Matrix Metalloproteinase-13/Collagenase-3 Deletion Promotes Collagen Accumulation and Organization in Mouse Atherosclerotic Plaques
http://www.100md.com
《循环学杂志》
the Donald W. Reynolds Cardiovascular Clinical Research Center, Brigham and Women’s Hospital, Harvard Medical School, Boston, Mass (J.D., E.A., P.L., J.R.V., M.A.)
Center for Immunology and Inflammatory Diseases, Massachusetts General Hospital and Harvard Medical School, Charlestown, Mass (M.I., S.M.K.)
Departments of Emergency Medicine and Anesthesiology, University of Massachusetts Medical School, Worcester (P.W.).
Abstract
Background— Interstitial collagen plays a crucial structural role in arteries. Matrix metalloproteinases (MMPs), including MMP-13/collagenase-3, likely contribute to collagen catabolism in atherosclerotic plaques.
Methods and Results— To test the hypothesis that a specific MMP-collagenase influences the development and structure of atherosclerotic plaques, this study used atherosclerosis-susceptible apolipoprotein E–deficient mice that lack MMP-13/collagenase-3 (Mmp-13–/–/apoE–/–) or express wild-type MMP-13/collagenase-3 (Mmp-13+/+/apoE–/–). Both groups consumed an atherogenic diet for 5 (n=8) or 10 weeks (n=9). Histological analyses of the aortic root of both groups revealed similar plaque size and accumulation of smooth muscle cells (a collagen-producing cell type) and macrophages (a major source of plaque collagenases) after 5 and 10 weeks of atherogenic diet. By 10 weeks, the plaques of Mmp-13–/–/apoE–/– mice contained significantly more interstitial collagen than those of Mmp-13+/+/apoE–/– mice (P<0.01). Furthermore, quantitative optical analyses revealed thinner and less aligned periluminal collagen fibers within the plaques of Mmp-13+/+/apoE–/– mice versus those from Mmp-13–/–/apoE–/– mice.
Conclusions— These data support the hypothesis that MMP-13/collagenase-3 plays a vital role in the regulation and organization of collagen in atherosclerotic plaques.
Key Words: atherosclerosis collagen metalloproteinases pathology plaque
Introduction
Interstitial collagen maintains structural integrity and influences cell functions in a wide variety of tissues.1–6 The regulation of collagen levels depends on several factors, including the number and activation state of collagen-producing cells and the expression of collagen-degrading enzymes. Collagenases of the matrix metalloproteinase (MMP) family, including MMP-1/collagenase-1, MMP-8/collagenase-2, and MMP-13/collagenase-3, can degrade triple helical fibrillar collagen at neutral pH, an initial step that permits further digestion by other MMPs.1–3,7,8 We proposed that an imbalance between collagen synthesis and degradation regulates critical aspects of atherosclerotic plaque structure.4,9 Fibrillar collagens confer tensile strength on the fibrous cap of the plaque. Disruption of a collagen-poor, thin-capped plaque frequently triggers acute coronary thrombosis in humans.10–13 Previous studies in humans and animals have localized MMPs including the interstitial collagenases in plaques.14–21 Nevertheless, we lack in vivo evidence that links collagenase action directly to collagen loss in atherosclerotic plaques.
Clinical Perspective page 2715
We recently provided evidence that collagenases can indeed regulate intimal collagen accumulation.22 We crossed collagenase-resistant mice (ColR/R), whose 1(I) chain of type I collagen has a "knock-in" mutation at the cleavage site shared by MMP-family interstitial collagenases, with atherosclerosis-susceptible apolipoprotein E–deficient (apoE–/–) mice. Atherosclerotic plaques of ColR/R/apoE–/– mice contained more interstitial collagen than those of apoE–/– mice with wild-type collagen type I. Our study also localized MMP-13/collagenase-3 and MMP-8/collagenase-2 in macrophages and atherosclerotic aortas in mice, a species that lacks the orthologue of human MMP-1/collagenase-1. "Unconventional" collagenases, MMP-2/gelatinase-A and membrane-type I MMP (MT1-MMP or MMP-14), may also cleave triple helical type I collagen.23–25 The relative contribution of various MMP-collagenases to collagen remodeling in atherosclerotic plaques, however, has not undergone rigorous evaluations.
We26 and others27 have recently reported effects of MMP-13 deletion on embryonic development of bone and cartilage. Nevertheless, the role of MMP-13 in arterial disease remains unexplored. We therefore used compound mutant Mmp-13–/–/apoE–/– mice to test the biological hypothesis that MMP-13/collagenase-3 regulates collagen content and structure in atherosclerotic plaques.
Methods
Animal Preparation
All experiments conformed to a protocol approved by the Standing Committee on Animals of Harvard Medical School. Mmp-13–/– mice were generated by gene targeting in embryonic stem cells.26 We backcrossed Mmp-13–/– mice (C57BL/6x129) 7 generations into congenic C57BL/6 mice. Mmp-13–/– mice (C57BL/6) were then crossed into apoE–/– mice (C57BL/6) to render Mmp-13–/– mice atherosclerosis susceptible, yielding Mmp-13+/+/apoE–/– and Mmp-13–/–/apoE–/– mice. Five-week-old male Mmp-13+/+/apoE–/– (n=17) and Mmp-13–/–/apoE–/– (n=17) littermates consumed an atherogenic diet (semipurified chow containing 1.25% cholesterol and 0% cholate, Research Diets) for 5 weeks (n=8 each) or 10 weeks (n=9 each).
Macrophage Culture
To determine whether MMP-13/collagenase-3 deficiency causes compensatory alterations in expression of other major matrix-degrading enzymes, which also could influence collagen accumulation in plaques, we examined mRNA expression by macrophages from Mmp-13+/+/apoE–/– and Mmp-13–/–/apoE–/– mice. Three days after intraperitoneal injection of 4.0% thioglycollate, we harvested peritoneal macrophages from mice and cultured them with Dulbecco’s modified Eagle’s medium (DMEM). After 24-hour incubation with DMEM containing 10% FCS, culture media of adherent cells were refreshed with DMEM with or without 10% FCS and incubated further for 72 hours.
Real-Time RT-PCR
Total RNA was extracted from peritoneal macrophage isolates (n=3 each) and mouse aortas (n=4 each, pooled) and reverse transcribed. Real-time RT-PCR used SYBR green PCR Master Mix and ABI PRISM 7900 Sequence Detection System (Applied Biosystems). Oligonucleotide primers used to recognize mouse RNAs include MMP-8: 5'-CAA-CCT-ATT-TCT-CGT-GGC-TG-3' and 5'-TGC-AGG-TCA-TAG-CCA-CTT-AG-3'; MMP-9: 5'-CGT-CGT-GAT-CCC-CAC-TTA-CT-3' and 5'-AAC-ACA-CAG-GGT-TTG-CCT-TC-3'; MMP-12: 5'-TTT-CTT-CCA-TAT-GGC-CAA-GC-3' and 5'-GGT-CAA-AGA-CAG-CTG-CAT-CA-3'; MMP-14: 5'-AGG-GTT-CCT-GGC-TCA-TGC-3' and 5'-ACA-GCG-GCC-GCA-CTC-ACA-3'; cathepsin K: 5'-CCA-GTG-GGA-GCT-ATG-GAA-GA-3' and 5'-AAG-TGG-TTC-ATG-GCC-AGT-TC-3'; transforming growth factor-1 (TGF-1): 5'-GCA-ACA-TGT-GGA-ACT-CTA-CCA-GAA-3' and 5'-GAC-GTC-AAA-AGA-CAG-CCA-CTC-3'; 1 procollagen-I: 5'-AAG-GTG-CTG-ATG-GTT-CTC-C-3' and 5'-TCT-TTC-TCC-TCT-CTG-ACC-G-3'; and GAPDH: 5'-TGG-GTG-TGA-ACC-ATG-AGA-A-3' and 5'-GCT-AAG-CAG-TTG GTG-GTG-C-3'.
Histological Assays
Histological evaluations of the aortic root used the method of Paigen et al.22,28 Hearts dissected in the region of the proximal aorta were embedded in paraffin, and 5-μm serial sections were cut. Histological analyses used sections approximately 50 μm above the beginning of aortic sinuses. Initial characterization of Mmp-13–/–/apoE–/– mice used hematoxylin and eosin staining and immunohistochemistry with a rabbit polyclonal antibody against human -smooth muscle actin (Laboratory Vision, Fremont, Calif) and rat monoclonal antibody against mouse Mac3 (Dako, Fort Collins, Colo) to determine whether the size of the intimal lesion and the amount of smooth muscle cell (SMC) and macrophage accumulation within the plaques changed depending on genotype. These 2 cell types have a particularly important role because their functional balance likely influences collagen accumulation; SMC produce the bulk of arterial interstitial collagen, whereas macrophages furnish most plaque collagenase.16,17,20 Immunohistochemistry for MMPs used a rabbit polyclonal anti-mouse MMP-9 and goat polyclonal anti-mouse and rat MMP-13 antibodies (Chemicon). Detection of interstitial collagen used picrosirius red staining with linear polarized light, as described previously.17,29,30
For quantification of histological assays, captured photomicrographs were transferred into an image analysis system (ImagePro Plus 5.1, Media Cybernetics).6,17,18,22 A color-threshold mask for immunostaining was established to detect red (positive) staining, and the same threshold was applied to all specimens. The percentage of area with positive color for each section was recorded. For picrosirius red staining, negative background (black) was chosen for thresholding, and the positive area was calculated by subtraction. This analysis was performed independently by 2 blinded investigators with excellent interobserver correlation (r=0.98).
Collagen Fiber Morphology
A subset of animals from each group (n=3) underwent a detailed structural analysis of collagen in the fibrous cap. Specifically, we exploited the optical properties of picrosirius red–stained fibers viewed with circularly polarized light to examine 3 structural parameters: fiber color, fiber orientation, and the fibers’ retardation of light. These optical properties derive from the anisotropic molecular configuration of birefringent materials such as collagen and furnish well-established tools to assess changes in collagen structure.31–34
Collagen Fiber Color
Collagen fiber color in picrosirius red–stained sections viewed with polarized light depends on thickness; as fiber thickness increases, the color changes from green to yellow to orange to red. Images from 3 different locations in each aortic sample were viewed with the use of a x40 objective lens on an Olympus BX51 polarizing microscope, recorded by a digital camera (DP 11, Olympus), displayed on a high-resolution Trinitron color monitor (Sony), and studied with the use of image analysis software (SigmaScan Pro 5.0, SPSS Inc). We assessed the relative amount and spatial distribution of each color fiber.34 Briefly, an automated software function separated each color image into its hue, saturation, and value components. In this way, colors are represented by a range of 256 hues. From the hue component image, we tabulated the frequency of each hue value within the region of interest and calculated the relative amount of each fiber color according to the following criteria; red, hue values 230 to 256 and 2 to 9; orange, 10 to 38; yellow, 39 to 51; and green, 52 to 128 (values from 129 to 229 represent interstitial space and noncollagen components, confirmed by inspection). The relative amount of each fiber color was expressed as a percentage of the total amount of collagen in the region. In turn, collagen content in the cap was determined by expressing the number of collagen pixels as a percentage of the total number of pixels in the region (mean pixel numbers per region: 103 963.4; mean pixel numbers per mouse: 311 890.3). Information about the spatial distribution of fiber colors was obtained through application of a color-threshold technique. According to the value ranges specified above, hue-threshold filters were applied sequentially to each original image. The resulting images, containing only fibers of a single color, were compared qualitatively.
Collagen Fiber Orientation
The other 2 morphological parameters—2-dimensional fiber orientation and the retardation of polarized light—were measured with the PolScope Imaging System (CRI Inc), which uses liquid crystal compensators with a CCD camera and image processing software to provide automated and precise measurement of these parameters. This approach, which functions as an attachment to a standard microscope, was originally developed for the analysis of cell cultures.35,36 We have previously confirmed the accuracy of the automated PolScope analysis versus established manual methods (P. Whittaker and L. Rich, unpublished data, 2005). For each cap region assessed in each sample, we measured 2-dimensional fiber orientation at 50 locations (selected by construction of a 10x5 grid over the image; 150 locations per mouse) and also measured the tangent to the vessel wall at the point of orientation measurement (to indicate the circumferential direction). For each orientation distribution obtained, we calculated the mean orientation angle, the angular deviation of the distribution (a measure of the spread of the distribution: the smaller the angular deviation, the more aligned the fibers), and the absolute difference between the mean orientation and the circumferential direction.
Collagen Fiber Retardation
The retardation () of polarized light by a birefringent material is determined by the equation =t(ne–no), where t is the thickness of the material and the term (ne–no) denotes the birefringence of the material with ne and no, the refractive indices of the orthogonal components into which the polarized light is resolved passing through it.37 These indices depend on the molecular anisotropy of the material; for example, thermal denaturation of collagen molecules decreases anisotropy, and hence retardation decreases.32 The retardation values, measured in nanometers, in plaque collagen were recorded at the same locations as the orientation angles. We then calculated the mean retardation of each sample and for each group.
Statistical Analysis
Differences between the 2 groups were determined with the use of the Mann-Whitney U test. The orientation data were analyzed with established tests to examine directional data.38
Results
Characteristics of Mice
Body mass and plasma lipid profile did not differ between Mmp-13+/+/apoE–/– and Mmp-13–/–/apoE–/– mice after 10 weeks on the atherogenic diet (Table).
Characteristics of Mice
MMP-13/Collagenase-3 Deficiency Did Not Alter Plaque Burden or Cell Content
The aortic intimal area of Mmp-13+/+/apoE–/– mice resembled that of the Mmp-13–/–/apoE–/– mice at 5 and 10 weeks (Figure 1). This result agrees with our previous finding that collagenase-resistant mice had atheroma burden similar to that of apoE–/– mice with wild-type collagen.22 Representative images show similar accumulation of plaque macrophages (Mac3) and SMC (-actin) in Mmp-13+/+/apoE–/– and Mmp-13–/–/apoE–/– mice at 10 weeks (Figure 2A). Quantitative image analysis demonstrated that MMP-13 deficiency altered neither macrophage nor SMC content of plaques compared with Mmp-13+/+/apoE–/– mice at 5 and 10 weeks of the atherogenic diet (Figure 2B). Immunoreactive MMP-13/collagenase-3 and MMP-9/gelatinase-B localized in the intima of Mmp-13+/+/apoE–/– mice that consumed an atherogenic diet for 10 weeks (Figure 3). In contrast, the aortic intima of Mmp-13–/–/apoE–/– mice did not contain MMP-13/collagenase-3 proteins but did express MMP-9/gelatinase-B.
Expression of Molecules Associated With Matrix Remodeling
MMP-13/collagenase-3 deficiency did not change levels of mRNAs that encode MMP-8/collagenase-2, MMP-9/gelatinase-B, MMP-12, or cathepsin K in thioglycollate-elicited peritoneal macrophages, demonstrated by real-time RT-PCR (Figure 4A). Additionally, we observed no change in mRNA expression of TGF-1 and Cbfa-1/Runx2, factors that regulate collagenase expression (Figure 4A). Mmp-13–/– macrophages did express higher levels of mRNA encoding MMP-14 mRNA, a more recently recognized putative collagenase.
We then examined the mRNA expression of these proteinases and regulators of MMP-13/collagenase-3 in the aortic tissue of the mice. Both groups had similar levels of mRNAs encoding MMP-8/collagenase-2, MMP-12, MMP-14, cathepsin K, TGF-1, and Cbfa-1 (Figure 4B). Furthermore, real-time RT-PCR showed similar levels of type I procollagen (1) mRNA expression in both groups. However, MMP-13/collagenase-3 deficiency reduced MMP-9/gelatinase-B mRNA expression.
MMP-13/Collagenase-3 Deficiency Increased Plaque Collagen Content
We tested the hypothesis that MMP-13/collagenase-3 deficiency increases collagen accumulation in atherosclerotic mouse aortas. The aortic intimas of both groups contained similar amounts of collagen after 5 weeks (data not shown). However, after 10 weeks on the atherosclerotic diet, the aortic intima of Mmp-13–/–/apoE–/– mice contained more collagen than that measured in Mmp-13+/+/apoE–/– mice in terms of both absolute and percent areas (Figure 5A, 5B).
MMP-13/Collagenase-3 Deficiency Influenced Collagen Fiber Morphology
Circularly rather than linearly polarized light permits the visualization of all picrosirius red–stained collagen fibers. Representative images show accumulation of more collagen in the intima of Mmp-13–/–/apoE–/– mice than in Mmp-13+/+/ apoE–/– mice (Figure 6A, top). The pseudocolor images illustrate results of retardation analysis (Figure 6A, middle). In these images, colors of increasing wavelength (ie, from purple through green to red) indicate greater retardation. Higher wavelength colors appeared with greater frequency in lesions from mice lacking MMP-13. The average retardation value in MMP-13 wild-type mice was significantly lower (22.3±1.9 nm) than in the Mmp-13–/– group (33.1±3.5 nm; P<0.05; Figure 6B, left).
Collagen color (hue) analysis revealed that the cap region of atherosclerotic plaques in wild-type mice had more green collagen (ie, thinner) fibers than those with the collagenase deficiency. Examination of the threshold images substantiated this finding, showing the relative abundance of green fibers in the wild-type sample (Figure 6A, bottom). Quantitative analysis indicates statistically significant differences (21.0±1.9% versus 12.3±2.1%; P<0.01; Figure 6B, right). Small differences between groups for the other fiber colors did not achieve statistical significance (data not shown).
The average angular deviation of the orientation distributions from the wild-type caps was significantly greater (20.0±2.0°) than that in the Mmp-13–/–/apoE–/– mice (13.6±1.5°; P<0.05). Representative examples demonstrate greater angular deviation in an Mmp-13+/+/apoE–/– mouse atheroma than in a plaque from the same location in an Mmp-13–/–/apoE–/– mouse (angular deviation=22.8° and 12.2°, respectively; Figure 6C). In addition, the mean orientation angle deviated further from the circumferential orientation in the MMP-13 wild-type group (14.3±2.6°) than in the Mmp-13–/– animals (8.7±1.8°; P<0.05).
Discussion
This study tested the hypothesis that a specific MMP-collagenase influences the development and structure of atherosclerotic plaques. We proposed more than a decade ago that a highly regulated balance of synthesis and degradation determines fibrillar collagen content in atherosclerotic plaques and that degradation by MMP-collagenases contributes significantly to arterial remodeling.4 We and others demonstrated overexpression of MMP-collagenases, including MMP-13, in atherosclerotic plaques.15–20 Moreover, Shah et al39 reported that macrophage-derived MMPs can digest human fibrous cap collagen. We also demonstrated that lipid lowering in hypercholesterolemic rabbits decreased collagenase expression and reciprocally increased collagen accumulation in plaques.9,17 Together, these studies provide compelling, albeit indirect, evidence for the important role of collagenases in collagen breakdown within inflamed plaques.
Our recent study on collagenase-resistant mutant "knock-in" mice in compound mutation with atherosclerosis susceptibility due to apoE deficiency provided direct evidence that collagenases critically determine plaque collagen content.22 Although this study underscored the importance of collagenases in the pathogenesis of atherosclerosis, it did not address the relative contribution of any specific collagenase in atherosclerosis. In the adult mouse, which lacks the orthologue of human MMP-1/collagenase-1, macrophages express both MMP-13/collagenase-3 and MMP-8/collagenase-2, although MMP-13/collagenase-3 appears more abundant.22
Several experimental and clinical studies have linked MMP-13/collagenase-3 with the pathogenesis of various diseases involving inflammation, angiogenesis, and tissue destruction.1–3,7,40,41 Mice transgenic for a constitutively active form of human MMP-13/collagenase-3 in cartilage exhibited erosion of the articular cartilage associated with augmented cleavage of type II collagen,42 the preferred substrate for this collagenase.8 This enzyme can also cleave type I collagen, a major component of the collagenous framework of arteries. In vitro experiments further suggest that MMP-13/collagenase-3 can degrade collagen type I as potently as MMP-1/collagenase-1 and MMP-8/collagenase-2.8 The collagenases of the MMP family share a cleavage site on type I collagen. However, unlike MMP-1/collagenase-1 and MMP-8/collagenase-2, MMP-13/collagenase-3 can also cleave type I collagen at another site in the N-telopeptide, downstream from the putative cross-linking lysine residue.43 The present study demonstrates that the aortic intima of Mmp-13–/–/apoE–/– mice contained more interstitial collagen than did that of Mmp-13+/+/apoE–/– mice, demonstrating definitively that this collagenase contributes to collagen remodeling during experimental atherogenesis.
Deletion of a gene in genetically altered mice sometimes causes "compensatory" increases in other genes that share similar functions with the targeted gene. Such responses hinder data interpretation and require careful examination. For example, our previous study on MMP-9–deficient mice unexpectedly revealed decreased myocardial collagen accumulation after experimental acute myocardial infarction compared with wild-type mice.44 This seemingly paradoxical result in MMP-9–deficient mice most likely resulted from an increase in MMP-13/collagenase-3. Therefore, the present study evaluated whether MMP-13/collagenase-3 deficiency altered the ability of macrophages to express other major matrix-degrading enzymes, particularly collagenases (eg, MMP-8/collagenase-2, MMP-14, cathepsin K), and levels of these proteinases in the atherosclerotic aorta (Figure 4). Although real-time RT-PCR revealed higher levels of MMP-14 mRNA expression in peritoneal macrophages of Mmp-13–/–/apoE–/– mice than Mmp-13+/+/apoE–/– mice, we found no substantial difference in levels of MMP-14 or other collagenases in aortas of mice of either genotype. This study documented a significant increase in interstitial collagen content in the intima of Mmp-13–/–/apoE–/– mice. Notably, MMP-13/collagenase-3 deficiency did not affect expression levels of procollagen-I mRNA. Furthermore, deletion of MMP-13/collagenase-3 did not change expression of TGF-1 (a regulator of production of MMPs and collagen) in either macrophages in vitro or aortas in vivo. The aortas of Mmp-13–/–/apoE–/– mice contained lower levels of MMP-9/gelatinase-B mRNA than those from Mmp-13+/+/apoE–/– mice. Because MMP-9/gelatinase-B alone cannot initiate degradation of native, undenatured interstitial collagen,23,27 it is unlikely that reduced MMP-9 expression played a major role in the accumulation of fibrillar collagen in lesions of MMP-13/collagenase-3–deficient mice. We therefore conclude that the observed increase in collagen content in the Mmp-13–/–/apoE–/– mice primarily resulted from MMP-13/collagenase-3 deficiency.
Previous animal studies have yielded contradictory results with regard to the role of MMPs in the acceleration or retardation of atheroma progression.45–50 Lemaitre et al49 demonstrated that macrophage-specific transgenic mice for human MMP-1/collagenase-1 had less advanced atherosclerosis. Rouis et al,45 however, reported that overexpression of human tissue inhibitor of metalloproteinase-1 (TIMP-1, an inhibitor of collagenases and other MMPs) decreased aortic lesion area in apoE–/– mice. In contrast, Silence et al48 showed reduced atherosclerotic plaques in mice with inactivated TIMP-1. Curiously, Lemaitre et al50 found no change in atheroma burden in Timp-1–/–/apoE–/– mice. Our recent study using ColR/R/apoE–/– mice demonstrated that interference with the ability of collagenases to digest type I collagen did not affect lesion size.22 Concordantly, the present study shows no significant difference in atheroma burden between Mmp-13+/+/apoE–/– and Mmp-13–/–/apoE–/– mice at 2 time points.
Because mouse atheromata do not reliably develop thrombotic complications seen clinically, this study did not seek to model the human disease. Rather, it tested a mechanistic hypothesis with regard to interstitial collagen metabolism in atherosclerosis. Indeed, the differences in MMP-collagenase utilization by mice and humans render inappropriate the direct extrapolation to human lesions. Hence, this study probed the mechanism of the regulation of collagen structure in atherosclerotic plaques rather than devising a model of the human disease.
In addition to increased collagen content, this study provides additional information about the effects of MMP-13/collagenase-3 deficiency on interstitial collagen fiber structure in atherosclerotic lesions. Collagen plays a vital structural role in various tissues through its fiber content, thickness, and orientation. The quantitative analysis in the present study, provided by combining picrosirius red staining and circularly polarized light, revealed that MMP-13/collagenase-3 deficiency yielded thicker, more aligned, and more circumferentially oriented collagen fibers in the cap region of the intimal lesions than in Mmp-13+/+/apoE–/– mice (Figure 6). These changes in collagen structure likely alter the mechanical properties of the plaque, a possibility that requires further study.
Our data support an in vivo role for MMP-13/collagenase-3 in the economy of interstitial collagen, a component of human atherosclerotic plaques that influences their clinical consequences. Particularly, our complementary approach using mice genetically altered in the enzyme (Mmp-13–/– mice) and substrate (ColR/R mice)22 strengthens the in vivo evidence that collagenases participate in arterial collagen remodeling. These results provide important insights into the pathogenesis of collagen breakdown in atherosclerosis as well as other inflammatory diseases.
Acknowledgments
This work was supported in part by grants from the National Institutes of Health (SCOR HL-56985 to Drs Libby and Aikawa; 1R01 HL-80472 to Dr Libby; 1R01 HL-66086 to Dr Aikawa; 5R01 AR44815 to Dr Krane) and the Donald W. Reynolds Foundation (to Dr Libby). Dr Deguchi received a fellowship from the Reynolds Foundation. We also acknowledge Karen E. Williams for her excellent editorial assistance and Karen Mendelson for her technical support.
References
Brinckerhoff CE, Matrisian LM. Matrix metalloproteinases: a tail of a frog that became a prince. Nat Rev Mol Cell Biol. 2002; 3: 207–214.
Visse R, Nagase H. Matrix metalloproteinases and tissue inhibitors of metalloproteinases: structure, function, and biochemistry. Circ Res. 2003; 92: 827–839.
Krane SM. Elucidation of the potential roles of matrix metalloproteinases in skeletal biology. Arthritis Res Ther. 2003; 5: 2–4.
Libby P. Molecular bases of the acute coronary syndromes. Circulation. 1995; 91: 2844–2850.
Whittaker P, Boughner DR, Kloner RA. Role of collagen in acute myocardial infarct expansion. Circulation. 1991; 84: 2123–2134.
Rabkin E, Aikawa M, Stone JR, Fukumoto Y, Libby P, Schoen FJ. Activated interstitial myofibroblasts express catabolic enzymes and mediate matrix remodeling in myxomatous heart valves. Circulation. 2001; 104: 2525–2532.
Freije JM, Diez-Itza I, Balbin M, Sanchez LM, Blasco R, Tolivia J, Lopez-Otin C. Molecular cloning and expression of collagenase-3, a novel human matrix metalloproteinase produced by breast carcinomas. J Biol Chem. 1994; 269: 16766–16773.
Knauper V, Lopez-Otin C, Smith B, Knight G, Murphy G. Biochemical characterization of human collagenase-3. J Biol Chem. 1996; 271: 1544–1550.
Aikawa M, Libby P. The vulnerable atherosclerotic plaque: pathogenesis and therapeutic approach. Cardiovasc Pathol. 2004; 13: 125–138.
Davies MJ, Thomas AC. Plaque fissuring: the cause of acute myocardial infarction, sudden ischaemic death, and crescendo angina. Br Heart J. 1985; 53: 363–373.
Falk E, Shah PK, Fuster V. Coronary plaque disruption. Circulation. 1995; 92: 657–671.
Kolodgie FD, Burke AP, Farb A, Gold HK, Yuan J, Narula J, Finn AV, Virmani R. The thin-cap fibroatheroma: a type of vulnerable plaque: the major precursor lesion to acute coronary syndromes. Curr Opin Cardiol. 2001; 16: 285–292.
Moreno PR, Fuster V. The year in atherothrombosis. J Am Coll Cardiol. 2004; 44: 2099–2110.
Henney AM, Wakeley PR, Davies MJ, Foster K, Hembry R, Murphy G, Humphries S. Localization of stromelysin gene expression in atherosclerotic plaques by in situ hybridization. Proc Natl Acad Sci U S A. 1991; 88: 8154–8158.
Galis ZS, Sukhova GK, Lark MW, Libby P. Increased expression of matrix metalloproteinases and matrix degrading activity in vulnerable regions of human atherosclerotic plaques. J Clin Invest. 1994; 94: 2493–2503.
Nikkari ST, O’Brien KD, Ferguson M, Hatsukami T, Welgus HG, Alpers CE, Clowes AW. Interstitial collagenase (MMP-1) expression in human carotid atherosclerosis. Circulation. 1995; 92: 1393–1398.
Aikawa M, Rabkin E, Okada Y, Voglic SJ, Clinton SK, Brinckerhoff CE, Sukhova GK, Libby P. Lipid lowering by diet reduces matrix metalloproteinase activity and increases collagen content of rabbit atheroma: a potential mechanism of lesion stabilization. Circulation. 1998; 97: 2433–2444.
Aikawa M, Rabkin E, Sugiyama S, Voglic SJ, Fukumoto Y, Furukawa Y, Shiomi M, Schoen FJ, Libby P. An HMG-CoA reductase inhibitor, cerivastatin, suppresses growth of macrophages expressing matrix metalloproteinases and tissue factor in vivo and in vitro. Circulation. 2001; 103: 276–283.
Fukumoto Y, Libby P, Rabkin E, Hill CC, Enomoto M, Hirouchi Y, Shiomi M, Aikawa M. Statins alter smooth muscle cell accumulation and collagen content in established atheroma of Watanabe heritable hyperlipidemic rabbits. Circulation. 2001; 103: 993–999.
Sukhova GK, Schonbeck U, Rabkin E, Schoen FJ, Poole AR, Billinghurst RC, Libby P. Evidence for increased collagenolysis by interstitial collagenases-1 and -3 in vulnerable human atheromatous plaques. Circulation. 1999; 99: 2503–2509.
Herman MP, Sukhova GK, Libby P, Gerdes N, Tang N, Horton DB, Kilbride M, Breitbart RE, Chun M, Schonbeck U. Expression of neutrophil collagenase (matrix metalloproteinase-8) in human atheroma: a novel collagenolytic pathway suggested by transcriptional profiling. Circulation. 2001; 104: 1899–1904.
Fukumoto Y, Deguchi JO, Libby P, Rabkin-Aikawa E, Sakata Y, Chin MT, Hill CC, Lawler PR, Varo N, Schoen FJ, Krane SM, Aikawa M. Genetically determined resistance to collagenase action augments interstitial collagen accumulation in atherosclerotic plaques. Circulation. 2004; 110: 1953–1959.
Aimes RT, Quigley JP. Matrix metalloproteinase-2 is an interstitial collagenase: inhibitor-free enzyme catalyzes the cleavage of collagen fibrils and soluble native type I collagen generating the specific 3/4- and 1/4-length fragments. J Biol Chem. 1995; 270: 5872–5876.
Ohuchi E, Imai K, Fujii Y, Sato H, Seiki M, Okada Y. Membrane type 1 matrix metalloproteinase digests interstitial collagens and other extracellular matrix macromolecules. J Biol Chem. 1997; 272: 2446–2451.
Sabeh F, Ota I, Holmbeck K, Birkedal-Hansen H, Soloway P, Balbin M, Lopez-Otin C, Shapiro S, Inada M, Krane S, Allen E, Chung D, Weiss SJ. Tumor cell traffic through the extracellular matrix is controlled by the membrane-anchored collagenase MT1-MMP. J Cell Biol. 2004; 167: 769–781.
Inada M, Wang Y, Byrne MH, Rahman MU, Miyaura C, Lopez-Otin C, Krane SM. Critical roles for collagenase-3 (Mmp13) in development of growth plate cartilage and in endochondral ossification. Proc Natl Acad Sci U S A. 2004; 101: 17192–17197.
Stickens D, Behonick DJ, Ortega N, Heyer B, Hartenstein B, Yu Y, Fosang AJ, Schorpp-Kistner M, Angel P, Werb Z. Altered endochondral bone development in matrix metalloproteinase 13-deficient mice. Development. 2004; 131: 5883–5895.
Paigen B, Morrow A, Holmes P, Mitchell D, Williams R. Quantitative assessment of atherosclerotic lesions in mice. Atherosclerosis. 1987; 68: 231–240.
Sweat F, Puchtler H, Rosenthal SI. Sirius red F3BA as a stain for connective tissue. Arch Pathol. 1964; 78: 69–72.
Junqueira LC, Bignolas G, Brentani RR. Picrosirius staining plus polarization microscopy, a specific method for collagen detection in tissue sections. Histochem J. 1979; 11: 447–455.
Whittaker P, Canham PB. Demonstration of quantitative fabric analysis of tendon collagen using two-dimensional polarized light microscopy. Matrix. 1991; 11: 56–62.
Whittaker P, Zheng S, Patterson MJ, Kloner RA, Daly KE, Hartman RA. Histologic signatures of thermal injury: applications in transmyocardial laser revascularization and radiofrequency ablation. Lasers Surg Med. 2000; 27: 305–318.
Whittaker P, Müller-Ehmsen J, Dow JS, Kedes LH, Kloner RA. Development of abnormal tissue architecture in transplanted neonatal rat myocytes. Ann Thorac Surg. 2003; 75: 1450–1456.
Rich L, Whittaker P. Collagen and picrosirius red staining: a polarized light assessment of fibrillar hue and spatial distribution. Braz J Morphol Sci. 2005; 22: 97–104.
Oldenbourg R, Mei G. New polarized light microscope with precision universal compensator. J Microsc. 1995; 180 (pt 2): 140–147.
Katoh K, Hammar K, Smith PJ, Oldenbourg R. Birefringence imaging directly reveals architectural dynamics of filamentous actin in living growth cones. Mol Biol Cell. 1999; 10: 197–210.
Wolman M. On the use of polarized light in pathology. Pathol Annu. 1970; 5: 381–416.
Batschelet E. Circular Statistics in Biology. London, UK: Academic Press; 1981.
Shah PK, Falk E, Badimon JJ, Fernandez-Ortiz A, Mailhac A, Villareal-Levy G, Fallon JT, Regnstrom J, Fuster V. Human monocyte-derived macrophages induce collagen breakdown in fibrous caps of atherosclerotic plaques: potential role of matrix-degrading metalloproteinases and implications for plaque rupture. Circulation. 1995; 92: 1565–1569.
Stahle-Backdahl M, Sandstedt B, Bruce K, Lindahl A, Jimenez MG, Vega JA, Lopez-Otin C. Collagenase-3 (MMP-13) is expressed during human fetal ossification and re-expressed in postnatal bone remodeling and in rheumatoid arthritis. Lab Invest. 1997; 76: 717–728.
Seandel M, Noack-Kunnmann K, Zhu D, Aimes RT, Quigley JP. Growth factor-induced angiogenesis in vivo requires specific cleavage of fibrillar type I collagen. Blood. 2001; 97: 2323–2332.
Neuhold LA, Killar L, Zhao W, Sung ML, Warner L, Kulik J, Turner J, Wu W, Billinghurst C, Meijers T, Poole AR, Babij P, DeGennaro LJ. Postnatal expression in hyaline cartilage of constitutively active human collagenase-3 (MMP-13) induces osteoarthritis in mice. J Clin Invest. 2001; 107: 35–44.
Krane SM, Byrne MH, Lemaitre V, Henriet P, Jeffrey JJ, Witter JP, Liu X, Wu H, Jaenisch R, Eeckhout Y. Different collagenase gene products have different roles in degradation of type I collagen. J Biol Chem. 1996; 271: 28509–28515.
Ducharme A, Frantz S, Aikawa M, Rabkin E, Lindsey M, Rohde LE, Schoen FJ, Kelly RA, Werb Z, Libby P, Lee RT. Targeted deletion of matrix metalloproteinase-9 attenuates left ventricular enlargement and collagen accumulation after experimental myocardial infarction. J Clin Invest. 2000; 106: 55–62.
Rouis M, Adamy C, Duverger N, Lesnik P, Horellou P, Moreau M, Emmanuel F, Caillaud JM, Laplaud PM, Dachet C, Chapman MJ. Adenovirus-mediated overexpression of tissue inhibitor of metalloproteinase-1 reduces atherosclerotic lesions in apolipoprotein E–deficient mice. Circulation. 1999; 100: 533–540.
Dollery CM, Humphries SE, McClelland A, Latchman DS, McEwan JR. Expression of tissue inhibitor of matrix metalloproteinases 1 by use of an adenoviral vector inhibits smooth muscle cell migration and reduces neointimal hyperplasia in the rat model of vascular balloon injury. Circulation. 1999; 99: 3199–3205.
Silence J, Lupu F, Collen D, Lijnen HR. Persistence of atherosclerotic plaque but reduced aneurysm formation in mice with stromelysin-1 (MMP-3) gene inactivation. Arterioscler Thromb Vasc Biol. 2001; 21: 1440–1445.
Silence J, Collen D, Lijnen HR. Reduced atherosclerotic plaque but enhanced aneurysm formation in mice with inactivation of the tissue inhibitor of metalloproteinase-1 (TIMP-1) gene. Circ Res. 2002; 90: 897–903.
Lemaitre V, O’Byrne TK, Borczuk AC, Okada Y, Tall AR, D’Armiento J. ApoE knockout mice expressing human matrix metalloproteinase-1 in macrophages have less advanced atherosclerosis. J Clin Invest. 2001; 107: 1227–1234.
Lemaitre V, Soloway PD, D’Armiento J. Increased medial degradation with pseudo-aneurysm formation in apolipoprotein E-knockout mice deficient in tissue inhibitor of metalloproteinases-1. Circulation. 2003; 107: 333–338.(Jun-O Deguchi, MD, PhD; E)
Center for Immunology and Inflammatory Diseases, Massachusetts General Hospital and Harvard Medical School, Charlestown, Mass (M.I., S.M.K.)
Departments of Emergency Medicine and Anesthesiology, University of Massachusetts Medical School, Worcester (P.W.).
Abstract
Background— Interstitial collagen plays a crucial structural role in arteries. Matrix metalloproteinases (MMPs), including MMP-13/collagenase-3, likely contribute to collagen catabolism in atherosclerotic plaques.
Methods and Results— To test the hypothesis that a specific MMP-collagenase influences the development and structure of atherosclerotic plaques, this study used atherosclerosis-susceptible apolipoprotein E–deficient mice that lack MMP-13/collagenase-3 (Mmp-13–/–/apoE–/–) or express wild-type MMP-13/collagenase-3 (Mmp-13+/+/apoE–/–). Both groups consumed an atherogenic diet for 5 (n=8) or 10 weeks (n=9). Histological analyses of the aortic root of both groups revealed similar plaque size and accumulation of smooth muscle cells (a collagen-producing cell type) and macrophages (a major source of plaque collagenases) after 5 and 10 weeks of atherogenic diet. By 10 weeks, the plaques of Mmp-13–/–/apoE–/– mice contained significantly more interstitial collagen than those of Mmp-13+/+/apoE–/– mice (P<0.01). Furthermore, quantitative optical analyses revealed thinner and less aligned periluminal collagen fibers within the plaques of Mmp-13+/+/apoE–/– mice versus those from Mmp-13–/–/apoE–/– mice.
Conclusions— These data support the hypothesis that MMP-13/collagenase-3 plays a vital role in the regulation and organization of collagen in atherosclerotic plaques.
Key Words: atherosclerosis collagen metalloproteinases pathology plaque
Introduction
Interstitial collagen maintains structural integrity and influences cell functions in a wide variety of tissues.1–6 The regulation of collagen levels depends on several factors, including the number and activation state of collagen-producing cells and the expression of collagen-degrading enzymes. Collagenases of the matrix metalloproteinase (MMP) family, including MMP-1/collagenase-1, MMP-8/collagenase-2, and MMP-13/collagenase-3, can degrade triple helical fibrillar collagen at neutral pH, an initial step that permits further digestion by other MMPs.1–3,7,8 We proposed that an imbalance between collagen synthesis and degradation regulates critical aspects of atherosclerotic plaque structure.4,9 Fibrillar collagens confer tensile strength on the fibrous cap of the plaque. Disruption of a collagen-poor, thin-capped plaque frequently triggers acute coronary thrombosis in humans.10–13 Previous studies in humans and animals have localized MMPs including the interstitial collagenases in plaques.14–21 Nevertheless, we lack in vivo evidence that links collagenase action directly to collagen loss in atherosclerotic plaques.
Clinical Perspective page 2715
We recently provided evidence that collagenases can indeed regulate intimal collagen accumulation.22 We crossed collagenase-resistant mice (ColR/R), whose 1(I) chain of type I collagen has a "knock-in" mutation at the cleavage site shared by MMP-family interstitial collagenases, with atherosclerosis-susceptible apolipoprotein E–deficient (apoE–/–) mice. Atherosclerotic plaques of ColR/R/apoE–/– mice contained more interstitial collagen than those of apoE–/– mice with wild-type collagen type I. Our study also localized MMP-13/collagenase-3 and MMP-8/collagenase-2 in macrophages and atherosclerotic aortas in mice, a species that lacks the orthologue of human MMP-1/collagenase-1. "Unconventional" collagenases, MMP-2/gelatinase-A and membrane-type I MMP (MT1-MMP or MMP-14), may also cleave triple helical type I collagen.23–25 The relative contribution of various MMP-collagenases to collagen remodeling in atherosclerotic plaques, however, has not undergone rigorous evaluations.
We26 and others27 have recently reported effects of MMP-13 deletion on embryonic development of bone and cartilage. Nevertheless, the role of MMP-13 in arterial disease remains unexplored. We therefore used compound mutant Mmp-13–/–/apoE–/– mice to test the biological hypothesis that MMP-13/collagenase-3 regulates collagen content and structure in atherosclerotic plaques.
Methods
Animal Preparation
All experiments conformed to a protocol approved by the Standing Committee on Animals of Harvard Medical School. Mmp-13–/– mice were generated by gene targeting in embryonic stem cells.26 We backcrossed Mmp-13–/– mice (C57BL/6x129) 7 generations into congenic C57BL/6 mice. Mmp-13–/– mice (C57BL/6) were then crossed into apoE–/– mice (C57BL/6) to render Mmp-13–/– mice atherosclerosis susceptible, yielding Mmp-13+/+/apoE–/– and Mmp-13–/–/apoE–/– mice. Five-week-old male Mmp-13+/+/apoE–/– (n=17) and Mmp-13–/–/apoE–/– (n=17) littermates consumed an atherogenic diet (semipurified chow containing 1.25% cholesterol and 0% cholate, Research Diets) for 5 weeks (n=8 each) or 10 weeks (n=9 each).
Macrophage Culture
To determine whether MMP-13/collagenase-3 deficiency causes compensatory alterations in expression of other major matrix-degrading enzymes, which also could influence collagen accumulation in plaques, we examined mRNA expression by macrophages from Mmp-13+/+/apoE–/– and Mmp-13–/–/apoE–/– mice. Three days after intraperitoneal injection of 4.0% thioglycollate, we harvested peritoneal macrophages from mice and cultured them with Dulbecco’s modified Eagle’s medium (DMEM). After 24-hour incubation with DMEM containing 10% FCS, culture media of adherent cells were refreshed with DMEM with or without 10% FCS and incubated further for 72 hours.
Real-Time RT-PCR
Total RNA was extracted from peritoneal macrophage isolates (n=3 each) and mouse aortas (n=4 each, pooled) and reverse transcribed. Real-time RT-PCR used SYBR green PCR Master Mix and ABI PRISM 7900 Sequence Detection System (Applied Biosystems). Oligonucleotide primers used to recognize mouse RNAs include MMP-8: 5'-CAA-CCT-ATT-TCT-CGT-GGC-TG-3' and 5'-TGC-AGG-TCA-TAG-CCA-CTT-AG-3'; MMP-9: 5'-CGT-CGT-GAT-CCC-CAC-TTA-CT-3' and 5'-AAC-ACA-CAG-GGT-TTG-CCT-TC-3'; MMP-12: 5'-TTT-CTT-CCA-TAT-GGC-CAA-GC-3' and 5'-GGT-CAA-AGA-CAG-CTG-CAT-CA-3'; MMP-14: 5'-AGG-GTT-CCT-GGC-TCA-TGC-3' and 5'-ACA-GCG-GCC-GCA-CTC-ACA-3'; cathepsin K: 5'-CCA-GTG-GGA-GCT-ATG-GAA-GA-3' and 5'-AAG-TGG-TTC-ATG-GCC-AGT-TC-3'; transforming growth factor-1 (TGF-1): 5'-GCA-ACA-TGT-GGA-ACT-CTA-CCA-GAA-3' and 5'-GAC-GTC-AAA-AGA-CAG-CCA-CTC-3'; 1 procollagen-I: 5'-AAG-GTG-CTG-ATG-GTT-CTC-C-3' and 5'-TCT-TTC-TCC-TCT-CTG-ACC-G-3'; and GAPDH: 5'-TGG-GTG-TGA-ACC-ATG-AGA-A-3' and 5'-GCT-AAG-CAG-TTG GTG-GTG-C-3'.
Histological Assays
Histological evaluations of the aortic root used the method of Paigen et al.22,28 Hearts dissected in the region of the proximal aorta were embedded in paraffin, and 5-μm serial sections were cut. Histological analyses used sections approximately 50 μm above the beginning of aortic sinuses. Initial characterization of Mmp-13–/–/apoE–/– mice used hematoxylin and eosin staining and immunohistochemistry with a rabbit polyclonal antibody against human -smooth muscle actin (Laboratory Vision, Fremont, Calif) and rat monoclonal antibody against mouse Mac3 (Dako, Fort Collins, Colo) to determine whether the size of the intimal lesion and the amount of smooth muscle cell (SMC) and macrophage accumulation within the plaques changed depending on genotype. These 2 cell types have a particularly important role because their functional balance likely influences collagen accumulation; SMC produce the bulk of arterial interstitial collagen, whereas macrophages furnish most plaque collagenase.16,17,20 Immunohistochemistry for MMPs used a rabbit polyclonal anti-mouse MMP-9 and goat polyclonal anti-mouse and rat MMP-13 antibodies (Chemicon). Detection of interstitial collagen used picrosirius red staining with linear polarized light, as described previously.17,29,30
For quantification of histological assays, captured photomicrographs were transferred into an image analysis system (ImagePro Plus 5.1, Media Cybernetics).6,17,18,22 A color-threshold mask for immunostaining was established to detect red (positive) staining, and the same threshold was applied to all specimens. The percentage of area with positive color for each section was recorded. For picrosirius red staining, negative background (black) was chosen for thresholding, and the positive area was calculated by subtraction. This analysis was performed independently by 2 blinded investigators with excellent interobserver correlation (r=0.98).
Collagen Fiber Morphology
A subset of animals from each group (n=3) underwent a detailed structural analysis of collagen in the fibrous cap. Specifically, we exploited the optical properties of picrosirius red–stained fibers viewed with circularly polarized light to examine 3 structural parameters: fiber color, fiber orientation, and the fibers’ retardation of light. These optical properties derive from the anisotropic molecular configuration of birefringent materials such as collagen and furnish well-established tools to assess changes in collagen structure.31–34
Collagen Fiber Color
Collagen fiber color in picrosirius red–stained sections viewed with polarized light depends on thickness; as fiber thickness increases, the color changes from green to yellow to orange to red. Images from 3 different locations in each aortic sample were viewed with the use of a x40 objective lens on an Olympus BX51 polarizing microscope, recorded by a digital camera (DP 11, Olympus), displayed on a high-resolution Trinitron color monitor (Sony), and studied with the use of image analysis software (SigmaScan Pro 5.0, SPSS Inc). We assessed the relative amount and spatial distribution of each color fiber.34 Briefly, an automated software function separated each color image into its hue, saturation, and value components. In this way, colors are represented by a range of 256 hues. From the hue component image, we tabulated the frequency of each hue value within the region of interest and calculated the relative amount of each fiber color according to the following criteria; red, hue values 230 to 256 and 2 to 9; orange, 10 to 38; yellow, 39 to 51; and green, 52 to 128 (values from 129 to 229 represent interstitial space and noncollagen components, confirmed by inspection). The relative amount of each fiber color was expressed as a percentage of the total amount of collagen in the region. In turn, collagen content in the cap was determined by expressing the number of collagen pixels as a percentage of the total number of pixels in the region (mean pixel numbers per region: 103 963.4; mean pixel numbers per mouse: 311 890.3). Information about the spatial distribution of fiber colors was obtained through application of a color-threshold technique. According to the value ranges specified above, hue-threshold filters were applied sequentially to each original image. The resulting images, containing only fibers of a single color, were compared qualitatively.
Collagen Fiber Orientation
The other 2 morphological parameters—2-dimensional fiber orientation and the retardation of polarized light—were measured with the PolScope Imaging System (CRI Inc), which uses liquid crystal compensators with a CCD camera and image processing software to provide automated and precise measurement of these parameters. This approach, which functions as an attachment to a standard microscope, was originally developed for the analysis of cell cultures.35,36 We have previously confirmed the accuracy of the automated PolScope analysis versus established manual methods (P. Whittaker and L. Rich, unpublished data, 2005). For each cap region assessed in each sample, we measured 2-dimensional fiber orientation at 50 locations (selected by construction of a 10x5 grid over the image; 150 locations per mouse) and also measured the tangent to the vessel wall at the point of orientation measurement (to indicate the circumferential direction). For each orientation distribution obtained, we calculated the mean orientation angle, the angular deviation of the distribution (a measure of the spread of the distribution: the smaller the angular deviation, the more aligned the fibers), and the absolute difference between the mean orientation and the circumferential direction.
Collagen Fiber Retardation
The retardation () of polarized light by a birefringent material is determined by the equation =t(ne–no), where t is the thickness of the material and the term (ne–no) denotes the birefringence of the material with ne and no, the refractive indices of the orthogonal components into which the polarized light is resolved passing through it.37 These indices depend on the molecular anisotropy of the material; for example, thermal denaturation of collagen molecules decreases anisotropy, and hence retardation decreases.32 The retardation values, measured in nanometers, in plaque collagen were recorded at the same locations as the orientation angles. We then calculated the mean retardation of each sample and for each group.
Statistical Analysis
Differences between the 2 groups were determined with the use of the Mann-Whitney U test. The orientation data were analyzed with established tests to examine directional data.38
Results
Characteristics of Mice
Body mass and plasma lipid profile did not differ between Mmp-13+/+/apoE–/– and Mmp-13–/–/apoE–/– mice after 10 weeks on the atherogenic diet (Table).
Characteristics of Mice
MMP-13/Collagenase-3 Deficiency Did Not Alter Plaque Burden or Cell Content
The aortic intimal area of Mmp-13+/+/apoE–/– mice resembled that of the Mmp-13–/–/apoE–/– mice at 5 and 10 weeks (Figure 1). This result agrees with our previous finding that collagenase-resistant mice had atheroma burden similar to that of apoE–/– mice with wild-type collagen.22 Representative images show similar accumulation of plaque macrophages (Mac3) and SMC (-actin) in Mmp-13+/+/apoE–/– and Mmp-13–/–/apoE–/– mice at 10 weeks (Figure 2A). Quantitative image analysis demonstrated that MMP-13 deficiency altered neither macrophage nor SMC content of plaques compared with Mmp-13+/+/apoE–/– mice at 5 and 10 weeks of the atherogenic diet (Figure 2B). Immunoreactive MMP-13/collagenase-3 and MMP-9/gelatinase-B localized in the intima of Mmp-13+/+/apoE–/– mice that consumed an atherogenic diet for 10 weeks (Figure 3). In contrast, the aortic intima of Mmp-13–/–/apoE–/– mice did not contain MMP-13/collagenase-3 proteins but did express MMP-9/gelatinase-B.
Expression of Molecules Associated With Matrix Remodeling
MMP-13/collagenase-3 deficiency did not change levels of mRNAs that encode MMP-8/collagenase-2, MMP-9/gelatinase-B, MMP-12, or cathepsin K in thioglycollate-elicited peritoneal macrophages, demonstrated by real-time RT-PCR (Figure 4A). Additionally, we observed no change in mRNA expression of TGF-1 and Cbfa-1/Runx2, factors that regulate collagenase expression (Figure 4A). Mmp-13–/– macrophages did express higher levels of mRNA encoding MMP-14 mRNA, a more recently recognized putative collagenase.
We then examined the mRNA expression of these proteinases and regulators of MMP-13/collagenase-3 in the aortic tissue of the mice. Both groups had similar levels of mRNAs encoding MMP-8/collagenase-2, MMP-12, MMP-14, cathepsin K, TGF-1, and Cbfa-1 (Figure 4B). Furthermore, real-time RT-PCR showed similar levels of type I procollagen (1) mRNA expression in both groups. However, MMP-13/collagenase-3 deficiency reduced MMP-9/gelatinase-B mRNA expression.
MMP-13/Collagenase-3 Deficiency Increased Plaque Collagen Content
We tested the hypothesis that MMP-13/collagenase-3 deficiency increases collagen accumulation in atherosclerotic mouse aortas. The aortic intimas of both groups contained similar amounts of collagen after 5 weeks (data not shown). However, after 10 weeks on the atherosclerotic diet, the aortic intima of Mmp-13–/–/apoE–/– mice contained more collagen than that measured in Mmp-13+/+/apoE–/– mice in terms of both absolute and percent areas (Figure 5A, 5B).
MMP-13/Collagenase-3 Deficiency Influenced Collagen Fiber Morphology
Circularly rather than linearly polarized light permits the visualization of all picrosirius red–stained collagen fibers. Representative images show accumulation of more collagen in the intima of Mmp-13–/–/apoE–/– mice than in Mmp-13+/+/ apoE–/– mice (Figure 6A, top). The pseudocolor images illustrate results of retardation analysis (Figure 6A, middle). In these images, colors of increasing wavelength (ie, from purple through green to red) indicate greater retardation. Higher wavelength colors appeared with greater frequency in lesions from mice lacking MMP-13. The average retardation value in MMP-13 wild-type mice was significantly lower (22.3±1.9 nm) than in the Mmp-13–/– group (33.1±3.5 nm; P<0.05; Figure 6B, left).
Collagen color (hue) analysis revealed that the cap region of atherosclerotic plaques in wild-type mice had more green collagen (ie, thinner) fibers than those with the collagenase deficiency. Examination of the threshold images substantiated this finding, showing the relative abundance of green fibers in the wild-type sample (Figure 6A, bottom). Quantitative analysis indicates statistically significant differences (21.0±1.9% versus 12.3±2.1%; P<0.01; Figure 6B, right). Small differences between groups for the other fiber colors did not achieve statistical significance (data not shown).
The average angular deviation of the orientation distributions from the wild-type caps was significantly greater (20.0±2.0°) than that in the Mmp-13–/–/apoE–/– mice (13.6±1.5°; P<0.05). Representative examples demonstrate greater angular deviation in an Mmp-13+/+/apoE–/– mouse atheroma than in a plaque from the same location in an Mmp-13–/–/apoE–/– mouse (angular deviation=22.8° and 12.2°, respectively; Figure 6C). In addition, the mean orientation angle deviated further from the circumferential orientation in the MMP-13 wild-type group (14.3±2.6°) than in the Mmp-13–/– animals (8.7±1.8°; P<0.05).
Discussion
This study tested the hypothesis that a specific MMP-collagenase influences the development and structure of atherosclerotic plaques. We proposed more than a decade ago that a highly regulated balance of synthesis and degradation determines fibrillar collagen content in atherosclerotic plaques and that degradation by MMP-collagenases contributes significantly to arterial remodeling.4 We and others demonstrated overexpression of MMP-collagenases, including MMP-13, in atherosclerotic plaques.15–20 Moreover, Shah et al39 reported that macrophage-derived MMPs can digest human fibrous cap collagen. We also demonstrated that lipid lowering in hypercholesterolemic rabbits decreased collagenase expression and reciprocally increased collagen accumulation in plaques.9,17 Together, these studies provide compelling, albeit indirect, evidence for the important role of collagenases in collagen breakdown within inflamed plaques.
Our recent study on collagenase-resistant mutant "knock-in" mice in compound mutation with atherosclerosis susceptibility due to apoE deficiency provided direct evidence that collagenases critically determine plaque collagen content.22 Although this study underscored the importance of collagenases in the pathogenesis of atherosclerosis, it did not address the relative contribution of any specific collagenase in atherosclerosis. In the adult mouse, which lacks the orthologue of human MMP-1/collagenase-1, macrophages express both MMP-13/collagenase-3 and MMP-8/collagenase-2, although MMP-13/collagenase-3 appears more abundant.22
Several experimental and clinical studies have linked MMP-13/collagenase-3 with the pathogenesis of various diseases involving inflammation, angiogenesis, and tissue destruction.1–3,7,40,41 Mice transgenic for a constitutively active form of human MMP-13/collagenase-3 in cartilage exhibited erosion of the articular cartilage associated with augmented cleavage of type II collagen,42 the preferred substrate for this collagenase.8 This enzyme can also cleave type I collagen, a major component of the collagenous framework of arteries. In vitro experiments further suggest that MMP-13/collagenase-3 can degrade collagen type I as potently as MMP-1/collagenase-1 and MMP-8/collagenase-2.8 The collagenases of the MMP family share a cleavage site on type I collagen. However, unlike MMP-1/collagenase-1 and MMP-8/collagenase-2, MMP-13/collagenase-3 can also cleave type I collagen at another site in the N-telopeptide, downstream from the putative cross-linking lysine residue.43 The present study demonstrates that the aortic intima of Mmp-13–/–/apoE–/– mice contained more interstitial collagen than did that of Mmp-13+/+/apoE–/– mice, demonstrating definitively that this collagenase contributes to collagen remodeling during experimental atherogenesis.
Deletion of a gene in genetically altered mice sometimes causes "compensatory" increases in other genes that share similar functions with the targeted gene. Such responses hinder data interpretation and require careful examination. For example, our previous study on MMP-9–deficient mice unexpectedly revealed decreased myocardial collagen accumulation after experimental acute myocardial infarction compared with wild-type mice.44 This seemingly paradoxical result in MMP-9–deficient mice most likely resulted from an increase in MMP-13/collagenase-3. Therefore, the present study evaluated whether MMP-13/collagenase-3 deficiency altered the ability of macrophages to express other major matrix-degrading enzymes, particularly collagenases (eg, MMP-8/collagenase-2, MMP-14, cathepsin K), and levels of these proteinases in the atherosclerotic aorta (Figure 4). Although real-time RT-PCR revealed higher levels of MMP-14 mRNA expression in peritoneal macrophages of Mmp-13–/–/apoE–/– mice than Mmp-13+/+/apoE–/– mice, we found no substantial difference in levels of MMP-14 or other collagenases in aortas of mice of either genotype. This study documented a significant increase in interstitial collagen content in the intima of Mmp-13–/–/apoE–/– mice. Notably, MMP-13/collagenase-3 deficiency did not affect expression levels of procollagen-I mRNA. Furthermore, deletion of MMP-13/collagenase-3 did not change expression of TGF-1 (a regulator of production of MMPs and collagen) in either macrophages in vitro or aortas in vivo. The aortas of Mmp-13–/–/apoE–/– mice contained lower levels of MMP-9/gelatinase-B mRNA than those from Mmp-13+/+/apoE–/– mice. Because MMP-9/gelatinase-B alone cannot initiate degradation of native, undenatured interstitial collagen,23,27 it is unlikely that reduced MMP-9 expression played a major role in the accumulation of fibrillar collagen in lesions of MMP-13/collagenase-3–deficient mice. We therefore conclude that the observed increase in collagen content in the Mmp-13–/–/apoE–/– mice primarily resulted from MMP-13/collagenase-3 deficiency.
Previous animal studies have yielded contradictory results with regard to the role of MMPs in the acceleration or retardation of atheroma progression.45–50 Lemaitre et al49 demonstrated that macrophage-specific transgenic mice for human MMP-1/collagenase-1 had less advanced atherosclerosis. Rouis et al,45 however, reported that overexpression of human tissue inhibitor of metalloproteinase-1 (TIMP-1, an inhibitor of collagenases and other MMPs) decreased aortic lesion area in apoE–/– mice. In contrast, Silence et al48 showed reduced atherosclerotic plaques in mice with inactivated TIMP-1. Curiously, Lemaitre et al50 found no change in atheroma burden in Timp-1–/–/apoE–/– mice. Our recent study using ColR/R/apoE–/– mice demonstrated that interference with the ability of collagenases to digest type I collagen did not affect lesion size.22 Concordantly, the present study shows no significant difference in atheroma burden between Mmp-13+/+/apoE–/– and Mmp-13–/–/apoE–/– mice at 2 time points.
Because mouse atheromata do not reliably develop thrombotic complications seen clinically, this study did not seek to model the human disease. Rather, it tested a mechanistic hypothesis with regard to interstitial collagen metabolism in atherosclerosis. Indeed, the differences in MMP-collagenase utilization by mice and humans render inappropriate the direct extrapolation to human lesions. Hence, this study probed the mechanism of the regulation of collagen structure in atherosclerotic plaques rather than devising a model of the human disease.
In addition to increased collagen content, this study provides additional information about the effects of MMP-13/collagenase-3 deficiency on interstitial collagen fiber structure in atherosclerotic lesions. Collagen plays a vital structural role in various tissues through its fiber content, thickness, and orientation. The quantitative analysis in the present study, provided by combining picrosirius red staining and circularly polarized light, revealed that MMP-13/collagenase-3 deficiency yielded thicker, more aligned, and more circumferentially oriented collagen fibers in the cap region of the intimal lesions than in Mmp-13+/+/apoE–/– mice (Figure 6). These changes in collagen structure likely alter the mechanical properties of the plaque, a possibility that requires further study.
Our data support an in vivo role for MMP-13/collagenase-3 in the economy of interstitial collagen, a component of human atherosclerotic plaques that influences their clinical consequences. Particularly, our complementary approach using mice genetically altered in the enzyme (Mmp-13–/– mice) and substrate (ColR/R mice)22 strengthens the in vivo evidence that collagenases participate in arterial collagen remodeling. These results provide important insights into the pathogenesis of collagen breakdown in atherosclerosis as well as other inflammatory diseases.
Acknowledgments
This work was supported in part by grants from the National Institutes of Health (SCOR HL-56985 to Drs Libby and Aikawa; 1R01 HL-80472 to Dr Libby; 1R01 HL-66086 to Dr Aikawa; 5R01 AR44815 to Dr Krane) and the Donald W. Reynolds Foundation (to Dr Libby). Dr Deguchi received a fellowship from the Reynolds Foundation. We also acknowledge Karen E. Williams for her excellent editorial assistance and Karen Mendelson for her technical support.
References
Brinckerhoff CE, Matrisian LM. Matrix metalloproteinases: a tail of a frog that became a prince. Nat Rev Mol Cell Biol. 2002; 3: 207–214.
Visse R, Nagase H. Matrix metalloproteinases and tissue inhibitors of metalloproteinases: structure, function, and biochemistry. Circ Res. 2003; 92: 827–839.
Krane SM. Elucidation of the potential roles of matrix metalloproteinases in skeletal biology. Arthritis Res Ther. 2003; 5: 2–4.
Libby P. Molecular bases of the acute coronary syndromes. Circulation. 1995; 91: 2844–2850.
Whittaker P, Boughner DR, Kloner RA. Role of collagen in acute myocardial infarct expansion. Circulation. 1991; 84: 2123–2134.
Rabkin E, Aikawa M, Stone JR, Fukumoto Y, Libby P, Schoen FJ. Activated interstitial myofibroblasts express catabolic enzymes and mediate matrix remodeling in myxomatous heart valves. Circulation. 2001; 104: 2525–2532.
Freije JM, Diez-Itza I, Balbin M, Sanchez LM, Blasco R, Tolivia J, Lopez-Otin C. Molecular cloning and expression of collagenase-3, a novel human matrix metalloproteinase produced by breast carcinomas. J Biol Chem. 1994; 269: 16766–16773.
Knauper V, Lopez-Otin C, Smith B, Knight G, Murphy G. Biochemical characterization of human collagenase-3. J Biol Chem. 1996; 271: 1544–1550.
Aikawa M, Libby P. The vulnerable atherosclerotic plaque: pathogenesis and therapeutic approach. Cardiovasc Pathol. 2004; 13: 125–138.
Davies MJ, Thomas AC. Plaque fissuring: the cause of acute myocardial infarction, sudden ischaemic death, and crescendo angina. Br Heart J. 1985; 53: 363–373.
Falk E, Shah PK, Fuster V. Coronary plaque disruption. Circulation. 1995; 92: 657–671.
Kolodgie FD, Burke AP, Farb A, Gold HK, Yuan J, Narula J, Finn AV, Virmani R. The thin-cap fibroatheroma: a type of vulnerable plaque: the major precursor lesion to acute coronary syndromes. Curr Opin Cardiol. 2001; 16: 285–292.
Moreno PR, Fuster V. The year in atherothrombosis. J Am Coll Cardiol. 2004; 44: 2099–2110.
Henney AM, Wakeley PR, Davies MJ, Foster K, Hembry R, Murphy G, Humphries S. Localization of stromelysin gene expression in atherosclerotic plaques by in situ hybridization. Proc Natl Acad Sci U S A. 1991; 88: 8154–8158.
Galis ZS, Sukhova GK, Lark MW, Libby P. Increased expression of matrix metalloproteinases and matrix degrading activity in vulnerable regions of human atherosclerotic plaques. J Clin Invest. 1994; 94: 2493–2503.
Nikkari ST, O’Brien KD, Ferguson M, Hatsukami T, Welgus HG, Alpers CE, Clowes AW. Interstitial collagenase (MMP-1) expression in human carotid atherosclerosis. Circulation. 1995; 92: 1393–1398.
Aikawa M, Rabkin E, Okada Y, Voglic SJ, Clinton SK, Brinckerhoff CE, Sukhova GK, Libby P. Lipid lowering by diet reduces matrix metalloproteinase activity and increases collagen content of rabbit atheroma: a potential mechanism of lesion stabilization. Circulation. 1998; 97: 2433–2444.
Aikawa M, Rabkin E, Sugiyama S, Voglic SJ, Fukumoto Y, Furukawa Y, Shiomi M, Schoen FJ, Libby P. An HMG-CoA reductase inhibitor, cerivastatin, suppresses growth of macrophages expressing matrix metalloproteinases and tissue factor in vivo and in vitro. Circulation. 2001; 103: 276–283.
Fukumoto Y, Libby P, Rabkin E, Hill CC, Enomoto M, Hirouchi Y, Shiomi M, Aikawa M. Statins alter smooth muscle cell accumulation and collagen content in established atheroma of Watanabe heritable hyperlipidemic rabbits. Circulation. 2001; 103: 993–999.
Sukhova GK, Schonbeck U, Rabkin E, Schoen FJ, Poole AR, Billinghurst RC, Libby P. Evidence for increased collagenolysis by interstitial collagenases-1 and -3 in vulnerable human atheromatous plaques. Circulation. 1999; 99: 2503–2509.
Herman MP, Sukhova GK, Libby P, Gerdes N, Tang N, Horton DB, Kilbride M, Breitbart RE, Chun M, Schonbeck U. Expression of neutrophil collagenase (matrix metalloproteinase-8) in human atheroma: a novel collagenolytic pathway suggested by transcriptional profiling. Circulation. 2001; 104: 1899–1904.
Fukumoto Y, Deguchi JO, Libby P, Rabkin-Aikawa E, Sakata Y, Chin MT, Hill CC, Lawler PR, Varo N, Schoen FJ, Krane SM, Aikawa M. Genetically determined resistance to collagenase action augments interstitial collagen accumulation in atherosclerotic plaques. Circulation. 2004; 110: 1953–1959.
Aimes RT, Quigley JP. Matrix metalloproteinase-2 is an interstitial collagenase: inhibitor-free enzyme catalyzes the cleavage of collagen fibrils and soluble native type I collagen generating the specific 3/4- and 1/4-length fragments. J Biol Chem. 1995; 270: 5872–5876.
Ohuchi E, Imai K, Fujii Y, Sato H, Seiki M, Okada Y. Membrane type 1 matrix metalloproteinase digests interstitial collagens and other extracellular matrix macromolecules. J Biol Chem. 1997; 272: 2446–2451.
Sabeh F, Ota I, Holmbeck K, Birkedal-Hansen H, Soloway P, Balbin M, Lopez-Otin C, Shapiro S, Inada M, Krane S, Allen E, Chung D, Weiss SJ. Tumor cell traffic through the extracellular matrix is controlled by the membrane-anchored collagenase MT1-MMP. J Cell Biol. 2004; 167: 769–781.
Inada M, Wang Y, Byrne MH, Rahman MU, Miyaura C, Lopez-Otin C, Krane SM. Critical roles for collagenase-3 (Mmp13) in development of growth plate cartilage and in endochondral ossification. Proc Natl Acad Sci U S A. 2004; 101: 17192–17197.
Stickens D, Behonick DJ, Ortega N, Heyer B, Hartenstein B, Yu Y, Fosang AJ, Schorpp-Kistner M, Angel P, Werb Z. Altered endochondral bone development in matrix metalloproteinase 13-deficient mice. Development. 2004; 131: 5883–5895.
Paigen B, Morrow A, Holmes P, Mitchell D, Williams R. Quantitative assessment of atherosclerotic lesions in mice. Atherosclerosis. 1987; 68: 231–240.
Sweat F, Puchtler H, Rosenthal SI. Sirius red F3BA as a stain for connective tissue. Arch Pathol. 1964; 78: 69–72.
Junqueira LC, Bignolas G, Brentani RR. Picrosirius staining plus polarization microscopy, a specific method for collagen detection in tissue sections. Histochem J. 1979; 11: 447–455.
Whittaker P, Canham PB. Demonstration of quantitative fabric analysis of tendon collagen using two-dimensional polarized light microscopy. Matrix. 1991; 11: 56–62.
Whittaker P, Zheng S, Patterson MJ, Kloner RA, Daly KE, Hartman RA. Histologic signatures of thermal injury: applications in transmyocardial laser revascularization and radiofrequency ablation. Lasers Surg Med. 2000; 27: 305–318.
Whittaker P, Müller-Ehmsen J, Dow JS, Kedes LH, Kloner RA. Development of abnormal tissue architecture in transplanted neonatal rat myocytes. Ann Thorac Surg. 2003; 75: 1450–1456.
Rich L, Whittaker P. Collagen and picrosirius red staining: a polarized light assessment of fibrillar hue and spatial distribution. Braz J Morphol Sci. 2005; 22: 97–104.
Oldenbourg R, Mei G. New polarized light microscope with precision universal compensator. J Microsc. 1995; 180 (pt 2): 140–147.
Katoh K, Hammar K, Smith PJ, Oldenbourg R. Birefringence imaging directly reveals architectural dynamics of filamentous actin in living growth cones. Mol Biol Cell. 1999; 10: 197–210.
Wolman M. On the use of polarized light in pathology. Pathol Annu. 1970; 5: 381–416.
Batschelet E. Circular Statistics in Biology. London, UK: Academic Press; 1981.
Shah PK, Falk E, Badimon JJ, Fernandez-Ortiz A, Mailhac A, Villareal-Levy G, Fallon JT, Regnstrom J, Fuster V. Human monocyte-derived macrophages induce collagen breakdown in fibrous caps of atherosclerotic plaques: potential role of matrix-degrading metalloproteinases and implications for plaque rupture. Circulation. 1995; 92: 1565–1569.
Stahle-Backdahl M, Sandstedt B, Bruce K, Lindahl A, Jimenez MG, Vega JA, Lopez-Otin C. Collagenase-3 (MMP-13) is expressed during human fetal ossification and re-expressed in postnatal bone remodeling and in rheumatoid arthritis. Lab Invest. 1997; 76: 717–728.
Seandel M, Noack-Kunnmann K, Zhu D, Aimes RT, Quigley JP. Growth factor-induced angiogenesis in vivo requires specific cleavage of fibrillar type I collagen. Blood. 2001; 97: 2323–2332.
Neuhold LA, Killar L, Zhao W, Sung ML, Warner L, Kulik J, Turner J, Wu W, Billinghurst C, Meijers T, Poole AR, Babij P, DeGennaro LJ. Postnatal expression in hyaline cartilage of constitutively active human collagenase-3 (MMP-13) induces osteoarthritis in mice. J Clin Invest. 2001; 107: 35–44.
Krane SM, Byrne MH, Lemaitre V, Henriet P, Jeffrey JJ, Witter JP, Liu X, Wu H, Jaenisch R, Eeckhout Y. Different collagenase gene products have different roles in degradation of type I collagen. J Biol Chem. 1996; 271: 28509–28515.
Ducharme A, Frantz S, Aikawa M, Rabkin E, Lindsey M, Rohde LE, Schoen FJ, Kelly RA, Werb Z, Libby P, Lee RT. Targeted deletion of matrix metalloproteinase-9 attenuates left ventricular enlargement and collagen accumulation after experimental myocardial infarction. J Clin Invest. 2000; 106: 55–62.
Rouis M, Adamy C, Duverger N, Lesnik P, Horellou P, Moreau M, Emmanuel F, Caillaud JM, Laplaud PM, Dachet C, Chapman MJ. Adenovirus-mediated overexpression of tissue inhibitor of metalloproteinase-1 reduces atherosclerotic lesions in apolipoprotein E–deficient mice. Circulation. 1999; 100: 533–540.
Dollery CM, Humphries SE, McClelland A, Latchman DS, McEwan JR. Expression of tissue inhibitor of matrix metalloproteinases 1 by use of an adenoviral vector inhibits smooth muscle cell migration and reduces neointimal hyperplasia in the rat model of vascular balloon injury. Circulation. 1999; 99: 3199–3205.
Silence J, Lupu F, Collen D, Lijnen HR. Persistence of atherosclerotic plaque but reduced aneurysm formation in mice with stromelysin-1 (MMP-3) gene inactivation. Arterioscler Thromb Vasc Biol. 2001; 21: 1440–1445.
Silence J, Collen D, Lijnen HR. Reduced atherosclerotic plaque but enhanced aneurysm formation in mice with inactivation of the tissue inhibitor of metalloproteinase-1 (TIMP-1) gene. Circ Res. 2002; 90: 897–903.
Lemaitre V, O’Byrne TK, Borczuk AC, Okada Y, Tall AR, D’Armiento J. ApoE knockout mice expressing human matrix metalloproteinase-1 in macrophages have less advanced atherosclerosis. J Clin Invest. 2001; 107: 1227–1234.
Lemaitre V, Soloway PD, D’Armiento J. Increased medial degradation with pseudo-aneurysm formation in apolipoprotein E-knockout mice deficient in tissue inhibitor of metalloproteinases-1. Circulation. 2003; 107: 333–338.(Jun-O Deguchi, MD, PhD; E)