The mechanism of Plg-dependent inflammatory cell recruitment was investigated in 2 models: thioglycollate-induced peritonitis and AAA, an atherosclerotic and inflammatory model. Our study provides in vivo evidence that Plg/plasmin is required for macrophage recruitment and this role is mediated through activation of MMP-9. These conclusions are based on the following results: (a) Plg deficiency markedly reduced macrophage recruitment in both models; (b) suppressed macrophage recruitment with unaffected monocyte blood level is due to macrophage accumulation in tissue in the thioglycollate model; (c) in the thioglycollate model, macrophage migration in vivo is consistent with the time course of activation of the primary proteolytic enzyme for collagen, MMP-9; (d) Plg–/– mice are protected from AAA formation; (e) macrophage infiltration and MMP-9 activity in the aorta are reduced in Plg–/– mice; and (f) actMMP-9 restored macrophage migration in Plg–/– mice in both models. These data establish that Plg-dependent macrophage migration results from Plg’s activation of MMP-9, which regulates the ability of inflammatory cells to invade ECM at sites of injury.
Trans-ECM migration is an important step in leukocyte migration. In the thioglycollate model, the collagen IV layer beneath the mesothelial cells acted as a barrier for macrophage emigration into the peritoneal cavity. In this model, Plg-dependent macrophage migration required MMP-9 activation and degradation of collagen IV. Other studies have also demonstrated that MMP-9–induced collage IV degradation is important for cell migration (35
). Although MMP-9 is not an interstitial collagenase, MMP-9 may participate in the degradation of interstitial collagen indirectly. Recent studies suggested that cleaved fragments of several matrixes (substrates of MMP-9) and MMP-9–activated cytokines were able to induce interstitial collagenases expression (38
). Also, MMP-9 may work in concert with other MMPs to degrade interstitial collagens. Therefore, MMP-9 may not be sufficient but is necessary for the Plg-regulated macrophage migration. In aorta, both elastin (a substrate of MMP-9) and interstitial collagen are important ECM components. During aneurysm formation, elastin degradation is thought to initiate aorta dilation, while loss of fibrillar collagen predisposes to aneurysm rupture (8
). Our data establish a mechanism (Plg/MMP-9) for the initiation and formation of aneurysm. In our CaCl2
-induced aneurysm model, rupture does not occur, so it remains to be determined whether Plg regulates degradation of interstitial collagens to induce rupture in the late phase of aneurysm progression.
Macrophage migration in Mmp9–/–
mice is slightly higher than in Plg–/–
mice, leading us to investigate whether other MMPs are involved in macrophage migration in the thioglycollate model. Mmp9+/+
mice stimulated with thioglycollate were treated with galardin (41
), a broad-spectrum MMP inhibitor. Galardin injection (Supplemental Figure 1) resulted in a reduction in macrophage migration in Mmp9–/–
mice, suggesting the potential involvement of other MMPs in macrophage recruitment. Given that Plg treatment is unable to increase the migration of Mmp9–/–
macrophages in vitro and that administration of actMMP-9 efficiently restores the macrophage migration suppressed by Plg deficiency in vitro and in vivo, other MMPs (not MMP-9) involved in the macrophage migration in peritonitis may be limited to non–Plg-mediated events during cell migration. This hypothesis is also supported by our data showing that MMP-9 deficiency fails to alter macrophage migration induced by MCP-1, indicating that the effect of MMP-9 on macrophage migration was independent of MCP-1–mediated macrophage migration.
The outcomes of studies designed to determine whether Plg activators (uPA and tPA) are required for macrophage migration have been variable, and this variation may depend on several factors, including the background of gene-targeted mice or the inflammatory model (13
). In the thioglycollate model, reduced macrophage migration is found in uPA-deficient mice in a B6 background but not in a mixed (B6 × 129) background (J. Hoover-Plow, unpublished observations). These inconsistencies may be due in part to the marked difference in macrophage migration in B6 and 129 mice in the thioglycollate model (46
). However, uPA rather than tPA plays the dominant role in macrophage infiltration in vascular injury models including AAA (15
), which implies that Plg may be involved in AAA development. However, uPA has been reported to induce leukocyte adhesion and migration as well as expression of MMPs independent of its role in the activation of Plg (43
). By using Plg–/–
mice, our study provides direct evidence that Plg-mediated MMP-9 activation is required for macrophage migration and AAA formation.
Studies have reported conflicting results regarding the dependence of macrophage infiltration on MMP-9 using Mmp9–/–
mice. Luttun et al. (7
) and Choi et al. (47
) reported fewer tissue macrophages in atherosclerosis lesion in Apoe–/–Mmp9–/–
mice, while Pyo et al. (48
) and Longo et al. (33
) reported that although MMP-9 deficiency protected against aneurysm formation, macrophage infiltration into aortas was not impaired in Mmp9–/–
mice. Our results showed that in CaCl2
-induced AAA model, Plg-mediated MMP-9 activation is responsible for macrophage infiltration into aorta. There are 2 possible explanations for these discrepancies: first, the background of Mmp9–/–
mice is different among these studies, and our own work (46
) as well as that of others (49
) shows that genetic background influences inflammatory responses; and second, Longo et al. (33
) suggested that MMP-2 and MMP-9 work in concert to produce AAA. MMP-2 expression was higher in Mmp9–/–
) and may maintain the macrophage infiltration to the aorta in Mmp9–/–
mice, while in Plg–/–
mice, activation of both MMP-9 and MMP-2 is inhibited, resulting in blockage of macrophage infiltration in AAA.
In summary, our findings demonstrate that MMP-9 activation by Plg is crucial to the Plg-dependent regulation of macrophage trans-ECM migration in inflammation and development of AAA. Targeting the Plg/MMP-9 pathway may offer a new approach for therapeutic intervention in inflammation-associated CVD.