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Accumulation of amyloid-b peptide (Aβ) aggregates in synapses may contribute to the profound synaptic loss characteristic of Alzheimer's disease (AD). The origin of synaptic Aβ aggregates remains elusive, but loss of endosomal proteostasis may trigger their formation. In this study, we identified the synaptic compartments where Aβ accumulates, and performed a longitudinal analysis of synaptosomes isolated from brains of TgCRND8 APP transgenic mice of either sex. To evaluate the specific contribution of Aβ-degrading protease endothelin-converting enzyme (ECE-1) to synaptic/endosomal Aβ homeostasis, we analyzed the effect of partial Ece1 KO in brain and complete ECE1 KO in SH-SY5Y cells. Global inhibition of ECE family members was used to further assess their role in preventing synaptic Aβ accumulation. Results showed that, before extracellular amyloid deposition, synapses were burdened with detergent-soluble Aβ monomers, oligomers, and fibrils. Levels of all soluble Aβ species declined thereafter, as Aβ42 turned progressively insoluble and accumulated in Aβ-producing synaptic endosomal vesicles with characteristics of multivesicular bodies. Accordingly, fibrillar Aβ was detected in brain exosomes. ECE-1-deficient mice had significantly increased endogenous synaptosomal Aβ42 levels, and protease inhibitor experiments showed that, in TgCRND8 mice, synaptic Aβ42 became nearly resistant to degradation by ECE-related proteases. Our study supports that Aβ accumulating in synapses is produced locally, within endosomes, and does not require the presence of amyloid plaques. ECE-1 is a determinant factor controlling the accumulation and fibrillization of nascent Aβ in endosomes and, in TgCRND8 mice, Aβ overproduction causes rapid loss of Aβ42 solubility that curtails ECE-mediated degradation. [ABSTRACT FROM AUTHOR]

Endothelin-1 (ET-1) induces contraction, proliferation, and collagen synthesis of activated hepatic stellate cells and is a potent mediator of portal hypertension. Endothelin-converting enzyme-1 (ECE-1) generates ET-1 from the inactive precursor big-endothelin-1. The cellular distribution and activity of ECE-1 in the liver is unknown. Hepatic fibrogenesis was induced in rats by CCl4 administration and secondary biliary cirrhosis after 6 weeks of complete bile duct occlusion (BDO). The tissue ET-1 and ET receptor protein levels were quantified, the ECE-1 isoform mRNAs were measured by RNase protection assay and ECE-1 activity was analyzed. ECE-1a and -b mRNA were upregulated in biliary cirrhosis and in CCl4-injured livers, whereas ECE-1c mRNA remained unchanged. ECE-1 activity was increased after BDO and peaked at 12 h after acute CCl4-intoxication. Tissue levels of ET-1, ETA- and ETB receptors were elevated 7-, 5-, and 4.6-fold in cirrhotic rats, respectively. ECE-1 activity increased following BDO and acute CCl4-intoxication. In conclusion, ECE-1a and -b RNAs are upregulated in fibrogenesis, indicating that these isoforms play a central role in ET-1 generation during fibrogenesis and portal hypertension.

Objective: To explore the mechanisms underlying endothelin-1 (ET-1) elevations induced by excessive fluoride exposure. Methods: We measured serum and bone fluoride ion content and plasma ET-1 levels and compared these parameters among different groups in an animal model. We also observed morphological changes in the aorta and endothelium of rabbits. In cell experiments, human umbilical vein endothelial cells (HUVECs) were treated with varying concentrations of NaF for 24h, with or without 10 µM U0126 pretreatment for 1 h. ET-1 levels in culture fluid and intracellular reactive oxygen species (ROS) levels, as well as ET1 gene, endothelin-converting enzyme-1 (ECE-1), extracellular signal-regulating kinase 1/2 (ERK1/2), pERK1/2 expression levels and RAS activation were measured and compared among the groups. Results: Plasma ET-1 levels of rabbits increased significantly in fluorinated groups compared with those in the control group. The rabbit thoracic aortas became slightly hardened in fluorinated groups compared with those in the control group, and some vacuoles were present in the endothelial cell cytoplasm of the rabbits in fluorinated groups. In our cell experiments, ET1 gene and ECE-1 expression levels in HUVECs and ET-1 expression levels in the cell culture supernatants increased significantly in some experimental groups compared with those in the control group. These trends paralleled the changes in intracellular ROS levels, RAS activation, and the pERK1/2-to-ERK1/2 ratio. After U0126 was added, ECE-1 expression and ET-1 levels decreased significantly. Conclusion: Excessive fluoride exposure leads to characteristic endothelial damage (vacuoles), thoracic aorta hardening, and plasma ET-1 level elevations in rabbits. In addition, the ROS-RAS-MEK1/2-pERK1/2/ERK1/2 pathway plays a crucial—and at least partial—role in ET-1 over-expression, which is promoted by excessive fluoride exposure.

Endothelin-1 (ET-1) induces contraction, proliferation, and collagen synthesis of activated hepatic stellate cells and is a potent mediator of portal hypertension. Endothelin-converting enzyme-1 (ECE-1) generates ET-1 from the inactive precursor big-endothelin-1. The cellular distribution and activity of ECE-1 in the liver is unknown. Hepatic fibrogenesis was induced in rats by CCl4 administration and secondary biliary cirrhosis after 6 weeks of complete bile duct occlusion (BDO). The tissue ET-1 and ET receptor protein levels were quantified, the ECE-1 isoform mRNAs were measured by RNase protection assay and ECE-1 activity was analyzed. ECE-1a and -b mRNA were upregulated in biliary cirrhosis and in CCl4-injured livers, whereas ECE-1c mRNA remained unchanged. ECE-1 activity was increased after BDO and peaked at 12 h after acute CCl4-intoxication. Tissue levels of ET-1, ETA- and ETB receptors were elevated 7-, 5-, and 4.6-fold in cirrhotic rats, respectively. ECE-1 activity increased following BDO and acute CCl4-intoxication. In conclusion, ECE-1a and -b RNAs are upregulated in fibrogenesis, indicating that these isoforms play a central role in ET-1 generation during fibrogenesis and portal hypertension. [ABSTRACT FROM AUTHOR]

Background: Endothelin‐converting enzyme‐1 (ECE‐1) primarily converts big endothelins (ETs) into active endothelin‐1 (ET‐1). However, the expression pattern and prognostication status of ECE‐1 in head and neck cancer (HNC) are enigmatic. In this study, we investigated ECE‐1 expression and assessed the roles of ECE‐1 as a predictor for HNC differentiation and prognosis. Materials and Methods: ECE‐1 expressions were evaluated by immunohistochemical analysis using a tissue microarray (TMA) composed of 100 cases of head and neck squamous cell carcinoma. The correlation of ECE‐1 expression with clinicopathologic variables and patient outcomes was analyzed. Results: ECE‐1 may be overexpressed in HNC carcinoma cells. Higher ECE‐1 level was detected more frequently in moderately to poorly differentiated tumors and showed a lower differentiation category compared to the G1 cases (p = 0.015); this finding was further confirmed by an adjusted odds ratio (OR) of 4.071 (p = 0.042). Moreover, Kaplan–Meier survival analyses showed that a higher ECE‐1 expression was associated with a poorer survival in patients with HNC (p < 0.0001). On multivariate Cox proportional hazards models analysis, ECE‐1 of high expression proved to be an independent prognostic factor with a hazard ratio (HR) of 3.985 (p < 0.001). Conclusion: Our data provide the first evidence that overexpression of ECE‐1 in HNC is a predictor of poor tumor differentiation and prognosis. [ABSTRACT FROM AUTHOR]

Patients with esophageal squamous cell carcinoma have poor survival and high recurrence rate, thus an effective prognostic biomarker is needed. Endothelin-converting enzyme-1 is responsible for biosynthesis of endothelin-1, which promotes growth and invasion of human cancers. The role of endothelin-converting enzyme-1 in esophageal squamous cell carcinoma is still unknown. Therefore, this study investigated the significance of endothelin-converting enzyme-1 expression in esophageal squamous cell carcinoma clinically. We enrolled patients with esophageal squamous cell carcinoma who provided pretreated tumor tissues. Tumor endothelin-converting enzyme-1 expression was evaluated by immunohistochemistry and was defined as either low or high expression. Then we evaluated whether tumor endothelin-converting enzyme-1 expression had any association with clinicopathological findings or predicted survival of patients with esophageal squamous cell carcinoma. Overall, 54 of 99 patients with esophageal squamous cell carcinoma had high tumor endothelin-converting enzyme-1 expression, which was significantly associated with lymph node metastasis (p = 0.04). In addition, tumor endothelin-converting enzyme-1 expression independently predicted survival of patients with esophageal squamous cell carcinoma, and the 5-year survival was poorer in patients with high tumor endothelin-converting enzyme-1 expression (p = 0.016). Among patients with locally advanced and potentially resectable esophageal squamous cell carcinoma (stage II and III), 5-year survival was poorer with high tumor endothelin-converting enzyme-1 expression (p = 0.003). High tumor endothelin-converting enzyme-1 expression also significantly predicted poorer survival of patients in this population. In patients with esophageal squamous cell carcinoma, high tumor endothelin-converting enzyme-1 expression might indicate high tumor invasive property. Therefore, tumor endothelin-converting enzyme-1 expression could be a good biomarker to identify patients with worse survival and higher risks of recurrence, who might benefit from the treatment by endothelin-converting enzyme-1 inhibitor. [ABSTRACT FROM AUTHOR]

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

Aim To analyse the ability of TWEAK to modify the endothelin system, particularly endothelin-1 (ET-1) and endothelinconverting enzyme-1 (ECE-1), studying the intracellular mechanisms implied. TNF-like weak inducer of apoptosis (TWEAK) is a member of TNF superfamily; it has different biological functions such as inflammation, angiogenesis, proliferation, and apoptosis. TWEAK and fibroblast growth-factor-inducible 14 are expressed in different cell types, including endothelial and smooth muscle cells. Despite their presence in endothelial cells, the effect of TWEAK on endothelial function is incompletely defined. Methods and results In cells, TWEAK induced protein (Western blot) and mRNA (quantitative polymerase chain reaction) expression of ECE-1. Results were related to transcriptional changes, as ECE-1 promoter activity (transfection assays) was also increased. Transfections with serial deletions of ECE-1 promoter suggest a potential role for AP-1 and NFkB, which were confirmed by electrophoretic mobility shift assays. When AP-1 or NFkB activations were inhibited by specific inhibitors of AP-1, PD-98059 (Erk1/2 inhibitor), or SP-600125 (JNK inhibitor), and also with an inhibitor of NFKB and PDTC, TWEAK effect was partially blocked in both cases, suggesting that both transcription factors are implied in ECE-1 regulation. Moreover, the endothelial changes induced by TWEAK were also tested in vivo, using 3-monthold male CD-1 mice treated with TWEAK 10 μg/kg body weight for 24h, finding similar effects, a rise in ET-1 production (enzyme-linked immunosorbent assay), and ECE-1 expression in aorta and lung tissues. Mice showed slight hypertension after 4 h of treatment, which disappeared at 24 h. Conclusions In pathological situations such as chronic inflammation, TWEAK could be more harmful through this effect at endothelial level. Pharmacological blockade of this cytokine could prevent the haemodynamic and structural changes related to an increased ET-1 synthesis. [ABSTRACT FROM AUTHOR]

Abstract: Although calcimimetics were developed to block parathyroid hormone synthesis, some reports suggest that they may also reduce blood pressure by unknown mechanisms. Calcimimetic-induced changes in the synthesis of endothelial vasoactive factors could be involved. Wistar rats were treated with the calcimimetic R-568, and systolic blood pressure (SBP) was registered with a tail-cuff sphygmomanometer, the content of endothelial nitric oxide synthase (eNOS) and endothelin-converting enzyme (ECE-1) in tissue was evaluated by immunohistochemistry and Western blot, circulating levels of endothelin-1 (ET-1) were measured by ELISA. R-568 reduced SBP and circulating levels of ET-1, without changes in eNOS expression. In contrast, R-568 increased the lung and vascular content of ECE-1. In order to analyze the mechanisms involved, we studied the effect of R-568 on human endothelial cells. R-568 did not modify neither eNOS protein content nor pre-pro-ET-1 mRNA expression, but increased ECE-1 protein content, and decreased ET-1 synthesis and ECE-1 activity. The inhibition of ECE-1 activity was very strong, similar to the classic ECE inhibitor phosphoramidon, the addition of exogenous zinc restored enzymatic activity. Moreover, the amount of zinc in immunoprecipitated ECE from R-568 treated cells was 3-fold less than in control cells. In conclusion, R-568 inhibits ECE by expelling zinc from the enzyme, with the subsequent decrease in enzymatic activity and reducing circulating levels of ET-1, which may be responsible for the lower SBP observed in R-568-treated rats. This descent would be partially compensated by the increased synthesis of the ECE-1 itself, and by other homeostatic mechanisms that regulate SBP. [Copyright &y& Elsevier]

Endothelin-converting enzyme-1b (ECE-1b) C-338A is a polymorphism located in ECE-1 gene promoter, which has been reported to correlate with the risk of carotid atherosclerosis. We conducted a hospital-based case-control study of 309 patients with ischemic stroke and 309 controls matched on age and gender. The frequencies of ECE-1b-338 CC, CA, and AA genotypes in cases (45.0, 40.4, and 14.6 %) were significantly different from those of controls (53.1, 39.1, and 7.8 %, χ = 8.519, P = 0.014), respectively. Compared with C carriers, A alleles showed correlation with a 42 % increased risk of ischemic stroke (adjusted odds ratio (OR) = 1.42, 95 % CI = 1.11-1.81). After adjustment for potential risk factors, the AA genotype still kept positive correlation with ischemic stroke (OR = 1.78; 95 % CI = 1.06-3.54). In stratified analyses, the significant association of the A allele with ischemic stroke was observed in female subjects (adjusted OR = 1.64, 95 % CI = 1.17-2.89) and the subjects with age ≥64 years old (adjusted OR = 2.09, 95 % CI = 1.38-3.23). The present study suggested that the C-338A polymorphism of the ECE-1b gene was associated with an increased risk of ischemic stroke in the Chinese Han population. [ABSTRACT FROM AUTHOR]

Background and Objective Endopeptidases, such as neutral endopeptidase ( NEP), endothelin-converting enzyme-1 ( ECE-1) and a disintegrin and metalloprotease 17 ( ADAM17), are believed to have various important roles in oral mucosal and epidermal tissue for the regulation of defensive biological responses in the oral cavity, and their expression and activity are influenced by various factors, including oral diseases. However, knowledge concerning these endopeptidases in the oral cavity has been minimal until now. This study focused on three metalloendopeptidases - NEP, ECE-1 and ADAM17 - in the oral buccal mucosal epithelium of patients with periodontal diseases and investigated the relationship between their gene-expression levels and periodontal disease. Material and Methods The levels of expression of NEP, ECE-1 and ADAM17 m RNAs in tissue samples collected from the oral buccal mucosal epithelium of 61 patients were investigated by relative quantification using real-time RT- PCR analysis. information on oral and systemic health was obtained from the clinical record of each patient. Results Among the three groups, classified based on the diagnosis of periodontal diseases (healthy/gingivitis, early periodontitis and moderate/advanced periodontitis), the relative expression level of NEP m RNA was significantly increased in the early periodontitis group and in the moderate/advanced periodontitis group compared with that in the healthy/gingivitis group. Moreover, the relative expression levels of ECE1 and ADAM17 m RNAs were significantly increased in the moderate/advanced periodontitis group compared with those in the healthy/gingivitis group. The correlation coefficients between the mean relative expression levels of NEP and ECE1 m RNAs, NEP and ADAM17 m RNAs, and ECE1 and ADAM17 m RNAs were r = 0.758, r = 0.707 and r = 0.934, respectively ( p < 0.001). Furthermore, among the oral-related factors, there was a significant correlation between the number of sites with probing pocket depths of more than 4 mm and of more than 6 mm and the relative expression levels of NEP, ECE1 and ADAM17 m RNAs. In stepwise logistic regression models, high relative expression levels of ECE1 and ADAM17 m RNAs were significantly associated with moderate/advanced periodontitis. Conclusion The present study suggests that the severity of periodontal disease may be associated with the expression of metalloendopeptidase genes, including NEP, ECE1 and ADAM17, in the buccal mucosal epithelium. [ABSTRACT FROM AUTHOR]

The endothelin (ET) system seems to play a pivotal role in hypertension and in proteinuric kidney disease, including the micro- and macro-vascular complications of diabetes. Endothelin-1 (ET-1) is a multifunctional peptide that primarily acts as a potent vasoconstrictor with direct effects on systemic vasculature and the kidney. ET-1 and ET receptors are expressed in the vascular smooth muscle cells, endothelial cells, fibroblasts and macrophages in systemic vasculature and arterioles of the kidney, and are associated with collagen accumulation, inflammation, extracellular matrix remodeling, and renal fibrosis. Experimental evidence and recent clinical studies suggest that endothelin receptor blockade, in particular selective ETR blockade, holds promise in the treatment of hypertension, proteinuria, and diabetes. Concomitant blockade of the ET receptor is not usually beneficial and may lead to vasoconstriction and salt and water retention. The side-effect profile of ET receptor antagonists and relatively poor antagonist selectivity for ET receptor are limitations that need to be addressed. This review will discuss what is currently known about the endothelin system, the role of ET-1 in the pathogenesis of hypertension and kidney disease, and summarize literature on the therapeutic potential of endothelin system antagonism. [ABSTRACT FROM AUTHOR]

Background and Purpose Calcitonin gene-related peptide ( CGRP) is a potent vasodilator, implicated in the pathogenesis of migraine. CGRP activates a receptor complex comprising, calcitonin receptor-like receptor ( CLR) and receptor activity-modifying protein 1 ( RAMP1). In vitro studies indicate recycling of CLR● RAMP1 is regulated by degradation of CGRP in early endosomes by endothelin-converting enzyme-1 ( ECE-1). However, it is not known if ECE-1 regulates the resensitization of CGRP-induced responses in functional arterial tissue. Experimental Approach CLR, ECE-1a-d and RAMP1 expression in rat mesenteric artery smooth muscle cells ( RMA- SMCs) and mesenteric arteries was analysed by RT-PCR and by immunofluorescence and confocal microscopy. CGRP-induced signalling in cells was examined by measuring cAMP production and ERK activation. CGRP-induced relaxation of arteries was measured by isometric wire myography. ECE-1 was inhibited using the specific inhibitor, SM-19712. Key Results RMA- SMCs and arteries contained mRNA for CLR, ECE-1a-d and RAMP1. ECE-1 was present in early endosomes of RMA- SMCs and in the smooth muscle layer of arteries. CGRP induced endothelium-independent relaxation of arteries. ECE-1 inhibition had no effect on initial CGRP-induced responses but reduced cAMP generation in RMA- SMCs and vasodilation in mesenteric arteries responses to subsequent CGRP challenges. Conclusions And Implications ECE-1 regulated the resensitization of responses to CGRP in RMA- SMCs and mesenteric arteries. CGRP-induced relaxation did not involve endothelium-derived pathways. This is the first report of ECE-1 regulating CGRP responses in SMCs and arteries. ECE-1 inhibitors may attenuate an important vasodilatory pathway, implicated in primary headaches and may represent a new therapeutic approach for the treatment of migraine. [ABSTRACT FROM AUTHOR]

Plasma concentrations of the mitogenic peptide endothelin-1 (ET-1) are significantly elevated in men with metastatic prostate cancer (PC). ET-1 also contributes to the transition of hormonally regulated androgen-dependent PC to androgen-independent disease. ET-1 is generated from big-ET-1 by endothelin-converting enzyme (ECE-1). ECE-1 is present in PC cell lines and primary tissue and is elevated in primary malignant stromal cells compared with benign. siRNA or shRNA-mediated knockdown of endogenous ECE-1 in either the epithelial or stromal compartment significantly reduced PC cell (PC-3) invasion and migration. The re-addition of ET-1 only partially recovered the effect, suggesting ET-1-dependent and -independent functions for ECE-1 in pPC. The ET-1-independent effect of ECE-1 on PC invasion may be due to modulation of downstream signalling events. Addition of an ECE-1 specific inhibitor to PC-3 cells reduced phosphorylation of focal adhesion kinase (FAK), a signalling molecule known to play a role in PC. siRNA-mediated knockdown of ECE-1 resulted in a significant reduction in FAK phosphorylation. Accordingly, transient ECE-1 overexpression in PNT1-a cells increased FAK phosphorylation. In conclusion, ECE-1 influences PC cell invasion via both ET-1-mediated FAK phosphorylation and ET-1 independent mechanisms. [ABSTRACT FROM AUTHOR]

Abstract: To investigate the association between endothelin-converting enzyme-1b (ECE-1b) C-338A polymorphism and gastric cancer risk, we conducted a hospital-based case–control study of 256 gastric cancer cases and 256 controls matched on age and gender. The genotypes were identified by polymerase chain reaction-restriction fragment length polymorphism. We found that the genotype frequencies were significantly different (P =0.005) between cases and controls. Compared with the wild genotype CC, the variant genotypes (CA+AA) were associated with a 64% increased risk of gastric cancer [adjusted odds ratio (OR)=1.64, 95% confidence interval (CI) 1.15–2.33]. Further stratification analyses indicated that the increased risk was especially noteworthy in older subjects (age ⩾58) (adjusted OR=1.91, 95% CI 1.18–3.09), women (adjusted OR=2.30, 95% CI 1.11–4.79) and non-smokers (adjusted OR=1.79, 95% CI 1.19–2.67). Our results suggest that the ECE-1b C-338A polymorphism may be associated with increased risk of gastric cancer. [Copyright &y& Elsevier]

Abstract: Background: Endothelin-converting enzyme-1 (ECE-1), the key enzyme responsible for endothelin-1 generation, has been linked to coronary artery disease (CAD). Recently, a genetic polymorphism (ECE-1b C-338A) located in ECE-1 gene promoter was identified. However, it is unclear whether this polymorphism is associated with the risk of CAD. Methods: We conducted a study with CAD patients and controls matched by age and sex to examine the prevalence of ECE-1b C-338A polymorphism in CAD. Results: The frequencies of ECE-1b-338CC, CA, and AA genotypes in cases (40.1%, 42.2%, and 17.7%) were significantly different from those of controls (50.6%, 40.5%, and 8.9%, χ 2 =9.989, P =0.007). Subjects with the variant genotypes (CA+ AA) had a 58% increased risk of CAD relative to CC carriers (adjusted OR=1.58, 95% CI=1.07–2.32). Furthermore, the adjusted OR of AA genotype for CAD was 2.33 (95% CI=1.25–4.35). In stratified analyses, the A allele was significantly associated with increased risk of CAD in female (adjusted OR=2.86, 95% CI=1.40–5.84) and subjects with age ≥ 64 y (adjusted OR=2.96, 95% CI=1.73–5.08). Moreover, the frequency of patients with variant genotypes increased gradually from single- to triple-vessel disease although without statistical significance (P =0.069 for trend). Conclusion: Our results suggested that ECE-1b-338C to A variant might be associated with increased risk of CAD in Chinese population. [Copyright &y& Elsevier]

Objective: To investigate the mechanism of vascular endothelial growth factor (VEGF)-induced endothelin-1 production in human umbilical vein endothelial cells (HUVECs). Methods: Endothelin-1 levels were measured in conditioned medium of women with preeclampsia HUVECs were treated with different concentrations of VEGF165 and at various time intervals. Next, we measured endothelin-1 levels after HUVECs were also incubated with VEGF and endothelin-converting enzyme-1 (ECE-1) inhibitor or tissue inhibitors of matrix metalloproteinase-2 (TIMP-2). Additionally, the circulating levels of total and free VEGF, matrix metalloproteinase-2 (MMP-2), and endothelin-1 were measured in 20 preeclamptic patients and 20 healthy pregnant controls. Results: HUVECs treated with VEGF increased their endothelin-1 production in a concentration and time-dependent manner. The production of endothelin-1 was inhibited by TIMP-2, but not by the ECE-1 inhibitor. Total VEGF, MMP-2, and endothelin-1 concentrations were higher in preeclampsia and showed significant positive correlations between them. Conclusion: These findings suggest that VEGF-induced endothelin-1 production might be mediated by MMP-2 rather than by ECE-1 upregulation. [ABSTRACT FROM AUTHOR]

A burgeoning body of evidence suggests that endothelin-1 (ET-1), the most potent endogenous vasoconstrictor yet identified, may be critical in the pathophysiology of various cardiovascular diseases. The ET system may also be implicated in the pathogenesis of cerebral vasospasm after aneurysmal subarachnoid hemorrhage (SAH). Clinical studies have shown that the levels of ET-1 are increased in the cerebrospinal fluid (CSF) of patients following SAH, suggesting that ET-1-mediated vasoconstriction plays a major role in the development of vasospasm after SAH. The potential involvement of ETs in SAH-induced vasospasm has triggered considerable interest in developing therapeutic strategies that inhibit the biologic effects of ET. One promising approach to block the biosynthesis of ETs is suppressing the proteolytic conversion of the precursor peptide (big ET-1) to its vasoactive form (ET-1) using metalloprotease as endothelin-converting enzyme (ECE) inhibitor. To date, three types of ECE-1 inhibitors have been synthesized: dual ECE-1/neutral endopeptidase 24.11 (NEP) inhibitors, triple ECE-1/NEP/angiotensin-converting enzyme (ACE) inhibitors and selective ECE-1 inhibitors. The therapeutic effects of ECE-1 inhibitors on the prevention and reversal of SAH-induced vasospasm in animal studies are reviewed and discussed. [ABSTRACT FROM AUTHOR]

Alterations of the expression of some peptidases in the pituitary gland of a fatigued rat model were identified. Rats were kept in a cage filled with water to a height of 1.5 cm to disturb deep sleep. After 24-h sleep disturbance, expression of neutral endopeptidase 24.11 (neprilysin) mRNA was increased in the intermediate lobe of the pituitary gland, whereas the mRNA expression of another family member, damage-induced neuronal endopeptidase, which is normally expressed in a subgroup of anterior pituitary cells, was significantly suppressed. These alterations were demonstrated by RT-PCR, northern blotting and in situ hybridization. Other family members, such as neprilysin 2 and endothelin converting enzyme-1, did not show any change in mRNA expression. An increase of neprilysin mRNA expression was not seen in any other tissues of the sleep-disturbed rats. The enzymatic activity of neprilysin was also increased in the pituitary. The augmentation of neprilysin expression and activity was prolonged as long as the sleep disturbance continued (up to 5 days), and returned to the basal level when rats were allowed to sleep freely. These results suggest that peptide processing and degradation in the pituitary may be an influential factor in fatigued states such as sleep disturbance. [ABSTRACT FROM AUTHOR]

Abstract: Modeling the three-dimensional structure of neprilysin 2 (NEP2) using the crystal structure of neprilysin as template revealed that their active sites share many common features, though slight differences therein cannot completely account for their specific pharmacological profiles. Recent evidence also suggest that residues outside the active site can play crucial functions in the maturation and enzymatic activity of these metalloproteases. To further explore the functions of amino acids in the acquisition and maintenance of the NEP2 structure, site-directed mutagenesis of conserved residues involved in the enzymatic activity of ECE-1 was performed. In particular, the ultimate tryptophan residue of ECE-1 was recently shown to be important in its activation. This residue was thus mutated in the secreted isoform of NEP2, as were proline residues located in its vicinity. Expression of these mutants in AtT20 cells and study of their secretion and catalytic activities shows that while the ultimate tryptophan residue of the NEP2 sequence is not essential to its proper and activity, structural changes in its vicinity can have a severe impact on the maturation processes involved in the activation of NEP2. [Copyright &y& Elsevier]

Abstract: This study addressed the question how different lipoproteins modulate the expression of endothelin-converting enzyme-1 (ECE-1) in human endothelial cells. The effect of native and oxidized low-density lipoproteins (nLDL, oxLDL) on expression of ECE-1, prepro-endothelin-1, and endothelin-1 peptide was studied in primary cultures of human endothelial cells. Native and oxidized LDL increased ECE-1 mRNA after 1h, reaching its maximum at 100μg/ml (1.9- and 2.5-fold, respectively). Furthermore, ECE-1 protein expression, prepro-endothelin-1 mRNA, and endothelin-1 peptide release were increased in response to nLDL or oxLDL. Induction of ECE-1 by nLDL and of prepro-endothelin-1 by oxLDL was reduced by protein kinase C inhibition. Increased expression of ECE-1 mRNA by oxLDL and of prepro-endothelin-1 by nLDL was blocked by an angiotensin II receptor type 1 antagonist. Our data provide evidence for a new mechanism how increased LDL plasma levels might contribute to enhanced endothelin-1 release in patients with hypercholesterolemia. [Copyright &y& Elsevier]

The progression of rat liver fibrosis induced by intraperitoneal administration of thioacetamide (TAA) was evaluated by immunocytochemistry using anti-α-smooth muscle actin (α-SMA), antiendothelin-converting enzyme (ECE)-1, and anti-monocyte chemotactic protein (MCP)-1 antibodies. The fibrous septal spaces gradually increased after administration of TAA, and pseudolobules were established in the 7-week TAA-treated groups. Immunoreactivities against α-SMA were not detected in hepatic stellate cells (HSCs) of the control group without TAA treatment, although they were observed in the HSCs around the fibrous septal spaces in all TAA-treated groups, indicating that activation of HSCs occurs during the establishment of pseudolobules. Immunoreactivities against ECE-1 and MCP-1 were seen in such HSCs of the TAA-treated groups, but few or no immunoreactivities were detected in the HSCs of the control group. The most significant increase in the ECE-1 immunoreactivities was detected in the 1-week TAA-treated group, whereas that in MCP-1 was observed in the 7-week TAA-treated group. The present immunocytochemistry indicated a difference in the accelerated expression period between immunoreactivities against ECE-1 and MCP-1 in the HSCs during the progression of TAA-induced liver fibrosis, suggesting that ECE-1 is involved in the early phase of liver fibrosis and that MCP-1 plays a role during the later phase. [ABSTRACT FROM AUTHOR]

Endothelin-converting enzyme-1 (ECE-1) is a membrane-bound zinc metallopeptidase that is homologous to neprilysin in amino acid sequence. A major in vivo function of ECE-1 is the generation of endothelin-1, a potent vasoconstrictor, from big endothelin-1. ECE-1 is also potentially involved in the processing or degradation of other peptide hormones. In this study we have used substrates based on the sequence of the COOH-terminal half of big endothelin-1 to examine the subsite specificity of recombinant ECE-1. The big endothelin-1 [16–38] peptides were systematically varied at either position 21 (P1) or position 22 (P1′) and used in steady-state kinetic analyses of ECE-1. The results indicate that the S1 pocket of ECE-1 is relatively nonselective, but that the S1′ subsite of ECE-1 has a preference for large hydrophobic side chains. The peptidyl carboxydipeptidase activity of ECE-1 was also characterized, revealing that substrates with COOH-terminal carboxylates are highly preferred over the cognate amides and esters. A site-directed mutagenesis study was carried out to identify the active-site amino acid residues specifically involved in binding to the COOH-terminal carboxylate of substrates. The data indicate that Arg133 of ECE-1, which corresponds to Arg102 of neprilysin that has been identified as an active-site residue of neprilysin involved in binding to the free carboxylate of some substrate peptides, may not play the same role. However, the low activity observed for an ECE-1 Arg726 mutant is consistent with a role for this arginine residue in the binding of substrates, a role which has been ascribed to arginine residues in both thermolysin (Arg203) and neprilysin (Arg717). [Copyright &y& Elsevier]

Patients with esophageal squamous cell carcinoma have poor survival and high recurrence rate, thus an effective prognostic biomarker is needed. Endothelin-converting enzyme-1 is responsible for biosynthesis of endothelin-1, which promotes growth and invasion of human cancers. The role of endothelin-converting enzyme-1 in esophageal squamous cell carcinoma is still unknown. Therefore, this study investigated the significance of endothelin-converting enzyme-1 expression in esophageal squamous cell carcinoma clinically. We enrolled patients with esophageal squamous cell carcinoma who provided pretreated tumor tissues. Tumor endothelin-converting enzyme-1 expression was evaluated by immunohistochemistry and was defined as either low or high expression. Then we evaluated whether tumor endothelin-converting enzyme-1 expression had any association with clinicopathological findings or predicted survival of patients with esophageal squamous cell carcinoma. Overall, 54 of 99 patients with esophageal squamous cell carcinoma had high tumor endothelin-converting enzyme-1 expression, which was significantly associated with lymph node metastasis ( p = 0.04). In addition, tumor endothelin-converting enzyme-1 expression independently predicted survival of patients with esophageal squamous cell carcinoma, and the 5-year survival was poorer in patients with high tumor endothelin-converting enzyme-1 expression ( p = 0.016). Among patients with locally advanced and potentially resectable esophageal squamous cell carcinoma (stage II and III), 5-year survival was poorer with high tumor endothelin-converting enzyme-1 expression ( p = 0.003). High tumor endothelin-converting enzyme-1 expression also significantly predicted poorer survival of patients in this population. In patients with esophageal squamous cell carcinoma, high tumor endothelin-converting enzyme-1 expression might indicate high tumor invasive property. Therefore, tumor endothelin-converting enzyme-1 expression could be a good biomarker to identify patients with worse survival and higher risks of recurrence, who might benefit from the treatment by endothelin-converting enzyme-1 inhibitor.