Murine abdominal aortic aneurysm model by orthotopic allograft transplantation of elastase-treated abdominal aorta Zhenjie Liu, MD, PhD, Qiwei Wang, MS, Jun Ren, MS, Carmel Rebecca Assa, Stephanie Morgan, PhD, Jasmine Giles, BS, Qi Han, BS, and Bo Liu, PhD, Madison, Wisc Objective: Murine models have proved instrumental in studying various aspects of abdominal aortic aneurysm (AAA), from identification of underlying pathophysiologic changes to the development of novel therapeutic strategies. In the current study, we describe a new model in which an elastase-treated donor aorta is transplanted to a recipient mouse and allowed to progress to aneurysm. We hypothesized that by transplanting an elastase-treated abdominal aorta of one genotype to a recipient mouse of a different genotype, one can differentiate pathophysiologic factors that are intrinsic to the aortic wall from those stemming from circulation and other organs. Methods: Elastase-treated aorta was transplanted to the infrarenal abdominal aorta of recipient mice by end-to-side microsurgical anastomosis. Heat-inactivated elastase-treated aorta was used as a control. Syngeneic transplants were performed with use of 12-week-old C57BL/6 littermates. Transplant grafts were harvested from recipient mice on day 7 or day 14 after surgery. The aneurysm outcome was measured by aortic expansion, elastin degradation, proinflammatory cytokine expression, and inflammatory cell infiltration and compared with that produced with the established, conventional elastase infusion model. Results: The surgical technique success rate was 75.6%, and the 14-day survival rate was 51.1%. By day 14 after surgery, all of the elastase-treated transplanted abdominal aortas had dilated and progressed to AAAs, defined as 100% or more increase in the maximal external diameter compared with that measured before elastase perfusion, whereas none of the transplanted aortas pretreated with inactive elastase became aneurysmal (percentage increase in maximum aortic diameter: 159.36% 6 23.27%, transplanted elastase, vs 41.46% 6 9.34%, transplanted inactive elastase). Aneurysm parameters, including elastin degradation and infiltration of macrophages and T lymphocytes, were found to be identical to those observed in the conventional elastase model. Quantitative polymerase chain reaction analysis revealed similarly increased levels of proinflammatory cytokines (relative changes of mRNA in the conventional elastase model vs transplant model: tumor necrosis factor a, 1.71 6 0.27 vs 2.93 6 0.86; monocyte chemoattractant protein 1, 2.36 6 0.58 vs 2.87 6 0.51; chemokine (C-C motif) ligand 5, 3.37 6 0.92 vs 3.46 6 0.83; and interferon g, 3.09 6 0.83 vs 5.30 6 1.69). Using green fluorescent protein transgenic mice as donors or recipients, we demonstrated that a small quantity of mononuclear leukocytes in the transplant grafts bared the genotype of the donors. Conclusions: Transplanted elastase-treated abdominal aorta could develop to aneurysm in recipient mice. This AAA transplant model can be used to examine how the microenvironment of a transplanted aneurysmal aorta may be altered by the contributions of the “global” environment of the recipient. (J Vasc Surg 2014;-:1-8.) Clinical Relevance: The elastase-induced mouse model of abdominal aortic aneurysm (AAA) has been widely accepted because of its pathologic similarities to human AAAs. In this study, we aimed to develop a permutation of this model to explore how particular cell types and molecular signaling pathways contribute to AAA. Using an orthotopic allograft transplantation model, we provide a method to elucidate etiopathogenetic mechanisms of AAA formation and to explore new therapeutic possibilities.
From the Division of Vascular Surgery, Department of Surgery, University of WisconsineMadison. This work was supported by the National Institutes of Health, United States R01HL088447 (B.L.). Author conflict of interest: none. Additional material for this article may be found online at www.jvascsurg.org. Reprint requests: Bo Liu, PhD, Department of Surgery, University of WisconsineMadison, 1111 Highland Ave, WIMR 5137, Madison, WI 53705 (e-mail: [email protected]). The editors and reviewers of this article have no relevant financial relationships to disclose per the JVS policy that requires reviewers to decline review of any manuscript for which they may have a conflict of interest. 0741-5214/$36.00 Copyright Ó 2014 by the Society for Vascular Surgery. http://dx.doi.org/10.1016/j.jvs.2014.05.019
Abdominal aortic aneurysm (AAA) is a common disease with a high prevalence of death.1,2 Whereas significant advances have been made in aneurysm imaging as well as in surgical intervention methods, effective pharmacologic treatments have remained elusive. Aggressive management of hypertension and hyperlipidemia is recommended in patients with AAA; however, therapies for these conditions have no proven effects on aneurysm growth and rupture.3 Elective aneurysm repair is generally not recommended for AAAs with a diameter .05; and 159.36% 6 23.27% vs 162.55% 6 21.44% at day 14, P > .05; Fig 2). These results suggest that the transplantation procedure itself has little impact on aneurysm development. Histopathologic characterization. Allografts harvested 7 or 14 days after surgery were used for histologic examination. Measurement of aortic cross sections confirmed luminal dilation regardless of transplantation in elastasetreated tissues. The aortic walls of transplanted elastasetreated and conventional elastase groups consistently displayed thinner medial layers but expanded adventitia
compared with aortas treated with inactivated elastase (the transplanted inactive elastase-treated and conventional inactivated elastase groups) (Supplementary Fig 2, online only). The elastase-induced degeneration and disruption of elastic lamina as well as the paucity of medial smooth muscle cells (SMCs) were similar between the transplanted elastase-treated and conventional elastase groups (Supplementary Fig 2, online only). There was no obvious intimal proliferation in either abdominal aortic allograft group (Supplementary Fig 2, online only). We examined vascular inflammation by immunohistochemical analysis with antibodies against CD3 (T-lymphocyte marker) and CD68 (macrophage marker). Similar to what has been reported in the conventional elastase model, abundant T-lymphocyte and macrophage populations were detected in adventitia and media of elastase-treated transplant grafts (Fig 3). Collectively, our data indicated that the orthotopic abdominal aortic transplant model produced aneurysmal dilation and histologic characteristics similar to those that have been observed in the conventional elastase model. Cytokine expression. We further compared the inflammatory response in the elastase-treated aorta grafts with that measured in the conventional elastase model by measuring local production of proinflammatory cytokines. Two weeks after transplantation, mRNA levels of tumor
JOURNAL OF VASCULAR SURGERY Volume -, Number -
Liu et al 5
Fig 3. Macrophage (CD68þ cells) and lymphocyte (CD3þ cells) infiltration in elastase-treated abdominal aortas (E) and transplanted elastase-treated abdominal aortas (Transplanted E) 7 days after surgery. Scale bar ¼ 50 mm. Quantification of macrophage and lymphocyte infiltration in aneurysm tissues as identified by CD68 and CD3 stains, respectively, expressed as CD68þ or CD3þ cells/nuclei. NS, Not significant, n ¼ 5.
necrosis factor a, monocyte chemoattractant protein 1, chemokine (C-C motif) ligand 5, and interferon g were significantly upregulated in the elastase-treated transplanted aorta compared with the inactive elastase-treated transplanted aorta (P < .05; Fig 4). The upregulation of those proinflammatory cytokines was similar to that observed in the conventional elastase model. Origins of the inflammatory cells. To demonstrate that our newly developed transplant model can be carried out between mice of different genotypes, we transplanted elastase-treated abdominal arteries from wild-type mice to GFP mice and vice versa. Both sets of transplant cohorts survived, and aortic grafts developed aneurysms. As shown in Fig 5, A, when wild-type aortas were transplanted to GFP recipients, the majority of medial cells in the grafts were negative of GFP. The converse was observed in the GFP to wild-type transplant cohort (Fig 5, B). As expected, the majority of CD68þ macrophages carried the genotype of the recipients. However, in both transplant cohorts, we did identify a small quantity of CD68þ cells that bared the genotype of the donor, demonstrating that some macrophages arose from a residential population rather than from the circulation (Fig 5, C). DISCUSSION Orthotopic transplantation of abdominal aorta, thoracic aortic arch, and carotid artery was established decades ago,19-26 but these procedures were designed to study chronic rejection, allograft vasculopathy, or atherosclerosis.
Orthotopic abdominal aorta transplantation has not been reported in studies of aneurysm. Thus, we developed a murine AAA model by orthotopic allograft transplantation of elastase-treated abdominal aorta. Data from our macroscopic and microscopic analyses indicate that this transplant AAA model displayed pathologic characteristics analogous to those of the established mouse elastase perfusion AAA model. The principal advantages of this transplant AAA model include the following: it generates a rapid-forming aneurysm through a well-established chemical induction; it permits exploitation of the rich genetic resources of murine species; it allows transplantation between donors and recipients of different genotypes; and it enables identification of cell origin without subjecting recipient mice to irradiation. Different from the thoracic aortic arch transplantation,19,27 anastomosis of the orthotopic abdominal aorta transplantation is more challenging because of the smaller diameter of the mouse abdominal aorta. Even under adequate magnification, the surgery requires highly skilled maneuvers. However, with a skilled operator, a high experimental success rate could be achieved. In our current study, the overall survival rate was higher than 70%. Two major causes of death were bleeding during the operation and postoperative aortic thrombosis, which could result in hind limb paralysis, followed by rapid deterioration to death within 72 hours. In our study, all elastase-treated transplanted abdominal aortas (six of six) became dilated and progressed to aneurysm on day 14 after surgery. We attribute the cause of aneurysm of aorta grafts to the elastase infusion because none of the aorta grafts that were pretreated with heat-
6 Liu et al
JOURNAL OF VASCULAR SURGERY --- 2014
Fig 4. Transplanted elastase-induced abdominal aortic aneurysm (AAA) had an inflammatory phenotype similar to that of the conventional elastase-treated AAA. At 14 days after surgery, the AAA tissues were harvested from different experimental groups: inactive elastase-treated (IE, n ¼ 8), transplanted inactive elastase-treated (Transplanted IE, n ¼ 6), elastase-treated (E, n ¼ 6), and transplanted elastase-treated (Transplanted E, n ¼ 7). Relative fold changes of mRNA levels were determined by real-time polymerase chain reaction (PCR). Data are represented as mean 6 standard error of the mean. *P < .05 (E vs IE, transplanted E vs transplanted IE). CCL5, Chemokine (C-C motif) ligand 5; IFN-g, interferon g; MCP-1, monocyte chemoattractant protein 1; NS, not significant; TNF-a, tumor necrosis factor a.
inactivated elastase developed aneurysm. On macroscopic and microscopic examination, the transplanted grafts exhibited morphologic features including progressive aortic dilation, disruption of elastic fibers, and vascular inflammation that resembled those of the murine elastase perfusion AAA model. In fact, we did not observe a statistically significant difference between our transplant model and the conventional elastase model in any of the parameters measured, suggesting that the transplant procedure alone has no significant impact on aneurysm development. We observed an abundant and diffuse infiltration of lymphocytes and mononuclear cells in the intima, media, and adventitial layers. In the media, elastic fibers were disrupted, and SMCs manifested focal degeneration. A considerable amount of monocyte and lymphocyte
infiltration was observed in the adventitia. The absence of intimal proliferation and thickening in these abdominal aortic allografts is different from carotid allograft transplantation and the murine thoracic aortic transplant model of chronic rejection22 but similar to isograft transplantation.28-30 We speculate that the proinflammatory microenvironment elicited by the elastase treatment favors cell death over proliferation. Allograft vasculopathy is caused by chronic immunemediated injury to the transplanted vasculature, leading to intimal SMC accumulation, luminal narrowing, and eventual ischemic graft failure.31 However, in our transplant AAA model, we observed inflammatory cell infiltration without intimal hyperplasia, SMC accumulation, or stenosis. This may be attributed to syngeneic transplantation or the
JOURNAL OF VASCULAR SURGERY Volume -, Number -
Liu et al 7
Fig 5. Both recipient- and donor-derived macrophages are present in elastase-treated transplant grafts. Elastase-treated abdominal aorta segments were allografted from C57B6 mice into GFPþ C57B6 mice (A) and vice versa (B). Representative confocal immunofluorescence images for macrophage marker CD68 (red) and GFP (green) in allografts 7 days after transplantation. Nuclei stained with DAPI (blue). The yellow arrowheads indicate CD68 and GFP double positive cells. GFP, Green fluorescent protein; Lu, lumen; WT, wild type. Scale bar ¼ 100 mm. C, Quantification of GFPþ/CD68þ or GFP/CD68þ cells in transplanted elastase-treated aneurysms, n ¼ 5.
short follow-up time in this model. Many rodent studies have shown that recipient cells repopulate allografts.19,32-34 Hagensen et al35 proved the recipient source of endothelial cells and SMCs in allograft to be the flanking segments of the recipient vasculature. Although we found that the majority of the mononuclear leukocytes were recruited from the recipient, a small but notable quantity of mononuclear leukocytes carried donor markers. These results suggest that in addition to circulating cells, a certain population of vascular wall resident cells, such as monocyte progenitors, may also differentiate to leukocytes and then proliferate or undergo self-renewal, thus contributing to vascular inflammation in AAA disease.36 The origin of macrophages or macrophage-like cells in atherosclerotic lesions is an actively pursued topic. In addition to bone marrow-derived myeloid cells, other cell types including SMCs are suggested to give rise to at least a portion of macrophage-like cells.37 Phenotypic switching of SMCs to a synthetic or proinflammatory state has recently been noted in mouse models of cerebral aneurysm as well as AAA,38-40 which may offer an alternative explanation for these donor-derived CD68þ cells. Finally, the GFPþ macrophages in the GFP to wild-type transplant group might have acquired their green fluorescent signals by engulfing apoptotic SMCs within the donor aorta. However, this is unlikely because we did not find evidence of phagocytosis, for example, multiple nuclei in a single cell. The reported elastase-induced transplantation AAA model has several limitations. Similar to the established elastase infusion AAA model, our transplant model lacks several prominent features of the human lesion, such as atherosclerosis and intraluminal thrombosis. It would be interesting to determine whether these atherosclerosislike features could be depicted if apolipoprotein E genee deficient mice are used as donors or recipients. Another limitation is that this model does not enable the identification of precise origin of recipient-derived cells.
CONCLUSIONS We have developed a murine AAA model by orthotopic transplantation of elastase-treated abdominal aorta. This new model is a rapid and feasible tool to study the pathophysiologic mechanism of AAA. As an alternative approach to BMC transplantation, the aorta transplant AAA model avoids irradiation and thus may have a higher survival rate. This is important, particularly considering the high lethality in many transgenic mouse lines after irradiation. The authors would like to thank Dr Greenspan for providing the microscope video recording system for this study. AUTHOR CONTRIBUTIONS Conception and design: ZL, QW, SM, BL Analysis and interpretation: ZL, QW, JR, CA, BL Data collection: ZL, QW, JR, CA, JG, QH Writing the article: ZL, QW, BL Critical revision of the article: ZL, QW, JR, CA, SM, BL Final approval of the article: ZL, QW, BL Statistical analysis: ZL, QW Obtained funding: BL Overall responsibility: BL ZL and QW contributed equally to this article and share co-first authorship. REFERENCES 1. Baxter BT, Terrin MC, Dalman RL. Medical management of small abdominal aortic aneurysms. Circulation 2008;117:1883-9. 2. Powell JT, Brady AR. Detection, management, and prospects for the medical treatment of small abdominal aortic aneurysms. Arterioscler Thromb Vasc Biol 2004;24:241-5. 3. Weintraub NL. Understanding abdominal aortic aneurysm. N Engl J Med 2009;361:1114-6. 4. Mortality results for randomised controlled trial of early elective surgery or ultrasonographic surveillance for small abdominal aortic
8 Liu et al
5.
6. 7. 8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21. 22.
23.
aneurysms. The UK Small Aneurysm Trial Participants. Lancet 1998;352:1649-55. Lederle FA, Wilson SE, Johnson GR, Reinke DB, Littooy FN, Acher CW, et al. Immediate repair compared with surveillance of small abdominal aortic aneurysms. N Engl J Med 2002;346:1437-44. Daugherty A, Cassis LA. Mouse models of abdominal aortic aneurysms. Arterioscler Thromb Vasc Biol 2004;24:429-34. Thompson M, Cockerill G. Matrix metalloproteinase-2: the forgotten enzyme in aneurysm pathogenesis. Ann N Y Acad Sci 2006;1085:170-4. Thompson RW. Basic science of abdominal aortic aneurysms: emerging therapeutic strategies for an unresolved clinical problem. Curr Opin Cardiol 1996;11:504-18. Ishibashi S, Goldstein JL, Brown MS, Herz J, Burns DK. Massive xanthomatosis and atherosclerosis in cholesterol-fed low density lipoprotein receptor-negative mice. J Clin Invest 1994;93:1885-93. Prescott MF, Sawyer WK, Von Linden-Reed J, Jeune M, Chou M, Caplan SL, et al. Effect of matrix metalloproteinase inhibition on progression of atherosclerosis and aneurysm in LDL receptor-deficient mice overexpressing MMP-3, MMP-12, and MMP-13 and on restenosis in rats after balloon injury. Ann N Y Acad Sci 1999;878:179-90. Gertz SD, Kurgan A, Eisenberg D. Aneurysm of the rabbit common carotid artery induced by periarterial application of calcium chloride in vivo. J Clin Invest 1988;81:649-56. Daugherty A, Manning MW, Cassis LA. Antagonism of AT2 receptors augments angiotensin II-induced abdominal aortic aneurysms and atherosclerosis. Br J Pharmacol 2001;134:865-70. Trollope A, Moxon JV, Moran CS, Golledge J. Animal models of abdominal aortic aneurysm and their role in furthering management of human disease. Cardiovasc Pathol 2011;20:114-23. Pyo R, Lee JK, Shipley JM, Curci JA, Mao D, Ziporin SJ, et al. Targeted gene disruption of matrix metalloproteinase-9 (gelatinase B) suppresses development of experimental abdominal aortic aneurysms. J Clin Invest 2000;105:1641-9. Johnston WF, Salmon M, Su G, Lu G, Stone ML, Zhao Y, et al. Genetic and pharmacologic disruption of interleukin-1b signaling inhibits experimental aortic aneurysm formation. Arterioscler Thromb Vasc Biol 2013;33:294-304. Morgan S, Yamanouchi D, Harberg C, Wang Q, Keller M, Si Y, et al. Elevated protein kinase C-d contributes to aneurysm pathogenesis through stimulation of apoptosis and inflammatory signaling. Arterioscler Thromb Vasc Biol 2012;32:2493-502. Wang Q, Ren J, Morgan S, Liu Z, Dou C, Liu B. Monocyte chemoattractant protein-1 (MCP-1) regulates macrophage cytotoxicity in abdominal aortic aneurysm. PLoS One 2014;9:e92053. Thompson RW, Curci JA, Ennis TL, Mao D, Pagano MB, Pham CT. Pathophysiology of abdominal aortic aneurysms: insights from the elastase-induced model in mice with different genetic backgrounds. Ann N Y Acad Sci 2006;1085:59-73. Shimizu K, Sugiyama S, Aikawa M, Fukumoto Y, Rabkin E, Libby P, et al. Host bone-marrow cells are a source of donor intimal smoothmuscle-like cells in murine aortic transplant arteriopathy. Nat Med 2001;7:738-41. Dambrin C, Calise D, Pieraggi MT, Thiers JC, Thomsen M. Orthotopic aortic transplantation in mice: a new model of allograft arteriosclerosis. J Heart Lung Transplant 1999;18:946-51. Shi C, Russell ME, Bianchi C, Newell JB, Haber E. Murine model of accelerated transplant arteriosclerosis. Circ Res 1994;75:199-207. Koulack J, McAlister VC, Giacomantonio CA, Bitter-Suermann H, MacDonald AS, Lee TD. Development of a mouse aortic transplant model of chronic rejection. Microsurgery 1995;16:110-3. Sun H, Valdivia LA, Subbotin V, Aitouche A, Fung JJ, Starzl TE, et al. Improved surgical technique for the establishment of a murine model of aortic transplantation. Microsurgery 1998;18:368-71.
JOURNAL OF VASCULAR SURGERY --- 2014
24. Chereshnev I, Trogan E, Omerhodzic S, Itskovich V, Aguinaldo JG, Fayad ZA, et al. Mouse model of heterotopic aortic arch transplantation. J Surg Res 2003;111:171-6. 25. Calise D, Dambrin C, Labat A, Pieraggi MT, Pons F, Benoist H, et al. Orthotopic aortic transplantation in rodents by the sleeve technique: a model system for the study of graft vascular disease. Transplant Proc 2001;33:2369-70. 26. Ailawadi G, Eliason JL, Roelofs KJ, Sinha I, Hannawa KK, Kaldjian EP, et al. Gender differences in experimental aortic aneurysm formation. Arterioscler Thromb Vasc Biol 2004;24:2116-22. 27. Ye P, Chen W, Wu J, Huang X, Li J, Wang S, et al. GM-CSF contributes to aortic aneurysms resulting from SMAD3 deficiency. J Clin Invest 2013;123:2317-31. 28. Pucci AM, Forbes RD, Billingham ME. Pathologic features in longterm cardiac allografts. J Heart Transplant 1990;9:339-45. 29. Adams DH, Russell ME, Hancock WW, Sayegh MH, Wyner LR, Karnovsky MJ. Chronic rejection in experimental cardiac transplantation: studies in the Lewis-F344 model. Immunol Rev 1993;134:5-19. 30. Mennander A, Raisanen A, Paavonen T, Hayry P. Chronic rejection in the rat aortic allograft. V. Mechanism of the angiopeptin (BIM23014C) effect on the generation of allograft arteriosclerosis. Transplantation 1993;55:124-8. 31. Mitchell RN, Libby P. Vascular remodeling in transplant vasculopathy. Circ Res 2007;100:967-78. 32. Hu Y, Davison F, Ludewig B, Erdel M, Mayr M, Url M, et al. Smooth muscle cells in transplant atherosclerotic lesions are originated from recipients, but not bone marrow progenitor cells. Circulation 2002;106:1834-9. 33. Hu Y, Davison F, Zhang Z, Xu Q. Endothelial replacement and angiogenesis in arteriosclerotic lesions of allografts are contributed by circulating progenitor cells. Circulation 2003;108:3122-7. 34. Hillebrands JL, Klatter FA, van den Hurk BM, Popa ER, Nieuwenhuis P, Rozing J. Origin of neointimal endothelium and aactin-positive smooth muscle cells in transplant arteriosclerosis. J Clin Invest 2001;107:1411-22. 35. Hagensen MK, Shim J, Falk E, Bentzon JF. Flanking recipient vasculature, not circulating progenitor cells, contributes to endothelium and smooth muscle in murine allograft vasculopathy. Arterioscler Thromb Vasc Biol 2011;31:808-13. 36. Psaltis PJ, Harbuzariu A, Delacroix S, Witt TA, Holroyd EW, Spoon DB, et al. Identification of a monocyte-predisposed hierarchy of hematopoietic progenitor cells in the adventitia of postnatal murine aorta. Circulation 2012;125:592-603. 37. Gomez D, Owens GK. Smooth muscle cell phenotypic switching in atherosclerosis. Cardiovasc Res 2012;95:156-64. 38. Starke RM, Chalouhi N, Ding D, Raper DM, McKisic MS, Owens GK, et al. Vascular smooth muscle cells in cerebral aneurysm pathogenesis. Transl Stroke Res 2014;5:338-46. 39. Airhart N, Brownstein BH, Cobb JP, Schierding W, Arif B, Ennis TL, et al. Smooth muscle cells from abdominal aortic aneurysms are unique and can independently and synergistically degrade insoluble elastin [published online ahead of print September 27, 2013]. J Vasc Surg doi: 10.1016/j.jvs.2013.07.097. 40. Branchetti E, Poggio P, Sainger R, Shang E, Grau JB, Jackson BM, et al. Oxidative stress modulates vascular smooth muscle cell phenotype via CTGF in thoracic aortic aneurysm. Cardiovasc Res 2013;100:316-24.
Submitted Mar 11, 2014; accepted May 6, 2014.
Additional material for this article may be found online at www.jvascsurg.org.
JOURNAL OF VASCULAR SURGERY Volume -, Number -
Liu et al 8.e1
Supplementary Fig 1 (online only). A, Kaplan-Meier survival curve shows the survival rate of the first 45 elastasetreated orthotopic abdominal aortic transplants, which were divided into three groups in sequence: 1 to 15, 16 to 30, and 31 to 45. AAA, Abdominal aortic aneurysm. B, Analysis of the death causes. The intraoperative mortality is 24.4%, mainly due to bleeding. Other postoperative complications of this procedure include thrombosis of the aorta, aneurysm rupture, hypovolemic shock, and unknown causes.
Supplementary Fig 2 (online only). Hematoxylin and eosin (H&E) staining of arterial tissues 7 days after surgery from different groups: inactive elastase-treated abdominal aorta (IE), n ¼ 4; elastase-treated abdominal aorta (E), n ¼ 5; transplanted inactive elastase-treated abdominal aorta (TIE), n ¼ 3; and transplanted elastase-treated abdominal aorta (TE), n ¼ 3. van Gieson stain shows elastin layer degradation in representative treated arteries 7 days after surgery. Smooth muscle alpha actin (SMaA) immunohistochemical stain shows increased smooth muscle degradation of the elastase-treated abdominal aorta transplant, which is similar to elastase-treated aorta in the conventional model. Scale bar ¼ 50 mm.
JOURNAL OF VASCULAR SURGERY --- 2014
8.e2 Liu et al
Supplementary Table (online only). Microsurgical instruments for the murine elastase-treated abdominal aorta transplantation Instruments Metzenbaum scissors Vannas micro dissecting spring scissors 1, curved Vannas micro dissecting spring scissors 2, angled Desmarres eyelid retractors
Rhoton forceps Dean knife Vessel dilation forceps 1 Vessel dilation forceps 2 Vessel cannulation forceps Micro needle holder Tubing Sutures
Size
Quantity
Corporation
500 300 300 5.12500 ; blade size: 16 mm
1 1 1 1
Roboz Roboz Roboz Anthony Products, Inc
14 mm 12 mm
1 1 1 1 1 1 1 1 1
700 500 ; blade: 1 7 mm 120 mm curved 120 mm straight 131 mm 5.2500 .01100 inner diameter .02400 outer diameter Polyamide monofilament: 9-0 MET 3/8R 140 mm 11-0 MET 3/8R 70 mm Nylon monofilament: 6-0, 15 mm 3/8C Silk braided: 6-0, 20 mm 3/8C
Catalog No. RS-6010SC RS-5621 RS-5619 60-05-13E
Roboz Roboz Roboz Roboz Roboz BRI Roboz
60-05-12E 60-05-11E RS-5262 RS-6220 RS-4927 RS-4929 RS-4990 SKU20-1050 IN-10
AROSurgical AROSurgical Hope Medical
LOT0001147503 LOT0411164003 LOT1205073
Hope Medical
LOT1205073
From the Division of Vascular Surgery, Department of Surgery, University of WisconsineMadison. This work was supported by the National Institutes of Health, United States R01HL088447 (B.L.). Author conflict of interest: none. Additional material for this article may be found online at www.jvascsurg.org. Reprint requests: Bo Liu, PhD, Department of Surgery, University of WisconsineMadison, 1111 Highland Ave, WIMR 5137, Madison, WI 53705 (e-mail: [email protected]). The editors and reviewers of this article have no relevant financial relationships to disclose per the JVS policy that requires reviewers to decline review of any manuscript for which they may have a conflict of interest. 0741-5214/$36.00 Copyright Ó 2014 by the Society for Vascular Surgery. http://dx.doi.org/10.1016/j.jvs.2014.05.019
Abdominal aortic aneurysm (AAA) is a common disease with a high prevalence of death.1,2 Whereas significant advances have been made in aneurysm imaging as well as in surgical intervention methods, effective pharmacologic treatments have remained elusive. Aggressive management of hypertension and hyperlipidemia is recommended in patients with AAA; however, therapies for these conditions have no proven effects on aneurysm growth and rupture.3 Elective aneurysm repair is generally not recommended for AAAs with a diameter .05; and 159.36% 6 23.27% vs 162.55% 6 21.44% at day 14, P > .05; Fig 2). These results suggest that the transplantation procedure itself has little impact on aneurysm development. Histopathologic characterization. Allografts harvested 7 or 14 days after surgery were used for histologic examination. Measurement of aortic cross sections confirmed luminal dilation regardless of transplantation in elastasetreated tissues. The aortic walls of transplanted elastasetreated and conventional elastase groups consistently displayed thinner medial layers but expanded adventitia
compared with aortas treated with inactivated elastase (the transplanted inactive elastase-treated and conventional inactivated elastase groups) (Supplementary Fig 2, online only). The elastase-induced degeneration and disruption of elastic lamina as well as the paucity of medial smooth muscle cells (SMCs) were similar between the transplanted elastase-treated and conventional elastase groups (Supplementary Fig 2, online only). There was no obvious intimal proliferation in either abdominal aortic allograft group (Supplementary Fig 2, online only). We examined vascular inflammation by immunohistochemical analysis with antibodies against CD3 (T-lymphocyte marker) and CD68 (macrophage marker). Similar to what has been reported in the conventional elastase model, abundant T-lymphocyte and macrophage populations were detected in adventitia and media of elastase-treated transplant grafts (Fig 3). Collectively, our data indicated that the orthotopic abdominal aortic transplant model produced aneurysmal dilation and histologic characteristics similar to those that have been observed in the conventional elastase model. Cytokine expression. We further compared the inflammatory response in the elastase-treated aorta grafts with that measured in the conventional elastase model by measuring local production of proinflammatory cytokines. Two weeks after transplantation, mRNA levels of tumor
JOURNAL OF VASCULAR SURGERY Volume -, Number -
Liu et al 5
Fig 3. Macrophage (CD68þ cells) and lymphocyte (CD3þ cells) infiltration in elastase-treated abdominal aortas (E) and transplanted elastase-treated abdominal aortas (Transplanted E) 7 days after surgery. Scale bar ¼ 50 mm. Quantification of macrophage and lymphocyte infiltration in aneurysm tissues as identified by CD68 and CD3 stains, respectively, expressed as CD68þ or CD3þ cells/nuclei. NS, Not significant, n ¼ 5.
necrosis factor a, monocyte chemoattractant protein 1, chemokine (C-C motif) ligand 5, and interferon g were significantly upregulated in the elastase-treated transplanted aorta compared with the inactive elastase-treated transplanted aorta (P < .05; Fig 4). The upregulation of those proinflammatory cytokines was similar to that observed in the conventional elastase model. Origins of the inflammatory cells. To demonstrate that our newly developed transplant model can be carried out between mice of different genotypes, we transplanted elastase-treated abdominal arteries from wild-type mice to GFP mice and vice versa. Both sets of transplant cohorts survived, and aortic grafts developed aneurysms. As shown in Fig 5, A, when wild-type aortas were transplanted to GFP recipients, the majority of medial cells in the grafts were negative of GFP. The converse was observed in the GFP to wild-type transplant cohort (Fig 5, B). As expected, the majority of CD68þ macrophages carried the genotype of the recipients. However, in both transplant cohorts, we did identify a small quantity of CD68þ cells that bared the genotype of the donor, demonstrating that some macrophages arose from a residential population rather than from the circulation (Fig 5, C). DISCUSSION Orthotopic transplantation of abdominal aorta, thoracic aortic arch, and carotid artery was established decades ago,19-26 but these procedures were designed to study chronic rejection, allograft vasculopathy, or atherosclerosis.
Orthotopic abdominal aorta transplantation has not been reported in studies of aneurysm. Thus, we developed a murine AAA model by orthotopic allograft transplantation of elastase-treated abdominal aorta. Data from our macroscopic and microscopic analyses indicate that this transplant AAA model displayed pathologic characteristics analogous to those of the established mouse elastase perfusion AAA model. The principal advantages of this transplant AAA model include the following: it generates a rapid-forming aneurysm through a well-established chemical induction; it permits exploitation of the rich genetic resources of murine species; it allows transplantation between donors and recipients of different genotypes; and it enables identification of cell origin without subjecting recipient mice to irradiation. Different from the thoracic aortic arch transplantation,19,27 anastomosis of the orthotopic abdominal aorta transplantation is more challenging because of the smaller diameter of the mouse abdominal aorta. Even under adequate magnification, the surgery requires highly skilled maneuvers. However, with a skilled operator, a high experimental success rate could be achieved. In our current study, the overall survival rate was higher than 70%. Two major causes of death were bleeding during the operation and postoperative aortic thrombosis, which could result in hind limb paralysis, followed by rapid deterioration to death within 72 hours. In our study, all elastase-treated transplanted abdominal aortas (six of six) became dilated and progressed to aneurysm on day 14 after surgery. We attribute the cause of aneurysm of aorta grafts to the elastase infusion because none of the aorta grafts that were pretreated with heat-
6 Liu et al
JOURNAL OF VASCULAR SURGERY --- 2014
Fig 4. Transplanted elastase-induced abdominal aortic aneurysm (AAA) had an inflammatory phenotype similar to that of the conventional elastase-treated AAA. At 14 days after surgery, the AAA tissues were harvested from different experimental groups: inactive elastase-treated (IE, n ¼ 8), transplanted inactive elastase-treated (Transplanted IE, n ¼ 6), elastase-treated (E, n ¼ 6), and transplanted elastase-treated (Transplanted E, n ¼ 7). Relative fold changes of mRNA levels were determined by real-time polymerase chain reaction (PCR). Data are represented as mean 6 standard error of the mean. *P < .05 (E vs IE, transplanted E vs transplanted IE). CCL5, Chemokine (C-C motif) ligand 5; IFN-g, interferon g; MCP-1, monocyte chemoattractant protein 1; NS, not significant; TNF-a, tumor necrosis factor a.
inactivated elastase developed aneurysm. On macroscopic and microscopic examination, the transplanted grafts exhibited morphologic features including progressive aortic dilation, disruption of elastic fibers, and vascular inflammation that resembled those of the murine elastase perfusion AAA model. In fact, we did not observe a statistically significant difference between our transplant model and the conventional elastase model in any of the parameters measured, suggesting that the transplant procedure alone has no significant impact on aneurysm development. We observed an abundant and diffuse infiltration of lymphocytes and mononuclear cells in the intima, media, and adventitial layers. In the media, elastic fibers were disrupted, and SMCs manifested focal degeneration. A considerable amount of monocyte and lymphocyte
infiltration was observed in the adventitia. The absence of intimal proliferation and thickening in these abdominal aortic allografts is different from carotid allograft transplantation and the murine thoracic aortic transplant model of chronic rejection22 but similar to isograft transplantation.28-30 We speculate that the proinflammatory microenvironment elicited by the elastase treatment favors cell death over proliferation. Allograft vasculopathy is caused by chronic immunemediated injury to the transplanted vasculature, leading to intimal SMC accumulation, luminal narrowing, and eventual ischemic graft failure.31 However, in our transplant AAA model, we observed inflammatory cell infiltration without intimal hyperplasia, SMC accumulation, or stenosis. This may be attributed to syngeneic transplantation or the
JOURNAL OF VASCULAR SURGERY Volume -, Number -
Liu et al 7
Fig 5. Both recipient- and donor-derived macrophages are present in elastase-treated transplant grafts. Elastase-treated abdominal aorta segments were allografted from C57B6 mice into GFPþ C57B6 mice (A) and vice versa (B). Representative confocal immunofluorescence images for macrophage marker CD68 (red) and GFP (green) in allografts 7 days after transplantation. Nuclei stained with DAPI (blue). The yellow arrowheads indicate CD68 and GFP double positive cells. GFP, Green fluorescent protein; Lu, lumen; WT, wild type. Scale bar ¼ 100 mm. C, Quantification of GFPþ/CD68þ or GFP/CD68þ cells in transplanted elastase-treated aneurysms, n ¼ 5.
short follow-up time in this model. Many rodent studies have shown that recipient cells repopulate allografts.19,32-34 Hagensen et al35 proved the recipient source of endothelial cells and SMCs in allograft to be the flanking segments of the recipient vasculature. Although we found that the majority of the mononuclear leukocytes were recruited from the recipient, a small but notable quantity of mononuclear leukocytes carried donor markers. These results suggest that in addition to circulating cells, a certain population of vascular wall resident cells, such as monocyte progenitors, may also differentiate to leukocytes and then proliferate or undergo self-renewal, thus contributing to vascular inflammation in AAA disease.36 The origin of macrophages or macrophage-like cells in atherosclerotic lesions is an actively pursued topic. In addition to bone marrow-derived myeloid cells, other cell types including SMCs are suggested to give rise to at least a portion of macrophage-like cells.37 Phenotypic switching of SMCs to a synthetic or proinflammatory state has recently been noted in mouse models of cerebral aneurysm as well as AAA,38-40 which may offer an alternative explanation for these donor-derived CD68þ cells. Finally, the GFPþ macrophages in the GFP to wild-type transplant group might have acquired their green fluorescent signals by engulfing apoptotic SMCs within the donor aorta. However, this is unlikely because we did not find evidence of phagocytosis, for example, multiple nuclei in a single cell. The reported elastase-induced transplantation AAA model has several limitations. Similar to the established elastase infusion AAA model, our transplant model lacks several prominent features of the human lesion, such as atherosclerosis and intraluminal thrombosis. It would be interesting to determine whether these atherosclerosislike features could be depicted if apolipoprotein E genee deficient mice are used as donors or recipients. Another limitation is that this model does not enable the identification of precise origin of recipient-derived cells.
CONCLUSIONS We have developed a murine AAA model by orthotopic transplantation of elastase-treated abdominal aorta. This new model is a rapid and feasible tool to study the pathophysiologic mechanism of AAA. As an alternative approach to BMC transplantation, the aorta transplant AAA model avoids irradiation and thus may have a higher survival rate. This is important, particularly considering the high lethality in many transgenic mouse lines after irradiation. The authors would like to thank Dr Greenspan for providing the microscope video recording system for this study. AUTHOR CONTRIBUTIONS Conception and design: ZL, QW, SM, BL Analysis and interpretation: ZL, QW, JR, CA, BL Data collection: ZL, QW, JR, CA, JG, QH Writing the article: ZL, QW, BL Critical revision of the article: ZL, QW, JR, CA, SM, BL Final approval of the article: ZL, QW, BL Statistical analysis: ZL, QW Obtained funding: BL Overall responsibility: BL ZL and QW contributed equally to this article and share co-first authorship. REFERENCES 1. Baxter BT, Terrin MC, Dalman RL. Medical management of small abdominal aortic aneurysms. Circulation 2008;117:1883-9. 2. Powell JT, Brady AR. Detection, management, and prospects for the medical treatment of small abdominal aortic aneurysms. Arterioscler Thromb Vasc Biol 2004;24:241-5. 3. Weintraub NL. Understanding abdominal aortic aneurysm. N Engl J Med 2009;361:1114-6. 4. Mortality results for randomised controlled trial of early elective surgery or ultrasonographic surveillance for small abdominal aortic
8 Liu et al
5.
6. 7. 8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21. 22.
23.
aneurysms. The UK Small Aneurysm Trial Participants. Lancet 1998;352:1649-55. Lederle FA, Wilson SE, Johnson GR, Reinke DB, Littooy FN, Acher CW, et al. Immediate repair compared with surveillance of small abdominal aortic aneurysms. N Engl J Med 2002;346:1437-44. Daugherty A, Cassis LA. Mouse models of abdominal aortic aneurysms. Arterioscler Thromb Vasc Biol 2004;24:429-34. Thompson M, Cockerill G. Matrix metalloproteinase-2: the forgotten enzyme in aneurysm pathogenesis. Ann N Y Acad Sci 2006;1085:170-4. Thompson RW. Basic science of abdominal aortic aneurysms: emerging therapeutic strategies for an unresolved clinical problem. Curr Opin Cardiol 1996;11:504-18. Ishibashi S, Goldstein JL, Brown MS, Herz J, Burns DK. Massive xanthomatosis and atherosclerosis in cholesterol-fed low density lipoprotein receptor-negative mice. J Clin Invest 1994;93:1885-93. Prescott MF, Sawyer WK, Von Linden-Reed J, Jeune M, Chou M, Caplan SL, et al. Effect of matrix metalloproteinase inhibition on progression of atherosclerosis and aneurysm in LDL receptor-deficient mice overexpressing MMP-3, MMP-12, and MMP-13 and on restenosis in rats after balloon injury. Ann N Y Acad Sci 1999;878:179-90. Gertz SD, Kurgan A, Eisenberg D. Aneurysm of the rabbit common carotid artery induced by periarterial application of calcium chloride in vivo. J Clin Invest 1988;81:649-56. Daugherty A, Manning MW, Cassis LA. Antagonism of AT2 receptors augments angiotensin II-induced abdominal aortic aneurysms and atherosclerosis. Br J Pharmacol 2001;134:865-70. Trollope A, Moxon JV, Moran CS, Golledge J. Animal models of abdominal aortic aneurysm and their role in furthering management of human disease. Cardiovasc Pathol 2011;20:114-23. Pyo R, Lee JK, Shipley JM, Curci JA, Mao D, Ziporin SJ, et al. Targeted gene disruption of matrix metalloproteinase-9 (gelatinase B) suppresses development of experimental abdominal aortic aneurysms. J Clin Invest 2000;105:1641-9. Johnston WF, Salmon M, Su G, Lu G, Stone ML, Zhao Y, et al. Genetic and pharmacologic disruption of interleukin-1b signaling inhibits experimental aortic aneurysm formation. Arterioscler Thromb Vasc Biol 2013;33:294-304. Morgan S, Yamanouchi D, Harberg C, Wang Q, Keller M, Si Y, et al. Elevated protein kinase C-d contributes to aneurysm pathogenesis through stimulation of apoptosis and inflammatory signaling. Arterioscler Thromb Vasc Biol 2012;32:2493-502. Wang Q, Ren J, Morgan S, Liu Z, Dou C, Liu B. Monocyte chemoattractant protein-1 (MCP-1) regulates macrophage cytotoxicity in abdominal aortic aneurysm. PLoS One 2014;9:e92053. Thompson RW, Curci JA, Ennis TL, Mao D, Pagano MB, Pham CT. Pathophysiology of abdominal aortic aneurysms: insights from the elastase-induced model in mice with different genetic backgrounds. Ann N Y Acad Sci 2006;1085:59-73. Shimizu K, Sugiyama S, Aikawa M, Fukumoto Y, Rabkin E, Libby P, et al. Host bone-marrow cells are a source of donor intimal smoothmuscle-like cells in murine aortic transplant arteriopathy. Nat Med 2001;7:738-41. Dambrin C, Calise D, Pieraggi MT, Thiers JC, Thomsen M. Orthotopic aortic transplantation in mice: a new model of allograft arteriosclerosis. J Heart Lung Transplant 1999;18:946-51. Shi C, Russell ME, Bianchi C, Newell JB, Haber E. Murine model of accelerated transplant arteriosclerosis. Circ Res 1994;75:199-207. Koulack J, McAlister VC, Giacomantonio CA, Bitter-Suermann H, MacDonald AS, Lee TD. Development of a mouse aortic transplant model of chronic rejection. Microsurgery 1995;16:110-3. Sun H, Valdivia LA, Subbotin V, Aitouche A, Fung JJ, Starzl TE, et al. Improved surgical technique for the establishment of a murine model of aortic transplantation. Microsurgery 1998;18:368-71.
JOURNAL OF VASCULAR SURGERY --- 2014
24. Chereshnev I, Trogan E, Omerhodzic S, Itskovich V, Aguinaldo JG, Fayad ZA, et al. Mouse model of heterotopic aortic arch transplantation. J Surg Res 2003;111:171-6. 25. Calise D, Dambrin C, Labat A, Pieraggi MT, Pons F, Benoist H, et al. Orthotopic aortic transplantation in rodents by the sleeve technique: a model system for the study of graft vascular disease. Transplant Proc 2001;33:2369-70. 26. Ailawadi G, Eliason JL, Roelofs KJ, Sinha I, Hannawa KK, Kaldjian EP, et al. Gender differences in experimental aortic aneurysm formation. Arterioscler Thromb Vasc Biol 2004;24:2116-22. 27. Ye P, Chen W, Wu J, Huang X, Li J, Wang S, et al. GM-CSF contributes to aortic aneurysms resulting from SMAD3 deficiency. J Clin Invest 2013;123:2317-31. 28. Pucci AM, Forbes RD, Billingham ME. Pathologic features in longterm cardiac allografts. J Heart Transplant 1990;9:339-45. 29. Adams DH, Russell ME, Hancock WW, Sayegh MH, Wyner LR, Karnovsky MJ. Chronic rejection in experimental cardiac transplantation: studies in the Lewis-F344 model. Immunol Rev 1993;134:5-19. 30. Mennander A, Raisanen A, Paavonen T, Hayry P. Chronic rejection in the rat aortic allograft. V. Mechanism of the angiopeptin (BIM23014C) effect on the generation of allograft arteriosclerosis. Transplantation 1993;55:124-8. 31. Mitchell RN, Libby P. Vascular remodeling in transplant vasculopathy. Circ Res 2007;100:967-78. 32. Hu Y, Davison F, Ludewig B, Erdel M, Mayr M, Url M, et al. Smooth muscle cells in transplant atherosclerotic lesions are originated from recipients, but not bone marrow progenitor cells. Circulation 2002;106:1834-9. 33. Hu Y, Davison F, Zhang Z, Xu Q. Endothelial replacement and angiogenesis in arteriosclerotic lesions of allografts are contributed by circulating progenitor cells. Circulation 2003;108:3122-7. 34. Hillebrands JL, Klatter FA, van den Hurk BM, Popa ER, Nieuwenhuis P, Rozing J. Origin of neointimal endothelium and aactin-positive smooth muscle cells in transplant arteriosclerosis. J Clin Invest 2001;107:1411-22. 35. Hagensen MK, Shim J, Falk E, Bentzon JF. Flanking recipient vasculature, not circulating progenitor cells, contributes to endothelium and smooth muscle in murine allograft vasculopathy. Arterioscler Thromb Vasc Biol 2011;31:808-13. 36. Psaltis PJ, Harbuzariu A, Delacroix S, Witt TA, Holroyd EW, Spoon DB, et al. Identification of a monocyte-predisposed hierarchy of hematopoietic progenitor cells in the adventitia of postnatal murine aorta. Circulation 2012;125:592-603. 37. Gomez D, Owens GK. Smooth muscle cell phenotypic switching in atherosclerosis. Cardiovasc Res 2012;95:156-64. 38. Starke RM, Chalouhi N, Ding D, Raper DM, McKisic MS, Owens GK, et al. Vascular smooth muscle cells in cerebral aneurysm pathogenesis. Transl Stroke Res 2014;5:338-46. 39. Airhart N, Brownstein BH, Cobb JP, Schierding W, Arif B, Ennis TL, et al. Smooth muscle cells from abdominal aortic aneurysms are unique and can independently and synergistically degrade insoluble elastin [published online ahead of print September 27, 2013]. J Vasc Surg doi: 10.1016/j.jvs.2013.07.097. 40. Branchetti E, Poggio P, Sainger R, Shang E, Grau JB, Jackson BM, et al. Oxidative stress modulates vascular smooth muscle cell phenotype via CTGF in thoracic aortic aneurysm. Cardiovasc Res 2013;100:316-24.
Submitted Mar 11, 2014; accepted May 6, 2014.
Additional material for this article may be found online at www.jvascsurg.org.
JOURNAL OF VASCULAR SURGERY Volume -, Number -
Liu et al 8.e1
Supplementary Fig 1 (online only). A, Kaplan-Meier survival curve shows the survival rate of the first 45 elastasetreated orthotopic abdominal aortic transplants, which were divided into three groups in sequence: 1 to 15, 16 to 30, and 31 to 45. AAA, Abdominal aortic aneurysm. B, Analysis of the death causes. The intraoperative mortality is 24.4%, mainly due to bleeding. Other postoperative complications of this procedure include thrombosis of the aorta, aneurysm rupture, hypovolemic shock, and unknown causes.
Supplementary Fig 2 (online only). Hematoxylin and eosin (H&E) staining of arterial tissues 7 days after surgery from different groups: inactive elastase-treated abdominal aorta (IE), n ¼ 4; elastase-treated abdominal aorta (E), n ¼ 5; transplanted inactive elastase-treated abdominal aorta (TIE), n ¼ 3; and transplanted elastase-treated abdominal aorta (TE), n ¼ 3. van Gieson stain shows elastin layer degradation in representative treated arteries 7 days after surgery. Smooth muscle alpha actin (SMaA) immunohistochemical stain shows increased smooth muscle degradation of the elastase-treated abdominal aorta transplant, which is similar to elastase-treated aorta in the conventional model. Scale bar ¼ 50 mm.
JOURNAL OF VASCULAR SURGERY --- 2014
8.e2 Liu et al
Supplementary Table (online only). Microsurgical instruments for the murine elastase-treated abdominal aorta transplantation Instruments Metzenbaum scissors Vannas micro dissecting spring scissors 1, curved Vannas micro dissecting spring scissors 2, angled Desmarres eyelid retractors
Rhoton forceps Dean knife Vessel dilation forceps 1 Vessel dilation forceps 2 Vessel cannulation forceps Micro needle holder Tubing Sutures
Size
Quantity
Corporation
500 300 300 5.12500 ; blade size: 16 mm
1 1 1 1
Roboz Roboz Roboz Anthony Products, Inc
14 mm 12 mm
1 1 1 1 1 1 1 1 1
700 500 ; blade: 1 7 mm 120 mm curved 120 mm straight 131 mm 5.2500 .01100 inner diameter .02400 outer diameter Polyamide monofilament: 9-0 MET 3/8R 140 mm 11-0 MET 3/8R 70 mm Nylon monofilament: 6-0, 15 mm 3/8C Silk braided: 6-0, 20 mm 3/8C
Catalog No. RS-6010SC RS-5621 RS-5619 60-05-13E
Roboz Roboz Roboz Roboz Roboz BRI Roboz
60-05-12E 60-05-11E RS-5262 RS-6220 RS-4927 RS-4929 RS-4990 SKU20-1050 IN-10
AROSurgical AROSurgical Hope Medical
LOT0001147503 LOT0411164003 LOT1205073
Hope Medical
LOT1205073
![[The abdominal aortic aneurysm].](/assets/img/hold.png)
