Endothelin-converting enzyme-1 increases in atherosclerotic mice: potential role of oxidized low density lipoproteins
2008; Elsevier BV; Volume: 50; Issue: 3 Linguagem: Inglês
10.1194/jlr.m800215-jlr200
ISSN1539-7262
AutoresPatricia Martínez‐Miguel, Viviana Raoch, Carlos Zaragoza, José Manuel Valdivielso, Manuel Rodríguez‐Puyol, Diego Rodrı́guez-Puyol, Susana López‐Ongil,
Tópico(s)Nitric Oxide and Endothelin Effects
ResumoThe 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 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. Atherosclerosis is a slowly evolutive age-linked disease of large arteries, characterized by local lipid deposition associated with chronic inflammatory response, leading potentially to acute plaque rupture, thrombosis, and ischemic diseases (1Berliner J.A. Navab M. Fogelman A.M. Frank J.S. Demer L.L. Edwards P.A. Watson A.D. Lusis A.J. Atherosclerosis: basic mechanisms. Oxidation, inflammation, and genetics.Circulation. 1995; 91: 2488-2496Crossref PubMed Scopus (1579) Google Scholar, 2Lusis A.J. Atherosclerosis.Nature. 2000; 407: 233-241Crossref PubMed Scopus (4640) Google Scholar). 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This increased synthesis of ET-1 may be related to the overexpression of prepro-ET-1 mRNA, as increased steady-state levels of this messenger have been demonstrated in the aorta from hypercholesterolemic mice (15d'Uscio L.V. Barton M. Shaw S. Lüscher T.F. Chronic ET(A) receptor blockade prevents endothelial dysfunction of small arteries in apolipoprotein E-deficient mice.Cardiovasc. Res. 2002; 53: 487-495Crossref PubMed Scopus (41) Google Scholar, 16Barton M. Haudenschild C.C. d'Uscio L.V. Shaw S. Munter K. Lüscher T.F. Endothelin ETA receptor blockade restores NO-mediated endothelial function and inhibits atherosclerosis in apolipoprotein E-deficient mice.Proc. Natl. Acad. Sci. USA. 1998; 95: 14367-14372Crossref PubMed Scopus (333) Google Scholar). However, changes in prepro-ET-1 intracellular levels are not the sole mechanism involved in the regulation of vascular ET-1 synthesis. Endothelin-converting enzyme (ECE-1), the enzyme that regulates the conversion from big ET-1 to ET-1, seems to be increased in atherosclerotic plaques (17Ihling C. Szombathy T. Bohrmann B. Brockhaus M. Schaefer H.E. Loeffler B.M. Coexpression of ECE-1 and ET-1 in different stages of human atherosclerosis.Circulation. 2001; 104: 864-869Crossref PubMed Scopus (116) Google Scholar), and its activity may be enhanced in patients with atherosclerosis (18Bohm F. Johansson B.L. Hedin U. Alving K. Pernow J. Enhanced vasoconstrictor effect of big-ET-1 in patients with atherosclerosis: relation to conversion to endothelin-1.Atherosclerosis. 2002; 160: 215-222Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). However, other studies propose that ECE-1 content and activity are decreased in hypercholesterolemic patients (19Ruschitzka F. Moehrlen U. Quaschning T. Lachat M. Noll G. Shaw S. Yang Z. Teupser D. Subkowski T. Turina M.I. et al.Tissue endothelin-converting enzyme activity correlates with cardiovascular risk factors in coronary artery disease.Circulation. 2000; 102: 1086-1092Crossref PubMed Scopus (32) Google Scholar). LDLs have been suggested to play a role in the modulation of the ET-1 synthesis. The oxidized form of these proteins increased the prepro-ET-1 mRNA expression in different cell types, with the subsequent increased ET-1 synthesis (20Tan M.S. Lee Y.J. Shin S.J. Tsai J.H. Oxidized low-density lipoprotein stimulates endothelin-1 release and mRNA expression from rat mesangial cells.J. Lab. Clin. Med. 1997; 129: 224-230Abstract Full Text PDF PubMed Scopus (17) Google Scholar, 21Boulanger C.M. Tanner F.C. Bea M.L. Hahn A.W. Werner A. Lüscher T.F. Oxidized low density lipoproteins induce mRNA expression and release of endothelin from human and porcine endothelium.Circ. Res. 1992; 70: 1191-1197Crossref PubMed Google Scholar). Moreover, native and oxLDL seem to stimulate ECE-1 expression in cultured endothelial cells (22Niemann B. Rohrbach S. Catar R.A. Muller G. Barton M. Morawietz H. Native and oxidized low-density lipoproteins stimulate endothelin-converting enzyme-1 expression in human endothelial cells.Biochem. Biophys. Res. Commun. 2005; 334: 747-753Crossref PubMed Scopus (24) Google Scholar), although indirect evidence from human studies suggests an inverse relationship between LDL levels and ECE-1 vascular activity (19Ruschitzka F. Moehrlen U. Quaschning T. Lachat M. Noll G. Shaw S. Yang Z. Teupser D. Subkowski T. Turina M.I. et al.Tissue endothelin-converting enzyme activity correlates with cardiovascular risk factors in coronary artery disease.Circulation. 2000; 102: 1086-1092Crossref PubMed Scopus (32) Google Scholar). Considering this information, we planned to analyze the possible role of ECE-1 in the pathogenesis of atherosclerosis, as well as the relationship between oxLDL and ECE-1, in order to clarify the previously published scarce and contradictory information. By a combined in vivo and in vitro approach, we tried to obtain consistent data and perform an analysis of the mechanisms responsible for the observed changes. Culture plates, SuperSignal detection system and secondary horseradish peroxidase-conjugated goat anti-mouse IgG were from Cultek (Pierce, Rockford, IL). Paragon electrophoresis system was from Beckamn Coulter Inc. (Fullerton, CA). Dual Luciferase Reporter Assay System, pGL3 vector, pRL-SV40 vector and T4 polynucleotide kinase were from Innogenetics (Walkersville, MD. Lipofectamine reagent and OptiMEM I media were from GIBCO-Invitrogen (Barcelona, Spain). Acrylamide-bisacrylamide was from Hispanlab-Pronadisa (Madrid, Spain). MXB films were from Kodak (Rochester, NY). Protein markers, BioRad protein assay kit, plates and electrophoresis equipment were from Bio-Rad Laboratories (Richmond, CA, USA). Protease inhibitor cocktail tablets were from Roche Diagnostics (Madrid, Spain). The ET-1 ELISA system, α-[32P]dCTP and γ-[32P]ATP were from GE Healthcare Bio-Sciences (Buckinghamshire, UK). Advantage Genomic PCR Kit was from Clontech Lab (Palo Alto, CA). Unless otherwise indicated, the rest of the drugs, culture media, antibodies, and reagents were from Sigma-Aldrich-Fluka Chemical Co. (St. Louis, MO). Male homozygous apoE and C57BL/6J control [wild-type (WT)] mice from 4 wks old were obtained from The Jackson Laboratory (Charles River España, Barcelona, Spain). WT and apoE were fed with a normal or Western type diet (TD88137, Harlan Teklad) to induce atherosclerosis for 8 wks. Animals had free access to water, were maintained at 24°C, and kept at a 12 h light/dark cycle. One week before the sacrifice, arterial blood pressure was measured in conscious animals by means of a tail-cuff sphygmomanometer (LE 5001 Pressure Meter, Letica Scientific Instruments, Hospitalet, Spain). Animals were trained for 3 days before starting the measurement to prevent stress and were prewarmed at 30°C with a heater (LE5660/6, Letica Scientific Instruments). Blood pressure was recorded in 2 consecutive days, with at least 20 determinations by day. In a subgroup of animals, blood pressure was also recorded at 7 wks, before and after the intraperitoneal administration of 1 mg/kg weight of FR-901533, a rather selective ECE antagonist (kindly provided by Dr. Yuriyo Yamamoto, Fujisawa Pharmaceutical Co.). After the 8 wks, animals were anesthetized with pentobarbital (50 mg/kg i.p.), and a blood sample was collected through puncture of the right ventricle. Plasma was separated (3,500 rpm, 10 min) and stored until biochemical determination (Hitachi 917). Plasma lipoproteins (LDL and HDL choslesterol), total cholesterol and triglycerides were determined using a colorimetric-based assay on a Cobas Mira Plus autoanalyzer (Roche Diagnostics, Basel, Switzerland) as described (15d'Uscio L.V. Barton M. Shaw S. Lüscher T.F. Chronic ET(A) receptor blockade prevents endothelial dysfunction of small arteries in apolipoprotein E-deficient mice.Cardiovasc. Res. 2002; 53: 487-495Crossref PubMed Scopus (41) Google Scholar). Aorta, lungs, kidneys, and heart were removed via a thoracic-abdominal incision and stored until analysis. Aorta, lung, kidney, and heart portions were collected in 4% paraformaldehyde for histological studies. Because of the scarce tissue it was impossible to assay ECE-1 mRNA expression in aortas. The investigation was conducted in conformity with the Public Health Service policy on the Humane Care and Use of Laboratory Animals incorporated in the Institute for Laboratory Animal Research (ILAR), Guide for the Care and Use of Laboratory Animals published by the US National Academies. All the studies were approved by our Institutional Committee of Alcala University. Preparations of aorta, lung, kidney, and heart tissues were subjected to immunostaining (23Saura M. Zaragoza C. Herranz B. Griera M. Diez-Marqués L. Rodríguez-Puyol D. Rodríguez-Puyol M. Nitric oxide regulates transforming growth factor-beta signalling in endothelial cells.Circ. Res. 2005; 97: 1115-1123Crossref PubMed Scopus (94) Google Scholar) with anti-ECE-1 antibody (mAb AEC32-236, generous gift from Dr. Kohei Shimada) (24Shimada K. Matsushita Y. Wakabayashi K. Takahashi M. Matsubara A. Iijima Y. Tanzawa K. Cloning and functional expression of human endothelin-converting enzyme cDNA.Biochem. Biophys. Res. Commun. 1995; 207: 807-812Crossref PubMed Scopus (122) Google Scholar). Antibody-protein complexes were detected with anti-HRP-horseradish secondary antibodies using diaminobenzidine reagent following manufacturer's instructions (Dako Cytometrix, Fort Collins, CO). At least four sections per animal were analyzed for each immunostaining. Blood samples were taken from six male patients, with ages between 52 and 70 years and hypercholesterolemia (range: 250–320 mg/dl), as well as from six normocholesterolemic males (range: 172–215 mg/dl) between 49 and 71 years of age. Everyone gave their informed consent to make the protocol approved by Institutional Committee from our hospital, in accordance with the Principles outlined in the Declaration of Helsinki (Cardiovascular Research 1997; 35: 2–4). Peripheral blood mononuclear cells (PBMC) were isolated from blood samples with Ficoll solution (Comercial Rafer, Madrid, Spain), in order to extract total proteins, as described below. Bovine aortic endothelial cells (BAEC) were isolated from bovine thoracic aortas using previously described methods (25Marsden P.A. Brock T.A. Ballermann B.J. Glomerular endothelial cells respond to calcium-mobilizing agonists with release of EDRF.Am. J. Physiol. 1990; 258: F1295-F1303PubMed Google Scholar). Characterization was based on their typical cobblestone appearance and uniform uptake of fluorescent acetylated LDL. Cells were maintained in RPMI 1,640 supplemented with 15% calf serum, 100 U/ml penicillin, and 100 μg/ml streptomycin in an atmosphere of 95% air and 5% CO2. Experiments were routinely performed on confluent monolayers at passages 2–5, made quiescent by serum deprivation. Cellular toxicity was evaluated in all experimental conditions by the trypan blue dye exclusion method and by measurement of lactic dehydrogenase (LDH) activity in the incubation media. No significant toxicity was detected. Human endothelial cell line, EA.hy926 (EA) were from Dr. Cora-Jean S. Edgell (Yale University School of Medicine, New Haven, CT), and they were grown in DMEM with high glucose supplemented with 10% fetal calf serum, 100 U/ml penicillin and 100 μg/ml streptomycin in an atmosphere of 95% air and 5% CO2. Mouse aortic endothelial cells (MAEC) were isolated from WT animals by Dr. Carlos Zaragoza (CNIC, Madrid, Spain), and they were grown in DMEM-F12 supplemented with 20% fetal calf serum, 100 U/ml penicillin, and 100 μg/ml streptomycin and endothelial growth factor. Human LDL (d = 1.019–1.063) was isolated from fresh plasma of healthy humans by sequential ultracentrifugation at 4°C as described (26Havel R.J. Eder H.A. Bragdon H.H. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum.J. Clin. Invest. 1955; 34: 1345-1353Crossref PubMed Scopus (6476) Google Scholar). Oxidative modification of LDL was performed by incubation with 25 μM CuSO4 in PBS for 24 h at room temperature (27Stanton L.W. White R.T. Bryant C.M. Protter A.A. Endemann G. A macrophage Fc receptor for IgG is also a receptor for oxidized low density lipoprotein.J. Biol. Chem. 1992; 267: 22446-22451Abstract Full Text PDF PubMed Google Scholar). Protein concentrations of lipoprotein preparations were determined using the BioRad protein assay kit. OxLDL was assessed by electrophoretic mobility under nondenaturing conditions using a Paragon electrophoresis system, showing a single band with a 2-fold faster migration rate than native LDL. A final concentration between 50–200 μg protein/ml of native LDL or oxLDL was used in cells. Proteins were obtained from tissue, human PBMC, and BAEC, by using the Lysis Buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 0.1% sodium deoxycholate, 1% Triton X-100, 10 mM sodium pyrophosphate) containing a protease inhibitor cocktail. Protein concentration was determined with BioRad protein assay kit. Proteins were separated on SDS-PAGE 6% (30 μg protein/lane), and transferred onto nitrocellulose membranes. Membranes were blocked with 5% (w/v) nonfat dry milk in Tween Tris buffered saline (TTBS) (20 mM Tris-HCl pH 7.5, 0.9% NaCl, 0.05% Tween 20) at room temperature, and then incubated for 90 min with 10 μg/ml of the monoclonal anti-ECE-1 antibody (mAb AEC32-236) or with 1:1,000 dilution of the monoclonal anti-LOX-1 antibody (mAb anti-LOX-1 #5-2 from Dr. Tatsuya Sawamura). After washing in TTBS, blots were incubated with 200-fold-diluted horseradish peroxidase-conjugated goat anti-mouse IgG. The immunoreactive bands were visualized with the SuperSignal detection system after 30 s of exposure to MXB film. Then blots were reblotted with a monoclonal anti-β-tubulin antibody for samples from mice tissues and BAEC or with a rabbit anti-actin antibody for human PBMC in order to normalize ECE-1 and LOX-1 levels. Total cellular RNA was isolated from tissues or BAEC with the guanidinium thiocyanate-phenol-chloroform method (28Chomczynski P. Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol chloroform extraction.Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63088) Google Scholar). For Northern analysis, blots of RNA were hybridized with α-[32P]dCTP labeled specific ECE-1 mice or bovine probes (29González-Santiago L. López-Ongil S. Quereda C. Rodríguez-Puyol M. Rodríguez-Puyol D. Imbalance in endothelial vasoactive factors as a possible cause of cyclosporin toxicity: a role for endothelin-converting enzyme.J. Lab. Clin. Med. 2000; 136: 395-401Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 30López-Ongil S. Diez-Marqués M.L. Griera M. Rodríguez-Puyol M. Rodríguez-Puyol D. Crosstalk between mesangial and endothelial cells: angiotensin II down-regulates endothelin-converting enzyme-1.Cell Physiol. Biochem. 2005; 15: 135-144Crossref PubMed Scopus (20) Google Scholar) in hybridization solution (50% Formamide, 5× Denhardt's solution, 5× SSPE, 0.5% SDS, and 100 μg herring sperm DNA) at 42°C. The filters were stripped by boiling in 0.1% SDS solution and reprobed with a 32P-labeled 18 S cDNA (5.8 kb fragment digested by EcoRI). The densitometric analysis of the films was performed with an image scanner using the public domain software package National Institutes of Health Image 1.55 (Bethesda, MD). Levels of ECE-1 were normalized by using 18 S expressions within the same lane, and expressed in relative densitometric units with respect to control values. Supernatants and membrane proteins from BAEC treated with LDL or oxLDL were collected as described (31Lopez-Ongil S. Senchak V. Saura M. Zaragoza C. Ames M. Ballermann B.J. Rodríguez-Puyol M. Rodríguez-Puyol D. Lowenstein C.J. Superoxide regulation of endothelin converting enzyme.J. Biol. Chem. 2000; 275: 26423-26427Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 32Ohnaka K. Takayanagi R. Yamauchi T. Okazaki H. Ohashi M. Umeda F. Nawata H. Identification and characterization of endothelin-converting enzyme activity in cultured bovine endothelial cells.Biochem. Biophys. Res. Commun. 1990; 168: 1128-1136Crossref PubMed Scopus (104) Google Scholar) in order to measure ET-1 production and ECE-1 activity, respectively, using an enzyme-linked immunosorbent assay (ELISA). The membrane proteins from BAEC treated for 24 h with 100 μg/ml OxLDL or LDL were isolated, and then 30 μg were incubated with a fixed amount of big ET-1 (100 ng) for 4 h at 37°C in the presence or not of 100 μM phosphoramidon. In order to generate a standard curve for ET-1 serial dilutions of ET-1 ranging from 1–16 fmol per well were used. A cubic-spline curve was fit to the standards and unknown values interpolated from the standard curves automatically. The cross-reactivity of the ELISA ET-1 antibody, as determined by the concentration giving 50% B/Bmax, was: ET-1 (100), ET-2 (100), ET-3 (<0.001), big-ET-1 human (<0.07), and atrial natriuretic peptide (<0.0006). To determine whether the effect of oxLDL on ECE-1 gene expression was mediated by the 5′-flanking region of the gene, a human ECE-1 promoter/luciferase reporter gene plasmid was constructed (pGL3-ECE-1) (33López-Ongil S. Saura M. Zaragoza C. González-Santiago L. Rodríguez-Puyol M. Lowenstein C.J. Rodríguez-Puyol D. Regulation of bovine endothelin-converting enzyme-1 by hydrogen peroxide.Free Radic. Biol. Med. 2002; 32: 406-413Crossref PubMed Scopus (29) Google Scholar). We used the PCR of HeLa cell genomic DNA to create serial deletion fragments of the human ECE-1 gene promoter with the 5′ ends at nucleotides −650 (AP-1), −596 (NF-κB), −542 (Acute phase), −483 (CAAT box), −444 (Shear stress), −328 (STAT), and −216 (Glucocorticoid receptor element), and the 3′ end nucleotide +1 using the Advantage Genomic PCR Kit. Fragments were subcloned in the Xho I-Hind III site of pGL3 vector, upstream from a luciferase reporter gene. BAEC were grown at 60–80% of confluence in 12-well plates and transfected with promoter/luciferase constructs, by mixing 0.1 μg/μL of pGL3-ECE-1 with 1 ng/μL of plasmid control from Renilla luciferase (pRL-SV40 vector) and 4 μg/ml of Lipofectamine into OptiMEM I media. After 24 h of transfection, cells were refed with complete RPMI 1,640 for at least 16 h, and then native LDL or oxLDL was added at different doses and times using RPMI without serum. Luciferase activity was assessed using a Dual Luciferase Reporter Assay System, and expressed as relative light units of pGL3-ECE-1/Renilla/mg protein of each well. BAEC were incubated with oxLDL at different times and electrophoretic mobility shift assays was displayed to check on the activation of NF-κB, as previously described (34López-Ongil S. Hernández-Perera O. Pérez de Lema G. Lamas S. Rodríguez-Puyol M. Rodríguez-Puyol D. Role of reactive oxygen species in the signaling cascade of Ciclosporine A-mediated up-regulation of NOS3 in vascular endothelial cells.Br. J. Pharmacol. 1998; 124: 447-454Crossref PubMed Scopus (115) Google Scholar). Oligonucleotide sequences were based on the putative NF-κB binding element in the ECE-1 promoter (from nucleotides −617 to −591) as follow: sense 5′-GGC TGG AGG GAT TTT TCC TCC TTT CA-3′ and antisense 5′-TGA AAG GAG GAA AAA TCC CTC CAG CC-3′ (35Valdenaire O. Rohrbacher E. Mattei M.G. Organization of the gene encoding the human endothelin-converting enzyme (ECE-1).J. Biol. Chem. 1995; 270: 29794-29798Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar). Oligonucleotides were labeled with γ-[32P]ATP at the 5′ end with T4 polynucleotide kinase and then incubated with nuclear extracts. Protein-DNA complexes were separated in a 6% nondenaturing polyacrylamide gel in 0.25 × Tris buffer EDTA. The gels were dried under vacuum and exposed to X-ray film. For competition experiments, 125-fold molar excess of competitor DNA (AP-1 oligonucleotide, NF-κB oligonucleotide) was coincubated with the labeled oligonucleotide probe (NF-κB). Sequences of the oligonucleotides for AP-1 were: sense 5′-CAT GGC TGT GTC ACC CTT GTC CC-3′ and antisense 5′-GGG ACA AGG GTG ACA CAG CCA TG-3′. Data are expressed as means ± SEM. Animal studies were analyzed by ANOVA, followed by the Scheffe multiple comparison test after confirming the normality of the distribution of data. Human studies were analyzed with the Mann-Whitney test. The in vitro studies include at least three separate experiments and are usually expressed as a percentage of the control values. Because the number of data in these experiments was never over 10, nonparametric statistics, particularly the Wilcoxon (two groups) or Friedman (more than two groups) tests, were selected to compare the paired results from the different experimental groups. The level of statistically significance was defined as P < 0.05. We used apoE mice, an animal model that resembles human atherosclerosis, to investigate the relationships between ECE-1 and vascular disease. ApoE mice developed atherosclerotic lesions in their aortas and had higher levels of cholesterol and lipids than WT animals (Fig. 1A). ApoE mice fed with the fat diet showed higher values of blood pressure than WT and apoE animals on the standard diet (Fig. 1A). LOX-1 protein content was increased in the aorta of apoE mice, a change that was also magnified by the fat diet (Fig. 1B). In apoE mice, we found an increased ECE-1 protein content in aorta, lungs, and kidneys, with respect to their wild-type counterparts, as detected by immunohistochemistry (Fig. 2, left part) and immunoblot (Fig. 2, right part). However, no significant differences were found in the heart (Fig. 2). The fat diet induced a slight but significant increase of ECE-1 protein content in aorta and lungs from apoE animals (Fig. 2, right part). The analysis of ECE-1 mRNA in these animals revealed an increased expression in the same organs as above, without significant differences in heart tissue (Fig. 3). When FR-901533, an ECE-1 inhibitor (36Pérez-Rivero G. Ruíz-Torres M.P. Rivas-Elena J.V. Jerkic M. Díez-Marqués M.L. López-Novoa J.M. Blasco M.A. Rodríguez-Puyol D. Mice deficient in telomerase activity develop hypertension because of an excess of endothelin production.Circulation. 2006; 114: 309-317Crossref PubMed Scopus (91) Google Scholar), was administered to mice via intraperitoneal at 1 mg/kg, blood pressure was reduced significantly in apoE mice on the fat diet versus WT mice and apoE on the normal diet (Fig. 4A).Fig. 3Changes in ECE-1 mRNA expression in aorta (A), lung (B), kidney cortex (C), and heart (D) from wild-type (WT) and apoE-deficient mice, o
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