Extracellular Matrix Remodeling Factors as Markers of Carotid Artery Atherosclerosis

Abstract

Materials and methods: 20 patients undergoing routine carotid endarterectomy and 40 healthy volunteers were enrolled in this study. MMPs activity and OPG and FN concentrations were measured in atherosclerotic plaques and nonchanged contiguous tissue after homogenization as well as in plasma from patients and reference group. The activity of MMPs was evaluated by gelatin zymography, and the concentration of OPG and FN was assessed by ELISA.

Results: OPG concentration and MMP-9 activity showed differences between plaque and nonchanged tissue; OPG was higher in adjacent tissue (P=0.0009), whereas MMP-9 was higher in plaque (proMMP-9 P=0.0003; MMP-9 P < 0.0001). The OPG plasma concentration and both MMPs plasma activity were higher in patients (OPG P < 0.001; proMMP-2 P=0.0292; and proMMP-9 P=0.0374), while FN plasma concentration was lower in patients than in the reference group (P=0.0004). The ROC curves analysis showed the highest AUC for OPG (0.943) with 85.0% sensitivity and 92.1% specificity.

Conclusions: The atherosclerotic plaque and the contiguous artery wall are biochemically different. OPG shows the highest potential to be a marker of advanced carotid atherosclerosis.

Figure 1

Key processes in atherosclerotic plaque formation.

Figure 2

Representative tissue fragments. The fragment A above the dotted line was separated and removed. Fragment B was used as atherosclerotic plaque.

Figure 3

Representative zymogram of different materials from one patient. A–capillary blood standard; B–plasma; C–reference tissue; and D–plaque.

Figure 4

Comparison of (a) osteoprotegerin (OPG), (b) fibronectin (FN), (c) matrix metalloproteinase 2 proenzyme (proMMP-2), (d) matrix metalloproteinase 9 proenzyme (proMMP-9), (e) matrix metalloproteinase 2 active form (MMP-2), and (f) matrix metalloproteinase 9 active form (MMP-9) in atherosclerotic plaque and reference tissue, from the same patients. The middle lines represent the median, and the whiskers extend from the 25th to the 75th percentiles.

Figure 5

Comparison of (a) osteoprotegerin (OPG), (b) fibronectin (FN), (c) matrix metalloproteinase 2 proenzyme (proMMP-2), and (d) matrix metalloproteinase 9 proenzyme (proMMP-9) in plasma of patients (study group) and reference group. The middle lines represent the median, and the whiskers extend from the 25th to the 75th percentiles.

Figure 6

ROC curve comparison for single markers. AUC for OPG is 0.943 (P < 0.001), with associated criterion >1.0349 ng/ml; sensitivity 85.0%, specificity 92.1%. AUC for FN is 0.712 for ≤316.9966 µg/ml; sensitivity 100%, specificity 47.4%. AUC for proMMP-2 is 0.676 for activity >0.6897 AU/mg; sensitivity 55.0%, specificity 87.5%. AUC for proMMP-9 is 0.625, with associated criterion >0.2637 AU/mg, sensitivity 42.1%, and specificity 100.0%.

Figure 7

ROC curve comparison for combined models. Model 1 consists of hypertension, current smoking, male sex, and total cholesterol, whereas model 2 consists of OPG, FN, proMMP-2, and proMMP-9. AUC for model 1 is 0.934 (P < 0.0001, sensitivity 90.0%, and specificity 90.0%), for model 1 + OPG is 0.974 (P < 0.0001, sensitivity 90.0%, and specificity 92.5%), and for model 2 it is 0.922 (P < 0.0001, sensitivity 80.0%, and specificity 92.5%).