ApoE knockout mice expressing human matrix metalloproteinase-1 in macrophages have less advanced atherosclerosis

Abstract

Matrix metalloproteinase-1 (MMP-1), or interstitial collagenase, has been hypothesized to contribute to the progression of the human atherosclerotic lesions by digesting the fibrillar collagens of the neointimal ECM. The apolipoprotein E knockout (apoE0) mouse model develops complex atherosclerotic lesions, but mice do not possess a homologue for MMP-1. To provide an in vivo evaluation of the role of MMP-1 in atherogenesis, we created a transgenic mouse model that expresses this enzyme specifically in the macrophage, under the control of the scavenger receptor A (SCAV) enhancer/promoter. The MMP-1 transgenic mice were crossed into the apoE0 background and fed an atherogenic diet for 16-25 weeks. Surprisingly, the transgenic mice demonstrated decreased lesion size compared with control littermates. The lesions of the transgenic animals were less extensive and immature, with fewer cellular layers and a diminished content of fibrillar collagen. There was no evidence of plaque rupture. Our data suggest that remodeling of the neointimal extracellular matrix by MMP-1 is beneficial in the progression of lesions.

Figure 1

Expression of human MMP-1 in transgenic macrophages. (a) Tissue expression pattern of human MMP-1 in transgenic mice. An RNase protection assay was performed using RNA isolated from peritoneal macrophages (Mφ), the heart, lung, aorta, liver, spleen, kidney, testis, and brain. Both wild-type (Wt) and transgenic (Tg) macrophages were analyzed. The unprotected probe is 842 nucleotides (nt), and the protected fragment is 585 nucleotides. (b) Western blot analysis of peritoneal macrophages culture media after activation with APMA. The activated human collagenase-1 (Mr 45,000) is detected only in the media from transgenic macrophages, not from wild-type. Macrophages from two transgenic and two wild-type mice were tested. Activated purified human interstitial collagenase (MMP-1) and culture medium alone (CM) were used as controls. (c) Collagenase activity in the culture media from transgenic and normal peritoneal macrophages. Culture media of peritoneal macrophages from two transgenic and two wild-type mice were activated with APMA and incubated with type I collagen. The characteristic 1/4 COOH-terminal fragments of the monomeric α1 and α2 chains from type I collagen, produced by MMP-1, are detected in the media from transgenic macrophages.

Figure 2

Migration properties of wild-type (Wt) and transgenic (Tg) peritoneal macrophages. Peritoneal macrophages (250,000 cells per well) were plated on Boyden chambers coated with human fibronectin (filled bars) or type I collagen (gray bars). The lower compartment of the chambers contained the monocyte chemotactic protein-1 at 10 ng/ml. Results are represented as the percentage of cells having penetrated the gel after 48 hours of incubation at 37°C.

Figure 3

Presence of human MMP-1 in the atherosclerotic lesions of transgenic mice. (a) Western blot analysis of the aortic proteins from apoE0 (C) and transgenic MMP-1/apoE0 (Tg) mice using a rabbit polyclonal antibody against human MMP-1. Fifty micrograms of proteins were loaded on the gel. A signal of Mr 55,000 corresponding to MMP-1 is detected in the extract from the transgenic mouse. (b) RT-PCR. One microgram of total RNA isolated from the aorta of a transgenic MMP-1/apoE0 mouse was used for RT-PCR using primers designed from exons 9 and 10 of the human MMP-1 gene. The expected size of 619 bp was detected (Tg). No signal was detected when the reverse transcriptase was replaced with Taq polymerase (C). The PCR product from genomic DNA shows the expected size of 819 bp. M = molecular weight marker. (c) Immunohistochemical detection of MMP-1. Sections of the proximal aortas from apoE0 and transgenic MMP-1/apoE0 mice that were fed a Western diet for 16 weeks were incubated with a mouse mAb raised against human MMP-1. Scale bar: 50 μm.

Figure 4

Immunohistochemical detection of cleaved type I collagen. Aortic sections from 30-week-old apoE0 and MMP-1/apoE0 mice were stained with the 9A4 mAb against collagenase-cleaved type I collagen. Sections were counterstained with methyl green. Scale bar: 100 μm.

Figure 5

Comparison of the aortic arch of apoE0 (a and c) and transgenic MMP-1/apoE0 (b and d) mice after 16 weeks of the Western diet. Longitudinal sections were stained with H&E (a and b) and Mallory trichrome (c and d). The areas shown in c and d correspond to the black boxes shown in aand b, respectively. Scale bar: (a and b) 200 μm; (c and d) 50 μm.

Figure 6

Quantitative comparison of the atherosclerotic lesions in apoE0 and transgenic MMP-1/apoE0 mice that were fed a Western diet for 16 weeks. Oil red-O–stained sections of proximal aortas from 21-week-old apoE0 (n = 10) and apoE0/MMP-1 (n = 10) were quantitated for neointimal lipid accumulation by video microscopy. Lesion sizes are presented as mean ± SEM.

Figure 7

Comparison of atherosclerotic lesions after 25 weeks of the Western diet. Histological sections at the same level of the proximal aortas of apoE0 (a and b) and transgenic MMP-1/apoE0 (c and d) mice were stained with silver impregnation (a and c), for collagen fibers, and Elastica van Gieson (b and d), for elastin. The media (M) and the lumen (L) of the aorta are indicated. Blood cells can be seen on the top of the lesion. Aortas from three MMP-1/apoE0 and four apoE0 mice were examined. Scale bar: 50 μm.