Neutrophil gelatinase associated lipocalin NGAL (or Lipocalin 2) was originally described as a 25 kDa protein, stored in the granules of human neutrophils, and was recognized as an acute phase protein , . NGAL gained considerable diagnostic and prognostic value in kidney disorders as a valuable marker of renal injury . Possible pathogenic roles of NGAL in cardiovascular disorders have also been intensively discussed .
Our group showed recently that the expression of NGAL in endarterectomy specimens is associated with symptomatic carotid atherosclerosis and revealed a pro-inflammatory capacity of NGAL in human macrophages, smooth muscle cells and endothelial cells in vitro . Another potential pro-atherosclerotic mechanism of NGAL includes the binding of MMP-9 and the formation of metalloproteinase (MMP)-9/NGAL complex, which protect MMP-9 from inactivation . Circulating NGAL reflects the degree of inflammation in patients with coronary artery disease (CAD)  and predicts mortality in patients with myocardial infarction . However, the association of circulating NGAL and MMP-9/NGAL levels with plaque vulnerability in patients with carotid artery stenosis has not been studied before.
Statins represent the main class of medication for a pharmacological approach to manage patients with carotid artery stenosis. The stabilizing effects of statins on plaque morphology were demonstrated . Mechanisms potentially responsible for anti-atherosclerotic effects of statins go far beyond their lipid-modifying properties. These so-called “pleiotropic” effects include anti-inflammatory, anti-thrombotic, antioxidant and immunomodulatory properties , , , , .
Carotid plaque vulnerability is a highly important prognostic marker for cerebrovascular events. However, its identification represents a significant challenge in the clinic. Therefore, the aim of our present study was to evaluate circulating NGAL and MMP-9/NGAL complex as potential biomarkers of atherosclerotic plaque vulnerability in patients with carotid artery stenosis. Moreover, as the link between statin treatment and decreased plaque vulnerability in patients with atherosclerosis was established previously  and statins were shown to reduce the concentration of circulating inflammatory mediators , we additionally investigated the association of NGAL and MMP-9/NGAL levels with statin treatment.
Materials and methods
One hundred and thirty-six consecutive patients, treated with carotid endarterectomy at the Department of Surgery, Division of Vascular Surgery at the Medical University of Vienna, Austria between October 2011 and October 2014 were included in the study. Patients with acute infection, autoimmune or neoplastic disease were excluded. All study subjects were Caucasian. The authors have complied with the World Medical Association Declaration of Helsinki regarding ethical conduct of research involving human subjects. The study was reviewed and approved by the Ethic Committee of the Medical University of Vienna (EK 269/2009), all study subjects gave written informed consent.
Patients were recognized as having symptomatic carotid stenosis if they had at least one neurological event directly connected to carotid stenosis within the last 6 months. The duplex sonography was used to classify carotid plaque according to echogenicity into “soft” (echolucent), calcified/hard (echogenic) and mixed. Histological classification of the endarterectomy specimens was performed according to the American Heart Association (AHA). Presence of peripheral artery disease (PAD) was defined according to Fontaine classification. CAD was defined by history of angina pectoris or known event of myocardial infarction.
Statin use was recorded at the time of enrollment. One hundred and eight patients were treated with statins (48 patients received simvastatin [Pfizer, Vienna, Austria] 40 mg, 44 atorvastatin [Pfizer, Vienna, Austria] 40 mg, and 16 rosuvastatin [AstraZeneca, Wedel, Austria] 40 mg), whereas 28 patients were not on statin treatment. All patients in the statin group took this medication for at least 6 months prior to carotid endarterectomy. Additional medication included antiaggregants and anti-coagulants such as acetylsalicylic acid (G.L. Pharma, Lannach, Austria), clopidogrel (Sanofi-Aventis, Vienna, Austria), marcoumar (Meda Pharma, Vienna, Austria) and low molecular weight heparin (Sanofi-Aventis, Vienna, Austria), and/or anti-hypertensive drugs such as angiotensin converting enzyme (ACE) inhibitors, β-blockers, calcium antagonists and diuretics.
Peripheral blood was drawn from the antecubital vein prior to surgery. Blood samples were centrifuged at 3000 g at 4°C for 15 min, serum was stored in aliquots at −80°C for further analysis.
NGAL and MMP-9/NGAL measurement
NGAL and MMP-9/NGAL levels in serum of patients were measured by specific enzyme-linked immunosorbent assay (ELISA, both from R&D Systems, Minneapolis, MN, USA; catalog number DLCN20 and DM9L20, respectively). The limit of detection was 0.012 μg/L for NGAL and 0.013 μg/L for MMP-9/NGAL assay. Coefficient of variation (CV) for intra-assay precision ranges from 3.1% to 4.4% and CV for inter-assay precision ranges from 5.6% to 7.9%. All laboratory measurements were performed by investigators blinded to patients’ data and characteristics.
Median (quartile) values were given to describe continuous variables, absolute numbers and percentages are used to describe categorical variables. Differences between patient groups with respect to NGAL and MMP-9/NGAL levels, respectively, were tested by the two-sample t-test. In the case of more than two groups, analysis of variance models were performed, and the Tukey method was applied to correct for multiple comparisons. Differences in continuous variables of statin patients compared to non-statin patients were tested using the two-sample t-test, non-normally distributed variables were compared by the Wilcoxon rank sum test. Categorical variables were compared by the χ2-test or the Fisher’s exact test, as appropriate. Correlations of continuous variables were characterized using the Spearman’s correlation coefficient. Univariate and multiple linear regression models were performed to evaluate the marginal and partial impact of the variables statins, creatinine, low density lipoproteins (LDL), neutrophils, body mass index (BMI), Fontaine classification, histological classification of the plaques and symptomatic presentation of carotid artery stenosis on the NGAL and MMP-9/NGAL levels. Rank-transformed serum creatinine levels and log-transformed values of LDL, neutrophils and MMP-9/NGAL were used for statistical analyses due to their skewed distributions. All p-values are results of two-sided tests and p-values <0.05 were considered as statistically significant. The SAS software version 9.4 (SAS Institute Inc. 2002–2012; Cary, NC, USA) was used for statistical analyses.
Characteristics of the patients
Demographic data of the total cohort of patients with carotid artery stenosis as well as statin-treated and non-treated subgroups are shown in Table 1. There were no differences with regard to most demographic or laboratory parameters between the two groups according to the use of statins. However, if patients were divided according to the Fontaine classification of PAD, significantly more patients with stage ≥II were present in the non-statin group (p=0.001). As for histological and ultrasound characteristics of the plaques, patients in the statin group had lower prevalence of type VI plaques (p=0.002) and “soft” (echolucent) plaques (p=0.0007) as compared to the patients in non-statin group.
NGAL and MMP-9/NGAL serum levels are higher in patients with vulnerable carotid plaques
In order to evaluate circulating NGAL and MMP-9/NGAL as possible biomarkers of plaque vulnerability, we first classified patients with carotid artery stenosis according to histological and ultrasound characteristics of the plaques. Patients with type VI plaques according to AHA had highest NGAL levels (126.6 [107.4–180.9] μg/L) as compared to the patients with type VII or more (54.0 [32.8–72.9] μg/L) or type V or less (76.8 [61.5–91.3] μg/L), as shown in Figure 1A (p<0.0001). Significant differences were also found for the level of MMP-9/NGAL complex (Figure 1B), if patients with type VI plaques (48.3 [28.6–73.4] μg/L) were compared to patients having plaques classified as type VII or more (23.3 [14.8–40.1] μg/L) or type V or less (27.7 [17.2–47.3] μg/L, p=0.004). High NGAL levels were moreover associated with “soft” carotid plaque in ultrasound (Figure 1C). These patients had significantly higher NGAL levels (113.2 [97.1–157.3] μg/L) as compared to the patients with calcified (61.5 [48.8–82.2] μg/L) or mixed plaque morphology (69.6 [61.4–78.6] μg/L, p<0.0001). As for MMP-9/NGAL complex and atherosclerotic plaque morphology at ultrasound, patients with “soft” carotid plaques had higher complex levels (45.9 [27.6–73.4] μg/L) as compared to the patients having calcified (26.5 [14.2–42.4] μg/L) and mixed plaque morphology (23.4 [16.1–47.5] μg/L, p=0.0004, Figure 1D). Classification of the lesions based on the combination of both histology and ultrasound confirmed our results. NGAL and MMP-9/NGAL levels (both p≤0.05, data not shown) were higher in patients with vulnerable (histological type VI and “soft” plaque in ultrasound, n=30) as compared with stable (histological class ≥VII and calcified plaque in ultrasound, n=31) lesions.
Circulating NGAL and MMP-9/NGAL in relation to symptomatic presentation and degree of carotid artery stenosis
Patients, who clinically presented with symptomatic carotid stenosis, had higher serum NGAL levels as compared to asymptomatic patients (97.1 [64.4–141.6] vs. 74.9 [54.1–99.4] μg/L; p=0.0007, Figure 2A). MMP-9/NGAL levels were not different between these two groups (28.6 [16.1–53.6] vs. 34.1 [19.5–54.1] μg/L, p=0.60, Figure 2B). Additional, symptomatic presentation of carotid stenosis is statistically significantly influencing circulating NGAL according to univariate (p=0.0007) and multiple linear regression models (p=0.0008), as shown in Tables 2 and 3.
If all 136 patients were divided into two groups according to their degree of stenosis as having ≥90% or <90%, higher NGAL levels were observed in the group with more pronounced degree of stenosis (81.3 [61.3–111.9] vs. 70.1 [58.3–104.6] μg/L, p=0.026, Figure 2C). MMP-9/NGAL complex levels were not different in these two groups (34.6 [18.8–56.5] vs. 31.8 [15.3–47.9] μg/L, p=0.216, Figure 2D).
NGAL and MMP-9/NGAL levels are decreased under statin treatment
The statin-treated group of patients demonstrated significantly lower NGAL (73.9 [54.1–100.1] vs. 128.0 [85.9–184.9] μg/L, p<0.0001) and MMP-9/NGAL levels (28.9 [16.2–49.2] vs. 40.6 [26.4–77.6] μg/L, p=0.046) as compared to the non-statin group (Figure 3A and B). Statins also demonstrate a significant influence on circulating NGAL levels both in univariate regression analyses and in multiple model (both p<0.0001, Table 2). For MMP-9/NGAL, the univariate regression models also exposed statins (p=0.046) as statistically significant explanatory variables (Table 3).
Moreover, we investigated the association of NGAL and MMP-9/NGAL to lesion morphology under statin treatment. We found that patients with vulnerable lesion, defined by having lesion type VI (Figure 3C) or “soft” plaques (Figure 3D), demonstrated lower NGAL levels (p=0.001 and p<0.0001, respectively) under statin treatment. MMP-9/NGAL did not differ significantly between these groups of patients (data not shown).
Additionally, in the subgroup of the non-statin patients, NGAL [157.3 (118.0–203.6) vs. 88.4 (61.4–107.3) μg/L, p=0.0005], but not MMP-9/NGAL levels [49.8 (27.3–82.5) vs. 32.2 (22.4–38.9) μg/L, p=0.29] were also higher in patients having stenosis grade of ≥90% as compared to the patients having <90%. In the subgroup of the patients on statin treatment, such differences were not observed. Patients on statin treatment had similar NGAL (73.9 [55.6–96.7] vs. 69.5 [48.9–104.6] μg/L, p=0.69) and MMP-9/NGAL (31.4 [17.6–51.8] vs. 29.1 [14.8–48.2] μg/L, p=0.46) levels, irrespective of their degree of stenosis.
We additionally compared, if different statins could be differentially associated with NGAL and MMP-9/NGAL levels. For this reason we compared three groups of statin-patients – treated with simvastatin, atorvastatin or rosuvastatin each at 40 mg – between each other and each of this statin-treated sub-group versus the non-statin group. We found no significant differences between the three patient groups on different statin treatment in regard to NGAL or MMP-9/NGAL levels (p>0.05, for all comparisons, data not shown). However, significant differences remained for NGAL levels between each statin group as compared to non-statin group (p<0.05, for all comparisons, data not shown). MMP-9/NGAL levels were not different between the patients on different statins and the non-statin group (p=0.146, data not shown).
Univariate regression analyses revealed that additionally to statins and symptomatic presentation of carotid stenosis, as described above, also neutrophils (p=0.003), serum creatinine (p=0.002), Fontaine classification (p<0.0001), and histological classification of the plaques (p<0.0001) are statistically significantly influencing the dependent variable NGAL (Table 2). However, in the multiple model, additionally to symptomatic presentation, LDL (p=0.005), serum creatinine (p=0.014), statin treatment (p<0.0001), Fontaine classification (p<0.0001), BMI (p=0.003), and histological classification (p<0.0001) demonstrated an independent statistically significant influence on levels of circulating NGAL (Table 2).
For MMP-9/NGAL, the univariate regression models exposed neutrophils (p<0.0001), Fontaine classification (p=0.023), and histological classification of the plaques (p=0.004), as significant explanatory variables. In the multiple analysis, only neutrophils (p=0.019) and BMI (p=0.043) were independently associated with NGAL/MMP-9 levels (Table 3).
Carotid atherosclerosis can lead to cerebrovascular events including stroke . Inflammatory cells and proteins trigger a chronic inflammatory state in atherosclerotic lesions in the vessel wall , , , , , , and their levels in the circulation could be used as biomarkers of plaque vulnerability . In our study, we identified circulating NGAL and MMP-9/NGAL as potential biomarkers of atherosclerotic plaque vulnerability in patients with carotid artery stenosis. NGAL and MMP-9/NGAL levels were higher in patients having atherosclerotic plaques type VI according to the AHA histological classification and plaques with decreased echogenicity at ultrasound. In addition, symptomatic patients and patients with more pronounced stenosis grade (≥90%) presented with higher NGAL levels as compared to asymptomatic or patients with stenosis grade <90%, respectively. Furthermore, we showed for the first time that statin-treated patients suffering from carotid atherosclerosis had lower levels of both serum NGAL and MMP-9/NGAL complex. This effect of the statins on the serum NGAL levels was independent of the type of medication, as patients taking simvastatin, atorvastatin or rosuvastatin equally exhibited decreased NGAL levels.
NGAL is being discussed more and more as a possible diagnostic and prognostic biomarker in different pathologies , , , . Although the association of NGAL with clinical and laboratory parameters in patients with carotid artery stenosis was investigated in several earlier studies , , , our present study is the first to investigate the level of circulating NGAL and MMP-9/NGAL in relation to atherosclerotic plaque vulnerability, defined by histological and ultrasound examination, in such patients. Previously, NGAL levels were shown to be higher in hypertensive as compared to normotensive patients in both early asymptomatic  and advanced carotid atherosclerosis . In the study of Giaginis et al. patients with advanced stenosis grade showed only a trend for correlation with increased NGAL and NGAL concentrations were not associated with the presence of the symptoms of carotid stenosis , contrary to our study here, which could be explained by the differences in the patients demographics and co-morbidities. In our patients’ cohort, serum creatinine and Fontaine classification of the PAD were identified as independent predictors for both NGAL and MMP-9/NGAL levels. In this respect, it should be noted that in our study the presence of PAD was classified clinically according to Fontaine’s classification. In order to define the possible clinical implication of NGAL and MMP-9/NGAL in PAD, studies on larger cohorts of patients with PAD should be performed and computed tomography angiography added to discriminate between patients without PAD and those with stage I PAD.
In human carotid endarterectomy specimens NGAL is expressed by macrophages, endothelial and smooth muscle cells . Tissue NGAL and MMP-9/NGAL expression was previously demonstrated to be associated with an unstable phenotype of atherosclerotic plaques , . Our group recently showed that NGAL mRNA expression in carotid plaque is associated with symptomatic presentation of carotid stenosis . However, those studies did not investigate levels of circulating NGAL and MMP-9/NGAL. Our present study identified circulating NGAL and MMP-9/NGAL as biomarkers of plaque instability. These results support previous publications on the association of NGAL and MMP-9/NGAL expression in carotid atherosclerotic tissue with unstable plaque phenotype , , , but additionally provide a new possibility to identify plaque vulnerability by measuring these proteins in the peripheral blood of the patients, which represents an only mildly invasive method. Based on our results we feel that a combination of a biomarker approach with visualized techniques could improve stratification of patients with carotid artery stenosis. We suggest that patients with higher levels of NGAL need shorter follow-up intervals compared to patients with lower NGAL levels. However, larger studies for the identification of proper and reliable cut-off values for NGAL in such patients are required.
Atherosclerotic plaque morphology is beneficially affected by statin administration , . Moreover, in symptomatic carotid artery stenosis, statin pre-treatment was associated with reduced risk of stroke , , In our study, patients with carotid artery stenosis treated with statins, demonstrated a lower prevalence of atherothrombotic (type VI) and echolucent or “soft” plaques, compared to the non-statin patients’ group. It remains a matter of debate if this is a LDL-associated effect or a “pleiotropic” mechanism of statins or both. A previous study by Giaginis et al. did not find any differences in NGAL levels in patients with carotid artery stenosis taking statins or no statins . However, in that study a high prevalence of patients with hyperlipidemia, defined as total cholesterol ≥5.18 mmol/L, has to be noted. Furthermore, Giaginis et al. in their study, did not specify how long the patients were on the statin treatment, and did not provide the characteristics of the patients in the statin and non-statin group in comparison. In our study, the median level of total cholesterol in the entire cohort was 166.0 mg/dL (4.29 mmol/L) and all patients in the statin-group were on the statin treatment for at least 6 months, similar to other clinical studies identifying the effects of statins on plaque morphology or circulating biomarker , , , . Moreover, the comparison of NGAL and MMP-9/NGAL levels in association with lesion morphology demonstrated lower NGAL levels under statin treatment in patients with vulnerable plaques as compared to non-statin patients with vulnerable plaques.
In conclusion, here we provide novel evidence that circulating NGAL and MMP-9/NGAL complex is associated with atherosclerotic plaque vulnerability in patients with carotid artery stenosis. Furthermore, we show that statin treatment reduces circulating NGAL and MMP-9/NGAL independently of the degree of stenosis. Considering the role of NGAL in low-level systemic inflammation , , , decreased levels of NGAL and MMP-9/NGAL complex under statin treatment could contribute to plaque stabilization in patients with carotid artery stenosis.
The authors wish to thank Mira Brekalo from the Department of Internal Medicine II, Division of Cardiology, Medical University of Vienna for the excellent technical support.
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About the article
Published Online: 2017-06-26
Published in Print: 2017-11-27
Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.
Financial support: Svitlana Demyanets has received grant support from the Herzfelder’sche Familienstiftung (Vienna, Austria). The other authors have no conflicts to report. Furthermore, this work was supported by the Association for the Promotion of Research in Atherosclerosis, Thrombosis and Vascular Biology.
Employment or leadership: None declared.
Honorarium: None declared.
Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.