A Genetic Variant Associated with Five Vascular Diseases Is a Distal Regulator of Endothelin-1 Gene Expression.

Genome-wide association studies (GWASs) implicate the PHACTR1 locus (6p24) in risk for five vascular diseases, including coronary artery disease, migraine headache, cervical artery dissection, fibromuscular dysplasia, and hypertension. Through genetic fine mapping, we prioritized rs9349379, a common SNP in the third intron of the PHACTR1 gene, as the putative causal variant. Epigenomic data from human tissue revealed an enhancer signature at rs9349379 exclusively in aorta, suggesting a regulatory function for this SNP in the vasculature. CRISPR-edited stem cell-derived endothelial cells demonstrate rs9349379 regulates expression of endothelin 1 (EDN1), a gene located 600 kb upstream of PHACTR1. The known physiologic effects of EDN1 on the vasculature may explain the pattern of risk for the five associated diseases. Overall, these data illustrate the integration of genetic, phenotypic, and epigenetic analysis to identify the biologic mechanism by which a common, non-coding variant can distally regulate a gene and contribute to the pathogenesis of multiple vascular diseases.

Epidermal ET-1 signal induces activation of resting hair follicles by upregulating the PI3K/AKT pathway in the dermis.

The prevalence of alopecia has increased recently. Hair loss is often accompanied by the resting phase of hair follicles (HFs). Dermal papilla (DP) plays a crucial role in HF development, growth, and regeneration. Activating DP can revive resting HFs. Augmenting WNT/ß-catenin signaling stimulates HF growth. However, the factors responsible for activating resting HFs effectively are unclear. In this study, we investigated epidermal cytokines that can activate resting HFs effectively. We overexpressed ß-catenin in both in vivo and in vitro models to observe its effects on resting HFs. Then, we screened potential epidermal cytokines from GEO DATASETs and assessed their functions using mice models and skin-derived precursors (SKPs). Finally, we explored the molecular mechanism underlying the action of the identified cytokine. The results showed that activation of WNT/ß-catenin in the epidermis prompted telogen-anagen transition. Keratinocytes infected with Ctnnb1-overexpressing lentivirus enhanced SKP expansion. Subsequently, we identified endothelin 1 (ET-1) expressed higher in hair-growing epidermis and induced the proliferation of DP cells and activates telogen-phase HFs in vivo. Moreover, ET-1 promotes the proliferation and stemness of SKPs. Western blot analysis and in vivo experiments revealed that ET-1 induces the transition from telogen-to-anagen phase by upregulating the PI3K/AKT pathway. These findings highlight the potential of ET-1 as a promising cytokine for HF activation and the treatment of hair loss.

Signals From Inflamed Perivascular Adipose Tissue Contribute to Small-Vessel Dysfunction in Women With Human Immunodeficiency Virus.

BACKGROUND: People with the human immunodeficiency virus (PWH) have microvascular disease. Because perivascular adipose tissue (PVAT) regulates microvascular function and adipose tissue is inflamed in PWH, we tested the hypothesis that PWH have inflamed PVAT that impairs the function of their small vessels. METHODS: Subcutaneous small arteries were dissected with or without PVAT from a gluteal skin biopsy from 11 women with treated HIV (WWH) aged < 50 years and 10 matched women without HIV, and studied on isometric myographs. Nitric oxide (NO) and reactive oxygen species (ROS) were measured by fluorescence microscopy. Adipokines and markers of inflammation and ROS were assayed in PVAT. RESULTS: PVAT surrounding the small arteries in control women significantly (P < .05) enhanced acetylcholine-induced endothelium-dependent relaxation and NO, and reduced contractions to thromboxane and endothelin-1. However, these effects of PVAT were reduced significantly (P < .05) in WWH whose PVAT released less adiponectin but more markers of ROS and inflammation. Moderation of contractions by PVAT were correlated positively with adipose adiponectin. CONCLUSIONS: PVAT from WWH has oxidative stress, inflammation, and reduced release of adiponectin, which may contribute to enhanced contractions and therefore could promote small-artery dysfunction.

Endothelin-1 influences mechanical properties and contractility of hiPSC derived cardiomyocytes resulting in diastolic dysfunction.

A better understanding of the underlying pathomechanisms of diastolic dysfunction is crucial for the development of targeted therapeutic options with the aim to increase the patients' quality of life. In order to shed light on the processes involved, suitable models are required. Here, effects of endothelin-1 (ET-1) treatment on cardiomyocytes derived from human induced pluripotent stem cells (hiPSCs) were investigated. While it is well established, that ET-1 treatment induces hypertrophy in cardiomyocytes, resulting changes in cell mechanics and contractile behavior with focus on relaxation have not been examined before. Cardiomyocytes were treated with 10 nM of ET-1 for 24 h and 48 h, respectively. Hypertrophy was confirmed by real-time deformability cytometry (RT-DC) which was also used to assess the mechanical properties of cardiomyocytes. For investigation of the contractile behavior, 24 h phase contrast video microscopy was applied. To get a deeper insight into changes on the molecular biological level, gene expression analysis was performed using the NanoString nCounter® cardiovascular disease panel. Besides an increased cell size, ET-1 treated cardiomyocytes are stiffer and show an impaired relaxation. Gene expression patterns in ET-1 treated hiPSC derived cardiomyocytes showed that pathways associated with cardiovascular diseases, cardiac hypertrophy and extracellular matrix were upregulated while those associated with fatty acid metabolism were downregulated. We conclude that alterations in cardiomyocytes after ET-1 treatment go far beyond hypertrophy and represent a useful model for diastolic dysfunction.

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.

Antecubital venous endothelial ETB receptor protein expression is preserved with aging in men.

Changes in endothelial function precede the development of cardiovascular disease (CVD). We have previously shown that age-related declines in endothelial function in women are due in part to a reduction in endothelial cell endothelin-B receptor (ETBR) protein expression. However, it is not known if ETBR protein expression changes with aging in men. The purpose of this study was to test the hypothesis that ETBR protein expression is attenuated in older men (OM) compared with younger men (YM). Primary endothelial cells were harvested from the antecubital vein of 14 OM (60 ± 6 yr; 26 ± 3 kg/m2) and 17 YM (24 ± 5 yr; 24 ± 2 kg/m2). Cells were stained with 4',6-diamidino-2-phenylindole, vascular endothelial cadherin, and ETBR. Images were quantified using immunocytochemistry. Endothelial function was assessed using brachial artery flow-mediated dilation (FMD). Systolic BP was similar (OM, 123 ± 11 vs. YM, 122 ± 10 mmHg) whereas diastolic BP was higher in OM (OM, 77 ± 7 vs. YM, 70 ± 6 mmHg; P < 0.01). Total testosterone was lower in OM (OM, 6.28 ± 4.21 vs. YM, 9.10 ± 2.68 ng/mL; P = 0.03). As expected, FMD was lower in OM (OM, 3.85 ± 1.51 vs. YM, 6.40 ± 2.68%; P < 0.01). However, ETBR protein expression was similar between OM and YM (OM, 0.39 ± 0.17 vs. YM, 0.42 ± 0.17 AU; P = 0.66). These data suggest that ETBR protein expression is not altered with age in men. These findings contrast with our previous data in women and further support sex differences in the endothelin system.NEW & NOTEWORTHY Our laboratory has previously shown that age-related declines in endothelial function are associated with a reduction in endothelial cell ETBR protein expression in women. However, it is unclear if endothelial cell ETBR protein expression is reduced with aging in men. This study demonstrates that endothelial cell ETBR protein expression is preserved with aging in men, and provides additional evidence for sex differences in the endothelin system.

EGR1 interacts with p-SMAD at the endothelin-1 gene promoter to regulate gene expression in TGFß1-stimulated IMR-90 fibroblasts.

Pulmonary fibrosis is a severe and progressive lung disease characterized by lung tissue scarring. Transforming growth factor beta 1 (TGFß1) is crucial in causing pulmonary fibrosis by promoting the activation of fibroblasts and their differentiation into myofibroblasts, which are responsible for excessive extracellular matrix deposition. This study aimed to identify genes activated by TGFß1 that promote fibrosis and to understand the regulatory pathway controlling myofibroblast. Endothelin-1 (ET-1) was identified as the top-ranking gene in the fibrosis-related gene set using quantitative PCR array analysis. TGFß1 upregulated EGR1 expression through the ERK1/2 and JNK1/2 MAPK pathways. EGR1 and p-SMAD2 proteins interacted with the ET-1 gene promoter region to regulate TGFß1-induced ET-1 expression in IMR-90 pulmonary fibroblasts. Mice lacking the Egr1 gene showed reduced ET-1 levels in a model of pulmonary fibrosis induced by intratracheal administration of bleomycin. These findings suggest that targeting EGR1 is a promising approach for treating pulmonary fibrosis, especially idiopathic pulmonary fibrosis, by affecting ET-1 expression and profibrotic reactions.

CD38/cADPR-mediated calcium signaling in a human myometrial smooth muscle cell line, PHM1.

Cyclic ADP-ribose (cADPR) has emerged as a calcium-regulating second messenger in smooth muscle cells. CD38 protein possesses ADP-ribosyl cyclase and cADPR hydrolase activities and mediates cADPR synthesis and degradation. We have previously shown that CD38 expression is regulated by estrogen and progesterone in the myometrium. Considering hormonal regulation in gestation, the objective of the present study was to determine the role of CD38/cADPR signaling in the regulation of intracellular calcium upon contractile agonist stimulation using immortalized pregnant human myometrial (PHM1) cells. Western blot, immunofluorescence, and biochemical studies confirmed CD38 expression and the presence of ADP-ribosyl cyclase (2.6 ± 0.1 pmol/mg) and cADPR hydrolase (26.8 ± 6.8 nmoles/mg/h) activities on the PHM1 cell membrane. Oxytocin, PGF2α, and ET-1 elicited [Ca2+]i responses, and 8-Br-cADPR, a cADPR antagonist significantly attenuated agonist-induced [Ca2+]i responses between 20% and 46% in average. The findings suggest that uterine contractile agonists mediate their effects in part through CD38/cADPR signaling to increase [Ca2+]i and presumably uterine contraction. As studies in humans are limited by the availability of myometrium from healthy donors, PHM1 cells form an in vitro model to study human myometrium.

Experimentally Induced Peripheral Venous Congestion Exacerbates Inflammation, Oxidative Stress, and Neurohormonal and Endothelial Cell Activation in Patients With Systolic Heart Failure.

BACKGROUND: Venous congestion (VC) is a hallmark of symptomatic heart failure (HF) requiring hospitalization; however, its role in the pathogenesis of HF progression remains unclear. We investigated whether peripheral VC exacerbates inflammation, oxidative stress and neurohormonal and endothelial cell (EC) activation in patients with HF with reduced ejection fraction (HFrEF). METHODS AND RESULTS: Two matched groups of patients with HFrEF and with no peripheral VC vs without recent HF hospitalization were studied. We modeled peripheral VC by inflating a cuff around the dominant arm, targeting ∼ 30 mmHg increase in venous pressure (venous stress test [VST]). Blood and ECs were sampled before and after 90 minutes of VST. We studied 44 patients (age 53 ± 12 years, 32% female). Circulating endothelin-1, tumor necrosis factor-α, interleukin-6, isoprostane, angiotensin II (ang-2), angiopoietin-2, vascular cell adhesion molecule-1, and CD146 significantly increased after the VST. Enhanced endothelin-1 and angiopoietin-2 responses to the VST were present in patients with vs without recent hospitalization and were prospectively associated with incident HF-related events; 6698 messenger ribonucleic acid (mRNA probe sets were differentially expressed in ECs after VST. CONCLUSIONS: Experimental VC exacerbates inflammation, oxidative stress, neurohormonal and EC activation and promotes unfavorable transcriptome remodeling in ECs of patients with HFrEF. A distinct biological sensitivity to VC appears to be associated with high risk for HF progression.

Short communications: Endothelin-1 in cardiac allograft vasculopathy.

INTRODUCTION: Cardiac allograft vasculopathy (CAV) is a leading cause of death following heart transplant. Endothelin-1 (ET-1) is a highly potent vasoconstrictor peptide derived from the vascular endothelium with multiple biological actions known to be relevant for CAV. We assessed the trans-myocardial gradient (TMG: coronary sinus minus coronary artery concentration: negative = extraction, positive = secretion) of ET-1 in heart transplant patients to determine correlations with angiographic, Intravascular Ultrasound (IVUS) and Optical Coherence Tomography (OCT) features of CAV. RESULTS: Vessels with more severe CAV demonstrated significantly higher (more positive) ET-1 TMG (IVUS Stanford Grade IV: -0.05 [-0.21, 0.13] pg/ml versus Stanford Grade I-III: -0.31 [-0.64, -0.11] pg/ml, p = 0.01). ET-1 TMG was positively correlated with mean intimal thickness on both IVUS and OCT (IVUS: Kendall's tau-b = 0.254, p = 0.02 and OCT: Kendall's tau-b = 0.344, p < 0.0001). Patients who died had net ET-1 release compared with surviving patients (died: 0.21 [0.19-0.24] versus surviving: -0.28 [-0.52, -0.17], p = 0.01). CONCLUSION: In heart transplant patients, coronary arteries with more intimal thickening are associated with a higher (more positive) trans-myocardial gradient of ET-1, suggesting that up-regulated ET-1 release in the coronary circulation may be permissive for the development of CAV.

Complex Regulation of Tau Phosphorylation by the Endothelin System in Brain Microvascular Endothelial Cells (BMVECs): Link to Barrier Function.

Endothelin-1 (ET-1), the most potent vasoconstrictor identified to date, contributes to cerebrovascular dysfunction. ET-1 levels in postmortem brain specimens from individuals diagnosed with Alzheimer's Disease (AD) and related dementias (ADRD) were shown to be related to cerebral hypoxia.  ET-1-mediated vascular dysfunction and ensuing cognitive deficits have also been reported in experimental models of AD and ADRD. Moreover, studies also showed that ET-1 secreted from BMVECs can affect neurovascular unit integrity in an autocrine and paracrine manner. Vascular contributions to cognitive impairment and dementia (VCID) is a leading ADRD cause known to be free of neuronal tau pathology, a hallmark of AD. However, a recent study reported cytotoxic hyperphosphorylated tau (p-tau) accumulation, which fails to bind or stabilize microtubules in BMVECs in VCID. Thus, the study aimed to determine the impact of ET-1 on tau pathology, microtubule organization, and barrier function in BMVECs. Cells were stimulated with 1uM ET-1 for 24 hours in the presence/absence of ETA (BQ123; 20uM) or ETB (BQ788; 20uM) receptor antagonists. Cell lysates were assayed for an array of phosphorylation site-specific antibodies and microtubule organization/stabilization markers. ET-1 stimulation increased p-tau Thr231 but decreased p-tau Ser199, Ser262, Ser396, and Ser214 levels only in the presence of ETA or ETB antagonism. ET-1 also impaired barrier function in the presence of ETA antagonism. These novel findings suggest that 1) dysregulation of endothelial tau phosphorylation may contribute to cerebral microvascular dysfunction and 2) the ET system may be an early intervention   target to prevent hyperphosphorylated tau-mediated disruption of BMVEC barrier function.

Endothelin-1 (ET-1) contributes to senescence and phenotypic changes in brain pericytes in diabetes-mimicking conditions.

Diabetes mediates endothelial dysfunction and increases the risk of Alzheimer's disease and related dementias. Diabetes also dysregulates the ET system. ET-1-mediated constriction of brain microvascular pericytes (BMVPCs) has been shown to contribute to brain hypoperfusion. Cellular senescence, a process that arrests the proliferation of harmful cells and instigates phenotypical changes and proinflammatory responses in endothelial cells that impact their survival and function. Thus, we hypothesized that ET-1 mediates BMVPC senescence and phenotypical changes in diabetes-like conditions. Human BMVPCs were incubated in diabetes-like conditions with or without ET-1 (1 µmol/L) for 3 and 7 days. Hydrogen peroxide (100 µmol/L H2O2) was used as a positive control for senescence and to mimic ischemic conditions. Cells were stained for senescence-associated ß-galactosidase or processed for immunoblotting and quantitative real-time PCR analyses. In additional experiments, cells were stimulated with ET-1 in the presence or absence of ETA receptor antagonist BQ-123 (20 µmol/L) or ETB receptor antagonist BQ-788 (20 µmol/L). ET-1 stimulation increased ß-galactosidase accumulation which was prevented by BQ-123. ET-1 also increased traditional senescence marker p16 protein and pericyte-specific senescence markers, TGFB1i1, PP1CA, and IGFBP7. Furthermore, ET-1 stimulated contractile protein α-SMA and microglial marker ostepontin in high glucose suggesting a shift toward an ensheathing or microglia-like phenotype. In conclusion, ET-1 triggers senescence, alters ETA and ETB receptors, and causes phenotypical changes in BMVPCs under diabetes-like conditions. These in vitro findings need to be further studied in vivo to establish the role of ETA receptors in the progression of pericyte senescence and phenotypical changes in VCID.

Endothelin-1 down-regulates nuclear factor erythroid 2-related factor-2 and contributes to perivascular adipose tissue dysfunction in obesity.

Perivascular adipose tissue (PVAT) negatively regulates vascular muscle contraction. However, in the context of obesity, the PVAT releases vasoconstrictor substances that detrimentally affect vascular function. A pivotal player in this scenario is the peptide endothelin-1 (ET-1), which induces oxidative stress and disrupts vascular function. The present study postulates that obesity augments ET-1 production in the PVAT, decreases the function of the nuclear factor erythroid 2-related factor-2 (Nrf2) transcription factor, further increasing reactive oxygen species (ROS) generation, culminating in PVAT dysfunction. Male C57BL/6 mice were fed either a standard or a high-fat diet for 16 weeks. Mice were also treated with saline or a daily dose of 100 mg·kg-1 of the ETA and ETB receptor antagonist Bosentan, for 7 days. Vascular function was evaluated in thoracic aortic rings, with and without PVAT. Mechanistic studies utilized PVAT from all groups and cultured WT-1 mouse brown adipocytes. PVAT from obese mice exhibited increased ET-1 production, increased ECE1 and ETA gene expression, loss of the anticontractile effect, as well as increased ROS production, decreased Nrf2 activity, and downregulated expression of Nrf2-targeted antioxidant genes. PVAT of obese mice also exhibited increased expression of Tyr216-phosphorylated-GSK3ß and KEAP1, but not BACH1 - negative Nrf2 regulators. Bosentan treatment reversed all these effects. Similarly, ET-1 increased ROS generation and decreased Nrf2 activity in brown adipocytes, events mitigated by BQ123 (ETA receptor antagonist). These findings place ET-1 as a major contributor to PVAT dysfunction in obesity and highlight that pharmacological control of ET-1 effects restores PVAT's cardiovascular protective role.

The endothelin-1-driven tumor-stroma feed-forward loops in high-grade serous ovarian cancer.

The high-grade serous ovarian cancer (HG-SOC) tumor microenvironment (TME) is constellated by cellular elements and a network of soluble constituents that contribute to tumor progression. In the multitude of the secreted molecules, the endothelin-1 (ET-1) has emerged to be implicated in the tumor/TME interplay; however, the molecular mechanisms induced by the ET-1-driven feed-forward loops (FFL) and associated with the HG-SOC metastatic potential need to be further investigated. The tracking of the patient-derived (PD) HG-SOC cell transcriptome by RNA-seq identified the vascular endothelial growth factor (VEGF) gene and its associated signature among those mostly up-regulated by ET-1 and down-modulated by the dual ET-1R antagonist macitentan. Within the ligand-receptor pairs concurrently expressed in PD-HG-SOC cells, endothelial cells and activated fibroblasts, we discovered two intertwined FFL, the ET-1/ET-1R and VEGF/VEGF receptors, concurrently activated by ET-1 and shutting-down by macitentan, or by the anti-VEGF antibody bevacizumab. In parallel, we observed that ET-1 fine-tuned the tumoral and stromal secretome toward a pro-invasive pattern. Into the fray of the HG-SOC/TME double and triple co-cultures, the secretion of ET-1 and VEGF, that share a common co-regulation, was inhibited upon the administration of macitentan. Functionally, macitentan, mimicking the effect of bevacizumab, interfered with the HG-SOC/TME FFL-driven communication that fuels the HG-SOC invasive behavior. The identification of ET-1 and VEGF FFL as tumor and TME actionable vulnerabilities, reveals how ET-1R blockade, targeting the HG-SOC cells and the TME simultaneously, may represent an effective therapeutic option for HG-SOC patients.

Mechanism of protective actions of sparsentan in the kidney: lessons from studies in models of chronic kidney disease.

Simultaneous inhibition of angiotensin II AT1 and endothelin ETA receptors has emerged as a promising approach for treatment of chronic progressive kidney disease. This therapeutic approach has been advanced by the introduction of sparsentan, the first dual AT1 and ETA receptor antagonist. Sparsentan is a single molecule with high affinity for both receptors. It is US Food and Drug Administration approved for immunoglobulin A nephropathy (IgAN) and is currently being developed as a treatment for rare kidney diseases, such as focal segmental glomerulosclerosis. Clinical studies have demonstrated the efficacy and safety of sparsentan in these conditions. In parallel with clinical development, studies have been conducted to elucidate the mechanisms of action of sparsentan and its position in the context of published evidence characterizing the nephroprotective effects of dual ETA and AT1 receptor inhibition. This review summarizes this evidence, documenting beneficial anti-inflammatory, antifibrotic, and hemodynamic actions of sparsentan in the kidney and protective actions in glomerular endothelial cells, mesangial cells, the tubulointerstitium, and podocytes, thus providing the rationale for the use of sparsentan as therapy for focal segmental glomerulosclerosis and IgAN and suggesting potential benefits in other renal diseases, such as Alport syndrome.

Blockade of the mineralocorticoid receptor improves markers of human endothelial cell dysfunction and hematological indices in a mouse model of sickle cell disease.

Increased endothelin-1 (ET-1) levels in patients with sickle cell disease (SCD) and transgenic mouse models of SCD contribute to disordered hematological, vascular, and inflammatory responses. Mineralocorticoid receptor (MR) activation by aldosterone, a critical component of the Renin-Angiotensin-Aldosterone-System, modulates inflammation and vascular reactivity, partly through increased ET-1 expression. However, the role of MR in SCD remains unclear. We hypothesized that MR blockade in transgenic SCD mice would reduce ET-1 levels, improve hematological parameters, and reduce inflammation. Berkeley SCD (BERK) mice, a model of severe SCD, were randomized to either sickle standard chow or chow containing the MR antagonist (MRA), eplerenone (156 mg/Kg), for 14 days. We found that MRA treatment reduced ET-1 plasma levels (p = .04), improved red cell density gradient profile (D50 ; p < .002), and increased mean corpuscular volume in both erythrocytes (p < .02) and reticulocytes (p < .024). MRA treatment also reduced the activity of the erythroid intermediate-conductance Ca2+ -activated K+ channel - KCa 3.1 (Gardos channel, KCNN4), reduced cardiac levels of mRNAs encoding ET-1, Tumor Necrosis Factor Receptor-1, and protein disulfide isomerase (PDI) (p < .01), and decreased plasma PDI and myeloperoxidase activity. Aldosterone (10-8 M for 24 h in vitro) also increased PDI mRNA levels (p < .01) and activity (p < .003) in EA.hy926 human endothelial cells, in a manner blocked by pre-incubation with the MRA canrenoic acid (1 µM; p < .001). Our results suggest a novel role for MR activation in SCD that may exacerbate SCD pathophysiology and clinical complications.

proANP Metabolism Provides New Insights Into Sacubitril/Valsartan Mode of Action.

BACKGROUND: Sacubitril/valsartan (S/V) treatment is beneficial in patients with heart failure with reduced ejection fraction (HFrEF), but its mode of action remains elusive, although it involves the increase in ANP (atrial natriuretic peptide). METHODS: Combining mass spectrometry and enzymatic assay in the plasma of 73 HFrEF patients treated with S/V and controls, we deciphered proANP processing that converts proANP into 4 vasoactive peptides. RESULTS: We found that proANP processing is sequential and involved meprin B, ECE (endothelin-converting enzyme) 1, and ANPEP (aminopeptidase N). This processing is limited in HFrEF patients via the downregulation of proANP production, corin, and meprin B activities by miR-425 and miR1-3p. S/V restored or compensated proANP processing by downregulating miR-425 and miR1-3p, hence increasing levels of proANP-derived bioactive peptides. In contrast, S/V directly and indirectly partially inhibited ECE1 and ANPEP. ECE1 partial inhibition resulted in a lower-than-expected increase in ET1 (endothelin 1), tilting the vasoactive balance toward vasodilation, and possibly hypotension. Furthermore, proANP glycosylation interferes with the midregional proANP assay -a clinical surrogate for proANP production, preventing any pathophysiological interpretation of the results. The analysis of S/V dose escalation with respect to baseline treatments suggests S/V-specific effects. CONCLUSIONS: These findings offer mechanistic evidence to the natriuretic peptide -defective state in HFrEF, which is improved by S/V. These data also strongly suggests that S/V increases plasma ANP by multiple mechanisms that involve 2 microRNAs, besides its protection from NEP (neprilysin) cleavage. Altogether, these data provide new insights on HFrEF pathophysiology and the mode of action of S/V.

Ang II Promotes ET-1 Production by Regulating NOX2 Activity Through Transcription Factor Oct-1.

BACKGROUND: Increasing evidence suggests that superoxide ions produced by NOX (nicotinamide adenine dinucleotide phosphate oxidases) mediate vascular effects of Ang II (angiotensin II) evoked by atherogenic diets. Here, we analyzed the mechanism by which NOX2 contributes to Ang II-induced ET-1 (endothelin 1) production in human microvascular endothelial cells. METHODS: The effects of high-fat diet were compared between WT (wild type) and Nox2 (mouse NOX2 gene)-deficient mice. ET-1 production and NOX2 expression by human microvascular endothelial cells in vitro were analyzed by ELISA, reverse transcription quantitative polymerase chain reaction, electrophoretic mobility shift assay, promoter deletions, RNA interference, and pharmacological inhibition. Production of superoxide anions was visualized by fluorescent cell labeling. RESULTS: Feeding mice high-fat diet for 10 weeks increased cardiac expression and plasma levels of Ang II and ET-1 in WT but not in Nox2-deficient animals. Exposure of human microvascular endothelial cells to Ang II resulted in increased ET-1 production, which could be blocked by silencing NOX2 (human NOX2 gene). Ang II promoted NOX2 expression through induction of the Oct-1 (human/mouse octamer binding transcription factor 1 protein) and activation of the NOX2 promoter region containing Oct-1-binding sites. Stimulation of NOX2 expression by Ang II was associated with increased production of superoxide anions. Inhibition of Oct-1 by small interfering RNA reduced Ang II-induced NOX2 expression and superoxide anion production, and neutralization of superoxide by SOD (superoxide dismutase) abolished Ang II-stimulated ET1 (human ET-1 gene) promoter activity, ET1 mRNA expression, and ET-1 release. CONCLUSIONS: Ang II may promote ET-1 production in the endothelium in response to atherogenic diets through a mechanism that involves the transcription factor Oct-1 and the increased formation of superoxide anions by NOX2.

Indigo Leaves-Induced Pulmonary Arterial Remodeling without Right Ventricular Hypertrophy in Rats.

Indigo naturalis (IN), derived from the leaves of the indigo plant, is a traditional Chinese medicine that has historically been used for its anti-inflammatory properties in the treatment of various diseases, including ulcerative colitis (UC). However, long-term use of IN in UC patients is incontrovertibly associated with the onset of pulmonary arterial hypertension (PAH). To investigate the mechanisms by which IN induces PAH, we focused on the raw material of IN, indigo leaves (IL). Only the condition of long-term chronic (6 months) and high-dose (containing 5% IL in the control diet) administration of IL induced medial thickening in the pulmonary arteries without right ventricular hypertrophy in our rat model. IL administration for a month did not induce pulmonary arterial remodeling but increased endothelin-1 (ET-1) expression levels within endothelial cell (EC) layers in the lungs. Gene Expression Omnibus analysis showed that ET-1 is a key regulator of PAH and that the IL component indican and its metabolite IS induced ET-1 mRNA expression via reactive oxygen species-dependent mechanism. We identified the roles of indican and IS in ET-1 expression in ECs, which were linked to pulmonary arterial remodeling in an animal model.

ET1 acts as a potential plasma biomarker and therapeutic target in deep venous thrombosis rat model.

Deep venous thrombosis (DVT) is the third leading cause of death in cardiovascular disease, following heart attacks and strokes. Early diagnosis and intervention are crucial for effective DVT therapy. We aim to investigate whether endothelin-1 (ET-1) could serve as an early diagnostic marker or a potential therapeutic target in a DVT rat model. CCK8 assay, invasion assay, and flow cytometry were used to detect the proliferation, migration and apoptosis of HUVECs, respectively. Elisa assay was used to detect ET-1 and coagulation factor VII in cell supernatant and rat?s plasma. Western blot was used to detect antioxidant signaling protein. Inferior vena cava stenosis was used to construct the DVT rat model. Lentivirus mediated overexpression of ET-1 in HUVECs impaired the cell proliferation and migration, increased cell apoptosis, inhibited the antioxidant signaling pathway proteins expression (e.g., NQO1, GCLC, Nrf-2), and upregulated coagulation factor VII. Furthermore, overexpression of ET-1 further impaired antioxidant signaling pathway protein in response to H2O2 treatment. However, lentivirus mediated ET-1 knockdown and BQ123 (an ET-1 inhibitor), showed the opposite results with ET-1 overexpression. We then established a DVT rat model by inferior vena cava stenosis. The stenosis induced early expression of ET-1 and coagulation factor VII in plasma at day 1 and restore their level at day 10. BQ123 could downregulate the coagulation factor VII to ameliorate the stenosis effects. Our findings suggest that ET-1 might serve as an early diagnostic marker for DVT rat model and a potential therapeutic target for treating DVT.