Exogenous Endothelin-1 Research Articles

Contribution of Mitochondrial Reactive Oxygen Species to Chronic Hypoxia-Induced Pulmonary Hypertension.

Pulmonary hypertension (PH) resulting from chronic hypoxia (CH) occurs in patients with chronic obstructive pulmonary diseases, sleep apnea, and restrictive lung diseases, as well as in residents at high altitude. Previous studies from our group and others demonstrate a detrimental role of reactive oxygen species (ROS) in the pathogenesis of CH-induced PH, although the subcellular sources of ROS are not fully understood. We hypothesized that mitochondria-derived ROS (mtROS) contribute to enhanced vasoconstrictor reactivity and PH following CH. To test the hypothesis, we exposed rats to 4 weeks of hypobaric hypoxia (PB ≈ 380 mmHg), with control rats housed in ambient air (PB ≈ 630 mmHg). Chronic oral administration of the mitochondria-targeted antioxidant MitoQ attenuated CH-induced decreases in pulmonary artery (PA) acceleration time, increases in right ventricular systolic pressure, right ventricular hypertrophy, and pulmonary arterial remodeling. In addition, endothelium-intact PAs from CH rats exhibited a significantly greater basal tone compared to those from control animals, as was eliminated via MitoQ. CH also augmented the basal tone in endothelium-disrupted PAs, a response associated with increased mtROS production in primary PA smooth muscle cells (PASMCs) from CH rats. However, we further uncovered an effect of NO synthase inhibition with Nω-nitro-L-arginine (L-NNA) to unmask a potent endothelial vasoconstrictor influence that accentuates mtROS-dependent vasoconstriction following CH. This basal tone augmentation in the presence of L-NNA disappeared following combined endothelin A and B receptor blockade with BQ123 and BQ788. The effects of using CH to augment vasoconstriction and PASMC mtROS production in exogenous endothelin 1 (ET-1) were similarly prevented by MitoQ. We conclude that mtROS participate in the development of CH-induced PH. Furthermore, mtROS signaling in PASMCs is centrally involved in enhanced pulmonary arterial constriction following CH, a response potentiated by endogenous ET-1.

Endogenous Endothelin‐1 Contributes to Enhanced Basal Pulmonary Arterial Tone Following Chronic Hypoxia: Role of Mitochondria‐Derived Reactive Oxygen Species

Previous studies from our laboratory demonstrate the importance of mitochondria-derived reactive oxygen species (mitoROS) in the development of pulmonary hypertension after chronic hypoxia (CH) exposure. Furthermore, we have identified a major role for the endothelium to augment mitoROS-dependent pulmonary vascular tone following CH. However, the mechanism of this response remains unclear. We hypothesized that the endogenous vasoconstrictor peptide, endothelin-1 (ET-1), facilitates mitoROS-dependent increases in basal tone after CH exposure. To test our hypothesis, we measured basal tone by videomicroscopy in isolated, pressurized, small pulmonary arteries [~150 μm inner diameter (i.d.)] from normoxic and CH (4 wk, 0.5 atm) rats with and without the ETA receptor and ETB receptor inhibitors, BQ123 and BQ788 (10 μM each), respectively. Parallel protocols were conducted in the presence of the mitochondria-targeted antioxidant, MitoQ (1 μM). All experiments were performed in the presence of the nitric oxide (NO) synthase inhibitor, Nω-nitro-L-arginine (300 μM), to limit the influence of endogenous NO. Pulmonary arterial tone was calculated as the percent difference in i.d. between Ca2+-free and Ca2+-containing conditions. To evaluate the contribution of mitoROS to ET-1-mediated vasoconstriction, we further assessed vasoconstrictor responses (percentage of baseline i.d.) to serial concentrations of exogenous ET-1 (10-11 to 10-7 M) in the presence or absence of MitoQ in endothelium-disrupted pulmonary arteries from each group. Additional experiments employed confocal microscopy to measure ET-1 (1 nM)-induced mitoROS production in primary cultures of pulmonary arterial smooth muscle cells (PASMCs) from control and CH rats using the fluorescent mitochondrial superoxide indicator MitoSOX (10 μM). Antimycin A (10 µM) treatment, which stimulates mitoROS production from complex III of electron transport chain, was used as a positive control. Both MitoQ and the combined administration of ETA and ETB receptor blockers abolished the effect of CH to increase basal tone (P<0.05). Arteries from CH rats also exhibited greater constriction to exogenous ET-1 (at 1 nM) compared to vessels from normoxic controls, which was prevented by MitoQ (P<0.05). Consistent with these findings, application of ET-1 significantly increased mitoROS production in PASMCs from CH rats (P<0.05), but not control animals. Application of antimycin A led to mitoROS generation in both control and CH groups (P<0.05). We conclude that endogenous ET-1 promotes pulmonary arterial constriction through mitoROS signaling in PASMCs following CH. These findings advance our understanding of the mechanistic basis for the vasoconstrictor component of CH-induced pulmonary hypertension.

Targeting the Endothelin-1 Receptors Curtails Tumor Growth and Angiogenesis in Multiple Myeloma.

The endothelin-1 (ET-1) receptors were recently found to mediate pro-survival functions in multiple myeloma (MM) cells in response to autocrine ET-1. This study investigated the effectiveness of macitentan, a dual ET-1 receptor antagonist, in MM treatment, and the mechanisms underlying its activities. Macitentan affected significantly MM cell (RPMI-8226, U266, KMS-12-PE) survival and pro-angiogenic cytokine release by down-modulating ET-1-activated MAPK/ERK and HIF-1α pathways, respectively. HIF-1α silencing abrogated the ET-1 mediated induction of genes encoding for pro-angiogenic cytokines such as VEGF-A, IL-8, Adrenomedullin, and ET-1 itself. Upon exposure to macitentan, MM cells cultured in the presence of the hypoxia-mimetic agent CoCl2, exogenous ET-1, or CoCl2 plus ET-1, down-regulated HIF-1α and the transcription and release of downstream pro-angiogenic cytokines. Consistently, macitentan limited significantly the basal pro-angiogenic activity of RPMI-8226 cells in chorioallantoic membrane assay. In xenograft mouse models, established by injecting NOG mice either via intra-caudal vein with U266 or subcutaneously with RPMI-8226 cells, macitentan reduced effectively the number of MM cells infiltrating bone marrow, and the size and microvascular density of subcutaneous MM tumors. ET-1 receptors targeting by macitentan represents an effective anti-proliferative and anti-angiogenic therapeutic approach in preclinical settings of MM.

Mast Cell Degranulation Increases Mouse Mast Cell Protease 4–Dependent Vasopressor Responses to Big Endothelin-1 But Not Angiotensin I

Mouse mast cell protease 4 (mMCP-4), the murine functional analog to the human chymase, is a serine protease synthesized and stored in mast cell secretory granules. Our previous studies reported physiologic and pathologic roles for mMCP-4 in the maturation and synthesis of the vasoactive peptide endothelin-1 (ET-1) from its precursor, big ET-1. The aim of this study was to investigate the impact of mast cell degranulation or stabilization on mMCP-4-dependent pressor responses after the administration of big ET-1 or angiotensin I (Ang I). In anesthetized mice, mast cell degranulation induced by compound 48/80 (C48/80) or stabilization by cromolyn enhanced or repressed, respectively, the dose-dependent vasopressor responses to big ET-1 in wild-type (WT) mice but not in mMCP-4 knockout mice in a chymase inhibitor (TY-51469)-sensitive fashion. In addition, mMCP-4-dependent hydrolysis of the fluorogenic substrate Suc-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin was depleted or enhanced in peritoneal mast cells isolated from mice pretreated with C48/80 or cromolyn, respectively. Furthermore, C48/80 or cromolyn markedly increased or abolished, respectively, ET-1 (1-31) conversion from exogenous big ET-1 in WT mice peritoneal fluid-isolated mast cells, in vitro. Finally, the vasopressor responses to Ang I were unaffected by mast cell activation or stabilization, whereas those induced by the angiotensin-converting enzyme-resistant Ang I analog, [Pro11, D-Ala12] Ang I, were potentiated by C48/80. Altogether, the present study shows that mast cell activation enhances the mMCP-4-dependent vasoactive properties of big ET-1 but not Ang I in the mouse model. SIGNIFICANCE STATEMENT: The current work demonstrates a significant role for mast cell stability in the cardiovascular pharmacology of big endothelin-1 but not angiotensin I in the murine systemic circulation.

Renomedullary Interstitial Cell Endothelin A Receptors Regulated Blood Pressure and Renal Function

Renomedullary interstitial cells (RMIC) may modulate blood pressure (BP) and renal function through release of natriuretic factors and through direct effects on medullary blood flow (RMIC encircle vasa recta). Endothelin‐1 (ET‐1) modulates RMIC vasoactive factor release and contraction via ETA receptors (ETAR) in vitro. Consequently, we tested the hypothesis that knockout of ETAR in RMIC regulates renal function and BP in vivo. We bred ETARf/f mice containing loxP‐flanked exons 6–8 of the EDNRA gene with tamoxifen‐inducible tenascin‐C‐CreER2 knockin mice. Tenascin‐C‐CreER2:ETARf/f mice were injected with tamoxifen (160 mg/kg IP) for 5 consecutive days at 3 months of age to obtain RMIC ETAR KO. Littermates of the same genotype without injection served as controls. EDNRA gene recombination occurred in inner medulla, but not cortex. Inner medulla ETAR mRNA content fell by 80%, while ETBR mRNA levels were unaffected. ETRA KO did not affect BP in mice on normal (0.3% Na+) or low (0.03% Na+) salt diets. In contrast, ETAR KO reduced salt‐sensitive systolic BP elevation in mice on a high (3.2% Na+) salt diet (ETAR KO 127±3 vs. control 134±2 mmHg, n=13, 15, p&lt;0.05). Urine volume and urinary Na+ and K+ excretion were higher in high‐salt diet‐treated ETAR KO mice compared with controls during the first 3 days of high salt intake. Urinary ET‐1, nitrite/nitrate, and prostaglandin E2 excretion were increased in high salt diet‐treated ETAR KO mice vs. controls. No significant differences were found in GFR, vasopressin, aldosterone, or renin levels between high salt fed ETAR KO and control mice. Inner medullary protein and mRNA levels of total NKCC2 and ENaC‐α and ‐β were decreased, and inner medullary cyclooxygenase‐2 (COX‐2), but not COX‐1, protein levels were increased, in high salt fed ETAR KO vs. control mice (n=8). Exogenous ET‐1 did not detectably alter RMIC intracellular Ca2+ concentration or cause RMIC contraction as assessed by ex vivo inner medulla confocal imaging. We conclude that disruption of RMIC ETAR reduces salt‐sensitive hypertension and this is associated with reductions in NKCC2 and ENaC together with elevations in several autocrine and paracrine natriuretic factors. These effects of RMIC ETAR, at least based on ex vivo studies, may not involve RMIC contraction. While further study is required, our data suggest a novel regulatory pathway for maintaining water/electrolyte homeostasis.Support or Funding InformationNHLBI P01 HL136267

A novel role for myeloid endothelin-B receptors in hypertension.

AimsHypertension is common. Recent data suggest that macrophages (Mφ) contribute to, and protect from, hypertension. Endothelin-1 (ET-1) is the most potent endogenous vasoconstrictor with additional pro-inflammatory properties. We investigated the role of the ET system in experimental and clinical hypertension by modifying Mφ number and phenotype.Methods and results In vitro, Mφ ET receptor function was explored using pharmacological, gene silencing, and knockout approaches. Using the CD11b-DTR mouse and novel mice with myeloid cell-specific endothelin-B (ETB) receptor deficiency (LysMETB−/−), we explored the effects of modifying Mφ number and phenotype on the hypertensive effects of ET-1, angiotensin II (ANG II), a model that is ET-1 dependent, and salt. In patients with small vessel vasculitis, the impacts of Mφ depleting and non-depleting therapies on blood pressure (BP) and endothelial function were examined. Mouse and human Mφ expressed both endothelin-A and ETB receptors and displayed chemokinesis to ET-1. However, stimulation of Mφ with exogenous ET-1 did not polarize Mφ phenotype. Interestingly, both mouse and human Mφ cleared ET-1 through ETB receptor mediated, and dynamin-dependent, endocytosis. Mφ depletion resulted in an augmented chronic hypertensive response to both ET-1 and salt. LysMETB−/− mice displayed an exaggerated hypertensive response to both ET-1 and ANG II. Finally, in patients who received Mφ depleting immunotherapy BP was higher and endothelial function worse than in those receiving non-depleting therapies.ConclusionMφ and ET-1 may play an important role in BP control and potentially have a critical role as a therapeutic target in hypertension.

Endothelin-1 inhibition improves the mBDNF/proBDNF ratio in endothelial cells and HT22 neurons under high glucose/palmitate growth conditions.

Diabetes increases the risk and worsens the progression of cognitive impairment. The hippocampus is an important domain for learning and memory. We previously showed that endothelin-1 (ET-1) reduced diabetes-induced inflammation in hippocampal neurons, suggesting a neuroprotective effect. Given that neurons and endothelial cells within the neurovascular unit depend on each other for proper function, we investigated the effect of ET-1 on brain-derived neurotrophic factor (BDNF) synthesis, a key neurotrophin and prosurvival factor, in neuronal (HT22 hippocampal neurons) and brain microvascular endothelial (BMEC-5i) cells under normal and diabetes-mimicking (high glucose plus palmitate) conditions. Cells were treated with exogenous ET-1 or ET receptor antagonists including ET(B) receptor selective antagonist BQ788 (1 microM) or dual-receptor antagonist bosentan (10 microM). Mature (m)BDNF, proBDNF and caspase-3 levels were measured by Western blotting. Diabetic conditions reduced the prosurvival mBDNF/proBDNF ratio in both HT22 and BMEC-5i cells. Addition of exogenous ET-1 had no effect on the BDNF system in HT22 cells in diabetic conditions. Both HT22 and BMEC-5i cells had an increase in the mBDNF/proBDNF ratio when grown in diabetes-simulating conditions in the presence of endothelin receptor inhibition. These data suggest that blockade of ET-1 may provide neuroprotection to hippocampal cells through the modulation of the BDNF system.

Lipopolysaccharide-induced necroptosis of brain microvascular endothelial cells can be prevented by inhibition of endothelin receptors.

Over activation of the endothelin-1 (ET-1) system in disease states contributes to endothelial dysfunction. On the other hand, ET-1 promotes proliferation and survival of endothelial cells. Regulation of programmed cell death (PCD) pathways is critical for cell survival. Recently discovered necroptosis (regulated necrosis) is a pathological PCD mechanism mediated by the activation of toll like receptor 4 (TLR4), which also happens to stimulate ET-1 production in dendritic cells. To establish the effect of ET-1 on PCD and survival of human brain microvascular endothelial cells (BMVECs) under control and inflammatory conditions, BMVECs were treated with ET-1 (10 nM, 100 nM and 1 microM) or lipopolysaccharide (LPS, 100 ng/ml). ET receptors were blocked with bosentan (10 microM). Under normal growth conditions, exogenous ET-1 reduced BMVEC viability and migration at a relatively high concentration (1 microM). This was accompanied with activation of necroptosis and apoptosis marker genes. LPS decreased endogenous ET-1 secretion, increased ET(B) receptor expression and activated necroptosis. Even though ET-1 levels were low (less than 10 nM levels used under normal growth conditions), blocking of ET receptors with bosentan inhibited the necroptosis pathway and improved the cell migration ability of BMVECs, suggesting that under inflammatory conditions, ET-1 activates PCD pathways in BMVECs even at physiological levels.

Rho kinase and Na+ /H+ exchanger mediate endothelin-1-induced pulmonary arterial smooth musclecell proliferation and migration.

Excessive production of endothelin‐1 (ET‐1) has been observed in almost all forms of pulmonary hypertension. ET‐1, a highly potent vasoconstrictor, can also potentiate pulmonary arterial smooth muscle cell (PASMC) growth and migration, both of which contribute to the vascular remodeling that occurs during the development of pulmonary hypertension. Increasing evidence indicates that alkalinization of intracellular pH (pH i), typically due to activation of Na+/H+ exchange (NHE), is associated with enhanced PASMC proliferation and migration. We recently demonstrated that application of exogenous ET‐1 increased NHE activity in murine PASMCs via a mechanism requiring Rho kinase (ROCK). However, whether ROCK and/or increased NHE activity mediate ET‐1‐induced migration and proliferation in PASMCs remains unknown. In this study, we used fluorescent microscopy in transiently cultured PASMCs from distal pulmonary arteries of the rat and the pH‐sensitive dye, BCECF‐AM, to measure changes in resting pH i and NHE activity induced by exposure to exogenous ET‐1 (10−8 mol/L) for 24 h. Cell migration and proliferation in response to ET‐1 were also measured using Transwell assays and BrdU incorporation, respectively. We found that application of exogenous ET‐1 had no effect on NHE1 expression, but increased pH i, NHE activity, migration, and proliferation in rat PASMCs. Pharmacologic inhibition of NHE or ROCK prevented the ET‐1‐induced changes in cell function (proliferation and migration). Our results indicate that ET‐1 modulates PASMC migration and proliferation via changes in pH i homeostasis through a pathway involving ROCK.

Effects Of Endothelin-1 On Intracellular Tetrahydrobiopterin Levels In Vascular Tissue

Objective: Tetrahydrobiopterin (BH4) is the essential cofactor of endothelial nitric oxide synthase (eNOS) and intracellular levels of BH4 is regulated by oxidative stress. The aim of this paper was to describe the influence of exogenous endothelin-1 on intracellular BH4 and its oxidation products dihydrobiopterin (BH2) and biopterin (B) in a wide range of vascular tissue.Design: Segments of internal mammary artery (IMA) and human saphenous vein (SV) from 41 patients undergoing elective surgery were incubated in ET-1 (0.1 μM). Aorta and lung from transgenic mice overexpressing ET-1 in the endothelium (ET-TG) were analysed with regards to intracellular biopterin levels. Human umbilical vein endothelial cells (HUVEC) were incubated in ET-1 (0.1 μM) and intracellular biopterin levels were analysed. From 6 healthy women undergoing caesarean section, subcutaneous fat was harvested and the resistance arteries in these biopsies were tested for ET-mediated endothelial dysfunction.Results: In HUVEC, exogenous ET-1 (0.1 μM) did not significantly change intracellular BH4, 1.54 ± 1.7 vs 1.68 ± 1.8 pmol/mg protein; p = .8. In IMA and SV, exogenous ET-1(0.1 μM) did not change intracellular BH4 n = 10, p = .4. In aorta from wild type vs ET-TG mice there was no significant difference in intracellular BH4 between the groups: 1.3 ± 0.49 vs 1.23 ± 0.3 pmol/mg protein; p = .6. In resistance arteries (n = 6) BH4 together with DTE (an antioxidant) was not able to prevent ET-mediated endothelial dysfunction.Conclusion: ET-1 did not significantly alter intracellular tetrahydrobiopterin levels in IMA, SV, HUVEC or aorta from ET-TG mice. These findings are important for future research in ET-1 mediated superoxide production and endothelial dysfunction.

Hippocampal Endothelin-1 decreases excitability of pyramidal neurons and produces anxiolytic effects

Anxiety disorders contribute to the pathophysiology of psychiatric diseases, including major depression, substance abuse, and schizophrenia. The hippocampus is important for anxiety modulation. However, the mechanisms that control the neuronal activity of the hippocampus in anxiety are still not clear. We found that Endothelin-1 (ET1) mRNA in the hippocampus was down-regulated in high-anxiety mice. Neutralizing endogenous ET1 in the hippocampal CA1 enhanced anxiety-like behaviors. We next revealed that most expression of ET1 and its receptors in the CA1 takes place in pyramidal neurons, and the ET1 signaling pathway directly regulated the excitability of CA1 pyramidal neurons and glutamatergic synaptic neurotransmission. Finally, we proved that neutralizing endogenous CA1 ET1 produces anxiogenic effects on low-anxiety mice, whereas infusing exogenous ET1 into the CA1 alleviates the anxiety susceptibility of high-anxiety mice. Together, these results indicate that ET1 signaling is critical in maintaining the excitability of glutamatergic neurons in the hippocampus and, thus, in modulating anxiety-like behaviors. Because ET1 is a risk factor for ischemic stroke, our findings might also help to explain the potential mechanism of emotional abnormality in stroke.

Mice, double deficient in lysosomal serine carboxypeptidases Scpep1 and Cathepsin A develop the hyperproliferative vesicular corneal dystrophy and hypertrophic skin thickenings

Vasoactive and mitogenic peptide, endothelin-1 (ET-1) plays an important role in physiology of the ocular tissues by regulating the growth of corneal epithelial cells and maintaining the hemodynamics of intraocular fluids. We have previously established that ET-1 can be degraded in vivo by two lysosomal/secreted serine carboxypeptidases, Cathepsin A (CathA) and Serine Carboxypeptidase 1 (Scpep1) and that gene-targeted CathAS190A /Scpep1-/- mice, deficient in CathA and Scpep1 have a prolonged half-life of circulating ET-1 associated with systemic hypertension. In the current work we report that starting from 6 months of age, ~43% of CathAS190A /Scpep1-/- mice developed corneal clouding that eventually caused vision impairment. Histological evaluation of these mice demonstrated a selective fibrotic thickening and vacuolization of the corneas, resembling human hyperproliferative vesicular corneal stromal dystrophy and coexisting with a peculiar thickening of the skin epidermis. Moreover, we found that cultured corneal epithelial cells, skin fibroblasts and vascular smooth muscle cells derived from CathA/Scpep1-deficient mice, demonstrated a significantly higher proliferative response to treatment with exogenous ET-1, as compared with cells from wild type mice. We also detected increased activation level of ERK1/2 and AKT kinases involved in cell proliferation in the ET-1-treated cultured cells from CathA/Scpep1 deficient mice. Together, results from our experimental model suggest that; in normal tissues the tandem of serine carboxypeptidases, Scpep1 and CathA likely constitutes an important part of the physiological mechanism responsible for the balanced elimination of heightened levels of ET-1 that otherwise would accumulate in tissues and consequently contribute to development of the hyper-proliferative corneal dystrophy and abnormal skin thickening.

Endothelin signaling regulates mineralization and posttranscriptionally regulates SOST in TMOb cells via miR 126-3p.

Previously, our laboratory identified ECE‐1, encoding endothelin‐converting enzyme‐1 (ECE‐1), as a positional candidate for a pleiotropic quantitative trait locus affecting femoral size, shape, and biomechanical performance. We hypothesized that endothelin‐1 (ET‐1) signaling promotes osteogenesis. Exposure of immortalized mouse osteoblast (TMOb) cells to big ET‐1 increased mineralization. Following big ET‐1 treatment, we measured the secretion of insulin‐like‐growth factor‐1 (IGF1), dickkopf‐homolog‐1 protein 1 (DKK1), and sclerostin (SOST). In each case, big ET‐1 signaling changed secretion in a manner that favored increased osteogenic activity. Treatment with ECE‐1, endothelin receptor A (EDNRA), or WNT receptor antagonists inhibited the big ET‐1‐mediated increase in mineralization. In the presence of big ET‐1, message levels of Runx2, Igf1, Dkk1, and Sost are uncoupled from protein production, suggesting posttranscriptional regulation. To evaluate the role of big ET‐1 in normal bone physiology, we inhibited EDNRA signaling during mineralization in the absence of exogenous ET‐1. EDNRA blockade reduced mineralization, decreased IGF1, and increased DKK1 and SOST secretion, responses opposite to those induced by exogenous big ET‐1. Pharmacological and siRNA knockdown to inhibit ECE‐1 reduced mineralization and IGF1 secretion with decreasing DKK1 and decreasing or stable SOST secretion, suggesting a further, unknown role of ECE‐1 in osteoblast maturation. Previously we identified miR 126‐3p as a potential ET‐1‐responsive regulator of SOST in murine cells. Overexpression of miR126‐3p increased mineralization in TMOb cells and decreased SOST secretion. Osteoblasts express the ET‐1 signaling pathway and ET‐1 signaling is necessary for normal osteoblast differentiation and mineralization, acting through regulation of miRs that target osteogenic molecules.

Salvianolic Acid A, as a Novel ETA Receptor Antagonist, Shows Inhibitory Effects on Tumor in Vitro.

Endothelin-1 (ET-1) autocrine and paracrine signaling modulate cell proliferation of tumor cells by activating its receptors, endothelin A receptor (ETAR) and endothelin B receptor (ETBR). Dysregulation of ETAR activation promotes tumor development and progression. The potential of ETAR antagonists and the dual-ETAR and ETBR antagonists as therapeutic approaches are under preclinical and clinical studies. Salvianolic acid A (Sal A) is a hydrophilic polyphenolic derivative isolated from Salvia miltiorrhiza Bunge (Danshen), which has been reported as an anti-cancer and cardio-protective herbal medicine. In this study, we demonstrate that Sal A inhibits ETAR activation induced by ET-1 in both recombinant and endogenous ETAR expression cell lines. The IC50 values were determined as 5.7 µM in the HEK293/ETAR cell line and 3.14 µM in HeLa cells, respectively. Furthermore, our results showed that Sal A suppressed cell proliferation and extended the doubling times of multiple cancer cells, including HeLa, DU145, H1975, and A549 cell lines. In addition, Sal A inhibited proliferation of DU145 cell lines stimulated by exogenous ET-1 treatment. Moreover, the cytotoxicity and cardio-toxicity of Sal A were assessed in human umbilical vein endothelial cells (HUVEC) and Human-induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs), which proved that Sal A demonstrates no cytotoxicity or cardiotoxicity. Collectively, our findings indicate that Sal A is a novel anti-cancer candidate through targeting ETAR.

Abstract 2943: Hypoxia induced resistance to chemotherapy is regulated by the endothelin receptor axis

Abstract Introduction Tumor cells growing in the brain are resistant to chemotherapy. We have recently demonstrated that the activation of the endothelin receptors A and B axis is involved in the induction of resistance to chemotherapy by primary brain tumor cells and by brain metastases. Treatment with a dual endothelin receptor antagonist in combination with chemotherapy produced significant therapy of orthotopically growing glioblastoma cells as well as breast cancer and lung cancer brain metastases. Since the milieu of tumors in the brain is hypoxic (0.5% - 1% O2), we investigated whether the chemo-resistance of the tumor cells is linked to hypoxia mediated activation of the endothelin receptor axis. Materials and Methods Human glioma (LN229), breast cancer (MDA231), and lung adenocarcinoma (PC14) cell lines were cultured in DMEM supplemented with 5% FBS. The endothelin receptor A (ETAR) and/or B (ETBR) of cancer cells were knocked down by shRNA. ETAR and ETBR were activated by incubating the tumor cells with exogenous endothelin-1 for 15 minutes or blocked by treating the parental or engineered cells with ETAR-specific antagonist, BQ123 (100nM), and/or ETBR-specific antagonist, BQ788 (100nM), for 2 hours prior to the addition of paclitaxel (5ng/ml), temozolomide (100μg/ml) or cisplatinum (5μg/ml). The cultures were placed into incubators with an atmosphere of 20%, 6% or 1% oxygen for 48 hours (MDA231 cells) or 72 hours (LN229 and PC14 cells). Tumor cells were then plated into 96-well plates to determine chemosensitivity by the MTT assay or into 6-well plates for western blots, or into chamber slides for immunohistochemical analyses. Expression of survival-related proteins such as pETAR, pETBR, pAkt, pMAPK, pNFκB, GSTA5, TWIST1 or Bcl2L1, were then determined. Results Stimulation of parental MDA-231, PC-14, or LN229 cells with exogenous ET-1 induced significant resistance against all tested chemotherapeutic agents. The resistance was abolished only when both the ETAR and ETBR were antagonized by a combination of BQ123 and BQ788. Parental cells cultured under hypoxia were also significantly resistant to chemotherapy and treatment of these cells with BQ123 and BQ788 reversed this resistance. The effects of exogenous ET-1 on the induction of chemo-resistance or BQ123 and BQ788 on restoration of chemo-sensitivity were not found in any cancer cells devoid of ETAR and ETBR. In all cases, the chemo-resistance of tumor cells was associated with increased level of expression of proteins related to cell survival. Conclusion These data suggest that the endothelin receptor axis plays a role in hypoxia induced resistance of cancer cells to chemotherapy. Additional studies are warranted to determine the functional changes of the endothelin receptor axis in hypoxia. Citation Format: Hyun Kyung Yu, Ho Jeong Lee, Fahao Zhang, Qiuyu Wu, Isaiah J. Fidler, Sun Jin Kim. Hypoxia induced resistance to chemotherapy is regulated by the endothelin receptor axis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2943.

Immature endothelial cells initiate endothelin-mediated constriction of newborn arteries.

Endothelial expression and the release of endothelin-1 (ET-1) in levels sufficient to initiate vasoconstriction is considered to be a hallmark feature of pathological endothelial dysfunction. During the immediate postnatal period, arterial endothelial cells undergo remarkable structural and functional changes as they transition to a mature protective cell layer, which includes a marked increase in NO dilator activity. The present study demonstrates that endothelial cells lining newborn central arteries express high levels of ET-1 peptides and, in response to endothelial stimulation, rapidly release ET-1 and initiate powerful ET-1-mediated constriction. This activity is lost as the endothelium matures in the postnatal period. Heightened activity of ET-1 in the neonatal endothelium might contribute to inappropriate responses of immature arteries to stress or injury. Indeed, the immature endothelium resembles dysfunctional endothelial cells, and retention or re-emergence of this phenotype may contribute to the development of vascular disease. Endothelial cells lining fetal and newborn arteries have an unusual phenotype, including reduced NO activity, prominent actin stress fibres and poorly developed cellular junctions. Experiments were performed to determine whether the immature endothelium of newborn arteries also expresses and releases endothelin-1 (ET-1) and initiates endothelium-dependent constriction. Carotid arteries were isolated from newborn (postnatal day 1; P1), postnatal day 7 (P7) and postnatal day 21 (P21) mice and assessed in a pressure myograph system. Endothelial stimulation with A23187 or thrombin caused constriction in P1 arteries, no significant change in diameter of P7 arteries, and dilatation in P21 arteries. In P1 arteries, constriction to thrombin or A23187 was inhibited by endothelial-denudation, by ET-1 receptor antagonists (BQ123 plus BQ788) or by inhibition of endothelin-converting enzyme (phosphoramidon or SM19712). ET-1 receptor antagonism did not affect responses to thrombin or A23187 in more mature arteries. Exogenous ET-1 caused similar concentration-dependent constrictions of P1, P7 and P21 arteries. Endothelial stimulation with thrombin rapidly increased the endothelial release of ET-1 from P1 but not P21 aortas. Endothelial expression of ET-1 peptides, as assessed by immunofluorescence analysis, was increased in P1 compared to P21 arteries. Therefore, newborn endothelial cells express high levels of ET-1 peptides, rapidly release ET-1 in response to endothelial stimulation, and initiate ET-1-mediated endothelium-dependent constriction. This activity is diminished as the endothelium matures in the immediate postnatal period. Heightened activity of ET-1 in neonatal endothelium probably reflects an early developmental role of the peptide, although this might contribute to inappropriate responses of immature arteries to stress or injury.

Endothelin-1 shifts the mediator of bradykinin-induced relaxation from NO to H2 O2 in resistance arteries from patients with cardiovascular disease.

We tested the hypothesis that in resistance arteries from cardiovascular disease (CVD) patients, effects of an endothelium-dependent vasodilator depend on the contractile stimulus. Arteries dissected from parietal pericardium of cardiothoracic surgery patients were studied by myography and imaging techniques. Segments were sub-maximally contracted by K(+) , the TxA2 analogue U46619 or endothelin-1 (ET-1). Relaxing effects of Na-nitroprusside were comparable, but those of bradykinin (BK) were bigger in the presence of ET-1 compared with K(+) or U46619. BK-induced relaxation was (i) abolished by L-NAME in K(+) -contracted arteries, (ii) partly inhibited by L-NAME in the presence of U46619 and (iii) not altered by indomethacin, L-NAME plus inhibitors of small and intermediate conductance calcium-activated K(+) channels, but attenuated by catalase, in ET-1-contracted arteries. This catalase-sensitive relaxation was unaffected by inhibitors of NADPH oxidases or allopurinol. Exogenous H2 O2 caused a larger relaxation of ET-1-induced contractions than those evoked by K(+) or U46619 in the presence of inhibitors of other endothelium-derived relaxing factors. Catalase-sensitive staining of cellular ROS with CellROX Deep Red was significantly increased in the presence of both 1μM BK and 2nM ET-1 but not either peptide alone. In resistance arteries from patients with CVD, exogenous ET-1 shifts the mediator of relaxing responses to the endothelium-dependent vasodilator BK from NO to H2 O2 and neither NADPH oxidases, xanthine oxidase nor NOS appear to be involved in this effect. This might have consequences for endothelial dysfunction in conditions where intra-arterial levels of ET-1 are enhanced.

Endothelin-1 Mediates Brain Microvascular Dysfunction Leading to Long-Term Cognitive Impairment in a Model of Experimental Cerebral Malaria.

Plasmodium falciparum infection causes a wide spectrum of diseases, including cerebral malaria, a potentially life-threatening encephalopathy. Vasculopathy is thought to contribute to cerebral malaria pathogenesis. The vasoactive compound endothelin-1, a key participant in many inflammatory processes, likely mediates vascular and cognitive dysfunctions in cerebral malaria. We previously demonstrated that C57BL6 mice infected with P. berghei ANKA, our fatal experimental cerebral malaria model, sustained memory loss. Herein, we demonstrate that an endothelin type A receptor (ETA) antagonist prevented experimental cerebral malaria-induced neurocognitive impairments and improved survival. ETA antagonism prevented blood-brain barrier disruption and cerebral vasoconstriction during experimental cerebral malaria, and reduced brain endothelial activation, diminishing brain microvascular congestion. Furthermore, exogenous endothelin-1 administration to P. berghei NK65-infected mice, a model generally regarded as a non-cerebral malaria negative control for P. berghei ANKA infection, led to experimental cerebral malaria-like memory deficits. Our data indicate that endothelin-1 is critical in the development of cerebrovascular and cognitive impairments with experimental cerebral malaria. This vasoactive peptide may thus serve as a potential target for adjunctive therapy in the management of cerebral malaria.

Endothelin-1 induces LIMK2-mediated programmed necrotic neuronal death independent of NOS activity.

BackgroundRecently, we have reported that LIM kinase 2 (LIMK2) involves programmed necrotic neuronal deaths induced by aberrant cyclin D1 expression following status epilepticus (SE). Up-regulation of LIMK2 expression induces neuronal necrosis by impairment of dynamin-related protein 1 (DRP1)-mediated mitochondrial fission. However, we could not elucidate the upstream effecter for LIMK2-mediated neuronal death. Thus, we investigated the role of endothelin-1 (ET-1) in LIMK2-mediated neuronal necrosis, since ET-1 involves neuronal death via various pathways.ResultsFollowing SE, ET-1 concentration and its mRNA were significantly increased in the hippocampus with up-regulation of ETB receptor expression. BQ788 (an ETB receptor antagonist) effectively attenuated SE-induced neuronal damage as well as reduction in LIMK2 mRNA/protein expression. In addition, BQ788 alleviated up-regulation of Rho kinase 1 (ROCK1) expression and impairment of DRP1-mediated mitochondrial fission in CA1 neurons following SE. BQ788 also attenuated neuronal death and up-regulation of LIMK2 expression induced by exogenous ET-1 injection.ConclusionThese findings suggest that ET-1 may be one of the upstream effectors for programmed neuronal necrosis through abnormal LIMK2 over-expression by ROCK1.

Bile Synthesis Peculiarities following Changes in the Functional State of the Endothelin Receptors

Endothelin-1(ET-1) regulates a variety of biochemical processes in liver. However, there is no clear view concerning endothelin receptors participation in the regulation of qualitative and quantitative characteristics of liver secretory function. The purpose of this study is to evaluate the choleretic effect of ET-1 and to determine the role of ЕТА receptors functional state in mediating the effect of ET-1 on bile and its organic components secretion. Endothelin-1 and BQ-123 (cyclo-Asppro-Val-Leu-Trp) were intraportally injected. Bile flow, bile acid concentration and content, hydroxylation and conjugation coefficients were estimated. The results of this study showed that the secreted bile volume was decreased under the effect of endothelin-1and BQ-123, although this decrease was more prolonged and profound in BQ-123 treated animals. Concentration of taurocholates, glycocholic acid and free bile acids was increased in the endothelin treated rats. When BQ123 was administered, an increase in glycochenodeoxycholic acid+glycodeoxycholic acid (GCDCA+GDCA) and the taurin-conjugated bile acids concentration was found, whereas the free bile acids concentration altered reversely. Coefficient of hydroxylation was diminished when entholelin receptors were blocked. Activation of endothelin receptors by exogenous endothelin-1 intensified bile acids biosynthesis via “neutral pathway,”involving microsomal oxidation enzymes. The present study concludes that the endothelin receptors blockade eliminated the regulatory function of endogenous endothelin and caused a shift in bile acids synthesis to mitochondrial enzymes through “acidic pathway”.