Published online before print January 12, 2009, doi: 10.1161/CIRCULATIONAHA.108.785949
(Circulation. 2009;119:426-435.)
© 2009 American Heart Association, Inc.
Molecular Cardiology |
Interferon-
and the Interferon-Inducible Chemokine CXCL10 Protect Against Aneurysm Formation and Rupture
From the Cardiovascular Research Center (V.L.K., A.P.O., D.A.H., A.D.), Graduate Center for Nutritional Sciences (V.L.K., A.D.), and Graduate Center for Toxicology (A.P.O., A.D.), University of Kentucky, Lexington, Ky; Center for Immunology & Inflammatory Diseases (A.Y.L., F.K., T.J.T.A., N.A., G.J.H., D.S., A.M.T., A.D.L., R.E.G.) and Cardiovascular Research Center (A.Y.L., F.K., T.J.T.A., N.A., G.J.H., D.S., R.E.G.), Massachusetts General Hospital, Charlestown, Mass, and Harvard Medical School, Boston, Mass.
Correspondence to Robert E. Gerszten, MD, Cardiology Division and Center for Immunology & Inflammatory Diseases, Massachusetts General Hospital–East Campus, 149 13th St, Room 8307, Charlestown, MA 02129. E-mail rgerszten{at}partners.org
Received April 23, 2008; accepted October 27, 2008.
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Abstract |
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Background— Vascular disease can manifest as stenotic plaques or ectatic aneurysms, although the mechanisms culminating in these divergent disease manifestations remain poorly understood. T-helper type 1 cytokines, including interferon-
and CXCL10, have been strongly implicated in atherosclerotic plaque development.
Methods and Results— Here, we specifically examined their role in the formation of abdominal aortic aneurysms in the angiotensin II–induced murine model. Unexpectedly, we found increased suprarenal aortic diameters, abdominal aortic aneurysm incidence, and aneurysmal death in apolipoprotein E– and interferon-–deficient (Apoe–/–/Ifng–/–) mice compared with Apoe–/– controls, although atherosclerotic luminal plaque formation was attenuated. The interferon-–inducible T-cell chemoattractant CXCL10 was highly induced by angiotensin II infusion in Apoe–/– mice, but this induction was markedly attenuated in Apoe–/–/Ifng–/– mice. Apoe–/–/Cxcl10–/– mice had decreased luminal plaque but also increased aortic size, worse morphological grades of aneurysms, and a higher incidence of death due to aortic rupture than Apoe–/– controls. Furthermore, abdominal aortic aneurysms in Apoe–/–/Cxcl10–/– mice were enriched for non–T-helper type 1–related signals, including transforming growth factor-β1. Treatment of Apoe–/–/Cxcl10–/– mice with anti-transforming growth factor-β neutralizing antibody diminished angiotensin II–induced aortic dilation.
Conclusions— The present study defines a novel pathway in which interferon- and its effector, CXCL10, contribute to divergent pathways in abdominal aortic aneurysm versus plaque formation, inhibiting the former pathology but promoting the latter. Thus, efforts to develop antiinflammatory strategies for atherosclerosis must carefully consider potential effects on all manifestations of vascular disease.
Key Words: aneurysm • atherosclerosis • immunology • inflammation • mice, knockout
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Introduction |
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The pathophysiological mechanisms that lead to stenotic plaques versus aneurysms, 2 distinct vascular lesions, remain poorly understood. Clinically, abdominal aortic aneurysms (AAAs) are more strongly correlated with a family history1 and smoking2 than are coronary stenoses. Diabetes mellitus, a strong risk factor for coronary plaques, actually protects against AAA formation in population-based studies.3,4
Clinical Perspective p 435
Chronic inflammation of the vascular wall is believed to contribute to both manifestations of arterial pathology.5,6 Atherosclerotic plaques are marked primarily by intimal infiltration of macrophages and T cells, at least at earlier disease stages. In contrast, aneurysmal segments are characterized by macrophage, T-cell, and B-cell accumulation primarily in the media and adventitia at all stages of disease evolution.6 Human atherosclerotic stenoses specifically express mediators characteristic of a T-helper type 1 (Th1) immune response, including interferon (IFN)-, as well as the IFN-–inducible T-cell chemoattractant IP-10 (IFN--inducible Protein of 10 kDa, now known as CXCL10).7 Characterization of mediators expressed by AAAs, however, has been inconsistent, which is potentially attributable to the different disease stages and anatomic areas studied. Tang et al8 found transmural accumulation of IFN-–producing T cells correlated with aortic dilation, although other groups have described a Th2-predominant immune response prevailing in human AAA.6,9
CD4+ T-cell deletion protects against AAA formation in a calcium chloride–induced AAA model in mice10; however, murine studies to date have not clarified whether adaptive cellular immunity of either the Th1 or Th2 system is detrimental or beneficial in aneurysmal disease. Although the Th1 cytokine IFN- contributes to atherosclerotic plaque formation,11 there are conflicting reports on the role of IFN- and its receptor in the development of AAAs. IFN- deficiency resulted in a modest reduction of disease pathology in the calcium chloride–induced AAA model, whereas IFN- infusion restored the severity of the disease.10 In contrast, IFN- receptor deficiency augmented AAA formation in an aortic allograft model of AAA formation.12 These seemingly contradictory studies may be less surprising in light of an emerging theme of proinflammatory and regulatory interplay of IFN- in inflammation and autoimmunity in other disease models, including arthritis13 and multiple sclerosis.14,15 IFN- appears to act as a master upstream regulator that modulates both proinflammatory and antiinflammatory processes depending on the disease stage and disease-specific cytokines.
Complete ablation of IFN- signaling, like global T-cell deletion, might thus disturb both the effector and regulatory arms of the immune system, potentially resulting in variable effects on vascular phenotypes. In contrast, a disruption that isolates specific downstream pathways might be particularly informative about the signals that contribute to AAAs. We therefore studied AAA formation both in IFN-–deficient mice and in mice deficient in the IFN-–inducible T-cell chemokine, CXCL10. These studies define a novel role for CXCL10 in AAA formation and more broadly suggest that cellular immunity may play different roles in 2 distinct manifestations of vascular disease, with important clinical implications.
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Methods |
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Mice
Apolipoprotein E–deficient (Apoe–/–) mice, interferon-
–deficient (Ifng–/–) mice (Jackson Laboratory, Bar Harbor, Me), and Cxcl10–/– mice16 were backcrossed 10 times into a C57BL/6J background and interbred to generate the experimental genotypes, which were confirmed by polymerase chain reaction (PCR) genotyping. All mice received a standard laboratory diet (Harlan Teklad, Harlan Laboratories, Indianapolis, Ind). All animal procedures were approved by university animal care protocols at their respective institutions (University of Kentucky and Massachusetts General Hospital).
Infusion of Angiotensin II
Experimental mice were treated with angiotensin (Ang) II (500 or 1000 ng · kg–1 · min–1 as indicated; Sigma Chemical Co, St Louis, Mo) or normal saline via ALZET model 2004 osmotic pumps (DURECT Corp, Cupertino, Calif) that were implanted subcutaneously as described previously.17 After 4 weeks of infusion, mice were euthanized for blood collection and aorta harvest.
Lipid Analysis
A total of 0.5 to 1.0 mL of blood was aspirated from experimental mice by right ventricular puncture on euthanasia. Serum cholesterol concentrations were measured by enzymatic colorimetric assay (Wako Chemical Co, Richmond, Va). Lipoprotein cholesterol distribution was determined in individual serum samples (50 µL) from mice after resolution on a Superose 6 column (GE Healthcare Biosciences, Piscataway, NJ).18
Blood Pressure Measurements
Systolic blood pressure was measured serially in conscious mice by use of a tail-cuff system (Visitech BP-2000, Visitech Systems Inc, Apex, NC, or Kent Scientific XBP1000, Torrington, Ct) during 3 training sessions at baseline and 4 weeks after placement of the Ang II pump.
Atherosclerotic Lesion Analysis
The size of atherosclerotic lesions was quantified with Image-Pro software (Media Cybernetics, Bethesda, Md).19 Discernable lesions on the luminal surface of the aorta were quantified from the aortic arch to the last intercostal artery branch in the thorax.
Quantification of Aneurysms and Morphometric Analysis
Aortic diameters and AAA incidence were determined as described previously.17 The maximum width of abdominal aortas was measured with computerized morphometry. Aneurysm incidence was quantified on the basis of a definition of an external suprarenal aorta width that was increased by 50% or more compared with saline-infused mice. In addition, we used a previously described classification system20,21 to categorize the morphological grade of the aneurysms: no aneurysm, type I (suprarenal dilation without thrombus), type II (remodeled suprarenal dilation with thrombus), type III (multiple aneurysms, including thoracic aneurysms and dissections), and death due to aneurysmal rupture. On necropsies of unexpected deaths, death due to rupture of an aneurysm was qualified by the presence of a retroperitoneal hematoma in addition to an AAA and/or the presence of a thoracic hematoma in addition to a thoracic aortic aneurysm or dissection. Measurements were conducted by 2 trained, independent observers blinded to genotype and treatment conditions.
Antibodies and Immunohistochemistry of Murine Lesions
For harvesting suprarenal aortas for immunohistochemistry, mice were perfused via left ventricular puncture with 4% paraformaldehyde under physiological pressure, and aortic segments were embedded in optimal cutting temperature compound (Tissue-Tek, Torrance, Calif). Serial 10-µm sections were cut surrounding the cross section of widest diameter, and every fifth section was stained. Tissues were stained with hematoxylin and eosin (Fisher Scientific, Hampton, NH) for morphology. Immunohistochemistry was performed with antibodies to identify macrophages (Mac3, 1:20, BD Biosciences, San Jose, Calif), CD4+ T cells (CD4, 1:50, BD Pharmingen, San Jose, Calif), Thy-1.2+ T cells (CD90.2; BD Pharmingen), and vascular smooth muscle cells (
-actin, 1:100, Abcam, Cambridge, Mass), as well as the chemokine CXCL10 (1:100, R&D Systems, Minneapolis, Minn). Negative controls were prepared with substitution of the primary antibody with an isotype-matched control antibody. Appropriate biotinylated secondary antibodies were used, followed by detection with an ABC Development Kit (Vector Laboratories, Burlingame, Calif) and color development with DAB (Chemicon, Temecula, Calif) or AEC (Dako, Glostrup, Denmark). High-powered fields of stained sections were captured randomly with a SPOT digitizer (Diagnostic Instruments, Sterling Heights, Mich), and quantitative analysis was performed with IPLab software (BD Biosciences, Rockville, Md) by a single observer blinded to genotype and condition. Areas that were positively stained were divided by total lesion area to account for variability in lesion size.
To assess transforming growth factor (TGF)-β activity, we used a polyclonal antibody specific for the free and active form of TGF-β.22,23 To test the role of TGF-β in AAA formation in Apoe–/– and Apoe–/–/Cxcl10–/– mice, mice were injected intraperitoneally 1 day before placement of the Ang II pump and 1 day after pump placement with a pan-specific neutralizing antibody against TGF-β1, -β2, and -β3 (1 mg/kg; R&D Systems)24 or isotype control.
RNA Isolation and Quantitative PCR
Total RNA was isolated from suprarenal aortas from mice perfused with buffer RLT (Applied Biosystems, Foster City, Calif) by mechanical homogenization with a roto-stator and RNeasy columns (Qiagen, Valencia, Calif).25,26 After DNaseI digestion, equivalent amounts of RNA from each sample were reverse transcribed with TaqMan reverse-transcription reagents, including oligo (dT)15, random hexamers, and MultiScribe reverse transcriptase (Applied Biosystems). Quantitative reverse-transcription PCRs were conducted with the Multiplex qPCR system as described previously.25,26 Amplification plots were analyzed with MX4000 software, version 3.0. Gene expression was normalized to GAPDH or β-actin as an internal control.
Vascular Contractility Experiments
Abdominal aortas were removed and cleaned of adventitia while immersed in Krebs buffer. Measurement of force contraction was performed at 37°C as described previously.27 Briefly, abdominal aortic segments (3 mm) were mounted by passing 2 tungsten wires through the arterial lumen and then were bathed in wells (Kent Scientific) filled with Krebs-Henseleit solution. Tension (1 g) was maintained continuously and recorded with a Tissue Force Analyzer 410 (Micro-Med Inc, Louisville, Ky). After 30 minutes of equilibration, abdominal aortic segments were immersed in potassium chloride (80 mmol/L) for 3 minutes. After washout, contractile activity was determined during incubation with phenylephrine (Sigma; 1 nmol/L to 1 mmol/L).
Statistical Analysis
Data were analyzed by 2-way ANOVA, Student t test, 
2, or Mann–Whitney rank sum with SigmaStat software (Aspire Software International, Ashburn, Va). Data were tested by use of parametric or nonparametric post hoc analysis, and multiple comparisons were performed with Tukey or Holm-Sidak tests, as appropriate. Percentage incidence of AAAs was analyzed by Fisher exact test. P<0.05 was considered statistically significant. All data are shown as mean±SEM.
The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.
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Results |
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Increased IFN-
and CXCL10 mRNA Expression in Ang II–Infused Apoe–/– MiceMice were placed on a normal diet for 20 weeks and then treated with Ang II (1000 ng · kg–1 · min–1) or vehicle control via ALZET osmotic pumps for 4 weeks, which generates AAA in a reproducible manner and mimics many of the important features of the human disease.17,28 Ang II infusion did not generate any AAAs in control C57BL/6J mice (data not shown). Quantitative real-time PCR analysis of suprarenal aortic segments revealed significantly higher expression of the Th1-associated cytokines IFN-
and CXCL10 (as well as its receptor, CXCR3) in the Ang II–exposed Apoe–/– mice than in the saline-treated controls (Figure 1). C57BL/6J mice had similar trends for CXCL10 and CXCR3, but overall expression levels were markedly lower.
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6 per condition; *P<0.05, **P<0.01).
IFN- Deficiency Has Contrasting Effects on Ang II–Induced AAA and Atherosclerosis Formation
To address the role of IFN- in AAA formation, we infused 20-week-old Apoe–/– mice that were either Ifng+/+ or Ifng–/– with Ang II (1000 ng · kg–1 · min–1) or saline. Unexpectedly, 50% of the Apoe–/–/Ifng–/– mice died due to rupture of the abdominal aorta within 2 to 10 days of Ang II infusion (Figure 2; online-only Data Supplement Figure I). In contrast, no deaths due to aneurysmal rupture occurred in the Apoe–/–/Ifng+/+ group. Because of the high incidence of mortality in Apoe–/–/Ifng–/– mice, the infusion of Ang II was decreased to 500 ng · kg–1 · min–1 for subsequent studies of this genotype.
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Total serum cholesterol concentrations were not altered by IFN- deficiency in either saline- or Ang II–infused mice (Table 1). IFN- deficiency did not alter systolic blood pressure before or during infusion of Ang II (data not shown). IFN- deficiency led to increased body weight in the Apoe–/– background, although body weight was unaffected by Ang II infusion (Table 1). Adiponectin levels were not different between Apoe–/–/Ifng–/– mice and Apoe–/– mice (n=9 of each; P=0.67), thus excluding one potentially confounding modifier of vascular pathology.29
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With the lower-dose infusion of Ang II (500 ng · kg–1 · min–1), we observed significantly increased suprarenal aortic diameters in Apoe–/–/Ifng–/– mice compared with Apoe–/– controls (Figure 3A; P<0.05). IFN-–deficient mice also had a concordant increase in the incidence of AAAs compared with Ifng+/+ controls (18 [62%] of 29 Apoe–/–/Ifng–/– mice versus 8 [32%] of 25 Apoe–/– mice, P<0.05; Figure 3B). No AAAs were present in saline-infused control mice. Of note, IFN- deficiency did not produce any discernible differences in medial area or thickness of the suprarenal aorta (online-only Data Supplement Figures IIA and IIB), which rules out preexisting vascular differences between these genotypes that might predispose Apoe–/–/Ifng–/– mice to AAA. Furthermore, in functional assays, IFN- deficiency did not impart any significant differences in the ability of the abdominal aorta to respond to KCl or phenylephrine (online-only Data Supplement Table I). Thus, despite IFN- deficiency leading to dramatic differences in aneurysm formation in the suprarenal aorta, this was not associated with discernible structural or functional changes in the vessel wall.
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Luminal atherosclerotic lesions were also quantified in mice infused with Ang II or saline. Minimal lesion development was noted in saline-infused mice fed a normal diet (Figure 4). Consistent with previous reports, Ang II infusion markedly enhanced atherosclerotic lesion size (Figure 4)17,30; however, as in hyperlipidemia-induced atherosclerosis, Ang II–induced lesion formation was attenuated by IFN- deficiency (P<0.05). Thus, IFN- deficiency had a differential effect on atherosclerotic lesion formation as opposed to AAA formation.
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CXCL10 Deficiency Has Contrasting Effects on Ang II–Induced AAA and Atherosclerosis Formation
CXCL10 is an IFN-–inducible effector T-cell chemokine that was highly upregulated by Ang II infusion in Apoe–/– mice (Figure 1). CXCL10 expression appeared highest in the media and adventitia, which suggests that its role in the recruitment of T cells likely occurred from the adventitia and neovessels and not from the aortic lumen. Consistent with our prior studies,31 we observed downregulation of CXCL10 in spleens and vascular lesions of hyperlipidemic, IFN-–deficient mice (Figures 5A and 5B). Like Apoe–/–/Ifng–/– mice,32 CXCL10-deficient mice in the ApoE background on a high-fat diet were also recently found to have a >2-fold reduction in atherosclerotic plaques compared with controls.26 We thus explored the role of CXCL10 in atherosclerotic plaque development and AAA formation in the Ang II model. Twenty-week-old Apoe–/– and Apoe–/–/Cxcl10–/– mice received Ang II (1000 ng · kg–1 · min–1) or saline for 28 days. After euthanasia, Apoe–/– and Apoe–/–/Cxcl10–/– mice had similar lipid profiles (Table 2). The Ang II–infused Apoe–/–/Cxcl10–/– mice weighed slightly more than their age-matched Apoe–/– controls (Table 2) but showed no differences in adiponectin levels (n=7 of each, P=0.21).
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CXCL10 deletion protected against atherosclerotic luminal plaque formation in the Ang II model (Figure 5C), consistent with prior results with a high-fat diet.26 Although luminal plaque formation was diminished, Apoe–/–/Cxcl10–/– mice had a significantly higher death rate due to aortic rupture than the Apoe–/– controls in the Ang II–triggered AAA model (42% versus 11%, P<0.01; Figure 6A; representative necropsy sample in online-only Data Supplement Figure III). We were interested to find that the rate of rupture in Apoe–/–/Cxcl10–/– mice was comparable to that observed in Apoe–/–/Ifng–/– mice. Concordant with the increased mortality observed in Apoe–/–/Cxcl10–/– double knockouts, we also documented more severe morphological changes throughout the aortas of these mice than in Apoe–/– controls (Figure 6B). Whereas Apoe–/– controls had localized suprarenal AAAs, the Apoe–/–/Cxcl10–/– mice had thoracic aneurysms and hematomas with and without abdominal aneurysms, as well as large aneurysms with spiral dissections. Consistently, the infrarenal aorta had no aneurysmal pathology in either genotype (although occasionally, the aortic segment between the renal arteries was involved in conjunction with a suprarenal AAA), which is in agreement with previous reports.34 We quantified these morphological differences using a previously reported classification grade that accounts for the complexity and multiplicity of the aneurysms.20,21 The distribution of grades was different between the 2 genotypes (Figure 6C; P<0.005), with the Apoe–/–/Cxcl10–/– mice having significantly more grade III aneurysms or death due to rupture of the aorta. Similar to the findings in IFN-–deficient mice, we also observed significantly increased suprarenal diameters and suprarenal/thoracic-to-infrarenal aortic area ratios in Apoe–/–/Cxcl10–/– mice compared with Apoe–/– controls (Figure 6D and 6E). Of note, these analyses may underestimate differences between the 2 genotypes, because mortality was substantially increased in Apoe–/–/Cxcl10–/– mice, and therefore, some aneurysms were not incorporated into the analyses represented by Figure 6D and 6E.
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Taken together, the Apoe–/–/Cxcl10–/– mice had qualitatively and quantitatively worse aneurysmal disease, with elements of remodeling, dilation, and rupture, which involved more of the aorta than previously reported with this model. Thus, deficiency of either IFN- or the IFN-–inducible chemokine, CXCL10, yielded exacerbation of AAA pathology, despite there being diminished plaque formation in the absence of either of these cytokines.
Reduction of T-Cell Accumulation in CXCL10-Deficient Aneurysms
We next performed immunohistochemical studies of aortic vessel-wall constituents to further delineate the effects of CXCL10 deletion on AAA formation. As previously reported, cross-sectional histology demonstrated lumen dilation, breaks in medial elastin, and thrombus formation.28 Consistent with deletion of the effector T-lymphocyte chemokine, CXCL10, we documented a significant decrease in CD4+ T-lymphocyte accumulation in the suprarenal AAAs of Apoe–/–/Cxcl10–/– mice compared with Apoe–/– controls, as assessed both by quantitative PCR and by immunohistochemical analysis (Figure 7A through 7C). A concordant reduction was found in mRNA for the CXCL10 receptor, CXCR3, consistent with decreased infiltration of effector T cells (P=0.004; Figure 7D).7,35 Also consistent with the decreased accumulation of activated T lymphocytes in particular, we observed a concomitant reduction in IFN- production within the vessel wall, as assessed by quantitative real-time PCR (Figure 7E). Interestingly, a significant decrease was also found in macrophage accumulation in the arterial wall of the Apoe–/–/Cxcl10–/– double-knockout mice compared with Apoe–/– controls (data not shown). Because CD4 is present at very low levels in macrophages, we also performed immunostaining with an anti-Thy-1.2 antibody, which confirmed the reduction in T cells in the lesions (online-only Data Supplement Figure IV).
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TGF-β1 Blockade Inhibits AAA Size in CXCL10-Deficient Mice
We hypothesized that in the absence of IFN- and CXCL10, the lesional cytokine milieu would be enriched for non–Th1-related signals, such as TGF-β1. Recent studies have also demonstrated that TGF-β1 activation appears to potentiate aortic root aneurysm formation in murine models of Marfan syndrome.24 In this context, we found that Apoe–/–/Cxcl10–/– aneurysmal sections contained significantly greater amounts of activated TGF-β, as assessed by immunohistochemical analysis with an activation-specific TGF-β antibody36 (Figure 8A and 8B). Furthermore, inhibition of TGF-β activity with a neutralizing antibody24 significantly diminished aortic area in CXCL10-deficient mice treated with Ang II for 2 weeks (Figure 8C).
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Discussion |
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Here, we specifically explored the roles of IFN-
and CXCL10 in the formation of AAAs. Although Ang II–induced atherosclerotic lesion formation was attenuated in IFN-–deficient mice, an unexpected increase was found in suprarenal aortic diameter and AAA incidence. The IFN-–inducible effector T-cell chemokine CXCL10, which is highly upregulated by Ang II infusion in Apoe–/– mice and downregulated in the setting of IFN- deficiency, also conferred protection from AAA formation. Compared with Apoe–/– control mice, compound-deficient Apoe–/–/Cxcl10–/– mice had increased aortic size, worse morphological grades of aneurysms, and a higher incidence of death due to aortic rupture. The aortas of Apoe–/–/Cxcl10–/– mice were characterized by downregulation of IFN- and upregulation of the proaneurysmal growth factor TGF-β1. Furthermore, inhibition of TGF-β with a neutralizing antibody diminished aortic area in the Ang II–treated Apoe–/–/Cxcl10–/– mice.
Although clinical evidence suggests that coronary atherosclerosis and AAA formation share some common features, important differences exist, such as the discordant effect of diabetes on the prevalence of these 2 disease manifestations. A prominent inflammatory component is common to both vascular pathologies, although histological analyses also show differences. Coronary atherosclerosis is marked specifically by the accumulation of a Th1-type immune response.7 In contrast, studies of AAA have found evidence for both Th1- and Th2-type responses.8,12 Prior studies have hinted at molecular signals that differ in aneurysmal versus stenotic vascular disease. For example, genetic deficiency of matrix metalloproteinase-3, tissue inhibitor of metalloproteinase-1, or 5-lipoxygenase and pharmacological inhibition of matrix metalloproteinases in hyperlipidemic mice have had variable effects on atherosclerosis compared with AAA formation.37–40 The present study extends prior work by defining a novel pathway in which IFN- and its effector, CXCL10, lead to discordant effects in the 2 vascular disease processes. Thus, although multiple lines of investigation have shown that Th1 cells and their related proinflammatory cytokines promote atherogenesis, we show that these same cytokines protect against the formation of AAAs in a well-characterized murine system.
An extensive literature documents the function of IFN- in potentiating the inflammatory response; however, more recent evidence suggests that IFN- also plays a role in the resolution of inflammatory processes. Data supporting the complex role of IFN- have come both from antibody-blocking experiments and from attempts to induce autoimmune inflammation in IFN- and IFN--receptor knockout mice. For example, increased disease severity was documented in mouse models of multiple sclerosis and collagen-induced arthritis with animals deficient for IFN- or the IFN- receptor.13–15 The present data are therefore consistent with the emerging notion of IFN- as a master regulator, upstream of multiple pathways that evolve during the disease process. With regard specifically to vascular biology, the contrasting effects of IFN- deficiency on AAA and stenotic vascular disease may be attributed to localization of these diseases to different layers of the aorta. Additionally, differences in the temporal accumulation of different cell types and the presence of different cytokines and growth factors might account for the opposing effect of IFN- deficiency on atherosclerosis and aneurysms.
We observed significantly increased levels of TGF-β1 in the Ang II–induced AAAs of Apoe–/–/Cxcl10–/– double-knockout mice compared with Apoe–/– controls. This finding is consistent with our prior studies in which we found enhanced expression of non-Th1 cytokines, including TGF-β1, in the diet-induced atherosclerotic plaques of CXCL10-deficient mice, despite overall diminished T-cell accumulation.33 We were particularly interested to find increases in TGF-β1 activation in Ang II–infused Apoe–/–/Cxcl10–/– double-knockout mice in light of recent studies demonstrating the role of TGF-β1 in aortic root aneurysm formation in murine models of Marfan syndrome.24 Investigators now postulate that fibrillin-1 tonically inhibits TGF-β1 signaling in the vessel wall. In Marfan syndrome, mutations in fibrillin-1 lead to enhanced TGF-β1 activation and ultimately vascular dilation. Furthermore, aneurysm formation in mice expressing a fibrillin mutation characteristic of human Marfan syndrome is inhibited by a TGF-β–blocking antibody or by the Ang II type 1 receptor blocker losartan,24 although there are likely to be important differences in pathways triggered in the hyperlipidemia and Ang II–induced aneurysm model as opposed to the genetically induced Marfan model. Our working model is that CXCL10, an IFN-–dependent chemokine, modulates the recruitment of effector T cells. The recruitment of T cells influences the local T-cell cytokine profile in the vessel wall, including the expression of additional IFN-. We have demonstrated that when CXCL10 is deleted, the recruitment of effector T cells is diminished, and the local cytokine milieu shifts away from a Th1 profile, which leads to an enrichment of signals including TGF-β1. Growth factors such as TGF-β1 activate fibroblasts and other cell types and elicit further TGF-β1 and cytokine generation,41 which may serve to amplify the initial changes in the cytokine profile. In atherosclerosis, shifting the balance away from Th1 cytokines with upregulation of TGF-β1 inhibits luminal plaque formation; however, TGF-β1 induction has been demonstrated to be a critical mechanism in aneurysmal dilation. Characterization of the downstream targets of TGF-β1 responsible for these discordant effects in the vasculature merits future investigation. Of note, precedent exists for TGF-β1 activity having dramatically different effects on specific aspects of disease pathology, such as mitigating inflammation but contributing to dysregulated tissue repair.42
Several findings of the present study potentially contrast with previous work. Although studies defining the effects of IFN- on atherosclerosis have been uniform, there are conflicting reports of the role of IFN- in aneurysm-associated disease models. Blockade of IFN- signaling with IFN- receptor–deficient mice increased AAA formation in an aortic allograft model of the disease,12 which is in agreement with the present study. However, investigators have demonstrated that increases in the abdominal aortic diameter of C57BL/6J mice triggered by intraperitoneal administration of calcium chloride were attenuated by IFN- deficiency.10 One other study has found that adenovirally mediated overexpression of TGF-β1 attenuated aortic dilation of SDS-treated guinea pig xenografts transplanted into Lewis rats.43 Finally, a recent report suggests that CXCR3 (the receptor for CXCL10) deficiency has no significant effect on calcium chloride–triggered aortic dilation.44 We would postulate that the marked differences between our model and prior systems account for the contrasting findings. The present studies used an Ang II–treated hyperlipidemic murine model on the C57BL/6J background, which differs significantly from the prior studies, which used either surgical intervention to apply calcium chloride to the external surface of the vessel or aortic transplantation to trigger AAA formation. We note that although the present data highlight a role for IFN- and effector T cells in AAA formation, recent findings have also suggested a role for other types of IFN-–producing cells, such as mast cells, in AAA generation.45
Limitations to the present study must also be considered. Debate about the fidelity of the most commonly used AAA mouse models (adventitial calcium chloride, intralumen porcine elastase, and subcutaneous Ang II infusion) for human disease is considerable. The task of assessing the relevance of any of these commonly used mouse models is made difficult by our lack of knowledge of the initiation and formative stages of human AAAs. Unfortunately, the most accessible human AAA tissue is from advanced lesions that have been resected during open surgical repair, which provides limited insight into the earlier stages. Despite these formidable barriers to validating any of the murine models, there are some indications that the renin-angiotensin system in particular is involved in the formation of human AAAs. Components of the renin-angiotensin system are highly expressed in human AAA tissue, particularly angiotensin-converting enzyme and chymase-3.46 Retrospective clinical analyses have revealed that angiotensin-converting enzyme inhibition is associated with reduced AAA rupture.47 Finally, genetic association studies are emerging that link specific Ang II type 1 receptor and angiotensin-converting enzyme polymorphisms with AAA as well.48,49
Experimental studies are only beginning to clarify the functional role of adaptive immunity in stenotic vascular occlusive disease and AAA formation. The prevailing dogma is that Th1 immune responses contribute in a causal manner to atherosclerosis in general, but particularly with regard to luminal atherosclerotic plaque buildup. In striking contrast, the present findings clearly demonstrate that 2 major Th1-associated cytokines, IFN- and CXCL10, play a protective role in AAA formation. The present data suggest that local modulation of CXCL10 represents a potential therapeutic strategy for AAA. Most importantly, the present study also suggests that efforts to develop antiinflammatory strategies for atherosclerosis must carefully consider the potential effects on all types of vascular disease manifestations and must consider both salutary and harmful aspects of the immune system.
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Acknowledgments |
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Sources of Funding
The authors gratefully acknowledge support from the National Institutes of Health to Dr Daugherty (HL62846 and HL 80100), Dr Luster (CA069212), and Dr Gerszten (HL65584) and from the American Heart Association (Grant-in-Aid to Dr Gerszten). Dr Gerszten is also supported by the Donald W. Reynolds Foundation and the Leducq Foundation.
Disclosures
None.
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References |
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1. Frydman G, Walker PJ, Summers K, West M, Xu D, Lightfoot T, Codd C, Dique T, Nataatmadja M. The value of screening in siblings of patients with abdominal aortic aneurysm. Eur J Vasc Endovasc Surg. 2003; 26: 396–400.[CrossRef][Medline] [Order article via Infotrieve]
2. Lederle FA, Nelson DB, Joseph AM. Smokers’ relative risk for aortic aneurysm compared with other smoking-related diseases: a systematic review. J Vasc Surg. 2003; 38: 329–334.[CrossRef][Medline] [Order article via Infotrieve]
3. Lederle FA, Johnson GR, Wilson SE, Chute EP, Hye RJ, Makaroun MS, Barone GW, Bandyk D, Moneta GL, Makhoul RG; Aneurysm Detection and Management Veterans Affairs Cooperative Study Investigators. The Aneurysm Detection and Management Study screening program: validation cohort and final results. Arch Intern Med. 2000; 160: 1425–1430.
4. Blanchard JF, Armenian HK, Friesen PP. Risk factors for abdominal aortic aneurysm: results of a case-control study. Am J Epidemiol. 2000; 151: 575–583.
5. Thompson RW, Geraghty PJ, Lee JK. Abdominal aortic aneurysms: basic mechanisms and clinical implications. Curr Probl Surg. 2002; 39: 110–230.[CrossRef][Medline] [Order article via Infotrieve]
6. Shimizu K, Mitchell RN, Libby P. Inflammation and cellular immune responses in abdominal aortic aneurysms. Arterioscler Thromb Vasc Biol. 2006; 26: 987–994.
7. Mach F, Sauty A, Iarossi AS, Sukhova GK, Neote K, Libby P, Luster AD. Differential expression of three T lymphocyte-activating CXC chemokines by human atheroma-associated cells. J Clin Invest. 1999; 104: 1041–1050.[Medline] [Order article via Infotrieve]
8. Tang PC, Yakimov AO, Teesdale MA, Coady MA, Dardik A, Elefteriades JA, Tellides G. Transmural inflammation by interferon-gamma-producing T cells correlates with outward vascular remodeling and intimal expansion of ascending thoracic aortic aneurysms. FASEB J. 2005; 19: 1528–1530.
9. Schonbeck U, Sukhova GK, Gerdes N, Libby P. T(H)2 predominant immune responses prevail in human abdominal aortic aneurysm. Am J Pathol. 2002; 161: 499–506.
10. Xiong 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: 2607–2612.
11. Hansson GK, Libby P. The immune response in atherosclerosis: a double-edged sword. Nat Rev Immunol. 2006; 6: 508–519.[CrossRef][Medline] [Order article via Infotrieve]
12. Shimizu K, Shichiri M, Libby P, Lee RT, Mitchell RN. Th2-predominant inflammation and blockade of IFN-gamma signaling induce aneurysms in allografted aortas. J Clin Invest. 2004; 114: 300–308.[CrossRef][Medline] [Order article via Infotrieve]
13. Ferber IA, Brocke S, Taylor-Edwards C, Ridgway W, Dinisco C, Steinman L, Dalton D, Fathman CG. Mice with a disrupted IFN-gamma gene are susceptible to the induction of experimental autoimmune encephalomyelitis (EAE). J Immunol. 1996; 156: 5–7.[Abstract]
14. Vermeire K, Heremans H, Vandeputte M, Huang S, Billiau A, Matthys P. Accelerated collagen-induced arthritis in IFN-gamma receptor-deficient mice. J Immunol. 1997; 158: 5507–5513.[Abstract]
15. Manoury-Schwartz B, Chiocchia G, Bessis N, Abehsira-Amar O, Batteux F, Muller S, Huang S, Boissier MC, Fournier C. High susceptibility to collagen-induced arthritis in mice lacking IFN-gamma receptors. J Immunol. 1997; 158: 5501–5506.[Abstract]
16. Tager AM, Dufour JH, Goodarzi K, Bercury SD, von Andrian UH, Luster AD. BLTR mediates leukotriene B(4)-induced chemotaxis and adhesion and plays a dominant role in eosinophil accumulation in a murine model of peritonitis. J Exp Med. 2000; 192: 439–446.
17. Daugherty A, Manning MW, Cassis LA. Angiotensin II promotes atherosclerotic lesions and aneurysms in apolipoprotein E-deficient mice. J Clin Invest. 2000; 105: 1605–1612.[Medline] [Order article via Infotrieve]
18. Heller EA, Liu E, Tager AM, Sinha S, Roberts JD, Koehn SL, Libby P, Aikawa ER, Chen JQ, Huang P, Freeman MW, Moore KJ, Luster AD, Gerszten RE. Inhibition of atherogenesis in BLT1-deficient mice reveals a role for LTB4 and BLT1 in smooth muscle cell recruitment. Circulation. 2005; 112: 578–586.
19. King VL, Szilvassy SJ, Daugherty A. Interleukin-4 deficiency decreases atherosclerotic lesion formation in a site-specific manner in female LDL receptor–/– mice. Arterioscler Thromb Vasc Biol. 2002; 22: 456–461.
20. Daugherty A, Manning MW, Cassis LA. Antagonism of AT2 receptors augments angiotensin II-induced abdominal aortic aneurysms and atherosclerosis. Br J Pharmacol. 2001; 134: 865–870.[CrossRef][Medline] [Order article via Infotrieve]
21. Wang YX, Cassi LA, Daugherty A. Angiotensin II-induced abdominal aortic aneurysms. In: Xu EQ, ed. A Handbook of Mouse Models for Cardiovascular Diseases. London, United Kingdom: Wiley; 2006: 125–146.
22. Flanders KC, Thompson NL, Cissel DS, Van Obberghen-Schilling E, Baker CC, Kass ME, Ellingsworth LR, Roberts AB, Sporn MB. Transforming growth factor-beta 1: histochemical localization with antibodies to different epitopes. J Cell Biol. 1989; 108: 653–660.
23. Ng CM, Cheng A, Myers LA, Martinez-Murillo F, Jie C, Bedja D, Gabrielson KL, Hausladen JM, Mecham RP, Judge DP, Dietz HC. TGF-beta-dependent pathogenesis of mitral valve prolapse in a mouse model of Marfan syndrome. J Clin Invest. 2004; 114: 1586–1592.[CrossRef][Medline] [Order article via Infotrieve]
24. Habashi JP, Judge DP, Holm TM, Cohn RD, Loeys BL, Cooper TK, Myers L, Klein EC, Liu G, Calvi C, Podowski M, Neptune ER, Halushka MK, Bedja D, Gabrielson K, Rifkin DB, Carta L, Ramirez F, Huso DL, Dietz HC. Losartan, an AT1 antagonist, prevents aortic aneurysm in a mouse model of Marfan syndrome. Science. 2006; 312: 117–121.
25. Bjorkbacka H, Kunjathoor VV, Moore KJ, Koehn S, Ordija CM, Lee MA, Means T, Halmen K, Luster AD, Golenbock DT, Freeman MW. Reduced atherosclerosis in MyD88-null mice links elevated serum cholesterol levels to activation of innate immunity signaling pathways. Nat Med. 2004; 10: 416–421.[CrossRef][Medline] [Order article via Infotrieve]
26. Heller EA, Liu E, Tager AM, Yuan Q, Lin AY, Ahluwalia N, Jones K, Koehn SL, Lok VM, Aikawa E, Moore KJ, Luster AD, Gerszten RE. Chemokine CXCL10 promotes atherogenesis by modulating the local balance of effector and regulatory T cells. Circulation. 2006; 113: 2301–2312.
27. Babamusta F, Rateri DL, Moorleghen JJ, Howatt DA, Li XA, Daugherty A. Angiotensin II infusion induces site-specific intra-laminar hemorrhage in macrophage colony-stimulating factor-deficient mice. Atherosclerosis. 2006; 186: 282–290.[CrossRef][Medline] [Order article via Infotrieve]
28. Saraff 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: 1621–1626.
29. Okamoto Y, Kihara S, Ouchi N, Nishida M, Arita Y, Kumada M, Ohashi K, Sakai N, Shimomura I, Kobayashi H, Terasaka N, Inaba T, Funahashi T, Matsuzawa Y. Adiponectin reduces atherosclerosis in apolipoprotein E-deficient mice. Circulation. 2002; 106: 2767–2770.
30. Daugherty A, Cassis L. Chronic angiotensin II infusion promotes atherogenesis in low density lipoprotein receptor –/– mice. Ann N Y Acad Sci. 1999; 892: 108–118.[CrossRef][Medline] [Order article via Infotrieve]
31. Medoff BD, Sauty A, Tager AM, Maclean JA, Smith RN, Mathew A, Dufour JH, Luster AD. IFN-gamma-inducible protein 10 (CXCL10) contributes to airway hyperreactivity and airway inflammation in a mouse model of asthma. J Immunol. 2002; 168: 5278–5286.
32. Whitman SC, Ravisankar P, Daugherty A. IFN-gamma deficiency exerts gender-specific effects on atherogenesis in apolipoprotein E-/- mice. J Interferon Cytokine Res. 2002; 22: 661–670.[CrossRef][Medline] [Order article via Infotrieve]
34. Daugherty A, Cassis LA. Mouse models of abdominal aortic aneurysms. Arterioscler Thromb Vasc Biol. 2004; 24: 429–434.
35. Dufour JH, Dziejman M, Liu MT, Leung JH, Lane TE, Luster AD. IFN-gamma-inducible protein 10 (IP-10; CXCL10)-deficient mice reveal a role for IP-10 in effector T cell generation and trafficking. J Immunol. 2002; 168: 3195–3204.
36. Ehrhart EJ, Segarini P, Tsang ML, Carroll AG, Barcellos-Hoff MH. Latent transforming growth factor beta1 activation in situ: quantitative and functional evidence after low-dose gamma-irradiation. FASEB J. 1997; 11: 991–1002.[Abstract]
37. Prescott MF, Sawyer WK, Von Linden-Reed J, Jeune M, Chou M, Caplan SL, Jeng AY. Effect of matrix metalloproteinase inhibition on progression of atherosclerosis and aneurysm in LDL receptor-deficient mice overexpressing MMP-3, MMP-12, and MMP-13 and on restenosis in rats after balloon injury. Ann N Y Acad Sci. 1999; 878: 179–190.[CrossRef][Medline] [Order article via Infotrieve]
38. Zhao L, Moos MP, Grabner R, Pedrono F, Fan J, Kaiser B, John N, Schmidt S, Spanbroek R, Lotzer K, Huang L, Cui J, Rader DJ, Evans JF, Habenicht AJ, Funk CD. The 5-lipoxygenase pathway promotes pathogenesis of hyperlipidemia-dependent aortic aneurysm. Nat Med. 2004; 10: 966–973.[CrossRef][Medline] [Order article via Infotrieve]
39. Lemaitre V, Soloway PD, D'Armiento J. Increased medial degradation with pseudo-aneurysm formation in apolipoprotein E-knockout mice deficient in tissue inhibitor of metalloproteinases-1. Circulation. 2003; 107: 333–338.
40. Silence J, Collen D, Lijnen HR. Reduced atherosclerotic plaque but enhanced aneurysm formation in mice with inactivation of the tissue inhibitor of metalloproteinase-1 (TIMP-1) gene. Circ Res. 2002; 90: 897–903.
41. Wynn TA. Common and unique mechanisms regulate fibrosis in various fibroproliferative diseases. J Clin Invest. 2007; 117: 524–529.[CrossRef][Medline] [Order article via Infotrieve]
42. Saxena V, Lienesch DW, Zhou M, Bommireddy R, Azhar M, Doetschman T, Singh RR. Dual roles of immunoregulatory cytokine TGF-beta in the pathogenesis of autoimmunity-mediated organ damage. J Immunol. 2008; 180: 1903–1912.
43. Dai J, Losy F, Guinault AM, Pages C, Anegon I, Desgranges P, Becquemin JP, Allaire E. Overexpression of transforming growth factor-beta1 stabilizes already-formed aortic aneurysms: a first approach to induction of functional healing by endovascular gene therapy. Circulation. 2005; 112: 1008–1015.
44. MacTaggart JN, Xiong W, Knispel R, Baxter BT. Deletion of CCR2 but not CCR5 or CXCR3 inhibits aortic aneurysm formation. Surgery. 2007; 142: 284–288.[CrossRef][Medline] [Order article via Infotrieve]
45. Sun 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: 3359–3368.[CrossRef][Medline] [Order article via Infotrieve]
46. Tsunemi K, Takai S, Nishimoto M, Yuda A, Hasegawa S, Sawada Y, Fukumoto H, Sasaki S, Miyazaki M. Possible roles of angiotensin II-forming enzymes, angiotensin converting enzyme and chymase-like enzyme, in the human aneurysmal aorta. Hypertens Res. 2002; 25: 817–822.[CrossRef][Medline] [Order article via Infotrieve]
47. Hackam DG, Thiruchelvam D, Redelmeier DA. Angiotensin-converting enzyme inhibitors and aortic rupture: a population-based case-control study. Lancet. 2006; 368: 659–665.[CrossRef][Medline] [Order article via Infotrieve]
48. Jones GT, Thompson AR, van Bockxmeer FM, Hafez H, Cooper JA, Golledge J, Humphries SE, Norman PE, van Rij AM. Angiotensin II type 1 receptor 1166C polymorphism is associated with abdominal aortic aneurysm in three independent cohorts. Arterioscler Thromb Vasc Biol. 2008; 28: 764–770.
49. Fatini C, Pratesi G, Sofi F, Gensini F, Sticchi E, Lari B, Pulli R, Dorigo W, Azas L, Pratesi C, Gensini GF, Abbate R. ACE DD genotype: a predisposing factor for abdominal aortic aneurysm. Eur J Vasc Endovasc Surg. 2005; 29: 227–232.[CrossRef][Medline] [Order article via Infotrieve]
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Footnotes |
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The online-only Data Supplement is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.108.785949/DC1.
*The first 2 authors contributed equally to this article. 
Guest Editor for this article was Daniel I. Simon, MD.
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