Matrix metalloproteinases 2 and 9 work in concert to produce aortic aneurysms

Abstract

Matrix metalloproteinases (MMPs) 9 and 2 are increased in human abdominal aortic aneurysm (AAA) tissue, but their precise role and potential interaction remain unclear. Experimental induction of aortic aneurysms in mice genetically deficient in these peptidases could provide new insight into AAA pathogenesis. Mice deficient in the expression of MMP-9 (MMP-9KO) or MMP-2 (MMP-2KO) and their corresponding wild-type background mice (WT) underwent AAA induction by abluminal application of calcium chloride (CaCl(2)). No aneurysm formation was observed at 10 weeks after treatment in either the MMP-9KO or the MMP-2KO mice, whereas the corresponding WT mice showed an average 74% and 52% increase in aortic diameter, respectively. Reinfusion of competent macrophages from the corresponding WT strains into knockout mice resulted in reconstitution of AAA in MMP-9KO but not MMP-2KO mice. These findings suggest that macrophage-derived MMP-9 and mesenchymal cell MMP-2 are both required and work in concert to produce AAA.

Figure 1

The time course of expression of MMP-9 and MMP-2 and corresponding aneurysm growth. WT mouse aortae were incubated with CaCl₂ or NaCl. Three days, 1 week, and 2, 3, and 4 weeks after treatment, the aortic diameters were measured and mice were sacrificed. (a) CaCl₂-treated mice; (b) NaCl-treated mice (control). (c) Aortic diameter increases in CaCl₂-treated mice at indicated time points. (d) H&E staining of CaCl₂-treated mouse aortic section, showing a significant inflammatory infiltrate at 3 days.

Figure 2

(a and b) Aortic diameter changes after NaCl and CaCl₂ treatment in C57BL/6 and MMP-2KO mice (a), and in 129/SvEv and MMP-9KO mice (b). Aortic diameters were measured before NaCl or CaCl₂ incubation (white bars) and at sacrifice (gray bars). Bars shown in each group represent the mean ± SE of 5–14 mice. The diameter in the CaCl₂-treated group was increased significantly at sacrifice in C57BL/6 and 129/SvEv mice (*P < 0.01). (c–m) Histologic changes of mouse aorta at 10 weeks after treatment. (c–f) Trichrome staining for elastic fibers. NaCl-treated (c) and CaCl₂-treated (d) aorta from C57BL/6 and NaCl-treated (e) or CaCl₂-treated (f) aorta from MMP-2KO mice. (g–j) Pentachrome staining for elastic fibers. NaCl-treated (g) and CaCl₂-treated (h) aorta from 129/SvEv and NaCl-treated (i) or CaCl₂-treated (j) aorta from MMP-9KO mice. (k–m) Immunoperoxidase staining for macrophages of mouse aorta 2 weeks after CaCl₂ incubation in 129/SvEv (k), MMP-9KO (l), and MMP-2KO (m). Mac3-positive cells are indicated by arrows. Elastic fibers are represented by arrowheads. (×100).

Figure 3

Gelatin zymography analysis of MMP activities expressed in experimental mice. Lane 1, WT; lanes 2 and 3, MMP-9KO; lanes 4 and 5, MMP-2KO.

Figure 4

(a) Aortic diameter changes after CaCl₂ treatment with WT macrophage infusion in MMP-2KO and MMP-9KO mice. Aortic diameters were measured before (white bars) and 8 weeks after (gray bars) CaCl₂ incubation. Bars shown in each group represent the mean ± SE of six to eight mice. The diameter in MMP-9KO mice increased significantly with macrophage infusion (*P < 0.01). (b) Gelatin zymography analysis of MMP activities expressed in WT peritoneal macrophages (lane 1) and aorta of MMP-9KO mouse infused with WT macrophages (lane 2). (c–e) Histologic analysis. Paraffin sections were stained for elastin fibers (Trichrome), and sections were photographed and shown with the lumen at the top. (c) The MMP-9KO mouse infused with MMP-9KO macrophages; (d) the MMP-9KO mouse infused with WT macrophages; (e) the MMP-2KO mouse infused with WT macrophages.