Cardiomyocyte-specific deletion of endothelin receptor A (ETA) obliterates cardiac aging through regulation of mitophagy and ferroptosis.

Advanced aging evokes unfavorable changes in the heart including cardiac remodeling and contractile dysfunction although the underlying mechanism remains elusive. This study was conducted to evaluate the role of endothelin-1 (ET-1) in the pathogenesis of cardiac aging and mechanism involved. Echocardiographic and cardiomyocyte mechanical properties were determined in young (5-6 mo) and aged (26-28 mo) wild-type (WT) and cardiomyocyte-specific ETA receptor knockout (ETAKO) mice. GSEA enrichment identified differentially expressed genes associated with mitochondrial respiration, mitochondrial protein processing and mitochondrial depolarization in cardiac aging. Aging elevated plasma levels of ET-1, Ang II and suppressed serum Fe2+, evoked cardiac remodeling (hypertrophy and interstitial fibrosis), contractile defects (fractional shortening, ejection fraction, cardiomyocyte peak shortening, maximal velocity of shortening/relengthening and prolonged relengthening) and intracellular Ca2+ mishandling (dampened intracellular Ca2+ release and prolonged decay), the effects with the exception of plasma AngII, ET-1 and Fe2+ were mitigated by ETAKO. Advanced age facilitated O2- production, carbonyl protein damage, cardiac hypertrophy (GATA4, ANP, NFATc3), ER stress, ferroptosis, compromised autophagy (LC3B, Beclin-1, Atg7, Atg5 and p62) and mitophagy (parkin and FUNDC1), and deranged intracellular Ca2+ proteins (SERCA2a and phospholamban), the effects of which were reversed by ETA ablation. ET-1 provoked ferroptosis in vitro, the response was nullified by the ETA receptor antagonist BQ123 and mitophagy inducer CsA. ETA but not ETB receptor antagonism reconciled cardiac aging, which was abrogated by inhibition of mitophagy and ferroptosis. These findings collectively denote promises of targeting ETA, mitophagy and ferroptosis in the management of aging-associated cardiac remodeling and contractile defect.

Role of Brain Endothelin Receptor Type B (ETB) in the Regulation of Tyrosine Hydroxylase in the Olfactory Bulb of DOCA-Salt Hypertensive Rats.

BACKGROUND: We previously reported that endothelins (ETs) regulate tyrosine hydroxylase (TH) activity and expression in the olfactory bulb (OB) of normotensive and hypertensive animals. Applying an ET receptor type A (ETA) antagonist to the brain suggested that endogenous ETs bind to ET receptor type B (ETB) to elicit effects. OBJECTIVE: The aim of the present work was to evaluate the role of central ETB stimulation on the regulation of blood pressure (BP) and the catecholaminergic system in the OB of deoxycorticosterone acetate (DOCA)-salt hypertensive rats. METHODS: DOCA-salt hypertensive rats were infused for 7 days with cerebrospinal fluid or IRL-1620 (ETB receptor agonist) through a cannula placed in the lateral brain ventricle. Systolic BP (SBP) and heart rate were recorded by plethysmography. The expression of TH and its phosphorylated forms in the OB were determined by immunoblotting, TH activity by a radioenzymatic assay, and TH mRNA by quantitative real-time polymerase chain reaction. RESULTS: Chronic administration of IRL-1620 decreased SBP in hypertensive rats but not in normotensive animals. Furthermore, the blockade of ETB receptors also decreased TH-mRNA in DOCA-salt rats, but it did not modify TH activity or protein expression. CONCLUSION: These findings suggest that brain ETs through the activation of ETB receptors contribute to SBP regulation in DOCA-salt hypertension. However, the catecholaminergic system in the OB does not appear to be conclusively involved although mRNA TH was reduced. Present and previous findings suggest that in this salt-sensitive animal model of hypertension, the OB contributes to chronic BP elevation.

Murine Cytomegalovirus Infection Induced miR-1929-3p Down-Regulation Promotes the Proliferation and Apoptosis of Vascular Smooth Muscle Cells in Mice by Targeting Endothelin A Receptor and Downstream NLRP3 Activation Pathway.

Our previous study demonstrated in vivo that mouse cytomegalovirus (MCMV) infection promoted vascular remodeling after downregulation of miR-1929-3p. This study aimed to investigate the role of miR-1929-3p/ETAR/NLRP3 pathway in mouse vascular smooth muscle cells (MOVAS) after MCMV infection. First, PCR was used to detect the success of the infection. Second, MOVAS were transfected with the miR-1929-3p mimic, inhibitor, and ETAR overexpressed adenovirus vector. Cell proliferation was detected using EdU, whereas apoptosis was detected using flow cytometry. The expression of miR-1929-3p and ETAR were detected using qRT-PCR. Western blot detected proteins of cell proliferation, apoptosis, and the NLRP3 inflammasome. Interleukin-1ß and interleukin-18 were determined using ELISA. The results revealed that after 48 h, MCMV infection promoted the proliferation of MOVAS when the MOI was 0.01. MCMV infection increased ETAR by downregulating miR-1929-3p. The miR-1929-3p mimic reversed the proliferation and apoptosis, whereas the miR-1929-3p inhibitor promoted this effect. ETAR overexpression further promoted MCMV infection by downregulating miR-1929-3p-mediated proliferation and apoptosis. MCMV infection mediates the downregulation of miR-1929-3p and the upregulation of ETAR, which activates NLRP3 inflammasome. In conclusion, MCMV infection promoted the proliferation of MOVAS, possibly by downregulating miR-1929-3p, promoting the upregulation of the target gene ETAR and activating NLRP3 inflammasome.

Blockade of endothelin-1 receptor B regulates molecules of the major histocompatibility complex in sickle cell disease.

Major Histocompatibility Complex (MHC) molecules have been proposed to play a role in Sickle Cell Disease (SCD) pathophysiology. Endothelial cells express MHC molecules following exposure to cytokines. SCD is characterized, in part, by vascular endothelial cell activation, increased oxidative stress, sickle cell adhesion, and excess levels of endothelin-1 (ET-1) contributing to vaso-occlusive crises. ET-1 activates endothelial cells, induces oxidative stress and inflammation, and alters erythrocyte volume homeostasis. However, the role of ET-1 on MHC regulation in SCD is unclear. We first studied two sickle transgenic knockout mouse models of moderate to severe disease phenotype, ßS-Antilles and Berkeley (BERK) mice. We observed significant increases in H2-Aa mRNA levels in spleens, lungs, and kidneys from transgenic sickle mice when compared to transgenic knockout mice expressing human hemoglobin A (HbA). Mice treated for 14 days with ET-1 receptor antagonists significantly reduced H2-Aa mRNA levels. We characterized the effect of ET-1 on MHC class II expression in the human endothelial cell line EA.hy926. We observed dose-dependent increases in the expression of MHC class II (HLA-DRA) and MHC transcription factor (CIITA) that were significantly blocked by treatment with BQ788, a selective blocker of ET-1 type B receptors. Chromatin immunoprecipitation studies in EA.hy926 cells showed that ET-1 increased Histone H3 acetylation of the HLA-DRA promoter, an event blocked by BQ788 treatment. These results implicate ET-1 as a novel regulator of MHC class II molecules and suggest that ET-1 receptor blockade represents a promising therapeutic approach to regulate both immune and vascular responses in SCD.

Low expression of miR-1929-3p mediates murine cytomegalovirus-induced fibrosis in cardiac fibroblasts via targeting endothelin a receptor/NLRP3 inflammasome pathway.

MicroRNAs are crucial in the development of myocardial remodeling in hypertension. Low miR-1929-3p expression induced by murine cytomegalovirus (MCMV) infection is closely related to hypertensive myocardial remodeling. This study investigated the molecular mechanism of miR-1929-3p-induced myocardial remodeling after MCMV infection. We modeled MCMV-infected mouse cardiac fibroblasts (MMCFs) as the primary cell model. First, MCMV infection reduced the expression of miR-1929-3p and increased the mRNA and protein expression of its target gene endothelin receptor type A (ETAR) in mouse cardiac fibroblasts (MCFs), which demonstrated an internal relationship with myocardial fibrosis (MF) based on high proliferation, phenotypic transformation (α-SMA), and collagen expression in MMCFs. The transfection of the miR-1929-3p mimic downregulated the high expression of ETAR and alleviated these adverse effects in MMCFs. Inversely, these effects were exacerbated by the miR-1929-3p inhibitor. Second, the transfection of endothelin receptor type A over-expressed adenovirus (adETAR) reversed these positive effects of the miR-1929-3p mimic on MF improvement. Third, the transfection of adETAR exhibited a strong inflammatory response in MMCFs with increased expression of NOD-like receptors pyrin domain containing 3 (NLRP3) and increased secretion of interleukin-18. However, we found that the ETAR antagonist BQ123 and the selected NLRP3 inflammasome inhibitor MCC950 effectively eliminated the inflammatory response induced by both MCMV infection and miR-1929-3p inhibitor. Moreover, the MCF supernatant was related to cardiomyocyte hypertrophy. Our findings suggest that MCMV infection promotes MF by inducing the downregulation of miR-1929-3p and the high expression of ETAR, which activates NLRP3 inflammasomes in MCFs.

Targeting tumor-stroma communication by blocking endothelin-1 receptors sensitizes high-grade serous ovarian cancer to PARP inhibition.

PARP inhibitors (PARPi) have changed the treatment paradigm of high-grade serous ovarian cancer (HG-SOC). However, the impact of this class of inhibitors in HG-SOC patients with a high rate of TP53 mutations is limited, highlighting the need to develop combinatorial therapeutic strategies to improve responses to PARPi. Here, we unveil how the endothelin-1/ET-1 receptor (ET-1/ET-1R) axis, which is overexpressed in human HG-SOC and associated with poor prognosis, instructs HG-SOC/tumor microenvironment (TME) communication via key pro-malignant factors and restricts the DNA damage response induced by the PARPi olaparib. Mechanistically, the ET-1 axis promotes the p53/YAP/hypoxia inducible factor-1α (HIF-1α) transcription hub connecting HG-SOC cells, endothelial cells and activated fibroblasts, hence fueling persistent DNA damage signal escape. The ET-1R antagonist macitentan, which dismantles the ET-1R-mediated p53/YAP/HIF-1α network, interferes with HG-SOC/stroma interactions that blunt PARPi efficacy. Pharmacological ET-1R inhibition by macitentan in orthotopic HG-SOC patient-derived xenografts synergizes with olaparib to suppress metastatic progression, enhancing PARPi survival benefit. These findings reveal ET-1R as a mechanistic determinant in the regulation of HG-SOC/TME crosstalk and DNA damage response, indicating the use of macitentan in combinatorial treatments with PARPi as a promising and emerging therapy.

The interaction of ß-arrestin1 with talin1 driven by endothelin A receptor as a feature of α5ß1 integrin activation in high-grade serous ovarian cancer.

Dissemination of high-grade serous ovarian cancer (HG-SOC) in the omentum and intercalation into a mesothelial cell (MC) monolayer depends on functional α5ß1 integrin (Intα5ß1) activity. Although the binding of Intα5ß1 to fibronectin drives these processes, other molecular mechanisms linked to integrin inside-out signaling might support metastatic dissemination. Here, we report a novel interactive signaling that contributes to Intα5ß1 activation and accelerates tumor cells toward invasive disease, involving the protein ß-arrestin1 (ß-arr1) and the activation of the endothelin A receptor (ETAR) by endothelin-1 (ET-1). As demonstrated in primary HG-SOC cells and SOC cell lines, ET-1 increased Intß1 and downstream FAK/paxillin activation. Mechanistically, ß-arr1 directly interacts with talin1 and Intß1, promoting talin1 phosphorylation and its recruitment to Intß1, thus fueling integrin inside-out activation. In 3D spheroids and organotypic models mimicking the omentum, ETAR/ß-arr1-driven Intα5ß1 signaling promotes the survival of cell clusters, with mesothelium-intercalation capacity and invasive behavior. The treatment with the antagonist of ETAR, Ambrisentan (AMB), and of Intα5ß1, ATN161, inhibits ET-1-driven Intα5ß1 activity in vitro, and tumor cell adhesion and spreading to intraperitoneal organs and Intß1 activity in vivo. As a prognostic factor, high EDNRA/ITGB1 expression correlates with poor HG-SOC clinical outcomes. These findings highlight a new role of ETAR/ß-arr1 operating an inside-out integrin activation to modulate the metastatic process and suggest that in the new integrin-targeting programs might be considered that ETAR/ß-arr1 regulates Intα5ß1 functional pathway.

Endothelin-3 is epigenetically silenced in endometrioid endometrial cancer.

PURPOSE: Changes in the activity of endothelins and their receptors may promote neoplastic processes. They can be caused by epigenetic modifications and modulators, but little is known about endothelin-3 (EDN3), particularly in endometrial cancer. The aim of the study was to determine the expression profile of endothelin family and their interactions with miRNAs, and to assess the degree of EDN3 methylation. METHODS: The study enrolled 45 patients with endometrioid endometrial cancer and 30 patients without neoplastic changes. The expression profile of endothelins and their receptors was determined with mRNA microarrays and RT-qPCR. The miRNA prediction was based on the miRNA microarray experiment and the mirDB tool. The degree of EDN3 methylation was assessed by MSP. RESULTS: EDN1 and EDNRA were overexpressed regardless of endometrial cancer grade, which may be due to the lack of regulatory effect of miR-130a-3p and miR-485-3p, respectively. In addition, EDN3 and EDNRB were significantly downregulated. CONCLUSION: The endothelial axis is disturbed in endometrioid endometrial cancer. The observed silencing of EDN3 activity may be mainly due to DNA methylation.

Effect and Mechanism of Endothelin Receptor A Inhibitor BQ-123 Combined with Electroacupuncture on Tibia Cancer Pain in Rats.

Objective: To research the impact and mechanism of endothelin receptor A inhibitor BQ-123 combined with electroacupuncture on tibia cancer pain in rats. Methods: Sprague-Dawley (SD) rats were randomly divided into sham group (SHAM group) and bone cancer pain model group (BCP group). The behavior of SD rats was measured. The histology of the right tibia was observed by hematoxylin-eosin (HE) staining. The remaining rats were randomly divided into model, BQ-123, electroacupuncture, and BQ-123+ electroacupuncture group. Behavioral tests were performed, and mechanical pain threshold (MWT) and thermal pain threshold (TWL) were measured. The expressions of α-smooth muscle actin (αSMA), ETAR (endothelin A receptor), ETB (End of Transmission Block), P-Phosphatidylinositol 3-kinase (PI3K), and P-Protein kinase B (Akt) were detected by real-time fluorescence quantitative PCR and western blot. Results: In the BCP group, bone structure was severely damaged, local tissue swelling was obvious, bone trabecula was missing, and bone cortex was discontinuous. The optical density of Glial fibrillary acidic protein (GFAP) and CD11b immunoreactive signal in BCP group was significantly increased, and most of the ETAR of endothelin receptor was comapped with NeuN, and a small part of GFAP was comapped with CD11b, but no comapped with CD11b. The AS score of BQ-123+ electroacupuncture group was significantly lower than that of BQ-123 group and electroacupuncture group (P < 0.05), whereas the MWT and TWL values were significantly higher than that of the BQ-123 group and electroacupuncture group (P < 0.05). The mRNA expression of α-SMA and ETAR in BQ-123+ electroacupuncture group was lower than that in BQ-123 and electroacupuncture group, and the protein expression of P-PI3K and P-Akt in BQ-123+ electroacupuncture group was lower as well. Conclusion: BQ-123 may inhibit the activation of PI3K/Akt signal path combined with electroacupuncture to alleviate the effects of tibia cancer pain in rats.

Endothelin A receptor antagonist attenuated renal iron accumulation in iron overload heme oxygenase-1 knockout mice.

Progressive iron accumulation and renal impairment are prominent in both patients and mouse models of sickle cell disease (SCD). Endothelin A receptor (ETA) antagonism prevents this iron accumulation phenotype and reduces renal iron deposition in the proximal tubules of SCD mice. To better understand the mechanisms of iron metabolism in the kidney and the role of the ETA receptor in iron chelation and transport, we studied renal iron handling in a nonsickle cell iron overload model, heme oxygenase-1 (Hmox-1-/-) knockout mice. We found that Hmox-1-/- mice had elevated plasma endothelin-1 (ET-1), cortical ET-1 mRNA expression, and renal iron content compared with Hmox-1+/+ controls. The ETA receptor antagonist, ambrisentan, attenuated renal iron deposition, without any changes to anemia status in Hmox-1-/- mice. This was accompanied by reduced urinary iron excretion. Finally, ambrisentan had an important iron recycling effect by increasing the expression of the cellular iron exporter, ferroportin-1 (FPN-1), and circulating total iron levels in Hmox-1-/- mice. These findings suggest that the ET-1/ETA signaling pathway contributes to renal iron trafficking in a murine model of iron overload.

Functional interaction between endothelin-1 and ZEB1/YAP signaling regulates cellular plasticity and metastasis in high-grade serous ovarian cancer.

BACKGROUND: Epithelial-to-mesenchymal transition (EMT) encompasses a highly dynamic and complex key process which leads to metastatic progression. In high-grade serous ovarian carcinoma (HG-SOC), endothelin-1 (ET-1)/endothelin A receptor (ETAR) signaling promotes EMT driving tumor progression. However, the complex nature of intertwined regulatory circuits activated by ET-1 to trigger the metastatic process is not fully elucidated. METHODS: The capacity of ET-1 pathway to guide a critical transcriptional network that is instrumental for metastatic growth was identified in patient-derived HG-SOC cells and cell lines through immunoblotting, q-RT-PCR, co-immunoprecipitation, in situ proximity ligation, luciferase reporter, chromatin immunoprecipitation assays and publicly available databases. Functional assays in HG-SOC cells and HG-SOC xenografts served to test the inhibitory effects of ET-1 receptors (ET-1R) antagonist in vitro and in vivo. RESULTS: We demonstrated that ET-1/ETAR axis promoted the direct physical ZEB1/YAP interaction by inducing their nuclear accumulation in HG-SOC cells. Moreover, ET-1 directed their engagement in a functional transcriptional complex with the potent oncogenic AP-1 factor JUN. This led to the aberrant activation of common target genes, including EDN1 (ET-1) gene, thereby creating a feed-forward loop that sustained a persistent ET-1/ZEB1 signaling activity. Notably, ET-1-induced Integrin-linked kinase (ILK) signaling mediated the activation of YAP/ZEB1 circuit driving cellular plasticity, invasion and EMT. Of therapeutic interest, treatment of HG-SOC cells with the FDA approved ET-1R antagonist macitentan, targeting YAP and ZEB1-driven signaling, suppressed metastasis in vivo in mice. High gene expression of ETAR/ILK/YAP/AP-1/ZEB1 was a strong predictor of poor clinical outcome in serous ovarian cancer patients, indicating the translational relevance of this signature expression. CONCLUSIONS: This study provides novel mechanistic insights of the ET-1R-driven mediators that support the ability of HG-SOC to acquire metastatic traits which include the cooperation of YAP and ZEB1 regulatory circuit paving the way for innovative treatment of metastatic ovarian cancer.

Association of polymorphisms in endothelin-1 and endothelin receptor a genes with vasovagal syncope.

The endothelin system may play a role in the pathogenesis of vasovagal syncope (VVS) because it is implicated in blood pressure regulation. We hypothesized that endothelin-related genetic polymorphisms might modulate susceptibility to VVS. This study aimed to evaluate the possible influence of endothelin-1 (EDN1) and endothelin receptor A (EDNRA) gene variants on the occurrence of tilt-induced VVS and autonomic nervous system activity during the head-up tilt test (HUT). Results were expressed as mean +/- SEM. In 254 patients with recurrent syncope (age 45.33+/-1.22 years, 94 males, 160 females), heart rate variability (HRV) was measured during HUT. EDN1 rs5370 G>T and EDNRA rs5333 T>C gene polymorphisms were assessed using high-resolution melting analysis. There was no statistically significant association between polymorphisms EDN1 rs5370 and EDNRA rs5333 and positivity of HUT or hemodynamic types of VVS. Patients with GT or TT genotypes at the rs5370 locus of the EDN1 had significantly higher values of high-frequency (HF) and the standard deviation of the average NN intervals at the time of the syncope, and they tended to have lower low-frequency (LF) and LF/HF ratio when compared to homozygotes (GG). No statistically significant differences were found in HRV parameters concerning the EDNRA rs5333 genotypes. Our findings suggest the potential role of EDN1 rs5370 variants in regulating autonomic nervous activity and pathogenesis of VVS.

Effects and prognostic values of miR-30c-5p target genes in gastric cancer via a comprehensive analysis using bioinformatics.

Gastric cancer (GC) is a common cancer and the leading cause of cancer-related death worldwide. To improve the diagnosis and treatment of GC, it is necessary to identify new biomarkers by investigating the cellular and molecular mechanisms. In this study, miR-30c-5p expression was significantly down-regulated in GC tissues by comprehensive analysis using multiple databases. The target genes of miR-30c-5p with up-regulated expression level in GC were identified, including ADAM12 (a disintegrin and metalloproteinase12), EDNRA (the Endothelin receptor type A), STC1 (stanniocalcin 1), and CPNE8 (the calcium-dependent protein, copine 8). The expression level of ADAM12 was significantly related to depth of invasion (p = 0.036) in GC patients. The expression level of EDNRA was significantly related to grade (P = 0.003), depth of invasion (P = 0.019), and lymphatic metastasis (P = 0.001). The expression level of CPNE8 was significantly related to grade (P = 0.043) and TNM stage (P = 0.027).Gene set enrichment analysis showed that they might participate in GC progression through cancer-related pathways. CIBERSORT algorithm analysis showed that their expressions were related to a variety of tumor-infiltrating immune cells. The higher expression of those target genes might be the independent risk factor for poor survival of GC patients, and they might be potential prognostic markers in GC patients.

Prenatal hypoxia induced ETBR activation and abnormal ROS signalling in pulmonary artery cells of rat offspring.

Pulmonary arterial hypertension is a progressive disorder characterized by remodeling and increased small pulmonary arteries resistance. Endothelin-1 (ET-1) was related to PAH and ET-1 receptors were up-regulated selectively in the lung when exposed to toxic factor hypoxia. However, the role of ET-1 signaling in the pathogenesis of prenatal hypoxia-induced pulmonary abnormalities remains to be elucidated. Pregnant rats were divided into prenatal hypoxia (10.5 % O2 from gestational day 4-21) and control group. Their three-month-old offspring male rats were tested for vascular functions and molecular analysis, DNA methylation was assessed for cellular hypoxia. Functional testing showed that ET-1-mediated vasoconstriction was enhanced, and the expressions of endothelin A receptor/B receptor (ETAR/ETBR), inositol 1,4,5-trisphosphate receptor, type 1, and the sensitivity of calcium channels were increased in the small pulmonary arteries following prenatal hypoxia. q-PCR and DHE staining showed that the expressions of NADPH oxidase 1/4 (Nox1/4) were up-regulated, along with the increased production of superoxide anion. Furthermore, superoxide anion promoted ET-1-mediated pulmonary artery contraction. In the pulmonary artery smooth muscle cell experiments, q-PCR, Western Blot, CCK8 and DHE staining showed that the expressions of ETBR, Nox1/4, and superoxide anion were increased by hypoxia, along with promoted cell proliferation. 2,2,6,6-Tetramethyl-1-piperidinyloxy reversed hypoxia-induced cell proliferation. ETBR antagonist BQ788 inhibited hypoxia-increased expressions of Nox1/4, superoxide anion production, and proliferation of cells. Moreover, methylation analysis indicated that hypoxia decreased the methylation levels of the ETBR promoter in the pulmonary artery smooth muscle cells. The results indicated that prenatal toxic factor hypoxia resulted in abnormal ETBR activation, which enhanced ET-1-mediated vasoconstriction of pulmonary arteries and pulmonary artery smooth muscle cell proliferation through ETBR/Nox1/4-derived ROS pathway.

Endothelin receptors in renal interstitial cells do not contribute to the development of fibrosis during experimental kidney disease.

Renal interstitial fibrosis is characterized by the development of myofibroblasts, originating from resident renal and immigrating cells. Myofibroblast formation and extracellular matrix production during kidney damage are triggered by various factors. Among these, endothelins have been discussed as potential modulators of renal fibrosis. Utilizing mouse models of adenine nephropathy (AN) and unilateral ureter occlusion (UUO), this study aimed to investigate the contribution of endothelin signaling in stromal mesenchymal resident renal interstitial cells. We found in controls that adenine feeding and UUO caused marked upregulations of endothelin-1 (ET-1) gene expression in endothelial and in tubular cells and a strong upregulation of ETA-receptor (ETA-R) gene expression in interstitial and mesangial cells, while the gene expression of ETB-receptor (ETB-R) did not change. Conditional deletion of ETA-R and ETB-R gene expression in the FoxD1 stromal cell compartment which includes interstitial cells significantly reduced renal ETA-R gene expression and moderately lowered renal ETB-R gene expression. ET receptor (ET-R) deletion exerted no apparent effects on kidney development nor on kidney function. Adenine feeding and UUO led to similar increases in profibrotic and proinflammatory gene expression in control as well as in ETAflflETBflfl FoxD1Cre+ mice (ET-Ko). In summary, our findings suggest that adenine feeding and UUO activate endothelin signaling in interstitial cells which is due to upregulated ETA-R expression and enhanced renal ET-1 production Our data also suggest that the activation of endothelin signaling in interstitial cells has less impact for the development of experimentally induced fibrosis.

Association of Gene Polymorphisms of Some Endothelial Factors with Stent Reendothelization after Elective Coronary Artery Revascularization.

Restenosis remains the main complication after percutaneous coronary interventions in patients with coronary heart disease. The causes of its development include, in particular, genetic factors. We studied polymorphic loci of genes encoding endothelin-1 (EDN1 rs5370), endothelin-1 receptor (EDNRA rs5333), endothelin-converting enzyme (ECE1 rs1076669), and endothelial NO synthase (eNOS rs1549758, eNOS rs1799983, and eNOS rs2070244) in the context of in-stent restenosis development. It was found that the analyzed polymorphisms of the endothelin system genes were more significant for patients aged ≥ 65 years, while the polymorphic loci of the endothelial NO synthase gene (eNOS rs1799983 and eNOS rs1549758) were predominantly associated with time of in-stent restenosis. The obtained results can be useful for comprehensive assessment of the restenosis risk factors and the choice of optimal treatment for patients with coronary heart disease before elective surgical intervention.

Prenatal endothelin or thromboxane receptor antagonism surpasses sympathoinhibition in improving cardiorenal malfunctions in preeclamptic rats.

Current therapies for preeclampsia (PE) and its complications are limited and defective. Considering the importance of endothelin (ET) and thromboxane A2 (TXA2) signaling in PE pathophysiology, we tested the hypothesis that prenatal blockade of endothelin ETA or thromboxane TXA2 receptors favorably reprograms preeclamptic cardiovascular and renal insults. PE was induced by daily oral administration of L-NAME (50 mg/kg) to pregnant rats for 7 consecutive days starting from gestational day 14. The effects of co-exposure to atrasentan (ETA receptor blocker, 10 mg/kg/day) or terutroban (TXA2 receptor blocker, 10 mg/kg/day) on cardiovascular and renal anomalies induced by PE were assessed on gestational day 20 (GD20) and at weaning time and compared with those evoked by the sympatholytic drug α-methyldopa (α-MD, 100 mg/kg/day), a prototypic therapy for PE management. Among all drugs, terutroban was basically the most potent in ameliorating PE-evoked increments in blood pressure and decrements in creatinine clearance. Cardiorenal tissues of PE rats exhibited significant increases in ETA and TXA2 receptor expressions and these effects disappeared after treatment with atrasentan and to a lesser extent by terutroban or α-MD. Atrasentan was also the most effective in reversing the reduced ETB receptor expression in renal tissues of PE rats. Signs of histopathological damage in cardiac and renal tissues of PE rats were mostly improved by all therapies. Together, pharmacologic elimination of ETA or TXA2 receptors offers a relatively better prospect than α-MD in controlling perinatal cardiorenal irregularities sparked by PE.

Endothelin-1 drives invadopodia and interaction with mesothelial cells through ILK.

Cancer cells use actin-based membrane protrusions, invadopodia, to degrade stroma and invade. In serous ovarian cancer (SOC), the endothelin A receptor (ETAR) drives invadopodia by a not fully explored coordinated function of ß-arrestin1 (ß-arr1). Here, we report that ß-arr1 links the integrin-linked kinase (ILK)/ßPIX complex to activate Rac3 GTPase, acting as a central node in the adhesion-based extracellular matrix (ECM) sensing and degradation. Downstream, Rac3 phosphorylates PAK1 and cofilin and promotes invadopodium-dependent ECM proteolysis and invasion. Furthermore, ETAR/ILK/Rac3 signaling supports the communication between cancer and mesothelial cells, favoring SOC cell adhesion and transmigration. In vivo, ambrisentan, an ETAR antagonist, inhibits the adhesion and spreading of tumor cells to intraperitoneal organs, and invadopodium marker expression. As prognostic factors, high EDNRA/ILK expression correlates with poor SOC clinical outcome. These findings provide a framework for the ET-1R/ß-arr1 pathway as an integrator of ILK/Rac3-dependent adhesive and proteolytic signaling to invadopodia, favoring cancer/stroma interactions and metastatic behavior.

G protein- and ß-arrestin Signaling Profiles of Endothelin Derivatives at the Type A Endothelin Receptor.

Background: Endothelin-1 (ET-1) is a potent vasoconstrictor in the cardiovascular system, an effect mediated through the type A endothelin receptor (ETAR), a G protein-coupled receptor (GPCR). Antagonists of the ETAR have shown promising results in randomized clinical trials. However, side effects limit widespread use. Biased agonists have been developed to mitigate the untoward effects of a number of GPCR antagonists. These agents block deleterious G-coupled pathways while stimulating protective ß-arrestin pathways. The goal of this study was to test whether there was any significant ligand bias between endothelin derivatives, and whether this could have any physiologic effects in the cardiovascular system. Methods: A panel of endothelin derivatives were tested in assays of G protein signaling and ß-arrestin 2 recruitment at the ETAR. We then tested the effects of ET-1 on the vasopressor response in wild-type and ß-arrestin 1 and 2 KO mice. Results: We found the endothelins activated a wide range of G proteins at the ETAR, but none of the endothelin derivatives demonstrated significant biased agonism. Endothelin derivatives did display structure-activity relationships with regards to their degrees of agonism. ß-arrestin 1 and 2 knockout mice did not display any differences to wild-type mice in the acute pressor response to ET-1, and ß-arrestin 2 knockout mice did not display any blood pressure differences to wild-type mice in the chronic responses to ET-1. Conclusions: Our findings are consistent with vasoconstriction being mediated by G proteins with a lack of significant desensitization by ß-arrestins at the ETAR. These findings suggest that G protein- and ß-arrestin-biased ETAR agonists could have distinct physiologic effects from balanced agonists, although the endothelin peptide scaffold does not appear suitable for designing such ligands.

CART decreases islet blood flow, but has no effect on total pancreatic blood flow and glucose tolerance in anesthetized rats.

Cocaine- and amphetamine-regulated transcript (CART) is a neurotransmitter and hormone, involved in the regulation of e.g. food intake, body weight, reward and addiction, and stress response. CART has also been found to affect insulin secretion and beta cell morphology, both in vivo and in vitro. Furthermore, CART affects regulation of the cardiovascular system and helps to modulate vascular tone. The present study evaluated the local effect of CART on the pancreatic and islet circulation and function. CART (25 µg/h) or saline, combinations of CART and endothelin-A receptor antagonist (BQ123; 100 µg/kg), and glucose (2 g/kg) were intravenously infused in Sprague Dawley rats followed by blood flow measurements using a microsphere technique. Separately, CART-infused animals underwent an intravenous glucose tolerance test (ivGTT). The direct effect of CART on insulin release was investigated using isolated islets from Sprague Dawley rats. CART reduced islet blood flow, without reduction in total pancreatic blood flow. The normal glucose-induced islet blood flow increase was diminished by CART, albeit still present. Simultaneously, CART had no effect on systemic-, intestinal- or renal blood flow. The endothelin-A receptor antagonist BQ123 together with CART had no pancreatic vascular effects. We found that CART has pronounced vascular constrictive actions restricted to the pancreatic islet circulation but had no effect on insulin release neither in vivo nor in vitro. The mechanisms behind the vascular effects are still unknown, but may reflect a direct action on pancreatic blood vessels.