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. Author manuscript; available in PMC: 2010 Apr 23.
Published in final edited form as: Circ Res. 2009 Sep 10;105(9):921–929. doi: 10.1161/CIRCRESAHA.109.200279

A role of matrix metalloproteinase-8 in atherosclerosis

Ross C Laxton 1,*, Yanhua Hu 1,*, Johan Duchene 1,*, Feng Zhang 1,*, Zhongyi Zhang 1, Kit-Yi Leung 1, Qingzhong Xiao 1, Ramona S Scotland 1, Conrad P Hodgkinson 1, Katherine Smith 1, Johann Willeit 1, Carlos López-Otín 1, Iain A Simpson 1, Stefan Kiechl 1, Amrita Ahluwalia 1, Qingbo Xu 1, Shu Ye 1
PMCID: PMC2853782  EMSID: UKMS27973  PMID: 19745165

Abstract

Rationale

Atherosclerotic lesions express matrix metalloproteinase-8 (MMP8) which possesses proteolytic activity on matrix proteins particularly fibrillar collagens and on non-matrix proteins such as angiotensin I (Ang I).

Objective

We studied whether MMP8 plays a role in atherogenesis.

Methods and Results

In atherosclerosis-prone apoE deficient mice, inactivating MMP8 resulted in a substantial reduction in atherosclerotic lesion formation. Immunohistochemical examinations showed that atherosclerotic lesions in MMP8 deficient mice had significantly fewer macrophages but increased collagen content. In line with results of in vitro assays showing Ang I cleavage by MMP8 generating angiotensin II (Ang II), MMP8 knockout mice had lower Ang II levels and lower blood pressure. In addition, we found that products of Ang I cleavage by MMP8 increased vascular cell adhesion molecule-1 (VCAM-1) expression and that MMP8 deficient mice had reduced VCAM-1 expression in atherosclerotic lesions. Intravital microscopy analysis showed that leukocyte rolling and adhesion on vascular endothelium was reduced in MMP8 knockout mice. Furthermore, we detected an association between MMP8 gene variation and extent of coronary atherosclerosis in patients with coronary artery disease. A relationship between MMP8 gene variation, plasma VCAM-1 level and atherosclerosis progression was also observed in a population-based, prospective study.

Conclusion

These results indicate that MMP8 is an important player in atherosclerosis.

Keywords: Atherosclerosis, matrix metalloproteinase, gene

Chronic inflammation plays an important role in the pathogenesis of atherosclerosis which in turn is the main underlying cause of the majority of coronary heart disease and stroke1. One of the earliest steps in atherogenesis is the attachment of circulating leukocytes to the vascular endothelium consequent to the expression of adhesion molecules. Following adhesion to endothelial cells, leukocytes penetrate the endothelium into the arterial intima, differentiate into macrophages, uptake modified lipoproteins and become lipid-laden foam cells which represent a major component of atherosclerotic lesions1. In addition, vascular smooth muscle cells and extracellular matrix proteins, particularly types I and III collagens and proteoglycans, are also major constituents of atherosclerotic plaques1.

The matrix metalloproteinases (MMPs) are zinc-dependent proteases that can degrade extracellular matrix proteins2. Some MMPs can also cleave non-matrix proteins such as cytokines and growth factors2. There is substantial evidence indicating that MMPs participate in a number of diseases with matrix remodeling and inflammation. Several MMPs have been demonstrated to be involved in atherosclerosis in which these different proteases appear to exert divergent effects3-14.

MMP8 (also known as collagenase-2) has also been suggested to be implicated in atherosclerosis15-17. MMP8 possesses proteolytic activity on several matrix proteins particularly type I collagen and on some non-matrix proteins such as angiotensin-I (Ang I)18. It has been shown that endothelial cells, macrophages and smooth muscle cells in atherosclerotic lesions express MMP815, that its expression is increased in rapidly progressing lesions16, and that elevated serum MMP8 concentrations are associated with the presence of atherosclerosis and with unfavorable cardiovascular outcome17. However, it has remained unknown whether MMP8 plays a causal role in atherogenesis or is simply a bystander marker. To address this issue, we studied whether inactivating the MMP8 gene would have an effect on atherogenesis in atherosclerosis-prone apoE deficient mice. We found that inactivating MMP8 resulted in a significant reduction in the extent of atherosclerosis, with a decrease in macrophages but an increase in collagen content in the atherosclerotic lesions. In addition, we found that MMP8 knockout mice had lower levels of angiotensin-II (Ang II), lower blood pressure, decreased vascular cell adhesion molecular-1 (VCAM-1) expression and reduced vascular leukocyte recruitment. Furthermore, we observed that in humans, MMP8 gene variation was associated with VCAM-1 level and severity and progression of atherosclerosis.

Methods

The methods are briefly described here, and further details are provided in the Online Supplements.

Animals

MMP8−/−/apoE+/+ mice19 were crossbred with MMP8+/+/apoE−/− mice to generate MMP8+/−/apoE+/− double heterozygous mice. MMP8+/−/apoE+/− double heterozygous mice were bred to produce MMP8−/−/apoE−/− double knockout mice and MMP8+/+/apoE−/− controls (littermates of MMP8−/−/apoE−/− double knockout mice). Six week old males of both types of mouse were fed a Western diet for 12 weeks.

Characterization of atherosclerotic lesions

The extent of aortic atherosclerotic lesions in MMP8−/−/apoE−/− double knockout mice and controls was analyzed by en face staining of aortas with oil red O, as previously described20. The contents of macrophages, smooth muscle cells, collagen type I, Ang II and VCAM-1 in aortic atherosclerotic lesions were examined by immunohistochemical analyses.

Real-time reverse-transcriptase-PCR (real-time RT-PCR)

MMP2, MMP9, MMP13 and MMP14 expression levels in MMP8−/−/ApoE−/− mice and control mice were quantified by real-time RT-PCR.

Measurements of cholesterol and triglycerides

Plasma cholesterol and triglyceride levels in the MMP8−/−/apoE−/− double knockout mice and controls were determined with the use of commercially available assay kits.

Angiotensin I cleavage by MMP8 and mass spectrometry

Ang I was incubated with purified human MMP8. An uncleaved Ang I control was generated by incubating Ang I in the assay buffer without MMP8. Ang I and cleavage products in the above solutions (from the cleavage reaction and the control, respectively) were extracted and analysed by liquid chromatography-electrospray tandem mass spectrometry. The mass spectral data were processed into peak lists and searched against the Swiss Prot database. The relative intensity of the angiotensin peptides was calculated using the total ion count of each peptide.

Enzyme-linked immuno-sorbent assay (ELISA)

Commercially available ELISA kits were used to measure plasma Ang I and Ang II. Concentrations of VCAM-1 in plasma samples were also measured by ELISA kits.

Blood pressure measurement

Blood pressure was measured in conscious mice using a volume pressure recording sensor and occlusion tail cuff.

Analyses of other angiotensin II forming enzymes

Tissue extracts were prepared from aortas from MMP8+/+/ApoE−/− or MMP8−/−/ApoE−/− mice and assayed for other angiotensin II forming enzymes including angiotensin-converting enzyme (ACE), chymase and cathespins.

Flow Cytometry

Mouse endothelial cells (C166) were incubated with Ang I that had been subjected to cleavage by MMP8 or uncleaved Ang I, in the absence or presence of the Ang II type 1 receptor (AT1R) antagonist losartan and the Ang II type 2 receptor (AT2R) antagonist PD123319, and then subjected to flow cytometric analysis of VCAM-1 expression.

Intravital microscopy

Mesenteries of anesthetized mice were superfused with bicarbonate-buffered solution at 37°C at a rate of 2ml/min. Cell rolling was determined by counting the number of leukocytes rolling per minute in each vessel. Cell adhesion was quantified by counting, for each vessel, the number of adherent leukocytes in a 100μm length.

Human subjects

We studied MMP8 gene variation in relation to extent of atherosclerosis in a group (n=2000) of patients with coronary artery disease (CAD) documented angiographically as having >50% diameter stenosis in at least one coronary artery21. We also examined MMP8 gene variation in relation to progression of atherosclerosis in subjects of the Bruneck Study, a population-based, prospective study of atherosclerosis22-25.

Functional analysis of MMP8 gene rs1940475 SNP

MMP8 zymogens with Glu87 or Lys87 were synthesized and subjected to activation by 4-aminophenylmercuric acetate, followed by Western blot analysis.

Statistical analyses

The statistical analyses performed in this study are described in Online Data Supplements.

Results

Reduced atherosclerosis in MMP8 deficient mouse

Extensive aortic atherosclerotic lesions were observed in MMP8+/+/apoE−/− mice (controls) fed a Western diet (Figure 1A). In contrast, MMP8−/−/apoE−/− double knockout mice had substantially less aortic atherosclerosis (36% reduction compared with controls, p=0.007, Figure 1A). Compared with the lesions in the controls, those in the double knockout mice had significantly fewer macrophages (70% reduction, p=0.002, Figure 1B). In addition, there was a trend towards lower smooth muscle cell content in the lesions in the double knockout mice than lesions in the controls (p=0.057, Figure 1C). On the other hand, collagen content was higher in the lesions in the double knockouts than lesions in the controls (differing by 27%, p=0.034, Figure 1D). There was no significant difference in plasma cholesterol and triglyceride levels between the two groups (p=0.887 for cholesterol and p=0.117 for triglyceride). The levels of expression of the other mouse collagenases including MMP2, MMP3, MMP13 and MMP14 did not differ between the two groups (p=0.108, p=0.611, p=0.509, and p=0.318, respectively, Online Supplement Figure 2).

Figure 1. Reduced atherosclerosis in MMP8−/−/apoE−/− mice.

Figure 1

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Panel A: representative images of Oil Red O stained en face preparations of aortas of MMP8+/+/apoE−/− and MMP8−/−/apoE−/− mice, and chart showing percentages of aortic surface area with atherosclerosis in the two groups of mice.

Panels B, C and D: representative images of immunostaining of macrophages (B), smooth muscle cells (C) and collagen (D) in aortic root atherosclerotic lesions of MMP8+/+/apoE−/− and MMP8−/−/apoE−/− mice, and charts showing percentages of mean macrophage, smooth muscle cell, and collagen contents in the two groups of mice.

Data presented are mean and standard error of mean.

Reduced Ang II level and VCAM-1 expression in MMP8 knockout mouse

MMP8 has been shown to cleave Ang I producing Ang II26, a well established atherogenic factor27-29, and angiotensin1-7 (Ang1-7). To verify this finding, we incubated Ang I with MMP8 and then performed liquid chromatography-electrospray tandem mass spectrometry analysis. The analysis showed cleavage of Ang I by MMP8, generating Ang II and Ang1-7, with the former being the major product (Figure 2 and Online Supplement Table 5).

Figure 2. Cleavage of Ang I by MMP8, producing Ang II and Ang1-7.

Figure 2

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Panel A: Chromatogram of mass spectrometry analysis of uncleaved Ang I, showing the detection of Ang I peptide ion (m/z value 432.84 3+). Panel B: Chromatogram of mass spectrometry analysis of products of Ang I cleavage by MMP8, showing the presence of Ang II (m/z value 523.79 2+) and Ang1-7 (450.25 2+) peptide ions, in addition to Ang I (m/z value 432.91 3+). The presence of Ang II, Ang1-7 and Ang I in the respective peaks were confirmed by peptide fragmentation sequencing.

We then examined whether there was a difference in Ang II level between MMP8−/−/apoE−/− double knockout mice and controls. Indeed, we found that Ang II levels in atherosclerotic lesions and blood were lower in double knockout mice than in controls (p=0.002 and p=0.026, respectively, Figure 3A and 3B). In contrast, plasma levels of Ang I, the processor of Ang II, were higher in the double knockout group than in the control group (p=0.018, Figure 3C), which might be explained by reduced Ang I cleavage in double knockout mice. Consistent with the finding of reduced Ang II levels in double knockout mice, we found that blood pressure was lower in double knockout mice than in controls (p=0.017 for systolic and p=0.023 for diastolic blood pressure, Figure 4). We found no difference between MMP8−/−/apoE−/− double knockout mice and controls in the levels of the other Ang II forming enzymes ACE, chymases and cathepsins (Online Supplement Figure 3).

Figure 3. Reduced Ang II levels in MMP8−/−/apoE−/− mice.

Figure 3

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Panel A: representative images of Ang II staining in atherosclerotic lesions in MMP8−/−/apoE−/− and MMP8+/+/apoE−/− mice respectively, and column chart showing lower relative intensity of Ang II staining in lesions in MMP8−/−/apoE−/− mice than in MMP8+/+/apoE−/− mice. Panels B and C: charts showing lower plasma Ang II level, but higher plasma Ang I level, in MMP8−/−/apoE−/− mice than in MMP8+/+/apoE−/− mice. Data presented as mean and standard error of mean.

Figure 4. Reduced blood pressure in MMP8−/−/apoE−/− mice.

Figure 4

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Chart showing lower systolic and diastolic blood pressure in MMP8−/−/ApoE−/− mice than in MMP8+/+/ApoE−/− mice. Data shown are mean ± standard error of mean.

Previous studies demonstrated that Ang II induced the expression of adhesion molecules28, 29 such as VCAM-130-34 which plays an important role in atherogenesis35. In agreement, our experiments showed that Ang II in a wide range of concentration increased VCAM-1 expression in endothelial cells (Online Supplement Figure 4). In addition, we found that the products of Ang I cleavage by MMP8 increased VCAM-1 expression. As shown in Figure 5A and 5B, flow cytometric analysis showed that cultured endothelial cells treated with products of Ang I cleavage by MMP8 expressed more VCAM-1 than endothelial cells treated with uncleaved Ang I (p=0.002). This effect was abolished in the presence of the AT1R antagonist losartan and the AT2R antagonist PD123319 (Figure 5C and 5D).

Figure 5. Products of Ang I cleavage by MMP8 increase VCAM-1 expression in endothelial cells.

Figure 5

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Panels A and B: representative plot (A) and mean fluorescence intensity (B) in flow cytometric analyses of VCAM-1 expression on endothelial cells incubated with uncleaved Ang I or with products of Ang I cleavage by MMP8. Panels C and D: representative plot (C) and mean fluorescence intensity (D) in flow cytometric analyses of VCAM-1 expression on endothelial cells incubated with uncleaved Ang I or with products of Ang I cleavage by MMP8, in the presence of the AT1R antagonist losartan and the AT2R antagonist PD123319. Error bars in the column charts are standard error of mean.

We also examined whether there was a difference in VCAM-1 expression level between MMP8−/−/apoE−/− double knockout mice and controls. We observed that VCAM-1 expression in endothelial and other cells in atherosclerotic lesions was substantially reduced in the double knockout mice compared with controls (Figure 6A, 6B and 6C). Plasma levels of soluble VCAM-1 were also lower in double knockout mice than in controls (Figure 6D).

Figure 6. Reduced VCAM-1 expression in MMP8−/−/apoE−/− mice compared with MMP8+/+/apoE−/− mice.

Figure 6

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Panels A, B and C show results of immunohistochemical analyses of VCAM-1 in aortic atherosclerotic lesions, (A), representative images; (B), mean VCMA-1 staining in lesions; (C), mean VCAM-1 staining in the endothelial layer. Panel D shows mean levels of soluble VCAM-1 in plasma. Error bars in the column charts are standard error of mean.

Reduced leukocyte recruitment in MMP8 knockout mice

Intravital microscopy examination showed that leukocyte rolling and adhesion on vascular endothelium was attenuated (by approximately 50%) in double knockout mice compared with controls (p=0.049 and p=0.009 respectively, Figure 7), which might be in part explained by the reduction of Ang II and VCAM-1 levels in the double knockout mice.

Figure 7. Reduced leukocyte recruitment in MMP8−/−/apoE−/− mice.

Figure 7

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Panel A: Intravital microscopic images showing leukocyte rolling (arrow point 1) and adhesion (arrow point 2) on mesenteric postcapillary venules in vivo. Panel B: Diagrams showing that leukocyte rolling and adhesion is reduced in MMP8−/−/apoE−/− mice compared with MMP8+/+/apoE−/− mice. Data presented as the mean and standard error of mean.

Association of MMP8 gene variation with VCAM-1 level and atherosclerosis in humans

Further to the animal and in vitro studies described above, we investigated whether in humans, there was a relationship between MMP8 gene variation and inter-individual differences in atherosclerosis. To address this question, we ascertained whether MMP8 gene variation was associated with extent of atherosclerosis in patients (n=2000) with CAD. In addition, we examined whether MMP8 gene variation was associated with atherosclerosis progression in a population-based, prospective study (the Bruneck study, n=782).

In the study of CAD patients, we initially genotyped 1000 subjects for a panel of 16 single nucleotide polymorphisms (SNPs) consisting of all common SNPs (with minor allele frequency ≥0.05) identified by re-sequencing the MMP8 gene proximal promoter (2kb) and coding regions and tagging SNPs selected from the HapMap database to capture (r2≥0.8) common SNPs (with minor allele frequency ≥0.05) in the introns, 5′ upstream sequence and 3′-untranslated region. An association was detected between the extent of coronary atherosclerosis and SNP rs1940475 (dbSNP database identification number) (p=0.01). We then genotyped 1000 additional CAD patients for this SNP and analyzed the data for this SNP in the entire sample (n=2000). The analysis showed a highly significant association between the SNP and extent of coronary atherosclerosis (p=0.0008), with the T allele having a lower frequency in patients who had >50% stenosis in two or three coronary arteries than in patients with >50% stenosis in one coronary artery (odds ratio=0.80, 95% confidence interval=0.70-0.91, Table 1). The association remained significant after taking into account the number of SNPs tested in this study, as the p-value (0.0008) for the association observed was smaller than the significance threshold (0.005) calculated using the SNPSpD method36, 37 to take into consideration of the number of SNPs tested and the degree of linkage disequilibrium between the SNPs.

Table 1. Association between MMP8 gene variation and extent of coronary atherosclerosis in patients with coronary artery disease (n=2000).

rs1940475 SNP Subjects with
significant atherosclerosis*
in one coronary artery		 Subjects with
significant atherosclerosis*
in two or three coronary arteries		 Odds ratio (95% confidence interval) †
for significant atherosclerosis*
in two or three coronary arteries
Genotype n (%) n (%)
C/C 170 (22.2) 342 (27.7) reference
C/T 398 (52.0) 632 (51.2) 0.77 (0.62-0.97), p=0.028
T/T 198 (25.8) 260 (21.1) 0.63 (0.49-0.83), p=0.0008
Total 766 (100.0) 1234 (100.0)
Allele frequency frequency
C 0.48 0.53 reference
T 0.52 0.47 0.80 (0.70-0.91), p=0.0008
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*

Signifcant atherosclerosis was defined by >50% stenosis.

†

calculated by logistic regression analysis with adjustment for age, gender, smoking, hypercholesterolemia, hypertension and diabetes.

In line with the finding in the above study of CAD patients, the population-based, prospective study (the Bruneck study) showed that the T allele of SNP rs1940475 was associated with a protective effect against carotid atherosclerosis progression in a 10 year follow-up [odds ratio 0.77 (95% confidence interval 0.60-0.98) for atherosclerosis progression between 1990 and 1995, and odds ratio 0.74 (95% confidence interval 0.56-0.99) for atherosclerosis progression between 1995 and 2000] (Online Supplement Table 6). In addition, we found that plasma levels of soluble VCAM-1 were lower in individuals who carried the T allele (p=0.014, Online Supplement Table 7).

MMP8 is secreted from cells as a latent form, and activation is achieved by removing the propeptide domain of the zymogen18. The rs1940475 is a non-synonymous substitution resulting in a change from glutamic acid (Glu) to lysine (Lys) at amino acid residue 87 in the propeptide domain of MMP8. We carried out in vitro analysis to investigate whether this SNP could have an effect on MMP8 activation. The analysis indicated that MMP8 zymogen with Lys87 (produced by the T allele) is less amenable to activation than the zymogen with Glu87 (produced by the C allele) (Online Supplement Figure 5).

Discussion

Our study shows that inactivating MMP8 causes a substantial reduction in the extent of atherosclerosis in apoE deficient mice fed a Western diet. The atherosclerotic lesions in the MMP8 knockout mice have fewer macrophages but higher collagen content. Compared with controls, MMP8 deficient mice have higher Ang I and lower Ang II levels, likely reflecting the ability of MMP8 to cleave Ang I and generate Ang II. Consistently, we found that blood pressure was lower in MMP8 knockout mice than in controls. In agreement with the finding that Ang II induces adhesion molecule expression28-34, we found that products of Ang I cleavage by MMP8 increases VCAM-1 expression in cultured endothelial cells. In addition, we observed reduced expression of VCAM-1 in MMP8 knockout mice than in controls. Further to the findings from the mouse study, we detected an association between MMP8 gene variation and extent of coronary atherosclerosis in CAD patients, and an association of the MMP8 gene variation with plasma VCAM-1 level and progression of atherosclerosis in a population-based, prospective cohort study (the Bruneck study). Functional analysis indicated that the MMP8 genetic variant associated with reduced atherosclerosis had an effect on MMP8 zymogen activation. Taken together, these results indicate that MMP8 is an important player in atherogenesis.

Classically MMPs are implicated in matrix remodeling through their matrix protein degrading activity. However, relatively recent studies have revealed that MMPs have other important biological functions arising from their proteolytic activity on non-matrix proteins2. In particular, an increasing number of cytokines, chemokines and growth factors have been shown to be processed by MMPs, and there is growing evidence indicating that the actions of MMPs on these non-matrix proteins play important roles in tumorigenesis and a number of inflammatory diseases2, 38. Although currently the most appreciated roles of MMPs in atherosclerosis are mainly related to matrix protein degradation that facilitates cell migration and undermines atherosclerotic plaque stability, it is conceivable that actions of MMPs on non-matrix proteins also play a part in the development and progression of atherosclerosis. The findings that MMP8 can cleave Ang I generating Ang II and that MMP8 knockout mice have reduced Ang II levels seem to support this notion. It is plausible that the role of MMP8 in atherogenesis is partly related to its proteolytic activity on matrix proteins particularly type I collagen as well as its ability to cleave Ang I generating Ang II. Thus, the reduction in atherosclerotic lesion formation in MMP8 knockout mice could be partly due to reduced blood pressure and reduced VCAM-1 expression, as a result of lower Ang II levels.

The best known Ang I convertor is ACE, but other enzymes (such as chymases, cathepsins and MMP8) have also been shown to be able to cleave Ang I to generate Ang II26, 28. ACE is a zinc-dependent metalloproteinase and belongs to the metalloproteinase super-family that also includes MMPs39, 40. The catalytic sites of ACE and collagenases share similarities in amino acid sequence and structure41, 42. Interestingly, it has been shown that the ACE inhibitors captopril, lisinopril, quinapril and enalapril also inhibit MMP activity43-47 through interacting with the MMP active site, analogous to their interaction with the active site of ACE42, 47. Although ACE is likely to be the main converter of Ang I, it is probable that other enzymes such as MMP8, chymases and cathepsins also contribute to Ang I conversion, particularly at locations where ACE levels are low.

Previous studies have examined the effects of several MMPs on atherogenesis in apoE deficient mice or LDL receptor deficient mice4-11, 13, 14. These studies have suggested divergent effects of these enzymes in atherosclerotic lesion formation and stability48. It was shown that expressing human MMP1 in macrophages in apoE deficient mice resulted in smaller aortic atherosclerotic lesions with reduced fibrillar collagen content4. Similarly, inactivating MMP3 increased aortic atherosclerotic lesion sizes and collagen content6. Inactivating MMP13 also increased, whilst overexpressing MMP14 decreased, collagen accumulation in aortic atherosclerotic lesions without an apparent effect on lesion size13, 14. In contrast, inactivating MMP2 resulted in decreased aortic atherosclerotic lesions with reduced numbers of smooth muscle cells5. Inactivating MMP9 also led to smaller aortic atherosclerotic lesions with reduced numbers of smooth muscle cells but also with fewer macrophages and lower collagen content8-10. Moreover, it was shown that overexpressing active MMP9 in macrophages in apoE deficient mice induced aortic atherosclerotic plaque disruption11. MMP12 has also been shown to exert an effect on atherosclerotic lesion stability7. In contrast, inactivating MMP7 had no effect in these characteristics7. Thus the findings from these studies suggest that these MMPs exert divergent effects, some impacting on lesion size, some affecting collagen content, and some being required for macrophage and/or smooth muscle cell accumulation. As described earlier, our study showed that MMP8 knockout resulted in reduced atherosclerotic lesions with fewer macrophages and increased collagen content. Thus, in apoE deficient mice, MMP8 has a different profile of activities from the other MMPs mentioned above. However, similar to MMP-1, -3, -13 and -144, 6, 13, 14, MMP8 reduced collagen accumulation in atherosclerotic lesions, which could potentially have implications on atherosclerotic lesion stability.

The present study has some limitations. The study only assessed atherosclerosis in the aorta following a Western diet for 12 weeks. Further work is warranted to study the effect of MMP8 deficiency on atherosclerosis in other vascular sites, in shorter and longer durations of Western diet, and in chow diet. For the CAD patients and the Bruneck cohort, no Ang II level data were available and therefore we were unable to determine whether there was an association between Ang II level and MMP8 genotype. This would also warrant investigating in future work. It would also be interesting to investigate the relative contributions of the reduction in Ang II generation and the increase in collagen content to atherosclerotic lesion development in MMP8 knockout mice in future studies

In summary, our study indicates that MMP8 is an important player in atherogenesis. Taken together with the aforementioned studies of other MMPs, this work provides further evidence of involvement of MMPs in atherosclerosis and further supports the notion that different MMPs have divergent effects in the atherogenic process.

Supplementary Material

supp1

Acknowledgments

Sources of funding This work was supported by grants from the British Heart Foundation (PG/08/051/25141 and PG/98183) and the European Union (LSHC-CT-2003-503297). JD was supported by a fellowship from the Barts and The London Charity. RSS is funded by a Wellcome Trust Career Development Fellowship

Non-standard Abbreviations and Acronyms

MMPs

matrix metalloproteinases

MMP8

matrix metalloproteinase-8

Ang I

angiotensin I

Ang II

angiotensin II

ACE

angiotensin converting enzyme

AT1R

Ang II type 1 receptor

AT2R

Ang II type 2 receptor

Ang1-7

angiotensin1-7

VCAM-1

vascular cell adhesion molecule-1

CAD

coronary artery disease

SNPs

single nucleotide polymorphisms

RT-PCR

reverse-transcriptase polymerase chain reaction

ELISA

Enzyme-linked immuno-sorbent assay

Footnotes

Disclosures None

References

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