Effect of MMP-2 Deficiency on Atherosclerotic Lesion Formation in ApoE-Deficient Mice
Objective— Although it has been reported that matrix metalloproteinase (MMP)-2 is a major proteinase in atherosclerotic plaque lesions, there is no direct evidence of the role of MMP-2 in atherosclerotic lesion formation. In the present study we determined the role of MMP-2 in atherosclerosis plaque development using apolipoprotein E-deficient (apoE−/−) mice.
Methods and Results— To generate MMP-2–deficient, apoE-deficient mice (MMP-2−/−:apoE−/−), MMP-2−/− mice were crossed with apoE−/− mice. After 8 weeks of feeding with a lipid-rich diet, morphological and biochemical studies of the aortic sinus and arch were conducted. A significant reduction of the atherosclerotic plaque in the aortic sinus and arch with the decrease in smooth muscle cell-positive area was observed in MMP-2−/−:apoE−/− mice compared with that of MMP-2+/+:apoE−/− mice. Macrophage- and collagen-positive areas were less in aortic sinus but not in aortic arch in MMP-2−/−:apoE−/− mice. There was no difference of MMP-9 mRNA expression in the plaque lesion between the 2 genotypes. A much lower level of mRNA expression of TIMP-1 and TIMP-2 was detected in the atherosclerotic plaque lesions of MMP-2−/−:apoE−/− mice than in those of MMP-2+/+:apoE−/− mice.
Conclusions— MMP-2 contributes to the development of atherosclerosis in apoE−/− mice.
In human or animal models of atherosclerosis, varying matrix metalloproteinases (MMPs) have been demonstrated to increase in atherosclerotic lesions, including MMP-1, -2, -3, -7, -9, -12, -13, and MT-MMPs.1–3 MMPs have been believed to contribute to the development and progression of atherosclerosis.1–3 However, there are only limited data providing direct evidence of the contribution of MMPs to the development of atherosclerotic lesions. Although MMP activity is commonly considered instrumental to the development of atherosclerotic lesions, this notion has been challenged by recent studies in gene-targeting mice. It has been reported that overexpression of the tissue inhibitor of metalloproteinases-1 (TIMP-1) reduced atherosclerotic lesions in apolipoprotein E-deficient (apoE−/−) mice.4 The deletion of the TIMP-1 gene resulted in either a reduction of or no change in the plaque size in the apoE−/− mice.5,6 Atherosclerotic lesions were significantly larger in mice with a combined deficiency of apoE and MMP-3 than in apoE−/− mice.7 Overexpression of MMP-1 in apoE−/− mice, which is not normally expressed in mice, decreased the extent of atherosclerosis.8 A more recent study demonstrated that MMP-9 deficiency but not MMP-12 deficiency reduced the atherosclerotic lesion growth in apoE−/− mice.9 MMP-13 deficiency had no effect on atherosclerotic plaque formation with similar accumulation of plaque macrophages and smooth muscle cells, but contained more interstitial collagen in apoE−/− mice.10 These results seemingly contradict a central tenet in our understanding of atherosclerosis—that increased MMP activity leads to the formation of a thicker neointima—suggesting that the role of individual MMPs in plaque lesions may actually have a different effect on the development of atherosclerotic plaque formation. The contribution of individual MMPs to plaque formation is only beginning to be investigated by the genetic manipulation of the expression of individual MMPs. Further work is necessary to determine the full spectrum of antiatherogenic or proatherogenic activities of the many MMPs that are expressed in atherosclerosis.
We previously demonstrated that MMP-2 deficiency significantly reduces neointimal lesion development in the carotid artery after ligation compared with control mice.11 Although it has been reported that MMP-2 is a major proteinase in atherosclerotic plaque lesions not only in human but also in animal models,12,13 there is no direct evidence of the role of MMP-2 in atherosclerotic lesion formation. In the present study we determined the role of MMP-2 in atherosclerosis plaque development using the apoE−/− mice.
Animals and the Experimental Protocol
All animal studies were conducted in accordance with the Animal Care and Use Committee guidelines of the Nagoya University School of Medicine. The generation of MMP-2-deficient (MMP-2 −/−) mice with the genetic background of C57BL/6 was described previously.14 ApoE −/− mice with the C57BL/6 genetic background were obtained from the Jackson Laboratory (Bar Harbor, Me). MMP-2 −/− mice were intercrossed with apoE −/− mice to generate breeding pairs with heterozygous deficiency of MMP-2 and apoE (MMP-2+/−:apoE+/−, which sired MMP-2−/−/apoE−/− and MMP-2+/+:apoE−/− littermate offspring. Genomic DNA was extracted from the tail tips for genotyping of offspring by Southern blotting for MMP-2 and by polymerase chain reaction for apoE (data not shown). At 8 weeks of age, male mice were started on a Western-type diet containing 21% fat and 0.15% cholesterol without sodium cholate, and they were maintained on this diet for 8 weeks.
Mice were euthanized by intraperitoneal pentobarbital injection, and blood samples of mice were collected into syringes that contained EDTA just before perfusion with isotonic saline from the left cardiac ventricle. The mice were perfused through the left cardiac ventricle with isotonic saline and 4% paraformaldehyde in 0.01 mol/L phosphate buffer (pH 7.4) under physiological pressure. The heart with ≈1 mm of proximal aorta attached and aortic arch were removed. The top half of the heart containing the aortic root or aortic arch was embedded and frozen in Tissue-Tek O.C.T. media. Sequential 20-μm sections were cut until the aortic valve leaflets appeared. From this point on, serial 6-μm sections were collected and stained routinely with hematoxylin and eosin (H&E), oil red O for lipid, and Masson’s trichrome for collagen. The corresponding sections on separate slides were used for immunohistochemical staining. The sections were preincubated with 5% serum and then incubated with antibodies against α smooth muscle actin (αSM actin; 1:50, Sigma Aldrich), macrophages (Mac-3, 1:40, BD Pharmingen), MMP-2 (MMP-2; 1:200, Fuji Chemical Co), and MMP-9 (MMP-9; 1:100, Chemicom). Immunohistochemical staining was visualized using an ABC kit (Vector Laboratories) according to the manufacturer’s instructions. Levamisole (Vector Laboratories) was used as the inhibitor of endogenous alkaline phosphatase. The counterstaining for the nucleus was performed with Mayer’s hematoxylin.
For the quantification of atherosclerotic lesions, all images were captured and analyzed by National Institutes of Health Image software. The images showed the area of intimal atherosclerotic plaque, lipid lesion assessed by the positive for oil red O staining, region containing macrophage as assessed by Mac-3 stating, and smooth muscle cell (SMC)-positive area as assessed by α-SM actin staining. The atherosclerotic plaque area of the aortic sinus or aortic arch was reported as the net area and the proportion of total intimal plaque lesion area to total cross-sectional vessel wall area, which was defined by the external elastic lamina. For quantification by image analysis, we set a threshold to automatically compute the areas positive for each antibody or histochemical stain and then computed the ratio of positively stained area to the total cross-sectional vessel wall area and intimal plaque lesion area studied.
Gelatin Zymography and mRNA Quantification
After perfusion with ice-cold phosphate-buffered saline, the aorta arch was dissected out and placed in ice-cold phosphate-buffered saline. After further mechanical rinsing under a dissection microscope, protein and total RNA were extracted from the tissue so that gelatin zymography and mRNA quantification could be performed.
For gelatin zymography, 20-μg protein extracts of the aortic arch were mixed with SDS sample buffer without a reducing agent and loaded onto a 10% SDS-polyacrylamide gel containing 1 mg/mL gelatin, as previously described in detail.15 Digestion bands were quantified by an image analyzer system (NIH image 1.62) and compared with a human MMP-2 standard (Oncogene Research Products).
The mRNA levels of MMPs and TIMPs in atherosclerotic lesions were quantified by real-time reverse-transcription and polymerase chain reaction. The total RNA was extracted from carotid arteries, and then reverse-transcribed. The synthesized cDNA was quantified by using TaqMan quantitative polymerase chain reaction analysis of each gene with the ABI PRISM 7700 Detection System according to the manufacturer’s protocol. Specifically, primer and probe sequences used for mouse MMP-2 were (forward) 5′-CCCCATGAAGCCTTGTTTACC, (reverse) 5′-TTGTAGGAGGTGCCCTGGAA, (probe) 5′-AATGC-TGATGGACAGCCCTGCA; for mouse MMP-9 were (forward) 5′-AGACCAAGGGTACAGCCTGTTC, (reverse) 5′-GGCACGCTG-GAATGATCTAAG (probe) 5′-TGGCTCATGCCTATGCACCTGGAC; for mouse TIMP-1 (forward) 5′-GCCTACACCCCAGTCATGGA, (reverse) 5′-GGCCCGTGATGAGAAACTCTT, (probe) 5′-TGGATATGCCCACAAGTCCCAGAACC; and for mouse TIMP-2 (forward) 5′-GTC-CCATGATCCCTTGCTACA, (reverse) 5′-TGCCCATTGATGCTCT-TCTCT, (probe) 5′-CTCCCCGGATGAGTGCCTCTGGA. Each RNA quantity was normalized to its respective glyceraldehydes-3-phosphate dehydrogenase (GAPDH) mRNA quantity.
Polyacrylamide Gel Disc Electrophoresis of Serum Lipoproteins
Polyacrylamide gel disc electrophoresis of serum (100 μL) was performed according to the method of Narayan et al.16 The pattern of electrophoresis was analyzed by densitometry.
Values were expressed as means±SD. Significant differences were analyzed using the Student t test. A value of P<0.05 was considered to be statistically significant.
Phenotype of MMP-2−/−:ApoE−/− Mouse
The MMP-2−/−:apoE−/− mouse did not show any gross anatomic abnormalities, including abnormalities in the blood vessels. However, this mutant mouse showed a slower growth rate at 16 weeks than the MMP-2+/+:apoE−/− mouse (n=12) with body weight of 19.9±2.9 grams and the MMP-2−/−:apoE−/− mouse (n=14) with a body weight of 18.0±2.3 grams. There were no differences in plasma total cholesterol levels between the MMP-2−/−:apoE−/− mouse and the MMP-2+/+:apoE−/− mouse after 8 weeks of a Western-type diet (56.9±9.9 mmol/L, n=12 versus 64.4.±9.5 mmol/L, n=14; P<0.08). The distribution of lipoproteins in serum assessed by the densitometric pattern of polyacrylamide gel disc electrophoresis was similar between 2 mutant mice (Figure 1).
Effect of MMP-2 Deficiency on Atherosclerotic Lesion Formation in ApoE-Deficient Mice
Cross-sectional analysis of the aortic sinus area as well as aortic arch showed that the total intimal lesion area was significantly less in MMP-2−/−:apoE−/− mice than in MMP-2+/+:apoE−/− mice, whether the lesion area was controlled for the total cross-sectional vessel wall area (Figures 2a, 2b, 3f, 3⇓h; Table). As shown in Figure 2c, 2d, 2e, 2f, 2g, and 2h and the Table, the areas that stained positive for lipid, macrophages, SMC, and collagen in the lesion of aortic sinus were less in MMP-2−/−:apoE−/− mice than those in MMP-2+/+: apoE−/− mice. However, the ratio of stained positive area for lipid, macrophage, and collagen to the intimal plaque area was similar in MMP-2−/−:apoE−/− and MMP-2+/+:apoE−/−. In aortic arch there was no difference of macrophage positive area between 2 genotypes, whether the lesion area was controlled for intimal lesion area (Figure 3g and 3i; Table). In contrast, SMC-positive area was less in MMP-2−/−: apoE−/− mice than that in MMP-2+/+:apoE−/− mice (Figure 3J, 3I; Table). As shown in Figure 3a 3b, 3j, and 3l, a much thicker fibrous cap region stained with SMC-specific antibody was observed in MMP-2+/+:apoE−/− mice than that of MMP-2−/−:apoE−/− mice both in aortic sinus and arch. The collagen-positive area was observed in thicker fibrous cap lesions in MMP-2+/+:apoE−/− mice but not in MMP-2−/−:apoE−/− mice (Figure 3k and 3m).
MMPs Expression and Localization
In the gelatin zymographic analysis of protein extracts from atherosclerotic plaque lesions, the latent forms of MMP-9 (92kDa) and MMP-2 (72kDa) and the active form of MMP-2 (62kDa) were detected in MMP-2+/+:apoE−/− mice (Figure 4A). As expected, a similar level of MMP-9 activity and no MMP-2 activity were observed in MMP-2−/−:apoE−/− mice.
No MMP-2 mRNA expression was found in atherosclerotic plaque lesions in MMP-2−/−:apoE−/− mice (Figure 4B). Although no significant difference in MMP-9 mRNA expression in plaque was observed between MMP-2−/−:apoE−/− and MMP-2+/+:apoE−/− mice, much lower mRNA expression of TIMP-1 and TIMP-2 was detected in the atherosclerotic plaque lesion of MMP-2−/−:apoE−/− mice (Figure 4C, 4D, 4E). Immunohistochemical analysis revealed that the staining for MMP-2 was observed at the fibrous cap region as well as atheromatous lesion containing foam cells of the aortic sinus of MMP-2+/+:apoE−/− mice (Figure 3c, 3d). MMP-2 was not detected in the lesion of MMP-2−/−:apoE−/− mice (Figure 3e). MMP-9 staining was observed in the plaque, and the staining pattern was not different between the 2 genotypes (data not shown).
Previous observations illustrate the potential complexities of manipulating a system in which MMPs have a dual role in plaque growth by means of SMC migration, matrix deposition, and instability caused by matrix destruction. In fact, MMP-1 overexpression to macrophages reduced the progression of atherosclerosis in apoE−/− mice, because it resulted in less collagenous matrix accumulation.8 MMP-3 deficiency was associated with increased collagen content in the plaques in apoE−/− mice, which is consistent with greater stability, although plaque size was increased overall.7 These previous observations may suggest that the activities of MMP-1 and MMP-3 in atherosclerotic lesions may contribute to a reduction of plaque size, possibly by causing the degradation of matrix components. In contrast, a deficiency of MMP-9 reduced the plaque size, macrophage content, and collagen deposition in aortic lesion of apoE−/− mice, but MMP-12 deficiency had no effect on plaque size or on the composition of the plaque.9 Interestingly, more recent report by Johnson et al suggested that MMP-9 deficiency increased plaque size with a increase in macrophage accumulation and a decrease in SMCs in brachiocephalic artery from apoE−/− mice.17 These inconsistent effects of MMP-9 deficiency on the atherosclerotic lesion formation as well as the changes in cellular composition at the lesion suggest that MMP-9 has different actions to the development of atherosclerosis at different regions of artery. This is also true for MMP-12 deficiency, as Johnson et al have also demonstrated that MMP-12 deficiency reduced the plaque size as well as macrophage content in brachiocephalic artery from apoE−/− mice.17 More recent findings suggested that MMP-13 deficiency had no effect on plaque development but decreased collagen content in the plaque in apoE−/− mice.10 These findings indicate the different contributions of individual MMPs to atherosclerotic plaque formation as well as to matrix protein accumulation in the plaque.
In the present study, we clearly demonstrated that MMP-2 deficiency reduces the atherosclerotic plaque lesion formation in apoE−/− mice. In aortic sinus, the reduction in plaque volume was associated with a significantly lower macrophage-positive and SMC-positive areas, as well as less collagenous matrix accumulation in the plaque lesions of MMP-2−/−:apoE−/− mice than those of MMP-2+/+:apoE−/− mice. In aortic arch, the reduction of lesion was associated with a lower SMC-positive area in MMP-2−/−:apoE−/− mice, but there were no differences in macrophage-positive and collagen-positive areas in the lesions between those genotypes. The reduction of the accumulation of SMCs in plaque lesions, common feature to aortic sinus and arch in MMP-2−/−:apoE−/− mice, is consistent with our previous findings that the targeted deletion of the MMP-2 gene reduced neointimal lesion formation after flow cessation in the murine carotid arteries, mainly because of the attenuation of the migration of SMCs from the medial to the intimal region through the reduction of proteolytic activities resulting from MMP-2 deficiency.11,18 In addition, a much thicker fibrous cap region that contains SMCs and collagen was observed in MMP-2+/+:apoE−/− mice than that of MMP-2−/−:apoE−/− mice, suggesting MMP-2 may induce plaque stability by accumulating SMC into the fibrous cap. We showed the different effects of MMP-2 on the macrophage accumulation in the atherosclerotic lesions of different regions of artery. It is possible that MMP-2 contributes to macrophage accumulation in the atherosclerotic lesions in different ways at different regions. This might be similar to the effect MMP-9 on macrophage accumulation in the atherosclerotic lesions, because MMP-9 deficiency increased macrophage accumulation in the lesions of brachiocephalic artery but reduced its accumulation in carotid artery and descending aorta in apoE−/− mice.9,17,21 The fact that MMP-2 colocalized with macrophage in atherosclerotic plaque lesion of aortic sinus, consistent with human atherosclerotic lesions19,20 may suggest that MMP-2 is involved in monocyte migration into the intima or that MMP-2 may affect macrophage proliferation in the intima. Further studies will be required to evaluate the role of MMP-2 on macrophage accumulation in the atherosclerotic plaque lesion.
MMPs have overlapping substrate specificities, so the effect of the loss of MMP-2 can be compensated for to some degree by another gelatinase, MMP-9. However, the MMP-9 mRNA level observed in MMP-2−/−:apoE−/− mice was not significantly higher than that observed in] MMP-2+/+:apoE−/− mice.
We also found a decreased expression of TIMPs mRNA in MMP-2–deficient mice, in agreement with our previous observation.11 These findings strengthen our hypothesis that MMP-2 may play a role in regulating the expression of TIMPs.
There are limitations in the present study. We have evaluated the atherosclerotic lesions only after 8 weeks of high-fat diet. This relative short period may represent early developing plaques. However, we demonstrated that some plaques already contain advanced lesions with lipid core surrounded by thick fibrous cap. This rapid growth of the plaque in the present study might be due to the higher serum cholesterol levels (≈60 mmol/L) than those of others previously reported. In addition, we did not demonstrate the net proteolytic activity in the lesions. It is ultimately the balance between the MMPs and TIMPs that determines the focal proteolysis around the cellular components in the lesion. Further studies will be required to examine the effect of MMP-2 deficiency on the net proteolytic activity in the atherosclerotic lesions.
In conclusion, we demonstrated that MMP-2 deficiency reduced atherosclerotic plaque lesions in apoE−/− mice. This reduction of the plaque lesions in MMP-2 deficiency was associated with the reduction of SMC accumulation in the plaque lesions. These results suggested that MMP-2 contributes to atherosclerotic plaque development in apoE−/− mice and that MMP-2 may induce plaque stability by accumulating SMC into the fibrous cap.
This work was supported by a research grant from the Scientific Research Fund of the Ministry of Education, Science, and Cultures, Japan (No. 13671182).
- Received November 1, 2005.
- Accepted March 9, 2006.
Newby AC. Dual role of matrix metalloproteinases (matrixins) in intimal thickening and atherosclerotic plaque rupture. Physiol Rev. 2005; 85: 1–31.
Galis ZS, Khatri JJ. Matrix metalloproteinases in vascular remodeling and atherogenesis: the good, the bad, and the ugly. Circ Res. 2002; 90: 251–262.
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.
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, 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.
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.
Luttun A, Lutgens E, Manderveld A, Maris K, Collen D, Carmeliet P, Moons L. Loss of matrix metalloproteinase-9 or matrix metalloproteinase-12 protects apolipoprotein E-deficient mice against atherosclerotic media destruction but differentially affects plaque growth. Circulation. 2004; 109: 1408–1414.
Deguchi JO, Aikawa E, Libby P, Vachon JR, Inada M, Krane SM, Whittaker P, Aikawa M. Matrix metalloproteinase-13/collagenase-3 deletion promotes collagen accumulation and organization in mouse atherosclerotic plaques. Circulation. 2005; 112: 2708–2715.
Kuzuya M, Kanda S, Sasaki T, Tamaya-Mori N, Cheng XW, Itoh T, Itohara S, Iguchi A. Deficiency of gelatinase a suppresses smooth muscle cell invasion and development of experimental intimal hyperplasia. Circulation. 2003; 108: 1375–1381.
Shi W, Brown MD, Wang X, Wong J, Kallmes DF, Matsumoto AH, Helm GA, Drake TA, Lusis AJ. Genetic backgrounds but not sizes of atherosclerotic lesions determine medial destruction in the aortic root of apolipoprotein E-deficient mice. Arterioscler Thromb Vasc Biol. 2003; 23: 1901–1906.
Itoh T, Ikeda T, Gomi H, Nakao S, Suzuki T, Itohara S. Unaltered secretion of beta-amyloid precursor protein in gelatinase A (matrix metalloproteinase 2)-deficient mice. J Biol Chem. 1997; 272: 22389–22392.
Cheng XW, Kuzuya M, Sasaki T, Kanda S, Tamaya-Mori N, Koike T, Maeda K, Nishitani E, Iguchi A. Green tea catechins inhibit neointimal hyperplasia in a rat carotid arterial injury model by TIMP-2 overexpression. Cardiovasc Res. 2004; 62: 594–602.
Johnson JL, George SJ, Newby AC, Jackson CL. Divergent effects of matrix metalloproteinases 3, 7, 9, and 12 on atherosclerotic plaque stability in mouse brachiocephalic arteries. Proc Natl Acad Sci U S A. 2005; 102: 15575–15580.
Kanda S, Kuzuya M, Ramos MA, Koike T, Yoshino K, Ikeda S, Iguchi A. Matrix metalloproteinase and alphavbeta3 integrin-dependent vascular smooth muscle cell invasion through a type I collagen lattice. Arterioscler Thromb Vasc Biol. 2000; 20: 998–1005.
Choi ET, Collins ET, Marine LA, Uberti MG, Uchida H, Leidenfrost JE, Khan MF, Boc KP, Abendschein DR, Parks WC. Matrix metalloproteinase-9 modulation by resident arterial cells is responsible for injury-induced accelerated atherosclerotic plaque development in apolipoprotein E-deficient mice. Arterioscler Thromb Vasc Biol. 2005; 25: 1020–1025.