ApoE knockout mice expressing human matrix metalloproteinase-1 in macrophages have less advanced atherosclerosis (original) (raw)
Generation of mice. ApoE0 mice on the C57Bl/6 genetic background were obtained from The Jackson Laboratory (Bar Harbor, Maine, USA). The generation of the transgenic mice was described previously (27). Briefly, the sequences upstream from the translation initiation codon of the human MMP-1 gene were removed. The collagenase genomic fragment (9.3 kb) was inserted downstream of the human scavenger receptor A enhancer/promoter sequence (4.5 kb) using a newly created _Sma_I restriction site (Figure 1). The transgene was isolated from the cloning plasmid by _Not_I/_Sal_I digestion, purified by CsCl gradient centrifugation, and microinjected into fertilized mouse eggs (F1[C57Bl/6xCBA/J]x F1[C57Bl/6xCBA/J]). Mice were crossed into the C57Bl/6 background for more than six generations and then crossbred with apoE0 mice. The animals were genotyped by PCR and Southern blot analysis as described elsewhere (28). All characterization studies were done on a second line of mice, and the data are identical to those presented here.
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.
RNase protection. Total RNA was prepared from mouse tissue and peritoneal macrophages using the guanidium thiocyanate-cesium chloride method (29), and RNase protection analysis was performed as described previously (30). Ten micrograms of RNA from a transgenic mouse line that overexpressed MMP-1 in the lung (30) were hybridized as a positive control.
Western blot analysis. Detection of human MMP-1 protein was performed by semiquantitative Western blot analysis. Mice were injected intraperitoneally with 1 ml of 4% thioglycolate, and the ascites fluid collected after 4 days. The macrophages were centrifuged in 5 ml of PBS, resuspended in 10 ml of Neuman and Tytell serumless medium (Life Technologies Inc., Gaithersburg, Maryland, USA), and plated on a 150-mm Petri dish. The medium was replaced after 2 hours of incubation at 37°C, and the cells were then incubated for another 48 hours. The culture supernatant was concentrated 12-fold. To analyze the proteins from the aorta, the tissue was homogenized in 2 ml of PBS containing 1% Triton X-100, 0.1% SDS, 0.2% NaN3, 1 mM EDTA, and serine- and cysteine-proteinases inhibitors (Complete Mini; Roche Molecular Biochemicals, Indianapolis, Indiana, USA). A bicinchoninic acid protein assay (Pierce Chemical Co., Rockford, Illinois, USA) was performed, to control for loading of protein for each sample on a SDS-PAGE (10%). After migration, the proteins were transferred to a nitrocellulose membrane (BioRad Laboratories Inc., Hercules, California, USA). The primary antibody used was a polyclonal rabbit antibody to human MMP-1 (Chemicon International Inc., Temecula, California, USA). Blots were developed by chemiluminescence as described by the manufacturer (NEN Life Science Products Inc., Boston, Massachusetts, USA).
Collagenase assay. Qualitative levels of MMP-1 protein were determined by a collagenase gel assay, to detect specific collagen degradation fragments. The macrophages were collected as already described here and were cultured in Neuman and Tytell serumless medium (Life Technologies Inc.) overnight. The culture medium was collected and concentrated 12-fold. Activation of latent MMP-1 was performed by incubation with 1.5 mM _p_-aminophenylmercuric acetate (APMA; Sigma Chemical Co., St. Louis, Missouri, USA) for 12 hours at 37°C. Ten microliters of each sample were incubated with 5 μL of 14C-labeled rat tail type I collagen (3 mg/ml) (31) in a total volume of 20 μL (50 mM Tris-HCl [pH 7.5], 0.15 M NaCl, 10 mM CaCl2, 0.05% Brij 35, and 0.02% NaN3) overnight at 37°C. The digestion products were analyzed by SDS-PAGE (9%).
Lipid and fast performance liquid chromatography analysis. Plasma samples from fasted apoE0 (n = 2) or transgenic apoE0/MMP-1 (n = 3) mice (on a Western diet for 16 weeks) were pooled to a final volume of 100 μl, and their lipoproteins were fractionated by fast performance liquid chromatography (FPLC) (Superose 6; Amersham Pharmacia Biotech Inc., Piscataway, New Jersey, USA). Phospholipids and total cholesterol levels of each fraction (1.5 ml) were determined by a 4-aminoantipyrine–based enzymatic assay (Wako Bioproducts, Richmond, Virginia, USA).
Quantitative cell migration assay. Peritoneal macrophages (250,000 cells per well) were plated on Boyden chambers coated with human fibronectin or type I collagen (Chemicon International Inc.) in DMEM medium with 10% FBS. The lower compartment of the chambers contained the same medium with murine monocyte chemotactic protein-1 (MCP-1) (R&D Systems Inc., Minneapolis, Minnesota, USA) at 10 ng/ml. After 48 hours of incubation (37°C, 5% CO2), the number of macrophages having penetrated the gel were quantified following the manufacturer’s instructions.
Histological analysis. Paraffin-embedded tissues were sectioned (4 μm) and stained with hematoxylin and eosin (H&E) for light microscopy. Serial sections were also stained by silver impregnation and Mallory trichrome, for collagen fibers, and Elastica van Gieson, for elastic fibers (32).
Immunohistochemistry. Immunohistochemical detection of human MMP-1 in the lesions was performed using the avidin-biotin-horseradish peroxidase method (ScyTek, Logan, Utah, USA) with specific mouse monoclonal antibodies (Fuji Chemicals, Toyama, Japan) at a final concentration of 6 μg/ml in PBS. The activity of the peroxidase was revealed by diaminobenzidine as a substrate, yielding a brown deposit. Sections were counterstained with hematoxylin. The detection of cleaved type I collagen neoepitopes was performed with the biotinylated monoclonal 9A4 antibody (33) at a concentration of 20 μg/ml in PBS, followed by a streptavidin-linked peroxidase revelation system (Zymed Laboratories Inc., South San Francisco, California, USA). Sections incubated with PBS alone were included as controls. Macrophages were detected using the rat anti-mouse Mac-3 monoclonal antibody (PharMingen, San Diego, California, USA).
Quantification of atherosclerosis. The mice were fed a high-fat Western-type diet (20% protein, 50% carbohydrate, 21% fat, 0.21% cholesterol; Research Diets, New Brunswick, New Jersey, USA) for 16 weeks. They were anesthetized with 2.5% avertin intraperitoneally, the inferior vena cava was nicked and the heart was pressure-perfused at 80 mmHg via left ventricular puncture. The heart was first perfused with PBS then with 10% neutral buffer formalin for 5 minutes to fix the aorta. The tissue was embedded in paraffin for histological analysis or in OCT. compound (Tissue-Tek; Miles Laboratories, Elkhart, Indiana, USA) and snap-frozen for quantitation studies. Transverse sections of 10 μm from the proximal aorta (covering a length of 1.2 mm) were stained with oil red-O. Every eighth section, for a total of six sections, was quantitated for accumulation of intimal lipid by video microscopy using the Image Pro software (version 3.0; Media Cybernetics, Silver Spring, Maryland, USA), and an average value was determined for each mouse (34). For matrix quantification, five transverse sections from each proximal aorta were stained with Masson trichrome, and the collagen content was measured using Image Pro.
Statistical analysis. Analysis were performed by the unpaired Student’s t test, with P < 0.05 considered significant. Lesion sizes are presented as mean ± SEM.