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Developmentof anovelmurinemodelofaortic aneurysms using peri-adventitialelastaseCastigliano M. Bhamidipati, DO, PhD, MSc,a Gaurav S. Mehta, MBBS,a Guanyi Lu, MD, PhD,b

Christopher W. Moehle, MS,a Carlos Barbery, BS,a Paul D. DiMusto, MD,c Adriana Laser, MD,c

Irving L. Kron, MD,a Gilbert R. Upchurch, Jr, MD,b and Gorav Ailawadi, MD,a Charlottesville, VA, andAnn Arbor, MI

Background. Our aim was to establish a novel model of abdominal aortic aneurysms (AAA) in miceusing application of peri-adventitial elastase.Methods. C57BL/6J male mice underwent infrarenal peri-adventitial application of either (1) sodiumchloride (control; n = 7), (2) porcine pancreatic elastase (PPE; n = 14), or (3) PPE and doxycycline(PPE + doxycycline 200 mg/kg; n = 11) for 14 days. Aortas were analyzed by video micrometry,immunohistochemistry, qualitative polymerase chain reaction, and zymography. Groups underwentMann–Whitney U comparisons.Results. At day 14 compared with baseline, control animals had minimal aortic dilation, whereasfusiform aneurysms were seen in PPE (control, 20 ± 3%; PPE, 82 ± 15%; P # .003). Doxycyclineabrogated aneurysm formation (PPE, 82 ± 15%; PPE + doxycycline, 37 ± 10%; P # .03). Comparedwith control and PPE + doxycycline, immunohistochemistry demonstrated greater elastin fiber degra-dation, macrophage infiltration, and matrix metalloproteinase-9 expression in PPE. Ki-67 and cleavedcaspase-3 were lower in control versus PPE. The loss of smooth muscle marker expression seen with PPEwas preserved in PPE + doxycycline. Zymography confirmed that both MMP-2 and -9 were more active inPPE than PPE + doxycycline.Conclusion. Peri-adventitial application of elastase is a simple, reproducible in vivo model of aneurysmformation leading to consistent infrarenal aortic aneurysm development by day 14, with inflammatory cellinfiltration and MMP upregulation. Doxycycline inhibits AAA progression in this model via limitingmatrix degradation and preserving differentiated smooth muscle cells. (Surgery 2012;152:238-46.)

From the Divisions of Thoracic and Cardiovascular Surgery,a and Vascular and Endovascular Surgery,b

Department of Surgery, University of Virginia School of Medicine, Charlottesville, VA; and the Department ofSurgery,c University of Michigan School of Medicine, Ann Arbor, MI

ABDOMINAL AORTIC ANEURYSMS (AAA) contribute to>15,000 operations a year in the United States,and is the thirteenth leading cause of death amongmen.1 The matrix of the abdominal aorta consists

ed by grants T32/ HL007849 (C.M.B.), R01/ HL081629), K08/HL098560 (G.A.) from theNationalHeart, Lung,d Institute. The content is solely the responsibility of theanddoesnotnecessarily represent theofficial views of thel Heart, Lung, and Blood Institute or the National Insti-Health. Supported by the Thoracic Surgery Foundationarch and Education Research Grant (G.A.).

d at the 6th Academic Surgical Congress, HuntingtonA.

d for publication February 10, 2012.

requests: Gorav Ailawadi, MD, Department of Surgery,ty of Virginia Health System, P.O. Box 800679, Charlot-VA 22908. E-mail: gorav@virginia.edu.

60/$ - see front matter

Mosby, Inc. All rights reserved.

016/j.surg.2012.02.010

URGERY

of collagen and elastin that degrades, and arethought to contribute to aneurysm formation viaseveral mechanisms.2-4 The destruction of the con-stitutive layers of the aortic wall by inflammatoryand mesenchymal cell infiltration, which leads tothe loss of smooth muscle cell function, are charac-teristic hallmarks of aneurysm formation.

Several small animal models of aortic aneurysmexist to help investigators study these mechanismsof disease.5 The ideal model to study aneurysmsshould allow a thorough evaluation of medial layerdegeneration, degradation by chronic inflamma-tory components, and mural thrombus contribu-tion to aneurysm formation. This type of uniquein vivo animal model that replicates human aneu-rysms has myriad redundant pathways, and makesfocused study challenging. As such, investigatorshave utilized combinations of isolated surgicaland/or toxic manipulation in vivo injury modelsand in vitro assays to understand the role of various

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inflammatory and smooth muscle cells in aneu-rysms. A landmark contribution by Anidjar et al6

first described the elastase perfusion model inrats in 1990 and documented similar chronic in-flammatory infiltrate as seen in humans. Thismodel requires in vivo isolation of the abdominalaorta, cannulation of the aorta to administer elas-tase, and careful repair of the aortotomy. Severalmodifications of the original rat model have al-lowed for adoption and use in transgenic andknockout mice.7,8

The elastase perfusion model has allowed muchof our understanding of the mechanisms leadingto AAA. This perfusion model, however, has limi-tations in that it is technically demanding with asteep learning curve. Specifically, isolation of themouse aorta can be difficult with the adjacentinferior vena cava and small lumbar branch vessels.Moreover, there can be variability in vessel injuryowing to variations in pressure during perfusionwith elastase. Additionally, careful aortotomy clo-sure while avoiding stenosis to reestablish ante-grade blood flow is crucial. These maneuvers canbe difficult to teach and may have limited its use toselect laboratories. Thus, a simpler model thatfacilitates aneurysm formation reproducibility thatis less challenging technically would provide aunique opportunity to augment aneurysm re-search. Herein, we describe a novel, simple, andefficient peri-adventitial elastase application in vivomurine aortic aneurysm model, and evaluate theprotective effect of doxycycline, a known potentmatrix metalloproteinase (MMP) inhibitor.9

METHODS

Murine peri-adventitial elastase model. Thismodel was initially conceived and validated byDr. Guanyi Lu while at the University of Michigan.Briefly, 8- to 12-week-old male C57BL/6J mice(WT, Jackson Laboratories, Bar Harbor, ME) thatweighed between 20 and 28 g were assignedrandomly to groups. Singly housed animals wereexposed to a 12-hour day–night cycle in 50%humidity and 708F temperature controlled rooms,and fed standard chow ad libitum (Harlan Labo-ratories, Indianapolis, IN).

We developed the model of peri-adventitialelastase application for experimental AAA forma-tion (Fig 1), where animals first underwent intra-peritoneal 75 mg/kg ketamine and 1 mg/kgmedetomidine anesthesia. A laparotomy was per-formed, and the abdominal aorta from just belowthe left renal vein to the iliac bifurcation was iden-tified. The abdominal aorta was isolated in situ af-ter retroperitoneal reflection. Circumferential

exposure of the infrarenal abdominal aorta wasachieved after careful dissection, and de novobranches of the aorta were left in situ. After ana-tomic identification, the aorta was bathed in either10 mL of 0.9% NaCl (control) or 100% porcinepancreatic elastase (PPE; Sigma-Aldrich Co., St.Louis, MO) for 10 minutes. After elastase expo-sure, the abdominal contents were replaced, thefascial layers were closed with interrupted 6-0coated undyed polyglactin 910 (Ethicon Inc., Som-erville, NJ), and skin with interrupted 6-0 polypro-pylene suture (Ethicon Inc.), after which micereceived 0.1 mg/kg subcutaneous buprenorphine,intraperitoneal 1 mg/kg atipamezole, and were re-covered after 1 mL subcutaneous 0.9% NaCladministration.

Video micrometry measurements of the aorticwall diameter were performed in situ before ap-plication, after application, and at the time ofharvest using a Q-Color3 Optical Camera (Olym-pus Corp., Center Valley, PA) attached to a LeicaMZ12 stereomicroscope (Leica Microsystems, Ban-nockburn, IL) using QCapture 6.0 Pro Software(QImaging Inc., Surrey, Canada). The entireinfrarenal aorta was removed at the time of har-vest. Aortas were divided and analyzed by histologyor immunohistochemistry, and gelatin zymogra-phy. Animal care and use were in accordance withthe Guide for the Care and Use of Laboratory Animals.10

Animal protocols were approved by the Universityof Virginia Institutional Animal Care and UseCommittee (#3634). Elastase–time associationswere anticipated to be reproducible with targetedaortic dilation between 30% and 40% when 10mL of elastase was applied to the abdominal aortafor 10 minutes (Fig 1, B).

Doxycycline treatment. Animals treated by dox-ycycline (PPE + doxycycline) were given 200 mg/kg oral doxycycline (Sigma Aldrich, Inc.) dissolvedin de-ionized water administered through photo-sensitive bottles, and changed every 48 hours asdescribed.11 Residual volumes were logged to rec-ord homogenous consumption across the group;no animals were excluded from analysis based ondoxycycline intake.

Histology and immunohistochemistry. Murineaortas were harvested at killing for histologicanalysis after left ventricular injection of 4% par-aformaldehyde solution in phosphate-buffered sa-line. Further fixation was achieved by overnightimmersion in 4% paraformaldehyde at 48C, andafter paraffin embedding, blocks were sectioned at5 mm. Every 10th section underwent screeninghematoxylin and eosin staining to identify theregion of interest (AAA) among individual

Fig 1. (A) The peri-adventitial elastase application model. The infra-renal murine aorta is first circumferentially dis-sected, and 10 mL of 100% PPE is topically applied to the adventitial aorta for 10 minutes. The aorta is monitored closelyfor phenotypic changes after which the animal is closed and recovered for 14 days. The AAA at day 14 is explanted foranalysis. The in situ, na€ıve aorta at the beginning of the procedure has a fibrous matrix (solid white arrows). (B) Effects ofelastase and time-dependent exposure on aortic dilation. Nonlinear regression curves for elastase volume and time ofexposure where intersected with targeted aortic dilation between 30% and 40%. When considered with visual aorticchanges during the operation, the most reproducible results were anticipated when 10 mL of elastase was applied for10 minutes.

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animals. Once the aneurysm segment was identi-fied, slide sections underwent serial immunohisto-chemistry staining.

After microwave antigen retrieval, antibodieswere bound and detected using VectaStain EliteKit (Vector Laboratories Inc., Burlingame, CA).Modified Russell-Movat Pentachrome (Movat) forelastin layers, and immunohistochemical stainingwere completed. Antibodies for immunohisto-chemical staining were anti-Mac2 for macrophages(Cedarlane Laboratories, Burlington, Canada),

anti–MMP-9 for MMP-9 (R&D Systems, Minneap-olis, MN), anti-SMaA (14A) for smooth muscle a-actin (Santa Cruz Biotechnology Inc., Santa Cruz,CA), anti–MHC-SM1 for smooth muscle myosinheavy chain (Kamiya Biomedical Company, Seattle,WA), anti–Ki-67 (M-19) for cell proliferation(Santa Cruz Biotechnology Inc.), and anti-cleavedcaspase-3 (Asp175) for apoptosis (Cell SignalingTechnology, Danvers, MA). Enzymatic color devel-opment was completed using diaminobenzidine(Dako Corporation, Carpentaria, CA). Appropriate

Fig 2. (A) Progression of aneurysm formation after peri-adventitial elastase application. The aorta at 5 minutesafter PPE application (A) develops wall thinning andearly qualitative changes. At 10 minutes after PPE appli-cation (B), the aorta develops an enriched red colora-tion, as the matrix seems to be visually degraded. Theinfrarenal murine abdominal aorta at 14 days afterPPE application, has a sheen that is noted with fusiformchanges (C). (B) Relative change in aortic diameterabove baseline. The relative change in the aortic diame-ter over baseline is highest in the PPE group. An aneu-rysm was defined as an increase in the aortic diameterby $50% above baseline. *P = .03; **P = .003.

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controls verified staining procedures, and imageswere acquired using AxioCam 4.6 Software with a103 objective on an AxioCamMRc (Carl Zeiss Inc.,Thornwood, NY). Threshold color gated imagequantification of the positive signal within an areaof interest was calculated in immunostained sec-tions using Image Pro Plus (Media Cybernetics Inc.,Bethesda, MD).12 Relative proportions of positivesignals were compared between groups.

Gelatin zymography. Snap-frozen murine aorticaneurysm samples were analyzed by gelatin zymog-raphy. Protein was extracted after harvest using1 mol/L tris (hydroxymethyl) aminomethanebuffer pH 7.5, and incubated for 30 minutes at378C, and concentration was determined usingBCA protein assay kit (Thermo Scientific, Rock-ford, IL). Electrophoresis was completed using0.8% gelatin in a 10% sodium dodecyl sulfatepolyacrylamide gel (SDS-PAGE) using equivalentvolume of each fraction. Enzymatic activity wasvisualized as negative staining with CoomassieBrilliant Blue R-250 (Thermo Scientific). Relativedensitometry analysis adjusted for background, oflytic bands indicating MMP activity was performedusing Gel DocXR+ System with a charge-coupleddevice camera and Image Lab software (BioRadLaboratories, Inc., Hercules, CA).

Statistical analysis. Pair-wise comparisons wereexamined after undergoing outlier evaluation asdefined by Grubbs.13 Data underwent unpairednonparametric analysis, with 2-tail probabilities atan alpha of 0.05 considered statistically significant.All calculations were performed using GraphPadPrism 5 (GraphPad Software Inc., La Jolla, CA).

RESULTS

Fusiform aneurysmal formation with elastaseapplication to adventitia of aorta. At the beginningof the operative procedure, the aorta was exposedafter retroperitoneal separation. With peri-adventitial elastase application, there was a notableand visible change within the initial 3–5 minutes inthe aortic wall with increasing exposure to elastase(Fig 2, A). During this period, the aorta was mon-itored for phenotypic changes, where adventitialdigestion was noted. The vessel sheath that enve-lopes the inferior vena cava and the distal aortaseparated after elastase exposure with minimal dis-section. Additionally, the aorta dilated by nearly30–40% after elastase application, which was thenfollowed subsequently by aneurysmal dilationover 14 days. Specifically, at the end of 10 minutes(10.4 ± 0.5), once 10 mL (9.4 ± 0.6 mL) of 100%elastase had bathed the abdominal aorta, early fu-siform dilation (33 ± 2% dilation over baseline)

was evident (Fig 2, B). This degree of initial injuryacross the elastase groups ensured adequate expo-sure, and was comparable to the elastase perfusionmodel from our laboratory.4

Peri-adventitial elastase creates reproducibleaneurysms. At day 14 compared with baseline,control animals exposed to saline had a 20 ± 3%increase in aortic diameter, whereas PPE animalsexposed to elastase had an 82 ± 15% increase inaortic size with 60% incidence (Fig 2, B). Addition-ally, compared with PPE mice, animals exposed toPPE + doxycycline for 14 days developed less aorticdilation (37 ± 10%; P = .03).

Fig 3. (A) Modified Russell-Movat Pentachrome (Movat). Movat staining (103) revealed degraded elastin and smoothmuscle layer(s) with PPE application (solid white arrows on anterior surface of aorta) that were preserved among doxy-cycline treated animals. (B) Activated macrophages (Mac2). Mac2 stain (103) show increased presence with elastaseadministration (solid white arrows). Activated macrophages are reduced with doxycycline treatment. (C) Matrixmetalloproteinase-9 (MMP-9). Total MMP-9 stain (103) shows increased presence with elastase administration. MMP-9 expression is attenuated with doxycycline treatment at day 14. (D) Quantified immunostaining for activated macro-phages (Mac2) and matrix metalloproteinase-9 (MMP-9). Threshold gated quantification of immunohistochemistryin aortic sections for Mac2 and MMP-9 show increased presence of macrophages, and MMP-9 at day 14 in the aorticwall after elastase administration. *P < .05; **P < .01.

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Peri-adventitial elastase application has greateraortic wall degradation, which is limited by doxy-cycline treatment.Modified Russell-Movat Pentach-rome stain in control animals showed preservedelastin fibers, which were degraded after PPEexposure (Fig 3, A). Importantly, degeneration ofelastin fibers was prevented with doxycycline treat-ment. The anterior surface of the aorta seems tohave greater elastic degradation than the posterioraspect of the aorta.

Immunostaining for activated macrophageswith Mac2 and total MMP in control animals hadlesser expression of inflammatory markers thanPPE (Fig 3, B and C). Importantly, doxycyclinetreatment attenuated macrophage infiltration andtotal MMP expression (Fig 3, D). Similarly, stainingfor smooth muscle marker proteins SM-22a, SMaA,and MHC-SM1 showed greater anterior smoothmuscle loss with elastase application (Fig 4, A–C).

Ki-67 protein to assess the active phase(s) of thecell cycle were higher in the PPE group comparedwith PPE + doxycycline (Fig 4, D). Caspase-3 is aninactive zymogen that, after apoptotic signaling,is cleaved by an initiator caspase, and this cleavedcaspase-3 expression that denotes apoptosis was in-creased from baseline only in PPE (Fig 4, E). Over-all, doxycycline treated mice had luminal smooth

muscle cell preservation similar to control animals(Fig 4, F).

Gelatin zymography confirms higher active-MMP-2 and –MMP-9 activity with elastase applica-tion. SDS-PAGE gel electrophoresis showed littleMMP activity in control animals, whereas moreintense MMP activity in PPE exposed mice wasnoted, and was attenuated after doxycyclinetreatment (Fig 5, A). Relative densitometry ofzymograms showed that compared with PPE, bothpro–MMP-9 (P = .049) and active MMP-9 (P = .04)were decreased after doxycycline treatment(Fig 5, B). There were no differences in pro–MMP-2 expression among groups (Fig 5, B). Active- MMP-2 was greater after PPE application and decreasedwith doxycycline treatment (Fig 5, B).

DISCUSSION

This study describes a novel experimental modelof aneurysm formation and evaluates the protectiveeffect of doxycycline as seen in other in vivo modelsof AAAs.9,14-19 Our model achieved aneurysmformation by degradation of the elastic lamina, in-creased presence of activated macrophages, re-duced smooth muscle protein expression, andincreased activeMMP activity as seen in other exper-imental models of aneurysm formation. Moreover,

Fig 4. (A) Smooth muscle 22-a (SM22a). SM22a immunostain (103) shows that the smooth muscle layer are preservedamong doxycycline-treated animals. The solid white arrows are on the anterior surface of the aorta. (B) Smooth muscle a-Actin (SMaA). SMaA immunostain (103) shows increased smooth muscle degradation after topical elastase exposure(solid white arrow), which is decreased after treatment with doxycycline. (C) myosin heavy chain (MHC-SM1). MHC-SM1immunostain (103) shows increased smooth muscle degradation after topical elastase exposure, which is reduced aftertreatment with doxycycline. (D) Ki-67. Ki-67 immunostaining (103) for cell proliferation, was greater in PPE group afterperi-adventitial elastase application as shown by the arrows and decreased with doxycycline treatment. (E) Cleavedcaspase-3 (Caspase). Caspase staining (103) showed increased apoptosis from baseline after peri-adventitial elastase ap-plication as highlighted by the solid black arrows. (F) Quantified immunostaining for smooth muscle 22-a (SM22a),Smooth muscle a-Actin (SMaA), myosin heavy chain (MHC-SM1), and antigen Ki-67. Threshold-gated quantificationof immunohistochemistry in aortic sections for smooth muscle cell markers were reduced among elastase administeredanimals. Cellular proliferation with marked increase in Ki-67 protein was noted with peri-adventitial elastase application.**P < .01; ***P < .001.

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themarginally increased and expected apoptosis onthe anterior surface of the aorta confirms that thecurrent model does not overexpose the aorta totoxic levels of pancreatic elastase with destructionof the adventitial surface andmechanical aortic dila-tion. Additionally, cell proliferation was higher inthe PPE-exposed, group as seen in small human aor-tic aneurysms, which supports the idea that inflam-matory cells, fibroblasts, and smooth muscle cellsare active around the adventitial wall and likelyhelp to remodel the aorta. The fusiform aneurysmsformed by this peri-adventitial application modelaremore consistent with those seen inhumanAAAs.

Matrix degradation enzymes are an importantcontributor to the study of aortic aneurysms, and inthe current model, elastase exposure led to in-creased active MMP activity. Historical models of

aneurysm research including the ‘‘Blotchy mousemodel,’’ acetrizoate-induced destruction of the me-dial layer of the aorta, and disruption of smoothmuscle cells during development by theophylline orb-aminopropionitrile have been supplanted withmore sophisticated murine in vivo models. More-over, de novo neutrophil elastasemay be involved inthe pathogenesis of aneurysms in humans, giventhe loss of elastic tissue and increased elastolyticactivity in the media of human aneurysms. Thesechanges suggest that the pathobiology of aorticaneurysms involve elastolysis of the aortic mediaand that elastase plays a major role in the destruc-tion of elastin within the aortic wall. The pressurecomponent of the perfusion model is importantwhen pancreatic elastase is administered, becausepressure perfusion with saline alone has been

Fig 5. (A) Gelatin zymography for MMP-2 and 9. Gelatin zymography was performed as described with Coomassie bril-liant blue for visualization of lytic bands. Faint bands are seen in the control and doxycycline-treated animals, whereasPPE-treated animals had intense bands. Relative densitometry was completed on all groups. B, Relative densitometry ofgelatin zymograms showed differences in protein expression between groups. Relative densitometry adjusting for back-ground influence shows that PPE exposure increases pro-MPP9 expression, which is attenuated with doxycycline treat-ment (A). Similarly, cleaving of the MMP-9 protein to the active form was greater after PPE (B). Doxycycline treatmentdecreased active MMP-9 expression. (C, D) Although pro–MMP-2 expression is not different between groups, the activeform of the protein is expressed in greater amounts after PPE, whereas doxycycline limited active MMP-2 expression.*P < .05; **P < .01.

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shown not to influence morphometry.2,4 Further-more, the use of elastase experimentally, more sothan papain, collagenase, trypsin, and chymotryp-sin, is critical to macrophage activation and subse-quent medial degeneration.6,20

Technically, the challenges in the well-established elastase perfusion model of excludingevery aortic branch in the infrarenal aorta by liga-tion, cannulating a 0.4-mm murine aorta, andsubsequently closing the aortotomy without creat-ing stenosis has limited the potential for widespreadadoption to select institutions. Although we haveextensive experience with the elastase perfusionmodel in our laboratory,2,4 we believe that there wasa real need for a less complex and reproduciblemodel of aortic aneurysm formation. For example,an important step in the elastase perfusion modelinvolves separating the distal inferior vena cavafrom the aorta to pass a ligature around the aorta fa-cilitating the stabilization of a retrograde perfusion

cannula. Our approach does not require individualvessel isolation or ligation, is easily teachable, anddoes not involve manipulation of the great vessels.Importantly, postoperative hind paw paralysis,which is lethal, is completely avoided.

Other murine models of experimental aorticaneurysms exist, including adventitial CaCl2 andangiotensin II infusion. These models provide cre-dence to the need for developing simpler experi-mental injury methods of aneurysm formation.These models have important limitations, specifi-cally the peri-aortic application of CaCl2 createsvery modest aneurysms at 4 weeks with only30–50% dilation without complete penetrance.5 Arecent combination procedure combining intralu-minal elastase and adventitial CaCl2 has been toutedto be a less difficult and reproducible rat model ofaneurysms.21 However, adaptation to the mousemodel is awaited. An alternate model whereosmotic pump delivered angiotensin II infusion is

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performed in apolipoprotein E knockout mice, re-sults in an aortic dissection with prominent throm-bus that is distinct from the laminated versionseen in mature human disease.22 Because thismodel is in an already genetically altered mouse, in-vestigations that require an additional genetic dele-tion become very challenging to perform.

Physiologically, our novel model presented hereis better suited to study the adventitial contribu-tion to aneurysm disease because elastase-induceddegradation of the abluminal surface inducesinflammatory homing. The prevailing theory dur-ing aneurysm development is that aneurysms de-velop owing to an inflammatory infiltrate thatinitiates at the adventitia. In the current study, weshow that adventitial inflammation is present sim-ilar to human disease. Both MMP-2 and -9 work inconcert to produce aortic aneurysms.23 MMP-2 isthought to contribute in large part in small aorticaneurysms and MMP-9 is believed to play a majorrole in larger aneurysms.24 Preventing smaller an-eurysms from disease progression is an importantclinical event that shown some promise in humaninvestigations.25,26 MMP-knockout mice have beenresistant to aneurysm formation,27 linking a cen-tral role of their contribution to disease progres-sion.2,28,29 Doxycycline, a potent MMP inhibitor,has been shown in multiple studies to prevent mu-rine aneurysm formation and progression.14,19 Weutilized this known mechanism of aneurysm treat-ment to evaluate our model and found that orallyadministering doxycycline to animals, attenuatedaneurysm development at 2 weeks.

This study has several limitations. First, the lackof atherosclerosis and intraluminal thrombus,which potentially contribute to human aneurysmaldisease are unaccounted; however, these are notseen in the elastase perfusion model either. Sec-ond, the dosing and incubation strategies are notconsistent compared with the other models ofaneurysmal disease, albeit optimized by multipledose response and time dependent preliminarywork. As such, varying stock elastase concentra-tions can influence the degree of injury. Finally,there are no comparisons against the conventionalelastase perfusion model presented, and so differ-ences in species, gender, or mechanism are notfully explained, although they are under investiga-tion by our laboratory.

The authors thank Melissa H. Bevard and John M.Sanders, Laboratory Research Specialist(s), HistologyCore, Robert M. Berne Cardiovascular Research Center,University of Virginia Health System, Charlottesville, VA,for their guidance in completing the immunostaining.

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