Endothelin-1, Outcomes in Patients With Heart Failure and Reduced Ejection Fraction, and Effects of Dapagliflozin: Findings From DAPA-HF.

BACKGROUND: ET-1 (endothelin-1) is implicated in the pathophysiology of heart failure and renal disease. Its prognostic importance and relationship with kidney function in patients with heart failure with reduced ejection fraction receiving contemporary treatment are uncertain. We investigated these and the efficacy of dapagliflozin according to ET-1 level in the DAPA-HF trial (Dapagliflozin and Prevention of Adverse Outcomes in Heart Failure). METHODS: We investigated the incidence of the primary outcome (cardiovascular death or worsening heart failure), change in kidney function, and the effect of dapagliflozin according to baseline ET-1 concentration, adjusting in Cox models for other recognized prognostic variables in heart failure including NT-proBNP (N-terminal pro-B-type natriuretic peptide). We also examined the effect of dapagliflozin on ET-1 level. RESULTS: Overall, 3048 participants had baseline ET-1 measurements: tertile 1 (T1; ≤3.28 pg/mL; n=1016); T2 (>3.28-4.41 pg/mL; n=1022); and T3 (>4.41 pg/mL; n=1010). Patients with higher ET-1 were more likely male, more likely obese, and had lower left ventricular ejection fraction, lower estimated glomerular filtration rate, worse functional status, and higher NT-proBNP and hs-TnT (high-sensitivity troponin-T). In the adjusted Cox models, higher baseline ET-1 was independently associated with worse outcomes and steeper decline in kidney function (adjusted hazard ratio for primary outcome of 1.95 [95% CI, 1.53-2.50] for T3 and 1.36 [95% CI, 1.06-1.75] for T2; both versus T1; estimated glomerular filtration rate slope: T3, -3.19 [95% CI, -3.66 to -2.72] mL/min per 1.73 m2 per y, T2, -2.08 [95% CI, -2.52 to -1.63] and T1 -2.35 [95% CI, -2.79 to -1.91]; P=0.002). The benefit of dapagliflozin was consistent regardless of baseline ET-1, and the placebo-corrected decrease in ET-1 with dapagliflozin was 0.13 pg/mL (95% CI, 0.25-0.01; P=0.029). CONCLUSIONS: Higher baseline ET-1 concentration was independently associated with worse clinical outcomes and more rapid decline in kidney function. The benefit of dapagliflozin was consistent across the range of ET-1 concentrations measured, and treatment with dapagliflozin led to a small decrease in serum ET-1 concentration. REGISTRATION: URL: https://www. CLINICALTRIALS: gov; Unique identifier: NCT03036124.

Gestational intermittent hypoxia induces endothelial dysfunction and hypertension in pregnant rats: role of endothelin type B receptor†.

Obstructive sleep apnea is a recognized risk factor for gestational hypertension, yet the exact mechanism behind this association remains unclear. Here, we tested the hypothesis that intermittent hypoxia, a hallmark of obstructive sleep apnea, induces gestational hypertension through perturbed endothelin-1 signaling. Pregnant Sprague-Dawley rats were subjected to normoxia (control), mild intermittent hypoxia (10.5% O2), or severe intermittent hypoxia (6.5% O2) from gestational days 10-21. Blood pressure was monitored. Plasma was collected and mesenteric arteries were isolated for myograph and protein analyses. The mild and severe intermittent hypoxia groups demonstrated elevated blood pressure, reduced plasma nitrate/nitrite, and unchanged endothelin-1 levels compared to the control group. Western blot analysis revealed decreased expression of endothelin type B receptor and phosphorylated endothelial nitric oxide synthase, while the levels of endothelin type A receptor and total endothelial nitric oxide synthase remained unchanged following intermittent hypoxia exposure. The contractile responses to potassium chloride, phenylephrine, and endothelin-1 were unaffected in endothelium-denuded arteries from mild and severe intermittent hypoxia rats. However, mild and severe intermittent hypoxia rats exhibited impaired endothelium-dependent vasorelaxation responses to endothelin type B receptor agonist IRL-1620 and acetylcholine compared to controls. Endothelium denudation abolished IRL-1620-induced vasorelaxation, supporting the involvement of endothelium in endothelin type B receptor-mediated relaxation. Treatment with IRL-1620 during intermittent hypoxia exposure significantly attenuated intermittent hypoxia-induced hypertension in pregnant rats. This was associated with elevated circulating nitrate/nitrite levels, enhanced endothelin type B receptor expression, increased endothelial nitric oxide synthase activation, and improved vasodilation responses. Our data suggested that intermittent hypoxia exposure during gestation increases blood pressure in pregnant rats by suppressing endothelin type B receptor-mediated signaling, providing a molecular mechanism linking intermittent hypoxia and gestational hypertension.

Porcine Model of Cerebral Ischemic Stroke Utilizing Intracortical Recordings for the Continuous Monitoring of the Ischemic Area.

PURPOSE: Our aim was to use intracortical recording to enable the tracking of ischemic infarct development over the first few critical hours of ischemia with a high time resolution in pigs. We employed electrophysiological measurements to obtain quick feedback on neural function, which might be useful for screening, e.g., for the optimal dosage and timing of agents prior to further pre-clinical evaluation. METHODS: Micro-electrode arrays containing 16 (animal 1) or 32 electrodes (animal 2-7) were implanted in the primary somatosensory cortex of seven female pigs, and continuous electrical stimulation was applied at 0.2 Hz to a cuff electrode implanted on the ulnar nerve. Ischemic stroke was induced after 30 min of baseline recording by injection of endothelin-1 onto the cortex adjacent to the micro-electrode array. Evoked responses were extracted over a moving window of 180 s and averaged across channels as a measure of cortical excitability. RESULTS: Across the animals, the cortical excitability was significantly reduced in all seven 30 min segments following endothelin-1 injection, as compared to the 30 min preceding this intervention. This difference was not explained by changes in the anesthesia, ventilation, end-tidal CO2, mean blood pressure, heart rate, blood oxygenation, or core temperature, which all remained stable throughout the experiment. CONCLUSIONS: The animal model may assist in maturing neuroprotective approaches by testing them in an accessible model of resemblance to human neural and cardiovascular physiology and body size. This would constitute an intermediate step for translating positive results from rodent studies into human application, by more efficiently enabling effective optimization prior to chronic pre-clinical studies in large animals.

Aerobic exercise training reduces ET-1-mediated vasoconstriction and improves endothelium-dependent vasodilation in postmenopausal women.

Endothelin-1 (ET-1) contributes to vascular dysfunction in postmenopausal women (PMW). Although aerobic exercise is beneficial in reducing ET-1-mediated vasoconstrictor tone in men, it is unknown whether this favorable vascular effect occurs in women. We tested the hypothesis that aerobic exercise training reduces ET-1-mediated vasoconstriction in PMW. We further hypothesized that reductions in ET-1 vasoconstrictor tone underly exercise-induced improvements in endothelium-dependent vasodilatation in PMW. Forearm blood flow (FBF) responses to intra-arterial infusion of selective ETA receptor blockade (BQ-123, 100 nmol/min for 60 min) and acetylcholine (4.0, 8.0, and 16.0 µg/100 mL tissue/min) in the absence and presence of ETA receptor blockade were determined before and after a 12-wk aerobic exercise training intervention in 18 healthy, sedentary PMW (58 ± 4 yr). Women exercised an average of 4.9 ± 0.7 day/wk for 51 ± 7 min/day at 71 ± 3% of maximal heart rate. Before exercise, BQ-123 significantly increased FBF (∼25%) in sedentary PMW; however, this effect was abolished following the exercise intervention. FBF responses to acetylcholine were also significantly higher after exercise training (from 4.2 ± 1.2 to 14.0 ± 3.8 mL/100 mL tissue/min) versus before (from 4.1 ± 1.0 to 11.4 ± 3.3 mL/100 mL tissue/min; ∼25% increase; P < 0.05). Before exercise training, coinfusion of BQ-123 with acetylcholine enhanced (∼25%; P < 0.05) the vasodilator response (from 4.4 ± 1.1 to 13.9 ± 4.2 mL/100 mL tissue/min) compared with acetylcholine alone; after exercise training, the presence of BQ-123 did not significantly affect the vasodilator response to acetylcholine. Aerobic exercise training reduces ET-1-mediated vasoconstriction in PMW. Furthermore, decreased ET-1-mediated vasoconstriction is an important mechanism underlying aerobic exercise-induced improvement in endothelium-dependent vasodilation in PMW.NEW & NOTEWORTHY Endothelin-1 (ET-1) contributes to declines in endothelial function in postmenopausal women. To our knowledge, we show for the first time that aerobic exercise reduces ET-1-mediated vasoconstriction in previously sedentary postmenopausal women. Moreover, aerobic exercise improved endothelial-dependent dilation due in part to the reductions in ET-1-mediated vasoconstriction.

Endothelin-1 acutely increases nitric oxide production via the calcineurin mediated dephosphorylation of Caveolin-1.

Endothelin (ET)-1 is an endothelial-derived peptide that exerts biphasic effects on nitric oxide (NO) levels in endothelial cells such that acute exposure stimulates-while sustained exposure attenuates-NO production. Although the mechanism involved in the decrease in NO generation has been identified but the signaling involved in the acute increase in NO is still unresolved. This was the focus of this study. Our data indicate that exposing pulmonary arterial endothelial cells (PAEC) to ET-1 led to an increase in NO for up to 30min after which levels declined. These effects were attenuated by ET receptor antagonists. The increase in NO correlated with significant increases in pp60Src activity and increases in eNOS phosphorylation at Tyr83 and Ser1177. The ET-1 mediated increase in phosphorylation and NO generation were attenuated by the over-expression of a pp60Src dominant negative mutant. The increase in pp60Src activity correlated with a reduction in the interaction of Caveolin-1 with pp60Src and the calcineurin-mediated dephosphorylation of caveolin-1 at three previously unidentified sites: Thr91, Thr93, and Thr95. The calcineurin inhibitor, Tacrolimus, attenuated the acute increase in pp60Src activity induced by ET-1 and a calcineurin siRNA attenuated the ET-1 mediated increase in eNOS phosphorylation at Tyr83 and Ser1177 as well as the increase in NO. By using a Caveolin-1 celluSpot peptide array, we identified a peptide targeting a sequence located between aa 41-56 as the pp60Src binding region. This peptide fused to the TAT sequence was found to decrease caveolin-pp60Src interaction, increased pp60Src activity, increased eNOS pSer1177 and NO levels in PAEC and induce vasodilation in isolated aortic rings in wildtype but not eNOS knockout mice. Together, our data identify a novel mechanism by which ET-1 acutely increases NO via a calcineurin-mediated dephosphorylation of caveolin-1 and the subsequent stimulation of pp60Src activity, leading to increases in phosphorylation of eNOS at Tyr83 and Ser1177.

Inhibition of Full Smooth Muscle Contraction in Isolated Human Detrusor Tissues by Mirabegron Is Limited to Off-Target Inhibition of Neurogenic Contractions.

Mirabegron is used for treatment of storage symptoms in overactive bladder (OAB) caused by spontaneous bladder smooth muscle contractions. However, owing to limitations in available studies using human tissues, central questions are still unresolved, including mechanisms underlying improvements by mirabegron and its anticontractile effects in the detrusor. Here, we assessed concentration-dependent mirabegron effects on contractions of human detrusor tissues in frequency-response curves and concentration-response curves for different cholinergic and noncholinergic agonists. Detrusor tissues were sampled from patients undergoing radical cystectomy. Contractions were induced by electric field stimulation (EFS) and by cumulative concentrations of cholinergic agonists, endothelin-1, and the thromboxane A2 analog U46619. EFS-induced contractions were inhibited using 10 µM mirabegron, but not using 1 µM. Inhibition by 10 µM mirabegron was resistant to the ß 3-adrenergic antagonist L-748,337. Concentration-dependent contractions by carbachol were not inhibited by 1 µM or 10 µM mirabegron. Concentration-response curves for methacholine were slightly right-shifted by 10 µM, but not 1 µM mirabegron. Concentration-dependent contractions by endothelin-1 or U46619 were not changed by mirabegron. In contrast, the muscarinic antagonist tolterodine right-shifted concentration-response curves for carbachol and methacholine and inhibited EFS-induced contractions. In conclusion, inhibition of neurogenic contractions in isolated detrusor tissues by mirabegron requires concentrations highly exceeding known plasma levels during standard dosing and the known binding constant (Ki values) for ß 3-adrenoceptors. Full contractions by cholinergic agonists, endothelin-1, and U46619 are not affected by therapeutic concentrations of mirabegron. Improvements of storage symptoms are most likely not imparted by inhibition of ß 3-adrenoceptors in the bladder wall itself. SIGNIFICANCE STATEMENT: Mirabegron is used for overactive bladder (OAB) treatment, but the underlying mechanisms are unclear, and preclinical and clinical findings are controversial due to limitations in available studies. Our findings suggest that inhibition of detrusor contractions by mirabegron is limited to neurogenic contractions, which requires unphysiologic concentrations and does not involve ß 3-adrenoceptors. Mechanisms accounting for improvements of OAB by mirabegron are located outside the urinary bladder.

Preservation of Adrenoceptor and Endothelin Receptor Mediated Vasoconstriction and of Endothelium-Dependent Relaxation after Cold Storage of Explanted Blood Vessels for ex vivo Analyses.

INTRODUCTION: Adrenoceptor and endothelin (ET) receptor-mediated vasoconstriction as well as endothelium-dependent vasodilation of human saphenous veins were compared before and after 20 h of cold storage. METHODS: Contractile responses to potassium chloride (KCl), norepinephrine (NE), and ET-1 as well as vasodilator responses to acetylcholine (ACh) were evaluated. RESULTS: Storage in HEPES-supplemented Dulbecco's modified Eagle's medium (HDMEM) diminished KCl induced contractile forces to 71% (p = 0.002) and NE induced contractions to 80% (p = 0.037), in contrast to HEPES-supplemented Krebs-Henseleit solution (HKH) and TiProtec solution. KCl-normalized NE contractions were not affected by storage. NE EC50 values were slightly lower (7.1E-8 vs. 7.5E-8, p = 0.019) after storage in HKH, with no changes after storage in the other solutions. Endothelium-dependent responses to ACh were not affected by storage. ET-1 induced contractions were attenuated after storage in HDMEM (77%, p = 0.002), HKH (75%, p = 0.020), and TiProtec (73%, p = 0.010) with no changes in normalized constrictions. ET-1 EC50 values were not affected by storage. CONCLUSION: Loss of contractility after storage in HDMEM may reflect the lower content of dextrose. There was no specific attenuation of adrenoceptor, ET-receptor, or ACh receptor mediated signal transduction after storage in any of the media. HKH or TiProtec are equally suitable cold storage solutions for ex vivo measurements.

PKG1α Cysteine-42 Redox State Controls mTORC1 Activation in Pathological Cardiac Hypertrophy.

RATIONALE: Stimulated PKG1α (protein kinase G-1α) phosphorylates TSC2 (tuberous sclerosis complex 2) at serine 1365, potently suppressing mTORC1 (mechanistic [mammalian] target of rapamycin complex 1) activation by neurohormonal and hemodynamic stress. This reduces pathological hypertrophy and dysfunction and increases autophagy. PKG1α oxidation at cysteine-42 is also induced by these stressors, which blunts its cardioprotective effects. OBJECTIVE: We tested the dependence of mTORC1 activation on PKG1α C42 oxidation and its capacity to suppress such activation by soluble GC-1 (guanylyl cyclase 1) activation. METHODS AND RESULTS: Cardiomyocytes expressing wild-type (WT) PKG1α (PKG1αWT) or cysteine-42 to serine mutation redox-dead (PKG1αCS/CS) were exposed to ET-1 (endothelin 1). Cells expressing PKG1αWT exhibited substantial mTORC1 activation (p70 S6K [p70 S6 kinase], 4EBP1 [elF4E binding protein-1], and Ulk1 [Unc-51-like kinase 1] phosphorylation), reduced autophagy/autophagic flux, and abnormal protein aggregation; all were markedly reversed by PKG1αCS/CS expression. Mice with global knock-in of PKG1αCS/CS subjected to pressure overload (PO) also displayed markedly reduced mTORC1 activation, protein aggregation, hypertrophy, and ventricular dysfunction versus PO in PKG1αWT mice. Cardioprotection against PO was equalized between groups by co-treatment with the mTORC1 inhibitor everolimus. TSC2-S1365 phosphorylation increased in PKG1αCS/CS more than PKG1αWT myocardium following PO. TSC2S1365A/S1365A (TSC2 S1365 phospho-null, created by a serine to alanine mutation) knock-in mice lack TSC2 phosphorylation by PKG1α, and when genetically crossed with PKG1αCS/CS mice, protection against PO-induced mTORC1 activation, cardiodepression, and mortality in PKG1αCS/CS mice was lost. Direct stimulation of GC-1 (BAY-602770) offset disparate mTORC1 activation between PKG1αWT and PKG1αCS/CS after PO and blocked ET-1 stimulated mTORC1 in TSC2S1365A-expressing myocytes. CONCLUSIONS: Oxidation of PKG1α at C42 reduces its phosphorylation of TSC2, resulting in amplified PO-stimulated mTORC1 activity and associated hypertrophy, dysfunction, and depressed autophagy. This is ameliorated by direct GC-1 stimulation.

Activation mechanism of endothelin ETB receptor by endothelin-1.

Endothelin, a 21-amino-acid peptide, participates in various physiological processes, such as regulation of vascular tone, humoral homeostasis, neural crest cell development and neurotransmission. Endothelin and its G-protein-coupled receptor are involved in the development of various diseases, such as pulmonary arterial hypertension, and thus are important therapeutic targets. Here we report crystal structures of human endothelin type B receptor in the ligand-free form and in complex with the endogenous agonist endothelin-1. The structures and mutation analysis reveal the mechanism for the isopeptide selectivity between endothelin-1 and -3. Transmembrane helices 1, 2, 6 and 7 move and envelop the entire endothelin peptide, in a virtually irreversible manner. The agonist-induced conformational changes are propagated to the receptor core and the cytoplasmic G-protein coupling interface, and probably induce conformational flexibility in TM6. A comparison with the M2 muscarinic receptor suggests a shared mechanism for signal transduction in class A G-protein-coupled receptors.

Endothelin-1 alters BMP signaling to promote proliferation of pulmonary artery smooth muscle cells.

Pulmonary arterial hypertension (PAH) is characterized by abnormal outgrowth of pulmonary artery smooth muscle cells (PASMCs) of the media. Abundant expression of endothelin-1 (ET-1) and activated p38 mitogen-activated protein kinase (p38MAPK) has been observed in PAH patients. p38MAPK has been implicated in cell proliferation. An unspecified disturbance in bone morphogenetic protein (BMP) signaling may be involved in the development of PAH. Type I receptors (BMPR1A and BMPR1B) and type II receptor (BMPR2) transduce signals via two distinct pathways, i.e., canonical and non-canonical pathways, activating Smad1/5/8 and p38MAPK, respectively. BMPR1B expression was previously reported to be enhanced in the PASMCs of patients with idiopathic PAH. BMP15 binds specifically to BMPR1B. We assessed the effects of ET-1 on BMP receptor expression and cell proliferation. BMP2 increased BMPR1B expression in human PASMCs after pretreatment with ET-1 in vitro. Although BMP2 alone did not affect PASMC proliferation, BMP2 treatment after ET-1 pretreatment significantly accelerated PASMC proliferation. PH-797804, a selective p38MAPK inhibitor, abrogated this proliferation. Similarly, after ET-1 pretreatment, BMP15 significantly accelerated the proliferation of PASMCs, whereas stimulation with BMP15 alone did not. In conclusion, in PASMCs, ET-1 exposure under pathological conditions alters BMP signaling to activate p38MAPK, resulting in cell proliferation.

GJD Modulates Cardiac/Vascular Inflammation and Decreases Blood Pressure in Hypertensive Rats.

Gedan Jiangya decoction (GJD) (aqueous ethanol extract), a traditional Chinese medicine formula which contain six botanical drugs (Uncaria rhynchophylla (Miq.) Miq., Salvia miltiorrhiza Bunge, Pueraria lobata (Willd.) Ohwi, Eucommia ulmoides Oliv., Prunella vulgaris L., and Achyranthes bidentata Blume) was designed to treat hypertension; however, the underlying mechanism of action is unclear. This study aimed to determine the mechanisms of action of GJD in the treatment of hypertension in spontaneously hypertensive rats (SHR). Male SHRs were randomly divided into five groups: GJD doses were low (1.36 g/kg/d), medium (2.72 g/kg/d), and high (5.44 g/kg/d), captopril (13.5 mg/kg/d), and SHR groups, with Wistar-Kyoto rats (WKY) serving as the control. Every rat was gavaged once a day. The ALC-NIBP, a noninvasive blood pressure device, measured systolic (SBP) and diastolic (DBP) blood pressures. Six weeks following treatment, all rats were anesthetized. The blood samples were obtained from the abdominal aorta and then serum isolated to assess endothelin-1 and angiotensin II, interleukin-1beta, interleukin-6, and TNF-alpha. The left ventricular and thoracic aortas were taken for HE staining, immunohistochemistry, RT-qPCR, and western blot examination. Following GJD therapy, SBP and DBP were significantly lowered, as were serum levels of endothelin-1 and angiotensin II. The thickness of the left ventricular and thoracic aorta walls reduced, as did type I collagen, type III collagen, and alpha-SMA expression in the left ventricular and aortic tissues. The GJD treatment significantly reduced serum levels of the inflammatory markers interleukin-1beta, interleukin-6, and TNF-alpha. Furthermore, interleukin-1 beta, interleukin-6, TNF-alpha, TAK1, and NF-κB/p65 levels were significantly reduced in left ventricular and aortic tissues, whereas IkB-alpha levels were significantly elevated. GJD has a dose-dependent effect on all parameters. In conclusion, GJD has been shown to lower blood pressure, improve cardiovascular remodeling, and reduce inflammation via regulating NF-κB in SHRs.

Tetrahydroxystilbene glucoside attenuated homocysteine-upregulated endothelin receptors in vascular smooth muscle cells via the ERK1 /2 /NF-κB signaling pathway.

2,3,5,4'-Tetrahydrostilbene-2-o-ß-d-glucoside (TSG) is the main active component of Polygonum multiflorum Thunb. It has effects on hypertension. However, the mechanism is unclear. Current research is devoted to exploring the mechanism of TSG improving HHcy-induced hypertension. The mice received a subcutaneous injection of Hcy in the presence or absence of TSG for 4 weeks. Blood pressure (BP) was measured using a noninvasive tail-cuff plethysmography method. Levels of plasma Hcy and endothelin-1 were measured using ELISA. Rat SMA without endothelium was cultured in a serum-free medium in the presence or absence of TSG with or without Hcy. The contractile response to sarafotoxin 6c or endothein-1 was studied using a sensitive myography. The levels of protein were detected using Western blotting. The results showed that TSG lowered HHcy-elevated BP and decreased levels of plasma Hcy and endothelin-1 in mice. Furthermore, the results showed that TSG inhibited Hcy-upregulated ET receptor expression and ET receptor-mediated contractile responses as well as the levels of p-ERK1/2 and p-p65 in SMA. In vivo results further validate the in vitro results. In conclusion, TSG can decrease the levels of plasma Hcy and ET-1 and downregulate Hcy-upregulated ET receptors in VSMCs by inhibiting the ERK1/2 /NF-κB/ETB2 pathway to lower the BP.

Salt, Not Always a Cardiovascular Enemy? A Mini-Review and Modern Perspective.

Dietary salt intake is a long-debated issue. Increased sodium intake is associated with high blood pressure, leading to salt-sensitive hypertension. Excessive salt intake leads to arterial stiffness in susceptible individuals via impaired nitric oxide action and increased endothelin-1 expression, overactivity of the renal sympathetic nervous system and also via aldosterone-independent activation of the mineralocorticoid receptor. Salt restriction in such individuals reduces blood pressure (BP) values. The optimal level of salt restriction that leads to improved cardiovascular outcomes is still under debate. Current BP and dietary guidelines recommend low sodium intake for the general population. However, a specific category of patients does not develop arterial hypertension in response to sodium loading. In addition, recent research demonstrates the deleterious effects of aggressive sodium restriction, even in heart failure patients. This mini review discusses current literature data regarding the advantages and disadvantages of salt restriction and how it impacts the overall health status.

Effect of endothelin on sex-dependent regulation of tone in coronary resistance vessels.

BACKGROUND/AIMS: Sex dependent differences in coronary artery vasoregulation may be due to variations in responses to endogenous vasoactive compounds including endothelin (ET-1) and nitric oxide (NO). METHODS: Septal coronary arteries (<200 µm) from healthy, sexually mature male, female and ovariectomized (i.e. surgical menopause) Sprague-Dawley rats were used. Myogenic tone, measured by pressure myography, was initially determined for all vessel segments studied before and after exposure to the nonselective ETA/ETB receptor blocker, bosentan (1 µM). Vasoconstrictor responses (vascular endothelium intact) to cumulative ET-1 (10-12 - 10-9 M) were assessed in a separate set of septal coronary vessels. Additional studies, examined the vasoconstrictor effects of ET-1 after NO blockade with L-NAME (200 µM). RESULTS: Myogenic tone was 26 ± 7% in male, 20 ± 7% in female (p = 0.04 versus male) and 24 ± 3% in ovariectomized (p = NS versus male/female) vessels. Antagonism of ET-1 receptors produced a greater reduction in myogenic tone in male, compared to female rats over a similar range of intraluminal pressure (20-80 mmHg). Robust constrictor responses to cumulative concentrations of ET-1 were observed in all vessels; however, male rats exhibited greater sensitivity to vasoconstrictor effects of ET-1. After exposure to L-NAME vessel responses to ET-1 were normalized in male and female (not studied in ovariectomized) groups. CONCLUSIONS: These findings confirm marked sex differences for myogenic tone and vessel constrictor responses to ET-1 in coronary resistance vessels. Results also suggest greater sensitivity to vasoconstrictor effects of ET-1 in male coronary resistance vessels.

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.

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

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

Renomedullary Interstitial Cell Endothelin A Receptors Regulate BP and Renal Function.

BACKGROUND: The physiologic role of renomedullary interstitial cells, which are uniquely and abundantly found in the renal inner medulla, is largely unknown. Endothelin A receptors regulate multiple aspects of renomedullary interstitial cell function in vitro. METHODS: To assess the effect of targeting renomedullary interstitial cell endothelin A receptors in vivo, we generated a mouse knockout model with inducible disruption of renomedullary interstitial cell endothelin A receptors at 3 months of age. RESULTS: BP and renal function were similar between endothelin A receptor knockout and control mice during normal and reduced sodium or water intake. In contrast, on a high-salt diet, compared with control mice, the knockout mice had reduced BP; increased urinary sodium, potassium, water, and endothelin-1 excretion; increased urinary nitrite/nitrate excretion associated with increased noncollecting duct nitric oxide synthase-1 expression; increased PGE2 excretion associated with increased collecting duct cyclooxygenase-1 expression; and reduced inner medullary epithelial sodium channel expression. Water-loaded endothelin A receptor knockout mice, compared with control mice, had markedly enhanced urine volume and reduced urine osmolality associated with increased urinary endothelin-1 and PGE2 excretion, increased cyclooxygenase-2 protein expression, and decreased inner medullary aquaporin-2 protein content. No evidence of endothelin-1-induced renomedullary interstitial cell contraction was observed. CONCLUSIONS: Disruption of renomedullary interstitial cell endothelin A receptors reduces BP and increases salt and water excretion associated with enhanced production of intrinsic renal natriuretic and diuretic factors. These studies indicate that renomedullary interstitial cells can modulate BP and renal function under physiologic conditions.

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

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

Endothelin-1 stimulates expression of cyclin D1 and S-phase kinase-associated protein 2 by activating the transcription factor STAT3 in cultured rat astrocytes.

Brain injury-mediated induction of reactive astrocytes often leads to glial scar formation in damaged brain regions. Activation of signal transducer and activator of transcription 3 (STAT3), a member of the STAT family of transcription factors, plays a pivotal role in inducing reactive astrocytes and glial scar formation. Endothelin-1 (ET-1) is a vasoconstrictor peptide, and its levels increase in brain disorders and promote astrocytic proliferation through ETB receptors. To clarify the mechanisms underlying ET-1-mediated astrocytic proliferation, here we examined its effects on STAT3 in cultured rat astrocytes. ET-1 treatment stimulated Ser-727 phosphorylation of STAT3 in the astrocytes, but Tyr-705 phosphorylation was unaffected, and ET-induced STAT3 Ser-727 phosphorylation was reduced by the ETB antagonist BQ788. ET-1 stimulated STAT3 binding to its consensus DNA-binding motifs. Monitoring G1/S phase cell cycle transition through bromodeoxyuridine (BrdU) incorporation, we found that ET-1 increases BrdU incorporation into the astrocytic nucleus, indicating cell cycle progression. Of note, STAT3 chemical inhibition (with stattic or 5,15-diphenyl-porphine (5,15-DPP)) or siRNA-mediated STAT3 silencing reduced ET-induced BrdU incorporation. Moreover, ET-1 increased astrocytic expression levels of cyclin D1 and S-phase kinase-associated protein 2 (SKP2), which were reduced by stattic, 5,15-DPP, and STAT3 siRNA. ChIP-based PCR analysis revealed that ET-1 promotes the binding of SAT3 to the 5'-flanking regions of rat cyclin D1 and SKP2 genes. Our results suggest that STAT3-mediated regulation of cyclin D1 and SKP2 expression underlies ET-induced astrocytic proliferation.

Glucose withdrawal induces Endothelin 1 release with significant angiogenic effect from first trimester (FTM), but not term human umbilical cord perivascular cells (HUCPVC).

BACKGROUND: Perivascular cells (PVC) and their "progeny," mesenchymal stromal cells (MSC), have high therapeutic potential for ischemic diseases. While hypoxia can increase their angiogenic properties, the other aspect of ischemic conditions-glucose shortage-is deleterious for MSC and limits their therapeutic applicability. Regenerative cells in developing vascular tissues, however, can adapt to varying glucose environment and react in a tissue-protective manner. Placental development and fetal insulin production generate different glucose fluxes in early and late extraembryonic tissues. We hypothesized that FTM HUCPVC, which are isolated from a developing vascular tissue with varying glucose availability react to low-glucose conditions in a pro-angiogenic manner in vitro. METHODS: Xeno-free (Human Platelet Lysate 2.5%) expanded FTM (n = 3) and term (n = 3) HUCPVC lines were cultured in low (2 mM) and regular (4 mM) glucose conditions. After 72 h, the expression (Next Generation Sequencing) and secretion (Proteome Profiler) of angiogenic factors and the functional angiogenic effect (rat aortic ring assay and Matrigel™ plug) of the conditioned media were quantified and statistically compared between all cultures. RESULTS: Low-glucose conditions had a significant post-transcriptional inductive effect on FTM HUCPVC angiogenic factor secretion, resulting in significantly higher VEGFc and Endothelin 1 release in 3 days compared to term counterparts. Conditioned media from low-glucose FTM HUCPVC cultures had a significantly higher endothelial network enhancing effect compared to all other experimental groups both in vitro aortic ring assay and in subcutan Matrigel™ plugs. Endothelin 1 depletion of the low-glucose FTM HUCPVC conditioned media significantly diminished its angiogenic effect CONCLUSIONS: FTM HUCPVC isolated from an early extraembryonic tissue show significant pro-angiogenic paracrine reaction in low-glucose conditions at least in part through the excess release of Endothelin 1. This can be a substantial advantage in cell therapy applications for ischemic injuries.