Carotid atherosclerotic plaque matrix metalloproteinase-12-positive macrophage subpopulation predicts adverse outcome after endarterectomy

Abstract

Background: Matrix metalloproteinase-12 (MMP-12) promotes atherosclerosis in animal models. MMP-12 is expressed in only a subset of foam-cell macrophages (FCMs) in human plaques. We investigated whether the prevalence of this MMP-12-expressing subpopulation is a prognostic indicator of adverse outcome in patients after carotid endarterectomy (CEA).

Methods and results: Serial sections of culprit lesions from 236 patients who underwent CEA and had undergone 3 years of clinical follow-up were stained immunocytochemically for MMP-12 and for CD68, and the MMP-12/CD68 ratio was used to quantify the MMP-12-expressing subpopulation. A high MMP-12/CD68 ratio correlated with a high content of lipid and total macrophages and a low content of vascular smooth muscle cells, as well as with MMP-8 (R=0.211, P=0.001), MMP-9 (R=0.251, P<0.001), and cleaved caspase-3 (R=0.142, P=0.036) activity measured in a neighboring segment. Dual immunohistochemical examination confirmed the location of MMP-12 in a subpopulation of MMP-8- and MMP-9-positive FCMs, whereas all apoptotic FCMs were MMP-12 positive. Patients who yielded plaques within the highest quartile compared with the lowest quartile of MMP-12/CD68 ratio had a 2.4-fold (hazard ratio, 2.4; 95% CI, 1.1- to 5.1-fold; adjusted P=0.027) increased risk of major adverse cardiovascular event and a 3.4-fold (3.4; 1.2- to 9.6-fold, P=0.024) increased risk for stroke.

Conclusions: The prevalence of an MMP-12-positive subset of FCMs is a prognostic marker for adverse clinical outcome after CEA.

Keywords: MMP-12; atherosclerosis; macrophages; outcome.

Figure 1.

Immunocytochemistry for MMP-12 and CD68 in human atherosclerotic plaques. A, A plaque section stained with antibodies against MMP-12 (magnification ×40). B, A section of a different plaque stained as in A. C, The boxed area of A (magnification ×400). D, The boxed area of B (magnification ×400). E, The corresponding area in C in a serial section stained with anti-CD68. F, The corresponding area in D in a serial section stained with nonimmune IgG. The bar within A and B represents 1 mm, and the bar within C to F represents 0.1 mm.

Figure 2.

Comparison of MMP-12– and CD68-stained areas in CEA specimens. A, An example of a CD68-positive area of interest selected for analysis while blind to the MMP-12 staining. B, The serial section stained for MMP-12. Most CD68 cells are also MMP-12 positive. C, A different CD68-positive area of interest selected for analysis. D, The area in C stained for MMP-12 in a serial section. Most CD68-positive cells are negative for MMP-12. E, The distribution of MMP-12–positive macrophages across all the patients. F, A box-whisker plot showing the values of MMP-12 positivity in the 4 quartiles. The bar within A to D represents 0.2 mm.

Figure 3.

Vulnerable plaque characteristics and the percentage of patients within the 4 quartiles of MMP-12–positive macrophages. A, The amount of lipid core (abbreviated to fat) within the plaque was semiquantitatively scored. B, The amount of macrophages, also semiquantitatively scored. ***P<0.001 ordinal regression.

Figure 4.

Immunohistochemical staining of adjacent segments for (A) MMP-8, (B) MMP-12, (C) 4′,6-diamidino-2-phenylindole (DAPI), and D merged and for (E) MMP-9, (F) MMP-12, (G) DAPI, and (H) merged: demonstrating colocalization of MMP-12 with MMP-8 and MMP-9.

Figure 5.

Immunohistochemical staining for (A) cleaved caspase-3 (CC3), (B) (MMP-12), (C) nuclear staining (4′,6-diamidino-2-phenylindole [DAPI]), and (D) merged, demonstrating colocalization of CC3 with MMP-12. All CC3-positive cells were also positive for MMP-12.

Figure 6.

Adjusted survival curves for the 4 different quartiles, based on MMP-12/CD68 ratio. A, MACE. B, Any recurrent stroke. Adjusted survival curves for the 4 different quartiles, based on quantitatively determined macrophages: C, MACE. D, Any recurrent stroke. Adjusted P-values for the first vs fourth quartile are shown.