Anti-CD 18 monoclonal antibody slows experimental aortic aneurysm expansion Michael A. Ricci, M D , Gail S t r indberg , M D , Jeffery M. Slaiby, M D , Rona ld Gu ibo rd , M D , Lisa J. Bergersen, BA, Patr icia Nichols , D V M , Edi th D. Hendley , P h D , and David B. Pilcher, M D , Burlington, Vt.

Purpose: Inflammation has been implicated as a contributing factor in the expansion of abdominal aortic aneurysms (AAA). To test this hypothesis, we examined the effects of a monoclonal antibody (MAB) to the leukocyte CD18 adhesion molecule on the expansion of experimental AAA. Methods: Aneurysms were induced by perfusion of an isolated segment of the infrarenal aorta with elastase in 22 normotensive (WKY) and 17 genetically hypertensive (WKHT) rats. Animals of both strains were randomly allocated to control or MAB-treated groups (MAB, 5 ~g/100 gm body weight intraperitoneally, daily, beginning on the operative day for a total of four doses). The activity of the MAB against rat leukocytes had first been determined by in vitro immunofluorescence flow cytometry. Aortic size was directly measttred initially and on day 14. At that time, a segment of aorta was stained with hematoxylin and eosin and mononuclear leukocytes and neutrophils were counted in each of 10 microscopic fields (400 x ). Results: The initial aortic size in all animals was 1.11 + 0.15 mm. All groups developed aneurysms significantly larger than the initial aortic size (p < 0.01). However, the MAB-treated animals had significantly smaller aneurysms than the tmtreated controls (mm): WKY: 3.63 + 1.26, WKY-MAB: 2.08 --- 0.30, WKHT: 4.54 + 1.86, WKHT- MAB: 2.37 - 0.40, p < 0.0001. There also were significantly fewer monocytes in the MAB-treated normotensive rats: WKY: 35.5 + 29.9, WKHT: 40.6 + 28.8, WKY-MAB: 8.9 + 8.5, WKHT-MAB: 32.3 + 25.7, p = 0.03. Neutrophil counts did not differ significantly between the groups. Conclusions: Treatment with anti-CD18 monoclonal antibody slows the expansion of AAA in this experimental model. The associated inflammatory process at day 14, as indicated by monocyte infiltration, is reduced, but this effect may be opposed by the presence of hypertension. Further evaluation of the role of leukocytes and adhesion molecules in the expansion of AAA is warranted. (J VAsc SURG 1996;23:301-7.)

Although the initial event leading to abdominal aortic aneurysm (AAA) formation remains a mystery, it seems clear that increased elastase activity and degradation of elastic fibers in the aortic wall is followed by progressive enlargement. Although pre-

From the Departments of Surgery, Pathology (Dr. Guibord), and Molecular Physiology and Biophysics (Dr. Hendley), The Uni- versity of Vermont College of Medicine, Burlington, Vermont.

Supported by the Vermont Affiliate of the American Heart Asso- ciation (Grant-in-Aid #93-06224S) and Vermont EPSCOR.

Presented at the Annual Joint Meeting for the Society for Vascular Surgery and the International Society for Cardiovascular Sur- gery, North American Chapter, New Orleans, La., June 11-14, 1995.

Reprint requests: Michael A. Ricci, MD, FAHC - UHC Campus, One South Prospect Street, Burlington, VT 05401.

Copyright © 1996 by The Society for Vascular Surgery and International Society for Cardiovascular Surgery, North Ameri- can Chapter.

0741-5214/96/$5.00 + 0 24/6/69712

vious experimental work has shown that hyperten- sion accelerates AAA expansion, 2,3 evidence is accu- mnlating that the inflammation plays a significant role in the development of AAA. 3-1~ Inflammation within the media and adventitia o f the aortic wall appears as a consistent component of aortic aneu- rysms. Rizzo et al. 4 demonstrated histologically that inflammatory cells are prominent in the adventitia and present in the media of most aneurysms, with little or none seen in control aortas taken at autopsy from patients without aneurysms. Koch and cowork- ers ~ compared the aortas of patients with aneurysms to those with occlusive disease and found increased inflammatory cells in the media and adventitia, increased amounts of macrophages, and increased numbers of lymphocytes in the aneurysmal aortas. Using a rat aneurysm model experimentally, Anidjar and colleagues 7 demonstrated a temporal correlation

301

JOURNAL OF VASCULAR SURGERY 3 0 2 Ricci et aL February 1996

between the development of the inflammatory infil- trate and the progressive enlargement of AAA.

To investigate the role of inflammation and hypertension on the expansion of experimental AAA, we hypothesized that inhibition of leukocyte adhesion would slow the growth of elastase-induced AAA in normotensive and hypertensive rats. Ad- ditionally, we hypothesized that AAA in the hy- pertensive rats would grow more than those in normotensive rats, even after inhibition of leukocyte adhesion, because it was expected that high blood pressure and inflammation would act independently on AAA expansion.

MATERIAL AND METHODS

In vitro testing ofmonoclonal antibody. Whole blood isolates were obtained from a normal human volunteer and a Wistar-Kyoto (WKY) rat. Leuko- cytes were isolated by centrifuging heparinized blood at 1500 rpm with Histopaque (Sigma Chemical Co., St. Louis) for 30 minutes. Plasma was removed and residual red blood cells were lysed with a water and 3.6% saline solution. The lenkocytes were washed and one million cells were suspended in i ml of Dulbecco's phosphate-buffered saline (PBS; without calcium and magnesium, Life Technologies, Grand Island, NY).

The CD18 monoclonal antibodies (MAB) used were similar to the TA-4 clone to the rat MAC-1 (CDllb/CD18) and LFA-1 (CDlla/CD18) be- longing to the [32-integrin group (#278004-1, Seika- gaku America, Rockville, Md.). This MAB has been shown to be effective in blocldng leukocyte aggrega- tion, endothelial cell adhesion, and in vivo lympho- cyte infiltration.14 For this portion of the experiment, the dose and reactivity of the TA-4 clone was determined by immunofluorescent flow cytometry. Twenty micrograms of TA-4 was added to a test tube of either rat or human leukocyte isolates. Sixty micrograms of the monoclonal anti-CD18 antibody 60.3 (Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, Wash.), a mouse antihuman leu- kocyte antibody, was added to additional test tubes of rat and human leulmcyte isolates. The cells were incubated for 30 minutes on ice, fixed with 0.5% paraformaldehyde for 5 minutes, washed twice with 5% heat-inactivated fetal calf serum, suspended in 25 txl offluorescein-detecting goat anti-mouse antibody, and incubated for 30 minutes on ice. They were washed with 5 % fetal calf serum then with 1% bovine serum albumin in PBS, and then resuspended in 0.5% paraformaldehyde. 15 Immunofluorescent flow cytometry was performed the day of isolation and

labeling using a fluorescence-activated cell sorter (Epich Elite, Coulter Electronics, Miami, Fla.).

Both human and rat cells were incubated with: (1) 60.3 and goat anti-mouse antibody, (2) goat anti-mouse antibody, and (3) TA-4 and goat anti- mouse antibody.

Flow cytometry confirmed TA-4 (CD18 MAB) binding to rat leukocytes and 60.3 to human leukocytes. With the TA-4, complete saturation of one million leukocytes (granulocytes and monocytes) was achieved with 20 >g (1:500 dilution). There are approximately 1.4 × 107 leukocytes in a milliliter of rat blood and approximately 6 ml of blood in a 100-gm rat (4.5-6.5 ml/100 gin), or 8.4 × 107 leukocytes per 100-gm rat. At the 1:500 dilution, there were 4.0 × 10 -1° gm MAB. The original test sample had 1.0 × 106 cells, so 3.4 × 106 gm MAB were needed to saturate the cells. Because absorption from intraperitoneal injection of MAB can be ex- pected to be 80% of the administered dose, 16 the final dose administered in these experiments was increased to 5 t,g/100 gm body weight (BW) to compensate for the intraperitoneal pharmacokinetics.

Animal model. Animals were housed at the University of Vermont and cared for according to the Guide for the Care and Use of Laboratory Animals (NIH Publication #86-23, revised 1985). The hy- pertensive animals were bred at the University of Vermont) 2 Two strains of rats (5.5 _+ 2.3 months) were used: normotensive Wistar-Kyoto rats and genetically hypertensive Wistar-Kyoto (WKHT) rats) 2 Rats weighed 217.8 + 22.5 gm (range, 169- 280 gin). Hypertensive animals had tail plethysmog- raptly weekly 2,12a3 and were not used in the experi- ment until sustained systolic hypertension was re- corded. In more than 13 years of working with these rats, spontaneous development of aortic aneurysms has not been observed.

AAA were induced in rats as previously de- scribed. 2,13 Briefly, anesthesia was induced with intraperitoneal chloral hydrate (300 mg/kg), the abdomen was opened in the midline, and with the aid of a binocular surgical microscope (9 × ), the infra- renal aorta was exposed and its diameter measured with a micrometer. Arterial branches from the level of the renal arteries to bifurcation were clamped. A polyethylene catheter (PE10) was inserted via the left iliac artery and advanced until the tip was within the aorta. Intraarterial blood pressure was measured. The catheter was secured temporarily with a ligature just above the aortic bifurcation and the aorta was occluded below the renal vessels with a clamp, producing an isolated 1-cm segment of aorta. The

JOURNAL OF VASCULAR SURGERY Volume 23, Number 2 Ricci et al. 303

isolated segment of aorta was infused with an elastase solution (15 units of porcine pancreatic elastase; Type I, E-1250, Sigma Chemical Co,, in 2 ml PBS, pH 8.6-8.8) for 2 hours. The residual elastase then was aspirated from the aorta, the catheter was removed, and the iliac vessel ligated. After surgery, the rats received four doses of buprenorphine (0.01 mg/kg subcutaneously every 12 hours).

After 14 days, the animals were reanesthetized with chloral hydrate and under physiologic condi- tions the aorta was measured again. The animals were then killed by exsanguination. The aneurysmal aorta was carefully dissected free of adherent tissue, rinsed, stored in formalin, and submitted for routine hema- toxylin and eosin and Leder's stain. Mononuclear cells and neutrophils were counted in each specimen per 10 high-powered fields (400 × ) by an experi- enced histopathologist (RG).

Experimental design. Before surgery, animals of both strains were allocated randomly to control or treatment groups. Animals in the treatment groups received 5 txg of the CD18 MAB per 100 gm of body weight intraperitoneally beginning on the operative day (before the start of anesthesia). The infusion was continued daily for 3 days (total, four doses). Thus four groups were studied (Table I).

Statistics. All data are expressed as the mean _+ standard deviation. Analysis between groups was by the one-way analysis of variance (ANOVA) with Bonferroni's adjustment to confirm points of sig- nificance and the Student's t test. A p value of 0.05 or less was considered significant. All statistical calculations were done on an IBM-compatible com- puter using In Star 2.05 (Graph Pad Software, San Diego, Calif.).

RESULTS

The initial aortic size in all animals was 1.11 _+ 0.15 mm. Mean intraarterial blood pres- sure (under anesthesia) was significantly higher in the WKHT animals: WKY: 94.2 ± 12.8, WKHT: 116.6 + 36.6 (p < 0.0001), as seen previously. 2,13

Aneurysms developed in all animals that were significantly larger than the initial aortic size (p < 0.01). Aneurysms were significantly smaller in both groups treated with MAB (Figure 1).

Control animals in both groups displayed a loss of the normal architecture of the aortic wall with flank necrosis in some cases. There was disruption of any remaining elastin fibers. Treated rats, conversely, showed preservation of the normal architecture with preservation of the elastin layers. Patchy inflamma- tory infiltrate was observed in the control animals,

Table 1. Study groups

Group No. of subjects

Control I WKY 11 II WKHT 9

Treatment III WKY-MAB 1 I IV WICHT-MAB 8

and this was markedly reduced in the MAB-treated groups.

Neutrophil and monocyte counts are shown in Table II. Representative specimens are shown in Figure 2. There was no significant difference in neutrophil counts at day 14. WKY-MAB rats had significantly fewer monocytes than WKY rats, but there was no difference between control and MAB- treated WKHT rats. There was also no difference between WKY and WKHT rats.

DISCUSSION

Using the elastase-gencrated model of AAA in rats, Anidjar and coworkcrs 7 correlated the expansion of AAA with inflammatory infiltration. Two and one half days after infusion with elastase, thc aortas dilated 300%, from 1.04 -+ 0.02 mm to 3.09 - 0.08 mm (p < 0.05). Immunohistologic studies showed macr0phages and T cells in the media. By day 6, the aorta was 421% larger (4.38 +_ 0.03 mm, p < 0.05) and the numbers ofmacrophages, neutro- phils, and T lymphocytes wcrc increased further. Additionally, in a second set of experiments, a nonspecific activator of the immune system (thiogly- colatc plus plasmin) was infused instead of clastin.

The aorta again enlarged with the infiltration of macrophages but more slowly. In the first set of experiments the aortas were devoid of stainable elastin and rapidly enlarged, with a "secondary" enlargement that coincided with the maximal inflam- matory infiltration. The second set of experiments saw principally a macrophage infiltration with de- layed but steady expansion.

This cellular infiltration also was sccn and furthcr characterized by Halpern et al~0 using this elastase- perfusion model. They examined the aortas at 1, 2, 3, and 6 days after infusion. Neutrophils, ED-2 positive macrophages, and CD4-positive T-helper cells had early increases (23 days) in the aneurysmal aortas (leading to our choice of MAB treatment up to day 4). In addition, IgM was detected on the external elastic lamina, suggesting that the cellular infiltration may be a specific immune response to disrupted

JOURNAL OF VASCULAR SURGERY 304 Ricci et al. February 1996

7

:t I 2

1

0 - WKY WKY-MAB

~ ' 4 - E

._N 3 03

WKHT WKHT-MAB

Fig. 1. Final aortic aneurysm size (mm) in all four groups at 14 days. ANOVA, p < 0.0001. * WKY versus WKY-MAB, p < 0.001, ** WKHT versus WKHT-MAB, p < 0.001.

Table II . White blood cell counts

Neutrophils Monocytes

WKY 19.4 _+ 21.6 35.5 _+ 29.9 WKHT 16.3 _+ 21.6 40.6 _+ 28.8 WKY-MAB 7.2 _+ 13.1 8.9 -+ 8.5 WKHT-MAB 36.3 _+ 33.3 32.3 _+ 25.7 A_NOVA p = 0.12 p = 0.032*

Average counts per 10 high-power fields. *WKY vs. WKY-MAB, p = 0.0125; WKHT vs. WKHT-MAB, p = 0.40.

elements within the aorta rather than a nonspecific response to injury. In both of these studies, the infiltration o f inflammatory cells coincided with the enlargement of the aorta, leading these authors to suggest this infiltration was responsible for aortic wall destruction and the formation o f AAA. 7,1°

In the present experiments we simply turned the question around, asking whether inhibition of in- flammatory cells would prevent AAA enlargement. Dobrin had demonstrated previously that inhibition of the inflammatory response with steroids and cyclosporin reduced aneurysm size. 16 Because Anid- jar et al7 demonstrated the involvement of lympho- cytes, macrophages, and neutrophils, the CD18 MAB was chosen to affect the widest possible inhibition of leukocytes. The [3-subunit CD18 is noncovalently linked to three ~-subunits, C D l l a , CD 11 b, or CD 1 l c. The CD 11/CD 18 heterodimer is expressed on all leukocytes) s The C D l l / C D 1 8 heterodimer complexes with intracellnlar adhesion molecules, and adhesion is primarily the result of conformational changes. Antibodies bind close to

adhesive sites and prevent adhesion by steric hin- drance) 9 It is likely that both unstimulated and stimulated neutrophils are inhibited by the CD18 MAB, whereas unstimulated monocytes are only partially inhibited by the MAB to CDlS. Stimulated monocytes, however, display increased inhibition by the MAB. 18 The CD18 MAB has been demonstrated to decrease leukocyte-endothelial cell adhesion. It also prevents neutrophil emigration in vitro as well as in a variety of experimental disease states. 14,18'19 Inhibition of leukocyte adhesion diminishes the inflammatory response by preventing further steps in the response. 19

As hypothesized, administration of the CD18 MAB reduced the magnitude of aortic dilation, corresponding to the "primary" dilation from the loss of elastin reported by Anidjar et al.7 The monocyte response was significantly decreased in normotensive animals at 14 days, and neutrophils showed a nonsignificant trend in this direction as well. It is possible that earlier inhibition ofneutrophil adhesion prevented the progressive steps in the inflammatory

JOURNAL OF VASCULAR SURGERY Volume 23, Number 2 Ricci et al. 3 0 5

Fig. 2. Histologic results of representative tissue specimen (hematoxylin and eosin stain, 400 × ). A, Control WKY rat aorta with a marked inflammatory infiltrate, primarily composed of lymphocytes but including polymorphonuclear leukocytes and plasma cells. The normal architecture of the aortic wall is disrupted, manifested by the disorder of the smooth muscle cells and elastic fiber bundles. B, Aortic wall from a CD18 MAB-treated WKY rat shows orderly, concentric layers of smooth muscle cells and layers of elastic fibers. Inflammatory cell infiltration is absent. C, Aortic wall from this WKHT control rat shows a marked inflammatory response consisting of polymorphonuclear leukocytes and mononuclear cells. The luminal aspect of the wall demonstrates intimal hyperplasia as well as disruption and loss of internal elastic lamina. D, Aorta from a MAB-treated WKHT rat shows orderly arrangement of smooth muscle cells, a prominent internal elastic lamina, and no appreciable inflammatory infiltrate.

response, including monocyte infiltration, as sug- gested above. TM This is presently being investigated.

Additionally, because it has previously been demonstrated that hypertension significantly in- creases aneurysm expansion, 2a3 we hypothesized that hypertension would act independently from the inflammatory response to increase aortic size. This was not confirmed. This would suggest that although hemodynamic forces exert a force on the expansion of AAA, these forces are secondary to the dilation seen in association with the inflammatory cell infiltration.

We have observed variation in the size of the AAA in our various studies, 2,13 particularly with the W K H T strain. Although this is likely due to slight, unidentified variations in technique, it speaks to the importance of concurrent controls with this model.

A corresponding significant decrease in the num- ber of monocytes at 14 days was not seen in hypertensive animals, an observation that is unex-

plained. It is known that elastin fragments are chemotactic for monocytes but offer less of a stimulus for neutrophils, 2° whereas collagen fragments are chemotactic for monocytes only. 21 It is possible that unidentified stimuli in the hypertensive rats (such as collagen fragmentation in the hypertensive aorta) led to monocyte chemotaxis, but that the early phase of neutrophil infiltration was blocked, preventing the "secondary" enlargement discussed above. Another explanation may relate to the reported finding that W K H T rats have greater numbers of circulating lymphocytes.22

Evidence of the role of the inflammatory response in human AAA is also accumulating. Brophy and co-workers 6 found a mononuclear infiltrate in 8 of 10 aortic aneurysms similar to that seen in this experi- mental study. They also found significant deposition of immunoglobullns (later seen in their experimental study as welll°). Workers at Northwestern University

JOURNAL OF VASCULAR SURGERY 306 Ricci et al. February 1996

used human AAA samples to investigate the signifi- cance of the inflammatory infiltration seen within the media and adventitia of aneurysmal aortas. 4,~ Few inflammatory cells were present in the normal aorta and only a mild infiltration was seen in atherosclerotic aortas, s Degenerative aortic aneurysms, however, had much greater inflammatory infiltration. The predominant cell type in the AAA tissue was the CD3-positive T lymphocytes (66%), which were found primarily in the adventitia, aggregating around the vasa vasorum. Additionally, a high proportion of CD4-positive T-helper cells was found in the aneu- rysmal aorta, compared with normal aortas and peripheral blood. CD19-positive lymphocytes, rare in the occlusive aorta, constituted about 25% of the infiltrate in AAA tissue, primarily in the adventitia. Frequently these lymphocytes were surrounded by clusters of CD3-positive cells. Macrophages tended to parallel the general inflammatory response, and were distributed in the media and adventitia with equal frequency. These authors postulated that the CD3 T-cells, particularly the CD4 subset, might be responsible for the release of interlenkin-2, which activates and causes the proliferation of CD-3 posi- tive T cells and CD19-positive B cells. These B cells, with macrophages and smooth muscle cells, can process antigen and perpetuate the immune response. Macrophages can release interleukin-l[3 (IL-I[3), which is chemotactic for CD3-positive and CD19- positive lymphocytes. They also can release tumor necrosis factor alpha (TNF-c~), which can influence the endothelial cell layer and affect lipolytic activity, perhaps playing a role in AAA development.

Subsequent work from this group has suggested that such cytokine activity (particularly IL-113) by macrophages and lymphocytes is responsible for regulating the increase in matrix protein turnover present in AAA. 11 Moreover, Newman and co- workers 8 have demonstrated increased TNF-a and IL- l[3in AAA tissue compared with control athero- sclerotic aortas. These investigators also found that tissue metalloproteinases (MMP), a diverse group of substances responsible for extracellular matrix de- struction, are increased in AAA, 24 and that the MMPs found in AAA tissue are localized to mononuclear cells (MMP-9) and macrophages (MAVIP-3, u - P A ) . 9

Considerable evidence exists to support an active role for the inflammatory response in the expansion of AAA, rather than merely that of an epi- phenomenon that accompanies AAA. By inhibiting the cellular components of the inflammatory re- sponse, this study, which is the first to demonstrate that AAA expansion can be limited with MAB

treatment, adds weight to this hypothesis. Although the inciting event remains a mystery, 8 it would seem that the elastase-generated AAA model, which mim- ics human aneurysms histologically, 2,23 also repro- duces the early events of aneurysm formation, especially the inflammatory response seen in hu- mans. 4,7,10 Thus it is a superior model for the study of AAA, particularly the secondary effects of inflamma- tion on extracellular matrix destruction.

The authors thank Lori Shatney and Beverly Batty for their assistance with these experiments.

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1. Dobrin PB. Pathophysiology and pathogenesis of aortic aneurysms. Surg Clin North Am 1989;69:687-703.

2. Gadowski GR, Ricci MA, Hendley ED, Pilcher DB. Hyper- tension accelerates the growth of experimentally induced abdominal aortic aneurysms, l Surg Res 1993;54:431-6.

3. Anidjar S, Dobrin PB, Cheifec G, Michel I-B. Experimental study of determinants of aneurysmal expansion of the abdominal aorta. Ann Vase Surg 1994;8:127-136.

4. Rizzo RJ, McCarthy WJ, Dixit SN, et al. Collagen types and matrix protein content in human abdominal aortic aneurysms. J VASC SURG 1989;10:365-73.

5. Koch AE, Haines GK, Rizzo RJ, et al. Human abdominal aortic aneurysms: immunophenotypic analysis suggesting an immune-mediated response. Am I Pathol 1990;137:1199- 1213.

6. Brophy CM, Reilly JM, Walker-Smith GJ, Tilson MD. The role of inflanamation in nonspecific abdominal aortic aneu- rysm disease. Ann Vase Surg 1991;5:229-33.

7. Anidjar S, Dobrin PB, Eichorst M, Graham GP, Cheifec G. Correlation of inflammatory infiltrate with the enlargement of experimental aortic aneurysms. J Vasc SURG 1992;16: 139,147.

8. Newman ICM, Jean-Claude J, Li H, Ramey WG, Tilson MD. Cytokines that activate proteolysis are increased in abdominal aortic aneurysms. Circulation 1994;90(part 2):II-224-II-227.

9. Newman KM, Jean-Claude 1, Li H, et al. Cellular localization of matrix metalloproteinases in the abdominal aortic aneurysm wall. J VASC SURG 1994;20:814-20.

10. Halpern VJ, Nackman GB, Gandhi RH, et al. The elastase infusion model of experimental aortic aneurysms: synchrony of induction of endogenous proteinases with matrix destruc- tion and inflammatory cell response. J VASC SURG 1994;20: 51-60.

11. Keen RR, Nolan KD, Cipollone M, et al. Interleukin-l[3 induces differential gene expression in aortic smooth muscle cells. J VASC SURG 1994;20:774-86.

12. Hendley ED, Ohlsson WG. Two new inbred Wistar-Kyoto strains: WKHA, hyperactive rats, and WK,HT, hypertensive rats. Am J Physiol 1991;261 (Heart Circ Physiol 30):H584- H590.

13. Slaiby L, Ricci MA, Gadowski G, Hendley E, Pilcher DB. Growth of aortic aneurysms is reduced by propranolol in a hypertensive rat model. J VASC SURG 1994;20:178-83.

14. Kameoka H, Ishibashi M, Tamatani T, et al. Comparative immunosuppressive effect of anti-CD18 and anti-CDll a monoclonal antibodies on rat heart allotransplantation. Trans- plant Proc 1993;25:833-36.

15. Wallis WJ, Hickstein DD, Schwartz BR, et al. Monoclonal

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antibody-defined functional epitopes on the adhesion- promoting glycoprotein complex (CDw18) of human neu- trophils. Blood 1986;67:1007-13.

16. Dobrin PB, In vivo model of aortic aneurysms. J VASC SURG 1994;20:150-152.

17. Barrett JS, Wagner JG, Fisher SJ, Wahl RL. Effect of intraperitoneal injection volume and antibody protein dose on the pharmacokinetics of intraperitoneally administered igG2aK murine monoclonal antibody in the rat. J Cancer Res 1991;51:3434-44.

18. Carlos TM, Harlan JM. Membrane proteins involved in phagocyte adherence to endothelium. Immun Rev 1990:114: 5-28.

19. Patarroyo M, Prieto 1, Pincon J, et al. Leukocyte-cell adhesion: a molecular process ftmdamental in leukocyte physiology. Immun Rev 1990:114:67-108.

20. Senior RM, Griffin CL, Mecham RP. Chemotactic activity of elastin-derived peptides, l Clin Invest 1980;66:859-62.

21. Postlethwaite AE, Kang AH. Collagen and collagen peptide- induced chemotaxis of human blood monocytes. I Exp Med 1976; 143:1299-1307.

22. Reed JP, Hendley ED. Blood cell changes in spontaneously hypertensive rats are not all associated with the hypertensive phenotype. J Hypertension 1994;12:391-399.

23. Newman KM, Malon AM, Shin RD, Scholes JV, RameyWG, Tilson MD. MatrLx metalloproteinases in abdominal aortic aneurysms: characterization, purification, and their possible sources. Connect Tissue Res 1994;30:1-12.

24. Anidjar S, Salzman J'L, Gentric D, et al. Elastase-induced experimental aneurysms in rats. Circulation 1990;82:973-81.

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