Endothelin-converting Enzyme-1 mRNA Research Articles

SiRNA Targeting ECE-1 Partially Reverses Pulmonary Arterial Hypertensionassociated Damage in a Monocrotaline Model.

The aim of this study was to develop a possible treatment for pulmonary arterial hypertension. Pulmonary arterial hypertension (PAH) is a rare disease characterised by a pulmonary arterial pressure greater than 20 mmHg. One of the factors that contribute to PAH is an increase in the production of endothelin-1, a polypeptide that increases vascular resistance in the pulmonary arteries, leading to increased pulmonary arterial pressure and right ventricular hypertrophy. The objective of this study was to design, synthesize, and evaluate two siRNAs directed against endothelin-1 in a rat model of PAH induced with monocrotaline. Wistar rats were administered monocrotaline (60 mg/kg) to induce a PAH model. Following two weeks of PAH evolution, the siRNAs were administered, and after two weeks, right ventricular hypertrophy was evaluated using the RV/LV+S ratio, blood pressure, weight, and relative expression of ECE-1 (Endothelin-converting enzyme-1) mRNA (messenger RNA) by RT-PCR (real-time PCR). The monocrotaline group showed an increase in the hypertrophy index and in ECE-1 mRNA, as well as a significant decrease in weight compared to the control group, while in the monocrotaline + siRNA group, a significant decrease was observed in the relative expression of ECE-1 mRNA, as well as in right ventricular hypertrophy. Based on the above information, we conclude that the administration of siRNAs directed to ECE-1 decreases the damage associated with PAH.

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 sAPPβ produced by β-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 β-secretase pathway and accumulation of the neurotoxic Aβ 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.

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-β1 (TGF-β1) 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-β1 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.

Modulation of Cytokine-Induced Astrocytic Endothelin-1 Production as a Possible New Approach to the Treatment of Multiple Sclerosis.

Background: In the human central nervous system (CN), resting astrocytes do not visually show endothelin-1 (ET-1)-like immunoreactivity. In patients with multiple sclerosis (MS), an inflammatory disorder of the CNS, high levels of ET-1 are found in reactive astrocytes in demyelinated plaques. ET-1 may contribute to the pathology of MS by interrupting the blood-brain-barrier, enhancing inflammatory responses, excitotoxicity and reducing cerebral blood flow. Methods: We used the human astrocytoma cell line 1321N1 to investigate the role of inflammatory cytokines involved in MS lesions (IL-1β, TNF-α, IFN-γ, LPS, IL-10, TGF-β) on astrocytic ET-1 upregulation. Prucalopride, rolipram, fenofibrate, fluoxetine, simvastatin, daglutril, and resveratrol were investigated as potential candidate drugs to suppress cytokine-induced astrocytic ET-1 production. Effects on ET-1 production were measured using both ELISA and RT-qPCR. Results and Conclusions: ET-1 secretion by astrocytoma cells was only stimulated by the pro-inflammatory cytokines IL-1β and TNF-α. Fluoxetine, simvastatin, and resveratrol significantly inhibited this IL-1β- and TNF-α-induced ET-1 production. Simvastatin and resveratrol significantly reduced ET-1 mRNA levels, indicating an effect at the level of transcription. Fluoxetine significantly reduced endothelin converting enzyme-1 mRNA levels, suggesting and effect at the level of protein-processing. The required concentrations of simvastatin (>0.1 µM) and resveratrol (>10 µM) cannot be achieved in humans using pharmacologically accepted doses. Fluoxetine exerted a significant inhibitory effect on ET-1 secretion at a concentration of 5 µM, which is pharmacologically achievable in human brain, but the effect was modest (<50% suppression) and probably not sufficient to obtain a clinically relevant ET-1 effect. Our in vitro model can be a useful screening tool in the development of new drugs to suppress astrocytic ET-1 production. The effect of simvastatin was for the most part mediated via the mevalonate pathway, suggesting that this might be an interesting target for further drug development.

Impact of Superoxide Dismutase Mimetic AEOL 10150 on the Endothelin System of Fischer 344 Rats.

Endothelin-1 is a potent vasoconstrictor and mitogenic peptide involved in the regulation of vasomotor tone and maintenance of blood pressure. Oxidative stress activates the endothelin system, and is implicated in pulmonary and cardiovascular diseases including hypertension, congestive heart failure, and atherosclerosis. Superoxide dismutase mimetics designed with the aim of treating diseases that involve reactive oxygen species in their pathophysiology may exert a hypotensive effect, but effects on the endothelin system are unknown. Our objective was to determine the effect of the superoxide dismutase mimetic AEOL 10150 on the basal endothelin system in vivo. Male Fischer-344 rats were injected subcutaneously with 0, 2 or 5 mg/kg body weight of AEOL 10150 in saline. Plasma oxidative stress markers and endothelins (bigET-1, ET-1, ET-2, ET-3) as well as lung and heart endothelin/nitric oxide system gene expressions were measured using HPLC-Coularray, HPLC-Fluorescence and RT-PCR respectively. AEOL 10150 reduced (p<0.05) the circulating levels of isoprostane (-25%) and 3-nitrotyrosine (-50%) measured in plasma 2h and 24h after treatment, confirming delivery of a physiologically-relevant dose and the potent antioxidant activity of the drug. The reduction in markers of oxidative stress coincided with sustained 24h decrease (p<0.05) of plasma levels of ET-1 (-50%) and ET-3 (-10%). Expression of preproET-1 and endothelin converting enzyme-1 mRNA were not altered significantly in the lungs. However preproET-1 (not significant) and ECE-1 mRNA (p<0.05) were increased (10–25%) in the heart. Changes in the lungs included decrease (p<0.05) of mRNA for the ET-1 clearance receptor ETB and the vasoconstriction-signaling ETA receptor (-30%), and an early surge of inducible nitric oxide synthase expression followed by sustained decrease (-40% after 24 hours). The results indicate that interception of the endogenous physiological flux of reactive nitrogen species and reactive oxygen species in rats impacts the endothelin/nitric oxide system, supporting a homeostatic relationship between those systems.

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.

Increased gene expression of the cardiac endothelin system in obese mice.

Obesity is a well-known risk factor of atherosclerosis and heart failure. In the human heart, a local endothelin system containing prepro-endothelin-1, endothelin-converting enzyme-1, and endothelin receptors A and B has been described. The endothelin system is activated in heart failure; however, the impact of obesity on the cardiac endothelin system is unknown. In this study, 18-week-old male C57BL/6 mice fed either a control diet or a high-fat diet for 10 weeks were analyzed. High-fat diet significantly increased the body weight of the animals and augmented low-density lipoprotein, high-density lipoprotein, and cholesterol plasma levels, compared to control. The animal groups showed no significant differences in left ventricular size or function (heart rate, ejection fraction, fractional shortening, left ventricular posterior wall thickness, cardiac output) after control or high-fat diet. We did not observe signs of cardiac hypertrophy or changes in markers of cardiac fibrosis in these heart samples. The cardiac expression of prepro-endothelin-1 mRNA, endothelin-converting enzyme-1 mRNA, and protein and endothelin receptors A and B mRNA was increased in 18-week-old obese C57BL/6 mice compared to animals with normal weight (p<0.05 vs. control). Furthermore, endothelin-1 plasma levels showed an increasing trend. In conclusion, an increased expression of genes of the endothelin system was observed in the hearts of 18-week-old mice after high-fat diet, possibly contributing to later cardiovascular complications of obesity.

Endothelin-converting enzyme-1 regulates trafficking and signalling of the neurokinin 1 receptor in endosomes of myenteric neurones

Neuropeptide signalling at the plasma membrane is terminated by neuropeptide degradation by cell-surface peptidases, and by β-arrestin-dependent receptor desensitization and endocytosis. However, receptors continue to signal from endosomes by β-arrestin-dependent processes, and endosomal sorting mediates recycling and resensitization of plasma membrane signalling. The mechanisms that control signalling and trafficking of receptors in endosomes are poorly defined. We report a major role for endothelin-converting enzyme-1 (ECE-1) in controlling substance P (SP) and the neurokinin 1 receptor (NK(1)R) in endosomes of myenteric neurones. ECE-1 mRNA and protein were expressed by myenteric neurones of rat and mouse intestine. SP (10 nM, 10 min) induced interaction of NK(1)R and β-arrestin at the plasma membrane, and the SP-NK(1)R-β-arrestin signalosome complex trafficked by a dynamin-mediated mechanism to ECE-1-containing early endosomes, where ECE-1 can degrade SP. After 120 min, NK(1)R recycled from endosomes to the plasma membrane. ECE-1 inhibitors (SM-19712, PD-069185) and the vacuolar H(+)ATPase inhibitor bafilomycin A(1), which prevent endosomal SP degradation, suppressed NK(1)R recycling by >50%. Preincubation of neurones with SP (10 nM, 5 min) desensitized Ca(2+) transients to a second SP challenge after 10 min, and SP signals resensitized after 60 min. SM-19712 inhibited NK(1)R resensitization by >90%. ECE-1 inhibitors also caused sustained SP-induced activation of extracellular signal-regulated kinases, consistent with stabilization of the SP-NK(1)R-β-arrestin signalosome. By degrading SP and destabilizing endosomal signalosomes, ECE-1 has a dual role in controlling endocytic signalling and trafficking of the NK(1)R: promoting resensitization of G protein-mediated plasma membrane signalling, and terminating β-arrestin-mediated endosomal signalling.

Nitric Oxide Decreases the Expression of Endothelin-Converting Enzyme-1 Through mRNA Destabilization

Endothelial function depends on the equilibrium in the synthesis of vasoactive endothelial factors. It is well known that endothelin and nitric oxide (NO) exhibit reciprocal regulation. We assessed the ability of NO to regulate endothelin-converting enzyme-1 (ECE-1) expression in vascular endothelial cells. Bovine aortic endothelial cells were incubated with 2 different NO donors as well as with a cyclic-GMP analog, dibutyryl-cGMP (dB-cGMP). ECE-1 protein content and mRNA expression were evaluated by Western blot and Northern blot, respectively, promoter activity by transfection experiments, ECE-1 activity by ELISA, and cGMP production by radioimmunoassay. Both NO donors decreased ECE-1 protein content, mRNA expression, and ECE-1 activity. ODQ, an inhibitor of soluble guanylyl cyclase, blocked those effects. NO donors raised cGMP levels, and dB-cGMP mimicked their effects on ECE-1 expression, which were blocked by KT5823, a nonspecific PKG inhibitor. The changes on ECE-1 expression were due to a destabilization on 3'-untranslated region (3'-UTR) of this mRNA, because the activity of a luciferase reporter construct containing the 3'-UTR of the ECE-1 gene was reduced by dB-cGMP in a PKG-dependent manner. The biological relevance of this regulation was confirmed in bovine aortic endothelial cells coincubated with macrophages in the presence of lipopolysaccharide, in eNOS-deficient mice, and in Wistar rats treated with NO donors. In every case, an inverse relationship was observed between NO and ECE-1 protein content. Our results support that NO regulates ECE-1 expression through a cGMP/PKG-dependent regulatory mechanism at the post-transcriptional level via the 3'-UTR of the ECE-1 gene.

Structure–Activity Relationship of Resveratrol and Its Analogue, 4,4′-Dihydroxy-Trans-Stilbene, Toward the Endothelin Axis in Human Endothelial Cells

Resveratrol inhibits endothelin-1, a vascular tension regulator. We synthesized the resveratrol analogue 4,4'-dihydroxy-trans-stilbene with 2 hydroxyl groups in the 4 and 4' position to obtain a molecule more active than resveratrol (3,4',5-trihydroxy-trans-stilbene). The results demonstrate that 4,4'-dihydroxy-trans-stilbene led to a significant decrease in total endothelin-1 secretion and in endothelin-1 messenger RNA (mRNA) levels in human endothelial cells. In addition, resveratrol and its analogue decreased endothelin-converting enzyme-1 mRNA levels and further reduced the activity of the enzyme. 4,4'-dihydroxy-trans-stilbene was more active than resveratrol because the new molecule exerted greater activity at the level of endothelin synthesis and conversion, even at a lower concentration. Although 4,4'-dihydroxy-trans-stilbene and resveratrol inhibited formation of reactive oxygen species and lipid peroxidation, the treatment of cells with different oxidant agents did not modify the endothelin-1 release. This finding suggests that the inhibition of endothelin-1 secretion is independent of the antioxidant properties of the 2 compounds. On the basis of these results, the resveratrol analogue 4,4'-dihydroxy-trans-stilbene could be a promising chemopreventive agent against cardiovascular diseases.

DsRNA activation of endothelin-1 and markers of vascular activation in endothelial cells and fibroblasts

BackgroundIn patients with systemic sclerosis (SSc), the relationship between innate immune activation, represented by increased expression of interferon (IFN)-regulated genes, and vascular injury/activation, manifest by increased endothelin-1 (ET-1), endothelin converting...

Blockade of Endothelin-1 with a Novel Series of 1,3,6-Trisubstituted-2-Carboxy-Quinol-4-Ones Controls Infection-Associated Preterm Birth

Preterm birth (PTB) currently accounts for 13% of all births in the United States, with the leading cause of PTB being maternal infection. Endothelin-1, an extremely potent vasoconstrictor capable of increasing myometrial smooth muscle tone, has been shown to be up-regulated in the setting of infection in pregnancy, ultimately leading to PTB. In previous work, we have shown that infection-associated PTB is controlled in our murine model by using phospharamidon, an endothelin-converting enzyme-1 inhibitor; knocking down endothelin-converting enzyme-1 mRNA; or blocking the binding of endothelin-1 to the endothelin-A (ET(A)) receptor with either BQ-123 or with HJP-272, the 6-OH compound of our series of novel synthetic (ET(A)) receptor antagonists. In the current study, we show that HJP-272, a highly selective ET(A) receptor antagonist with an IC(50) of 70.1 nmol/L, binds in a noncompetitive manner to the ET(A) receptor. Additionally, we introduce n-propyl (HJP-286) and n-butyl (HJP-278) analogs of HJP-272. We find that the LD(50) of HJP-272, the analog in the series most effective in controlling preterm birth, is more than 20-fold higher than its therapeutic dose. Acute exposure to high doses of these compounds produces no histological changes in any organ, while chronic exposure produces only a rare hepatotoxic effect. These findings may be of clinical significance, as there is currently no FDA-approved therapy for women presenting with threatened preterm delivery.

Endothelin-converting enzyme-1 increases in atherosclerotic mice: potential role of oxidized low density lipoproteins

The aim of our study was to analyze the relationships between atherosclerosis and endothelin-converting enzyme-1 (ECE-1). Four-week-old C57BL/6J [wild-type (WT)] and apolipoprotein E-deficient (apoE) mice were fed with a standard or Western-type fat diet for 8 wks. ApoE showed atherosclerotic lesions in the aorta, higher blood pressure and vascular lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) protein content than WT. ApoE showed a significant increase in ECE-1 protein content and mRNA expression in aorta, lung, and kidney, without changes in heart. When an ECE-1 inhibitor, FR-901533, was administered to them, blood pressure decreased in apoE on fat diet versus apoE on normal diet and WT. ECE-1 and LOX-1 protein content were elevated in peripheral blood mononuclear cells (PBMC) from hypercholesterolemic patients. In order to study the mechanism involved in this ECE-1 up-regulation, bovine aortic endothelial cells (BAEC) were treated with oxidized-low density lipoproteins (oxLDL). OxLDL, but not LDL, increased ECE-1 protein content, mRNA expression and promoter activity. Our results demonstrate that ECE-1 increases in different atherosclerosis situations. Up-regulation of ECE-1 could contribute, at least partially, to the development of hypertension seen in apoE mice, because FR-901533 avoided it. Probably, atherosclerotic situations course with an increase of oxLDL, which is able to induce ECE-1 expression with the subsequent potential pathological effects.

The Endothelin-Converting Enzyme-1/Endothelin-1 Pathway Plays a Critical Role in Inflammation-Associated Premature Delivery in a Mouse Model

Premature delivery occurs in 12% of all births and accounts for nearly half of long-term morbidity. Current therapeutic approaches to preterm delivery are ineffective and present serious risks to both mother and fetus. The single most common cause of preterm birth is infection. Previous in vitro investigations have shown that endothelin-1 (ET-1) is induced by inflammatory cytokines and that it increases myometrial smooth muscle tone. Furthermore, we have previously shown that both the endothelin-converting enzyme-1 (ECE-1) inhibitor, phosphoramidon, as well as a novel ET-1 receptor A antagonist synthesized by our group, control premature delivery in a mouse model of inflammation-associated preterm delivery. In the current work, we show that levels of both ET-1 and ECE-1 are increased in gestational tissues in E16.5 mice induced to deliver prematurely after lipopolysaccharide administration. We also show that premature delivery is controlled by treatment with the selective endothelin receptor A antagonist BQ-123 in a dose-dependent manner. Finally, we show here for the first time that premature delivery can be controlled using RNA silencing, by hydrodynamic transfection of E15 mice with ECE-1 RNAi. Taken together, these data support a critical role for the ECE-1/ET-1 system in inflammation-associated premature delivery. The ability to control premature delivery by antagonizing or silencing the ECE-1/ET-1 system offers a novel approach to an unmet clinical need.

Expression of human endothelin-converting enzyme isoforms: role of angiotensin IIThis article is one of a selection of papers published in the special issue (part 1 of 2) on Forefronts in Endothelin.

A key step in endothelin-1 (ET-1) synthesis is the proteolytic cleavage of big ET-1 by the endothelin-converting enzyme-1 (ECE-1). Four alternatively spliced isoforms, ECE-1a to ECE-1d, have been discovered; however, regulation of the expression of specific ECE-1 isoforms is not well understood. Therefore, we stimulated primary human umbilical vein endothelial cells (HUVECs) with angiotensin II (Ang II). Furthermore, expression of ECE-1 isoforms was determined in internal mammary arteries of patients undergoing coronary artery bypass grafting surgery. Patients had received one of 4 therapies: angiotensin-converting enzyme inhibitors (ACE-I), Ang II type 1 receptor blockers (ARB), HMG-CoA reductase inhibitors (statins), and a control group that had received neither ACE-I, ARB (that is, treatment not interfering in the renin-angiotensin system), nor statins. Under control conditions, ECE-1a is the dominant isoform in HUVECs (4.5+/-2.8 amol/microg RNA), followed by ECE-1c (2.7+/-1.0 amol/microg), ECE-1d (0.49+/-0.17 amol/microg), and ECE-1b (0.17+/-0.04 amol/microg). Stimulation with Ang II did not change the ECE-1 expression pattern or the ET-1 release. We found that ECE-1 mRNA expression was higher in patients treated with statins than in patients treated with ARB therapy (5.8+/-0.76 RU versus 3.0+/-0.4 RU), mainly attributed to ECE-1a. In addition, ECE-1a mRNA expression was higher in patients receiving ACE-I therapy than in patients receiving ARB therapy (1.68+/-0.27 RU versus 0.83+/-0.07 RU). We conclude that ECE-1a is the major ECE-1 isoform in primary human endothelial cells. Its expression in internal mammary arteries can be regulated by statin therapy and differs between patients with ACE-I and ARB therapy.

Endothelin-1 and endothelin-converting enzyme-1 in human granulomatous pathology of eyelid: an immunohistochemical and in situ hybridization study in chalazia.

Endothelin-1 (ET-1), a potent vasoconstrictor peptide, is involved in several functions of eye pathophysiology, such as regulation of intraocular tension and retinal reactive vasoconstriction. As ET-1 pro-inflammatory and fibrosing activity is emerging in different fields of pathology, we investigated the expression of ET-1 and endothelin-converting enzyme-1 (ECE-1) in chalazia, granulomatous lesions of the eyelid. ET-1 and ECE-1 were analyzed by immunohistochemistry (IHC) in twenty surgically removed chalazia, with regard to expression in eyelid structures and inflammatory infiltrate. Phenotype of ET-1 expressing inflammatory cells was established by double immunofluorescence. The cellular localization of prepro-ET-1 (pp-ET-1) mRNA and ECE-1 mRNA was studied by nonisotopic in situ hybridization (ISH). Neutrophils (PMNs), macrophages and T-lymphocytes were scattered in stroma, around alveoli and grouped in lipogranulomas. PMNs, macrophages, basal epithelium of meibomian adenomers and central ducts immunostained for ET-1. ECE-1 protein was found in meibomian adenomers, conjunctival epithelium, tarsal mucous glands and in inflammatory cells. Hybridization signals for pp-ET-1 mRNA and ECE-1 mRNA were recognized in healthy and degenerating meibomian ducts, adenomers, inflammatory cells, as well as in vessel walls. ECE-1 mRNA was also present in conjunctival epithelium and Henle's crypts. Our findings suggest that the multifunctional peptide ET-1 may have a role in molecular genesis of tissue damage in chalazia. ET-1 cytokine activity is likely to support the migration of inflammatory cells and the setting of lipogranulomas; ET-1 stimulation might contribute to proliferation of fibroblasts and collagen synthesis. ET-1 upregulation on meibomian adenomers and ducts may further enhance granulomas formation by stimulating lipid release.

Bovine Endothelial Cells Interact with Fully‐luteinized, but Not Luteinizing, Granulosa Cells in the mRNA Expression of Endothelin‐1 System in Response to Prostaglandin F2α

The corpus luteum (CL) undergoes regression by prostaglandin (PG)F(2alpha) from uterus and endothelin-1 (ET-1) plays an important role during luteolysis as a local mediator of PGF(2alpha) in the cow. Endothelial cells (EC) and luteal cells are main cell types making up the CL and their interactions are vital for CL function. We aimed to examine the relevance of interactions between EC and luteal cells on stimulation of genes which involved ET-1 synthesis by PGF(2alpha). We further focused the impact of maturity of luteal cells on the stimulation of the genes. To make a microenvironment which resembles the CL, we used bovine aortic endothelial cells (BAEC) and luteinizing or fully-luteinized granulosa cells (GC) and evaluated the effect of PGF(2alpha) on the expression for mRNA of ET-1 system by using real-time RT-PCR. PGF(2alpha) stimulated the expression of preproET-1 and endothelin converting enzyme-1 mRNA only in the co-cultures of BAEC with fully-luteinized GC, but not with luteinizing GC. The data suggest that interactions between BAEC and fully-luteinized GC enhance the capability of BAEC to produce ET-1 in response to PGF(2alpha). This mechanism may contribute to the local induction of luteolytic action of PGF(2alpha) which is dependent on the age/maturation of the CL.

Atrasentan Treatment of Pulmonary Vascular Disease in Piglets With Increased Pulmonary Blood Flow

We studied the effect of chronic endothelin A receptor blockade by atrasentan on the pulmonary endothelin-1 system and vascular endothelial growth factor (VEGF) expression in piglets with high pulmonary blood flow. Twenty-five 4-week-old piglets with high pulmonary blood flow were randomized to three groups: sham operated (n = 8), placebo (water) (n = 7), or treatment with atrasentan (2 mg/kg per day) (n = 10). After 3 months, mean pulmonary arterial pressure (PAP) was higher in the placebo group than in the sham group [18 +/- 2 mm Hg versus 14 +/- 1 mm Hg; P < 0.05 (ANOVA)]. Atrasentan treatment was associated with lower cardiac output, PAP (14 +/- 1 mm Hg), and medial wall thickness of pulmonary arteries (diameter: 50-150 microM) compared with placebo [13.6 +/- 3.0% versus 18.1 +/- 4.2%; P < 0.05 (ANOVA)]. Quantitative real-time polymerase chain reaction for endothelin-1, endothelin B receptor, and endothelin-converting enzyme-1 mRNA in lung tissue did not differ. However, immunostaining as well as mRNA for VEGF were lower in atrasentan-treated animals (relative gene expression: atrasentan versus placebo: 0.8 +/- 0.3 versus 1.5 +/- 0.3; P = 0.009). Atrasentan treatment effectively reduces medial hypertrophy in piglets with chronic pulmonary hyperperfusion. Chronic endothelin A receptor blockade by atrasentan may interfere with the expression of VEGF.

Tanshinone IIA inhibits endothelin-1 production in TNF-?-induced brain microvascular endothelial cells through suppression of endothelin-converting enzyme-1 synthesis

To investigate the effects of tanshinone IIA (Tan IIA) on the regulation of the production of endothelin (ET)-1 (including large ET-1), mRNA levels of ET-1, endothelin-converting enzyme-1 (ECE-1), endothelin-A receptor (ETA) and endothelin-B receptor (ETB) induced by TNF-alpha in rat brain microvascular endothelial cells (BMVEC). The ET-1 release (including large ET-1) into the culture medium was determined by enzyme immunoassay. The levels of ET-1, ECE-1, ETA, and ETB mRNA were measured by RT-PCR. Endothelin receptor binding was also tested. The induction of ET-1 release by TNF-alpha from cultured BMVEC was dose-dependently reduced by Tan IIA, but large ET-1 levels progressively increased in response to Tan IIA; the mRNA expression of ET-1 was unaffected. Tan IIA also caused a decrease in ETA receptor mRNA and ECE-1 expression in a dose-dependent manner. Endothelin receptor binding was unaltered in BMVEC stimulated with TNF-alpha alone or a combination of TNF-alpha and Tan IIA. These findings suggest that Tan IIA may inhibit ET-1 production in TNF-alpha-induced BMVEC through the suppression of ECE-1 synthesis.

Effect of eicosapentaenoic acid on the different endothelin system components in endothelin-1-induced hypertrophied cardiomyocytes.

The cardiovascular benefit of fish oil, including eicosapentaenoic acid (EPA), in humans and experimental animals has been reported. The role of endothelin-1 (ET-1) in cardiac hypertrophy is well known. Endothelin-1 stimulates prepro-ET-1 mRNA expression in cardiomyocytes, and the autocrine/paracrine system of ET-1 is important for cardiomyocyte hypertrophy. Although many studies link EPA to cardiac protection, the effect of EPA on cardiac hypertrophy has yet to be clarified. Recently, we demonstrated that ET-1-induced cardiomyocytic change could be prevented by pretreatment with EPA. The present study investigated the changes of different components of the ET system at the mRNA level in ET-1-administered cardiomyocytes, and examined the effect of EPA pretreatment. Ventricular cardiomyocytes were isolated from 2-day-old Sprague-Dawley rats, cultured in Dulbecco's modified Eagle's medium and Ham F12 supplemented with 0.1% fatty acid-free bovine serum albumin for 3 days. At Day 4 of culture, the cardiomyocytes were divided into 3 groups: control group, ET-1-treated (0.1 nM) group, and ET-1-treated group pretreated with EPA (10 microM). Twenty-four hours after treatment, the gene expressions of different components of the endothelin system in three experimental groups were evaluated by real-time polymerase chain reaction. Prepro-ET-1 mRNA expression was 53% upregulated in ET-1-induced hypertrophied cardiomyocytes and suppressed in the EPA-pretreated group. Endothelin-converting enzyme-1 (ECE-1) was also increased in ET-1-administered cardiomyocytes by 42% compared with the control group and was reversed in the EPA-pretreated group. The two receptors of ET system, ET(A) and ET(B), tended to be increased in the ET-1-treated group, but no statistical significance was seen among study groups. Endothelin-1 increased prepro-ET-1 and ECE-1 mRNA expression in hypertrophied-neonatal cardiomyocytes, and this was reversed with EPA pretreatment. Thus, EPA may play a crucial role in the regression of ET-1-induced cardiomyocyte hypertrophy, partly through the suppression of ET-1 and ECE-1 expression.