Treatment With Simvastatin Suppresses the Development of Experimental Abdominal Aortic Aneurysms in Normal and Hypercholesterolemic Mice

Abstract

Objective:

To determine if treatment with hydroxymethylglutaryl-coenzyme A reductase inhibitors (statins) can influence the development of experimental abdominal aortic aneurysms (AAAs).

Summary Background Data:

AAAs are associated with atherosclerosis, chronic inflammation, and matrix metalloproteinase (MMP)-mediated connective tissue destruction. Because statins exert antiinflammatory activities independent of their lipid-lowering effects, these agents may help suppress aneurysmal degeneration.

Methods:

C57Bl/6 wild-type and hypercholesterolemic apoE-deficient mice underwent transient perfusion of the aorta with elastase followed by subcutaneous treatment with either 2 mg/kg simvastatin per day or vehicle. Aortic diameter (AD) was measured before and 14 days after elastase perfusion. The extent of aortic dilatation (ΔAD) was determined with AAAs defined as ΔAD >100%.

Results:

Wild-type mice treated with simvastatin exhibited a 21% reduction in ΔAD and a 33% reduction in AAAs compared with vehicle-treated controls. Suppression of AAAs in simvastatin-treated mice was associated with preservation of medial elastin and vascular smooth muscle cells, as well as a relative reduction in aortic wall expression of MMP-9 and a relative increase in expression of TIMP-1. In hypercholesterolemic apoE-deficient mice, treatment with simvastatin was associated with a 26% reduction in ΔAD and a 30% reduction in AAAs. Treatment with simvastatin had no effect on serum cholesterol levels in either normal or hypercholesterolemic mice.

Conclusions:

Treatment with simvastatin suppresses the development of experimental AAAs in both normal and hypercholesterolemic mice. The mechanisms of this effect are independent of lipid-lowering and include preservation of medial elastin and smooth muscle cells, as well as altered aortic wall expression of MMPs and their inhibitors.

Simvastatin suppressed the development of experimental abdominal aortic aneurysms in normal mice and hypercholesterolemic apoE-deficient animals while having no effect on serum cholesterol levels. Aortas from simvastatin-treated mice exhibited preservation of medial elastin and vascular smooth muscle cells, as well as alterations in aortic wall expression of MMP-9 and TIMP-1.

Abdominal aortic aneurysms (AAAs) are a common and potentially life-threatening disorder associated with aging and atherosclerosis.¹ Despite these associations, there is considerable uncertainty regarding the precise role of atherosclerosis in the etiology and pathophysiology of AAAs, with opinion ranging from the view that aneurysms arise as a direct consequence of advanced atheromatous disease to speculation that aneurysmal degeneration might be an independent disease process only coincidentally related to atherosclerosis.^2–4 This debate has been fueled by the discrepancy between strong evidence linking hypercholesterolemia with atherogenesis and the lack of a compelling relationship between altered lipid metabolism and AAAs.^5–7

It has become evident over the past decade that many of the cellular and molecular mechanisms involved in aneurysmal degeneration are analogous to those involved in the clinical complications of atherosclerosis such as rupture of atheromatous plaques.^8–10 The most important of these mechanisms include: 1) arterial wall accumulation and activation of mononuclear inflammatory cells, including both macrophages and lymphocytes; 2) increased local expression of proinflammatory cytokines, chemokines, and matrix-degrading proteinases; 3) accelerated degradation of structurally important matrix proteins (ie, elastin and collagen); 4) pronounced oxidative and hemodynamic stresses; and 5) depletion of medial smooth muscle cells (SMC) through accelerated senescence and apoptosis. The occurrence of these pathophysiological processes within both AAAs and vulnerable atherosclerotic plaques suggests that these conditions likely share many potential targets for pharmacologic therapy, whether they represent distinct diseases or simply divergent aspects of the same heterogeneous disease process.

Three-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) are widely used in the treatment of hypercholesterolemia, atherosclerosis, and coronary artery disease. Large clinical trials consistently demonstrate significant reductions in cardiac events and mortality associated with statin therapy, but discrepancies between observed clinical benefits and the degree of angiographic improvement has raised the possibility that statins might exert additional therapeutic effects beyond those attributable solely to cholesterol-lowering.^11–18 In support of this notion, recent laboratory studies have demonstrated a host of antiinflammatory and other “pleiotropic” effects associated with statin therapy.^19–21 Because statins influence pathophysiological mechanisms in atherosclerosis that appear similar to those involved in aneurysmal degeneration, we postulated that treatment with statins might also have an impact on the development and progression of AAAs.

The primary purpose of this study was to determine if treatment with an HMG-CoA reductase inhibitor would favorably influence the development of experimental AAAs, and second, to explore whether any effects of statins on AAA development could be considered independent of changes in circulating cholesterol. To address these questions, we applied a previously characterized mouse model of aortic aneurysms to normocholesterolemic animals and to mice with genetically determined hypercholesterolemia, and assessed whether treatment with simvastatin has an influence on aneurysmal degeneration under each of these conditions. We examined the effects of simvastatin because this drug is one of the most commonly used agents in clinical practice, and chose apoE-deficient mice as a model for hypercholesterolemia because these animals do not exhibit reductions in serum cholesterol during treatment with statins.^22–27 This experimental strategy thereby allowed us to examine the effects of statin therapy on aneurysm development in both normocholesterolemic and hypercholesterolemic animals in a manner independent of changes in blood cholesterol.

MATERIALS AND METHODS

Animals and Experimental Groups

C57BL/6J wild-type mice and apolipoprotein E (apoE)-deficient mice on a C57Bl/6 background were purchased from The Jackson Laboratory (Bangor, ME). All experimental procedures were performed in male animals that had reached maturity (8–10 weeks of age) according to a protocol approved by the Animal Studies Committee at Washington University School of Medicine.

Group I (normocholesterolemia) consisted of 35 wild-type mice that had been maintained on normal laboratory chow beginning at 3 weeks of age (PicoLab Rodent Diet 5053; PMI Nutrition International/El Mel, St. Charles, MO). Group II (hypercholesterolemia) consisted of 53 apoE-deficient mice that had been placed on a high-fat Western diet beginning at 3 weeks of age. The composition of this diet included 0.15% (wt/wt) cholesterol, 21% fat, 17% protein, and 48% carbohydrate (Harlan Teklad TD88137; Madison, WI). To verify circulating lipid levels, blood was drawn from 6 animals in each experimental group at the time the mice were killed and total serum cholesterol was measured using the Infinity Cholesterol Reagent kit from Sigma Diagnostics (St. Louis, MO).

Drug Treatment

Simvastatin tablets (40 mg) were purchased from Merck, Sharp and Dohme (West Point, PA). Because orally administered simvastatin requires metabolism to its pharmacologically active form by hepatic lactonases, the drug was chemically activated by alkaline hydrolysis before administration by subcutaneous injection in accord with a previously reported protocol.^23,28 Preactivated simvastatin was diluted with phosphate-buffered saline (PBS) to a final working concentration of 0.4 mg/mL. Beginning 7 days before the induction of experimental AAAs, a subset of animals in each group underwent treatment with 2 mg/kg per day preactivated simvastatin by daily subcutaneous injection with the remaining animals in each group treated with PBS vehicle. Treatment with drug or vehicle was continued for 14 days after elastase perfusion, at which time all animals were killed by intravenous pentobarbital overdose. The dose regimen for simvastatin was based on previous studies demonstrating beneficial effects of the drug in mouse models of atherosclerosis and cerebral ischemia, and the recognition that treatment with simvastatin does not decrease serum cholesterol levels in wild-type or apoE-deficient mice.^23–27

Elastase-Induced Abdominal Aortic Aneurysms and Aortic Diameter Measurements

Transient elastase perfusion was used to induce the development of experimental AAAs as previously described.^29–31 Briefly, mice (20–35 g) were anesthetized with intraperitoneal sodium pentobarbital (55–60 mg/kg) and a laparotomy was performed under sterile conditions. The abdominal aorta was isolated with the assistance of an operating stereomicroscope (Leica, Deerfield, IL) and the preperfusion aortic diameter (AD) was measured with a calibrated ocular grid. Temporary ligatures were placed around the proximal and distal aorta, and an aortotomy was created at the bifurcation using the tip of a 30-gauge needle. A heat-tapered segment of PE-10 polyethylene tubing was introduced through the aortotomy and secured, and the aortic lumen was perfused for 5 minutes at 100 mm Hg with saline containing 0.414 U/mL type I porcine pancreatic elastase (Sigma Chemical Co., St. Louis, MO). After removing the perfusion catheter, the aortotomy was repaired without constriction of the lumen, and the postperfusion AD was measured at least 5 minutes after restoration of flow to the lower extremities. Animals were allowed free access to food and water for 14 days, when the aorta was reexposed by laparotomy under anesthesia. Final AD measurements were obtained before euthanasia and tissue procurement. AAAs were defined as an interval increase in diameter (ΔAD) greater than 100% between day 0 (preperfusion) and day 14 (final).

Light Microscopy

Abdominal aortic tissues were perfusion-fixed with 10% neutral-buffered formalin (120 mm Hg for 10 minutes). Specimens were embedded in paraffin and 5-μm cross-sections of the aortic wall were stained with hematoxylin and eosin (H&E), Verhoeff-Van Gieson (VVG) for elastin, and picrosirius red for collagen. Additional sections were stained with immunoperoxidase techniques using antialpha-SMC actin monoclonal antibodies (clone 1A4, catalog #A2547; Sigma Chemical Co.), as previously described.³²

Quantitative (Real-Time) Reverse Transcriptase–Polymerase Chain Reaction

Aortic wall mRNA expression was measured by real-time reverse transcriptase–polymerase chain reaction (RT-PCR) as previously described.^30,31 Briefly, 3 to 4 pooled tissue samples were pulverized under liquid nitrogen and total RNA was isolated with Trizol reagent (Gibco BRL, Grand Island, NY). Each sample was normalized to 1 μg of total RNA and cDNA synthesis was performed by reverse transcription on a GeneAmp 2400 thermal cycler system using reagents provided by the manufacturer (Applied Biosystems, Foster City, CA). The reverse transcription products served as the template for real-time PCR analysis using reagents and protocols provided in the SYBR Green PCR kit and the GeneAmp 5700 Sequence Detection System (Applied Biosystems). The gene-specific primers used were as follows: mouse matrix metalloproteinase-9 (MMP-9) (GenBank Ref NM013599), forward primer: 5′-ACA ATC CTT GCA ATG TGG ATG TT-3′, reverse primer: 5′-CGC CCT GGA TCT CAG GAA TA-3′; mouse tissue inhibitor of metalloproteinases-1 (TIMP-1) (GenBank Ref NM011593), forward primer: 5′-ACG CCT ACA CCC CAG TCA TG-3′, reverse primer: 5′-GGG ACT TGT GGG CAT ATC CA-3′; and mouse β-actin (GenBank Ref M12481), forward primer: 5′-CCC TAA GGC CAA CCG TGA A-3′, reverse primer: 5′-GTT GAG GTC TCA AAC ATG ATC TG-3′. PCR reactions (40 cycles) were performed in quadruplicate with SYBR Green PCR Core Reagents (Applied Biosystems), and fluorescence signals were analyzed using the GeneAmp 5700 Sequence Detection System software (version 1.3; Applied Biosystems). Results for each sample were normalized to the concentration of β-actin mRNA.

Statistics

Statistical analysis was performed with the InStat 3.0a program (GraphPad Software, Inc., San Diego, CA). Within-group comparisons of AD at different intervals after elastase perfusion were performed by repeated-measures analysis of variance (ANOVA) with the Bonferroni multiple comparisons test. Between-group comparisons of AD at a given interval were performed by nonparametric testing using the Mann-Whitney rank-sum U test, as were comparisons of the extent of aortic dilatation (ΔAD) on day 14. Differences between groups in the incidence of AAAs were evaluated using 2 × 2 contingency tables and the 2-tailed Fisher exact test. Relative differences in gene expression were analyzed using the paired Student t test. In all cases, a P value <0.05 was considered significant.

RESULTS

Effects of Simvastatin in Normocholesterolemic Mice

Consistent with our previous studies,^29–31 wild-type C57Bl/6 mice treated with vehicle (n = 15) exhibited a significant expansion in aortic size after transient elastase perfusion, with AD increasing from 0.51 ± 0.01 mm (mean ± standard error of mean [SEM]) before perfusion to 1.40 ± 0.06 mm on day 14 (P <0.001) (Table 1). The overall extent of aortic dilatation in this group was 174 ± 12%, with 14 of 15 animals (93%) developing AAAs (Fig. 1).

TABLE 1. Aortic Diameter Measurements in Normocholesterolemic Mice

FIGURE 1. Effect of simvastatin on the development of elastase-induced abdominal aortic aneurysms (AAAs) in normocholesterolemic mice. C57Bl/6 wild-type mice were subjected to transient elastase perfusion of the abdominal aorta and treatment with either vehicle (n = 15) or simvastatin (n = 20) for up to 14 days. (A) The extent of aortic dilatation for each animal was determined as the percent increase from the normal (preperfusion) aortic diameter. Data shown represent the mean ± standard error of mean. Aneurysmal dilatation is defined as an increase >100% (indicated by the horizontal line). *Vehicle versus simvastatin, Mann-Whitney rank-sum U test. (B) Incidence of AAAs. **Vehicle versus Simvastatin, 2-tailed Fisher exact test.

Normocholesterolemic (wild-type) animals treated with simvastatin (n = 20) also exhibited a significant increase in AD after elastase perfusion, from 0.51 ± 0.01 mm before perfusion to 1.20 ± 0.06 mm on day 14 (P <0.001). Although there was no difference between the vehicle-treated and simvastatin-treated groups with regard to AD measurements before or immediately after elastase perfusion, treatment with simvastatin was associated with a 17% reduction in median AD on day 14 compared with vehicle-treated controls (P <0.05) (Table 1). The overall extent of aortic dilatation in simvastatin-treated animals was 137% ± 12%, representing a 21% reduction from that observed in the vehicle-treated control group (P <0.05), and the incidence of AAAs was reduced by 33% (12 of 20 mice treated with simvastatin [60%] vs. 14 of 15 mice treated with vehicle [93%]; P <0.05) (Fig. 1). There was no difference in total serum cholesterol between wild-type mice treated with vehicle (93 ± 5 mg/dL) and those treated with simvastatin (95 ± 3 mg/dL).

Morphologic Changes

To elucidate the structural basis for decreased aneurysm development in simvastatin-treated mice, morphologic changes in the aortic wall were evaluated by histochemical staining for elastin, collagen, and vascular smooth muscle cells. As shown in Figure 2, the development of AAAs in vehicle-treated mice was accompanied by marked thickening of the aortic wall, a dense transmural inflammatory response, and complete destruction of the elastic media. Although simvastatin-treated animals exhibited inflammatory cell infiltration similar to vehicle controls (Fig. 2H, I), the most remarkable and consistent difference was preservation of intact elastic lamellae within the aortic media. As shown in Figure 3, staining with sirius red for collagen revealed a somewhat more diffuse appearance of interstitial collagen fibers within the media and adventitia of AAAs compared with normal aorta, but there was no detectable difference in the overall collagen content between simvastatin-treated mice and vehicle-treated controls. Sections stained for alpha-smooth muscle actin revealed a marked depletion of medial SMC in elastase-induced AAAs, with significant preservation of vascular SMC in simvastatin-treated mice (Fig. 4).

FIGURE 2. Effect of simvastatin on aortic wall elastin after elastase perfusion. Aortic tissues were obtained from normocholesterolemic wild-type mice and stained with VVG for elastin (A–E, G, and H) or hematoxylin & eosin (C, F, and I). (A–C) Normal aorta before elastase perfusion (day 0). (D–F) Vehicle-treated, 14 days after elastase perfusion. (G–I) Simvastatin-treated, 14 days after elastase perfusion. Original magnification, 20× (A, D, and G); 60× (all other panels). Arrows indicate mononuclear phagocytes infiltrating the aortic wall. Results shown are representative of at least 3 animals in each group.

FIGURE 3. Effect of simvastatin on aortic wall collagen after elastase perfusion. Aortic tissues obtained from normocholesterolemic wild-type mice were stained with picrosirius red for interstitial (types I and III) collagen. (A–C) Normal aorta before elastase perfusion (day 0). (D–F) Vehicle-treated control 14 days after elastase perfusion. (G–I) Simvastatin-treated, 14 days after elastase perfusion. Light-field views (A, D, and G) are compared with dark-field views taken under polarized light (B, C, E, F, H, and I) to visualize interstitial collagens. Original magnification, 20× (A, B, D, E, G, and H); 60× (C, F, and I). Results shown are representative of at least 3 animals in each group.

FIGURE 4. Effect of simvastatin on aortic wall smooth muscle cells after elastase perfusion. Aortic tissues were obtained from normocholesterolemic wild-type mice and stained with immunoperoxidase techniques for alpha-smooth muscle cell actin (brown reaction product). (A and B) Normal aorta before elastase perfusion (day 0). (C and D) Vehicle-treated, 14 days after elastase perfusion. (E and F(Simvastatin-treated, 14 days after elastase perfusion. Original magnification, 20× (A, C, and E); 60× (B, D, and F). Results shown are representative of at least 3 animals in each group.

Aortic Wall Gene Expression

To examine molecular mechanisms that might underlie the reduction in aneurysmal dilatation in mice treated with simvastatin, real-time RT-PCR was used to measure aortic wall expression of MMP-9 and its endogenous inhibitor, TIMP-1. As shown in Figure 5, vehicle-treated animals exhibited an 8-fold increase in MMP-9 mRNA between day 0 and development of AAAs on day 14; in contrast, there was a 4-fold decrease in the expression of TIMP-1 mRNA. Treatment with simvastatin for 1 week before elastase perfusion was associated with a 34-fold reduction in expression of MMP-9 in the normal (unperfused) aorta. Although MMP-9 expression in simvastatin-treated animals was increased by 13-fold on day 14 after elastase perfusion, the overall level of MMP-9 expression at this interval was 20-fold less than that observed in vehicle-treated controls. Treatment with simvastatin was associated with a 2-fold reduction in the expression of TIMP-1 expression in normal (unperfused) aortic tissue; however, in this group, there was a 4-fold increase in the expression of TIMP-1 by 14 days after elastase perfusion. At 14 days after elastase perfusion, TIMP-1 mRNA levels in simvastatin-treated mice were therefore increased 9-fold as compared with those observed in vehicle-treated controls at the same interval. These results indicate that animals treated with simvastatin exhibited a relative decrease in aortic wall expression of MMP-9 and a relative increase in expression of TIMP-1 during the response to elastase-induced injury, with the MMP-9:TIMP-1 ratio increasing 37-fold between days 0 and 14 in vehicle-treated controls but only 3-fold in simvastatin-treated mice. Simvastatin treatment was therefore associated with a 16-fold decrease in the MMP-9:TIMP-1 ratio on day 0 and a 182-fold decrease in the MMP-9:TIMP-1 ratio on day 14.

FIGURE 5. Effect of simvastatin on mouse aortic wall gene expression during elastase-induced aneurysmal degeneration. Aortic wall RNA samples were obtained at the indicated intervals from normocholesterolemic C57BL/6 wild-type mice treated with either vehicle or simvastatin, and mRNA transcript levels were measured by real-time reverse transcriptase–polymerase chain reaction. Data shown indicate the mean ± standard error of mean transcript levels for MMP-9 (A) and TIMP-1 (B) as normalized to β-actin (n = 3). *P <0.05, day 14 versus day 0 for the group indicated. **P <0.05, simvastatin versus vehicle for the interval indicated.

Effects of Simvastatin in Hypercholesterolemic Mice

To determine if the aneurysm-suppressing effects of simvastatin might be altered in the presence of hypercholesterolemia, the induction of AAAs was also examined in fat-fed apoE-deficient mice that had markedly elevated total serum cholesterol levels (mean ± SEM: 846 ± 66 mg/dL). Similar to wild-type mice, hypercholesterolemic apoE-deficient mice developed a significant increase in AD after transient elastase perfusion, with mean AD increasing from 0.50 ± 0.01 mm before perfusion to 1.37 ± 0.08 mm on day 14 (P <0.001) (Table 2). The overall extent of aortic dilatation was 176 ± 16%, with 20 of 25 animals (80%) developing AAAs (Fig. 6).

TABLE 2. Aortic Diameter Measurements in Hypercholesterolemic Mice

FIGURE 6. Effect of simvastatin on the development of elastase-induced abdominal aortic aneurysms (AAAs) in hypercholesterolemic mice. C57Bl/6 apoE-deficient mice were subjected to transient elastase perfusion of the abdominal aorta and treatment with either vehicle (n = 25) or simvastatin (n = 28) for up to 14 days. (A) The extent of aortic dilatation for each animal was determined as the percent increase from the normal (preperfusion) aortic diameter. Data shown represent the mean ± standard error of mean. Aneurysmal dilatation is defined as an increase >100% as indicated by the horizontal line. *Vehicle versus simvastatin, Mann-Whitney rank-sum U test. (B) Incidence of AAAs. **Vehicle versus simvastatin, 2-tailed Fisher exact test.

In apoE-deficient mice treated with simvastatin, AD increased significantly from 0.50 ± 0.08 mm before perfusion to 1.14 ± 0.07 mm on day 14 (P <0.05) (Table 2). There was no difference between groups in AD measured before or immediately after elastase perfusion, yet the median AD on day 14 was 28% lower in simvastatin-treated apoE-deficient animals compared with vehicle-treated apoE-deficient controls (P <0.05). The overall extent of aortic dilatation in simvastatin-treated apoE-deficient animals was 131% ± 14%, a 26% reduction from that observed in the vehicle-treated apoE-deficient control group (P <0.05), and the incidence of AAAs was 50% in apoE-deficient mice treated with simvastatin compared with 80% in apoE-deficient controls (P <0.05) (Fig. 6). There was no difference in total serum cholesterol in apoE-deficient mice treated for 3 weeks with simvastatin (836 ± 43 mg/dL) versus vehicle (846 ± 66 mg/dL).

DISCUSSION

The most important finding of this study is that treatment with simvastatin reduced the development of elastase-induced experimental AAAs in both normocholesterolemic and apoE-deficient hypercholesterolemic mice and that this occurred in the absence of drug-induced alterations in total serum cholesterol. Treatment with simvastatin was also associated with structural preservation of aortic wall elastin and medial smooth muscle cells despite the presence of a chronic inflammatory response, and there was a relative reduction in aortic wall expression of MMP-9 and a relative increase in expression of TIMP-1. These results provide the first experimental evidence that treatment with statins can suppress aneurysmal degeneration in an animal model of AAAs and that the underlying mechanism is independent of serum cholesterol levels. These findings have potentially important clinical implications as a result of the widespread use of statins in treatment of patients with atherosclerosis and the frequency of small asymptomatic AAAs in this population.

In addition to the inhibition of AAAs, perhaps the most striking observation in this study was the structural preservation of the medial elastic lamellae that occurred in mice treated with simvastatin. Elastin degradation in AAAs is thought to require proteinases elaborated by mononuclear phagocytes, including MMPs, cathepsins, and serine proteases. Despite the presence of mononuclear phagocytes within the elastase-injured aorta, we found that treatment with simvastatin was associated with alterations in the relative balance of aortic wall gene expression that might favor diminished MMP-9 activity. Although the results regarding MMP-9 and TIMP-1 expression need to be further verified at the protein and enzyme activity levels, these findings indicate that simvastatin-mediated preservation of elastin may have been the result of suppression of elastolytic activities (ie, MMP-9), thereby serving to limit structural damage. Previous reports indicate that suppression of MMP-9 is a consistent effect of HMG-CoA reductase inhibitors, because statins can inhibit MMP-9 activity produced by monocytes,^33,34 macrophages,^35,36 fibroblasts,³⁷ vascular SMC,³⁸ and cultured tumor cell lines.³⁹ The functional importance of this mechanism is illustrated by studies showing that simvastatin can suppress migration of THP-1 human monocytic cells in response to MCP-1, in part through a reduction in MMP-9.³³ The potential clinical relevance of this mechanism was also highlighted by a recent study using cultured human AAA tissue explants, in which exposure to cerivastatin suppressed production of MMP-9 up to 5-fold in vitro.⁴⁰ Overproduction of TIMP-1 might also contribute to suppression of MMP-9 activity as well as other MMPs involved in the degradation of matrix proteins such as interstitial collagenases, thereby maintaining aortic wall tensile strength and diminishing aneurysmal dilatation. This conclusion is also consistent with observations on the mechanisms by which HMG-CoA reductase inhibitors favorably influence the behavior of atheromatous plaques by nonlipid-lowering activities, including a recent report in which pravastatin affected a number of markers related to plaque stabilization, including TIMP-1 expression, in human carotid artery atherosclerosis.⁴¹ Finally, although we observed no measurable difference in overall collagen content in aortas from simvastatin-treated mice and vehicle controls, simvastatin treatment may still be associated with functionally important differences in aortic collagen such as changes in the distribution, alignment, or crosslinking of collagen fibers that we were unable to assess in this study.

One of the mechanisms suspected to underlie the activities of HMG-CoA reductase inhibitors in atherosclerosis is a prominent suppressive effect on inflammatory cell function, which may serve to diminish the propensity of vulnerable plaques to rupture.21,24,42,43 This includes effects on monocyte/macrophage growth,^8,44 MMP expression and secretion,⁴⁵ expression of tissue factor, adhesion molecules, and proinflammatory cytokines,^35,46,47 secretion of MCP-1 and interleukin-8,⁴⁸ and activation of NFκB.⁴⁹ Although we observed no measurable decrease in the intensity of aortic wall inflammation associated with simvastatin, our finding that simvastatin reduced mouse aortic wall expression of MMP-9 likely reflects important drug effects on mononuclear phagocytes because we have previously shown that inflammatory cell production of MMP-9 is critical for AAA formation in the same mouse model.²⁹ Because inhibition of protein isoprenylation has emerged as a key cellular mechanism by which statins might exert their antiinflammatory activities in atherosclerosis and other conditions, our results suggest that alterations in protein isoprenylation may also be an important pathway to be examined in aortic aneurysms.⁵⁰

A second mechanism potentially explaining the clinical benefit of statins is a direct action on vascular SMC. It has been demonstrated that statins can inhibit SMC migration and proliferation,^28,51,52 decrease SMC expression of plasminogen activator inhibitor-1,⁵³ and decrease SMC free radical production induced by angiotensin II.⁵⁴ The importance of this mechanism is illustrated by the demonstration that simvastatin reduces neointima formation in human saphenous vein explants in association with inhibition of SMC proliferation and migration.⁵⁵ We have previously demonstrated that medial SMC depletion is a prominent feature of human AAAs in association with accelerated replicative senescence and increased apoptosis.^32,56 The preservation of medial SMC we observed in simvastatin-treated animals may thereby reflect the effects of statin treatment on some of these molecular mechanisms; moreover, these medial SMC may have an important function in the synthesis and secretion of structurally important matrix proteins (ie, interstitial collagens), thereby contributing to the prevention of aneurysmal dilatation.

Hypercholesterolemia is an important risk factor for atherosclerosis, and the apoE-deficient mouse has provided an important model to investigate the development of complex atheromatous lesions.⁵⁷ Although it is still unclear if hypercholesterolemia has a specific role in development of AAAs, we did not observe any deleterious effects of severe hypercholesterolemia on elastase-induced aneurysmal degeneration. Because the apoE-deficient mice used here were 8 to 10 weeks of age and exposed to a high-fat diet for only 5 weeks at the time of the experimental procedures, the duration of hypercholesterolemia was not sufficient for the development of the severe and widespread atherosclerotic lesions typically observed in older apoE-deficient animals. It therefore remains possible that aneurysm formation might have been more significantly affected by a longer period of hypercholesterolemia or in the presence of established atheromatous lesions within the abdominal aorta. Another point worth repeating is that apoE-deficient mice are known to be resistant to cholesterol-lowering by treatment with statins as opposed to the effects of these drugs in different mouse models of hypercholesterolemia or in humans.^22–27 It is therefore possible that in other experimental or clinical settings in which statins cause a reduction in serum cholesterol, drug treatment might have additional effects on aneurysmal degeneration beyond those observed in this study.

The results of this investigation suggest that treatment with statins might have clinical potential in suppressing the development and growth of AAAs. Previous studies in rats and mice have demonstrated effective suppression of elastase-induced aneurysm formation by treatment with antiinflammatory agents,⁵⁸ hydroxamate- and tetracycline-based MMP inhibitors,^59,60 and angiotensin-converting enzyme antagonists,⁶¹ and similar results have been observed in mice with genetic deficiencies in either MMP-9 or MMP-2.^29,62 Although it remains difficult to compare these different studies, we have consistently observed reductions in aneurysmal dilatation of approximately 60% in MMP-9-deficient mice or after treatment of wild-type mice with doxycycline, an agent that suppresses both expression and proteolytic activity of MMP-9.^29,60,63 Because this degree of suppression is somewhat greater than that observed with simvastatin, more direct comparisons will be necessary to evaluate the relative efficacy of these different pharmacologic strategies. Moreover, because tetracyclines, statins, and other agents act by distinct but potentially overlapping mechanisms, it remains possible that combination therapies might be even more effective than any single agent alone. Because the cellular and molecular mechanisms underlying aneurysmal degeneration appear to parallel those involved in the vulnerability of atheromatous plaques to rupture, studies with the elastase-induced mouse model of AAAs can be expected to yield further insights into the pathogenesis and treatment of these conditions.