Cystatin C Deficiency Promotes Inflammation in Angiotensin II–Induced Abdominal Aortic Aneurisms in Atherosclerotic Mice
2010; Elsevier BV; Volume: 177; Issue: 1 Linguagem: Inglês
10.2353/ajpath.2010.090381
ISSN1525-2191
AutoresStephanie Schulte, Jiusong Sun, Peter Libby, Lindsey A. MacFarlane, Chongxiu Sun, Marco López-Ilasaca, Guo‐Ping Shi, Galina K. Sukhova,
Tópico(s)Abdominal vascular conditions and treatments
ResumoAn imbalance between cysteinyl cathepsins and their principal endogenous inhibitor cystatin C (CystC) may favor proteolysis in the pathogenesis of human abdominal aortic aneurysms (AAA), yet a direct role of CystC in AAA remains unproven. This study used CystC and apolipoprotein E (ApoE) compound mutant (CystC−/−ApoE−/−) mice to examine directly the role of cysteine protease/protease inhibitor imbalance in AAA formation in angiotensin II–induced AAA. CystC-deficiency increased lumenal diameter and lesion size compared with control mice. CystC−/− ApoE−/− lesions also demonstrated enhanced inflammatory cell accumulation, more severe elastin fragmentation, and fewer smooth muscle cells in the tunica media. Macrophage content, measured as percent positive area (23.2 ± 1.4% versus 11.2 ± 1.4%; P = 0.0003) and number of the CD4+ T cells (ninefold; P = 0.048), increased significantly in CystC−/−ApoE−/− lesions. CystC deficiency increased cathepsin activity (5.5 fold; P = 0.001) in AAA, yielding greater elastin degradation and proangiogenic laminin-5 γ2 peptide production, which may account for increased microvascularization in CystC−/−ApoE−/− compared with ApoE−/− lesions. Increased leukocyte adhesion molecule VCAM-1 expression and leukocyte proliferation might also promote inflammation in CystC-deficient AAA. These data indicate that CystC contributes to experimental AAA pathogenesis and that enhanced cysteine protease activity, due to the lack of CystC, favors inflammation in AAA lesions induced in atherosclerotic mice by promoting microvascularization and smooth muscle cell apoptosis as well as leukocytes adhesion and proliferation. An imbalance between cysteinyl cathepsins and their principal endogenous inhibitor cystatin C (CystC) may favor proteolysis in the pathogenesis of human abdominal aortic aneurysms (AAA), yet a direct role of CystC in AAA remains unproven. This study used CystC and apolipoprotein E (ApoE) compound mutant (CystC−/−ApoE−/−) mice to examine directly the role of cysteine protease/protease inhibitor imbalance in AAA formation in angiotensin II–induced AAA. CystC-deficiency increased lumenal diameter and lesion size compared with control mice. CystC−/− ApoE−/− lesions also demonstrated enhanced inflammatory cell accumulation, more severe elastin fragmentation, and fewer smooth muscle cells in the tunica media. Macrophage content, measured as percent positive area (23.2 ± 1.4% versus 11.2 ± 1.4%; P = 0.0003) and number of the CD4+ T cells (ninefold; P = 0.048), increased significantly in CystC−/−ApoE−/− lesions. CystC deficiency increased cathepsin activity (5.5 fold; P = 0.001) in AAA, yielding greater elastin degradation and proangiogenic laminin-5 γ2 peptide production, which may account for increased microvascularization in CystC−/−ApoE−/− compared with ApoE−/− lesions. Increased leukocyte adhesion molecule VCAM-1 expression and leukocyte proliferation might also promote inflammation in CystC-deficient AAA. These data indicate that CystC contributes to experimental AAA pathogenesis and that enhanced cysteine protease activity, due to the lack of CystC, favors inflammation in AAA lesions induced in atherosclerotic mice by promoting microvascularization and smooth muscle cell apoptosis as well as leukocytes adhesion and proliferation. The incidence of abdominal aortic aneurysm (AAA) continues to rise, necessitating 46,000 operations and causing about 15,000 deaths annually in the United States.1Stanley JC Barnes RW Ernst CB Hertzer NR Mannick JA Moore WS Vascular surgery in the United States: workforce issues. 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Common morphological characteristics of AAA include chronic transmural inflammation characterized by abundant leukocyte infiltrates, degradation of medial elastin, depletion of medial smooth muscle cells (SMCs), and extensive neovascularization.4Anidjar S Dobrin PB Eichorst M Graham GP Chejfec G Correlation of inflammatory infiltrate with the enlargement of experimental aortic aneurysms.J Vasc Surg. 1992; 16: 139-147Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 5Shimizu K Mitchell RN Libby P Inflammation and cellular immune responses in abdominal aortic aneurysms.Arterioscler Thromb Vasc Biol. 2006; 26: 987-994Crossref PubMed Scopus (436) Google Scholar, 6Thompson RW Liao S Curci JA Vascular smooth muscle cell apoptosis in abdominal aortic aneurysms.Coron Artery Dis. 1997; 8: 623-631Crossref PubMed Scopus (123) Google Scholar, 7Henderson EL Geng YJ Sukhova GK Whittemore AD Knox J Libby P Death of smooth muscle cells and expression of mediators of apoptosis by T lymphocytes in human abdominal aortic aneurysms.Circulation. 1999; 99: 96-104Crossref PubMed Scopus (317) Google Scholar, 8Holmes DR Liao S Parks WC Thompson RW Medial neovascularization in abdominal aortic aneurysms: a histopathologic marker of aneurysmal degeneration with pathophysiologic implications.J Vasc Surg. 1995; 21 (discussion 771–762): 761-771Abstract Full Text PDF PubMed Scopus (106) Google Scholar Inflammatory cells including macrophages, lymphocytes, dendritic cells, neutrophils, and mast cells accumulate in AAA lesions in humans and mice and contribute prominently to AAA development.5Shimizu K Mitchell RN Libby P Inflammation and cellular immune responses in abdominal aortic aneurysms.Arterioscler Thromb Vasc Biol. 2006; 26: 987-994Crossref PubMed Scopus (436) Google Scholar, 9Sun J Sukhova GK Yang M Wolters PJ MacFarlane LA Libby P Sun C Zhang Y Liu J Ennis TL Knispel R Xiong W Thompson RW Baxter BT Shi GP Mast cells modulate the pathogenesis of elastase-induced abdominal aortic aneurysms in mice.J Clin Invest. 2007; 117: 3359-3368Crossref PubMed Scopus (178) Google Scholar Positive correlation between inflammatory infiltrates and aneurysmal enlargement,4Anidjar S Dobrin PB Eichorst M Graham GP Chejfec G Correlation of inflammatory infiltrate with the enlargement of experimental aortic aneurysms.J Vasc Surg. 1992; 16: 139-147Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 9Sun J Sukhova GK Yang M Wolters PJ MacFarlane LA Libby P Sun C Zhang Y Liu J Ennis TL Knispel R Xiong W Thompson RW Baxter BT Shi GP Mast cells modulate the pathogenesis of elastase-induced abdominal aortic aneurysms in mice.J Clin Invest. 2007; 117: 3359-3368Crossref PubMed Scopus (178) Google Scholar as well as regression of established AAA by limiting proinflammatory signaling in mice,10Aoki H Yoshimura K Matsuzaki M Turning back the clock: regression of abdominal aortic aneurysms via pharmacotherapy.J Mol Med. 2007; 85: 1077-1088Crossref PubMed Scopus (42) Google Scholar suggests an important role of inflammation in AAA pathogenesis. Loss of elastin relates closely to aneurysm dilatation.11Dobrin PB Baker WH Gley WC Elastolytic and collagenolytic studies of arteries. 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We have demonstrated overexpression of these elastolytic and collagenolytic enzymes in both human atherosclerotic and AAA lesions.16Sukhova GK Shi GP Simon DI Chapman HA Libby P Expression of the elastolytic cathepsins S and K in human atheroma and regulation of their production in smooth muscle cells.J Clin Invest. 1998; 102: 576-583Crossref PubMed Scopus (536) Google Scholar, 17Liu J Sukhova GK Yang JT Sun J Ma L Ren A Xu WH Fu H Dolganov GM Hu C Libby P Shi GP Cathepsin L expression and regulation in human abdominal aortic aneurysm, atherosclerosis, and vascular cells.Atherosclerosis. 2006; 184: 302-311Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar Cystatin C (CystC), the dominant endogenous inhibitor of cysteine proteases (CPs), inhibits cysteinyl cathepsins competitively.18Chapman Jr, HA Reilly Jr, JJ Yee R Grubb A Identification of cystatin C, a cysteine proteinase inhibitor, as a major secretory product of human alveolar macrophages in vitro.Am Rev Respir Dis. 1990; 141: 698-705Crossref PubMed Scopus (77) Google Scholar Inflammation diminishes CystC expression and/or secretion; for example, alveolar macrophages from people who smoke cigarettes, a prominent risk factor for AAA, release 10 to 50% less CystC in vitro than macrophages from nonsmokers.19Rodriguez RJ White RR Senior RM Levine EA Elastase release from human alveolar macrophages: comparison between smokers and nonsmokers.Science. 1977; 198: 313-314Crossref PubMed Scopus (72) Google Scholar We demonstrated that aneurysmal lesions have substantially decreased CystC levels compared with nondiseased arteries. Therefore, altered counterbalance between cysteinyl cathepsins and CystC may promote proteolysis within the arterial wall.20Shi GP Sukhova GK Grubb A Ducharme A Rhode LH Lee RT Ridker PM Libby P Chapman HA Cystatin C deficiency in human atherosclerosis and aortic aneurysms.J Clin Invest. 1999; 104: 1191-1197Crossref PubMed Scopus (390) Google Scholar, 21Sukhova G Shi G-P Libby P Role of elastolytic cathepsins in vascular remodeling.in: Matsuzawa YKT Nagai R Teramoto T Elsevier Science BV, Amsterdam, Kyoto, Japan2004: 498-501Google Scholar Consistent with our findings, evaluation of expression of different proteases from growing and ruptured human AAAs revealed a five-fold increase of cathepsins K and L, and a 30-fold increase in cathepsin S proteins, but 80% lower CystC protein levels, compared with nondiseased aortas.22Abdul-Hussien H Soekhoe RG Weber E von der Thusen JH Kleemann R Mulder A van Bockel JH Hanemaaijer R Lindeman JH Collagen degradation in the abdominal aneurysm: a conspiracy of matrix metalloproteinase and cysteine collagenases.Am J Pathol. 2007; 170: 809-817Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar Furthermore, low serum CystC levels in AAA patients and their inverse correlation with AAA progression and expansion highlight the importance of CPs and inhibitor balance for AAA pathogenesis.20Shi GP Sukhova GK Grubb A Ducharme A Rhode LH Lee RT Ridker PM Libby P Chapman HA Cystatin C deficiency in human atherosclerosis and aortic aneurysms.J Clin Invest. 1999; 104: 1191-1197Crossref PubMed Scopus (390) Google Scholar, 23Lindholt JS Erlandsen EJ Henneberg EW Cystatin C deficiency is associated with the progression of small abdominal aortic aneurysms.Br J Surg. 2001; 88: 1472-1475Crossref PubMed Scopus (88) Google Scholar, 24Sukhova GS Shi GP Do cathepsins play a role in AAA pathogenesis? Blackwell Publishing, New York2006: 161-169Google Scholar This study tested the hypothesis that cathepsin/cystatin imbalance regulates the formation of angiotensin II (Ang II)–induced AAA in mice.25Daugherty A Manning MW Cassis LA Angiotensin II promotes atherosclerotic lesions and aneurysms in apolipoprotein E-deficient mice.J Clin Invest. 2000; 105: 1605-1612Crossref PubMed Scopus (979) Google Scholar The results provide further support for dysregulated proteinase activity as a critical influence on arterial remodeling. ApoE−/− mice (C57BL/6, the Jackson Laboratories, Bar Harbor, ME) were initially crossbred with the CystC−/− mice (C57BL/6/129S)26Huh CG Hakansson K Nathanson CM Thorgeirsson UP Jonsson N Grubb A Abrahamson M Karlsson S Decreased metastatic spread in mice homozygous for a null allele of the cystatin C protease inhibitor gene.Mol Pathol. 1999; 52: 332-340Crossref PubMed Scopus (102) Google Scholar to generate CystC+/−ApoE+/− mice, which were used subsequently to generate three littermate-controlled experimental groups: CystC+/+ApoE−/−, CystC−/−ApoE−/−, and CystC−/−ApoE+/+ mice. Six-month-old male mice underwent subcutaneous implantation of an osmotic minipump (ALZET Scientific Products, Cupertino, CA, Model 1002) filled either with saline or an Ang II solution (500 ng/min/kg; Sigma, St. Louis, MO). After 28 days on a chow diet, systolic blood pressure was measured, animals were sacrificed, plasma collected, aortic diameter measured, and aortic lesions harvested for lesion characterization.27Daugherty A Manning MW Cassis LA Antagonism of AT2 receptors augments angiotensin II-induced abdominal aortic aneurysms and atherosclerosis.Br J Pharmacol. 2001; 134: 865-870Crossref PubMed Scopus (230) Google Scholar, 28Saraff K Babamusta F Cassis LA 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 (312) Google Scholar Apoptosis was evaluated in mice infused with Ang II (1000 ng/min/kg) for 5 days. All procedures were approved by the Animal Research Committee of Harvard Medical School. Serum total cholesterol, lipoprotein cholesterol, and triglyceride concentrations were determined using enzymatic assay kits according to the manufacturer (Pointe Scientific, Inc., Canton MI). We measured systolic blood pressure using a noninvasive tail cuff method and a PC-based data analyzer (BP 2000, Visitech Systems, Inc., Apex, NC). Three to four measurements were obtained from each nonanesthetized mouse (6 mice per group). This study used a previously published approach to quantification of experimental aneurysms.27Daugherty A Manning MW Cassis LA Antagonism of AT2 receptors augments angiotensin II-induced abdominal aortic aneurysms and atherosclerosis.Br J Pharmacol. 2001; 134: 865-870Crossref PubMed Scopus (230) Google Scholar, 28Saraff K Babamusta F Cassis LA 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 (312) Google Scholar We measured the percentage of incidence per group, evaluated lesion severity with grading keys,27Daugherty A Manning MW Cassis LA Antagonism of AT2 receptors augments angiotensin II-induced abdominal aortic aneurysms and atherosclerosis.Br J Pharmacol. 2001; 134: 865-870Crossref PubMed Scopus (230) Google Scholar measured maximal external AAA diameter under physiological blood pressure while mice were anesthetized. We used OCT-embedded aneurysmal tissue for immunohistochemical analysis and snap-frozen AAA segments for protein extraction. Saraff et al28Saraff K Babamusta F Cassis LA 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 (312) Google Scholar previously described the complex pathology of Ang II–induced mouse AAA lesions. Intramural hematoma develops in the dissection area, inducing leukocyte infiltration in the hematoma and tunica media between 14 and 28 days.28Saraff K Babamusta F Cassis LA 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 (312) Google Scholar Because the influence of CP activity on the inflammatory response during AAA development was a key interest, we used 28 days of Ang II infusion for AAA analysis. All AAA tissue segments for immunohistochemistry were obtained from the maximal external aneurysm diameter (Figure 1A, double-headed arrows)27Daugherty A Manning MW Cassis LA Antagonism of AT2 receptors augments angiotensin II-induced abdominal aortic aneurysms and atherosclerosis.Br J Pharmacol. 2001; 134: 865-870Crossref PubMed Scopus (230) Google Scholar at the level of medial elastica breaks. Ten slides per sample (three sections per slide) were numbered consecutively, and slides from identical levels were used for staining with each antibody. Serial cryostat cross sections (6 μm) were used for immunostaining for macrophages (Mac-3, 1:900), T cells (CD4, 1:90), endothelial cells (ECs) (CD31, 1:1500) (BD Pharmingen, Franklin Lakes, NJ), SMCs (α-actin, FITC-conjugated, 1:750, Sigma), apoptosis (TUNEL, Chemicon, Billerica, MA), proliferation (Ki-67, Novocastra Laboratories Ltd., Newcastle upon Tyne, UK), and laminin-5 γ2 (1:250).29Wang B Sun J Kitamoto S Yang M Grubb A Chapman HA Kalluri R Shi GP Cathepsin S controls angiogenesis and tumor growth via matrix-derived angiogenic factors.J Biol Chem. 2006; 281: 6020-6029Crossref PubMed Scopus (200) Google Scholar Data were analyzed as we described previously.9Sun J Sukhova GK Yang M Wolters PJ MacFarlane LA Libby P Sun C Zhang Y Liu J Ennis TL Knispel R Xiong W Thompson RW Baxter BT Shi GP Mast cells modulate the pathogenesis of elastase-induced abdominal aortic aneurysms in mice.J Clin Invest. 2007; 117: 3359-3368Crossref PubMed Scopus (178) Google Scholar We used incubation with appropriate IgG as a control of antibody specificity (not shown). The percentage of lesion area with positive color to total area for each section was recorded. Two observers blinded to the origin of the samples analyzed all data. For double fluorescent immunohistochemistry, we mixed mouse cell type–specific antibodies conjugated with fluorochrome: CD11b-FITC or CD3-PE with rabbit anti-Ki-67 (cell proliferation). Staining for proliferation used biotinylated anti-rabbit secondary antibody followed by streptavidin conjugated with Texas Red (for costaining with CD11b) or with FITC (for costaining with CD3). Data were presented as mean ± SE. Difference between groups was analyzed statistically by the nonparametric Mann–Whitney test. The degree of correlation between two variables was evaluated by linear regression analysis and by calculating the respective coefficient of determination r2. P values <0.05 were considered significant. To quantify cathepsin activity, we labeled aortic tissue extracts with biotinylated JPM, an active site-directed probe that irreversibly binds to active sites of cathepsins.9Sun J Sukhova GK Yang M Wolters PJ MacFarlane LA Libby P Sun C Zhang Y Liu J Ennis TL Knispel R Xiong W Thompson RW Baxter BT Shi GP Mast cells modulate the pathogenesis of elastase-induced abdominal aortic aneurysms in mice.J Clin Invest. 2007; 117: 3359-3368Crossref PubMed Scopus (178) Google Scholar, 16Sukhova GK Shi GP Simon DI Chapman HA Libby P Expression of the elastolytic cathepsins S and K in human atheroma and regulation of their production in smooth muscle cells.J Clin Invest. 1998; 102: 576-583Crossref PubMed Scopus (536) Google Scholar Microdissected AAA tissues were pulverized and lysed in a pH 5.5 buffer.16Sukhova GK Shi GP Simon DI Chapman HA Libby P Expression of the elastolytic cathepsins S and K in human atheroma and regulation of their production in smooth muscle cells.J Clin Invest. 1998; 102: 576-583Crossref PubMed Scopus (536) Google Scholar Protein extracts (2 μg/sample) were incubated with 1 μl of biotin-JPM (5 μmol/L) for 1 hour at 37°C, followed by separation on a 14% SDS-PAGE for immunoblot analysis with horseradish peroxidase-conjugated avidin (1:10,000, Sigma) and visualization using the ECL-detection kit (Amersham Pharmacia, Buckinghamshire, UK). Individual cysteinyl cathepsins were identified by their relative molecular weights. Densitometric analysis measured the relative amount of total cathepsin activities of each sample. Data were presented as fold increase in cathepsin activity compared with the ApoE−/− control aortae (mean ± SE, n = 3 per group). An aortic ring assay was used to test CPs function in endothelial tubule formation as previously described.9Sun J Sukhova GK Yang M Wolters PJ MacFarlane LA Libby P Sun C Zhang Y Liu J Ennis TL Knispel R Xiong W Thompson RW Baxter BT Shi GP Mast cells modulate the pathogenesis of elastase-induced abdominal aortic aneurysms in mice.J Clin Invest. 2007; 117: 3359-3368Crossref PubMed Scopus (178) Google Scholar We determined elastolytic activity in situ in 8-μM unfixed cryostat sections from aneurysmal lesions using elastin conjugated with quenched fluorescein, which requires cleavage by elastolytic enzymes to become fluorescent, as a substrate (DQ-elastin; Molecular Probes, Eugene, OR).9Sun J Sukhova GK Yang M Wolters PJ MacFarlane LA Libby P Sun C Zhang Y Liu J Ennis TL Knispel R Xiong W Thompson RW Baxter BT Shi GP Mast cells modulate the pathogenesis of elastase-induced abdominal aortic aneurysms in mice.J Clin Invest. 2007; 117: 3359-3368Crossref PubMed Scopus (178) Google Scholar Elastolytic activities were determined in two separate experiments with conditions optimized for CP or matrix metalloproteinase (MMP). CP relative input was determined using an EDTA-containing pH-5.5 buffer, with or without the nonselective cathepsin inhibitor E64d (20 μmol/L); MMP activity was determined in an E64d-containing pH-7.4 buffer, with or without the metalloenzyme inhibitor EDTA (20 mmol/L). Sections were examined under a fluorescent microscope with all images captured under the same settings and shutter conditions. The elastic laminae in the tunica media showed autofluorescence, this region was therefore excluded from analysis of elastolytic activity. SMCs were isolated from mouse aortas as described previously.30Sukhova GK Zhang Y Pan JH Wada Y Yamamoto T Naito M Kodama T Tsimikas S Witztum JL Lu ML Sakara Y Chin MT Libby P Shi GP Deficiency of cathepsin S reduces atherosclerosis in LDL receptor-deficient mice.J Clin Invest. 2003; 111: 897-906Crossref PubMed Scopus (297) Google Scholar We plated 1 × 104 SMC/cm2 onto an 8-chamber slide and incubated overnight with or without 0.06 mmol/L pyrrolidinedithiocarbamate (PDCT, Sigma).9Sun J Sukhova GK Yang M Wolters PJ MacFarlane LA Libby P Sun C Zhang Y Liu J Ennis TL Knispel R Xiong W Thompson RW Baxter BT Shi GP Mast cells modulate the pathogenesis of elastase-induced abdominal aortic aneurysms in mice.J Clin Invest. 2007; 117: 3359-3368Crossref PubMed Scopus (178) Google Scholar Staining for apoptosis (TUNEL) was performed according to the manufacturer’s recommendations (Intergen Discovery Products). Slides were mounted with DAPI-containing media (Vectashield, Vector, Laboratories, Burlingame, CA). AAA incidence and severity evaluated 28 days after Ang II infusion27Daugherty A Manning MW Cassis LA Antagonism of AT2 receptors augments angiotensin II-induced abdominal aortic aneurysms and atherosclerosis.Br J Pharmacol. 2001; 134: 865-870Crossref PubMed Scopus (230) Google Scholar, 28Saraff K Babamusta F Cassis LA 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 (312) Google Scholar did not differ significantly between groups. External AAA diameter, measured by a micrometer ocular at the areas of maximal aortic thickness under physiological blood pressure (Figure 1A, double-headed arrows),27Daugherty A Manning MW Cassis LA Antagonism of AT2 receptors augments angiotensin II-induced abdominal aortic aneurysms and atherosclerosis.Br J Pharmacol. 2001; 134: 865-870Crossref PubMed Scopus (230) Google Scholar was significantly larger in mice deficient in CystC (2.4 ± 0.2 versus 1.9 ± 0.2 mm, P < 0.04) compared with the control ApoE−/− group (Figure 1B). Further evaluation of cryostat cross-sections, taken from aortic segments with maximal external AAA diameters, could yield smaller (shrunk) luminal diameters due to the manipulations required for frozen tissue processing. However, CystC-deficient mice had larger lesion size (1.9 ± 0.3 versus 0.9 ± 0.2 mm2, P < 0.03) and aortic lumen diameter (1.2 ± 0.09 versus 0.8 ± 0.03 mm, P < 0.02) compared with the controls (Figure 1C). Ang II–infused CystC−/−ApoE+/+ mice and saline-infused CystC−/−ApoE−/− mice did not develop AAA (controls). Ang II can affect systolic blood pressure. However, we did not detect any systolic blood pressure differences between the mice with and without CystC expression 28 days after Ang II infusion (122 ± 9 in CystC−/−ApoE−/− versus 122 ± 18 mm Hg in control group, P = 0.8, n = 6 per group). Thus, increased AAA size in CystC−/−ApoE−/− mice did not result from altered blood pressure. Serum cholesterol levels correlate strongly with atherosclerosis. In mice with diet-induced atherosclerosis, absence of CystC increased serum lipid levels.31Sukhova GK Wang B Libby P Pan JH Zhang Y Grubb A Fang K Chapman HA Shi GP Cystatin C deficiency increases elastic lamina degradation and aortic dilatation in apolipoprotein E-null mice.Circ Res. 2005; 96: 368-375Crossref PubMed Scopus (128) Google Scholar However, CystC deficiency did not affect serum lipid levels among chow diet–fed Ang II–infused ApoE−/− mice (data not shown). These data correspond with the findings in humans that serum lipid levels do not correlate with AAA expansion.32Lindholt JS Heegaard NH Vammen S Fasting H Henneberg EW Heickendorff L Smoking, but not lipids, lipoprotein(a) and antibodies against oxidised LDL, is correlated to the expansion of abdominal aortic aneurysms.Eur J Vasc Endovasc Surg. 2001; 21: 51-56Abstract Full Text PDF PubMed Scopus (107) Google Scholar Leukocytes likely promote AAA formation by releasing cytokines, chemokines, proteases, and growth factors and mediating immune responses. Mice lacking these cells resist experimental AAA.9Sun J Sukhova GK Yang M Wolters PJ MacFarlane LA Libby P Sun C Zhang Y Liu J Ennis TL Knispel R Xiong W Thompson RW Baxter BT Shi GP Mast cells modulate the pathogenesis of elastase-induced abdominal aortic aneurysms in mice.J Clin Invest. 2007; 117: 3359-3368Crossref PubMed Scopus (178) Google Scholar, 33Xiong W Zhao Y Prall A Greiner TC Baxter BT Key roles of CD4+ T cells and IFN-gamma in the development of abdominal aortic aneurysms in a murine model.J Immunol. 2004; 172: 2607-2612PubMed Google Scholar, 34Eliason JL Hannawa KK Ailawadi G Sinha I Ford JW Deogracias MP Roelofs KJ Woodrum DT Ennis TL Henke PK Stanley JC Thompson RW Upchurch Jr, GR Neutrophil depletion inhibits experimental abdominal aortic aneurysm formation.Circulation. 2005; 112: 232-240Crossref PubMed Scopus (170) Google Scholar Immunostaining revealed much larger macrophage-positive areas in AAA lesions from CystC−/−ApoE−/− mice than in those from CystC+/+ApoE−/− mice (23.2 ± 1.4% versus 11.2 ± 1.4%; P < 0.0003) (Figure 2, A and B). CystC−/−ApoE−/− lesions also had ninefold more CD4+ T cells compared with CystC+/+ApoE−/− mice (343.4 ± 121.7 versus 39.1 ± 7.2 cells/mm2; P < 0.05) (Figure 2C). The majority of leukocytes localized in the periadventitial space but also in the hematoma and the tunica media, where leukocyte accumulation colocalized with elastica fragmentation and loss. Within AAA lesions, leukocytes probably furnish most of the elastolytic cathepsins35Hall A Ekiel I Mason RW Kasprzykowski F Grubb A Abrahamson M Structural basis for different inhibitory specificities of human cystatins C and D.Biochemistry. 1998; 37: 4071-4079Crossref PubMed Google Scholar that may facilitate fragmentation of the medial elastic laminae, a hallmark of human and mouse AAA. We tested the hypothesis that increased accumulation of leukocytes in CystC−/−ApoE−/− mice promotes elastin degradation and rupture. Elastin integrity was graded after Verhoeff–van Gieson staining as follows: grade 1, noticeable interruption in the medial elastic laminae without change of luminal shape; grade 2, small disruption in elastic laminae with bulbous extension of the aortic lumen; grade 3, prolonged disruption in the elastic laminae; and grade 4, ruptured medial elastica with extensive increase of the aortic lumen (Figure 3A). In agreement with our hypothesis, AAA lesions from CystC−/−ApoE−/− mice had significantly greater elastica loss than those from CystC+/+ApoE−/− mice (3.5 ± 0.3 versus 1.9 ± 0.4; P < 0.004) (Figure 3B). Higher grades of elastica rupture in CystC-deficient AAA lesions corresponded to greater length of these breaks on longitudinal sections (299.3 ± 21.3 versus 93 ± 16.5 μm; P < 0.005, n = 11 per group). Elastica degradation and disruption in the tunica media contribute directly to
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