Macrophage elastase derived from adventitial macrophages modulates aortic remodeling

Abstract

Abdominal aortic aneurysm (AAA) is pathologically characterized by intimal atherosclerosis, disruption and attenuation of the elastic media, and adventitial inflammatory infiltrates. Although all these pathological events are possibly involved in the pathogenesis of AAA, the functional roles contributed by adventitial inflammatory macrophages have not been fully documented. Recent studies have revealed that increased expression of matrix metalloproteinase-12 (MMP-12) derived from macrophages may be particularly important in the pathogenesis of both atherosclerosis and AAA. In the current study, we developed a carrageenan-induced abdominal aortic adventitial inflammatory model in hypercholesterolemic rabbits and evaluated the effect of adventitial macrophage accumulation on the aortic remodeling with special reference to the influence of increased expression of MMP-12. To accomplish this, we compared the carrageenan-induced aortic lesions of transgenic (Tg) rabbits that expressed high levels of MMP-12 in the macrophage lineage to those of non-Tg rabbits. We found that the aortic medial and adventitial lesions of Tg rabbits were greater in degree than those of non-Tg rabbits, with the increased infiltration of macrophages and prominent destruction of elastic lamellae accompanied by the frequent appearance of dilated lesions, while the intimal lesions were slightly increased. Enhanced aortic lesions in Tg rabbits were focally associated with increased dilation of the aortic lumens. RT-PCR and Western blotting revealed high levels of MMP-12 in the lesions of Tg rabbits that were accompanied by elevated levels of MMP-2 and -3, which was caused by increased number of macrophages. Our results suggest that adventitial inflammation constitutes a major stimulus to aortic remodeling and increased expression of MMP-12 secreted from adventitial macrophages plays an important role in the pathogenesis of vascular diseases such as AAA.

Keywords: MMP-12; abdominal aortic aneurysm; atherosclerosis; elastin; macrophage; transgenic rabbits.

FIGURE 1

Schematic illustration of carrageenan-induced abdominal aortic lesions. Rabbits were fed a cholesterol-diet for 4 weeks before the surgery. A piece of sterilized gauze pre-soaked with λ-carrageenan was placed along the isolated abdominal aorta between the inferior mesenteric artery and lumbar artery and wrapped with a piece of polyethylene film. At the end of experiments, aortic rings were sectioned as shown on the right. The abdominal aorta was cut into 10 cross-sections.

FIGURE 2

Micrographs of abdominal aortic lesions from non-Tg and Tg rabbits. Cross-sections were stained with H&E, with EVG, or immunohistochemically with Abs against macrophages and MMP-12. Adventitial macrophage infiltration and MMP-12 staining of a Tg rabbit aorta are shown at higher magnification at the bottom. Macrophage infiltration was quantitated and showed as percentage. 4–10 immunostained sections from each rabbit (n = 8 for non-Tg and 9 for Tg) were used. The data were expressed as the mean ± SE. **p < .01 vs. non-Tg.

FIGURE 3

Disruption of elastin fibers of the aortic wall (A) and quantitation of the elastin contents of each section (B). A: Histological features of destroyed elastic fibers in carrageenan-induced lesions. Cross-sections were stained with EVG. IEL (arrows): internal elastic lamina. B: The data were expressed as the mean ± SE (n = 12 for each group). *p < .05 **p < .01 vs. non-Tg.

FIGURE 4

Representative micrographs of the focal dilation in the abdominal aortas of Tg rabbits (A) and quantitation of aortic dilation (B). A: The lesions were stained with either H&E or EVG. Arrows indicate the protruded lumens. B: The aortic dilation was evaluated by measuring a defined area of the internal elastic lamina and calculating the diameter in each segment of the aorta. The data were expressed as the mean ± SE (n = 12 for each group). *p < .05 vs. non-Tg.

FIGURE 5

Representative intimal atherosclerotic lesions of Tg and non-Tg rabbits (A) and quantitation of intimal lesions (B). A: Cross-sections of the aorta were stained with H&E or immunohistochemically stained with Abs against macrophages (Mϕ) and smooth muscle cells (SMC). B: The area occupied by intimal lesions was measured using an image analysis system as described in the Materials and Methods. The data were expressed as the mean ± SE (n = 12 for each group). *p < .05, **p < .01 vs. non-Tg.

FIGURE 6

Western blot analysis (A) and real-time RT-PCR analysis (B). A: Proteins isolated from the aortic lesions were subjected to 10% SDS-PAGE under reducing conditions, and probed with Abs against MMP-12, MMP-2, MMP-3, and MMP-9. The same membrane was re-probed with mAb against β-actin to indicate that equal amounts of proteins were loaded. The relative level of each MMP was quantitated by calculating the optical density (OD) of each signal on the films using a densitometer (GS-700, Bio-Rad) and normalized relative to the amount of protein for β-actin. All analyses were performed in triplicate and the values are expressed as the mean ± SE (n = 3 for each group). *p < .05 vs. non-Tg. B: Expression of MMP-12, -2, -3, and -9, MCP-1, and TIMP-1 were analyzed with real-time RT-PCR. Expression levels of each gene are expressed as a percentage of the control value. Data are expressed as the mean ± SE (n = 5 for each group). *p < .05 vs. non-Tg.