Exome sequencing identifies novel genetic variants associated with varicose veins.

BACKGROUND: Varicose veins (VV) are one of the common human diseases, but the role of genetics in its development is not fully understood. METHODS: We conducted an exome-wide association study of VV using whole-exome sequencing data from the UK Biobank, and focused on common and rare variants using single-variant association analysis and gene-level collapsing analysis. FINDINGS: A total of 13,823,269 autosomal genetic variants were obtained after quality control. We identified 36 VV-related independent common variants mapping to 34 genes by single-variant analysis and three rare variant genes (PIEZO1, ECE1, FBLN7) by collapsing analysis, and most associations between genes and VV were replicated in FinnGen. PIEZO1 was the closest gene associated with VV (P = 5.05 × 10-31), and it was found to reach exome-wide significance in both single-variant and collapsing analyses. Two novel rare variant genes (ECE1 and METTL21A) associated with VV were identified, of which METTL21A was associated only with females. The pleiotropic effects of VV-related genes suggested that body size, inflammation, and pulmonary function are strongly associated with the development of VV. CONCLUSIONS: Our findings highlight the importance of causal genes for VV and provide new directions for treatment.

Comprehensive analysis of the endothelin system in the kidneys of mice, rats, and humans.

The intrarenal endothelin (ET) system is an established moderator of kidney physiology and mechanistic contributor to the pathophysiology and progression of chronic kidney disease in humans and rodents. The aim of the present study was to characterize ET system by combining single cell RNA sequencing (scRNA-seq) data with immunolocalization in human and rodent kidneys of both sexes. Using publicly available scRNA-seq data, we assessed sex and kidney disease status (human), age and sex (rats), and diurnal expression (mice) on the kidney ET system expression. In normal human biopsies of both sexes and in rodent kidney samples, the endothelin-converting enzyme-1 (ECE1) and ET-1 were prominent in the glomeruli and endothelium. These data agreed with the scRNA-seq data from these three species, with ECE1/Ece1 mRNA enriched in the endothelium. However, the EDN1/Edn1 gene (encodes ET-1) was rarely detected, even though it was immunolocalized within the kidneys, and plasma and urinary ET-1 excretion are easily measured. Within each species, there were some sex-specific differences. For example, in kidney biopsies from living donors, men had a greater glomerular endothelial cell endothelin receptor B (Ednrb) compared with women. In mice, females had greater kidney endothelial cell Ednrb than male mice. As commercially available antibodies did not work in all species, and RNA expression did not always correlate with protein levels, multiple approaches should be considered to maintain required rigor and reproducibility of the pre- and clinical studies evaluating the intrarenal ET system.

Brachial Plexus Root Avulsion Injury-Induced Endothelin-Converting Enzyme-Like 1 Overexpression Is Associated with Injured Motor Neurons Survival.

Brachial plexus root avulsion (BPRA) injury arises from challenging delivery during childbirth, sports-related incidents, or car accidents, leading to extensive loss of motor neurons (MNs) and subsequent paralysis, including both motor and sensory impairment. Surgical nerve re-implantation cannot effectively restore motor function, and the survival of injured MNs is vital for axon regeneration and re-innervating the target muscles. Therefore, identifying novel molecular targets to improve injured MNs survival is of great significance in the treatment of BPRA injuries. Endothelin-converting enzyme-like 1 (ECEL1), a membrane-bound metallopeptidase, was initially identified as a molecule associated with nerve injuries. Damaged neurons exhibit a significant increase in the expression of ECEL1 following various types of nerve injuries, such as optic nerve injury and sciatic nerve injury. This study aimed to investigate the relationship between ECEL1 overexpression and the survival of injured MNs following BPRA injury. Our results observed a significant elevation in ECEL1 expression in injured MNs and positively correlated with MNs survival following BPRA injury. The transcription of ECEL1 is regulated by the transcription factors c-Jun and ATF3 in the context of BPRA injury, which is consistent with previous other nerve injuries study. In addition, the expression of TrkA gradually decreases in ECEL1-positive MNs and ECEL1 possibly preserves the activity of downstream AKT-GSK3ß pathway of TrkA in injured MNs. In conclusion, our results introduce a promising therapeutic molecular target to assist re-implantation surgery for the treatment of BPRA injury.

A non-canonical role of endothelin converting enzyme 1 (ECE1) in promoting lung cancer development via directly targeting protein kinase B (AKT).

BACKGROUND: Lung cancer is the second most common malignancy in the world, and lung adenocarcinoma (LUAD) in particular is the leading cause of cancer death worldwide. Endothelin converting enzyme 1 (ECE1) is a membrane-bound metalloprotease involved in endothelin-1 (ET-1) processing and regulates vasoconstriction. However, very few studies have reported the involvement of ECE1 in regulating tumor cell proliferation, and the mechanism remains poorly understood. Therefore, we aimed to determine the role of ECE1 in lung cancer development. METHODS: The Cancer Genome Atlas database and Kaplan-Meier plotter were used to assess the association between ECE1 and lung cancer. The expression of ECE1 was detected using immunohistochemistry staining and western blotting. A variety of in vitro assays were performed to evaluate the effects of ECE1 on the colony formation, proliferation, migration and invasion using ECE1 knockdown lung cancer cells. The gene expression profiles regulated by ECE1 were investigated by RNA sequencing. An immunoprecipitation assay and immunofluorescence assay were used to evaluate the mechanism underlying the regulatory effect of ECE1 on protein kinase B (AKT). The effect of ECE1 on tumor development was assessed by xenografted lung cancer cells in either C57BL/6 mice or nude mice. RESULTS: ECE1 was upregulated in LUAD and correlated with the poor prognosis of patients with LUAD. Functional studies showed that knockdown of ECE1 retarded the progression of tumors formed by lung cancer cells at least partly by inhibiting tumor cell proliferation. Moreover, ECE1 accelerated tumor cell proliferation through promoting AKT activation dispensable of its canonical target ET-1. Mechanically, ECE1 interacted with the pleckstrin homology (PH) domain of AKT and facilitated its translocation to the plasma membrane for activation. Furthermore, the inhibition of AKT activity counteracted the lung cancer cell growth inhibition observed both in vitro and in xenografts caused by ECE1 suppression. CONCLUSIONS: The present study reveals a non-canonical function of ECE1 in regulating AKT activation and cell proliferation, which provides the basis for the development of a novel strategy for the intervention of cancer including LUAD by abrogating ECE1-AKT signaling.

Mutation analysis of the ECE1 gene in late-onset Alzheimer's disease.

We recently identified a rare coding mutation (R186C) in the ECE2 gene in a late-onset AD (LOAD) family, and demonstrated ECE2 is a risk gene for AD development. ECE1 is a homologous enzyme that shares catalytic activity with ECE2. Although ECE1 has been regarded as a potential candidate gene for AD, few studies have investigated the role of ECE1 variants in patients with AD. In this study, we aimed to investigate rare variants in ECE1 in a cohort of 610 patients with LOAD (age of onset ≥65 years). The summary data of ECE1 variants from ChinaMAP database were used as controls (n = 10,588). We found four rare variants (p.R50W, p.A166=, p.R650Q, and p.P751=) in the patients with sporadic LOAD, while we identified a large number of controls carrying rare variants in ECE1. Moreover, there was no significant association between LOAD and non-synonymous rare damaging variants at the gene level. Our results suggest rare coding variants of ECE1 might not play an important role in AD risk in the Chinese population.

Cancer Stem Cell and Aggressiveness Traits Are Promoted by Stable Endothelin-Converting Enzyme-1c in Glioblastoma Cells.

Glioblastoma (GBM) is the most common and aggressive type of brain tumor due to its elevated recurrence following treatments. This is mainly mediated by a subpopulation of cells with stemness traits termed glioblastoma stem-like cells (GSCs), which are extremely resistant to anti-neoplastic drugs. Thus, an advancement in the understanding of the molecular processes underlying GSC occurrence should contribute significantly towards progress in reducing aggressiveness. High levels of endothelin-converting enzyme-1 (ECE1), key for endothelin-1 (ET-1) peptide activation, have been linked to the malignant progression of GBM. There are four known isoforms of ECE1 that activate ET-1, which only differ in their cytoplasmic N-terminal sequences. Isoform ECE1c is phosphorylated at Ser-18 and Ser-20 by protein kinase CK2, which increases its stability and hence promotes aggressiveness traits in colon cancer cells. In order to study whether ECE1c exerts a malignant effect in GBM, we designed an ECE1c mutant by switching a putative ubiquitination lysine proximal to the phospho-serines Lys-6-to-Arg (i.e., K6R). This ECE1cK6R mutant was stably expressed in U87MG, T98G, and U251 GBM cells, and their behavior was compared to either mock or wild-type ECE1c-expressing clone cells. ECE1cK6R behaved as a highly stable protein in all cell lines, and its expression promoted self-renewal and the enrichment of a stem-like population characterized by enhanced neurospheroid formation, as well as increased expression of stem-like surface markers. These ECE1cK6R-derived GSC-like cells also displayed enhanced resistance to the GBM-related chemotherapy drugs temozolomide and gemcitabine and increased expression of the ABCG2 efflux pump. In addition, ECE1cK6R cells displayed enhanced metastasis-associated traits, such as the modulation of adhesion and the enhancement of cell migration and invasion. In conclusion, the acquisition of a GSC-like phenotype, together with heightened chemoresistance and invasiveness traits, allows us to suggest phospho-ECE1c as a novel marker for poor prognosis as well as a potential therapeutic target for GBM.

ECE2 is a prognostic biomarker associated with m6A modification and involved in immune infiltration of lung adenocarcinoma.

Background: The targeted therapy for lung cancer relies on prognostic genes and requires further research. No research has been conducted to determine the effect of endothelin-converting enzyme 2 (ECE2) in lung cancer. Methods: We analyzed the expression of ECE2 in lung adenocarcinoma (LUAD) and normal adjacent tissues and its relationship with clinicopathological characteristics from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus database (GEO). Immunohistochemical staining was used to further validate the findings. GO/KEGG enrichment analysis and gene set enrichment analysis (GSEA) of ECE2 co-expression were performed using R software. Data from TIMER, the GEPIA database, and TCGA were analyzed to determine the relationship between ECE2 expression and LUAD immune infiltration. To investigate the relationship between ECE2 expression levels and LUAD m6A modification, TCGA data and GEO data were analyzed. Results: ECE2 is highly expressed in various cancers including LUAD. ECE2 showed high accuracy in distinguishing tumor and normal sample results. The expression level of ECE2 in LUAD was significantly correlated with tumor stage and prognosis. GO/KEGG enrichment analysis showed that ECE2 was closely related to mitochondrial gene expression, ATPase activity and cell cycle. GSEA analysis showed that ECE2-related differential gene enrichment pathways were related to mitotic cell cycle, MYC pathway, PLK1 pathway, DNA methylation pathway, HIF1A pathway and Oxidative stress-induced cellular senescence. Analysis of the TIMER, GEPIA database, and TCGA datasets showed that ECE2 expression levels were significantly negatively correlated with B cells, CD4+ cells, M2 macrophages, neutrophils, and dendritic cells. TCGA and GEO datasets showed that ECE2 was significantly associated with m6A modification-related genes HNRNPC, IGF2BP1, IGF2BP3 and RBM1. Conclusion: ECE2 is associated with m6A modification and immune infiltration and is a prognostic biomarker in LUAD.

Association of Endothelin-Converting Enzyme and Endothelin-1 Gene Polymorphisms with Essential Hypertension in Malay Ethnics.

Objectives: Endothelin-1 (ET-1), the most potent endogenous vasoconstrictor, generated by enzymatic cleavage catalyzed by an endothelin-converting enzyme (ECE), plays a significant role in the regulation of hypertension. Methods: This study investigates the effect of endothelin-1 (Lys198Asn/rs5370) and ECE (rs212526 C/T) gene polymorphisms with essential hypertension (EH) among Malay ethnics. To determine the association of gene polymorphism, 177 hypertensives and controls (196) were genotyped using Taqman method. Results: A significant difference was observed in ET-1 rs5370 and ECE rs212526 gene polymorphisms between EH and control subjects (P < 0.001). A significantly high body mass index (BMI), waist-to-hip ratio, fasting plasma glucose, hemoglobin A1c, systolic and diastolic blood pressure, and lipid profiles were observed among the EH patients when compared to controls (P < 0.05). Moreover, T allele (rs5370) carriers in males have a high risk for EH. There was no significant association between gender in ECE C/T polymorphisms (P > 0.05). Conclusion: Based on our result, it is evident that the T allele of ET-1 rs5370 polymorphism and C allele of ECE rs212526 have a significant genetic risk factor in EH among Malay subjects, and BMI and age are associated with hypertension.

Effect of Global Brain Ischemia on Amyloid Precursor Protein Metabolism and Expression of Amyloid-Degrading Enzymes in Rat Cortex: Role in Pathogenesis of Alzheimer's Disease.

The incidence of Alzheimer's disease (AD) increases significantly following chronic stress and brain ischemia which, over the years, cause accumulation of toxic amyloid species and brain damage. The effects of global 15-min ischemia and 120-min reperfusion on the levels of expression of the amyloid precursor protein (APP) and its processing were investigated in the brain cortex (Cx) of male Wistar rats. Additionally, the levels of expression of the amyloid-degrading enzymes neprilysin (NEP), endothelin-converting enzyme-1 (ECE-1), and insulin-degrading enzyme (IDE), as well as of some markers of oxidative damage were assessed. It was shown that the APP mRNA and protein levels in the rat Cx were significantly increased after the ischemic insult. Protein levels of the soluble APP fragments, especially of sAPPß produced by ß-secretase, (BACE-1) and the levels of BACE-1 mRNA and protein expression itself were also increased after ischemia. The protein levels of APP and BACE-1 in the Cx returned to the control values after 120-min reperfusion. The levels of NEP and ECE-1 mRNA also decreased after ischemia, which correlated with the decreased protein levels of these enzymes. However, we have not observed any changes in the protein levels of insulin-degrading enzyme. Contents of the markers of oxidative damage (di-tyrosine and lysine conjugates with lipid peroxidation products) were also increased after ischemia. The obtained data suggest that ischemia shifts APP processing towards the amyloidogenic ß-secretase pathway and accumulation of the neurotoxic Aß peptide as well as triggers oxidative stress in the cells. These results are discussed in the context of the role of stress and ischemia in initiation and progression of AD.

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.

Identification of novel therapeutic targets for contrast induced acute kidney injury (CI-AKI): alpha blockers as a therapeutic strategy for CI-AKI.

Iodinated contrast is used for imaging and invasive procedures and it can cause contrast induced acute kidney injury (CI-AKI), which is the third leading hospital-acquired health problem. The purpose of the present study was to determine the effect of α-adrenergic receptor-1b (Adra1b) inhibition by using terazosin on change in kidney function, gene, and protein expression in C57BL/6J male mice, 6-8 weeks with chronic kidney disease (CKD). CKD was induced by surgical nephrectomy. Twenty eight days later, 100-µL of iodinated contrast (CI group) or saline (S group) was given via the carotid artery. Whole-transcriptome RNA-sequencing (RNA-Seq) analysis of the kidneys was performed at day 2. Mice received either 50-µL of saline ip or terazosin (2 mg/kg) in 50-µL of saline ip 1 hour before contrast administration which was continued every 12 hours until the animals were euthanized 2 and 7 days later. The kidneys were removed for gene expression, immunohistochemical analysis, and blood serum analyzed for kidney function. Differential gene expression analysis identified 21 upregulated and 436 downregulated genes (fold change >2; P < 0.05) that were common to all sample (n = 3 for both contrast and saline). We identified Adra1b using bioinformatic analysis. Mice treated with terazosin had a significant decrease in serum creatinine, urinary Kim-1 levels, HIF-1α, apoptosis, and downstream Adrab1 genes including Ece1, Edn1, pMAPK14 with increased cell proliferation. Contrast exposure upregulated Adra1b gene expression in HK-2 cells. Inhibition of Adra1b with terazosin abrogated Ece1, Edn1, and contrast-induced Fsp-1, Mmp-2, Mmp-9 expression, and caspase-3/7 activity in HK-2 cells.

CircECE1 activates energy metabolism in osteosarcoma by stabilizing c-Myc.

BACKGROUND: Osteosarcoma (OS) is the most common malignant bone tumor and has a poor prognosis. The potential involvement of circular RNAs (circRNAs) in OS progression remains unexplored. Here, we report that CircECE1, a circular RNA derived from human ECE1, plays a critical role in energy metabolism in OS. METHODS: The RIP chip sequence assay was performed to confirm CircECE1, through overexpression or knockdown of CircECE1 to verify its function in 143B and U2OS. RNA immunoprecipitation and immunoprecipitation were used to verify CircECE1's regulation of protein c-Myc and co- immunoprecipitation was used to verified the competitive binding relationship between CircECE1 and SPOP. The influence of CircECE1 on energy metabolism was evaluated by seahorse experiment, western blot, and immunohistochemistry. RESULTS: We found that CircECE1 is highly expressed in OS tissues and cells and that CircECE1 knockdown suppresses tumor proliferation and metastasis both in vitro and in vivo. Further, CircECE1 significantly promotes glucose metabolism in OS cells in vitro and in vivo. Mechanistically, CircECE1 interacts with c-Myc to prevent speckle-type POZ-mediated c-Myc ubiquitination and degradation. C-Myc inhibits thioredoxin binding protein (TXNIP) transcription and subsequently activates the Warburg effect. CONCLUSIONS: CircECE1 regulates the Warburg effect through the c-Myc/TXNIP axis. CircECE1 mediated signal transduction plays a important role in OS process and energy metabolism. These findings may identify novel targets for OS molecular therapy.

Endothelin converting enzyme-1 (ECE-1) deletion in association with Endothelin-1 downregulation ameliorates kidney fibrosis in mice.

Kidney fibrosis is a common final pathway of chronic kidney diseases, which are characterized by renal architecture damage, inflammation, fibroblast expansion and myofibroblast formation. Endothelin converting enzyme-1 (ECE-1) contributes to activation of Endothelin-1 (ET-1), a potent vasoconstrictor and pro-fibrotic substance. This study elucidated the effect of ECE-1 knockout in kidney fibrosis model in mice in association of ET-1 downregulation. Kidney fibrosis was performed in ECE-1 knockout (ECE-1 KO) and vascular endothelial derived ET-1 KO (VEETKO) mice (2 months, 20-30 g, n = 30) and their wild type (WT) littermates using unilateral ureteral obstruction (UUO) procedure. Mice were euthanized on day-7 and day-14 after UUO. Histopathological analysis was conducted for fibrosis and tubular injury. Immunostainings were done to quantify macrophages (F4/80), fibroblasts (FSP-1) and myofibroblasts (α-SMA). Monocyte Chemoattractant Protein-1 (MCP-1), ECE-1 and preproET-1 (ppET-1) mRNA expression were quantified with qRT-PCR, while Transforming Growth Factor-ß1 (TGF-ß1) and α-SMA protein level were quantified with Western blot. ECE-1 KO mice demonstrated reduction of ECE-1 and ppET-1 mRNA expression, attenuation of kidney fibrosis, tubular injury, MCP-1 mRNA expression and macrophage number compared to WT. Double immunostaining revealed fibroblast to myofibroblast formation after UUO, while ECE-1 KO mice had significantly lower fibroblast number and myofibroblast formation compared to WT, which were associated with significantly lower TGF-ß1 and α-SMA protein levels in day-14 of UUO. VEETKO mice also demonstrated attenuation of ET-1 protein level, fibrosis and myofibroblast formation. In conclusion, ECE-1 knockout and ET-1 downregulation attenuated kidney fibrosis.

Association of ECE1 gene polymorphisms and essential hypertension risk in the Northern Han Chinese: A case-control study.

BACKGROUND: The ECE1 gene polymorphisms have been studied as a candidate gene in essential hypertension, but no consensus has been reached. To systematically explore their possible association, a case-control study was conducted. METHODS: This study included 398 hypertensive subjects and 596 healthy volunteers as control subjects in the Northern Han Chinese. A total of 10 tag SNPs of ECE1 gene were genotyped successfully by TaqMan assay. RESULTS: A total of 10 SNPs (rs212544, rs2076280, rs115071, rs2076283, rs9426748, rs11590928, rs212515, rs2236847, rs2282715, and rs2774028) were identified as the tag SNPs for ECE1 gene. Although no positive connection has been found in general population, several SNPs have been found to be related to EH risk in gender-stratified subgroup analysis. In males, rs115071 T allele influenced EH risk in a protective manner, with dominant model (TT+TC vs. CC: p = .032, OR = 0.655, 95% CI = 0.445-0.965), additive model (TT vs. TC vs. CC: p = .019, OR = 0.616, 95% CI = 0.411-0.924), as well as allele comparison (T vs. C: p = .045, OR = 0.702, 95% CI = 0.496-0.992). While, in females, rs212544 AA genotype would increase the onset risk of EH (recessive model: AA vs. GA+GG, p = .024, OR = 1.847, 95% CI = 1.086-3.142). In the three haplotype blocks identified, rs2076283-rs2236847 C-T haplotype was associated with a decreased risk of EH (OR = 0.558, p = .046). CONCLUSION: The current case-control study suggested that several SNPs and related haplotypes on ECE1 gene might be associated with the susceptibility of EH in certain gender subgroups in the Northern Han Chinese population.

Identification of Alzheimer's disease-associated rare coding variants in the ECE2 gene.

Accumulation of amyloid ß protein (Aß) due to increased generation and/or impaired degradation plays an important role in Alzheimer's disease (AD) pathogenesis. In this report, we describe the identification of rare coding mutations in the endothelin-converting enzyme 2 (ECE2) gene in 1 late-onset AD family, and additional case-control cohort analysis indicates ECE2 variants associated with the risk of developing AD. The 2 mutations (R186C and F751S) located in the peptidase domain in the ECE2 protein were found to severely impair the enzymatic activity of ECE2 in Aß degradation. We further evaluated the effect of the R186C mutation in mutant APP-knockin mice. Overexpression of wild-type ECE2 in the hippocampus reduced amyloid load and plaque formation, and improved learning and memory deficits in the AD model mice. However, the effect was abolished by the R186C mutation in ECE2. Taken together, the results demonstrated that ECE2 peptidase mutations contribute to AD pathogenesis by impairing Aß degradation, and overexpression of ECE2 alleviates AD phenotypes. This study indicates that ECE2 is a risk gene for AD development and pharmacological activation of ECE2 could be a promising strategy for AD treatment.

Irreversible and sustained upregulation of endothelin axis during oncogene-associated pancreatic inflammation and cancer.

Endothelin-1 (ET-1) and its two receptors, endothelin receptor A (ETAR) and endothelin receptor B (ETBR) exhibit deregulated overexprerssion in pancreatic ductal adenocarcinoma (PDAC) and pancreatitis. We examined the expression pattern of endothelin (ET) axis components in the murine models of chronic and acute inflammation in the presence or absence of oncogenic K-ras. While the expression of endothelin converting enzyme-1 (ECE-1), ET-1, ETAR and ETBR in the normal pancreas is restricted predominantly to the islet cells, progressive increase of ET receptors in ductal cells and stromal compartment is observed in the KC model (Pdx-1 Cre; K-rasG12D) of PDAC. In the murine pancreas harboring K-rasG12D mutation (KC mice), following acute inflammation induced by cerulein, increased ETAR and ETBR expression is observed in the amylase and CK19 double positive cells that represent cells undergoing pancreatic acinar to ductal metaplasia (ADM). As compared to the wild type (WT) mice, cerulein treatment in KC mice resulted in significantly higher levels of ECE-1, ET-1, ETAR and ETBR, transcripts in the pancreas. Similarly, in response to cigarette smoke-induced chronic inflammation, the expression of ET axis components is significantly upregulated in the pancreas of KC mice as compared to the WT mice. In addition to the expression in the precursor pancreatic intraepithelial neoplasm (PanIN lesions) in cigarette smoke-exposure model and metaplastic ducts in cerulein-treatment model, ETAR and ETBR expression is also observed in infiltrating F4/80 positive macrophages and α-SMA positive fibroblasts and high co-localization was seen in the presence of oncogenic K-ras. In conclusion, both chronic and acute pancreatic inflammation in the presence of oncogenic K-ras contribute to sustained upregulation of ET axis components in the ductal and stromal cells suggesting a potential role of ET axis in the initiation and progression of PDAC.

Transfer and Vascular Effect of Endothelin Receptor Antagonists in the Human Placenta.

Increasing evidence suggests a role for the ET (endothelin) system in preeclampsia. Hence, blocking this system with endothelin receptor antagonists (ERAs) could be a therapeutic strategy. Yet, clinical studies are lacking due to possible teratogenic effects of ERAs. In this study, we investigated the placental transfer of ERAs and their effect on ET-1-mediated vasoconstriction. Term placentas were dually perfused with the selective ETAR (ET type A receptor) antagonists sitaxentan and ambrisentan or the nonselective ETAR/ETBR antagonist macitentan and subsequently exposed to ET-1 in the fetal circulation. ET-1 concentration-response curves after incubation with sitaxentan, ambrisentan, macitentan, or the selective ETBR antagonist BQ-788 were also constructed in isolated chorionic plate arteries using wire-myography, and gene expression of the ET-system was quantified in healthy and early onset preeclamptic placentas. At steady state, the mean fetal-to-maternal transfer ratios were 0.32±0.05 for sitaxentan, 0.21±0.02 for ambrisentan, and 0.05±0.01 for macitentan. Except for BQ-788, all ERAs lowered the response to ET-1, both in the perfused cotyledon and isolated chorionic plate arteries. Placental gene expression of ECE-1, ETAR, and ETBR were comparable in healthy and preeclamptic placentas, while ET-1 expression was higher in preeclampsia. Our study is the first to show direct transfer of ERAs across the term human placenta. Furthermore, ETAR exclusively mediates ET-1-induced constriction in the fetoplacental vasculature. Given its limited transfer, macitentan could be considered as potential preeclampsia therapy. Extending knowledge on placental transfer to placentas of preeclamptic pregnancies is required to determine whether ERAs might be applied safely in preeclampsia.

Endothelin-converting enzyme-1c promotes stem cell traits and aggressiveness in colorectal cancer cells.

Endothelin-1 is a mitogenic peptide that activates several proliferation, survival, and invasiveness pathways. The effects of endothelin-1 rely on its activation by endothelin-converting enzyme-1 (ECE1), which is expressed as four isoforms with different cytoplasmic N termini. Recently, isoform ECE1c has been suggested to have a role in cancer aggressiveness. The N terminus of ECE1c is phosphorylated by protein kinase CK2 (also known as casein kinase 2), and this enhances its stability and promotes invasiveness in colorectal cancer cells. However, it is not known how phosphorylation improves stability and why this is correlated with increased aggressiveness. We hypothesized that CK2 phosphorylation protects ECE1c from N-terminal ubiquitination and, consequently, from proteasomal degradation. Here, we show that lysine 6 is the bona fide residue involved in ubiquitination of ECE1c and its mutation to arginine (ECE1cK6R ) significantly impairs proteasomal degradation, thereby augmenting ECE1c stability, even in the presence of the CK2 inhibitor silmitasertib. Furthermore, colorectal cancer cells overexpressing ECE1cK6R displayed enhanced cancer stem cell (CSC) traits, including increased stemness gene expression, chemoresistance, self-renewal, and colony formation and spheroid formation in vitro, as well as enhanced tumor growth and metastasis in vivo. These findings suggest that CK2-dependent phosphorylation enhances ECE1c stability, promoting an increase in CSC-like traits. Therefore, phospho-ECE1c may be a biomarker of poor prognosis and a potential therapeutic target for colorectal cancer.

Hypoxic regulation of EDN1, EDNRA, EDNRB, and ECE1 gene expressions in ERN1 knockdown U87 glioma cells.

OBJECTIVE: The aim of the present investigation was to study the effect of hypoxia on the expression of genes encoding endothelin-1 (EDN1) and its cognate receptors (EDNRA and EDNRB) as well as endothelin converting enzyme 1 (ECE1) in U87 glioma cells in response to inhibition of endoplasmic reticulum stress signaling mediated by ERN1/IRE1 (endoplasmic reticulum to nucleus signaling 1) for evaluation of their possible significance in the control of glioma growth through ERN1 and hypoxia. METHODS: The expression level of EDN1, EDNRA, EDNRB, and ECE1 genes as well as micro-RNA miR-19, miR-96, and miR-206 was studied in control and ERN1 knockdown U87 glioma cells under hypoxia by quantitative polymerase chain reaction. RESULTS: It was shown that the expression level of EDN1, EDNRA, EDNRB, and ECE1 genes was up-regulated in ERN1 knockdown glioma cells in comparison with the control glioma cells, being more significant for endothelin-1. We also observed down-regulation of microRNA miR-206, miR-96, and miR-19a, which have specific binding sites in mRNA EDN1, EDNRA, and EDNRB, correspondingly, and can participate in posttranscriptional regulation of these mRNA expressions. Furthermore, inhibition of ERN1 endoribonuclease lead to up-regulation of EDNRA and ECE1 gene expressions and down-regulation of the expression level of EDN1 and EDNRB genes in glioma cells. Thus, the expression of EDNRA and ECE1 genes is regulated by ERN1 endoribonuclease, but EDN1 and EDNRB genes preferentially by ERN1 protein kinase. We have also shown that hypoxia enhanced the expression of EDN1, EDNRA, and ECE1 genes and that knockdown of ERN1 signaling enzyme function significantly modified the response of all studied gene expressions to hypoxia. Thus, effect of hypoxia on the expression level of EDN1 and ECE1 genes was significantly or completely reduced in ERN1 knockdown glioma cells since the expression of EDNRA gene was down-regulated under hypoxia. Moreover, hypoxia is induced the expression of EDNRB gene in ERN1 knockdown glioma cells. CONCLUSIONS: Results of this investigation demonstrate that ERN1 knockdown significantly increased the expression of endothelin-1 and its receptors as well as ECE1 genes by different mechanisms and that all studied gene expressions were sensitive to hypoxia. It is possible that hypoxic regulation of the expression of these genes is a result of complex interaction of variable ERN1 related transcription and regulatory factors with HIF1A and possibly contributed to the control of glioma growth.

Endothelin-converting enzyme-1 regulates glucagon-like peptide-1 receptor signalling and resensitisation.

Following nutrient ingestion, glucagon-like peptide 1 (GLP-1) is secreted from intestinal L-cells and mediates anti-diabetic effects, most notably stimulating glucose-dependent insulin release from pancreatic ß-cells but also inhibiting glucagon release, promoting satiety and weight reduction and potentially enhancing or preserving ß-cell mass. These effects are mediated by the GLP-1 receptor (GLP-1R), which is a therapeutic target in type 2 diabetes. Although agonism at the GLP-1R has been well studied, desensitisation and resensitisation are perhaps less well explored. An understanding of these events is important, particularly in the design and use of novel receptor ligands. Here, using either HEK293 cells expressing the recombinant human GLP-1R or the pancreatic ß-cell line, INS-1E with endogenous expressesion of the GLP-1R, we demonstrate GLP-1R desensitisation and subsequent resensitisation following removal of extracellular GLP-1 7-36 amide. Resensitisation is dependent on receptor internalisation, endosomal acidification and receptor recycling. Resensitisation is also regulated by endothelin-converting enzyme-1 (ECE-1) activity, most likely through proteolysis of GLP-1 in endosomes and the facilitation of GLP-1R dephosphorylation and recycling. Inhibition of ECE-1 activity also increases GLP-1-induced activation of extracellular signal-regulated kinase and generation of cAMP, suggesting processes dependent upon the lifetime of the internalised ligand-receptor complex.