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Differential Roles of Endothelin-1 in Angiotensin II-Induced Atherosclerosis and Aortic Aneurysms in Apolipoprotein E-Null Mice

2011; Elsevier BV; Volume: 179; Issue: 3 Linguagem: Inglês

10.1016/j.ajpath.2011.05.014

ISSN

1525-2191

Autores

Renée S. Suen, Sarah Rampersad, Duncan J. Stewart, David W. Courtman,

Tópico(s)

Protease and Inhibitor Mechanisms

Resumo

Because both endothelin-1 (ET-1) and angiotensin II (AngII) are independent mediators of arterial remodeling, we sought to determine the role of ET receptor inhibition in AngII-accelerated atherosclerosis and aortic aneurysm formation. We administered saline or AngII and/or bosentan, an endothelin receptor antagonist (ERA) for 7, 14, or 28 days to 6-week- and 6-month-old apolipoprotein E-knockout mice. AngII treatment increased aortic atherosclerosis, which was reduced by ERA. ET-1 immunostaining was localized to macrophage-rich regions in aneurysmal vessels. ERA did not prevent AngII-induced aneurysm formation but instead may have increased aneurysm incidence. In AngII-treated animals with aneurysms, ERA had a profound effect on the non-aneurysmal thoracic aorta via increasing wall thickness, collagen/elastin ratio, wall stiffness, and viscous responses. These observations were confirmed in acute in vitro collagen sheet production models in which ERA inhibited AngII's dose-dependent effect on collagen type 1 α 1 (COL1A1) gene transcription. However, chronic treatment reduced matrix metalloproteinase 2 mRNA expression but enhanced COL3A1, tissue inhibitor of metalloproteinase 1 (TIMP-1), and TIMP-2 mRNA expressions. These data confirm a role for the ET system in AngII-accelerated atherosclerosis but suggest that ERA therapy is not protective against the formation of AngII-induced aneurysms and can paradoxically stimulate a chronic arterial matrix remodeling response. Because both endothelin-1 (ET-1) and angiotensin II (AngII) are independent mediators of arterial remodeling, we sought to determine the role of ET receptor inhibition in AngII-accelerated atherosclerosis and aortic aneurysm formation. We administered saline or AngII and/or bosentan, an endothelin receptor antagonist (ERA) for 7, 14, or 28 days to 6-week- and 6-month-old apolipoprotein E-knockout mice. AngII treatment increased aortic atherosclerosis, which was reduced by ERA. ET-1 immunostaining was localized to macrophage-rich regions in aneurysmal vessels. ERA did not prevent AngII-induced aneurysm formation but instead may have increased aneurysm incidence. In AngII-treated animals with aneurysms, ERA had a profound effect on the non-aneurysmal thoracic aorta via increasing wall thickness, collagen/elastin ratio, wall stiffness, and viscous responses. These observations were confirmed in acute in vitro collagen sheet production models in which ERA inhibited AngII's dose-dependent effect on collagen type 1 α 1 (COL1A1) gene transcription. However, chronic treatment reduced matrix metalloproteinase 2 mRNA expression but enhanced COL3A1, tissue inhibitor of metalloproteinase 1 (TIMP-1), and TIMP-2 mRNA expressions. These data confirm a role for the ET system in AngII-accelerated atherosclerosis but suggest that ERA therapy is not protective against the formation of AngII-induced aneurysms and can paradoxically stimulate a chronic arterial matrix remodeling response. Atherosclerosis and aneurysmal vascular diseases lead to substantial morbidity and mortality, and they share a number of clinical risk factors.1Reed D. Reed C. Stemmermann G. Hayashi T. Are aortic aneurysms caused by atherosclerosis?.Circulation. 1992; 85: 205-211Crossref PubMed Scopus (276) Google Scholar Both diseases are associated with inflammation, and, although some believe that advanced atherosclerosis may be a prerequisite for abdominal aortic aneurysm (AAA), not all patients with AAA have evidence of substantial atherosclerosis nor does atherosclerosis always lead to aneurysm formation. Indeed, several studies have suggested that aneurysms and atherosclerosis are influenced by distinctly different inflammatory processes with T helper (Th)1 cytokines predominating in atherosclerosis and Th2 responses predominating in aneurysms.2Shimizu K. Shichiri M. Libby P. Lee R.T. Mitchell R.N. Th2-predominant inflammation and blockade of IFN-gamma signaling induce aneurysms in allografted aortas.J Clin Invest. 2004; 114: 300-308Crossref PubMed Scopus (187) Google Scholar, 3Shimizu K. Mitchell R.N. Libby P. Inflammation and cellular immune responses in abdominal aortic aneurysms.Arterioscler Thromb Vasc Biol. 2006; 26: 987-994Crossref PubMed Scopus (493) Google Scholar, 4Schonbeck U. Sukhova G.K. Gerdes N. Libby P. 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Role of oxidative stress in the pathogenesis of abdominal aortic aneurysms.Arterioscler Thromb Vasc Biol. 2007; 27: 461-469Crossref PubMed Scopus (264) Google Scholar that suggest the utility of a common therapeutic approach toward each condition. However, it is also possible that potential therapeutic approaches may have divergent effects on both AAA and atherosclerosis. Angiotensin II (AngII) has been associated with atherosclerosis and aneurysm rupture in humans8Dimmeler S. Rippmann V. Weiland U. Haendeler J. Zeiher A.M. Angiotensin II induces apoptosis of human endothelial cells Protective effect of nitric oxide.Circ Res. 1997; 81: 970-976Crossref PubMed Scopus (347) Google Scholar, 9Keidar S. 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Ho J.J. Wilson D.W. Rutledge J.C. Vergona R. Sullivan M.E. Wang Y.X. Enalapril attenuates angiotensin II-induced atherosclerosis and vascular inflammation.Atherosclerosis. 2005; 178: 9-17Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 13Daugherty A. Manning M.W. Cassis L.A. Angiotensin II promotes atherosclerotic lesions and aneurysms in apolipoprotein E-deficient mice.J Clin Invest. 2000; 105: 1605-1612Crossref PubMed Scopus (1073) Google Scholar, 14Gavrila D. Li W.G. McCormick M.L. Thomas M. Daugherty A. Cassis L.A. Miller Jr, F.J. Oberley L.W. Dellsperger K.C. Weintraub N.L. Vitamin E inhibits abdominal aortic aneurysm formation in angiotensin II-infused apolipoprotein E-deficient mice.Arterioscler Thromb Vasc Biol. 2005; 25: 1671-1677Crossref PubMed Scopus (145) Google Scholar, 15King V.L. Lin A.Y. Kristo F. Anderson T.J. Ahluwalia N. Hardy G.J. Owens III, A.P. Howatt D.A. Shen D. Tager A.M. Luster A.D. Daugherty A. Gerszten R.E. Interferon-gamma and the interferon-inducible chemokine CXCL10 protect against aneurysm formation and rupture.Circulation. 2009; 119: 426-435Crossref PubMed Scopus (94) Google Scholar, 16Manning M.W. Cassis L.A. Daugherty A. Differential effects of doxycycline, a broad-spectrum matrix metalloproteinase inhibitor, on angiotensin II-induced atherosclerosis and abdominal aortic aneurysms.Arterioscler Thromb Vasc Biol. 2003; 23: 483-488Crossref PubMed Scopus (257) Google Scholar, 17Ni W. Kitamoto S. Ishibashi M. Usui M. Inoue S. Hiasa K. Zhao Q. Nishida K. Takeshita A. Egashira K. Monocyte chemoattractant protein-1 is an essential inflammatory mediator in angiotensin II-induced progression of established atherosclerosis in hypercholesterolemic mice.Arterioscler Thromb Vasc Biol. 2004; 24: 534-539Crossref PubMed Scopus (95) Google Scholar, 18Saraff K. Babamusta F. Cassis L.A. Daugherty A. Aortic dissection precedes formation of aneurysms and atherosclerosis in angiotensin II-infused apolipoprotein E-deficient mice.Arterioscler Thromb Vasc Biol. 2003; 23: 1621-1626Crossref PubMed Scopus (340) Google Scholar, 19Tham D.M. Martin-McNulty B. Wang Y.X. da C V. Wilson D.W. Athanassious C.N. Powers A.F. Sullivan M.E. Rutledge J.C. Angiotensin II injures the arterial wall causing increased aortic stiffening in apolipoprotein E-deficient mice.Am J Physiol Regul Integr Comp Physiol. 2002; 283: R1442-R1449Crossref PubMed Scopus (54) Google Scholar, 20Tham D.M. Martin-McNulty B. Wang Y.X. Wilson D.W. Vergona R. Sullivan M.E. Dole W. Rutledge J.C. Angiotensin II is associated with activation of NF-kappaB-mediated genes and downregulation of PPARs.Physiol Genomics. 2002; 11: 21-30PubMed Google Scholar, 21Weiss D. Kools J.J. Taylor W.R. Angiotensin II-induced hypertension accelerates the development of atherosclerosis in Apo E-deficient mice.Circulation. 2001; 103: 448-454Crossref PubMed Scopus (301) Google Scholar The AngII/apoE−/− mouse model is well characterized and represents the best model of combined atherosclerosis and aneurysm initiation; the striking features of this model are a marked infiltration of macrophages into the adventitia and media, the breakdown of elastin lamellae, and the development of marked dilation of the suprarenal aorta.13Daugherty A. Manning M.W. Cassis L.A. Angiotensin II promotes atherosclerotic lesions and aneurysms in apolipoprotein E-deficient mice.J Clin Invest. 2000; 105: 1605-1612Crossref PubMed Scopus (1073) Google Scholar, 22Daugherty A. Rateri D.L. Cassis L.A. Role of the renin-angiotensin system in the development of abdominal aortic aneurysms in animals and humans.Ann N Y Acad Sci. 2006; 1085: 82-91Crossref PubMed Scopus (46) Google Scholar Of interest, atherosclerosis and AAA in this model occur independent of increased blood pressure, a risk factor traditionally associated with both diseases.10Cassis L.A. Gupte M. Thayer S. Zhang X. Charnigo R. Howatt D.A. Rateri D.L. Daugherty A. ANG II infusion promotes abdominal aortic aneurysms independent of increased blood pressure in hypercholesterolemic mice.Am J Physiol Heart Circ Physiol. 2009; 296: H1660-H1665Crossref PubMed Scopus (166) Google Scholar, 16Manning M.W. Cassis L.A. Daugherty A. Differential effects of doxycycline, a broad-spectrum matrix metalloproteinase inhibitor, on angiotensin II-induced atherosclerosis and abdominal aortic aneurysms.Arterioscler Thromb Vasc Biol. 2003; 23: 483-488Crossref PubMed Scopus (257) Google Scholar, 21Weiss D. Kools J.J. Taylor W.R. Angiotensin II-induced hypertension accelerates the development of atherosclerosis in Apo E-deficient mice.Circulation. 2001; 103: 448-454Crossref PubMed Scopus (301) Google Scholar Although hyperlipidemia itself has been shown to induce the development of AAA in aged apoE−/− mice,23Daugherty A. Cassis LA: Mouse models of abdominal aortic aneurysms.Arterioscler Thromb Vasc Biol. 2004; 24: 429-434Crossref PubMed Scopus (385) Google Scholar the incidence remains low but is markedly increased by AngII infusion. Pathologic differences may exist between young and old animals, and an appropriate comparison of the two under identical experimental conditions may help elucidate the importance of age and/or the presence of pre-existing lesions on AAA development. Endothelin (ET)-1 is known to play an important role in atherogenesis,24Barton M. Haudenschild C.C. d'Uscio L.V. Shaw S. Munter K. Luscher T.F. Endothelin ETA receptor blockade restores NO-mediated endothelial function and inhibits atherosclerosis in apolipoprotein E-deficient mice.Proc Natl Acad Sci U S A. 1998; 95: 14367-14372Crossref PubMed Scopus (334) Google Scholar, 25Babaei S. Picard P. Ravandi A. Monge J.C. Lee T.C. Cernacek P. Stewart D.J. Blockade of endothelin receptors markedly reduces atherosclerosis in LDL receptor deficient mice: role of endothelin in macrophage foam cell formation.Cardiovasc Res. 2000; 48: 158-167Crossref PubMed Scopus (72) Google Scholar, 26Bousette N. Giaid A. Endothelin-1 in atherosclerosis and other vasculopathies.Can J Physiol Pharmacol. 2003; 81: 578-587Crossref PubMed Scopus (39) Google Scholar yet its contribution to the formation of aneurysms is largely unknown. ET-1 is an important downstream mediator of many of the biological effects of AngII, having a pivotal role in vascular remodeling.27Sung C.P. Arleth A.J. Storer B.L. Ohlstein E.H. Angiotensin type 1 receptors mediate smooth muscle proliferation and endothelin biosynthesis in rat vascular smooth muscle.J Pharmacol Exp Ther. 1994; 271: 429-437PubMed Google Scholar Moreau et al28Moreau P. d'Uscio L.V. Shaw S. Takase H. Barton M. Luscher T.F. Angiotensin II increases tissue endothelin and induces vascular hypertrophy: reversal by ET(A)-receptor antagonist.Circulation. 1997; 96: 1593-1597Crossref PubMed Scopus (309) Google Scholar reported that increased arterial smooth muscle cell (SMC) hypertrophy in AngII-infused rats was completely inhibited with the administration of a selective ET(A)-receptor antagonist, LU135252. In this study, we have examined young and old apoE-null mice, confirming a modest contribution of ET-1 to atherogenesis in the AngII-infused model but also providing evidence of a previously unrecognized protective role for the ET-1 pathway in limiting the extent of fibrosis and remodeling of the aneurysmal matrix. Young (4 weeks) and old (6 months) apoE−/− mice (The Jackson Laboratory, Bar Harbor, ME) were examined for atherosclerotic progression and aneurysm formation after infusion with AngII with or without bosentan (Tracleer; Actelion Pharmaceuticals Ltd., Allschwil, Switzerland), a dual ET-1 receptor antagonist (ERA). Four-week-old mice (n = 180) were fed a Western-type diet (TD 88137; Harlan Teklad, Madison, WI) beginning 2 weeks before experimentation (mean cholesterol, 27.49 ± 1.63 mmol/L; mean triglyceride, 1.42 ± 0.17 mmol/L at sacrifice) to encourage lesion progression, and 6-month-old mice (n = 194) received normal chow. Invasive procedures were performed under anesthesia (xylazine 20 mg/kg and ketamine 100 mg/kg). All animal studies were conducted under protocols approved by the local animal care committee in accordance with guidelines from the Canadian Council of Animal Care. Mice were randomly assigned to receive a 4-week infusion of either AngII (1000 ng · kg−1 · minute−1; A9525; Sigma-Aldrich, St. Louis, MO) or 0.9% NaCl by a subcutaneous osmotic minipump (Model 2004; Durect Corporation, Cupertino, CA). Mice assigned ERA received 10 mg · kg−1 · day−1 bosentan in their drinking water (a dose that significantly inhibited acute ET-1–induced rise in systemic blood pressure). After 4 weeks of treatment blood pressure was measured from the right common carotid artery with a fluid-filled catheter. Blood was extracted through cardiac puncture into EDTA vials, spun down (250 × g) and the plasma stored at −80°C. The arterial system was perfusion fixed with 4% paraformaldehyde at 70 mmHg pressure, and the aorta (arch to the iliac bifurcation) was removed, stripped of its adventitia, and photographed. Aneurysms were determined to be present if the widest region of the suprarenal aorta was 1.5 times greater than the upstream descending aorta. En face oil red O staining (O0625; Sigma-Aldrich)25Babaei S. Picard P. Ravandi A. Monge J.C. Lee T.C. Cernacek P. Stewart D.J. Blockade of endothelin receptors markedly reduces atherosclerosis in LDL receptor deficient mice: role of endothelin in macrophage foam cell formation.Cardiovasc Res. 2000; 48: 158-167Crossref PubMed Scopus (72) Google Scholar was used to evaluate the percentage of luminal surface area occupied by lipid (atherosclerosis) in non-aneurysmal vessels with the use of ImageJ64 analysis software version 1.44 (National Institutes of Health, Bethesda, MD). Serum total cholesterol and triglyceride concentrations were measured with an enzymatic cholesterol assay in a colorimetric procedure on a Technicon RA1000 analyzer (Bayer, Tarrytown, NY; n = 6). ET-1 levels from precipitated plasma samples after 2 and 4 weeks of treatment were analyzed through enzyme-linked immunosorbent assay (ELISA) with the use of commercially available kits [Endothelin (1–21) ELISA Immunoassay; ALPCO Diagnostics, Salem, NH] according to manufacturer's recommendations. Aortas from a subset of the 6-month-old animals underwent uniaxial mechanical testing (n = 10 to 20). A 2-mm ring of the thoracic aorta was excised between the aortic arch and intercostal arteries and mounted between two orthodontic wire loops (0.4-mm diameter) to avoid gripping artifacts. The loops were mounted on a Mach-1 Micromechanical Test System (Biosyntech, Montreal, QC, Canada) to allow for the precise actuator-based circumferential extension of the tissue while load was recorded with a 1000-g load cell. During testing tissue was submersed in oxygenated Krebs solution (pH 7.4, and 37°C) containing 0.8 g/L papaverine (P3510; Sigma-Aldrich). A digital image of the tissue at 0.1-g resting tension was captured to determine segment (gauge) length and width. Stress was calculated from the wall thickness values measured from digital images of sections adjacent to the excised ring. Tissues were preconditioned at 20 cycles to 7-g load (approximately 15% of maximum load) with a strain rate of 10 mm/minute. For stress relaxation testing, tissues were loaded to 7 g, and the load decay was observed for 100 seconds. Finally, tissues were preconditioned as above before loading at 10 mm/minute to fracture. All chemicals are from Sigma-Aldrich unless specified. Tissues were dried and digested in cyanogen bromide (50 mg/mL in 70% in formic acid), and the supernatant fluid, along with the washed pellet, was collected and separately hydrolyzed overnight at 110°C in 6N HCl. Total collagen was determined with the hydroxyproline assay modified from Huszar et al.29Huszar G. Maiocco J. Naftolin F. Monitoring of collagen and collagen fragments in chromatography of protein mixtures.Anal Biochem. 1980; 105: 424-429Crossref PubMed Scopus (212) Google Scholar The supernatant fluid was reconstituted in collagen assay buffer (0.26 mol/L citric acid, 0.21 mol/L glacial acetic acid, 0.88 mol/L sodium acetate.3H2O, 0.85 mol/L sodium hydroxide; pH 6.0) and free hydroxyproline in the sample was analyzed by reacting with Chloramine T (0.5 mol/L in n-propanol; Fisher Scientific, Ottawa, ON, Canada) for 20 minutes, then with Ehrlich solution and perchloric acid (15 minutes at 65°C), and followed with absorbance reading at 550 nm against a hydroxyproline standard (pH 6). Total collagen was calculated by assuming a 12.7% hydroxyproline content. Elastin content was determined as total protein (Ninhydrin assay) in the cyanogen bromide–insoluble pellet with absorbance read at 570 nm against a hydrolyzed elastin standard. Elastin purity was confirmed in four samples through amino acid analysis by quadrupole time-of-flight mass spectrometry (Advanced Protein Technology Centre, Hospital for Sick Children, Toronto, ON, Canada). Cross-sections spanning the length of the aorta were stained with H&E, Verhoeff's van Gieson, and picosirius red. Routine immunohistochemistry (IHC) was performed with the following primary antibodies: AngII (rabbit polyclonal, 1:1000; IHC7002; Peninsula Laboratories, Inc., San Carlos, CA), mouse ET-1 (rabbit polyclonal, 1:600; IHC6901; Peninsula Laboratories, Inc.), and mouse macrophage (rat polyclonal Mac-3, 1:10; 550292; BD Pharmingen, Franklin Lakes, NJ). A substitution with pre-immune serum in place of the primary and/or secondary antibodies was used as negative controls. Quantification of Mac-3 staining in intimal areas was performed with digital planimetry. Apoptosis was detected with the DeadEnd Fluorometric TUNEL System (G3250; Promega, Madison, WI), and data were confirmed by immunostaining with fluorescein-conjugated cleaved caspase-3 (Asp175) antibody (1:100; 9667; Cell Signaling Technology, Danvers, MA). Slides were also stained with cyanine 3–conjugated monoclonal anti–α-smooth muscle actin (1:200; C6198; Sigma-Aldrich) and ToPro3 nuclear counterstain (1:5000; T3605; Invitrogen, Carlsbad, CA). Fluorescence intensity was quantified by a blinded observer, using a fixed gain and power setting on a Nikon (Melville, NY) fluorescent microscope equipped with an epi-illumination single-band emitter filter cassette for the separate illumination of green (543 nm), red (633 nm), and blue (488 nm) fluorescence. Rat A10 SMC (CRL-1476; American Type Culture Collection, Manassas, VA) from passages 14 to 17 were cultured at an initial density of 10,000 cells/cm2 in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and penicillin/streptomycin (100 U/100 μg/mL). At confluence, cultures were fed daily with freshly prepared 50 μg/mL L-ascorbic acid (A7631; Sigma-Aldrich) to encourage collagen production for a total of 28 days. At 15 days of ascorbate supplementation flasks were randomized to receive supplementation with AngII (10−7 mol/L; Sigma-Aldrich), ERA (10−5 mol/L), both compounds, or vehicle (n = 9 to 10 separate experiments). Total RNA was extracted from collagen sheets with the use of the RNeasy MiniKit (74104; Qiagen, Valencia, CA) with the on-column DNase digestion step (RNase-Free DNase Set; 79254; Qiagen). Total RNA (1 μg) was reverse transcribed to first-strand cDNA with the use of the Omniscript RT Kit (205111; Qiagen). Primer sequences used for collagen type 3 α 1 (COL3A1),30Zhang L. Tran N. Chen H.Q. Kahn C.J. Marchal S. Groubatch F. Wang X. Time-related changes in expression of collagen types I and III and of tenascin-C in rat bone mesenchymal stem cells under co-culture with ligament fibroblasts or uniaxial stretching.Cell Tissue Res. 2008; 332: 101-109Crossref PubMed Scopus (42) Google Scholar membrane type-1 matrix metalloproteinase (MMP), tissue inhibitor of metalloproteinase (TIMP)-1, and TIMP-231He J.Z. Quan A. Xu Y. Teoh H. Wang G. Fish J.E. Steer B.M. Itohara S. Marsden P.A. Davidge S.T. Ward M.E. Induction of matrix metalloproteinase-2 enhances systemic arterial contraction after hypoxia.Am J Physiol Heart Circ Physiol. 2007; 292: H684-H693Crossref PubMed Scopus (13) Google Scholar are the same as previously described. Primer sets used for collagen type 1 α 1 (COL1A1) was 5′-AAGGTTCTCCTGGTGAAGCTG-3′ and 5′-ATCACACCAGCCTGTCCACGG-3′, MMP-2 was 5′-ACACTGGGACCTGTCACTCC-3′ and 5′-ACACGGCATCAATCTTTTCC-3′, and interferon (IFN)-γ was 5′-GCCCTCTCTGGCTGTTACTG-3′ and 5′-CCAAGAGGAGGCTCTTTCCT-3′. cDNA amplification for each gene of interest was monitored with 2× SYBR Green PCR Master Mix (4309155; Applied Biosystems, Carlsbad, CA) with the use of the ABI PRISM Sequence Detection System (model 7900HT SDS, Applied Biosystems). All results were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression (TaqMan Rodent GAPDH Control Reagents kit; 4308313; Applied Biosystems). mRNA levels were calculated with the ΔΔCt formula: target gene expression/GAPDH expression = 2(ΔΔCt) and are reported in arbitrary units. Rat A10 cells were treated with AngII, ERA, both compounds, or vehicle (n = 3 to 4 per group) for 24 hours before being serum starved overnight. Cells were then harvested with trypLE (12563029; Invitrogen), incubated in serum-free Dulbecco's modified Eagle's medium for 1 hour, and plated at 26,000 cells/cm2 on plates precoated with type I collagen (PICL24P05; Millipore, Bellerica, MA). Cells were incubated for 1.5 hours at 37°C, and nonadherent cells were removed by washing with PBS. Six to eight images per treatment group were taken at ×10 magnification under an inverted light microscope, and the number of adherent cells per field were counted. Rat A10 SMCs pretreated for 24 hours with AngII, ERA, both compounds, or vehicle (n = 3 to 4) were harvested with 2 mmol/L EDTA (E9884; Sigma-Aldrich) and resuspended at 1000,000 cells/mL in staining buffer (1% bovine serum albumin, 0.1% sodium azide in PBS) containing anti-β1-integrin antibody (mouse monoclonal, 1:500; MAB1965; Millipore) or mouse IgG1 isotype control (human, 1:500; 11-632-C100; Axxora, San Diego, CA) for 1 hour. Cells were centrifuged (400 × g, 5 minutes), washed, and incubated with Alexa Fluor 488 (goat polyclonal, 1:1000; A11001; Invitrogen) for 1 hour in the dark. Cells were washed, resuspended in staining buffer, and analyzed with a Cell Lab Quanta SC system (Beckman Coulter, Mississauga, ON, Canada). χ2 testing was used to determine the significance of aneurysm incidence. Quantitative results are expressed as mean ± SEM unless otherwise stated, and differences were determined by nonpaired t-test, or one-way analysis of variance with post hoc tests performed with Bonferroni's or Dunnet's multiple comparison tests when appropriate. For tissue mechanics, the nonparametric Kruskal-Wallis test was used. Analyses were performed with GraphPad Prism version 5 (GraphPad Software, Inc., San Diego, CA). P values < 0.05 were considered significant. Mean arterial blood pressure was not significantly different in any of the treatment groups of the same age when measured under anesthetic conditions. In vivo telemetric blood pressure measurements were also performed in a subset of 8-week-old mice with the use of implantable telemetry (DSI PhysioTel PA-C10; Data Sciences International, St. Paul, MN) by carotid catheter placement. Measurements were taken for 1 hour, twice daily for 7 days to establish a baseline control, then treated with AngII (n = 3) or combined AngII and ERA (n = 4) for 7 days. These animals showed a modest increase in blood pressure measured after 7 days of treatment compared with baseline (101.2 ± 2.75 mmHg versus 124.9 ± 5.71 mmHg; P < 0.05; n = 7 for control versus treatment); however, no differences in mean arterial blood pressure between AngII and AngII/ERA treatment groups were observed (Figure 1). Plasma cholesterol and triglyceride concentrations were not significantly different in any of the treatment groups of the same age. Plasma concentrations of ET-1 from AngII-treated young mice were not elevated after 2 weeks (10.98 ± 1.92 fmol/mL) but trended higher by week 4; however, this was not statistically significant over controls (young: 15.36 ± 3.38 fmol/mL versus 7.94 ± 1.98 fmol/mL, P = 0.10; old: 12.92 ± 2.97 fmol/mL versus 7.68 ± 2.20 fmol/mL, P = 0.25; n = 10 to 14 for AngII versus control); old mice treated with ERA did show a significant increase in plasma ET-1 (young: 13.27 ± 2.20 fmol/mL, P = 0.09, n = 12; old: 19.87 ± 1.85 fmol/mL, P < 0.01, n = 5 for ERA versus control). ET-1 immunostaining was seen mainly in the intima and media of aortas from AngII-treated animals, regions that often stained positive for the macrophage marker Mac-3 (Figure 2A). ERA was able to diminish ET-1 staining in aortic cross-sections from old AngII-treated animals (Figure 2A).Figure 2ET antagonism in AngII-accelerated atherosclerosis. A: Representative photomicrographs of ET-1 and Mac-3 immunostaining in control, AngII, or AngII/ERA mouse thoracic aortas (brown indicates positive immunostaining; arrows, blue, hematoxylin for cell nuclei). Scale bar = 50 μm. En face oil red O–stained aortas from mice 6 weeks old (B) and 6 months old (C), and percentage of en face intimal surface area positive for oil red O staining measured in both age groups. Quantification was performed by two independent observers. Data are presented as means ± SEM; n = 6 to 10. *P < 0.01 versus age-matched control; **P < 0.001 versus all groups; and ***P < 0.01 AngII/ERA versus AngII.View Large Image Figure ViewerDownload Hi-res image Download (PPT) AngII-treated 6-week-old mice showed an eightfold (P < 0.001) increase in en face lesion area (Figure 2B), whereas 6-month-old mice had a threefold increase (P < 0.01) compared with age-matched controls (Figure 2C). Administration of ERA reduced AngII-induced lesion area in the young mice (P < 0.01) but not in the old animals, possibly because of the presence of pre-existing lesions in the aortas of older apoE−/− mice.32Reddick R.L. Zhang S.H. Maeda N. Atherosclerosis in mice lacking apo E Evaluation of lesional development and progression.Arterioscler Thromb. 1994; 14 ([published erratum appears in Arterioscler Thromb 1994, 14(5):839]): 141-147Crossref PubMed Scopus (549) Google Scholar, 33Zhang S.H. Reddick R.L. Piedrahita J.A. Maeda N. Spontaneous hypercholesterolemia and arterial lesions in mice lacking apolipoprotein E.Science. 1992; 258: 468-471Crossref PubMed Scopus (1836) Google Scholar Saline-treated apoE−/− mice did not develop AAA (Figure 3A). In all groups of AngII-infused mice, a subset exhibited histologic evidence of aneurysm formation in the suprarenal aorta, including thickened media and adventitial layers, and disruptions to the elastic lamina (Figure 3). To our surprise, ERA failed to protect against AngII

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