Neutrophil Proteases Promote Experimental Abdominal Aortic Aneurysm via Extracellular Trap Release and Plasmacytoid Dendritic Cell Activation.

OBJECTIVE: We previously established that neutrophil-derived dipeptidyl peptidase I (DPPI) is essential for experimental abdominal aortic aneurysm (AAA) development. Because DPPI activates several neutrophil serine proteases, it remains to be determined whether the AAA-promoting effect of DPPI is mediated by neutrophil serine proteases. APPROACH AND RESULTS: Using an elastase-induced AAA model, we demonstrate that the absence of 2 neutrophil serine proteases, neutrophil elastase and proteinase-3, recapitulates the AAA-resistant phenotype of DPPI-deficient mice. DPPI and neutrophil serine proteases direct the in vitro and in vivo release of extracellular structures termed neutrophil extracellular traps (NETs). Administration of DNase1, which dismantles NETs, suppresses elastase-induced AAA in wild-type animals and in DPPI-deficient mice reconstituted with wild-type neutrophils. NETs also contain the cathelicidin-related antimicrobial peptide that complexes with self-DNA in recruiting plasmacytoid dendritic cells (pDCs), inducing type I interferons (IFNs) and promoting AAA in DPPI-deficient mice. Conversely, depletion of pDCs or blockade of type I IFNs suppresses experimental AAA. Moreover, we find an abundance of human cathelicidin peptide, a 37 amino acid sequence starting with 2 leucines and the human orthologue of cathelicidin-related antimicrobial peptide, in the vicinity of pDCs in human AAA tissues. Increased type I IFN mRNA expression is observed in human AAA tissues and circulating IFN-α is detected in ≈50% of the AAA sera examined. CONCLUSIONS: These results suggest that neutrophil protease-mediated NET release contributes to elastase-induced AAA through pDC activation and type I IFN production. These findings increase our understanding of the pathways underlying AAA inflammatory responses and suggest that limiting NET, pDC, and type I IFN activities may suppress aneurysm progression.

Angiotensin-converting enzyme 2 decreases formation and severity of angiotensin II-induced abdominal aortic aneurysms.

OBJECTIVE: Angiotensin-converting enzyme 2 (ACE2) cleaves angiotensin II (AngII) to form angiotensin-(1-7) (Ang-(1-7)), which generally opposes effects of AngII. AngII infusion into hypercholesterolemic male mice induces formation of abdominal aortic aneurysms (AAAs). This study tests the hypothesis that deficiency of ACE2 promotes AngII-induced AAAs, whereas ACE2 activation suppresses aneurysm formation. APPROACH AND RESULTS: ACE2 protein was detectable by immunostaining in mice and human AAAs. Whole-body deficiency of ACE2 significantly increased aortic lumen diameters and external diameters of suprarenal aortas from AngII-infused mice. Conversely, ACE2 deficiency in bone marrow-derived cells had no effect on AngII-induced AAAs. In contrast to AngII-induced AAAs, ACE2 deficiency had no significant effect on external aortic diameters of elastase-induced AAAs. Because ACE2 deficiency promoted AAA formation in AngII-infused mice, we determined whether ACE2 activation suppressed AAAs. ACE2 activation by administration of diminazene aceturate (30 mg/kg per day) to Ldlr(-/-) mice increased kidney ACE2 mRNA abundance and activity and elevated plasma Ang-(1-7) concentrations. Unexpectedly, administration of diminazene aceturate significantly reduced total sera cholesterol and very low-density lipoprotein-cholesterol concentrations. Notably, diminazene aceturate significantly decreased aortic lumen diameters and aortic external diameters of AngII-infused mice resulting in a marked reduction in AAA incidence (from 73% to 29%). None of these effects of diminazene aceturate were observed in the Ace2(-/y) mice. CONCLUSIONS: These results demonstrate that ACE2 exerts a modulatory role in AngII-induced AAA formation, and that therapeutic stimulation of ACE2 could be a benefit to reduce AAA expansion and rupture in patients with an activated renin-angiotensin system.

Smooth muscle cells from abdominal aortic aneurysms are unique and can independently and synergistically degrade insoluble elastin.

BACKGROUND: The purpose of this study was to further elucidate the role of the vascular smooth muscle cells (SMCs) in abdominal aortic aneurysm (AAA) disease. We hypothesized that that AAA SMCs are unique and actively participate in the process of degrading the aortic matrix. METHODS: Whole-genome expression profiles of SMCs from AAAs, nondilated abdominal aorta (NAA), and carotid endarterectomy (CEA) were compared. We quantified elastolytic activity by culturing SMCs in [(3)H]elastin-coated plates and measuring solubilized tritium in the media after 7 days. Matrix metalloproteinase (MMP)-2 and MMP-9 production was assessed using real-time polymerase chain reaction, zymography, and Western blotting. RESULTS: Each SMC type exhibited a unique gene expression pattern. AAA SMCs had greater elastolytic activity than NAA-SMCs (+68%; P < .001) and CEA-SMCs (+45%; P < .001). Zymography showed an increase of active MMP-2 (62 kD) in media from AAA SMCs. AAA SMCs demonstrated twofold greater expression of MMP-2 messenger (m)RNA (P < .05) and 7.3-fold greater MMP-9 expression (P < .01) than NAA-SMCs. Culture with U937 monocytes caused a synergistic increase of elastolysis by AAA SMCs (41%; P < .001) but not NAA-SMCs or CEA-SMCs (P = .99). Coculture with U937 caused a large increase in MMP-9 mRNA in AAA-SMCs and NAA-SMCs (P < .001). MMP-2 mRNA expression was not affected. Western blots of culture media showed a fourfold increase of MMP-9 (92 kD) protein only in AAA-SMCs/U937 but not in NAA-SMCs/U937 (P < .001) and a large increase in active-MMP2 (62 kD), which was less apparent in NAA-SMCs/U937 media (P < .01). CONCLUSIONS: AAA-SMCs have a unique gene expression profile and a proelastolytic phenotype that is augmented by macrophages. This may occur by a failure of post-transcriptional control of MMP-9 synthesis.

Novel mechanism of aortic aneurysm development in mice associated with smoking and leukocytes.

OBJECTIVE: The purpose of this study was to evaluate potential mechanisms promoting abdominal aortic aneurysm development with tobacco smoke (TS) exposure. METHODS AND RESULTS: Experiments used the elastase perfusion model of abdominal aortic aneurysms with smoke-free controls. The effect of TS exposure was evaluated in C57/Bl6 mice, after broad-spectrum matrix metalloproteinase inhibition with doxycycline and in mice deficient in matrix metalloproteinase-9, matrix metalloproteinase-12, Cathepsin-S, and Neutrophil Elastase. Preparations of washed marrow, spleen, and peripheral blood leukocytes were transferred to smoke-free mice from 6-week TS-exposed mice or smoke-free mice. All mice were euthanized 14 days after elastase perfusion, and the percentage of change in aortic diameter (%Δ aortic diameter) was calculated. Electron microscopy of aortic tissue from animals exposed to TS without elastase exposure did not demonstrate any ultrastructural changes. Neither doxycycline nor any specific elastase deficiency was effective at preventing an increase in %Δ aortic diameter in TS-exposed animals. Smoke exposure for 6 weeks increased the %Δ aortic diameter after a smoke-free interval of up to 6 weeks before elastase perfusion. Leukocyte preparations from TS-exposed mice localized to abdominal aortic aneurysms and increased the %Δ aortic diameter in smoke-free mice. CONCLUSIONS: The effect of TS on the development of abdominal aortic aneurysms is not dependent on the activity of elastolytic enzymes and persists for long periods despite cessation of TS. Alterations in leukocyte response to aortic injury appear to mediate this effect.

Complement-dependent neutrophil recruitment is critical for the development of elastase-induced abdominal aortic aneurysm.

BACKGROUND: We previously established that neutrophils play a critical role in the development of experimental abdominal aortic aneurysm (AAA). The signal that initiates the influx of neutrophils to the aortic wall, however, remains unknown. In this study, we tested the hypothesis that complement participates in the development of AAA by providing the necessary chemotactic signal that recruits neutrophils to the aortic wall. METHODS AND RESULTS: Using an elastase-induced model of AAA, we showed that pretreatment of C57BL/6 mice with cobra venom factor, which depleted serum of complement activity, protected mice from AAA development. Whereas control mice exhibited a mean aortic diameter of 156+/-2% on day 14 after elastase perfusion, mice treated with cobra venom factor exhibited a mean aortic diameter of 90+/-4% (P<0.001). Examination of mice deficient in factor B further indicated that the alternative pathway of complement played a major role in this process (mean aortic diameter of 105+/-4% in factor B-deficient mice, P<0.001 compared with controls). Activation of the alternative pathway led to generation of the anaphylatoxins C3a and C5a, which recruited neutrophils to the aortic wall. Moreover, antagonism of both C3a and C5a activity was required to block AAA, which suggests that each can independently promote the aneurysmal phenotype. In addition, we demonstrated that complement alternative-pathway involvement was not restricted to this experimental model but was also evident in human AAAs. CONCLUSIONS: The identification of involvement of the complement system in the pathophysiology of AAA provides a new target for therapeutic intervention in this common disease.

Critical role of mast cell chymase in mouse abdominal aortic aneurysm formation.

BACKGROUND: Mast cell chymase may participate in the pathogenesis of human abdominal aortic aneurysm (AAA), yet a direct contribution of this serine protease to AAA formation remains unknown. METHODS AND RESULTS: Human AAA lesions had high numbers of chymase-immunoreactive mast cells. Serum chymase level correlated with AAA growth rate (P=0.009) in a prospective clinical study. In experimental AAA produced by aortic elastase perfusion in wild-type (WT) mice or those deficient in the chymase ortholog mouse mast cell protease-4 (mMCP-4) or deficient in mMCP-5 (Mcpt4(-/-), Mcpt5(-/-)), Mcpt4(-/-) but not Mcpt5(-/-) had reduced AAA formation 14 days after elastase perfusion. Even 8 weeks after perfusion, aortic expansion in Mcpt4(-/-) mice fell by 50% compared with that of the WT mice (P=0.0003). AAA lesions in Mcpt4(-/-) mice had fewer inflammatory cells and less apoptosis, angiogenesis, and elastin fragmentation than those of WT mice. Although Kit(W-sh/W-sh) mice had protection from AAA formation, reconstitution with mast cells from WT mice, but not those from Mcpt4(-/-) mice, partially restored the AAA phenotype. Mechanistic studies suggested that mMCP-4 regulates expression and activation of cysteine protease cathepsins, elastin degradation, angiogenesis, and vascular cell apoptosis. CONCLUSIONS: High chymase-positive mast cell content in human AAA lesions, greatly reduced AAA formation in Mcpt4(-/-) mice, and significant correlation of serum chymase levels with human AAA expansion rate suggests participation of mast cell chymase in the progression of human and mouse AAA.

Mast cells modulate the pathogenesis of elastase-induced abdominal aortic aneurysms in mice.

Abdominal aortic aneurysm (AAA), an inflammatory disease, involves leukocyte recruitment, immune responses, inflammatory cytokine production, vascular remodeling, neovascularization, and vascular cell apoptosis, all of which contribute to aortic dilatation. This study demonstrates that mast cells, key participants in human allergic immunity, participate in AAA pathogenesis in mice. Mast cells were found to accumulate in murine AAA lesions. Mast cell-deficient KitW-sh/KitW-sh mice failed to develop AAA elicited by elastase perfusion or periaortic chemical injury. KitW-sh/KitW-sh mice had reduced aortic expansion and internal elastic lamina degradation; decreased numbers of macrophages, CD3+ T lymphocytes, SMCs, apoptotic cells, and CD31+ microvessels; and decreased levels of aortic tissue IL-6 and IFN-gamma. Activation of mast cells in WT mice via C48/80 injection resulted in enhanced AAA growth while mast cell stabilization with disodium cromoglycate diminished AAA formation. Mechanistic studies demonstrated that mast cells participated in angiogenesis, aortic SMC apoptosis, and matrix-degrading protease expression. Reconstitution of KitW-sh/KitW-sh mice with bone marrow-derived mast cells from WT or TNF-alpha-/- mice, but not from IL-6-/- or IFN-gamma-/- mice, caused susceptibility to AAA formation to be regained. These results demonstrate that mast cells participate in AAA pathogenesis in mice by releasing proinflammatory cytokines IL-6 and IFN-gamma, which may induce aortic SMC apoptosis, matrix-degrading protease expression, and vascular wall remodeling, important hallmarks of arterial aneurysms.

The creation of an infrarenal aneurysm within the native abdominal aorta of swine.

BACKGROUND: Models of native abdominal aortic aneurysms (AAA) have been created in rodents using elastase and calcium chloride perfusion. These models, however, do not permit the evaluation of endovascular devices. This study describes the use of mechanical and enzymatic techniques to create native AAA in swine. METHODS: Surgically exposed abdominal aortas of ten male Yorkshire swine (25-35 kg) were dilated, then perfused for 20 min with a 50-mL solution of elastase (30 units) and collagenase (8000 units). Serial magnetic resonance imaging (MRI) at 1, 3, and 6 wk was used to evaluate postoperative aortic diameter. Animals were euthanized at 24 h, 48 h and 1, 2, and 6 wk for histological evaluation. RESULTS: MRI demonstrated an increase in mean aortic diameter by 73.3% +/- 30.2% (33.3-116.7%), which gradually increased postoperatively. Partial endothelial loss, mural neutrophil infiltrate, and elastin disruption were evident (1, 3, and 7 days). Smooth muscle cell attrition occurred within the inner tunica media (7 days). Collagen deposition, limited SMC repopulation and luminal reendothelialization appeared at 3-6 wk. Elastin injury persisted. CONCLUSIONS: The creation of an infrarenal aneurysm is possible within the native aorta of swine. After aneurysm creation, progressive increase in aortic diameter was detectable. Further evaluation will be necessary to more completely characterize the nature and extent of elastase-induced porcine aortic aneurysmal degeneration.

Neutrophil depletion inhibits experimental abdominal aortic aneurysm formation.

BACKGROUND: Neutrophils may be an important source of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9), two matrix-degrading enzymes thought to be critical in the formation of an abdominal aortic aneurysm (AAA). The purpose of this investigation was to test the hypothesis that neutrophil depletion would limit experimental AAA formation by altering one or both of these enzymes. METHODS AND RESULTS: Control, rabbit serum-treated (RS; n=27) or anti-neutrophil-antibody-treated (anti-PMN; n=25) C57BL/6 mice underwent aortic elastase perfusion to induce experimental aneurysms. Anti-PMN-treated mice became neutropenic (mean, 349 cells/microL), experiencing an 84% decrease in the circulating absolute neutrophil count (P<0.001) before elastase perfusion. Fourteen days after elastase perfusion, control mice exhibited a mean aortic diameter (AD) increase of 104+/-14% (P<0.0001), and 67% developed AAAs, whereas anti-PMN-treated mice exhibited a mean AD increase of 42+/-33%, with 8% developing AAAs. The control group also had increased tissue neutrophils (20.3 versus 8.6 cells per 5 high-powered fields [HPFs]; P=0.02) and macrophages (6.1 versus 2.1 cells per 5 HPFs, P=0.005) as compared with anti-PMN-treated mice. There were no differences in monocyte chemotactic protein-1 or macrophage inflammatory protein-1alpha chemokine levels between groups by enzyme-linked immunosorbent assay. Neutrophil collagenase (MMP-8) expression was detected only in the 14-day control mice, with increased MMP-8 protein levels by Western blotting (P=0.017), and MMP-8-positive neutrophils were seen almost exclusively in this group. Conversely, there were no statistical differences in MMP-2 or MMP-9 mRNA expression, protein levels, enzyme activity, or immunostaining patterns between groups. When C57BL/6 wild-type (n=15) and MMP-8-deficient mice (n=17) were subjected to elastase perfusion, however, ADs at 14 days were no different in size (134+/-7.9% versus 154+/-9.9%; P=0.603), which suggests that MMP-8 serves only as a marker for the presence of neutrophils and is not critical for AAA formation. CONCLUSIONS: Circulating neutrophils are an important initial component of experimental AAA formation. Neutrophil depletion inhibits AAA development through a non-MMP-2/9-mediated mechanism associated with attenuated inflammatory cell recruitment.

Pathophysiology of abdominal aortic aneurysms: insights from the elastase-induced model in mice with different genetic backgrounds.

Abdominal aortic aneurysms (AAAs) represent a complex degenerative disorder involving chronic aortic wall inflammation and destructive remodeling of structural connective tissue. Studies using human AAA tissues have helped identify a variety of molecular mediators and matrix-degrading proteinases, which contribute to aneurysm disease, thereby providing a sound foundation for understanding AAAs; however, these human tissue specimens represent only the "end stage" of a long and progressive disease process. Further progress in understanding the pathophysiology of AAAs is therefore dependent in part on the development and application of effective animal models that recapitulate key aspects of the disease. Based on original studies in rats, transient perfusion of the abdominal aorta with porcine pancreatic elastase has provided a reproducible and robust model of AAAs. More recent applications of this model to mice have also opened new avenues for investigation. In this review, we summarize investigations using the elastase-induced mouse model of AAAs including results in animals with targeted deletion of specific genes and more general differences in mice on different genetic backgrounds. These studies have helped us identify genes that are essential to the development of AAAs (such as MMP9, IL6, and AT1R) and to reveal other genes that may be dispensable in aneurysm formation. Investigations on mice from different genetic backgrounds are also beginning to offer a novel approach to evaluate the genetic basis for susceptibility to aneurysm development.

Accelerated enlargement of experimental abdominal aortic aneurysms in a mouse model of chronic cigarette smoke exposure.

BACKGROUND: Cigarette smoking and pulmonary emphysema are strongly associated with abdominal aortic aneurysms (AAAs), but the biologic mechanisms linking these conditions are undefined. STUDY DESIGN: To determine if exposure to cigarette smoke influences formation and growth of experimental AAAs, 129/SvEv mice were acclimated to daily cigarette smoke exposure for 2 weeks followed by transient elastase perfusion of the abdominal aorta to induce aneurysmal degeneration. Smoking was continued for intervals of either 2 or 12 weeks (8 mice per group). Nonsmoking 129/SvEv controls (n = 29) underwent elastase perfusion and followup evaluation at the same time intervals. In all animals, abdominal aortic diameter (AD) was measured to determine interval increases in AD (Delta AD), with AAAs defined as a Delta AD > 100%. RESULTS: Preperfusion and immediate postperfusion ADs were not significantly different between experimental groups. Aneurysmal dilatation was present 2 weeks after elastase perfusion in both smoking mice and nonsmoking controls, with no significant difference in final AD (mean +/- SEM: smoking, 1.23 +/- 0.11 mm versus nonsmoking, 1.22 +/- 0.05 mm). There were also no differences in the overall extent of aortic dilatation (Delta AD smoking, 136 +/- 24% versus nonsmoking, 138 +/- 10%), or the incidence of AAAs (smoking, 75% versus nonsmoking, 79%). Although all animals had developed AAAs by 12 weeks after elastase perfusion, the overall extent of aortic dilatation was 50% greater in smoking mice compared with nonsmoking controls (Delta AD smoking, 204 +/- 23% versus nonsmoking, 135 +/- 17%; p < 0.05). CONCLUSIONS: Short-term exposure to cigarette smoke did not alter initial development of experimental AAAs, but chronic smoke exposure was associated with a substantial increase in the late progression of aneurysmal dilatation. This novel combination of in vivo experimental models offers a new approach to investigate mechanisms by which cigarette smoking promotes aneurysmal degeneration.

Periadventitial adipose-derived stem cell treatment halts elastase-induced abdominal aortic aneurysm progression.

AIM: Demonstrate that periadventitial delivery of adipose-derived mesenchymal stem cells (ADMSCs) slows aneurysm progression in an established murine elastase-perfusion model of abdominal aortic aneurysm (AAA). MATERIALS & METHODS: AAAs were induced in C57BL/6 mice using porcine elastase. During elastase perfusion, a delivery device consisting of a subcutaneous port, tubing and porous scaffold was implanted. Five days after elastase perfusion, 100,000 ADMSCs were delivered through the port to the aorta. After sacrifice at day 14, analyzed metrics included aortic diameter and structure of aortic elastin. RESULTS: ADMSC treated aneurysms had a smaller diameter and less fragmented elastin versus saline controls. CONCLUSION: Periadventitial stem cell delivery prevented the expansion of an established aneurysm between days 5 and 14 after elastase perfusion.

Effect of novel limited-spectrum MMP inhibitor XL784 in abdominal aortic aneurysms.

BACKGROUND: Inhibiting the growth of small abdominal aortic aneurysms (AAAs) is a clinically valuable goal and fills an important therapeutic void. Based on studies in animals and humans, inhibition of the activity of elastolytic matrix metalloproteinases (MMPs) has the potential to slow AAA expansion and limit morbidity and the need for surgery. Previous attempts to make use of the synthetic MMP inhibitors in the treatment of chronic conditions have been limited by intolerable side effects. The limited-spectrum synthetic MMP inhibitor, XL784, was well tolerated and devoid of side-effects associated with other nonspecific MMP inhibitors in phase I studies. We hypothesized that clinically relevant doses of XL784 would be effective at inhibiting aneurysm development in a mouse model. METHODS: The 14-day elastase-perfusion model of AAA in mice was used. An initial screening study of XL784 (50 [n = 17], 125 [n = 17], and 250 mg/kg [n = 18]) administered via gavage daily until harvest. Controls received diluent alone (n = 18) or doxycycline in drinking water (n = 19). Aortic diameter was measured pre-perfusion (AD(pre)) and at harvest (AD(har)). A second study used XL784 (250 [n = 9]; 375 [n = 9], and 500 mg/kg [n = 14]) and diluent alone (n = 9) administered via gavage. The percentage dilatation [%ΔAD = [(AD(har) - AD(pre))/AD(pre)] ×100] was calculated and elastin and inflammatory content was scored. RESULTS: All mice tolerated the treatments similarly. Control mice all developed aneurysms with a mean %ΔAD of 158.5% ± 4.3%. Treatment with all doses of XL784 and doxycycline were effective in inhibiting aortic dilatation. There was a clear dose-response relationship between XL784 and reductions in aortic dilatation at harvest (50 mg/kg 140.4% ± 3.2%; 125 mg/kg 129.3% ± 5.1%; 250 mg/kg 119.2% ± 3.5%; all Ps < .01 compared to control). This continued with the higher doses (375 mg/kg 88.6% ± 4.4%; 500 mg/kg 76.0% ± 3.5%). The highest 2 doses of XL784 tested were more effective than doxycycline (112.2% ± 2.0%, P < .05) in inhibiting maximal dilatation of the aorta after elastase perfusion.

Cigarette smoking increases aortic dilatation without affecting matrix metalloproteinase-9 and -12 expression in a modified mouse model of aneurysm formation.

OBJECTIVE: The development of abdominal aortic aneurysms (AAA) is presumed to result from multiple genetic and environmental factors, with exposure to tobacco smoke the single largest known factor predisposing to aneurysm growth. We have attempted to adapt the elastase-perfused animal model to determine whether tobacco exposure can lower the threshold of aortic injury necessary for AAA development. METHODS: Adult C57BL/6 mice underwent transient perfusion of the infrarenal aorta with an active solution of elastase: high-dose (HDE, 0.19 U/mL, n=9), standard-dose (SDE, 0.16 U/mL, n=21) or low-dose (LDE, 0.07 U/mL, n=24). Control animals (n=24) were treated with heat inactivated elastase (HIE). Twenty LDE perfused mice were exposed to cigarette smoke (LDE-S) beginning 2 weeks before perfusion and continuing until aortic harvest. Aortic diameter (AD) was measured preperfusion, postperfusion, and at harvest on day 14. AAA was defined as %DeltaAD>or=100% between preperfusion and harvest. Aortas from each group (except HDE) were analyzed for matrix metalloproteinase-9 (MMP-9) and MMP-12 expression by real-time polymerase chain reaction normalized to glyceraldehyde-3-phosphate dehydrogenase. RESULTS: All SDE mice developed large AAA by %DeltaAD (189.3%+/-16.9%, mean+/-standard error of the mean), but control mice had only a small dilatation (69.7%+/-3.7%, P<.01). Higher doses of elastase did not produce larger aneurysms in HDE mice. In contrast, only 63% of LDE mice showed aneurysmal dilatation, and these were significantly smaller (104.3%+/-4.2%, P<.01). When exposed to cigarette smoke, LDE animals developed significantly larger aneurysms (%DeltaAD, 134.5%+/-7.9%, P=.0021). There was no difference in normalized aortic MMP-9 and MMP-12 expression between elastase doses or between smoke-exposed and unexposed animals. Histologic analysis revealed that smoking increased the extent of aortic elastin degradation when compared with LDE-S animals. CONCLUSION: Aneurysm development in the elastase model is dependent on the quantity of active elastase infused. Exposure of animals to tobacco smoke after a relatively minor aortic elastase injury produces increases in elastin degradation and aneurysm size without affecting MMP-9 or MMP-12 expression. To our knowledge, this is the first demonstration in an animal model that smoking can act as a synergistic factor in AAA development. Further understanding of the relationship between smoking and AAA in this model may help unveil the pathophysiologic pathways involved between cigarette smoke and AAAs.

Critical role of dipeptidyl peptidase I in neutrophil recruitment during the development of experimental abdominal aortic aneurysms.

Dipeptidyl peptidase I (DPPI) is a lysosomal cysteine protease critical for the activation of granule-associated serine proteases, including neutrophil elastase, cathepsin G, and proteinase 3. DPPI and granule-associated serine proteases have been shown to play a key role in regulating neutrophil recruitment at sites of inflammation. It has recently been suggested that neutrophils and neutrophil-associated proteases may also be important in the development and progression of abdominal aortic aneurysms (AAAs), a common vascular disease associated with chronic inflammation and destructive remodeling of aortic wall connective tissue. Here we show that mice with a loss-of-function mutation in DPPI are resistant to the development of elastase-induced experimental AAAs. This is in part because of diminished recruitment of neutrophils to the elastase-injured aortic wall and impaired local production of CXC-chemokine ligand (CXCL) 2. Furthermore, adoptive transfer of wild-type neutrophils is sufficient to restore susceptibility to AAAs in DPPI-deficient mice, as well as aortic wall expression of CXCL2. In addition, in vivo blockade of CXCL2 by using neutralizing antibodies directed against its cognate receptor leads to a significant reduction in aortic dilatation. These findings suggest that DPPI and/or granule-associated serine proteases are necessary for neutrophil recruitment into the diseased aorta and that these proteases act to amplify vascular wall inflammation that leads to AAAs.

Oral administration of diferuloylmethane (curcumin) suppresses proinflammatory cytokines and destructive connective tissue remodeling in experimental abdominal aortic aneurysms.

Chronic transmural inflammation and proteolytic destruction of medial elastin are key mechanisms in the development of abdominal aortic aneurysms (AAAs). Diferuloylmethane (curcumin) is a major component of the food additive tumeric, which has been shown to have anti-inflammatory properties. To determine if ingestion of curcumin influences aneurysmal degeneration, C57Bl/6 mice underwent transient elastase perfusion of the abdominal aorta to induce the development of AAAs, followed by daily oral gavage with 100 mg/kg curcumin (n = 36) or water alone (n = 31). By 14 days, mice in the control group developed a mean increase in aortic diameter of 162.8 +/- 4.6% along with a dense mononuclear inflammation and destruction of medial elastin. By comparison, the mean increase in aortic diameter in the curcumin-treated group was only 133.2 +/- 5.2% (p < 0.0001). Although aortic wall inflammation was similar between the groups, the structural integrity of medial elastin was significantly greater in curcumin-treated mice. Curcumin-treated mice also exhibited relative decreases in aortic tissue activator protein-1 and nuclear factor kappaB DNA binding activities and significantly lower aortic tissue concentrations of interleukin-1beta (IL-1beta), IL-6, monocyte chemoattractant protein-1, and matrix metalloproteinase-9 (all p < 0.05). These data demonstrate for the first time that oral administration of curcumin can suppress the development of experimental AAAs, along with structural preservation of medial elastin fibers and reduced aortic wall expression of several cytokines, chemokines, and proteinases known to mediate aneurysmal degeneration. The possibility that dietary ingestion of curcumin may have a beneficial effect in degenerative aortic aneurysms warrants further consideration.

Temporal changes in mouse aortic wall gene expression during the development of elastase-induced abdominal aortic aneurysms.

OBJECTIVE: To characterize temporal changes in mouse aortic wall gene expression associated with the development of experimental abdominal aortic aneurysms. METHODS: C57BL/6 mice underwent transient perfusion of the abdominal aorta with either elastase (n = 61) or heat-inactivated elastase as a control (n = 68). Triplicate samples of radiolabeled aortic wall complementary DNA were prepared at intervals of 0, 3, 7, 10, and 14 days, followed by hybridization to nylon microarrays (1181 genes). Autoradiographic intensity data were normalized by conversion to z scores, and differences in gene expression were defined by two-tailed z tests at a significance threshold of P < .01. RESULTS: Elastase perfusion caused a progressive increase in aortic diameter up to 14 days accompanied by transmural inflammation and destructive remodeling of the elastic media. No aneurysms occurred in the control group. Compared with healthy aorta, 336 genes exhibited significant alterations during at least 1 interval after elastase perfusion (135 at more than 1 interval and 14 at all intervals), with pronounced increases for interleukin 6, cyclin E2, interleukin 1beta, osteopontin, CD14/lipopolysaccharide receptor, P-selectin glycoprotein ligand 1, and gelatinase B/matrix metalloproteinase 9 (all >20-fold on day 3). Sixty-two genes exhibited synchronous alterations in the elastase and control groups, thus suggesting a nonspecific response. By direct comparisons between the elastase and control groups, there were 384 genes with significant differences in expression for at least 1 interval after aortic perfusion, including 234 with differential upregulation (eg, p44MAPK/ERK1, osteopontin, heat shock protein 84, hypoxia-inducible factor 1alpha, apolipoprotein E, monocyte chemotactic protein 3, MIG (monokine induced by gamma interferon), and interleukin 2 receptor gamma) and 163 with differential downregulation (eg, prothrombin, granzyme B, ataxia telangiectasia mutated, and interleukin-converting enzyme). CONCLUSIONS: Development of elastase-induced abdominal aortic aneurysms in mice is accompanied by altered aortic wall expression of genes associated with acute and chronic inflammation, matrix degradation, and vascular tissue remodeling. Knowledge of these alterations will facilitate further studies on the functional molecular mechanisms that underlie aneurysmal degeneration.

Localized administration of doxycycline suppresses aortic dilatation in an experimental mouse model of abdominal aortic aneurysm.

Treatment with doxycycline suppresses the development of abdominal aortic aneurysms (AAAs) in experimental animal models, but its use in humans can be accompanied by dose-related side effects. We sought to determine if localized administration of doxycycline can achieve inhibition of AAAs equivalent to that achieved by systemic treatment. C57BL/6 mice underwent transient elastase perfusion of the abdominal aorta to induce the development of AAAs. After 14 days, the mean increase in aortic diameter was reduced from 167.2+/-7.8% in untreated mice to only 129.7+/-13.8% in mice treated with 100 mg/kg/day oral doxycycline (p<0.05). Using osmotic minipumps to provide continuous periaortic infusion of doxycycline, localized infusion at rates of 0.75 to 1.0 mg/kg/day suppressed AAAs to an equivalent or even greater extent than systemic treatment [mean increase in aortic diameter 131.5+/-14.4% at 0.75 mg/kg/day, p<0.05; 103.2+/-13.5% at 1.0 mg/kg/day, p<0.01). Mean plasma doxycycline levels reached 332+/- 62 ng/mL during oral administration, but the drug was undetectable in the circulation during localized infusion. The doxycycline concentration in aortic tissue extracts was 22+/- 6 ng/mL during systemic treatment compared to only 5.6+/- 2.2 ng/mL [at 0.75 mg/kg/day] and 7.8+/- 4.0 ng/mL [at 1.0 mg/kg/day] during localized infusion (p<0.05). Localized administration of doxycycline can effectively suppress experimental AAAs with undetectable plasma drug levels, even at doses 100-fold lower than those used during oral drug administration. Localized delivery of doxycycline holds promise as a novel strategy to inhibit the progressive expansion of aortic aneurysms, perhaps as a pharmacological adjunct to endovascular (stent graft) treatment.

Suppression of experimental abdominal aortic aneurysms in mice by treatment with pyrrolidine dithiocarbamate, an antioxidant inhibitor of nuclear factor-kappaB.

OBJECTIVE: Proinflammatory cytokines and matrix metalloproteinases (MMPs) are prominent mediators of the connective tissue destruction that characterizes abdominal aortic aneurysms (AAAs), and nuclear factor (NF)-kappaB is a cytokine-responsive transcription factor that promotes macrophage MMP expression. The purpose of this study was to determine whether aneurysmal degeneration is influenced by pyrrolidine dithiocarbamate (PDTC), a pharmacologic inhibitor of NF-kappaB. METHODS: Adult male C57BL/6 mice underwent transient elastase perfusion of the abdominal aorta to induce the development of AAAs. Animals were treated every 48 hours by intraperitoneal injection with either saline (n = 34) or PDTC 20 mg/kg (n = 49). Aortic diameter (AD) measurements were used to determine the extent of aortic dilatation before and immediately after elastase perfusion and again at day 14. RESULTS: All saline-treated mice developed AAAs associated with mononuclear inflammation and destruction of medial elastin (overall increase in AD, mean +/- SEM, 169.1% +/- 7.5%). In contrast, the incidence of AAAs was only 63% in PDTC-treated mice, with a reduction in the overall increase in AD to 109.8% +/- 4.2% ( P < .0001 vs saline), decreased inflammation, and structural preservation of aortic wall connective tissue. Although aneurysm development in saline-treated mice was associated with a marked increase in aortic tissue NF-kappaB and activator protein 1 DNA-binding activities, both activities were substantially reduced in PDTC-treated animals. PDTC-treated mice also exhibited significantly lower serum and aortic wall concentrations of interleukin 1beta and interleukin 6, as well as lower amounts of aortic wall MMP-9, as compared with saline-treated controls. CONCLUSIONS: Treatment with PDTC inhibits elastase-induced experimental AAAs in the mouse, along with suppression of aortic wall NF-kappaB and activator protein 1 transcription factor activities, reduced expression of proinflammatory cytokines, and suppression of MMP-9. NF-kappaB is therefore a potentially important therapeutic target for the suppression of aneurysmal degeneration. CLINICAL RELEVANCE: Development and progression of human AAAs is associated with inflammation and enzymatic degradation of connective tissue proteins. MMP-9 is one of the enzymes involved in aneurysm disease, and its production may be induced in part by activation of the transcription factor NF-kappaB. In this mouse model, treatment with pyrrolidine dithiocarbamate (a pharmacologic inhibitor of NF-kappaB) acted to suppress MMP-9 and aneurysm development. It is hoped that treatment strategies that target NF-kappaB may eventually be shown to suppress the growth of small aortic aneurysms in patients.

Treatment with simvastatin suppresses the development of experimental abdominal aortic aneurysms in normal and hypercholesterolemic mice.

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 (DeltaAD) was determined with AAAs defined as DeltaAD >100%. RESULTS: Wild-type mice treated with simvastatin exhibited a 21% reduction in DeltaAD 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 DeltaAD 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.