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Associations of matrix metalloproteinase-9 and monocyte chemoattractant protein-1 concentrations with carotid atherosclerosis, based on measurements of plaque and intima–media thickness

  • Author Footnotes
    1 Changhong Tan and Yi Liu contributed equally to this study and should be considered as co-first authors.
    Changhong Tan
    Footnotes
    1 Changhong Tan and Yi Liu contributed equally to this study and should be considered as co-first authors.
    Affiliations
    Department of Neurology, the Second Affiliated Hospital of Chong Qing Medical University, Number 76, LinJiang Road, YuZhong District, 400010 Chong Qing, China
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  • Author Footnotes
    1 Changhong Tan and Yi Liu contributed equally to this study and should be considered as co-first authors.
    Yi Liu
    Footnotes
    1 Changhong Tan and Yi Liu contributed equally to this study and should be considered as co-first authors.
    Affiliations
    Department of Neurology, the Second Affiliated Hospital of Chong Qing Medical University, Number 76, LinJiang Road, YuZhong District, 400010 Chong Qing, China
    Search for articles by this author
  • Weina Li
    Affiliations
    Department of Neurology, the Second Affiliated Hospital of Chong Qing Medical University, Number 76, LinJiang Road, YuZhong District, 400010 Chong Qing, China
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  • Fen Deng
    Affiliations
    Department of Neurology, the Second Affiliated Hospital of Chong Qing Medical University, Number 76, LinJiang Road, YuZhong District, 400010 Chong Qing, China
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  • Xi Liu
    Affiliations
    Department of Neurology, the Second Affiliated Hospital of Chong Qing Medical University, Number 76, LinJiang Road, YuZhong District, 400010 Chong Qing, China
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  • Xin Wang
    Affiliations
    Department of Neurology, the Second Affiliated Hospital of Chong Qing Medical University, Number 76, LinJiang Road, YuZhong District, 400010 Chong Qing, China
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  • Yuejiang Gui
    Affiliations
    Department of Neurology, the Second Affiliated Hospital of Chong Qing Medical University, Number 76, LinJiang Road, YuZhong District, 400010 Chong Qing, China
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  • Lu Qin
    Affiliations
    Department of Neurology, the Second Affiliated Hospital of Chong Qing Medical University, Number 76, LinJiang Road, YuZhong District, 400010 Chong Qing, China
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  • Changlin Hu
    Affiliations
    Department of Neurology, the Second Affiliated Hospital of Chong Qing Medical University, Number 76, LinJiang Road, YuZhong District, 400010 Chong Qing, China
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  • Lifen Chen
    Correspondence
    Corresponding author. Tel.: +86 23 63693088.
    Affiliations
    Department of Neurology, the Second Affiliated Hospital of Chong Qing Medical University, Number 76, LinJiang Road, YuZhong District, 400010 Chong Qing, China
    Search for articles by this author
  • Author Footnotes
    1 Changhong Tan and Yi Liu contributed equally to this study and should be considered as co-first authors.

      Highlights

      • MMP-9 was independently associated with high total carotid artery plaque score.
      • MMP-9 was independently associated with plaque instability.
      • MMP-9 was independently associated with large intima–media thickness (IMT) value.
      • MCP-1 was independently associated with IMT, but not with plaque morphology.
      • High serum uric acid level was independently associated with plaque instability.

      Abstract

      Purpose

      To examine associations of matrix metalloproteinase-9 (MMP-9) and monocyte chemoattractant protein-1 (MCP-1) concentrations with the severity of carotid atherosclerosis, based on measurements of carotid plaque and intima–media thickness (IMT).

      Methods

      This cross-sectional study included 116 stroke-free participants (45.7% males, 54.3% females; mean age, 64.73 ± 14.53 years). Serum MMP-9 and MCP-1 concentrations were measured, and plaque morphology, including total plaque score (PS), plaque stability, and IMT, was assessed ultrasonographically. Participants were grouped according to total PS (0, 1–2, ≥3), plaque stability (no plaque, stable, unstable) and IMT tertiles (<0.8 mm, 0.8–1 mm, >1 mm). Multinomial logistic regression models were used to assess the associations of MMP-9 and MCP-1 concentrations with plaque and IMT values after adjusting for vascular risk factors.

      Results

      MMP-9 quartiles (vs. quartile 1) were significantly associated with a greater prevalence of plaque instability [Q2: odds ratio (OR) = 5.13, 95% confidence interval (CI) = 1.01–24.9, p = 0.042; Q3: OR = 15.5, 95% CI = 3.1–78.1, p = 0.001; Q4: OR = 13.2, 95% CI = 2.7–64.97, p = 0.001] and high total PS (Q3: OR = 10.02, 95% CI = 1.5–65.33, p = 0.016; Q4: OR = 21.5, 95% CI = 3.5–132.1, p = 0.001). MCP-1 concentration was significantly associated with IMT (OR = 22.94, 95% CI = 2.14–245.66, p = 0.01).

      Conclusions

      Elevated serum MMP-9 concentration was independently associated with high total carotid artery PS, plaque instability, and large IMT value. MCP-1 concentration was independently associated with IMT, but not with plaque morphology.

      Keywords

      1. Introduction

      Carotid atherosclerosis, detected noninvasively by high-resolution ultrasound imaging, has been used as a marker for the development of cardiovascular and cerebrovascular diseases. The severity of carotid atherosclerosis can be assessed using intima–media thickness (IMT), total plaque area (TPA), and plaque stability. Previous studies have shown that increased IMT [
      • Chambless L.E.
      • Folsom A.R.
      • Clegg L.X.
      • et al.
      Carotid wall thickness is predictive of incident clinical stroke: the Atherosclerosis Risk in Communities (ARIC) study.
      ,
      • Iglesias del Sol A.
      • Bots M.L.
      • Grobbee D.E.
      • Hofman A.
      • Witteman J.C.
      Carotid intima-media thickness at different sites: relation to incident myocardial infarction; the Rotterdam Study.
      ,
      • Hollander M.
      • Hak A.E.
      • Koudstaal P.J.
      • et al.
      Comparison between measures of atherosclerosis and risk of stroke: the Rotterdam Study.
      ,
      • Kitamura A.
      • Iso H.
      • Imano H.
      • et al.
      Carotid intima-media thickness and plaque characteristics as a risk factor for stroke in Japanese elderly men.
      ,
      • Lorenz M.W.
      • von Kegler S.
      • Steinmetz H.
      • Markus H.S.
      • Sitzer M.
      Carotid intima-media thickening indicates a higher vascular risk across a wide age range: prospective data from the Carotid Atherosclerosis Progression Study (CAPS).
      ,
      • Cao J.J.
      • Arnold A.M.
      • Manolio T.A.
      • et al.
      Association of carotid artery intima-media thickness, plaques, and C-reactive protein with future cardiovascular disease and all-cause mortality: the Cardiovascular Health Study.
      ] and plaque instability [
      • Cao J.J.
      • Arnold A.M.
      • Manolio T.A.
      • et al.
      Association of carotid artery intima-media thickness, plaques, and C-reactive protein with future cardiovascular disease and all-cause mortality: the Cardiovascular Health Study.
      ] predict future vascular events independently of conventional vascular risk factors. The results of a large-scale 10-year follow-up study suggested that TPA is a stronger predictor than IMT of first ischemic stroke [
      • Mathiesen E.B.
      • Johnsen S.H.
      • Wilsgaard T.
      • Bønaa K.H.
      • Løchen M.L.
      • Njølstad I.
      Carotid plaque area and intima-media thickness in prediction of first-ever ischemic stroke: a 10-year follow-up of 6584 men and women: the Tromsø Study.
      ].
      Atherosclerosis is a chronic inflammatory process in the arterial wall involving matrix metalloproteinases (MMPs) and monocyte chemotactic protein-1 (MCP-1). It is characterized by extracellular matrix (ECM) remodeling, a complex process in which MMPs play an important role [
      • Sapienza P.
      • di Marzo L.
      • Borrelli V.
      • et al.
      Metalloproteinases and their inhibitors are markers of plaque instability.
      ,
      • Johnson J.L.
      • Jackson C.L.
      • Angelini G.D.
      • et al.
      Activation of matrix-degrading metalloproteinases by mast cell proteases in atherosclerotic plaques.
      ,
      • Loftus I.M.
      • Goodall S.
      • Crowther M.
      • et al.
      Increased MMP9 activity in acute carotid plaques: therapeutic avenues to prevent stroke.
      ,
      • Ghilardi G.
      • Biondi M.L.
      • DeMonti M.
      • et al.
      Matrix metalloproteinase-1 and matrix metalloproteinase-3 gene promoter polymorphisms are associated with carotid artery stenosis.
      ,
      • Alvarez B.
      • Ruiz C.
      • Chacon P.
      • et al.
      Serum values of metalloproteinase-2 and metalloproteinase-9 as related to unstable plaque and inflammatory cells in patients with greater than 70% carotid artery stenosis.
      ,
      • Formato M.
      • Farina M.
      • Spirito R.
      • et al.
      Evidence for a proinflammatory and proteolytic environment in plaques from endarterectomy segments of human carotid arteries.
      ]. Increased MMP-9 expression has been found in carotid atherosclerotic plaques rendered unstable by carotid endarterectomy [
      • Sluijter J.P.
      • Pulskens W.P.
      • Schoneveld A.H.
      • 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.
      ,
      • Stintzing S.
      • Heuschmann P.
      • Barbera L.
      • et al.
      Overexpression of MMP9 and tissue factor in unstable carotid plaques associated with Chlamydia pneumoniae, inflammation, and apoptosis.
      ,
      • Heo S.H.
      • Cho C.H.
      • Kim H.O.
      • et al.
      Plaque rupture is a determinant of vascular events in carotid artery atherosclerotic disease: involvement of matrix metalloproteinases 2 and 9.
      ]. However, total blood MMP-9 levels have been reported variously to be positively [
      • Romero J.R.
      • Vasan R.S.
      • Beiser A.S.
      • et al.
      Association of carotid artery atherosclerosis with circulating biomarkers of extracellular matrix remodeling: the Framingham Offspring Study.
      ] or negatively [
      • Olson F.J.
      • Schmidt C.
      • Gummesson A.
      • et al.
      Circulating matrix metalloproteinase9 levels in relation to sampling methods, femoral and carotid atherosclerosis.
      ,
      • Lien L.M.
      • Hsieh Y.C.
      • Bai C.H.
      • et al.
      Association of blood active matrix metalloproteinase-3 with carotid plaque score from a community population in Taiwan.
      ] correlated with carotid plaque stability or IMT. The initiation and progression of atherosclerosis depend on the recruitment of monocytes to sites of active inflammation; for example, MCP-1 initiates the development of atheroma. Elevated serum MCP-1 concentrations have been documented in patients with cardio-cerebrovascular disease [
      • de Lemos J.A.
      • Morrow D.A.
      • Sanatine M.S.
      • et al.
      Association between plasma levels of monocyte chemoattractant protein-1 and long-term clinical outcomes in patients with acute coronary syndromes.
      ,
      • Arakelyan A.
      • Petrkova J.
      • Hermanova Z.
      • Boyajyan A.
      • Lukl J.
      • Petrek M.
      Serum levels of the MCP-1 chemokine in patients with ischemic stroke and myocardial infarction.
      ], especially in those undergoing hemodialysis [
      • Uchida E.
      • Anan F.
      • Masaki T.
      • et al.
      Monocyte chemoattractant protein-1 is associated with silent cerebral infarction in patients on haemodialysis.
      ]. However, a large-scale study failed to prove that MCP-1 level was an independent predictor of ischemic stroke and cardiovascular disease [
      • Canouï-Poitrine F.
      • Luc G.
      • Mallat Z.
      • et al.
      PRIME Study Group
      Systemic chemokine levels, coronary heart disease, and ischemic stroke events: the PRIME study.
      ]. To our knowledge, the relationship between MCP-1 and the severity of carotid atherosclerosis has not been reported. For this reason, and given the conflicting results regarding the relationship between MMP-9 and carotid atherosclerosis severity, we examined blood concentrations of MMP-9 and MCP-1 in relation to the severity of carotid atherosclerosis, assessed using the carotid plaque score (PS), IMT, and plaque stability.

      2. Methods

      2.1 Study participants

      Study participants were recruited from among consecutive patients aged ≥30 years who were referred for carotid ultrasound examination at the Neurology Department of the Second Affiliated Hospital of Chongqing Medical University (China) in 2011–2012. Exclusion criteria were as follows: 1) histories of stroke, 2) histories of transient ischemic attack, 3) recent acute coronary syndrome, 4) cancer, and 5) inflammatory disease (e.g., rheumatoid arthritis and gingivitis). The institutional ethics committee approved this study and all participants provided informed consent.

      2.2 Baseline clinical data

      All participants underwent baseline clinical examination, including medical history documentation, physical examination, laboratory testing, and carotid ultrasound examination. Collected data included patients' age and blood pressure; histories of cigarette and alcohol use, hypertension, and/or diabetes mellitus; and measurement of total cholesterol, triglyceride, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, uric acid (UA), and C-reactive protein (CRP) levels in fasting blood samples. Blood samples were promptly centrifuged at 3000× g for 10 min at 4 °C. Aliquots of serum were stored in a central laboratory at −80 °C.
      Hypertension was defined as the use of anti-hypertensive medication, average systolic blood pressure ≥140 mmHg, or average diastolic blood pressure ≥90 mmHg. Diabetes was defined as the use of anti-diabetic medication or fasting glucose level ≥7.0 mmol/L. Smokers were classified on the basis of a history of smoking >100 cigarettes, using the simplified definition of the United States Centers for Disease Control. Alcohol use was defined as alcohol intake ≥100 g/d for at least 5 years.

      2.3 Carotid ultrasound examination

      The carotid arteries were evaluated with high-resolution B-mode ultrasonography (model iE33; Philips Medical Systems, Eindhoven, The Netherlands). A reader blinded to all clinical information determined measurements at a central reading facility. TPA was indirectly represented using a previously reported PS method [
      • Lien L.M.
      • Hsieh Y.C.
      • Bai C.H.
      • et al.
      Association of blood active matrix metalloproteinase-3 with carotid plaque score from a community population in Taiwan.
      ,
      • Sutton-Tyrrell K.
      • Wolfson Jr., S.K.
      • Thompson T.
      • Kelsey S.F.
      Measurement variability in duplex scan assessment of carotid atherosclerosis.
      ,
      • Crouse J.R.
      • Harpold G.H.
      • Kahl F.R.
      • Toole J.F.
      • McKinney W.M.
      Evaluation of a scoring system for extracranial carotid atherosclerosis extent with B-mode ultrasound.
      ]. In brief, longitudinal images of the bilateral carotid arteries, including the proximal (>10 mm proximal to bulb bifurcation) and distal common carotid artery, bulb, and internal and external carotid arteries (10 segments), were acquired. The presence of plaque was scored according to the encroachment of localized echoic structures into the arterial lumen, representing at least 50% of the surrounding IMT value [
      • Touboul P.J.
      • Hennerici M.G.
      • Meairs S.
      • et al.
      Mannheim carotid intima–media thickness consensus (2004–2006). An update on behalf of the Advisory Board of the 3rd and 4thwatching the risk symposium, 13th and 15th European stroke conferences.
      ]. PSs were assigned as follows: 0, normal/no plaque; 1, one small plaque (<30% stenosis); 2, one medium plaque (30–49% stenosis) or multiple small plaques; 3, one large plaque (50–99% stenosis) or multiple plaques with more than one medium plaque; and 4, 100% occlusion [
      • Lien L.M.
      • Hsieh Y.C.
      • Bai C.H.
      • et al.
      Association of blood active matrix metalloproteinase-3 with carotid plaque score from a community population in Taiwan.
      ,
      • Sutton-Tyrrell K.
      • Wolfson Jr., S.K.
      • Thompson T.
      • Kelsey S.F.
      Measurement variability in duplex scan assessment of carotid atherosclerosis.
      ,
      • Crouse J.R.
      • Harpold G.H.
      • Kahl F.R.
      • Toole J.F.
      • McKinney W.M.
      Evaluation of a scoring system for extracranial carotid atherosclerosis extent with B-mode ultrasound.
      ]. PSs for the 10 segments were summed to obtain a total PS for each patient. The study participants were divided into three groups based on total PS (0, 1–2, ≥3) [
      • Lien L.M.
      • Hsieh Y.C.
      • Bai C.H.
      • et al.
      Association of blood active matrix metalloproteinase-3 with carotid plaque score from a community population in Taiwan.
      ].
      Carotid IMT was measured in three segments: the distal common carotid artery (1 cm proximal to the carotid bulb), the carotid artery bifurcation (1 cm proximal to the flow divider), and the proximal internal carotid artery (1 cm length). The mean of all IMT measurements from the right and left sides was calculated for each patient [
      • Nambi V.
      • Chambless L.
      • Folsom A.R.
      • et al.
      Carotid intima-media thickness and presence or absence of plaque improves prediction of coronary heart disease risk: the ARIC (Atherosclerosis Risk in Communities) study.
      ]. The participants were divided into three groups based on IMT tertiles (T1, <0.8 mm; T2, 0.8–1 mm; T3, >1 mm).
      Plaque stability was classified as absent, stable, or unstable according to surface characteristics, echogenicity, and texture, as reported previously [
      • Cao J.J.
      • Arnold A.M.
      • Manolio T.A.
      • et al.
      Association of carotid artery intima-media thickness, plaques, and C-reactive protein with future cardiovascular disease and all-cause mortality: the Cardiovascular Health Study.
      ]. The absence of plaque was characterized by a smooth intimal surface with no focal thickening. Unstable plaque was defined as the presence of a markedly irregular or ulcerated surface or hypodense or heterogeneous plaques occupying >50% of the TPA. Stable plaque was defined as the presence of isoechoic, hyperdense, calcified, or homogeneous plaques or a slightly irregular surface. When more than one type of plaque was detected in an individual, plaque risk was determined using the more severe type.

      2.4 Enzyme-linked immunosorbent assay

      A researcher blinded to the clinical data measured serum MMP-9 and MCP-1 levels using commercially available enzyme-linked immunosorbent assay kits (4A Biotech Co., Beijing, China). Samples were handled in an identical and blinded fashion throughout the study. They were analyzed in duplicate and in random order to reduce systemic bias and interassay variation. Mean intra-assay coefficients of variation for the method were 9.5% for MMP-9 and 8.6% for MCP-1. The mean inter-assay coefficients of variation for the method were 11.3% for MMP-9 and 9.1% for MCP-1. MMP-9 and MCP-1 concentrations were examined as continuous variables and in quartiles.

      2.5 Statistical analyses

      Continuous data, expressed as means ± standard deviations or medians and interquartile ranges, were analyzed using a general linear model or a nonparametric test. Categorical data, expressed as numbers and percentages, were compared using the chi-squared test. Multinomial logistic regression models with no plaque as the reference were constructed to examine associations between MMP-9 (as a continuous variable and in quartiles) and plaque phenotypes after adjusting for age, UA level, and vascular risk factors (diabetes mellitus, hypertension, HDL and LDL cholesterol levels, smoking history, and alcohol use). All analyses were performed with SPSS software (ver. 11.5 for Windows; SPSS Inc., Chicago, IL, USA). Two-tailed p values <0.05 were considered to indicate statistical significance.

      3. Results

      3.1 Cohort characteristics

      The study sample comprised 116 participants (53 men, 63 women) with a mean age of 64 ± 14.5 years. Baseline cohort characteristics according to total PS, plaque stability, and IMT tertile are shown in Table 1, Table 2, Table 3. Total PS and plaque stability were significantly affected by age (p = 0.008 and p = 0.003, respectively) and hypertension (p = 0.001 and p = 0.044, respectively; Table 1, Table 2). IMT was significantly affected by hypertension (p = 0.009) and LDL cholesterol (p = 0.014) and MCP-1 (p = 0.049) levels (Table 3). In general linear models, UA was significantly associated with plaque instability (p = 0.013) and large IMT value (p = 0.028). The association of UA with plaque instability persisted after adjustment for age, hypertension, diabetes mellitus, and LDL cholesterol level [odds ratio (OR) = 8.36, 95% confidence interval (CI) = 1.46–47.90, p = 0.017], but that with IMT did not (OR = 1.01, 95% CI = 1.00–1.02, p = 0.052). We found no relationship between CRP level and the severity of carotid atherosclerosis.
      Table 1Characteristics of study participants according to total carotid artery plaque score.
      VariableTotal plaque scorep
      0 n = 431–2 n = 37≥3 n = 36
      Age (years)
      Mean ± standard deviation.
      60 ± 8.765 ± 16.770 ± 160.008
      Males (%)39.551.447.20.558
      Alcohol use (%)4.716.213.90.211
      Hypertension (%)34.943.2750.001
      Diabetes mellitus (%)18.624.333.30.32
      Cigarette smoking (%)1429.722.20.23
      TC (mg/dL)
      Mean ± standard deviation.
      189.9 ± 35.5197.8 ± 47.3201.1 ± 45.40.488
      LDL cholesterol (mg/dL)
      Mean ± standard deviation.
      105.3 ± 31.6112.0 ± 32.7119.9 ± 39.20.179
      HDL cholesterol (mg/dL)
      Median (interquartile range). TC: total cholesterol; LDL: low-density lipoprotein; HDL: high-density lipoprotein; TG: triglycerides; CRP: C-reactive protein; MMP: matrix metalloproteinase; MCP-1: monocyte chemoattractant protein-1.
      48 (39–60)45 (39–53)44 (38–52)0.397
      TG (mg/dL)
      Median (interquartile range). TC: total cholesterol; LDL: low-density lipoprotein; HDL: high-density lipoprotein; TG: triglycerides; CRP: C-reactive protein; MMP: matrix metalloproteinase; MCP-1: monocyte chemoattractant protein-1.
      114 (72–188)147 (84–186)136 (90–179)0.539
      Uric acid (umol/L)
      Mean ± standard deviation.
      266 ± 76.7289 ± 104.9329 ± 152.60.053
      CRP (mg/L)
      Median (interquartile range). TC: total cholesterol; LDL: low-density lipoprotein; HDL: high-density lipoprotein; TG: triglycerides; CRP: C-reactive protein; MMP: matrix metalloproteinase; MCP-1: monocyte chemoattractant protein-1.
      1.6 (0.8–3.6)1.1 (0.5–3.8)2 (0.9–8.3)0.289
      MMP-9 (ng/mL)
      Median (interquartile range). TC: total cholesterol; LDL: low-density lipoprotein; HDL: high-density lipoprotein; TG: triglycerides; CRP: C-reactive protein; MMP: matrix metalloproteinase; MCP-1: monocyte chemoattractant protein-1.
      244 (78–440)479 (308–680)762 (427–1232)<0.001
      MCP-1 (pg/mL)
      Mean ± standard deviation.
      64 ± 3978 ± 5383 ± 820.376
      a Mean ± standard deviation.
      b Median (interquartile range). TC: total cholesterol; LDL: low-density lipoprotein; HDL: high-density lipoprotein; TG: triglycerides; CRP: C-reactive protein; MMP: matrix metalloproteinase; MCP-1: monocyte chemoattractant protein-1.
      Table 2Characteristics of study participants according to plaque stability.
      VariablePlaque stabilityp
      No plaque n = 43Stable n = 32Unstable n = 41
      Age (years)
      Mean ± standard deviation.
      60 ± 8.771 ± 12.464 ± 18.60.003
      Males (%)39.543.853.70.432
      Alcohol use (%)4.712.517.10.184
      Hypertension (%)34.959.458.50.044
      Diabetes mellitus (%)18.634.424.40.294
      Cigarette smoking (%)1431.3220.204
      TC (mg/dL)
      Mean ± standard deviation.
      190 ± 36203 ± 51197 ± 430.437
      LDL cholesterol (mg/dL)
      Mean ± standard deviation.
      105 ± 32113 ± 36118 ± 360.245
      HDL cholesterol (mg/dL)
      Median (interquartile range). TC: total cholesterol; LDL: low-density lipoprotein; HDL: high-density lipoprotein; TG: triglycerides; CRP: C-reactive protein; MMP: matrix metalloproteinase; MCP-1: monocyte chemoattractant protein-1.
      48 (39–60)46 (41–58)41 (38–49)0.145
      TG (mg/dL)
      Median (interquartile range). TC: total cholesterol; LDL: low-density lipoprotein; HDL: high-density lipoprotein; TG: triglycerides; CRP: C-reactive protein; MMP: matrix metalloproteinase; MCP-1: monocyte chemoattractant protein-1.
      115 (72–188)127 (77–168)151 (102–194)0.273
      Uric acid (umol/L)
      Mean ± standard deviation.
      266 ± 76.7275 ± 103.3335 ± 145.40.013
      CRP (mg/L)
      Median (interquartile range). TC: total cholesterol; LDL: low-density lipoprotein; HDL: high-density lipoprotein; TG: triglycerides; CRP: C-reactive protein; MMP: matrix metalloproteinase; MCP-1: monocyte chemoattractant protein-1.
      1.6 (0.8–3.6)1.7 (0.6–5.6)1.8 (0.7–3.3)0.923
      MMP-9 (ng/mL)
      Median (interquartile range). TC: total cholesterol; LDL: low-density lipoprotein; HDL: high-density lipoprotein; TG: triglycerides; CRP: C-reactive protein; MMP: matrix metalloproteinase; MCP-1: monocyte chemoattractant protein-1.
      244 (78–440)449 (316–786)658 (394–1138)<0.001
      MCP-1 (pg/mL)
      Mean ± standard deviation.
      64 ± 3981 ± 5680 ± 780.399
      a Mean ± standard deviation.
      b Median (interquartile range). TC: total cholesterol; LDL: low-density lipoprotein; HDL: high-density lipoprotein; TG: triglycerides; CRP: C-reactive protein; MMP: matrix metalloproteinase; MCP-1: monocyte chemoattractant protein-1.
      Table 3Characteristics of study participants according to tertiles of carotid intima–media thickness.


      Variable
      Carotid IMT tertilep
      T1 n = 43T2 n = 37T3 n = 36
      Age (years)
      Mean ± standard deviation.
      58 ± 12.465 ± 15.068 ± 13.10.058
      Males (%)36.444.6600.293
      Alcohol use (%)4.59.5250.106
      Hypertension (%)36.445.9800.009
      Diabetes mellitus (%)18.224.3350.443
      Cigarette smoking (%)9.121.6350.139
      TC (mg/dL)
      Mean ± standard deviation.
      179 ± 43197 ± 36208 ± 590.078
      LDL cholesterol (mg/dL)
      Mean ± standard deviation.
      97 ± 36112 ± 30128 ± 420.014
      HDL cholesterol (mg/dL)
      Median (interquartile range). IMT: intima–media thickness; TC: total cholesterol; LDL: low-density lipoprotein; HDL: high-density lipoprotein; TG: triglycerides; CRP: C-reactive protein; MMP: matrix metalloproteinase; MCP-1: monocyte chemoattractant protein-1.
      45 (37–63)46 (40–56)42 (33–51)0.153
      TG (mg/dL)
      Median (interquartile range). IMT: intima–media thickness; TC: total cholesterol; LDL: low-density lipoprotein; HDL: high-density lipoprotein; TG: triglycerides; CRP: C-reactive protein; MMP: matrix metalloproteinase; MCP-1: monocyte chemoattractant protein-1.
      76 (59–140)138 (81–191)162 (122–206)0.005
      Uric acid (umol/L)
      Mean ± standard deviation.
      245 ± 88.3294 ± 89.6339 ± 90.80.028
      CRP (mg/L)
      Median (interquartile range). IMT: intima–media thickness; TC: total cholesterol; LDL: low-density lipoprotein; HDL: high-density lipoprotein; TG: triglycerides; CRP: C-reactive protein; MMP: matrix metalloproteinase; MCP-1: monocyte chemoattractant protein-1.
      1.8 (0.7–3.8)1.6 (0.6–3.7)2.3 (1.4–3.3)0.340
      MMP-9 (ng/mL)
      Median (interquartile range). IMT: intima–media thickness; TC: total cholesterol; LDL: low-density lipoprotein; HDL: high-density lipoprotein; TG: triglycerides; CRP: C-reactive protein; MMP: matrix metalloproteinase; MCP-1: monocyte chemoattractant protein-1.
      194 (70–375)456 (261–757)796 (426–1313)<0.001
      MCP-1 (pg/mL)
      Mean ± standard deviation.
      46 ± 2880 ± 6084 ± 760.049
      a Mean ± standard deviation.
      b Median (interquartile range). IMT: intima–media thickness; TC: total cholesterol; LDL: low-density lipoprotein; HDL: high-density lipoprotein; TG: triglycerides; CRP: C-reactive protein; MMP: matrix metalloproteinase; MCP-1: monocyte chemoattractant protein-1.

      3.2 Associations of MMP-9 with plaque and IMT

      When MMP-9 was examined as a continuous variable, clear dose–response relationships were observed between increased MMP-9 level and plaque instability (p = 0.010; Table 2), high total PS (p = 0.000; Table 1), and increased IMT (p = 0.000; Table 3).
      MMP-9 quartiles were ≤201.5 ng/mL (Q1), 201.6–427.9 ng/mL (Q2), 428.0–786.7 ng/mL (Q3), and ≥786.8 ng/mL (Q4). Table 4 shows the relationships between MMP-9 quartile and total PS, IMT, and plaque stability in multivariate adjusted models. MMP-9 quartiles (vs. quartile 1) were significantly associated with a greater prevalence of plaque instability (Q2: OR = 5.13, 95% CI = 1.01–24.9, p = 0.042; Q3: OR = 15.5, 95% CI = 3.1–78.1, p = 0.001; Q4: OR = 13.2, 95% CI = 2.7–64.97, p = 0.001) and high total PS (Q3: OR = 10.02, 95% CI = 1.5–65.33, p = 0.016; Q4: OR = 21.5, 95% CI = 3.5–132.1, p = 0.001). No significant association was observed between MMP-9 Q2 and total PS (OR = 5.15, 95% CI = 0.79–33.3, p = 0.085).
      Table 4Multinomial logistic regression model of the associations of MMP-9 with total plaque score and plaque stability.


      Outcome
      MMP-9 quartile
      Q1Q2Q3Q4
      Or (95% CI)pOr (95% CI)pOr (95% CI)p
      Total plaque score
      1–2 vs. 0Reference2.36 (0.66–8.49)0.1875.86 (1.52–22.55)0.012.04 (0.47–8.79)0.339
      >3 vs. 0Reference5.15 (0.79–33.3)0.08510.02 (1.5–65.33)0.01621.5 (3.5–132.1)0.001
      Plaque stability
      Stable plaque vs. no plaqueReference1.68 (0.4–7.06)0.4762.37 (0.52–10.85)0.2642.34 (0.53–10.33)0.263
      Unstable plaque vs. no plaqueReference5.13 (1.01–24.9)0.04215.5 (3.1–78.1)0.00113.2 (2.7–64.97)0.001
      MMP: matrix metalloproteinase; Q: quartile; OR: adjusted odds ratio; CI: confidence interval.
      Because unstable plaque was observed more frequently in patients with more cardiovascular risk factors and regression analysis might not allow proper exclusion of the effects of these factors on serum markers, we performed a power analysis to ascertain the relationship between MMP-9 and unstable plaque. A receiver operating characteristic curve was used to determine a cutoff value of 510.15 ng/mL related to unstable plaque (area under the curve = 0.732). This cutoff value was then applied to the unstable and no plaque groups. We used the chi-squared test to determine the association between elevated MMP-9 and unstable plaque (OR = 8.179). The Power and Sample Size Calculation program (version 3.0) [
      • Dupont W.D.
      • Plummer W.D.
      Power and sample Size calculations: a review and computer program.
      ] provided a power of 0.997.

      3.3 Associations of MCP-1 with plaque and IMT

      In general linear models, high serum MCP-1 level was significantly associated with increased IMT (p = 0.049; Table 3), but not with plaque stability (p = 0.399; Table 2) or total PS (p = 0.376; Table 1). MCP-1 quartiles were ≤32.4 pg/mL (Q1), 32.5–62.5 pg/mL (Q2), 62.6–106.4 pg/mL (Q3), and ≥106.5 pg/mL (Q4). The association with IMT remained stable in a multinomial logistic regression model adjusted for covariates, including age, hypertension, diabetes mellitus, cigarette smoking, and LDL cholesterol and UA levels (OR = 22.94, 95% CI = 2.14–245.66, p = 0.01; Table 5).
      Table 5Multinomial logistic regression model of IMT associations with traditional vascular risk factors and MCP-1.
      VariableCarotid IMT tertile
      T1T2T3
      Or (95% CI)pOr (95% CI)p
      AgeReference1.07 (1.01–1.14)0.031.04 (1.00–1.09)0.03
      SexReference0.54 (0.07–3.78)0.530.83 (0.20–3.34)0.79
      HypertensionReference1.60 (0.39–6.56)0.526.08 (1.07–34.53)0.04
      Diabetes mellitusReference0.59 (0.09–4.05)0.590.53 (0.11–2.69)0.45
      Cigarette smokingReference0.63 (0.05–7.79)0.720.96 (0.12–7.60)0.97
      LDL cholesterolReference4.68 (1.69–12.92)0.0032.65 (1.15–6.13)0.022
      Uric acidReference1.01 (0.99–1.02)0.0961.01 (1.00–1.02)0.052
      MCP-1 (Q4 vs. Q1)Reference12.37 (0.80–191.94)0.07222.94 (2.14–245.66)0.01
      MCP-1: monocyte chemoattractant protein-1; IMT: intima–media thickness; T: tertile; OR: adjusted odds ratio; CI: confidence interval; LDL: low-density lipoprotein; Q: quartile.

      4. Discussion

      In this study, we sought to relate serum biomarkers to carotid atherosclerosis parameters predictive of vascular events [IMT, plaque instability, and TPA (indirectly represented by total PS)]. Our results demonstrate relationships between circulating biomarkers of ECM remodeling and the severity of carotid atherosclerosis, assessed using duplex ultrasound. First, we found significant associations between high MMP-9 level and plaque instability, high total PS, and increased IMT. The associations with plaque instability and high total PS remained significant after adjustment for traditional vascular risk factors. Second, high MCP-1 concentration was associated with increased IMT, but not with plaque instability or high total PS. Third, high UA level was associated with plaque instability. To our knowledge, this study is the first to demonstrate a dose–response relationship between serum MMP-9 level and carotid plaque stability.
      Elevated MCP-1 is a predictor of cardio-cerebrovascular disease, perhaps due to its effect on atherosclerosis. A previous study found a positive correlation between MCP-1 level and coronary atherosclerosis, but this association was not independent of traditional vascular risk factors, especially age [
      • Deo R.
      • Khera A.
      • McGuire D.K.
      • et al.
      Association among plasma levels of monocyte chemoattractant protein-1,traditional cardiovascular risk factors, and subclinical atherosclerosis.
      ]. Thakore et al. [
      • Thakore A.H.
      • Guo C.Y.
      • Larson M.G.
      • et al.
      Association of multiple inflammatory markers with carotid intimal medial thickness and stenosis (from the Framingham Heart Study).
      ] reported that blood inflammatory markers, including CRP, interleukin-6, soluble intercellular adhesion molecule-1, MCP-1, CD40 ligand, and P-selectin, assessed as a group, were significantly associated with internal carotid artery IMT (p = 0.01). However, in contrast to our results, MCP-1 showed no significant positive relationship with IMT when assessed individually [
      • Thakore A.H.
      • Guo C.Y.
      • Larson M.G.
      • et al.
      Association of multiple inflammatory markers with carotid intimal medial thickness and stenosis (from the Framingham Heart Study).
      ]. These findings may reflect the distinct determinants of different phenotypes [
      • Touboul P.J.
      • Hennerici M.G.
      • Meairs S.
      • et al.
      Mannheim carotid intima–media thickness consensus (2004–2006). An update on behalf of the Advisory Board of the 3rd and 4thwatching the risk symposium, 13th and 15th European stroke conferences.
      ].
      Cao et al. [
      • Cao J.J.
      • Arnold A.M.
      • Manolio T.A.
      • et al.
      Association of carotid artery intima-media thickness, plaques, and C-reactive protein with future cardiovascular disease and all-cause mortality: the Cardiovascular Health Study.
      ,
      • Cao J.J.
      • Thach C.
      • Manolio T.A.
      • et al.
      C-reactive protein, carotid intima-media thickness, and incidence of ischemic stroke in the elderly: the Cardiovascular Health Study.
      ] noted that elevated CRP level was associated with increased risks of cardiovascular and cerebrovascular diseases and all-cause mortality in patients with atherosclerosis detectable with carotid ultrasound. Moreover, they confirmed that CRP level was closely associated with IMT [
      • Cao J.J.
      • Thach C.
      • Manolio T.A.
      • et al.
      C-reactive protein, carotid intima-media thickness, and incidence of ischemic stroke in the elderly: the Cardiovascular Health Study.
      ], in agreement with Alizadeh Dehnavi et al. [
      • Alizadeh Dehnavi R.
      • de Roos A.
      • Rabelink T.J.
      • et al.
      Elevated CRP levels are associated with increased carotid atherosclerosis independent of visceral obesity.
      ]. In contrast, we failed to demonstrate a relationship between CRP and atherosclerosis severity. The existence of such an association remains controversial. Equivalent or more severe atherosclerotic lesions were observed in transgenic CRP-deficient mice compared with controls [
      • Teupser D.
      • Weber O.
      • Rao T.N.
      • Sass K.
      • Thiery J.
      • Fehling H.J.
      No reduction of atherosclerosis in C-reactive protein (CRP)-deficient mice.
      ], and transgenic rabbits with low and high CRP expression fed a high-cholesterol diet developed similar degrees of aortic atherosclerosis [
      • Koike T.
      • Kitajima S.
      • Yu Y.
      • et al.
      Human C-reactive protein does not promote atherosclerosis in transgenic rabbits.
      ]. Moreover, Rozalski et al. [
      • Rozalski R.
      • Migdalski A.
      • Gackowski D.
      • et al.
      Does morphology of carotid plaque depend on patient's oxidative stress?.
      ] detected the highest CRP levels in patients with the most stable plaque. These data challenge the atherogenic effects of CRP and are consistent with the results of a large-scale genetic epidemiology study [
      • Dehghan A.
      • Dupuis J.
      • Barbalic M.
      • et al.
      Meta-analysis of genome-wide association studies in >80,000 subjects identifies multiple loci for C-reactive protein levels.
      ]. Thus, CRP may have no effect on the severity of atherosclerosis.
      Our finding that high UA level was associated with plaque instability is in accord with those of previous studies [
      • Li Q.
      • Yang Z.
      • Lu B.
      • et al.
      Serum uric acid level and its association with metabolic syndrome and carotid atherosclerosis in patients with type 2 diabetes.
      ,
      • Takayama S.
      • Kawamoto R.
      • Kusunoki T.
      • Abe M.
      • Onji M.
      Uric acid is an independent risk factor for carotid atherosclerosis in a Japanese elderly population without metabolic syndrome.
      ]. The mechanisms by which UA reflects the risk of carotid atherosclerosis are incompletely understood. UA regulates critical pro-inflammatory pathways in vascular smooth-muscle cells [
      • Kanellis J.
      • Watanabe S.
      • Li J.H.
      • et al.
      Uric acid stimulates monocyte chemoattractant protein-1 production in vascular smooth muscle cells via mitogen-activated protein kinase and cyclooxygenase-2.
      ,
      • Baldwin W.M.S.
      • Marek G.
      • Wymer D.
      • et al.
      Hyperuricemia as a mediator of the proinflammatory endocrine imbalance in the adipose tissue in a murine model of the metabolic syndrome.
      ], resulting in low-grade inflammation and insulin resistance in subjects with metabolic syndrome, as well as endothelial cell dysfunction and atherosclerosis.
      Our study has some limitations. First, we measured total serum MMP-9 levels, not those of tissue inhibitors of metalloproteinases (TIMPs). MMP-9/TIMPs or active MMP-9 concentrations may be better indicators than total MMP-9 levels of ongoing vascular remodeling activities. Second, carotid IMT values can be biased by subjective measurement, and automated image-processing software can provide more objective values.
      In conclusion, our results indicate that blood MMP-9 and MCP-1 levels are associated with carotid atherosclerosis. MMP-9 concentration showed a dose–response relationship with carotid plaque stability. Our findings also support a dose–response relationship between MCP-1 level and IMT. Moreover, we found that high UA level was associated with plaque instability. These serum biomarkers can be used to distinguish unstable from stable plaque, reflect the extent of carotid atherosclerosis beyond carotid ultrasound findings, and can help to target vulnerable patients and monitor the beneficial effects of pharmacological agents.

      Acknowledgments

      This study was supported by the Natural Science Foundation project of CQ CSTC (no. 2011BB5140 ). The funding source had no influence on study design; data collection, analysis, or interpretation; the writing of the manuscript; or the decision to submit this manuscript for publication. The contents are solely the responsibility of the authors and do not necessarily represent the views of the funding source.

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