Homeostatic, Non-Canonical Role of Macrophage Elastase in Vascular Integrity

Abstract

Background: Matrix metalloproteinase (MMP)-12 is highly expressed in abdominal aortic aneurysms and its elastolytic function has been implicated in the pathogenesis. This concept is challenged, however, by conflicting data. Here, we sought to revisit the role of MMP-12 in abdominal aortic aneurysm.

Methods: Apoe^-/- and Mmp12^-/-/Apoe^-/- mice were infused with Ang II (angiotensin). Expression of neutrophil extracellular traps (NETs) markers and complement component 3 (C3) levels were evaluated by immunostaining in aortas of surviving animals. Plasma complement components were analyzed by immunoassay. The effects of a complement inhibitor, IgG-FH_1-5 (factor H-immunoglobulin G), and macrophage-specific MMP-12 deficiency on adverse aortic remodeling and death from rupture in Ang II-infused mice were determined.

Results: Unexpectedly, death from aortic rupture was significantly higher in Mmp12^-/-/Apoe^-/- mice. This associated with more neutrophils, citrullinated histone H3 and neutrophil elastase, markers of NETs, and C3 levels in Mmp12^-/- aortas. These findings were recapitulated in additional models of abdominal aortic aneurysm. MMP-12 deficiency also led to more pronounced elastic laminae degradation and reduced collagen integrity. Higher plasma C5a in Mmp12^-/- mice pointed to complement overactivation. Treatment with IgG-FH_1-5 decreased aortic wall NETosis and reduced adverse aortic remodeling and death from rupture in Ang II-infused Mmp12^-/- mice. Finally, macrophage-specific MMP-12 deficiency recapitulated the effects of global MMP-12 deficiency on complement deposition and NETosis, as well as adverse aortic remodeling and death from rupture in Ang II-infused mice.

Conclusions: An MMP-12 deficiency/complement activation/NETosis pathway compromises aortic integrity, which predisposes to adverse vascular remodeling and abdominal aortic aneurysm rupture. Considering these new findings, the role of macrophage MMP-12 in vascular homeostasis demands re-evaluation of MMP-12 function in diverse settings.

Keywords: angiotensin II; gene deletion; inflammation; macrophages; neutrophils.

Figure 1.

Effect of Mmp12 deletion on Ang II–induced abdominal aortic aneurysms (AAA) development and progression in Apoe-/- mice. A, Kaplan-Meier survival curves of Ang II–induced AAA in Apoe^-/- (n=66) and Mmp12^-/-/Apoe^-/- (n=36) mice. The curves were compared using log-rank (Mantel-Cox) test; P<1x10^-4. B through D, Flow cytometric evaluation of CD45⁺ cells (B), CD11b⁺/Ly6G^- mononuclear myeloid cells, primarily macrophages (C), and CD11b⁺/Ly6G⁺ neutrophils (D) as % among live cells in aortas of Apoe^-/- and Mmp12^-/-/Apoe^-/- mice after 18 to 24 hours of Ang II or saline (control) infusion. Values are expressed as means±SD; n=4 to 9 per group; ns, not significant. E and F, Representative images of immunofluorescence staining and quantification of neutrophils (Ly6G, red), neutrophil extracellular traps (Cit-H3, red), and NE (red) in surviving Apoe^-/- and Mmp12^-/-/Apoe^-/- mice after 7 days of Ang II infusion (E) and control animals (F). Nuclei are stained blue with 4′,6-diamidino-2-phenylindole (DAPI). n=4 per group. Scale bar: 500 µm. *P<0.05 by Kruskal-Wallis with Dunn multiple comparisons post hoc test (B–D) or by 2-tailed Mann-Whitney U test (E and F). Ang II indicates angiotensin II; Cit-H3, citrullinated histone 3; DAPI, 4′,6-diamidino-2-phenylindole; MMP, matrix metalloprotease; and NE, neutrophil elastase.

Figure 2.

Effect of Mmp12 deletion on Ang II–induced AAA development and progression in hyperlipidemic WT mice. A. Kaplan-Meier survival curves of Ang II–induced AAA in hyperlipidemic WT (n=17) and Mmp12^-/- (n=20) mice. The curves were compared using log-rank (Mantel-Cox) test; P<0.05. B, Quantification of maximal suprarenal abdominal aorta external diameter at baseline (no Ang II) and in animals surviving 4 weeks of Ang II infusion; n=4 to 9. C and D, Representative immunofluorescence staining images and quantification of neutrophils (Ly6G, red), neutrophil extracellular traps (Cit-H3, red), and NE (red) in surviving hyperlipidemic WT and Mmp12^-/- mice after 28 days of Ang II infusion (C) and saline-infused control animals (D). Nuclei are stained blue with DAPI. n=4 per group. Scale bar: 500 µm. *P<0.05, **P<0.01, by Kruskal-Wallis test with Dunn multiple comparisons post hoc test (B), or *P<0.05, by 2-tailed Mann-Whitney U test (C and D). Ang II indicates angiotensin II; Cit-H3, citrullinated histone 3; DAPI, 4′,6-diamidino-2-phenylindole; HFD, high-fat diet; MMP, matrix metalloprotease; NE, neutrophil elastase; SAA, suprarenal abdominal aorta; and WT, hyperlipidemic wild type.

Figure 3.

Effect of Mmp12 deficiency on aortic wall extracellular matrix composition. A, Representative examples of Verhoeff–van Gieson (VVG) staining of SAA in Apoe^-/- and Mmp12^-/-/Apoe^-/- (left column) and WT and Mmp12^-/- (right column) mice. Scale bar: 250 µm. B through G, Representative images of immunofluorescence staining and quantification of tropoelastin (red, B and C), collagen type I (red, D and E) and single-stranded collagen using R-CHP (red, F and G) in Apoe^-/- and Mmp12^-/-/Apoe^-/- (B, D, and F) and WT and Mmp12^-/- (C, E, and G) mice. Nuclei are stained blue with DAPI. n=4 per group. Scale bar: 500 µm. H, Burst pressure, as a functional readout of wall strength, of DTA and SAA in Apoe^-/- and Mmp12^-/-/Apoe^-/- mice. n=5 to 6 per group. *P<0.05, **P<0.01 by 2-tailed Mann-Whitney U test (B through H). DAPI indicates 4′,6-diamidino-2-phenylindole; DTA, descending thoracic aorta; MMP, matrix metalloprotease; R-CHP, collagen hybridizing peptide, Cy3 conjugate; SAA, suprarenal abdominal aorta; and WT, wild-type.

Figure 4.

Effect of Mmp12 deletion on aortic wall complement deposition. A and B, Representative images of immunofluorescence staining and quantification of C3 deposits (red) in suprarenal abdominal aorta of Apoe^-/- and Mmp12^-/-/Apoe^-/- (A), and WT and Mmp12^-/- (B) mice. Nuclei are stained with DAPI in blue. n=5 per group. Scale bar: 500 µm. C and D, Plasma C3 and C5a levels of Apoe^-/- and Mmp12^-/-/Apoe^-/- (C), and WT and Mmp12^-/- (D) mice. n=5 to 10 per group. E and F, Plasma C3 levels in Mmp12^-/-/Apoe^-/- (E) and Mmp12^-/- (F) mice at baseline and 9 days of treatment with IgG-FH_1-5 (factor H-immunoglobulin G) or MOPC antibody (control). n=5 per group. G and H, Representative images of immunofluorescence staining and quantification of C3 (red) in suprarenal abdominal aorta of Mmp12^-/-/Apoe^-/- (G) and Mmp12^-/- (H) mice after 9 days of treatment with IgG-FH_1-5 or MOPC antibody (control). n=5 per group; Scale bar: 500 µm. I and J, Representative images of immunofluorescence staining and quantification of Cit-H3 (red) in suprarenal abdominal aorta of Mmp12^-/-/Apoe^-/- (I) and Mmp12^-/- (J) mice after 9 days of treatment with IgG-FH_1-5 or MOPC antibody (control). n=4 to 5 per group. Scale bar: 500 µm. *P<0.05, **P<0.01, ****P<1x10^-4 by 2-tailed Mann-Whitney U test (A through J). Cit-H3 indicates citrullinated histone H3; C3, complement component 3; C5a, complement component 5a; DAPI, 4′,6-diamidino-2-phenylindole; IgG-FH_1-5, factor H-immunoglobulin G; MMP, matrix metalloprotease; and WT, wild-type.

Figure 5.

Effect of IgG-FH1-5 on (factor H-immunoglobulin G) survival during Ang II (angiotensin II) infusion in Mmp12^-/- mice. A. Kaplan-Meier survival curves of Ang II–infused Mmp12^-/- mice following Pcsk9-Ad injection and HFD, treated with either IgG-FH_1-5 or MOPC antibody as control (n=10 per group). The curves were compared using log-rank (Mantel-Cox) test, P<0.05. B, Quantification of maximal suprarenal abdominal aorta external diameter after 4 weeks of Ang II infusion in surviving Mmp12^-/- mice treated with either IgG-FH_1-5 or MOPC antibody, n=4 to 8 per group. C, Representative images of immunofluorescence staining and quantification of complement component 3d (C3d, red) deposition in suprarenal abdominal aorta of surviving IgG-FH_1-5- or MOPC-treated Mmp12^-/- mice after 28 days of Ang II infusion. Nuclei are stained blue with DAPI. n=4 to 7 per group; Scale bar: 500 µm. D, Representative images of immunofluorescence staining and quantification of neutrophils (Ly6G, red), NETs (Cit-H3, red), and NE (red) in suprarenal abdominal aorta of surviving IgG-FH_1-5- or MOPC-treated Mmp12^-/- mice after 28 days of Ang II infusion. Nuclei are stained blue with DAPI. n=4 to 7 per group; Scale bar: 500 µm. *P<0.05, **P<0.01 by 2-tailed Mann-Whitney U test (B through D). Cit-H3 indicates citrullinated histone 3; DAPI, 4′,6-diamidino-2-phenylindole; HFD, high-fat diet; IgG-FH_1-5, factor H-immunoglobulin G; NE, neutrophil elastase; and NET, neutrophil extracellular trap.

Figure 6.

Effect of macrophage Mmp12 deficiency on aortic composition and Ang II (angiotensin II)–induced abdominal aortic aneurysms (AAA) development and survival. A and B, Relative β-actin-normalized Mmp12 gene expression in bone marrow-derived macrophages from Mmp12^flox/flox/Apoe^-/- and Mmp12^flox/flox/Apoe^-/-/Csf1r-iCre (A), and Mmp12^flox/flox and Mmp12^flox/flox/Csf1r-iCre (B) mice at 5 to 6 weeks post-tamoxifen administration. n=4 to 5 per group; *P<0.05. C and D, Representative images of immunofluorescence staining and quantification of Neutrophils (Ly6G, red), NETs (Cit-H3, red), and complement component 3d (C3d, red) in suprarenal abdominal aorta of Mmp12^flox/flox/Apoe^-/- and Mmp12^flox/flox/Apoe^-/-/Csf1r-iCre (C) and Mmp12^flox/flox and Mmp12^flox/flox/Csf1r-iCre (D) mice at 5 to 6 weeks post-tamoxifen administration. Nuclei are stained blue with DAPI. n=4 per group. Scale bar: 500 µm. *P<0.05, 2-tailed Mann-Whitney U test (A through D). E, Kaplan-Meier survival curves of Ang II–infused Mmp12 ^flox/flox/Apoe^-/- (n=9) and Mmp12 ^flox/flox/Apoe^-/-/Csf1r-iCre (n=10) mice post-tamoxifen administration. The curves were compared using log-rank (Mantel-Cox) test. P<0.05. F, Quantification of maximal suprarenal abdominal aorta external diameter of the surviving mice. **P<0.01 by 2-tailed Mann-Whitney U test. Cit-H3 indicates citrullinated histone 3; DAPI, 4′,6-diamidino-2-phenylindole; MMP, matrix metalloprotease; NE, neutrophil elastase; and NET, neutrophil extracellular trap.