. This study firstly suggests that Pin1 determines the size of hypertrophied cardiomyocyte by regulating the activity of phosphorylated molecules and that statins exert their pleiotropic effects partly via Pin1 inactivation.

Aims: The purpose of the current study was to evaluate the significance of the endothelin system in catecholamine release and synthesis in the adrenal gland. Main Methods: Primary cultures of isolated bovine adrenal medullary cells were prepared. Gene expression, intracellular calcium changes, epinephrine release, and other factors were investigated. Key findings: Expression of endothelin receptors in the rat adrenal gland was confirmed. Intravenous infusion of endothelin-1 (1.0 nmol) increased blood pressure (systolic and diastolic). Endothelin-1 stimulated intracellular calcium changes, resulting in increased nuclear factor of T cell (NFAT) activity and epinephrine release from cultured adrenal medullary cells. Furthermore, endothelin-1 increased catecholamine synthesis and caused hypertrophic changes in the cell size. Significance: Our findings indicate involvement of the endothelin system in the sympathetic regulation of the adrenal medulla., 弘前医学. 65(2-4), 2014, p.218-226

Endothelin-1 (ET1) is a peptide that was initially identified as a strong inductor of vascular contraction. In the last 25 years, there have been several biological processes identified in which ET1 seems to play a critical role. In particular, genetic studies have unveiled that ET1 is important for neuronal development, growth and function. Experimental studies identified ET1 as a regulator of the interaction between sympathetic neurons and cardiac myocytes. This might be of clinical importance since patients suffering from heart failure are characterized by disrupted norepinephrine homeostasis in the heart. This review summarizes the important findings on the role of ET1 for sympathetic neurons and norepinephrine homeostasis in the heart.