Background-Neutrophils may be an important source of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9), two matrix-degrading enzymes thought to be critical in the formation of an abdominal aortic aneurysm (AAA). The purpose of this investigation was to test the hypothesis that neutrophil depletion would limit experimental AAA formation by altering one or both of these enzymes. Methods and Results-Control, rabbit serum-treated (RS; nϭ27) or anti-neutrophil-antibody-treated (anti-PMN; nϭ25) C57BL/6 mice underwent aortic elastase perfusion to induce experimental aneurysms. Anti-PMN-treated mice became neutropenic (mean, 349 cells/L), experiencing an 84% decrease in the circulating absolute neutrophil count (PϽ0.001) before elastase perfusion. Fourteen days after elastase perfusion, control mice exhibited a mean aortic diameter (AD) increase of 104Ϯ14% (PϽ0.0001), and 67% developed AAAs, whereas anti-PMN-treated mice exhibited a mean AD increase of 42Ϯ33%, with 8% developing AAAs. The control group also had increased tissue neutrophils (20.3 versus 8.6 cells per 5 high-powered fields [HPFs]; Pϭ0.02) and macrophages (6.1 versus 2.1 cells per 5 HPFs, Pϭ0.005) as compared with anti-PMN-treated mice. There were no differences in monocyte chemotactic protein-1 or macrophage inflammatory protein-1␣ chemokine levels between groups by enzyme-linked immunosorbent assay. Neutrophil collagenase (MMP-8) expression was detected only in the 14-day control mice, with increased MMP-8 protein levels by Western blotting (Pϭ0.017), and MMP-8 -positive neutrophils were seen almost exclusively in this group. Conversely, there were no statistical differences in MMP-2 or MMP-9 mRNA expression, protein levels, enzyme activity, or immunostaining patterns between groups. When C57BL/6 wild-type (nϭ15) and MMP-8 -deficient mice (nϭ17) were subjected to elastase perfusion, however, ADs at 14 days were no different in size (134Ϯ7.9% versus 154Ϯ9.9%; Pϭ0.603), which suggests that MMP-8 serves only as a marker for the presence of neutrophils and is not critical for AAA formation. Conclusions-Circulating
Background-This investigation tested the hypothesis that L-selectin is important in experimental abdominal aortic aneurysm (AAA) formation in rodents. Methods and Results-Rat abdominal aortas were perfused with saline (control) or porcine pancreatic elastase and studied on postperfusion days 1, 2, 4, 7, and 14 (nϭ5 per treatment group per day). Neutrophil (polymorphonucleur leukocyte, PMN) and macrophage counts per high-powered field (HPF) were performed on fixed sections. L-selectin expression and protein levels in aortic tissue were determined by polymerase chain reaction and Western blot, respectively. Elastase-perfused aortic diameters were significantly increased compared with control aortas at all time points except day 1 (PϽ0.05). PMN counts significantly increased in elastase-perfused aortas compared with control aortas at days 1, 2, and 4, reaching maximum levels at day 7 (40.8 versus 0.3 PMNs/HPF, Pϭ0.001). L-selectin mRNA expression in elastase-perfused aortas was 18 (Pϭ0.018), 17 (PϽ0.001), and 8 times (Pϭ0.02) times greater than control aortas at days 1, 2, and 4, respectively. Western blot demonstrated a significant 69% increase in L-selectin protein at day 7 in elastase-as compared with saline-perfused aortas (Pϭ0.005). Subsequent experiments involved similar studies on postperfusion days 4, 7, and 14 of aortas from C57BL/6 wild-type (WT) mice (nϭ21) and L-selectin-knockout (LKO) mice (nϭ19). LKO mice had significantly smaller aortic diameters at day 14 as compared with WT mice (88% versus 123%, Pϭ0.02). PMN counts were significantly greater in elastase-perfused WT mouse aortas as compared with LKO mouse aortas at day 4 after perfusion (12.8 versus 4.8 PMNs/HPF, Pϭ0.02). Macrophage counts were significantly greater at all time points after perfusion in elastase-perfused WT mouse aortas compared with elastase-perfused LKO mouse aortas, with a maximum difference at day 7 after perfusion (13.3 versus 0.5 macrophages/HPF, PϽ0.001). Conclusion-L-selectin-mediated neutrophil recruitment may be a critical early step in AAA formation. (Circulation.2005;112:241-247.)
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