Prostaglandin Receptor EP4 in Abdominal Aortic Aneurysms
2012; Elsevier BV; Volume: 181; Issue: 1 Linguagem: Inglês
10.1016/j.ajpath.2012.03.016
ISSN1525-2191
AutoresRichard Y. Cao, Tim St. Amand, Xinzhi Li, Sung-Hee Yoon, Carol P. Wang, Hui Song, Takayuki Maruyama, Peter Brown, David Zelt, Colin Funk,
Tópico(s)Aortic Disease and Treatment Approaches
ResumoAbdominal aortic aneurysm (AAA) pathogenesis is distinguished by vessel wall inflammation. Cyclooxygenase (COX)-2 and microsomal prostaglandin E synthase-1, key components of the most well-characterized inflammatory prostaglandin pathway, contribute to AAA development in the 28-day angiotensin II infusion model in mice. In this study, we used this model to examine the role of the prostaglandin E receptor subtype 4 (EP4) and genetic knockdown of COX-2 expression (70% to 90%) in AAA pathogenesis. The administration of the prostaglandin receptor EP4 antagonist AE3-208 (10 mg/kg per day) to apolipoprotein E (apoE)–deficient mice led to active drug plasma concentrations and reduced AAA incidence and severity compared with control apoE-deficient mice (P < 0.01), whereas COX-2 genetic knockdown/apoE-deficient mice displayed only a minor, nonsignificant decrease in incidence of AAA. EP4 receptor protein was present in human and mouse AAA, as observed by using Western blot analysis. Aortas from AE3-208–treated mice displayed evidence of a reduced inflammatory phenotype compared with controls. Atherosclerotic lesion size at the aortic root was similar between all groups. In conclusion, the prostaglandin E2–EP4 signaling pathway plays a role in the AAA inflammatory process. Blocking the EP4 receptor pharmacologically reduces both the incidence and severity of AAA in the angiotensin II mouse model, potentially via attenuation of cytokine/chemokine synthesis and the reduction of matrix metalloproteinase activities. Abdominal aortic aneurysm (AAA) pathogenesis is distinguished by vessel wall inflammation. Cyclooxygenase (COX)-2 and microsomal prostaglandin E synthase-1, key components of the most well-characterized inflammatory prostaglandin pathway, contribute to AAA development in the 28-day angiotensin II infusion model in mice. In this study, we used this model to examine the role of the prostaglandin E receptor subtype 4 (EP4) and genetic knockdown of COX-2 expression (70% to 90%) in AAA pathogenesis. The administration of the prostaglandin receptor EP4 antagonist AE3-208 (10 mg/kg per day) to apolipoprotein E (apoE)–deficient mice led to active drug plasma concentrations and reduced AAA incidence and severity compared with control apoE-deficient mice (P < 0.01), whereas COX-2 genetic knockdown/apoE-deficient mice displayed only a minor, nonsignificant decrease in incidence of AAA. EP4 receptor protein was present in human and mouse AAA, as observed by using Western blot analysis. Aortas from AE3-208–treated mice displayed evidence of a reduced inflammatory phenotype compared with controls. Atherosclerotic lesion size at the aortic root was similar between all groups. In conclusion, the prostaglandin E2–EP4 signaling pathway plays a role in the AAA inflammatory process. Blocking the EP4 receptor pharmacologically reduces both the incidence and severity of AAA in the angiotensin II mouse model, potentially via attenuation of cytokine/chemokine synthesis and the reduction of matrix metalloproteinase activities. Abdominal aortic aneurysm (AAA), characterized by a dilatation exceeding the normal diameter by >50%,1Upchurch Jr, G.R. Schaub T.A. Abdominal aortic aneurysm.Am Fam Physician. 2006; 73: 1198-1204PubMed Google Scholar is associated with advanced age, male sex, cigarette smoking, atherosclerosis, hypertension, and genetic predisposition.2Lloyd-Jones D. Adams R.J. Brown T.M. Carnethon M. Dai S. De Simone G. Ferguson T.B. Ford E. Furie K. Gillespie C. Go A. Greenlund K. Haase N. Hailpern S. Ho P.M. Howard V. Kissela B. Kittner S. Lackland D. Lisabeth L. Marelli A. McDermott M.M. Meigs J. Mozaffarian D. Mussolino M. Nichol G. Roger V.L. Rosamond W. Sacco R. Sorlie P. Thom T. Wasserthiel-Smoller S. Wong N.D. Wylie-Rosett J. Heart disease and stroke statistics–2010 update: a report from the American Heart Association.Circulation. 2010; 121: e46-e215Crossref PubMed Scopus (1408) Google Scholar, 3Golledge J. Muller J. Daugherty A. Norman P. Abdominal aortic aneurysm: pathogenesis and implications for management.Arterioscler Thromb Vasc Biol. 2006; 26: 2605-2613Crossref PubMed Scopus (466) Google Scholar, 4van Vlijmen-van Keulen C.J. Pals G. Rauwerda J.A. Familial abdominal aortic aneurysm: a systematic review of a genetic background.Eur J Vasc Endovasc Surg. 2002; 24: 105-116Abstract Full Text PDF PubMed Scopus (74) Google Scholar The histopathological features of AAAs are characterized by chronic inflammatory cell recruitment to the aortic wall, with tissue degeneration and remodeling, and depletion of medial smooth muscle cells.5McCormick M.L. Gavrila D. Weintraub N.L. Role of oxidative stress in the pathogenesis of abdominal aortic aneurysms.Arterioscler Thromb Vasc Biol. 2007; 27: 461-469Crossref PubMed Scopus (264) Google Scholar, 6Thompson R.W. Reflections on the pathogenesis of abdominal aortic aneurysms.Cardiovasc Surg. 2002; 10: 389-394Crossref PubMed Scopus (48) Google Scholar AAAs are a common vascular condition with life-threatening implications from aortic rupture, which has been reported to have a mortality rate as high as 90%2Lloyd-Jones D. Adams R.J. Brown T.M. Carnethon M. Dai S. De Simone G. Ferguson T.B. Ford E. Furie K. Gillespie C. Go A. Greenlund K. Haase N. Hailpern S. Ho P.M. Howard V. Kissela B. Kittner S. Lackland D. Lisabeth L. Marelli A. McDermott M.M. Meigs J. Mozaffarian D. Mussolino M. Nichol G. Roger V.L. Rosamond W. Sacco R. Sorlie P. Thom T. Wasserthiel-Smoller S. Wong N.D. Wylie-Rosett J. Heart disease and stroke statistics–2010 update: a report from the American Heart Association.Circulation. 2010; 121: e46-e215Crossref PubMed Scopus (1408) Google Scholar and causes >15,000 deaths per year in the United States.7Baxter B.T. Terrin M.C. Dalman R.L. Medical management of small abdominal aortic aneurysms.Circulation. 2008; 117: 1883-1889Crossref PubMed Scopus (284) Google Scholar Surgical repair by standard means or interventional endovascular stent placement is the only option for treatment. Therefore, developing pharmacological prevention strategies to block AAA progression is a high priority. Cyclooxygenase (COX)-2, an enzyme that generates inflammatory mediators, such as prostaglandin E2 (PGE2), was highly expressed in human AAA specimens, and the enzyme was proposed as a potential target for pharmacotherapy.8Holmes D.R. Wester W. Thompson R.W. Reilly J.M. Prostaglandin E2 synthesis and cyclooxygenase expression in abdominal aortic aneurysms.J Vasc Surg. 1997; 25: 810-815Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar To test this assertion in animals, experiments involving both genetic and pharmacological inhibition of COX-2 were conducted using the well-characterized angiotensin II (AngII)–induced mouse AAA model. Results revealed that COX-2 contributed significantly to AAA formation.9King V.L. Trivedi D.B. Gitlin J.M. Loftin C.D. Selective cyclooxygenase-2 inhibition with celecoxib decreases angiotensin II-induced abdominal aortic aneurysm formation in mice.Arterioscler Thromb Vasc Biol. 2006; 26: 1137-1143Crossref PubMed Scopus (110) Google Scholar, 10Gitlin J.M. Trivedi D.B. Langenbach R. Loftin C.D. Genetic deficiency of cyclooxygenase-2 attenuates abdominal aortic aneurysm formation in mice.Cardiovasc Res. 2007; 73: 227-236Crossref PubMed Scopus (75) Google Scholar Because of the potential renal and cardiovascular risks of COX-2 selective inhibition,11Morham S.G. Langenbach R. Loftin C.D. Tiano H.F. Vouloumanos N. Jennette J.C. Mahler J.F. Kluckman K.D. Ledford A. Lee C.A. Smithies O. Prostaglandin synthase 2 gene disruption causes severe renal pathology in the mouse.Cell. 1995; 83: 473-482Abstract Full Text PDF PubMed Scopus (1025) Google Scholar, 12Dinchuk J.E. Car B.D. Focht R.J. Johnston J.J. Jaffee B.D. Covington M.B. Contel N.R. Eng V.M. Collins R.J. Czerniak P.M. Gorry S.A. Trzaskos J.M. Renal abnormalities and an altered inflammatory response in mice lacking cyclooxygenase II.Nature. 1995; 378: 406-409Crossref PubMed Scopus (895) Google Scholar, 13Fitzgerald G.A. Coxibs and cardiovascular disease.N Engl J Med. 2004; 351: 1709-1711Crossref PubMed Scopus (834) Google Scholar, 14Grosser T. Fries S. FitzGerald G.A. Biological basis for the cardiovascular consequences of COX-2 inhibition: therapeutic challenges and opportunities.J Clin Invest. 2006; 116: 4-15Crossref PubMed Scopus (835) Google Scholar downstream blockade in the PGE2 arm of the pathway was tested using mice with disruption of microsomal prostaglandin E synthase-1 (mPGES-1) expression. This resulted in suppressed AAA formation in the AngII model with mice on a low-density lipoprotein receptor–deficient background.15Wang M. Lee E. Song W. Ricciotti E. Rader D.J. Lawson J.A. Pure E. FitzGerald G.A. Microsomal prostaglandin E synthase-1 deletion suppresses oxidative stress and angiotensin II-induced abdominal aortic aneurysm formation.Circulation. 2008; 117: 1302-1309Crossref PubMed Scopus (114) Google Scholar mRNA-encoding PGE2 receptor subtypes EP2, EP3, and EP4 have been detected in human AAA specimens, and parallel in vitro experiments demonstrated that PGE2 stimulated secretion of IL-6 from aortic macrophages.16Bayston T. Ramessur S. Reise J. Jones K.G. Powell J.T. Prostaglandin E2 receptors in abdominal aortic aneurysm and human aortic smooth muscle cells.J Vasc Surg. 2003; 38: 354-359Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar Taken together, results from these studies implicate a COX-2/mPGES-1/EP receptor pathway potentially linked via cytokines/chemokines, as suggested by others,17Wang Y. Ait-Oufella H. Herbin O. Bonnin P. Ramkhelawon B. Taleb S. Huang J. Offenstadt G. Combadiere C. Renia L. Johnson J.L. Tharaux P.L. Tedgui A. Mallat Z. TGF-beta activity protects against inflammatory aortic aneurysm progression and complications in angiotensin II-infused mice.J Clin Invest. 2010; 120: 422-432Crossref PubMed Scopus (304) Google Scholar, 18Tieu B.C. Lee C. Sun H. Lejeune W. Recinos 3rd, A. Ju X. Spratt H. Guo D.C. Milewicz D. Tilton R.G. Brasier A.R. An adventitial IL-6/MCP1 amplification loop accelerates macrophage-mediated vascular inflammation leading to aortic dissection in mice.J Clin Invest. 2009; 119: 3637-3651Crossref PubMed Scopus (339) Google Scholar, 19Shimizu 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 (186) Google Scholar to inflammatory circuits in AAA formation. The purposes of the present study were to study the role of one EP receptor subtype, EP4, in this AAA signaling axis using a selective EP4 antagonist in the AngII AAA murine model; to examine if EP4 receptor protein is present in human AAA; and to determine if genetic COX-2 knockdown (COX2KD) differs from COX-2 knockout in terms of phenotype in the murine model. Because PGE2-EP4 signaling promotes type 17 helper T-cell expansion and IL-17 production,20Yao C. Sakata D. Esaki Y. Li Y. Matsuoka T. Kuroiwa K. Sugimoto Y. Narumiya S. Prostaglandin E2-EP4 signaling promotes immune inflammation through Th1 cell differentiation and Th17 cell expansion.Nat Med. 2009; 15: 633-640Crossref PubMed Scopus (431) Google Scholar, 21Sheibanie A.F. Yen J.H. Khayrullina T. Emig F. Zhang M. Tuma R. Ganea D. The proinflammatory effect of prostaglandin E2 in experimental inflammatory bowel disease is mediated through the IL-23→IL-17 axis.J Immunol. 2007; 178: 8138-8147PubMed Google Scholar a pleiotropic cytokine, which mediates pro-inflammatory responses, we also investigated the presence of IL-17 in AAA samples and other aspects of the inflammatory process. The AngII-induced mouse AAA model, using an atherosclerotic-susceptible strain [either apolipoprotein E deficient (apoE−/−) or low-density lipoprotein receptor deficient], has become an exceedingly popular tool because of its simplicity and because certain facets of the model resemble human disease acquisition, including male sex preponderance in the setting of mild hypertension with enhanced incidence in the presence of hyperlipidemia.22Daugherty A. Cassis L.A. Mouse models of abdominal aortic aneurysms.Arterioscler Thromb Vasc Biol. 2004; 24: 429-434Crossref PubMed Scopus (385) Google Scholar, 23Daugherty 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 In this study, apoE−/− mice on a C57BL/6 genetic background (Jackson Laboratory, Bar Harbor, ME) were crossbred with COX2KD mice in which COX-2 expression was reduced by 70% to 90%, but not eliminated.24Seta F. Chung A.D. Turner P.V. Mewburn J.D. Yu Y. Funk C.D. Renal and cardiovascular characterization of COX-2 knockdown mice.Am J Physiol Regul Integr Comp Physiol. 2009; 296: R1751-R1760Crossref PubMed Scopus (43) Google Scholar Alzet osmotic minipumps (model 2004; Durect Corporation, Cupertino, CA), loaded with AngII (Sigma-Aldrich, St Louis, MO), were implanted s.c. into 3-month-old chow-fed male mice in the dorsal region under isoflurane anesthesia (delivered in 100% O2) to obtain a delivery rate of 1 μg/kg per minute during the course of 4 weeks, as previously described.25Cao R.Y. Adams M.A. Habenicht A.J. Funk C.D. Angiotensin II-induced abdominal aortic aneurysm occurs independently of the 5-lipoxygenase pathway in apolipoprotein E-deficient mice.Prostaglandins Other Lipid Mediat. 2007; 84: 34-42Crossref PubMed Scopus (30) Google Scholar The plane of anesthesia was monitored by lack of response to toe pinch and even respiration rate. Mice with comparable body weights were divided into three groups: group 1, control (apoE−/−); group 2, COX2KD on an apoE−/− background; and group 3, EP4 antagonist–treated apoE−/− (Table 1). The EP4 antagonist, ONO-AE3-208 (Ono Pharmaceutical Co, Ltd, Osaka, Japan), was administered via the drinking water at 10 mg/kg per day, starting 1 week before AngII infusion and for the complete 4-week duration of AngII infusion. Fresh water with drug was replaced at weekly intervals. The Animal Use Committee at Queen's University (Kingston, ON, Canada) approved the animal protocols described herein (Funk-2009-086), and experiments conform to the NIH guidelines.Table 1Mice in the Study GroupsGroupsGenotypesTreatmentBody weight (g)⁎Data are given as mean ± SEM.ControlapoE−/−AngII31.5 ± 0.5 (n = 31)COX2KDCOX2KD/apoE−/−AngII32.3 ± 0.7 (n = 27)AE3-208apoE−/−AngII + AE3-20831.0 ± 0.7 (n = 23)P > 0.05 for control versus either COX2KD or AE3-208. Data are given as mean ± SEM. Open table in a new tab P > 0.05 for control versus either COX2KD or AE3-208. Abdominal ultrasonographic imaging was performed under isoflurane anesthesia using a Vevo 770 high-resolution ultrasound system (VisualSonics, Toronto, ON) with a 40-MHz frequency real-time microvisualization scan head (RMV 704) and a 10 × 10-mm field of view in two-dimensional and three-dimensional (3D) modes. This method was used to monitor AAA progression at 1 and 2 weeks after AngII infusion initiation and just before euthanasia by CO2 asphyxiation at 4 weeks. The 3D data were further reconstructed into volume measurements using VisualSonics software version 3.0 (Vevo 770; Toronto, ON, Canada). After mice were euthanized by CO2 asphyxiation on day 28 of AngII infusion, blood was collected from the inferior vena cava for drug concentration and lipid profile analysis. Hearts were prepared for aortic root atherosclerotic lesion analysis, and aortic trees from the distal iliac bifurcation to the proximal aortic root were carefully dissected free from surrounding tissue. Abdominal aortas with aneurysms were measured at the greatest suprarenal diameter, whereas aortas without aneurysms were measured approximately 2 mm above the right renal artery, where most AAAs develop; they were measured with a micrometer and scored according to a previous classification.26Daugherty A. Manning M.W. Cassis L.A. Antagonism of AT2 receptors augments angiotensin II-induced abdominal aortic aneurysms and atherosclerosis.Br J Pharmacol. 2001; 134: 865-870Crossref PubMed Scopus (242) Google Scholar These specimens were embedded in optimal cutting temperature medium and stored at −80°C until sections were cut for further histological and IHC analysis. Some mice did not have the AAA diameter measured because of aortic rupture and other reasons (eg, a mouse died 2 days before end point because of unidentified reasons or nonrupture related). AAA sections were stained with Movat's pentachrome for morphological analysis. CD90.2, CD4, CD68, CD80, CD163, interferon-γ, IL-2, IL-17, and macrophage inflammatory protein (MIP)-1α expression levels were tested by immunofluorescence methods, as previously described.27Cao R.Y. Amand T. Ford M.D. Piomelli U. Funk C.D. The murine angiotensin II-induced abdominal aortic aneurysm model: rupture risk and inflammatory progression patterns.Front Pharmacol. 2010; 1: 1-9Google Scholar Briefly, AAA sections were fixed with acetone for 5 minutes, washed, blocked with 3% normal goat serum for 30 minutes and then incubated with primary antibodies against CD90.2 (BD Pharmingen, Mississauga, ON), CD163 (Santa Cruz Biotechnology, Santa Cruz, CA), CD68 and CD80 (Serotec, Oxford, UK), IL-17 (Epitomics, Burlingame, CA), and MIP-1α (Novus Biologicals, Littleton, CO) for 2 hours. After washing with PBS, specimens were incubated with appropriate fluorescently labeled secondary antibodies (Jackson ImmunoResearch, West Grove, PA) for 1 hour. Coverslips were mounted with VECTASHIELD plus DAPI (Vector, Burlingame, CA). Visualization was performed with a DM-IRB fluorescent microscope (Leica, Richmond Hill, ON). Quantification of positively stained cells was conducted with Image-Pro Plus software version 5.1 (Media Cybernetics, Silver Spring, MD), counting an equal number of defined fields on multiple slides between each of the three treatment groups/genotypes, summing the total positive cells, and generating a percentage (relative to DAPI-stained cells). Atherosclerotic lesions at the aortic root were quantified by counting the total lesion area stained by oil red O (Sigma-Aldrich). Aortic matrix metalloproteinase (MMP)-2 and MMP-9 activities were analyzed using gelatin zymography, as previously described.17Wang Y. Ait-Oufella H. Herbin O. Bonnin P. Ramkhelawon B. Taleb S. Huang J. Offenstadt G. Combadiere C. Renia L. Johnson J.L. Tharaux P.L. Tedgui A. Mallat Z. TGF-beta activity protects against inflammatory aortic aneurysm progression and complications in angiotensin II-infused mice.J Clin Invest. 2010; 120: 422-432Crossref PubMed Scopus (304) Google Scholar Briefly, aortic samples from control and AE3-208–treated mice were homogenized and equal amounts of protein (Bio-Rad protein assay; Bio-Rad, Mississauga, ON, Canada) were loaded into wells of a 10% SDS-polyacrylamide gel containing 0.1% gelatin and electrophoresed under nonreducing conditions. Samples from a mouse embryo fibroblast cell line (a generous gift from Dr. Alan Mak's laboratory, Department of Biomedical and Molecular Sciences, Queen's University) were loaded as positive control. After electrophoresis, the proteins were renatured by soaking the gel in renaturing buffer and then placed into developing buffer. Gels were stained with Coomassie Blue and then destained with acetic acid until areas of protease activity appeared as clear bands against a dark blue background, where proteases had digested the substrate. The plasma concentrations of drug were analyzed at study end point by liquid chromatography–tandem mass spectrometry with multiple reaction monitoring in electrospray ionization-positive ion mode using a Prominence_XR (Shimadzu, Kyoto, Japan) high performance liquid chromatography/API4000 (AB Sciex Tokyo, Japan) mass spectrometer with a Shim-pack XR-ODSII analytical column (2.0-mm internal diameter × 75 mm length of column; Shimadzu). The mobile phase consisted of solvent A (water containing 0.1% formic acid) and solvent B (acetonitrile containing 0.1% formic acid) starting at a 9:1 ratio, with a gradient over 1.5 minutes to a 1:9 ratio, and constant at this ratio until 3 minutes, with a flow rate of 0.5 mL/minute. Candesartan was used as an internal standard. Twenty-five AAA specimens from the infrarenal segment of the abdominal aorta in patients (17 males and 8 females; aged 57 to 84 years) undergoing surgery for AAA repair were obtained, along with three control nonaneurysm tissues (3 males; aged 43 to 48 years) removed from the infrarenal segment of the abdominal aorta postmortem (within 24 hours). Demographics are found in Supplemental Table S1 (available at http://ajp.amjpathol.org). All tissues were washed with one times PBS to remove blood, then snap frozen in liquid nitrogen and kept at −80°C. Tissues were obtained with approval from the Research Ethics Board of the Faculty of Health Sciences, Queen's University and Kingston General Hospital, and the investigation conforms to the principles outlined in the Declaration of Helsinki. Pieces of aortic tissues were fixed in 10% neutral-buffered formalin and embedded in paraffin. Sections (8 μm thick) were prepared, consisting of the entire thickness of the vessel wall and stained with modified Movat's pentachrome, and were subsequently mounted in Permount (Fisher Scientific, Fair Lawn, NJ) and coverslip protected. Additional pieces were resuspended in tissue protein extraction reagent (ThermoScientific, Rockford, IL) plus a protease inhibitor (Roche Diagnostics, Indianapolis, IN) mixture, followed by sonication on ice (three times, 5 seconds each). The sonicated homogenate was centrifuged at 13,000 × g for 5 minutes, and the supernatant fraction was assayed (Bradford method) to determine protein concentration. Proteins (30 μg) were subjected to 12% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Millipore Corporation, Billerica, MA) via a semidry transfer technique. The membranes were blocked overnight in 5% milk (suspended in Tris-buffered saline) at 4°C. Membranes were treated with an anti-human EP4 receptor polyclonal antibody (1:200 dilution; catalogue number sc20677; Santa Cruz Biotechnology, Santa Cruz, CA) or anti-β-actin monoclonal antibody (1:5000 dilution; Sigma-Aldrich Co, Oakville, ON) for 1 hour at room temperature, followed by washing and incubation with the respective secondary antibodies (1:2000 anti-rabbit and 1:10,000 anti-mouse) in 5% milk and Tris-buffered saline. Protein bands were visualized on a FluorChem 8900 instrument (Alpha Innotech, San Leandro, CA) after the application of a chemiluminescence detection reagent (GE Healthcare, Buckinghamshire, UK). ImageJ software version 1.45s (NIH, Bethesda, MA) was used to quantify the protein band intensities. The intensities of the protein of interest were normalized to β-actin to generate an expression ratio. Data are expressed as mean ± SEM. Differences between two groups were analyzed by the Student's t-test. Correlation was tested by linear regression and Pearson coefficient analyses. Differences of two proportions were analyzed by Fisher's exact test. The calculation of power for the detection of observed difference was performed using the formula of Rosner,28Rosner B. Fundamentals of Biostatistics.ed 7. Duxbury Press, Belmont, CA2011Google Scholar which was based on the normal approximation for the distribution of the test statistics. P < 0.05 was considered significant. This study was designed to test the impact of either genetic COX2KD or pharmacological inhibition of the PGE2 receptor EP4 on AAA pathogenesis using three groups of male apoE-deficient mice (Table 1). Abdominal aortic enlargements and aneurysm complications, such as aortic dissections and ruptures, were monitored by both ultrasonographic screening and end point dissection. Suspected abdominal aortic expansions >50% of the original aortic size were considered as aortic aneurysms. COX2KD mice had a lower AAA incidence compared with control mice [16 (59%) of 27 versus 24 (77%) of 31], which did not reach statistical significance (Figure 1A). AAA severity based on the classification of Daugherty et al23Daugherty 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 and aortic diameter (1.5 ± 0.15 versus 1.5 ± 0.13 mm) of COX2KD mice did not differ from control mice (Figure 1, B and C). On the other hand, administration of the PGE2 receptor EP4 antagonist ONO-AE3-208 via the drinking water resulted in significantly lower AAA incidence [6 (26%) of 23, P < 0.01], with no severe type III aneurysms, and a smaller average abdominal aortic diameter (1.1 ± 0.1 mm, P < 0.05) versus control mice. To ensure that the mice were receiving adequate drug via the drinking water, plasma concentrations of drug were evaluated. Drug-treated mice had measurable levels at the 28-day end point (average, 56 ± 20 ng/mL; n = 17), whereas all selected control and COX2KD mouse plasma samples were lower than the lowest limit of detection (<1 ng/mL, n = 10). Representative aortas at the gross dissection level revealed grossly remodeled vessel walls, frequently with thrombus formation, especially in the control and COX-2 genetic knockdown groups (Figure 1D). The development and progression of AAA were followed by two-dimensional B-mode high-resolution ultrasonographic imaging at three intervals (days 7, 14, and 28). We detected most of these AAAs (representative images are in Figure 2A), and the measured diameters at day 28 correlated well with those measured at dissection by micrometer measurements. Thoracic or ascending aortic arch aneurysms were missed by ultrasonographic screening because these areas were not scanned. Power Doppler measurements were performed to obtain geometric parameters of the AAA for reconstruction into aortic volumes. We found that the aortic volume measurements correlated nicely with the AAA severity score (Figure 2B, r2 = 0.69, P < 0.0001). Although aortic rupture occurred in close to one third of control animals [9 (29%) of 31], it was lower in the other two groups [4 (15%) of 27 COX2KD and 2 (9%) of 23 EP4 antagonist treated; Figure 3A ]. Based on power analysis, significantly more animals would have to be studied/treated to judge the genetic effects of COX2KD/EP4 antagonist efficacy on rupture rate because of the low incidence (see Discussion). We, however, made some qualitative assessments about the rupture rate. As we27Cao R.Y. Amand T. Ford M.D. Piomelli U. Funk C.D. The murine angiotensin II-induced abdominal aortic aneurysm model: rupture risk and inflammatory progression patterns.Front Pharmacol. 2010; 1: 1-9Google Scholar and others10Gitlin J.M. Trivedi D.B. Langenbach R. Loftin C.D. Genetic deficiency of cyclooxygenase-2 attenuates abdominal aortic aneurysm formation in mice.Cardiovasc Res. 2007; 73: 227-236Crossref PubMed Scopus (75) Google Scholar, 15Wang M. Lee E. Song W. Ricciotti E. Rader D.J. Lawson J.A. Pure E. FitzGerald G.A. Microsomal prostaglandin E synthase-1 deletion suppresses oxidative stress and angiotensin II-induced abdominal aortic aneurysm formation.Circulation. 2008; 117: 1302-1309Crossref PubMed Scopus (114) Google Scholar, 26Daugherty A. Manning M.W. Cassis L.A. Antagonism of AT2 receptors augments angiotensin II-induced abdominal aortic aneurysms and atherosclerosis.Br J Pharmacol. 2001; 134: 865-870Crossref PubMed Scopus (242) Google Scholar have previously noted, most of the aortic ruptures occurred within the first week of AngII infusion and they occurred at the suprarenal abdominal aorta region and also at the aortic arch (Figure 3, B and C). Previously, gene expression for the prostaglandin EP4 receptor subtype was confirmed by RT-PCR analysis in human AAA specimens.16Bayston T. Ramessur S. Reise J. Jones K.G. Powell J.T. Prostaglandin E2 receptors in abdominal aortic aneurysm and human aortic smooth muscle cells.J Vasc Surg. 2003; 38: 354-359Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar We collected 25 AAA specimens from patients undergoing elective/emergency surgical repair, along with three normal aorta sections from the same infrarenal segment (see Supplemental Table S1 at http://ajp.amjpathol.org), and examined protein expression for EP4 by using Western blot analysis. A histological assessment revealed the differences in vessel wall structure between the normal and AAA samples (Figure 4, A and B). The latter displayed disruptions of the elastic laminae, along with collagen deposition, vascularization, and inflammatory infiltrates, and the intimal layer was often obliterated and replaced by a fibrin-rich nonocclusive intraluminal thrombus. The adventitial layer revealed aneurysmal granulomas with follicle-like aggregates of inflammatory infiltrates in close proximity to vessels of the vasa vasorum. Aortic layers from the control samples were normal in structure. Western blot analysis revealed that a band consistent with EP4 expression (53 kDa) was detected in both AAA and non-AAA tissues (Figure 4C) but not in lipopolysaccharide-challenged leukocytes, in which EP4 receptor expression was suppressed29Ikegami R. Sugimoto Y. Segi E. Katsuyama M. Karahashi H. Amano F. Maruyama T. Yamane H. Tsuchiya S. Ichikawa A. The expression of prostaglandin E receptors EP2 and EP4 and their different regulation by lipopolysaccharide in C3H/HeN peritoneal macrophages.J Immunol. 2001; 166: 4689-4696PubMed Google Scholar and COX-2 was induced. From a qualitative assessment, differences in EP4 expression between AAA and non-AAA samples were not apparent and quantitative densitometry analysis between AAA (n =
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