Endothelin-converting Enzyme Inhibitor Research Articles

The Role of NOX2-Derived Reactive Oxygen Species in the Induction of Endothelin-Converting Enzyme-1 by Angiotensin II.

Endothelin-1 is a key regulator of vascular tone and blood pressure in health and disease. We have recently found that ET-1 production in human microvascular endothelial cells (HMECs) can be promoted by angiotensin II (Ang II) through a novel mechanism involving octamer-binding transcription factor-1 (Oct-1), NADPH oxidase-2 (NOX2), and superoxide anions. As the formation of bioactive ET-1 also depends on endothelin-converting enzyme-1 (ECE-1), we investigated the transcriptional regulation of the ECE1 gene. We found that exposure of HMECs to Ang II resulted in a concentration- and time-dependent increase in ECE1 mRNA expression. Pharmacological inhibition of ECE-1 reduced Ang II-stimulated ET-1 release to baseline values. The effect of Ang II on ECE1 mRNA expression was associated with Oct-1 binding to the ECE1 promoter, resulting in its increased activity. Consequently, the Ang II-stimulated increase in ECE1 mRNA expression could be prevented by siRNA-mediated Oct-1 inhibition. It could also be abolished by silencing the NOX2 gene and neutralizing superoxide anions with superoxide dismutase. In mice fed a high-fat diet, cardiac expression of Ece1 mRNA increased in wild-type mice but not in Nox2-deficient animals. It can be concluded that Ang II engages Oct-1, NOX2, and superoxide anions to stimulate ECE1 expression in the endothelium.

The Effect of Tauroursodeoxycholic Acid (TUDCA) Treatment on Pregnancy Outcomes and Uterine Artery Function in a Rat Model of Advanced Maternal Age

IntroductionAdvanced maternal age (AMA; ≥35 years) increases the risk of pregnancy complications and adverse pregnancy outcomes such as fetal growth restriction. Endoplasmic reticulum (ER) and oxidative stress have been linked to vascular dysfunction (i.e. reduced nitric oxide [NO] & increased endothelin‐1 [ET‐1] levels) in aging. Tauroursodeoxycholic acid (TUDCA) is an ER and oxidative stress inhibitor and has been shown to improve embryo development in vitro. However, whether treatment with TUDCA can help improve uterine vascular function and pregnancy outcomes in AMA has not been investigated. We hypothesize that TUDCA treatment will improve pregnancy outcomes and vascular function in a rat model of AMA.MethodsYoung (4 months) and aged (9.5 months of age; ~35 years in humans) pregnant control or TUDCA‐treated rats were studied on gestational day 20 (term=22 days). Following euthanasia, pregnancy outcomes (litter size, resorption sites, fetal weight, and placental weight) were recorded, and main uterine arteries were isolated. Endothelium‐dependent vasorelaxation to methacholine (MCh) was assessed by wire myography in the presence/absence of pan NO synthase inhibitor (L‐NAME). In addition, big‐endothelin‐1 (bET‐1) vasoconstriction responses were evaluated, in the presence/absence of endothelin converting enzyme (ECE) inhibitor (CGS). Data summarized as maximum vasodilation response (Emax) and analyzed by two‐way ANOVA with Sidak’s post‐test, p<0.05 was considered significant.ResultsFetal body weights were reduced in AMA control rats compared to young dams (p<0.017), while TUDCA treatment tended to increase fetal body weight in AMA rats (p=0.067); TUDCA did not affect fetal body weight in the young dams. Placental weights, litter size and number of resorptions were similar in all the groups. The maximum relaxation (Emax) to MCh was impaired in the AMA dams compared to young dam (p=0.047), and this reduced relaxation was not evident in the TUDCA treated AMA dams. Ex vivo treatment with L‐ NAME showed an increased contribution of NO in young TUDCA treated rats (ΔEmax [differences in Emax]; p=0.043) compared to young control rats but not in AMA rats. Ex vivo treatment with CGS demonstrated reduced ECE conversion in young TUDCA treated rats (Emax; p<0.001), while CGS had no effect in the young control dams or both AMA groups.ConclusionOur study assessed TUDCA as an intervention to improve the pregnancy outcomes in a rat model of AMA. An increased fetal body weight suggests a beneficial effect of TUDCA in AMA pregnancies. However, contrary to our hypothesis, in young dams, TUDCA increased NO contribution and reduced ECE effect, but did not affect uterine artery function in AMA dams. Nevertheless, TUDCA has the potential to improve pregnancy outcomes in complicated pregnancies, but further studies are warranted.

Endothelin-1 down-regulated vascular endothelial growth factor A is involved in trichloroethene-induced kidney injury.

The mechanism of kidney injury in occupational medicamentosa-like dermatitis due to trichloroethylene exposure is not well understood. This study aimed to investigate the role of endothelin-1 (ET-1)/vascular endothelial-derived growth factor-A (VEGF-A) in trichloroethylene (TCE)-induced renal injury. Forty BALB/c female mice were used in this study to build the TCE-sensitization mouse model. Transmission electron microscopic observation, histological examination, periodic acid-Schiff staining, serum urea nitrogen, creatinine, and urinary total protein levels were used to reflect renal injury. Glypican1, syndecan1, ET-1 and VEGF-A protein levels were measured by western blot. Serum ET-1 level was also measured. Tumor necrosis factor alpha (TNF-α) and vascular cell adhesion molecule-1 (VCAM-1) were detected by immunohistochemistry. The results showed that TCE-sensitized mouse kidneys were damaged and accompanied by increased serum ET-1. After treatment with CGS 35066, the inhibitor of endothelin converting enzyme-1 (ECE-1), kidney ET-1, TNF-α and VCAM-1 levels decreased, and renal function improved in TCE+CGS 35066-sensitized positive mice. In addition, kidney VEGF-A, glomerular endothelial cell glypican1 and syndecan1 levels increased, and endothelial cell damage was alleviated after treatment with CGS 35066. The results suggest that inhibiting ECE-1 could alleviate glomerular endothelial cell injury by inhibiting ET-1 expression, thus promoting endothelial cell repair by upregulating VEGF-A.

Altered Vascular Adaptations to Pregnancy in a Rat Model of Advanced Maternal Age.

Advanced maternal age (≥35 years old) increases the risk of pregnancy complications such as preeclampsia and fetal growth restriction. We previously demonstrated vascular dysfunction and abnormal pregnancy outcomes in a rat model of advanced maternal age. However, vascular adaptations to pregnancy in aging were not studied. We hypothesize that advanced maternal age is associated with a more vasoconstrictive phenotype due to reduced nitric oxide (NO) and increased activity of matrix metalloproteinases (MMPs), contributing to impaired vascular adaptations to pregnancy. A rat model of advanced maternal age was used: young (4 months) and aged (9.5 months; ∼35 years in humans) non-pregnant and pregnant rats. On gestational day 20 (term = 22 days; non-pregnant rats were aged-matched), blood pressure and heart rate were measured (tail cuff plethysmography) and vascular function was assessed in mesenteric arteries (wire myography). Endothelium-dependent relaxation to methylcholine (MCh) was assessed in the presence/absence of nitric oxide synthase inhibitor (L-NAME), or inhibitors of endothelium-dependent hyperpolarization (EDH; apamin and TRAM-34). Vasoconstriction responses to big endothelin-1 (bigET-1), in the presence/absence of MMPs-inhibitor (GM6001) or endothelin converting enzyme (ECE-1) inhibitor (CGS35066), in addition, ET-1 responsiveness, were measured. Blood pressure was elevated only in aged non-pregnant rats (p < 0.001) compared to all other groups. MCh responses were not different, however, L-NAME decreased maximum vasodilation in young (p < 0.01) and aged pregnant rats (p < 0.001), and decreased MCh sensitivity in young non-pregnant rats (p < 0.01), without effects in aged non-pregnant rats. EDH contribution to relaxation was similar in young non-pregnant, and aged non-pregnant and pregnant rats, while EDH-mediated relaxation was absent in young pregnant rats (p < 0.001). BigET-1 responses were enhanced in aged non-pregnant (p < 0.01) and pregnant rats (p < 0.05). No significant changes in bigET-1 conversion occurred in the presence of MMP-inhibitor, whereas ECE-1 inhibition reduced bigET-1 constriction in aged rats (p < 0.01). No differences in ET-1 sensitivity were observed. In conclusion, contrary to our hypothesis, reduced blood pressure, and an increased EDH-dependent contribution to vasodilation suggest a compensatory mechanism that may reflect beneficial adaptations in these aged rats that were able to maintain pregnancy. These data increase our understanding of how the vascular adaptive pathways in pregnancy compensate for advanced maternal age.

Therapeutic Effect of Endothelin-Converting Enzyme Inhibitor on Chronic Kidney Disease through the Inhibition of Endoplasmic Reticulum Stress and the NLRP3 Inflammasome.

Chronic inflammation and oxidative stress significantly contribute to the development and progression of chronic kidney disease (CKD). The NOD-like receptor family pyrin containing domain-3 (NLRP3) inflammasome plays a key role in the inflammatory response. The renal endothelin (ET) system is activated in all cases of CKD. Furthermore, ET-1 promotes renal cellular injury, inflammation, fibrosis and proteinuria. Endothelin-converting enzymes (ECEs) facilitate the final processing step of ET synthesis. However, the roles of ECEs in CKD are not clear. In this study, we investigated the effects of ETs and ECEs on kidney cells. We found that ET-1 and ET-2 expression was significantly upregulated in the renal tissues of CKD patients. ET-1 and ET-2 showed no cytotoxicity on human kidney tubular epithelial cells. However, ET-1 and ET-2 caused endoplasmic reticulum (ER) stress and NLRP3 inflammasome activation in tubular epithelial cells. The ECE inhibitor phosphoramidon induced autophagy. Furthermore, phosphoramidon inhibited ER stress and the NLRP3 inflammasome in tubular epithelial cells. In an adenine diet-induced CKD mouse model, phosphoramidon attenuated the progression of CKD by regulating autophagy, the NLRP3 inflammasome and ER stress. In summary, these findings showed a new strategy to delay CKD progression by inhibiting ECEs through autophagy activation and restraining ER stress and the NLRP3 inflammasome.

Discovery of a novel analogue of FR901533 and the corresponding biosynthetic gene cluster from Streptosporangium roseum No. 79089.

FR901533 (1, also known as WS79089B), WS79089A (2), and WS79089C (3) are polycyclic aromatic natural products with promising inhibitory activity to endothelin-converting enzymes. In this work, we isolated five tridecaketide products from Streptosporangium roseum No. 79089, including 1-3, benaphthamycin (4) and a novel FR901533 analogue (5). The structure of 5 was characterized based on spectroscopic data. Compared with the major product 2, the new compound 5 has an additional hydroxyl group at C-12 and an extra methyl group at the 13-OH. The configuration of C-19 of these compounds was determined to be R using Mosher's method. A putative biosynthetic gene cluster for compounds 1-5 was discovered by analyzing the genome of S. roseum No. 79089. This 38.6-kb gene cluster contains 38 open reading frames, including a minimal polyketide synthase (wsaA-C), an aromatase (wsaD), three cyclases (wsaE, F, and W), and a series of tailoring enzymes such as monooxygenases (wsaO1-O7) and methyltransferases (wsaM1 and M2). Disruption of the ketosynthase gene (wsaA) in this gene cluster abolished the production of 1-5, confirming that this gene cluster is indeed responsible for the biosynthesis of 1-5. A type II polyketide biosynthetic pathway was proposed for this group of natural endothelin-converting enzyme inhibitors. KEY POINTS: • Five aromatic tridecaketides were isolated from Streptosporangium roseum No. 79089. • A novel FR901533 analogue, 12-hydroxy-13-O-methyl-WS79089A, was characterized. • The absolute configuration of C-19 of FR901533 and analogues was determined. • The biosynthetic gene cluster of FR901533 and analogues was discovered.

Endothelins contribute to early renal insufficiency engendered by acute urinary tract obstruction in neonatal pigs

Urinary tract obstruction reduces kidney functions, which, if left uncorrected, may lead to irreversible renal loss, especially in infants. Despite the removal of obstruction, a decline in steady‐state renal blood flow (RBF) persists, vascular mechanisms of which are poorly understood. Urinary tract obstruction‐induced increase in the levels of vasoactive endothelin (ET)‐1 has been reported in humans and rodents. However, whether obstructive renal vasculopathy is associated with increased renal production of all ET isoforms (ET1‐3) remains unresolved. Also, the mechanisms that underlie ET‐induced renal vasoregulation in neonates are unclear. Here, we show that ET receptor (ETR)‐operated calcium entry via TRPC3 channels increases renal vascular resistance and reduces RBF in neonatal pigs. Acute unilateral urinary tract obstruction (UUO) in neonatal pigs increased peptidase endothelin converting enzyme 1 (ECE1)‐dependent renal production of ET1‐3 and diacylglycerol (DAG). Despite the removal of obstruction, acute UUO reduced kidney perfusion and caused an increase in renal vascular resistance (RVR) and the urinary levels of NGAL, the early predictive biomarker of acute kidney injury. The protein expression levels of ETRs (ETA and ETB) and TRPC3 channels in renal microvessels were unaltered in neonatal pigs subjected to acute UUO. However, pharmacological inhibition of ECE1, ETR, and TRPC3 mitigated acute UUO‐induced increase in RVR and urine NGAL in the pigs. These data suggest that increased ECE1 production and proteolytic processing of all big ETs to their vasoactive isoforms contribute to early neonatal renal insufficiency engendered by UUO. ET‐derived DAG activates renal vascular smooth muscle cell TRPC3 channels, leading to extracellular calcium entry, vasoconstriction, increased RVR and sustained hypoperfusionSupport or Funding InformationNIH/NIDDK: R01DK101668 and R56DK120595

Human Placental Stem Cell Therapy in Stroke: Endothelial / Smooth Muscle Mechanisms Underlying Protection?

Following thrombolytic therapy for stroke, ischemia/reperfusion (I/R) mediated inflammation often disrupts the blood brain barrier (BBB). This can enhance expression of endothelial adhesion markers and perturb normal blood flow regulation. Proposed benefits of stem cell therapy (SCT) in stroke include secretion of protective and trophic factors which limit acute brain I/R injury. We evaluated human placenta mesenchymal stem cells (hPMSC) as potential ameliorative SCT in an acute ischemic stroke model. We hypothesized that potential benefits of hPMSCs, in acute stroke injury, reflect site‐specific suppression of post‐ischemic immune cell adhesion, transvascular leukocyte emigration and preservation of the BBB. This can limit the progression of post –ischemic stroke injury. We found that intraperitoneal (IP) administration of hPMSCs at the time of reperfusion, using the transient middle cerebral artery occlusion (MCAO)/reperfusion model, produced highly significant protection of the ipsilateral hemisphere. We then examined hPMSC support of BBB integrity via mobilization of junctional proteins in human brain endothelial cells (hCMEC‐D3) in conjunction with a biotin/FITC‐avidin permeability assay coupled with western blotting and immunolocalization of junctional proteins. We also assessed the ability of hPMSC to suppress inflammation‐driven hCMEC‐D3 binding of macrophages (RAW264.7) and transmigration of PMNs. In addition, we used collagen gels to study the influence of hPMSC on human brain vascular smooth muscle cells (hBVSMC) tonic contractility.We observed that hPMSC administration significantly (p&lt;0.001) enhanced the barrier integrity of brain endothelial monolayers, at 24h, in an oxygen/glucose deprived/reperfused (OGDR) condition. We found that hCMEC‐D3, which were independently co‐cultured with hPMSCs, expressed significantly higher levels of VE‐cadherin and α‐catenin under OGDR conditions (p&lt;0.001). Additionally, the spatial localization of VE‐cadherin and α‐catenin at hCMEC‐D3 junctions was increased, in the OGDR state, following hPMSC treatment. We also observed that hPMSC treatment significantly (p&lt;0.0001) reduced binding of RAW264.7 to TNF‐α/ LPS activated hCMEC‐D3 monolayers. The expression of VCAM‐1 was increased in TNF‐α/ LPS activated hCMEC‐D3 while this was significantly (p&lt;0.01, P&lt;0.05) decreased by hPMSC treatment. hPMSC also inhibited (p&lt;0.05) PMN transmigration across hCMEC‐D3 monolayers. Lastly, we found that hBVSMC contraction was significantly increased by hPMSC treatment (p=0.0001). This effect was reversed by angiotensin II Type 1 receptor blockade (Losartan, 50μM), angiotensin converting enzyme (Captopril, 50μM), thromboxane A2 synthesis (Ozagrel, 10μM), and endothelin converting enzyme inhibition (SM19712, 100μM).We conclude that pleiotropic factors associated with hPMSC administration can have a favorable impact on BBB integrity, immune cell interaction, and vasoregulation potentially alleviating the detrimental effects of ischemic stroke.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Ligand-Specific Signaling Profiles and Resensitization Mechanisms of the Neuromedin U2 Receptor.

The structurally related, but distinct neuropeptides, neuromedin U (NmU) and neuromedin S (NmS) are ligands of two G protein-coupled NmU receptors (NMU1 and NMU2). Hypothalamic NMU2 regulates feeding behavior and energy expenditure and has therapeutic potential as an anti-obesity target, making an understanding of its signaling and regulation of particular interest. NMU2 binds both NmU and NmS with high affinity, resulting in receptor-ligand co-internalization. We have investigated whether receptor trafficking events post-internalization are biased by the ligand bound and can therefore influence signaling function. Using recombinant cell lines expressing human NMU2, we demonstrate that acute Ca2+ signaling responses to NmU or NmS are indistinguishable and that restoration of responsiveness (resensitization) requires receptor internalization and endosomal acidification. The rate of NMU2 resensitization is faster following NmU compared with NmS exposure, but is similar if endothelin-converting enzyme-1 activity is inhibited or knocked down. Although acute activation of extracellular signal-regulated kinase (ERK) is also similar, activation by NMU2 is longer lasting if NmS is the ligand. Furthermore, when cells are briefly challenged before removal of free, but not receptor-bound ligand, activation of ERK and p38 mitogen-activated protein kinase by NmS is more sustained. However, only NmU responses are potentiated and extended by endothelin-converting enzyme-1 inhibition. These data indicate that differential intracellular ligand processing produces different signaling and receptor resensitization profiles and add to the findings of other studies demonstrating that intracellular ligand processing can shape receptor behavior and signal transduction.

Role of Myocardial Endothelin‐Converting Enzyme in Promoting Coronary Arteriolar Constriction during Diabetes

Coronary blood flow is regulated, by adjusting the diameters of coronary arterioles, to meet the oxygen demand of the myocardium. The release of local metabolites by the myocardium upon metabolic activation can trigger nitric oxide (NO)‐dependent coronary arteriolar dilation. In diabetes, coronary blood flow regulation is compromised by impairing endothelium‐dependent NO‐mediated vasodilation. However, it is unclear whether the myocardium plays a role in altering coronary arteriolar constriction to endothelin‐1 (ET‐1), which has been shown to contribute to the development of diabetic cardiomyopathy. To address this issue, pig coronary arterioles were isolated with the surrounding myocardial tissue, cannulated and pressurized for functional study. The vasomotor function was analyzed using the videomicroscopic technique and compared with isolated arterioles without myocardium. Streptozocin‐injected pigs (2 wk of hyperglycemia; 524 ± 48 mg/dl; n = 7) or age‐matched control pigs (euglycemia; 91 ± 7 mg/dl; n = 7) were used to study the influence of myocardium on coronary vasomotor function under diabetes. Coronary arterioles with and without myocardial tissue from control pigs constricted to ET‐1 (0.1 nM) in a comparable manner. Without myocardium, incubating the arterioles with ET‐1 precursor big ET‐1 (0.1 μM) for 40 minutes caused a 32% constriction of the vessels. The vasoconstriction reached 53% in the presence of myocardial tissue. The enhanced big ET‐1‐induced vasoconstriction was inhibited 74% by ETA receptor antagonist BQ123, 24% by ETB receptor antagonist BQ788, and 87% by endothelin‐converting enzyme (ECE) inhibitor CGS35066. In diabetic pigs, coronary arteriolar constriction in response to big ET‐1 was enhanced by 26% compared with that from the euglycemic pigs, and the vasoconstriction was further increased in the presence of myocardial tissue. However, ET‐1‐induced coronary arteriolar constriction without surrounding myocardium was not altered by diabetes. The ECE1 protein expression was increased in the myocardium in diabetic pigs. In conclusion, myocardial ECE is capable of converting big ET‐1 to ET‐1 for coronary arteriolar constriction. Under diabetic condition, upregulated myocardial ECE1 expression appears to promote ET‐1 synthesis for coronary arteriolar constriction, and may contribute to coronary blood flow dysregulation and lead to insufficient blood supply to the heart.Support or Funding InformationSupported by Baylor Scott &amp; White Kruse Chair Endowment Fund, and NIH Grants R01EY023335 and R01EY024624This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

High endothelin-converting enzyme-1 expression independently predicts poor survival of patients with esophageal squamous cell carcinoma.

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.

Immature endothelial cells initiate endothelin-mediated constriction of newborn arteries.

Endothelial expression and the release of endothelin-1 (ET-1) in levels sufficient to initiate vasoconstriction is considered to be a hallmark feature of pathological endothelial dysfunction. During the immediate postnatal period, arterial endothelial cells undergo remarkable structural and functional changes as they transition to a mature protective cell layer, which includes a marked increase in NO dilator activity. The present study demonstrates that endothelial cells lining newborn central arteries express high levels of ET-1 peptides and, in response to endothelial stimulation, rapidly release ET-1 and initiate powerful ET-1-mediated constriction. This activity is lost as the endothelium matures in the postnatal period. Heightened activity of ET-1 in the neonatal endothelium might contribute to inappropriate responses of immature arteries to stress or injury. Indeed, the immature endothelium resembles dysfunctional endothelial cells, and retention or re-emergence of this phenotype may contribute to the development of vascular disease. Endothelial cells lining fetal and newborn arteries have an unusual phenotype, including reduced NO activity, prominent actin stress fibres and poorly developed cellular junctions. Experiments were performed to determine whether the immature endothelium of newborn arteries also expresses and releases endothelin-1 (ET-1) and initiates endothelium-dependent constriction. Carotid arteries were isolated from newborn (postnatal day 1; P1), postnatal day 7 (P7) and postnatal day 21 (P21) mice and assessed in a pressure myograph system. Endothelial stimulation with A23187 or thrombin caused constriction in P1 arteries, no significant change in diameter of P7 arteries, and dilatation in P21 arteries. In P1 arteries, constriction to thrombin or A23187 was inhibited by endothelial-denudation, by ET-1 receptor antagonists (BQ123 plus BQ788) or by inhibition of endothelin-converting enzyme (phosphoramidon or SM19712). ET-1 receptor antagonism did not affect responses to thrombin or A23187 in more mature arteries. Exogenous ET-1 caused similar concentration-dependent constrictions of P1, P7 and P21 arteries. Endothelial stimulation with thrombin rapidly increased the endothelial release of ET-1 from P1 but not P21 aortas. Endothelial expression of ET-1 peptides, as assessed by immunofluorescence analysis, was increased in P1 compared to P21 arteries. Therefore, newborn endothelial cells express high levels of ET-1 peptides, rapidly release ET-1 in response to endothelial stimulation, and initiate ET-1-mediated endothelium-dependent constriction. This activity is diminished as the endothelium matures in the immediate postnatal period. Heightened activity of ET-1 in neonatal endothelium probably reflects an early developmental role of the peptide, although this might contribute to inappropriate responses of immature arteries to stress or injury.

Abstract A58: Expression and function of endothelin converting enzyme 1 and endothelin receptors in breast cancer invasion

Abstract The endothelin axis, comprised of the active peptides endothelin 1-3 (ET1-3), the endothelin A receptor (ETAR) and endothelin B receptor (ETBR), and endothelin converting enzyme 1 (ECE1) has been studied in numerous cancers. In clinical samples of breast cancer, increased levels of ET1, ECE1, and the endothelin receptors have been reported, and several studies correlate expression of these proteins with poor prognosis. The endothelin pathway is known to induce mitogen activated protein kinase activation, leading to increased survival, proliferation, and migration, but the precise role of ECE1 and the endothelin receptors in breast cancer invasion is not fully understood. We hypothesized that the endothelin axis is instrumental in modulating breast cancer cell invasion. To initially confirm that the endothelin axis regulates breast cancer cell invasion, we measured MDA-MB-231 breast cancer cell invasion using an in vitro assay in the presence of an ECE1 inhibitor, CGS 35066, and found that abrogating ECE1 decreases breast cancer invasion. Next, we measured expression of ECE1 mRNA and protein using RT-PCR and Western blots in normal human mammary epithelial cells (HMECs), low-invading MCF-7 breast cancer cells, and highly invasive MDA-MB-231 breast cancer cells. We found that the major ECE1 isoform, ECE-1c, was most highly expressed on MCF-7 cells and that HMECs expressed more of the intracellular ECE1 isoform, ECE-1b, than either cancer cell line. Western blot analysis confirmed these results, as total ECE1 protein expression was highest in the MCF-7 cells. While multiple studies have been published on the expression of the endothelin receptors ETAR and ETBR in clinical breast cancer samples, less is known about the specific expression and function of these receptors in these breast cancer cell lines. Western blot analysis demonstrated notable up-regulation of ETAR expression in the MCF-7 and MDA-MB-231 cells compared to the HMECs, which is consistent with clinical findings. Interestingly, ETBR expression was expressed in HMECs but its expression was low or negative in both breast cancer cell lines. Previous studies have demonstrated that knockdown of ETAR inhibits breast cancer cell invasion, but whether ETBR functions similarly in breast cancer is unknown. To determine if ETBR might be regulating invasion in mammary cells, we transfected MCF7 cells with ETBR-specific siRNA and found that knocking down the gene encoding for ETBR (EDNRB) resulted in significantly increased cell invasion. This result suggests that while ETAR positively regulates breast cancer cell invasion, as reported in other studies, ETBR may be negatively regulating invasion through the endothelin axis. Together, our findings support an important role for the endothelin axis in breast cancer invasion, and specifically implicate ETAR and ETBR as performing complimentary and opposing roles in the regulation of breast cancer cell invasion. Citation Format: Mary Elizabeth Belles, Meena Halaka, Jessica M. Do, Parth Majmudar, Reem Sidani, Hannah Stephen, Molly Watson, Rebecca E. Conway. Expression and function of endothelin converting enzyme 1 and endothelin receptors in breast cancer invasion. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A58.

Inflammation-induced abnormalities in the subcellular localization and trafficking of the neurokinin 1 receptor in the enteric nervous system.

Activated G protein-coupled receptors traffic to endosomes and are sorted to recycling or degradative pathways. Endosomes are also a site of receptor signaling of sustained and pathophysiologically important processes, including inflammation. However, the mechanisms of endosomal sorting of receptors and the impact of disease on trafficking have not been fully defined. We examined the effects of inflammation on the subcellular distribution and trafficking of the substance P (SP) neurokinin 1 receptor (NK1R) in enteric neurons. We studied NK1R trafficking in enteric neurons of the mouse colon using immunofluorescence and confocal microscopy. The impact of inflammation was studied in IL10(-/-)-piroxicam and trinitrobenzenesulfonic acid colitis models. NK1R was localized to the plasma membrane of myenteric and submucosal neurons of the uninflamed colon. SP evoked NK1R endocytosis and recycling. Deletion of β-arrestin2, which associates with the activated NK1R, accelerated recycling. Inhibition of endothelin-converting enzyme-1 (ECE-1), which degrades endosomal SP, prevented recycling. Inflammation was associated with NK1R endocytosis in myenteric but not submucosal neurons. Whereas the NK1R in uninflamed neurons recycled within 60 min, NK1R recycling in inflamed neurons was delayed for >120 min, suggesting defective recycling machinery. Inflammation was associated with β-arrestin2 upregulation and ECE-1 downregulation, which may contribute to the defective NK1R recycling. We conclude that inflammation evokes redistribution of NK1R from the plasma membrane to endosomes of myenteric neurons through enhanced SP release and defective NK1R recycling. Defective recycling may be secondary to upregulation of β-arrestin2 and downregulation of ECE-1. Internalized NK1R may generate sustained proinflammatory signals that disrupt normal neuronal functions.

Endothelin-1 upregulation mediates aging-related cardiac fibrosis

Endothelin-1 (ET-1) plays a major role in regulating myocardial fibrosis in several pathological conditions, such as hypertension and diabetes. Aging is an independent risk factor for myocardial fibrosis. We hypothesized that ET-1 upregulation may be a basis of enhanced collagen synthesis in the senescent fibroblasts resulting in cardiac fibrosis with aging. To examine this hypothesis, we cultured mouse cardiac fibroblasts to passage-30 (P30). β–Galactosidase activity and several other aging markers were markedly increased in P30 (vs. P3) fibroblasts, indicating that these cells were indeed undergoing senescence. Importantly, ET-1 expression was markedly upregulated in P30 (vs. P3) fibroblasts. Of note, estrogen receptor-α (ER-α), an important negative regulator of ET-1, was downregulated in P30 fibroblasts. We also studied aged (130-weeks old, female) mice hearts, and observed that ET-1 was upregulated and ER-α was downregulated in these hearts (vs. 6-week old mice hearts, female). Similar observations were made in the fibroblasts isolated from aged mice hearts. ET-1 upregulation with aging was also seen in ≈70-year old (vs. ≈30-year old) human heart sections. In concert with ET-1 upregulation, the expression of fibronectin and collagens was found to be markedly increased in P30 cardiac fibroblasts in culture, fibroblasts isolated from the aged mice hearts, and in aged human hearts. Interestingly, inhibition of ET-1 in the senescent P30 fibroblasts by 2 different strategies (the use of siRNA and the use of endothelin converting enzyme inhibitors) markedly suppressed expression of fibrosis signals. Further, treatment with synthetic ET-1 enhanced fibronectin and collagen expression in P3 cardiac fibroblasts. These observations in mice and human hearts suggest that aging-related cardiac fibrosis is, at least partially, dependent on the upregulation of ET-1.

Aspects of the endothelin system in colorectal cancer

Endothelin system members including endothelin-1, endothelin-2, endothelin-3, endothelin receptors, and endothelin converting enzyme are considered major regulating factors in cancer cell biology and cancer microenvironment. Endothelins are members of the matrix metalloproteinase superfamily of proteases, which are released from pre-proteins, bind to their receptors with differential affinity, and are degraded following cellular uptake. For their structural similarity, endothelin-2 and endothelin-3 can be regarded as natural competitors for the endothelin-1 receptors and as natural antagonists of endothelin-1. Endothelin-1 is regulated at several levels, primarily at the level of transcription. Remarkably, endothelin-1 is overexpressed in colorectal cancer, and elevated plasma levels were found in colorectal cancer patients. Endothelin receptor type A has an unequal distribution in the colon, as it is over-expressed in the proximal and distal segments of the colon. Compared with normal mucosal tissue, there is high expression of endothelin receptor type A and low expression of endothelin receptor type B in colorectal cancer at all Dukes stages. By binding to endothelin receptor type A, endothelin-1 leads to down-regulation of epithelial and increased expression of mesenchymal markers. Also, endothelin-1 acts as anti-apoptotic factor through multiple pathways like PI3K-dependent AKT activation or NF-κB signaling. Members of the endothelin system might be used as cancer biomarker and from a therapeutic point of view, targeting the endothelin axis is a promising aim. In effect, potential drugs may include endothelin converting enzyme inhibitors as well as selective and non-selective antagonists of endothelin receptor types A and B. Biomedical Reviews 2014; 25: 1-13.

Neural peptidase endothelin-converting enzyme 1 regulates endothelin 1-induced pruritus.

In humans, pruritus (itch) is a common but poorly understood symptom in numerous skin and systemic diseases. Endothelin 1 (ET-1) evokes histamine-independent pruritus in mammals through activation of its cognate G protein-coupled receptor endothelin A receptor (ETAR). Here, we have identified neural endothelin-converting enzyme 1 (ECE-1) as a key regulator of ET-1-induced pruritus and neural signaling of itch. We show here that ETAR, ET-1, and ECE-1 are expressed and colocalize in murine dorsal root ganglia (DRG) neurons and human skin nerves. In murine DRG neurons, ET-1 induced internalization of ETAR within ECE-1-containing endosomes. ECE-1 inhibition slowed ETAR recycling yet prolonged ET-1-induced activation of ERK1/2, but not p38. In a murine itch model, ET-1-induced scratching behavior was substantially augmented by pharmacological ECE-1 inhibition and abrogated by treatment with an ERK1/2 inhibitor. Using iontophoresis, we demonstrated that ET-1 is a potent, partially histamine-independent pruritogen in humans. Immunohistochemical evaluation of skin from prurigo nodularis patients confirmed an upregulation of the ET-1/ETAR/ECE-1/ERK1/2 axis in patients with chronic itch. Together, our data identify the neural peptidase ECE-1 as a negative regulator of itch on sensory nerves by directly regulating ET-1-induced pruritus in humans and mice. Furthermore, these results implicate the ET-1/ECE-1/ERK1/2 pathway as a therapeutic target to treat pruritus in humans.

Soluble and Catalytically Active Endothelin Converting Enzyme-1 is Present in Cerebrospinal Fluid of Subarachnoid Hemorrhage Patients

Endothelin converting Enzyme-1 (ECE-1) is essential for the production of Endothelin-1 (ET-1), which is associated with vasospasm following subarachnoid hemorrhage (SAH). We have previously demonstrated the presence of a catalytically active soluble form of ECE-1 in the media of endothelial cells. We aimed to determine if this form of ECE-1 exists in vivo, in cerebrospinal fluid (CSF) of SAH patients. We examined CSF taken from SAH subjects for the presence of soluble ECE-1 using a bradykinin based quenched fluorescent substrate assay. We obtained further confirmation by characterizing the CSF mediated cleavage products of BigET-1 and BigET₁₈₋₃₄ (6 μg/ml) using mass spectrometry. The specificity of cleavage was confirmed using the ECE-1 inhibitor CGS35066 5 nmol/L. SAH CSF samples had mean ECE-1 activity of 0.127 ± 0.037 μmols of substrate cleaved/μl of CSF/24 h. The C-terminal peptides generated upon the cleavage of BigET-1 and BigET₁₈₋₃₄ were detected 48 h after incubation of these substrates with CSF. Cleavage of these substrates was inhibited by CGS35066. Results of Western blots also produced strong evidence for the presence of truncated soluble ECE-1 in CSF. These results strongly suggest the presence of a truncated but catalytically active form of ECE-1 in the CSF of SAH subjects. Further studies are necessary to determine the biological significance of soluble ECE-1 in CSF of SAH subjects, including an association with vasospasm after SAH.

Alteration of basilar artery rho-kinase and soluble guanylyl cyclase protein expression in a rat model of cerebral vasospasm following subarachnoid hemorrhage.

Background and Purpose. The vasoconstrictor endothelin-1 (ET-1) has been implicated in the pathogenesis of cerebral vasospasm following subarachnoid hemorrhage (SAH). Previous results showed that CGS 26303, an endothelin converting enzyme (ECE) inhibitor, effectively prevented and reversed arterial narrowing in animal models of SAH. In the present study, we assessed the effect of CGS 26303 on neurological deficits in SAH rats. The involvement of vasoactive pathways downstream of ET-1 signaling in SAH was also investigated. Methods. Sprague-Dawley rats were divided into five groups (n = 6/group): (1) normal control, (2) SAH, (3) SAH+vehicle, (4) SAH+CGS 26303 (prevention), and (5) SAH+CGS 26303 (reversal). SAH was induced by injecting autologous blood into cisterna magna. CGS 26303 (10 mg/kg) was injected intravenously at 1 and 24 hr after the initiation of SAH in the prevention and reversal protocols, respectively. Behavioral changes were assessed at 48 hr after SAH. Protein expression was analyzed by Western blots. Results. Deficits in motor function were obvious in the SAH rats, and CGS 26303 significantly improved the rate of paraplegia. Expressions of rho-kinase-II and membrane-bound protein kinase C-δ and rhoA were significantly increased, while those of soluble guanylyl cyclase α 1 and β 1 as well as protein kinase G were significantly decreased in the basilar artery of SAH rats. Treatment with CGS 26303 nearly normalized these effects. Conclusions. These results demonstrate that the rhoA/rho-kinase and sGC/cGMP/PKG pathways play pivotal roles in cerebral vasospasm after SAH. It also shows that ECE inhibition is an effective strategy for the treatment of this disease.

Endothelin converting enzyme inhibition attenuates early albuminuria and late renal failure in streptozotocin induced diabetic mice

It has recently been established that a high salt diet leads to sodium storage within the skin of rats and mice. This increase in sodium in the highly vascularized skin results in macrophage infiltration and lymphangiogenesis. While dysfunction in this process has been implicated in salt sensitive hypertension, the mechanisms are poorly understood. Because vascular endothelin-1 (ET-1) is upregulated in response to a high salt diet or hypertonicity, and is a potent chemoattractant, we hypothesized that endothelial derived ET-1 mediates infiltration of immune cells into the skin during chronic high salt intake, thereby allowing sodium clearance from the skin and preventing accumulation. Our data indicate that increasing extracellular concentration of human endothelial cells by 40 mOsm with NaCl, similar to what is seen in the interstitial space of rats placed on a high salt diet, leads to a 50% increase in ET-1 production, a mechanism likely mediated by TonEBP. Furthermore, in response to one week of high salt diet, skin Na/water ratio was elevated in vascular endothelial cell ET-1 knockout mice compared to wild type mice (0.112 ± 0.007 vs. 0.096 ± 0.004 mmol/ml). These data suggest a critical role for ET-1 in preventing the accumulation of sodium in the skin during a high salt intake, an emerging mechanism of the body's ability to buffer blood pressure changes in response to increases in sodium intake.