Abstract

Background: Matrix metalloproteinase-9 (MMP-9) has a potential role in arterial plaque rupture, but its relation to risk of coronary heart disease (CHD) is uncertain.

Aim: To determine whether circulating levels of serum MMP-9 are prospectively related to the risk of CHD in the general population.

Methods: We measured baseline MMP-9 levels in stored serum samples of subjects in a case-control study nested within a prospective study of 5661 men followed up for 16 years for CHD events (465 cases, 1076 controls).

Results: MMP-9 values were associated with cigarette smoking, and with several inflammatory and haemostatic markers, but not with age, body mass index, blood pressure or lipid measurements. Men in the top third of baseline MMP-9 levels had an age-adjusted odds ratio (OR) for CHD of 1.37 (95% CI 1.04–1.82) compared with those in the bottom third. Adjustment for conventional risk factors (smoking in particular) reduced the odds ratio to borderline significance: OR 1.28 (95% CI 0.95–1.74), while additional adjustment for two markers of generalized inflammation, interleukin-6 and C-reactive protein, further attenuated the association: OR 1.13 (0.82–1.56).

Conclusion: Serum MMP-9 has a modest association with incident CHD in the general population, which is not independent of cigarette smoking exposure and circulating markers of generalized inflammation. MMP-9 is unlikely to be a clinically useful biomarker of CHD risk, but may still play a role in the pathogenesis of CHD.

Introduction

The mechanisms by which a low-grade chronic inflammatory state brings about atherosclerotic plaque development and subsequent rupture are the subject of much current interest.1 Although circulating markers of general inflammation associated with the acute phase response, such as fibrinogen2 and C-reactive protein (CRP),3 are associated with risk of coronary heart disease (CHD) among healthy populations and those with prevalent disease, their roles in the biology of atherosclerosis remain uncertain.4,5

Matrix metalloproteinases (MMPs) are a family of zinc-containing zymogen endoproteinases, which have functions in the normal and injury-induced turnover of the extracellular matrix.6 MMP-9 (gelatinase B, 92 kDa collagenase) degrades interstitial collagens, proteoglycan core protein, elastin and type IV collagen in the basement membrane.6,7 It has been proposed that localized inflammation may promote production and activation of MMP-9, and bring about de-stabilizing structural changes in vulnerable atherosclerotic plaques.8–13 MMP-9 also potentiates the chemokine interleukin (IL)-8 and modifies the local chemokine profile,14 and hence may promote cellular infiltration of the plaque, increasing the size of the necrotic core. These processes render the plaque susceptible to rupture due to reduced mechanical strength, and hence increase the probability of atherothrombotic ischaemia. Tissue studies have shown that active MMP-9 can be found in plaques in regions of foam cell accumulation15 and that production of MMP-9 is higher in phenotypically unstable plaques than in stable ones.10,15,16

Epidemiological studies have shown that MMP-9 levels are increased in the circulation of patients with acute coronary syndromes.17,18 However, there is very limited information on whether circulating MMP-9 is a risk factor for future cardiovascular events among healthy men. To our knowledge, the only prospective studies reported to date are based on cohorts with established baseline CHD.19–22 Data from these cohorts are conflicting, although the largest study to date (the Atherogene study,19n = 137 cases) reported significant independent associations between circulating MMP-9 levels with risk of cardiovascular disease (CVD) death or myocardial infarction (MI). In order to clarify the relationship between systemic circulating serum MMP-9 levels and incident CHD in the general population, we report here the results of a case-control study of healthy middle-aged British men, nested within a large prospective study (the British Regional Heart Study).

Methods

In 1978–80, 7735 males aged 40–59 were randomly selected from general practice registers in each of 24 British towns and invited to take part in the British Regional Heart Study (response rate 78%). Nurses administered questionnaires, made physical measurements including blood pressure, weight, height and body mass index (BMI), recorded an electrocardiograph (ECG) and collected non-fasting venous blood samples. In 5661 men in 18 of the study towns, serum was stored at −20°C for subsequent analysis.23 Pre-existing CHD was defined on the basis of a history of a doctor's diagnosis of CHD, a positive history of angina or possible MI on the Rose (WHO) chest pain questionnaire or an ECG consistent with definite or possible CHD. All men were monitored subsequently for all-cause mortality and for cardiovascular morbidity, with a follow-up loss of <1%.24 Fatal CHD cases were ascertained through National Health Service Central Registers on the basis of a death certificate with ICD-9 codes 410–414. The diagnosis of non-fatal myocardial infarction was based on reports from general practitioners, supplemented by regular reviews of general practice records and diagnosed in accordance with WHO criteria.24 A nested case-control study was established, based on all 643 CHD cases (279 CHD deaths and 364 cases of non-fatal MI) occurring between 1978 and 1996, and a total of 1278 controls (2 per case) ‘frequency-matched’ to cases on town of residence and age in 5-year bands was randomly selected from among men who had survived to the end of the study period free from incident coronary heart disease. Due to attrition of samples as a result of use in previous studies,25–30 residual serum samples were available for 465 of these cases and 1067 controls for MMP-9 measurements to be carried out in 2004–05. The baseline characteristics of subjects for whom serum samples were and were not available for the present study were not appreciably different from the original case-control study (P > 0.10 for case-control proportion, age distribution, residential town proportions and social class proportions).

Laboratory methods

P. Welsh, who was blind to the case-control status of participants, measured serum concentrations of MMP-9 using a commercially available sandwich ELISA (R&D Systems, Oxon, UK) (intra-assay CV 4.4%, inter-assay CV 10.4%). Markers of systemic inflammation, vascular adhesion molecules, and haematological variables were assayed as previously reported.25–30

Statistical analysis

Highly skewed variables were log-transformed where necessary. We pre-specified case-control analyses by thirds of MMP-9 values in controls. Unmatched stratified logistic regression was fitted using unconditional maximum likelihood methods (SAS version 8.1). The associations between MMP-9 and a variety of known and suspected CHD risk factors were examined using unmatched logistic regressions and analysis of covariance for categorical and continuous variables respectively; emphasis was mainly given to differences more extreme than 2.6 SD (2P > 0.01) to make allowance for multiple comparisons.

Results

Established vascular risk factors showed the expected differences between the case and control populations (Table 1). Cases were more likely to have evidence of baseline CHD, to be current cigarette smokers, or to have diabetes. Cases also had a higher average BMI, and had a higher average systolic and diastolic blood pressure. They had higher average total cholesterol and triglyceride levels and lower high density lipoprotein cholesterol level, as well as elevated CRP levels at baseline. For MMP-9, geometric mean levels were higher among cases than controls (age-adjusted proportional difference 7%, 95% CI 1–13%; P = 0.03).

Table 1

Baseline characteristics of the case and control populations

 Cases (n = 465) Controls (n = 1076) P-value 

 
Questionnaire    
    Age (years) 52.5 (5.1) 5.24 (5.3) Matched 
    Current smoker (n, %) 238 (51.3) 451 (42.0) 0.0007 
    Evidence of coronary disease (n, %) 197 (42.4) 276 (25.7) <0.0001 
    History of diabetes 14 (3.0) 16 (1.5) 0.05 
    >2 drinks alcohol/day (n, %) 86 (18.5) 239 (22.2) 0.10 
    Non-manual occupation (n, %) 144 (32.3) 400 (38.2) 0.03 
    Physical activity (inactive/occasional) (n, %) 218 (47.3) 430 (40.6) 0.02 
Physical Measurements    
    Body mass index (kg/m226.0 (3.4) 25.4 (3.3) 0.002 
    Systolic blood pressure (mmHg) 151.2 (21.4) 146.5 (21.0) <0.0001 
    Diastolic blood pressure (mmHg) 85.5 (13.2) 82.9 (13.4) 0.0003 
    FEV1 (l/min) 3.13 (0.64) 3.22 (0.70) 0.02 
Blood Sample    
    Total cholesterol (mmol/l) 6.67 (1.04) 6.19 (0.99) <0.0001 
    HDL cholesterol (mmol/l) 1.10 (0.28) 1.15 (0.27) 0.0001 
    Triglyceride (mmol/l)a 1.95 (0.69, 5.51) 1.66 (0.56, 4.90) <0.0001 
    C-reactive protein (mg/l)a 2.34 (0.29, 19.69) 1.41 (0.32, 14.76) <0.0001 
    MMP-9 (ng/ml)a 741.3 (274.7, 2000.8) 691.8 (245.0, 1953.4) 0.026 
 Cases (n = 465) Controls (n = 1076) P-value 

 
Questionnaire    
    Age (years) 52.5 (5.1) 5.24 (5.3) Matched 
    Current smoker (n, %) 238 (51.3) 451 (42.0) 0.0007 
    Evidence of coronary disease (n, %) 197 (42.4) 276 (25.7) <0.0001 
    History of diabetes 14 (3.0) 16 (1.5) 0.05 
    >2 drinks alcohol/day (n, %) 86 (18.5) 239 (22.2) 0.10 
    Non-manual occupation (n, %) 144 (32.3) 400 (38.2) 0.03 
    Physical activity (inactive/occasional) (n, %) 218 (47.3) 430 (40.6) 0.02 
Physical Measurements    
    Body mass index (kg/m226.0 (3.4) 25.4 (3.3) 0.002 
    Systolic blood pressure (mmHg) 151.2 (21.4) 146.5 (21.0) <0.0001 
    Diastolic blood pressure (mmHg) 85.5 (13.2) 82.9 (13.4) 0.0003 
    FEV1 (l/min) 3.13 (0.64) 3.22 (0.70) 0.02 
Blood Sample    
    Total cholesterol (mmol/l) 6.67 (1.04) 6.19 (0.99) <0.0001 
    HDL cholesterol (mmol/l) 1.10 (0.28) 1.15 (0.27) 0.0001 
    Triglyceride (mmol/l)a 1.95 (0.69, 5.51) 1.66 (0.56, 4.90) <0.0001 
    C-reactive protein (mg/l)a 2.34 (0.29, 19.69) 1.41 (0.32, 14.76) <0.0001 
    MMP-9 (ng/ml)a 741.3 (274.7, 2000.8) 691.8 (245.0, 1953.4) 0.026 

HDL: high density lipoprotein; FEV: forced expiratory volume.

Values are mean (SD) unless otherwise specified.

aIndicates geometric mean (95% CI).

Baseline MMP-9 levels in the control population were significantly positively associated with markers of systemic inflammation (CRP, IL-6, serum amyloid A, white cell count), vascular adhesion molecules [E- and P-selectin and intracellular adhesion molecule (ICAM)-1, although not vascular cell adhesion molecule (VCAM)-1] and some haematological variables [haematocrit and fibrin D-dimer, but not tissue-plasminogen activator (t-PA) or von Willebrand factor (VWF) antigens] (Table 2). There was no association with homocysteine levels. MMP-9 levels were inversely related to forced expiratory volume (FEV1) and strongly related to cigarette smoking habits, as well as showing increasing trends with alcohol intake. Although it was weakly related to non-fasting glucose, MMP-9 showed no relationship to insulin and was not associated with other established vascular risk factors such as age, evidence of CHD at entry, blood pressure, BMI, physical activity or any lipid measurements (Table 2).

Table 2

Relation of MMP-9 (thirds) with other cardiovascular risk factors

 Tertiles of MMP-9
 
  
Tertile Low Middle High   
Range (ng/ml) (70–555) n = 358
 
(556–872) n = 359
 
(873–3500) n = 359
 
  
 Mean SE Mean SE Mean SE P-trend 

 
Age (years) 52.1 0.3 52.6 0.3 52.5 0.3 0.27 
Current smoker (n, %) 123 (34) 34.4 134 (37) 37.3 194 (54) 54.2 <0.0001 
>2 drinks alcohol/day (n, %)  74 (21) 20.7  71 (20) 19.8  94 (26) 26.3 0.04 
Evidence of CHD at entry (n, %)  91 (25) 25.4  81 (23) 22.6 104 (29) 29.0 0.40 
History of diabetes at baseline (n0.6 2.0 2.0 n/a 
Non-manual occupation (n, %) 137 (38) 39.3 133 (37) 37.7 130 (36) 37.7 0.44 
Physical activity (inactive/occasional) (n, %) 138 (38) 38.9 143 (40) 40.4 149 (42) 42.5 0.51 
Body mass index (kg/m2) 25.5 0.2 25.5 0.2 25.1 0.2 0.11 
Systolic blood pressure (mmHg) 146.9 1.1 147.6 1.1 145.0 1.1 0.41 
Diastolic blood pressure (mmHg) 83.5 0.7 83.2 0.7 81.9 0.7 0.23 
FEV1 (l/min) 3.30 0.04 3.25 0.04 3.09 0.04 0.002 
Total cholesterol (mmol/l) 6.24 0.05 6.18 0.05 6.14 0.05 0.18 
HDL cholesterol (mmol/l) 1.15 0.01 1.15 0.01 1.16 0.01 0.63 
Triglyceride (mmol/l)a 0.21 0.01 0.22 0.01 0.24 0.01 0.12 
Insulin (mU/l)a 1.08 0.02 1.10 0.02 1.05 0.02 0.21 
Glucose (mmol/l)a 0.745 0.004 0.743 0.004 0.73 0.004 0.03 
Von Willebrand factor (IU/dl)a 2.01 0.01 2.02 0.01 2.02 0.01 0.49 
Fibrin D-dimer (ng/ml)a 1.83 0.02 1.86 0.02 1.96 0.02 <0.0001 
t-PA (ng/ml)a 1.00 0.01 1.00 0.01 1.03 0.01 0.61 
Haematocrit (%) 43.10 0.17 44.20 0.17 44.70 0.17 <0.0001 
Albumin (g/l) 44.7 0.1 44.5 0.1 44.3 0.1 0.07 
Globulin (g/l) 29.1 0.2 29.0 0.2 29.0 0.2 0.48 
White cell count (109/l)a 0.79 0.01 0.84 0.01 0.90 0.01 <0.0001 
Serum amyloid A protein (mg/l)a 0.80 0.02 0.85 0.02 0.90 0.02 <0.0001 
Homocysteine (mmol/l)a 1.12 0.01 1.13 0.02 1.12 0.02 0.61 
E-selectin (ng/ml)a 1.76 0.01 1.77 0.01 1.80 0.01 0.002 
P-selectin (ng/ml)a 1.94 0.01 2.04 0.01 2.09 0.01 <0.0001 
ICAM-1 (ng/ml)a 2.45 0.01 2.46 0.01 2.50 0.01 <0.0001 
VCAM-1 (ng/ml)a 2.64 0.01 2.63 0.01 2.62 0.01 0.14 
CRP (mg/l)a 0.09 0.03 0.13 0.03 0.28 0.03 <0.0001 
IL-6 (pg/ml)a 0.27 0.01 0.36 0.01 0.44 0.01 <0.0001 
 Tertiles of MMP-9
 
  
Tertile Low Middle High   
Range (ng/ml) (70–555) n = 358
 
(556–872) n = 359
 
(873–3500) n = 359
 
  
 Mean SE Mean SE Mean SE P-trend 

 
Age (years) 52.1 0.3 52.6 0.3 52.5 0.3 0.27 
Current smoker (n, %) 123 (34) 34.4 134 (37) 37.3 194 (54) 54.2 <0.0001 
>2 drinks alcohol/day (n, %)  74 (21) 20.7  71 (20) 19.8  94 (26) 26.3 0.04 
Evidence of CHD at entry (n, %)  91 (25) 25.4  81 (23) 22.6 104 (29) 29.0 0.40 
History of diabetes at baseline (n0.6 2.0 2.0 n/a 
Non-manual occupation (n, %) 137 (38) 39.3 133 (37) 37.7 130 (36) 37.7 0.44 
Physical activity (inactive/occasional) (n, %) 138 (38) 38.9 143 (40) 40.4 149 (42) 42.5 0.51 
Body mass index (kg/m2) 25.5 0.2 25.5 0.2 25.1 0.2 0.11 
Systolic blood pressure (mmHg) 146.9 1.1 147.6 1.1 145.0 1.1 0.41 
Diastolic blood pressure (mmHg) 83.5 0.7 83.2 0.7 81.9 0.7 0.23 
FEV1 (l/min) 3.30 0.04 3.25 0.04 3.09 0.04 0.002 
Total cholesterol (mmol/l) 6.24 0.05 6.18 0.05 6.14 0.05 0.18 
HDL cholesterol (mmol/l) 1.15 0.01 1.15 0.01 1.16 0.01 0.63 
Triglyceride (mmol/l)a 0.21 0.01 0.22 0.01 0.24 0.01 0.12 
Insulin (mU/l)a 1.08 0.02 1.10 0.02 1.05 0.02 0.21 
Glucose (mmol/l)a 0.745 0.004 0.743 0.004 0.73 0.004 0.03 
Von Willebrand factor (IU/dl)a 2.01 0.01 2.02 0.01 2.02 0.01 0.49 
Fibrin D-dimer (ng/ml)a 1.83 0.02 1.86 0.02 1.96 0.02 <0.0001 
t-PA (ng/ml)a 1.00 0.01 1.00 0.01 1.03 0.01 0.61 
Haematocrit (%) 43.10 0.17 44.20 0.17 44.70 0.17 <0.0001 
Albumin (g/l) 44.7 0.1 44.5 0.1 44.3 0.1 0.07 
Globulin (g/l) 29.1 0.2 29.0 0.2 29.0 0.2 0.48 
White cell count (109/l)a 0.79 0.01 0.84 0.01 0.90 0.01 <0.0001 
Serum amyloid A protein (mg/l)a 0.80 0.02 0.85 0.02 0.90 0.02 <0.0001 
Homocysteine (mmol/l)a 1.12 0.01 1.13 0.02 1.12 0.02 0.61 
E-selectin (ng/ml)a 1.76 0.01 1.77 0.01 1.80 0.01 0.002 
P-selectin (ng/ml)a 1.94 0.01 2.04 0.01 2.09 0.01 <0.0001 
ICAM-1 (ng/ml)a 2.45 0.01 2.46 0.01 2.50 0.01 <0.0001 
VCAM-1 (ng/ml)a 2.64 0.01 2.63 0.01 2.62 0.01 0.14 
CRP (mg/l)a 0.09 0.03 0.13 0.03 0.28 0.03 <0.0001 
IL-6 (pg/ml)a 0.27 0.01 0.36 0.01 0.44 0.01 <0.0001 

aLog-transformed to base 10.

In comparisons of men in the highest third vs. the lowest third of the MMP-9 control distribution, the age-adjusted odds ratio (OR) for CHD was 1.37 (95% CI 1.04–1.82) (Table 3). Adjustment for smoking status attenuated the result to borderline significance: OR 1.27 (0.96–1.69), although further adjustment for other cardiovascular risk factors did not attenuate the result further [OR 1.28 (0.95–1.74)]. After adjusting for CRP and IL-6 levels in the final model the association was attenuated towards the null: OR 1.13 (0.82–1.56). Only the age and town-adjusted model yielded a significant trend across thirds (P = 0.039). Exclusion of men with evidence of baseline CHD had little material effect on the odds ratios observed, although the age and town-adjusted association with CHD risk was no longer statistically significant.

Table 3

OR (95% CI) of coronary heart disease in men who had values of MMP-9 in the top thirds of the distribution of controls relative to those who had values in the bottom third

MMP-9 values (ng/ml) Cases Controls Odds ratio (95% CI) adjusted for
 
Tertiles N N Age and town only Age, town and smoking status Age, town, smoking and risk factorsa Age, town smoking, risk factorsa and inflammatory factorsb 

 
All 465 cases and 1076 controls 
873–3500 184 359 1.37 (1.04, 1.82) 1.27 (0.96, 1.69) 1.28 (0.95, 1.74) 1.13 (0.82, 1.56) 
556–872 146 359 1.03 (0.77, 1.38) 1.01 (0.75, 1.35) 1.00 (0.73, 1.36) 1.01 (0.74, 1.14) 
70–555 135 358 
Total 465 1076     
   P-value (trend) = 0.039 0.12 0.11 0.38 
268 cases and 800 controls without baseline evidence of coronary heart disease 
873–3500 104 255 1.29 (0.94, 1.84) 1.17 (0.81, 1.67) 1.24 (0.85, 1.82) 1.07 (0.72, 1.61) 
556–872 78 278 0.80 (0.55, 1.16) 0.79 (0.54, 1.14) 0.81 (0.54, 1.19) 0.82 (0.55, 1.23) 
70–555 86 267 
Total 268 800     
   P-value (trend) = 0.15 0.37 0.19 0.48 
MMP-9 values (ng/ml) Cases Controls Odds ratio (95% CI) adjusted for
 
Tertiles N N Age and town only Age, town and smoking status Age, town, smoking and risk factorsa Age, town smoking, risk factorsa and inflammatory factorsb 

 
All 465 cases and 1076 controls 
873–3500 184 359 1.37 (1.04, 1.82) 1.27 (0.96, 1.69) 1.28 (0.95, 1.74) 1.13 (0.82, 1.56) 
556–872 146 359 1.03 (0.77, 1.38) 1.01 (0.75, 1.35) 1.00 (0.73, 1.36) 1.01 (0.74, 1.14) 
70–555 135 358 
Total 465 1076     
   P-value (trend) = 0.039 0.12 0.11 0.38 
268 cases and 800 controls without baseline evidence of coronary heart disease 
873–3500 104 255 1.29 (0.94, 1.84) 1.17 (0.81, 1.67) 1.24 (0.85, 1.82) 1.07 (0.72, 1.61) 
556–872 78 278 0.80 (0.55, 1.16) 0.79 (0.54, 1.14) 0.81 (0.54, 1.19) 0.82 (0.55, 1.23) 
70–555 86 267 
Total 268 800     
   P-value (trend) = 0.15 0.37 0.19 0.48 

aBlood pressure, total cholesterol, HDL, body mass index, history of diabetes, CHD, social class, physical activity.

bC-reactive protein, IL-6 (fitted as logged variables).

Discussion

To our knowledge, this is the first report of circulating levels of MMP-9 with risk of CHD in a generally healthy population, and is the largest case-control study of MMP-9 associations with risk of CHD to date. MMP-9 showed a significant association with risk of CHD; men in the highest third of MMP-9 had an increase in CHD risk of about 40%. However, the association was attenuated following adjustment for cigarette smoking and for other inflammatory markers.

Findings of elevated levels of MMP-9 among current smokers (who are at increased CHD risk) are of particular interest, because they may reflect greater expression of MMP-9 in tissues including arterial plaques, thus increasing the risk of plaque rupture. Increased levels of MMP-9 expression among current smokers may suggest that MMP-9 is one possible pathophysiological link between cigarette smoke exposure and increased risk of CHD events. This speculative link would be consistent with data in animal and tissue models showing that MMP-9 is associated with and may confer plaque instability,8–13,15,16 and with at least one mouse model showing that smoking exposure raises pulmonary MMP-9 levels.31 We have also shown that serum MMP-9 is associated not only with circulating inflammatory markers (in agreement with previous findings19) but also with circulating levels of adhesion molecules ICAM-1, P-selectin and E-selectin. This association with circulating adhesion molecules may be more specific than a broad range association of MMP-9 with activated vascular endothelium, since MMP-9 shows no associations with two other potential markers of activated endothelium, VWF and t-PA,32 or with VCAM-1. The mechanisms behind these differing associations are currently unclear.

Consistent with MMP-9's association with smoking habits, its association with CHD risk was attenuated after adjusting for smoking habit (OR 1.28, 95% CI 0.95–1.74), although not by adjustment for other conventional risk markers. This residual borderline association was further reduced after adjustment for IL-6 and CRP [OR 1.13 (0.82–1.56)]. These findings suggest that the association between MMP-9 and incident CHD in the general population is confounded by smoking habit and by markers of generalized inflammation. It therefore seems unlikely that MMP-9 would add further discrimination to CHD risk prediction models for the general population. More data in generally healthy populations are required to establish its association with CHD risk more reliably.

The present study suggests that the association between MMP-9 and incident CHD is modest; our best estimate is of an increase of about 40% in the highest third, with the upper 95% confidence effectively excluding a doubling of risk in this group. This association appears weaker than that previously reported in a cohort of patients with unstable angina [hazard risk ratio comparing extreme quartiles of MMP-9 in age and sex-adjusted model 2.66 (95% CI 1.42–4.99)].19 The difference between the studies could reflect the imprecision of the relative risk estimates (95% confidence intervals are relatively wide in both studies). Alternatively this could reflect a real difference, possibly because patients with unstable angina have a higher prevalence of unstable coronary artery plaques than the general population in the present study. MMP-9 may be a weaker marker of CHD event risk in a generally healthy cohort rather than those with established arterial disease due to the ability of MMP-9 to de-stabilize established plaques,8–13 rather than to directly stimulate atherogenesis. This possibility is in agreement with other epidemiological data showing that production of MMP-9 is higher in phenotypically unstable than stable plaques.10,15,16 Other potential explanations for the difference in these reports include that the present study was performed using serum samples, whilst the Atherogene report used plasma.19 The ELISA we used did not recommend use of plasma samples due to chelation issues for MMPs.

Although MMP-9 is unlikely to prove a clinically useful biomarker of CHD risk based on these results, an OR of 1.4 for CHD comparing extreme thirds of a general population can be described as moderate. To put these results in context, a 1 mmol/l increase in LDL total cholesterol in the general population results in a 12% increase in relative risk.33 Therefore the potential role of MMP-9 in the aetiology of CHD in the general population should not be discounted, and its potential as a therapeutic target requires further consideration.

The major strength of this study is that it is a large prospective study nested within a well-maintained cohort study with high follow-up rates. Limitations of the study include the age of the samples (∼24 years at time of assay), and we cannot exclude the possibility that degradation may have had some bearing on our results. MMP-9 is known to be labile even at low temperatures of storage, and as such degradation of samples over even short epidemiological time frames is unavoidable. One study has shown that plasma MMP-9 levels fall by 65% after 2 years storage at −80°C.34 Our own analysis of 10 serum samples stored at −50°C for 1 year is consistent with this, showing a 28.5% drop in median detectable levels, although the fall in levels was uniform (log-transformed Spearman correlation at 1 year vs. baseline = 0.93) (Welsh et al., unpublished observations). It is difficult to extrapolate these results to storage conditions over ∼24 years, and studies of MMP-9 stability over such a long period have not yet been performed to our knowledge. However, in the present study all cases and controls were treated in the same manner and, based on the limited available data, we would anticipate reasonably uniform effects of storage conditions on the samples MMP-9 levels. Our observations regarding significant MMP-9 associations with smoking habits and CRP but not with lipid measurements are consistent with the Atherogene study report of samples stored for a shorter time period,19 and a recent cross-sectional study which also used plasma.35 Furthermore, the long-stored samples used in the present nested case-control study have been used in studies of several other novel potential cardiovascular risk factors,25–30 including the cytokine IL-6,30 and yielded similar results to other studies in which samples were stored for shorter periods. Finally, it may be important to note that circulating active MMP-9 could be a better marker of risk of CHD than MMP-9 antigen (both pre-MMP-9 and active MMP-9) measured here. This possibility requires further epidemiological study.

Conclusion

There is a significant association between serum MMP-9 and incident CHD in the general population. This is largely related to cigarette smoking exposure and to circulating markers of generalized inflammation. The association is of biological interest and is consistent with a potential role for MMP-9 in pathogenesis of CHD. However, MMP-9 is unlikely to be a clinically useful additional risk predictor for CHD in the general population.

Acknowledgements

We thank Helen Mosson for secretarial assistance with manuscript preparation.

PW, AR and GDOL are supported by project and programme grants from the British Heart Foundation. Professor A.G. Shaper established the British Regional Heart Study, which is a British Heart Foundation Research Group. The views expressed in this article are those of the authors and not necessarily those of the funding agencies.

Conflict of interest: None declared.

References

1
Hansson
GK
Inflammation, atherosclerosis, and coronary artery disease
N Engl J Med
 , 
2005
, vol. 
352
 (pg. 
1685
-
95
)
2
Fibrinogen Studies Collaboration
Plasma fibrinogen level and the risk of major cardiovascular diseases and nonvascular mortality: an individual participant meta-analysis
JAMA
 , 
2005
, vol. 
294
 (pg. 
1799
-
809
)
3
Danesh
J
Wheeler
JG
Hirschfield
GM
Eda
S
Eiriksdottir
G
Rumley
A
, et al.  . 
C-reactive protein and other circulating markers of inflammation in the prediction of coronary heart disease
N Engl J Med
 , 
2004
, vol. 
350
 (pg. 
1387
-
97
)
4
Trion
A
de Maat
M
Jukema
W
Maas
A
Offerman
E
Havekes
L
, et al.  . 
No effect of C-reactive protein on early atherosclerosis development in apolipoprotein E*3-leiden/human C-reactive protein transgenic mice
Arterioscler Thromb Vasc Biol
 , 
2005
, vol. 
25
 (pg. 
1635
-
40
)
5
Gulledge
AA
McShea
C
Schwartz
T
Koch
G
Lord
ST
Effects of hyperfibrinogenemia on vasculature of C57BL/6 mice with and without atherogenic diet
Arterioscler Thromb Vasc Biol
 , 
2003
, vol. 
23
 (pg. 
130
-
5
)
6
Van den Steen
PE
Dubois
B
Nelissen
I
Rudd
PM
Dwek
RA
Opdenakker
G
Biochemistry and molecular biology of gelatinase B or matrix metalloproteinase-9 (MMP-9)
Crit Rev Biochem Mol Biol
 , 
2002
, vol. 
37
 (pg. 
375
-
536
)
7
Opdenakker
G
Van den Steen
PE
Dubois
B
Nelissen
I
Van Coillie
E
Masure
S
, et al.  . 
Gelatinase B functions as regulator and effector in leukocyte biology
J Leukoc Biol
 , 
2001
, vol. 
69
 (pg. 
851
-
9
)
8
Shah
PK
Pathophysiology of coronary thrombosis: role of plaque rupture and plaque erosion
Prog Cardiovasc Dis
 , 
2002
, vol. 
44
 (pg. 
357
-
68
)
9
Galis
ZS
Khatri
JJ
Matrix metalloproteinases in vascular remodeling and atherogenesis: the good, the bad, and the ugly
Circ Res
 , 
2002
, vol. 
90
 (pg. 
251
-
62
)
10
Sluijter
JP
Pulskens
WP
Schoneveld
AH
Velema
E
Strijder
CF
Moll
F
, et al.  . 
Matrix metalloproteinase 2 is associated with stable and matrix metalloproteinases 8 and 9 with vulnerable carotid atherosclerotic lesions: a study in human endarterectomy specimen pointing to a role for different extracellular matrix metalloproteinase inducer glycosylation forms
Stroke
 , 
2006
, vol. 
37
 (pg. 
235
-
9
)
11
Pasterkamp
G
Schoneveld
AH
Hijnen
DJ
de Kleijn
DP
Teepen
H
van der Wal
AC
, et al.  . 
Atherosclerotic arterial remodeling and the localization of macrophages and matrix metalloproteases 1, 2 and 9 in the human coronary artery
Atherosclerosis
 , 
2000
, vol. 
150
 (pg. 
245
-
53
)
12
van der Wal
AC
Becker
AE
van der Loos
CM
Das
PK
Site of intimal rupture or erosion of thrombosed coronary atherosclerotic plaques is characterised by an inflammatory process irrespective of dominant plaque morphology
Circulation
 , 
1994
, vol. 
89
 (pg. 
36
-
44
)
13
Shah
PK
Falk
E
Badimon
JJ
Fernandez-Ortiz
A
Mailhac
A
Villareal-Levy
G
, et al.  . 
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
, vol. 
92
 (pg. 
1565
-
9
)
14
Van den Steen
PE
Proost
P
Wuyts
A
Van Damme
J
Opdenakker
G
Neutrophil gelatinase B potentiates interleukin-8 tenfold by aminoterminal processing, whereas it degrades CTAP-III, PF-4 and GRO-α and leaves RANTES and MCP-2 intact
Blood
 , 
2000
, vol. 
96
 (pg. 
2673
-
81
)
15
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
, vol. 
94
 (pg. 
2493
-
503
)
16
Loftus
IM
Naylor
AR
Goodall
S
Crowther
M
Jones
L
Bell
PR
, et al.  . 
Increased matrix metalloproteinase-9 activity in unstable carotid plaques. A potential role in acute plaque disruption
Stroke
 , 
2000
, vol. 
31
 (pg. 
40
-
7
)
17
Kai
H
Ikeda
H
Yasukawa
H
Kai
M
Seki
Y
Kuwahara
F
, et al.  . 
Peripheral blood levels of matrix metalloproteinases-2 and -9 are elevated in patients with acute coronary syndromes
J Am Coll Cardiol
 , 
1998
, vol. 
32
 (pg. 
368
-
72
)
18
Inokubo
Y
Hanada
H
Ishizaka
H
Fukushi
T
Kamada
T
Okumura
K
Plasma levels of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 are increased in the coronary circulation in patients with acute coronary syndrome
Am Heart J
 , 
2001
, vol. 
141
 (pg. 
211
-
7
)
19
Blankenberg
S
Rupprecht
HJ
Poirier
O
Bickel
C
Smieja
M
Hafner
G
, et al.  . 
AtheroGene Investigators. Plasma concentrations and genetic variation of matrix metalloproteinase 9 and prognosis of patients with cardiovascular disease
Circulation
 , 
2003
, vol. 
107
 (pg. 
1579
-
85
)
20
Wu
TC
Leu
HB
Lin
WT
Lin
CP
Lin
SJ
Chen
JW
Plasma matrix metalloproteinase-3 level is an independent prognostic factor in stable coronary artery disease
Eur J Clin Invest
 , 
2005
, vol. 
35
 (pg. 
537
-
45
)
21
Cavusoglu
E
Ruwende
C
Chopra
V
Yanamadala
S
Eng
C
Clark
LT
, et al.  . 
Tissue inhibitor of metalloproteinase-1 (TIMP-1) is an independent predictor of all-cause mortality, cardiac mortality, and myocardial infarction
Am Heart J
 , 
2006
, vol. 
151
 (pg. 
1101.e1
-
8
)
22
Eldrup
N
Gronholdt
ML
Sillesen
H
Nordestgaard
BG
Elevated matrix metalloproteinase-9 associated with stroke or cardiovascular death in patients with carotid stenosis
Circulation
 , 
2006
, vol. 
114
 (pg. 
1847
-
54
)
23
Shaper
AG
Pocock
SJ
Walker
M
Cohen
NM
Wale
CJ
Thomson
AG
British Regional Heart Study: cardiovascular risk factors in middle-aged men in 24 towns
Br Med J (Clin Res Ed)
 , 
1981
, vol. 
283
 (pg. 
179
-
86
)
24
Walker
M
Whincup
PH
Shaper
AG
The British Regional Heart Study 1975-2004
Int J Epidemiol
 , 
2004
, vol. 
33
 (pg. 
1185
-
92
)
25
Danesh
J
Whincup
P
Walker
M
Lennon
L
Thomson
A
Appleby
P
, et al.  . 
Low grade inflammation and coronary heart disease: prospective study and updated meta-analyses
BMJ
 , 
2000
, vol. 
321
 (pg. 
199
-
204
)
26
Danesh
J
Whincup
P
Walker
M
Lennon
L
Thomson
A
Appleby
P
, et al.  . 
Fibrin D-dimer and coronary heart disease: prospective study and meta-analysis
Circulation
 , 
2001
, vol. 
103
 (pg. 
2323
-
7
)
27
Malik
I
Danesh
J
Whincup
P
Bhatia
V
Papacosta
O
Walker
M
, et al.  . 
Soluble adhesion molecules and prediction of coronary heart disease: a prospective study and meta-analysis
Lancet
 , 
2001
, vol. 
358
 (pg. 
971
-
6
)
28
Whincup
PH
Danesh
J
Walker
M
Lennon
L
Thomson
A
Appleby
P
, et al.  . 
von Willebrand factor and coronary heart disease: prospective study and meta-analysis
Eur Heart J
 , 
2002
, vol. 
23
 (pg. 
1764
-
70
)
29
Lowe
GD
Danesh
J
Lewington
S
Walker
M
Lennon
L
Thomson
A
, et al.  . 
Tissue plasminogen activator antigen and coronary heart disease. Prospective study and meta-analysis
Eur Heart J
 , 
2004
, vol. 
25
 (pg. 
252
-
9
)
30
Danesh
J
Kaptoge
S
Mann
AG
Sarwar
N
Wood
A
Angleman
SB
, et al.  . 
Long-term interleukin-6 levels and subsequent risk of coronary heart disease: two new prospective studies and a systematic review
PLoS Medicine
 , 
2008
, vol. 
5
 (pg. 
e78:0001
-
0011
)
31
Wright
JL
Tai
H
Wang
R
Wang
X
Churg
A
Cigarette smoke upregulates pulmonary vascular matrix metalloproteinases via TNF-alpha signaling
Am J Physiol Lung Cell Mol Physiol
 , 
2007
, vol. 
292
 (pg. 
L125
-
33
)
32
Van den Eijnden-Schrauwen
Y
Atsma
DE
Lupu
F
de Vries
RE
Kooistra
T
Emeis
JJ
Involvement of calcium and G proteins in the acute release of tissue-type plasminogen activator and von Willebrand factor from cultured human endothelial cells
Arterioscler Thromb Vasc Biol
 , 
1997
, vol. 
17
 (pg. 
2177
-
87
)
33
Baigent
C
Keech
A
Kearney
PM
Blackwell
L
Buck
G
Pollicino
C
, et al.  . 
Cholesterol Treatment Trialists’ (CTT) Collaborators. Efficacy and safety of cholesterol-lowering treatment: prospective meta-analysis of data from 90,056 participants in 14 randomised trials of statins
Lancet
 , 
2005
, vol. 
366
 (pg. 
1267
-
78
)
34
Rouy
D
Ernens
I
Jeanty
C
Wagner
DR
Plasma storage at −80 degrees C does not protect matrix metalloproteinase-9 from degradation
Anal Biochem
 , 
2005
, vol. 
338
 (pg. 
294
-
8
)
35
Garvin
P
Nilsson
L
Carstensen
J
Jonasson
L
Kristenson
M
Circulating matrix metalloproteinase-9 is associated with cardiovascular risk factors in a middle-aged normal population
PLoS ONE
 , 
2008
, vol. 
3
 pg. 
e1774