Elevated circulating levels of matrix metalloproteinase-9 and its association with cardiovascular risk in young smokers

Dinesh Nath; Meera Shivasekar; VM Vinodhini

doi:10.4103/jms.jms_98_21

ORIGINAL ARTICLE

Year : 2022 | Volume : 36 | Issue : 3 | Page : 94-100

Elevated circulating levels of matrix metalloproteinase-9 and its association with cardiovascular risk in young smokers

Dinesh Nath, Meera Shivasekar, VM Vinodhini
Department of Biochemistry, SRM Medical College Hospital and Research Centre, SRMIST, Kattankulthur, Tamil Nadu, India

Date of Submission	26-Jul-2021
Date of Decision	07-Mar-2022
Date of Acceptance	05-Sep-2022
Date of Web Publication	25-Feb-2023

Correspondence Address:
Dr. Meera Shivasekar
Department of Biochemistry, SRM Medical College Hospital and Research Centre, SRMIST, Kattankulthur - 603 203, Tamil Nadu
India

Source of Support: None, Conflict of Interest: None

Check

DOI: 10.4103/jms.jms_98_21

Abstract

Background: Smoking causes cardiovascular risk which may alter the stability between the production and degradation of the extracellular matrix. Matrix metalloproteinase-9 (MMP-9) is a zinc-containing endopeptidase that degrades extracellular matrix and plays a vital role in tissue remodeling. As a result, elevated serum MMP-9 levels produced by smoking, particularly in younger age, raise the risk of future coronary heart disease (CHD).
Aim and Objective: Our aim is to find out the possible relationship between circulating MMP-9 and the risk of cardiovascular disease in young smokers.
Materials and Methods: The study contains three groups. Group 1 includes 60 young active smokers with diabetic CHD, Group 2 includes 60 young active smokers with CHD, and Group 3 includes 60 healthy controls as nonsmokers who were attending SRM Medical College Hospital in Tamil Nadu for cardiology and medicine OP. Enzyme-linked immune sorbent assay was used to measure serum MMP-9, high-sensitivity C-reactive protein (hsCRP), and apolipoprotein E (APO-E) levels, and enzymatic techniques were employed to quantify lipid levels.
Results: When compared to the controls, the mean serum MMP-9, hsCRP, and APO-E levels were significantly higher in both the groups. The study also shows a significant positive association between MMP-9 with hsCRP, APO-E, smoking burden, and smoking intensity.
Conclusion: The study concludes that increased MMP-9 levels, particularly in inflammatory conditions caused by smoking, are associated with an increased risk of future cardiovascular disease.

Keywords: Apolipoprotein E, cardiovascular disease, high-sensitivity C-reactive protein, matrix metalloproteinase-9, smoking

How to cite this article:
Nath D, Shivasekar M, Vinodhini V M. Elevated circulating levels of matrix metalloproteinase-9 and its association with cardiovascular risk in young smokers. J Med Soc 2022;36:94-100

How to cite this URL:
Nath D, Shivasekar M, Vinodhini V M. Elevated circulating levels of matrix metalloproteinase-9 and its association with cardiovascular risk in young smokers. J Med Soc [serial online] 2022 [cited 2023 Jun 9];36:94-100. Available from: https://www.jmedsoc.org/text.asp?2022/36/3/94/370597

Introduction

Smoking is regarded as an environmental risk factor which is associated with different genetic factors and multifactorial disorders.^[1] The mechanism by which smoking induces cardiovascular risk is still unclear to some extent.^[2] It is well established that smoking can increase cardiovascular disease which is adversely related to the duration of cigarette smoke and the number of smoking per day.^[3] Other than active smoking, passive smoking also increases cardiovascular risk by altering the serum lipid levels in young smokers.^[4] The chemical components in cigarette smoke, especially nicotine, can increase the fatty acid level by stimulating the release of adrenaline, leading to an increase in the cardiovascular risk.^[5] Free fatty acid can increase the triglyceride level (TGL) and low-density lipoprotein (LDL) secretion and release cholesterol from the hepatic circulation.^[6] Smoking also produces free radicals which can alter the coagulation system,^[7] and may accelerate the formation of plaque in the arteries.^[8] Smoking also induces inflammation which may lead atherosclerotic plaque formation and plays an important role for the development of cardiovascular disease. Most recently, different studies show that the formation of atherosclerotic plaque and subsequent rupture happens due to the development of chronic inflammation.^[8] Although for the predictor of cardiovascular disease, the markers for general inflammation are related to acute phase reactants and pro-inflammatory cytokines including high-sensitivity C-reactive protein (hsCRP).^[9] Most particularly, the tissue necrosis factor-α and pro-inflammatory cytokines such as interleukin-1 (IL-1) upregulate matrix metalloproteinase-9 (MMP-9) synthesis by mesenchymal stem cells.^[10],[11] Recent studies reveal that smoking may alter the stability of extracellular matrix by altering the balance between the production and degradation of MMP, which may lead to the development of cardiovascular disease.^[12] Matrix metalloproteinase are the families of zinc-containing zymogene endopeptidase that degrade extracellular matrix proteins, play a pivotal role in extracellular matrix for tissue remodeling, and contribute in a multiplicity physiological process.^{[13],[14],[15]} Among the MMP family, MMP-9 is a 92 KDa collagenase enzyme. In the basement membrane, MMP-9 breaks proteoglycan proteins, type 4 collagen, and interstitial proteins.^[16],[17] Till date, it is the most studied enzyme in relation to cardiovascular disease. Different studies show that, at high inflammatory condition, the MMP-9 activity has been increased in the formation of atherosclerotic plaque.^[18],[19] Hence, the increased level of MMP-9, especially in the inflammatory condition caused by smoking, is at a higher risk of future cardiovascular disease. Thus, MMP-9 can be used as a therapeutic target as well as a biomarker for future coronary heart disease (CHD).^[20],[21] Hence, being a part of India in the southern state, the smoking rate in Tamil Nadu is vulnerably increased, which may increase the prevalence of CHD patients in recent times, which may be due to the modification of lifestyle and the increase rate of smoking in early age. Thus, the current study intends to investigate the link between circulating MMP-9 and the risk of CHD in young smokers.

Materials and Methods

Study design and subjects

The present cross-sectional study was conducted at SRM Medical College Hospital and Research Center, SRMIST, Tamil Nadu, India, between October 2019 and September 2021. The study group was divided into three groups. Group 1 includes 60 young active smokers with diabetic CHD, Group 2 includes 60 young active smokers with CHD, and Group 3 includes 60 healthy controls as nonsmokers who were attending SRM Medical College Hospital in Tamil Nadu for cardiology and medicine OP. A standard questionnaire is used during regular cardiovascular health assessments to acquire information about the patient's history and lifestyle characteristics. Patients who smoked on a regular basis and had been diagnosed with CHD met the inclusion criteria. The diagnosis of CHD was based on abnormal coronary angiography with more than 50% stenosis in one or more major arteries. The study excludes the participants with cardiomyopathy, chronic diseases such as liver failure, cancer patients, heart failure, pregnancy, cardiovascular accidents, significant systemic sickness, and systemic inflammatory disease.

Ascertainment of smoking exposure

The Smoking habits were ascertained through self-reporting. Males aged between (20-55 years) who were either non-smoker or at least 5 cigarettes per day for more than one-year duration of smoking.^[22]

Ascertainment of covariates

Self-reporting is used to determine sociodemographic factors such as age, gender, educational history, and other health and medical histories. Standard equipment and technique were used to measure anthropometric characteristics such as weight and height. After 2 min of rest, resting blood pressure (BP) was measured three times with 1-min intervals in the sat posture; the average of the second and third measurements was used for analysis. A systolic BP of more than 140 mmHg and a diastolic BP of more than 90 mmHg were defined as hypertension. Diabetes mellitus was defined as a previous medical diagnosis of diabetes mellitus or meeting diagnostic criteria for diagnosis based on fasting plasma glucose levels of >126 mg/dl, 2-h plasma glucose levels obtained as part of a 75-g oral glucose tolerance test >200, or the glycated hemoglobin test of >6.5%.

Assays

A sandwich enzyme-linked immunosorbent assay (ELISA) was used to determine the concentrations of serum MMP-9, hsCRP, and apolipoprotein-E (ELISA). The AU480 automated analyzer was used to assess fasting serum glucose levels, total cholesterol (TC), TGLs, serum high-density lipoprotein cholesterol (HDL-C) levels, low-density cholesterol levels, and serum very LDL levels.

Ethical consideration

Ethical approval for the study was obtained from the Human Research Ethical Committee of SRM Medical College Hospital and Research Center, SRMIST (approval number IEC No: 1763). All study participants provided informed consent.

Statistical analysis

The statistical analysis was performed using IBM Corp.'s Statistical Package for the Social Sciences (SPSS), version 22 (Armonk, NY, USA). The quantitative variables are expressed as mean and standard deviation (SD). Data were examined using one-way analysis of variance to compare the differences between the three groups. Differences were considered to be highly significant, significant, or nonsignificant for P < 0.001, P < 0.05, or P > 0.05, respectively. The associations between variables were determined using Pearson's correlation coefficient (r).

Results

Baseline and biochemical characteristics of the study groups

[Table 1] and [Table 2] show the demographic and baseline characteristic data of all the three groups. The subjects' weight, body mass index (BMI), waist-to-hip (W/H) ratio, BP, number of cigarettes per day (smoking intensity), and smoking duration all showed a significant difference (smoking burden). When compared to controls, smokers in Groups 1 and 2 had substantially higher fasting blood glucose (FBG) and lipid levels. The study group was not receiving any kind of a lipid-lowering treatment.

Table 1: Anthropometric measurements of smokers with coronary heart disease and normal controls

Click here to view

Table 2: Biochemical characteristics of Group 1, Group 2, and normal controls (nonsmokers)

Click here to view

The serum MMP-9, hsCRP, and apolipoprotein E (APO-E) levels were represented as mean ± SD in [Table 2]. The result from the study revealed a gradual increase in serum MMP-9, hsCRP, and APO-E levels in smokers with CHD followed by the diabetic CHD subjects when compared to controls (P < 0.0001), indicating that smokers induce inflammation.

Correlation analysis

As shown in [Table 3], the study shows Pearson's correlation between serum MMP-9, hsCRP, and APO-E in diabetic CHD subjects. We found a positive correlation between MMP-9 and hsCRP (r = 0.3776), APO-E (r = 0.4614), BMI (r = 0.4673), FBG (r = 0.3978), TC (r = 0.4858), TGL (r = 0.3917), LDL (r = 0.4689), TC/HDL (r = 0.3225), number of smoking/day (r = 0.4287), duration of smoking (r = 0.3638), and a negative correlation was found with HDL-C levels.

Table 3: Correlation of all different parameters in smokers with diabetic coronary heart disease subjects

Click here to view

As shown in [Table 4], the study also shows Pearson's correlation between serum MMP-9, hsCRP, and APO-E in nondiabetic CHD subjects. We also found a positive correlation between MMP-9 and hsCRP (r = 0.3776), APO-E (r = 0.4039), BMI (r = 0.734), W/H ratio (r = 0.4089), FBG (r = 0.3105), TC (r = 0.3204), TGL (r = 0.3881), LDL-C (r = 0.5003), TC/HDL-C (r = 0.4071), LDL-C/HDL-C (r = 0.4926), number of smoking/day (r = 0.3411), and duration of smoking (r = 0.3175).

Table 4: Correlation of all different parameters in smokers with nondiabetic coronary heart disease subjects

Click here to view

Along with this, we also performed a linear regression analysis to see whether the relationship between MMP-9 with hsCRP, APO-E, and smoking intensity (number of smoking per day) was affected by smoking status [Figure 1].

Figure 1: Linear regression analysis of MMP-9 levels with other biochemical parameters in smokers with diabetic CHD subjects, MMP-9: Matrix metalloproteinase-9, CHD: Coronary heart disease. (a) MMP-9 with APO-E, (b) MMP-9 with hsCRP, (c) MMP-9 with BMI, (d) MMP-9 with HDL, (e) MMP-9 with LDL, (f) MMP-9 with smoking/day

Click here to view

Discussion

To our information, this report is the first-ever report on MMP-9 with respect to young smokers. Smoking habit continuously elevates the risk of cardiovascular disease and peripheral vascular disease.^[23] Modifiable risk factor such as high BP or high cholesterol level does not explain clearly the relation between cigarette smoke and the occurrence of cardiovascular disease risk.^[24],[25] However, certain studies give evidence that smoking increases the circulating MMP-9 concentration, which increases the risk of cardiovascular disease.^[26]

This current study shows a significant positive correlation between the cardiovascular risk factors, especially smoking, and the inflammatory marker (hsCRP) with MMP-9 in young smokers as it is compared to nonsmokers with a significant P < 0.05. Moreover, different studies show that the elevated levels of MMP-9 were positively associated with smoking status and inflammatory markers (including hsCRP and IL-6).^[27],[28] The study also shows that MMP-9 is significantly correlated with APO-E levels. This may be due to the shedding of the lipoprotein receptor by MMP-9. MMP-9 is an endopeptidase that can bind and proteolysis (i.e., shedding) lipoprotein receptors.

In this study, the serum level of MMP-9 was significantly increased in smokers with CHD (with and without diabetes) than the control group (P < 0.0001). MMP-9 levels were significantly increased in Group 1 when compared to Group 2 with a significant value of (<0.001), which may reflect abnormal extracellular matrix metabolism in the diabetic CHD group.^[29],[30] The study also identifies MMP-9 as a new modulator of cholesterol metabolism. Furthermore, the results suggest that dysregulation of MMP-9 activity can alter the hepatic transcriptional responses to dietary cholesterol, resulting in metabolic disorders that could lead to atherosclerosis and CHD.^[31]

Smoking accelerates inflammation and oxidative modification of lipids and prospectively slows down the matrix metalloproteinase activity at a various different level. By activating inflammatory transcription factors, smoking increases MMP expression.^[12] Along with this, smoking also elevates the monocyte expression of IL-beta cells.^[32] Cigarette smoke contains nicotine and predominant metabolite cotinine increases the production of vascular smooth muscle cell collagenase and gelatinase may lead to plaque rupture.^[33]

Different mechanisms have been anticipated to explain by which smoking induces the stimulation of MMP, both in vitro and in vivo.^[34],[35] In vitro tobacco smoke induces MMP-9 expression through the endothelial cell.^[36] Similarly, exposure of smoking induces MMP-1 expression through human fibroblast. While tobacco smoke also induces proteolysis by inhaling the cadmium present in smoke,^[13] which may lead to cardiovascular disease as it increases in the aorta of smokers.^[37]

The major limitation of this study is the limited number of sample size. Other than this, we did not see the genetic polymorphisms of the complete set of data which may be able to explain in some more extent in the distribution of the variance of MMP-9 in young smokers. Nevertheless, we need further study on smoking which may reflect the MMP-9 concentration in young smokers. It is very important to analyze the activity of endogenous tissue inhibitors of metalloproteinase 1 (TIMP-1), because it creates a balance between the MMP-9 and TIMP-1.^{[38],[39],[40]} However, our study did not analyze the TIMP-1 concentration.

Hence, our finding on MMP-9 in young smokers gives evidence that cigarette smoking accelerates the circulating MMP-9 levels in young age.

Conclusion

The current investigation revealed a substantial link between serum MMP-9 and the risk of CHD among young smokers. According to the findings, an increase in MMP-9 levels, particularly in inflammatory conditions produced by smoking, is associated with a greater risk of future cardiovascular disease

Acknowledgment

The authors acknowledge the Department of Medicine, Cardiology and Master Health Check-up Unit for the permitting and supporting.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

References

1.	Zeng J, Luo S, Huang Y, Lu Q. Critical role of environmental factors in the pathogenesis of psoriasis. J Dermatol 2017;44:863-72.
2.	Páramo JA, Beloqui O, Rodríguez JA, Diez J, Orbe J. Association between matrix metalloproteinase-10 concentration and smoking in individuals without cardiovascular disease. Rev Esp Cardiol 2008;61:1267-73.
3.	Wilhelmsen L. Coronary heart disease: Epidemiology of smoking and intervention studies of smoking. Am Heart J 1988;115:242-9.
4.	Yano K, Reed DM, McGee DL. Ten-year incidence of coronary heart disease in the Honolulu Heart Program. Relationship to biologic and lifestyle characteristics. Am J Epidemiol 1984;119:653-66.
5.	Shepherd J, Packard CJ, Patsch JR, Gotto AM Jr., Taunton OD. Effects of dietary polyunsaturated and saturated fat on the properties of high density lipoproteins and the metabolism of apolipoprotein A-I. J Clin Invest 1978;61:1582-92.
6.	Bonanome A, Pagnan A, Biffanti S, Opportuno A, Sorgato F, Dorella M, et al. Effect of dietary monounsaturated and polyunsaturated fatty acids on the susceptibility of plasma low density lipoproteins to oxidative modification. Arterioscler Thromb 1992;12:529-33.
7.	Sreekanth KS, Sabu MC, Varghese L, Manesh C, Kuttan G, Kuttan R. Antioxidant activity of smoke shield in-vitro and in-vivo. J Pharm Pharmacol 2003;55:847-53.
8.	Hansson GK. Inflammation, atherosclerosis, and coronary artery disease. N Engl J Med 2005;352:1685-95.
9.	Welsh P, Whincup PH, Papacosta O, Wannamethee SG, Lennon L, Thomson A, et al. Serum matrix metalloproteinase-9 and coronary heart disease: A prospective study in middle-aged men. QJM 2008;101:785-91.
10.	Ridker PM, Hennekens CH, Buring JE, Rifai N. C-reactive protein and other markers of inflammation in the prediction of cardiovascular disease in women. N Engl J Med 2000;342:836-43.
11.	Ridker PM, Glynn RJ, Hennekens CH. C-reactive protein adds to the predictive value of total and HDL cholesterol in determining risk of first myocardial infarction. Circulation 1998;97:2007-11.
12.	Perlstein TS, Lee RT. Smoking, metalloproteinases, and vascular disease. Arterioscler Thromb Vasc Biol 2006;26:250-6.
13.	Lagente V, Boichot E. Role of matrix metalloproteinases in the inflammatory process of respiratory diseases. J Mol Cell Cardiol 2010;48:440-4.
14.	Busti C, Falcinelli E, Momi S, Gresele P. Matrix metalloproteinases and peripheral arterial disease. Intern Emerg Med 2010;5:13-25.
15.	Rydlova M, Holubec L Jr., Ludvikova M Jr., Kalfert D, Franekova J, Povysil C, et al. Biological activity and clinical implications of the matrix metalloproteinases. Anticancer Res 2008;28:1389-97.
16.	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;37:375-536.
17.	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;69:851-9.
18.	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;94:2493-503.
19.	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;31:40-7.
20.	Ramos-Fernandez M, Bellolio MF, Stead LG. Matrix metalloproteinase-9 as a marker for acute ischemic stroke: A systematic review. J Stroke Cerebrovasc Dis 2011;20:47-54.
21.	Muroski ME, Roycik MD, Newcomer RG, Van den Steen PE, Opdenakker G, Monroe HR, et al. Matrix metalloproteinase-9/gelatinase B is a putative therapeutic target of chronic obstructive pulmonary disease and multiple sclerosis. Curr Pharm Biotechnol 2008;9:34-46.
22.	Morris P. Research Protocol a Randomized, Controlled, Crossover Study Comparing Exercise Capacity in Healthy Adult Male Smokers Smoking Electrically Heated Cigarettes, Conventional Cigarettes, and No-Smoking. Bates No: 2067327075/2067327092. A Report of the Surgeon General; 2002.
23.	Barquera S, Pedroza-Tobías A, Medina C, Hernández-Barrera L, Bibbins-Domingo K, Lozano R, et al. Global Overview of the Epidemiology of Atherosclerotic Cardiovascular Disease. Archives of Medical Research 2015:46:328-38.
24.	Khot UN, Khot MB, Bajzer CT, Sapp SK, Ohman EM, Brener SJ, et al. Prevalence of conventional risk factors in patients with coronary heart disease. JAMA 2003;290:898-904.
25.	Yusuf S, Hawken S, Ounpuu S, Dans T, Avezum A, Lanas F, et al. Effect of potentially modifiable risk factors associated with myocardial infarction in 52 countries (the INTERHEART study): Case-control study. Lancet 2004;364:937-52.
26.	Liu PY, Chen JH, Li YH, Wu HL, Shi GY. Synergistic effect of stromelysin-1 (matrix metallo-proteinase-3) promoter 5A/6A polymorphism with smoking on the onset of young acute myocardial infarction. Thromb Haemost 2003;90:132-9.
27.	Blankenberg S, Rupprecht HJ, Poirier O, Bickel C, Smieja M, Hafner G, et al. Plasma concentrations and genetic variation of matrix metalloproteinase 9 and prognosis of patients with cardiovascular disease. Circulation 2003;107:1579-85.
28.	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;35:537-45.
29.	Signorelli SS, Malaponte G, Libra M, Di Pino L, Celotta G, Bevelacqua V, et al. Plasma levels and zymographic activities of matrix metalloproteinases 2 and 9 in type II diabetics with peripheral arterial disease. Vasc Med 2005;10:1-6.
30.	Derosa G, D'Angelo A, Tinelli C, Devangelio E, Consoli A, Miccoli R, et al. Evaluation of metalloproteinase 2 and 9 levels and their inhibitors in diabetic and healthy subjects. Diabetes Metab 2007;33:129-34.
31.	Hernandez-Anzaldo S, Brglez V, Hemmeryckx B, Leung D, Filep JG, Vance JE, et al. Novel role for Matrix metalloproteinase 9 in modulation of Cholesterol metabolism. J Am Heart Assoc 2016;5:e004228.
32.	Ryder MI, Saghizadeh M, Ding Y, Nguyen N, Soskolne A. Effects of tobacco smoke on the secretion of interleukin-1beta, tumor necrosis factor-alpha, and transforming growth factor-beta from peripheral blood mononuclear cells. Oral Microbiol Immunol 2002;17:331-6.
33.	Carty CS, Soloway PD, Kayastha S, Bauer J, Marsan B, Ricotta JJ, et al. Nicotine and cotinine stimulate secretion of basic fibroblast growth factor and affect expression of matrix metalloproteinases in cultured human smooth muscle cells. J Vasc Surg 1996;24:927-34.
34.	Morimoto Y, Tsuda T, Nakamura H, Hori H, Yamato H, Nagata N, et al. Expression of matrix metalloproteinases, tissue inhibitors of metalloproteinases, and extracellular matrix mRNA following exposure to mineral fibers and cigarette smoke in vivo. Environ Health Perspect 1997;105 Suppl 5:1247-51.
35.	Nakamura T, Ebihara I, Shimada N, Koide H. Effect of cigarette smoking on plasma metalloproteinase-9 concentration. Clin Chim Acta 1998;276:173-7.
36.	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;292:L125-33.
37.	Abu-Hayyeh S, Sian M, Jones KG, Manuel A, Powell JT. Cadmium accumulation in aortas of smokers. Arterioscler Thromb Vasc Biol 2001;21:863-7.
38.	Vu TH, Werb Z. Matrix metalloproteinases: Effectors of development and normal physiology. Genes Dev 2000;14:2123-33.
39.	Brew K, Dinakarpandian D, Nagase H. Tissue inhibitors of metalloproteinases: Evolution, structure and function. Biochim Biophys Acta 2000;1477:267-83.
40.	Hansson J, Vasan RS, Ärnlöv J, Ingelsson E, Lind L, Larsson A, et al. Biomarkers of extracellular matrix metabolism (MMP-9 and TIMP-1) and risk of stroke, myocardial infarction, and cause-specific mortality: Cohort study. PLoS One 2011;6:e16185.