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Background: Plasma biomarkers may be useful to detect healthy individuals at increased risk for atherosclerotic manifestations, such as carotid artery stenosis. The aim of this longitudinal cohort study was to evaluate new biomarkers in relation to C-reactive protein and conventional risk factors for carotid artery stenosis during long term follow-up
Methods
The following markers were measured in 5550 middle-aged subjects: C-reactive protein, lipoprotein-associated phospholipase A2, proneurotensin, midregional pro-adrenomedullin, midregional pro-atrial natriuretic peptide, N-terminal pro B-type natriuretic peptide, copeptin, and cystatin C. Subjects with prevalent carotid artery stenosis were excluded. Subjects were followed in national patient registers for 23.4 (interquartile range 19.5–24.3) years regarding incident carotid artery stenosis, both operated and non-operated.
Results
When including conventional risk markers in Cox regression, N-terminal pro B-type natriuretic peptide (Hazard ratio 1.36; 95% confidence interval 1.12–1.65; p = 0.002) was independently associated with incident carotid artery stenosis, whereas there were trends for C-reactive protein (HR 1.20; 95% confidence interval 0.98–1.48; p = 0.071), and midregional pro-adrenomedullin (Hazard ratio 1.21; 95% confidence interval 0.99–1.47; p = 0.061). Midregional pro-adrenomedullin (Hazard ratio 1.30; 95% confidence interval 1.03–1.65; p = 0.029) was independently associated with incident surgery for carotid artery stenosis, whereas there was a trend for N-terminal pro B-type natriuretic peptide (Hazard ratio 1.31; 95% confidence interval 1.00–1.72; p = 0.052).
Conclusions
N-terminal pro B-type natriuretic peptide and midregional pro-adrenomedullin can be used as predictors for clinically detected carotid artery stenosis during long-term follow-up of healthy subjects.
Atherosclerosis is a systemic inflammatory disease which can affect the carotid arteries leading to stenosis, embolization and subsequent neurological damage. Carotid artery stenosis is estimated to occur in 4.4–7% of the elderly population and to cause around 7% of ischaemic strokes.
In addition to traditional atherosclerotic risk factors, plasma biomarkers measured in healthy individuals may be useful to detect individuals at increased risk for developing carotid artery disease, and may also serve to identify factors enhancing or halting development of carotid disease. Several novel promising candidate plasma biomarkers for cardiovascular disease have been identified. Lipoprotein-associated phospholipase A2 (Lp-PLA2) is an enzyme produced by macrophages, considered as a biomarker for vascular inflammation and progression of atherosclerosis,
Interpretation of lipoprotein-associated phospholipase A2 levels is influenced by cardiac disease, comorbidities, extension of atherosclerosis and treatments.
Lp-PLA Studies Collaboration Lipoprotein-associated phospholipase A (2) and risk of coronary disease, stroke, and mortality: collaborative analysis of 32 prospective studies.
a precursor of the hormone neurotensin, has also been proposed as a marker for cardiovascular disease development, and the midregional peptides pro-adrenomedullin (MR-proADM), and proatrial natriuretic peptide (MR-proANP) are potentially associated with microvascular endothelial dysfunction
et al; ESC study group on biomarkers in cardiology of the acute cardiovascular care association. use of copeptin for rapid rule-out of acute myocardial infarction.
Cystatin C is an endogenous inhibitor of cathepsins, and elevated levels of this marker have been cross-sectionally associated with the presence of extracranial carotid artery stenosis in the acute stage of noncardioembolic stroke.
and conventional risk factors for prediction of incident carotid artery stenosis in a large population-based study of middle-aged individuals. The secondary aim was to evaluate if any of the mentioned biomarkers were independently associated with incident carotid artery surgery.
Material and methods
Study sample
The longitudinal population-based prospective cohort Malmö Diet and Cancer study (MDCS)
Relation between insulin resistance and carotid intima-media thickness and stenosis in non-diabetic subjects. results from a cross-sectional study in Malmo, Sweden.
included 30,447 middle-aged individuals from Malmö, Sweden. Study subjects underwent baseline examinations in 1991–1996. A random subsample (n = 6103) from this cohort was included in the MDCS cardiovascular cohort,
(Fig 1). Current smoking was defined as self-reported regular smoking or smoking cessation within the last year. Diabetes mellitus was defined as self-reported physician´s diagnosis, use of anti-diabetic medication, or fasting blood glucose >6.0 mmol/L. Hypertension was defined as use of antihypertensive medication or blood pressure ≥140/90 mmHg. Ultrasound examination was performed on the right carotid, and individuals with carotid artery stenosis at baseline were excluded. Written informed consent to long-term follow-up for incident cardiovascular disease was obtained from all participants. The Regional ethical review board in Lund, Sweden (LU 51/90 and 2013/566) approved the study.
Fig. 1Descriptive flow diagram of study participants and plasma biomarker data.
and Outpatient Register and the Cause of Death Register) by linkage of the ten-digit personal identification number unique to each Swedish resident. Follow-up was extended until December 31st, 2016, and median follow up was 23.4 years (IQR 19.5–24.3). In both the Inpatient and Cause of Death registers, the diagnosis carotid artery stenosis is coded using a Swedish revision of the International Classification of Disease (ICD),
version 8 (432,00; 432,90), version 9 (443B), and version 10 (I65.2). Surgical procedures performed on carotid arteries are coded using a Swedish classification system
Relation between insulin resistance and carotid intima-media thickness and stenosis in non-diabetic subjects. results from a cross-sectional study in Malmo, Sweden.
with a diagnosis of carotid artery stenosis were randomly selected for the validation procedure using patient record data. A stenosis degree of ≥ 60% measured with ultrasound, computer tomography (CT-), or magnetic resonance (MR-) angiography was considered as validation of the diagnosis. Among these 100 patients, 57 had symptomatic carotid artery stenosis and
The influence of age, sex and other variables on the plasma level of N-terminal pro brain natriuretic peptide in a large sample of the general population.
asymptomatic carotid artery stenosis. The proportions of operated patients with symptomatic and asymptomatic stenosis were 85.9% (49/57) and 14.3% (6/42), respectively. One patient had coronary artery disease and was misdiagnosed. The diagnosis of carotid artery stenosis could therefore be confirmed in 99% of cases, and was symptomatic in 57%.
Laboratory measurements
Fasting total cholesterol and triglycerides were measured according to standard procedures at the Department of Clinical Chemistry, Skåne University Hospital Malmö. HbA1c was determined by ion exchange chromatography, using the Swedish Mono-S standardization system; reference values were 3.9–5.3% in non-diabetic individuals.
Plasma biomarkers were measured from fasting plasma samples that had been frozen at - 80° C immediately after collection.
CRP was measured by a high-sensitivity Tina-quant® latex assay (Roche Diagnostics, Basel, Switzerland). The average coefficient of variation (CV) was 4.59%.
Lp-PLA2 mass measurements were performed using the commercially available second generation PLAQTM test (diaDexus Inc., South San Francisco, CA, USA) enzyme-linked immunosorbent assay (ELISA) kit.
Plasma-EDTA samples are stable for Lp-PLA2 activity and mass measurements within 7 days of collection for refrigerated samples and for more than 10 years from collection when stored at -70 °C.
. Levels of MR-proADM were measured using immunoluminometric sandwich assays targeted against amino acids in the midregions of the peptide (BRAHMS AG, Henningsdorf, Germany).
Lower and upper limits of detection were 0.08 and 25 nmol/L, respectively. MR-proANP was measured using immunoluminometric sandwich assays targeted against amino acids in the midregion of the peptide (BRAHMS, Berlin, Germany). NT pro-BNP was measured using the automated Dimension Vista Intelligent Lab System method (Siemens Diagnostics, Nürnberg, Germany).
Mean inter-assay CVs were ≤10% for MR-proADM, ≤10% for MR-pro-ANP, and 2.7% for NT pro-BNP. Copeptin was measured using a commercially available assay in the chemiluminescence/coated tube format (BRAHMS AG, Henningsdorf, Germany). Lower detection limit was 0.4 pmol/L and functional assay sensitivity (<20% interassay CV) was <1 pmol/L.
Cystatin C was measured using a particle-enhanced immune-nephelometric assay (N Latex Cystatin, Siemens Diagnostics, Dade Behring, Deerfield, IL) with a mean inter-assay CV of 4.3%.
Statistical analysis
Quantitative normal and skewed distributed variables are presented as mean with standard deviation and median with interquartile range (IQR), respectively. Dichotomous variables are presented as count and proportion. Individuals with a diagnosis of carotid artery stenosis at baseline were excluded from the current study and prospective analyses included only incident carotid artery stenosis. Plasma biomarkers and confounders for incident clinically detected carotid artery stenosis were assessed using Cox regression models, and hazard ratios (HRs) were expressed per one standard deviation (SD) increment of each respective log transformed plasma biomarker (skewed distributed) entered one at a time in the Cox regression models. Analyses were performed using SPSS for Windows, version 24.0 (SPSS Inc, Chicago, IL). A p-value less than 0.05 was considered significant.
Results
Baseline conventional risk factor assessment
Seven individuals with either carotid stenosis at the baseline examination or with a previously registered diagnosis of carotid artery stenosis were excluded. The cumulative incidence of carotid artery stenosis was 2.3% (125/5543), 3.4% (75/2227) for men versus 1.5% (50/3316) for women (p < 0.001), during median follow up of 23.4 years (IQR 19.5–24.3). Sixty-five patients were operated for carotid artery stenosis, 38/2227 men (1.7%) and 27/3316 women (0.8%). Baseline risk factor characteristics for individuals in the cohort with or without carotid artery stenosis are shown in Table 1. When including the conventional risk markers listed in table 1 into a Cox regression analysis, age at baseline (HR 1.04; 95% CI 1.00–1.07), male gender (HR 2.71; 95% CI 1.79–4.09), current smoking (HR 2.33; 95% CI 1.55–3.50), hypertension (HR 2.63; 95% CI 1.54–4.50), and cholesterol (HR 1.28; 95% CI 1.07–1.54), were independently associated with incident carotid artery stenosis, whereas diabetes mellitus (HR 1.49; 95% CI 0.53–4.26), triglycerides (HR 0.97; 95% CI 0.78–1.21), and HbA1c (HR 1.21; 95% CI 0.99–1.49) were not.
Table 1Baseline characteristics in participants with and without development of carotid stenosis (CS) during follow-up.
Plasma biomarker levels at baseline in individuals with and without incident carotid artery stenosis are shown in table 1. In the Cox regression analysis, NT pro-BNP (HR 1.36; 95% CI 1.12–1.65; p = 0.002) was independently associated with incident carotid artery stenosis (table 2), whereas there were trends towards such associations for CRP (HR 1.20; 95% CI 0.98–1.48; p = 0.071) and MR-proADM (HR 1.21; 95% CI 0.99–1.47; p = 0.061). When the 55 individuals undergoing open surgery or stenting for carotid artery stenosis were analyzed separately, MR-proADM (HR 1.30; 95% CI 1.03–1.65; p = 0.029) was independently associated with incident operation for carotid artery stenosis, and there was a trend towards such an association for NT pro-BNP (HR 1.31; 95% CI 1.00–1.72; p = 0.052) (table 3).
Table 2Multi-variable adjusted hazards ratios for incident carotid artery stenosis in relation to plasma biomarkers.
Hazard ratios (HR) were expressed per one SD increment of each respective log transformed plasma biomarker biomarker entered one at a time in the analysis.
The following baseline variables were entered in the multivariable analysis besides each respective plasma biomarker: age, sex, body mass index, current smoking, diabetes mellitus, hypertension, cholesterol.
Hazard ratios (HR) were expressed per one SD increment of each respective log transformed plasma biomarker biomarker entered one at a time in the analysis.
The following baseline variables were entered in the multivariable analysis besides each respective plasma biomarker: age, sex, body mass index, current smoking, diabetes mellitus, hypertension, cholesterol.
Hazard ratios (HR) were expressed per one SD increment of each respective log transformed plasma biomarker entered one at a time in the analysis.
In our study, incident carotid artery stenosis could be independently predicted by NT pro-BNP during a median follow up of 23.4 years of middle-aged subjects from the background population. This variable therefore seems to not only be relevant as a risk marker in coronary ischaemia and heart failure,
but can also be used to predict development of carotid artery stenosis during long-term follow-up. In fact, however, our observation is a corroboration of previously presented relationships between NT pro-BNP and subclinical indices of carotid artery disease. High levels of NT pro-BNP levels have been related to height adjusted interadventitial diameter of the common carotid artery in elderly subjects without established cardiovascular disease,
The plasma protein profile and cardiovascular risk differ between intima-media thickness of the common carotid artery and the bulb: a meta-analysis and a longitudinal evaluation.
Such measures are seldom assessed in clinical practice, however, and no relationships could be demonstrated between NT pro-BNP and the presence of carotid plaques.
The plasma protein profile and cardiovascular risk differ between intima-media thickness of the common carotid artery and the bulb: a meta-analysis and a longitudinal evaluation.
regardless of whether the TIA was related to carotid artery stenosis or not, but it has been pointed out that such an association might be even more relevant for cardioembolic stroke.
Furthermore, the marker has also been studied in relation to the endpoint used in the present study, carotid artery stenosis. When NT pro-BNP was evaluated in patients after surgery for high-grade asymptomatic carotid artery stenosis, it was found to predict higher mortality during a mean follow up of 5.5 years in male patients,
N-terminal pro B-type natriuretic peptide (NT pro-BNP) is a predictor of long-term survival in male patients of 75 years and older with high-grade asymptomatic internal carotid artery stenosis.
whereas no such relationship could be demonstrated in women. This discrepancy is of special interest, as NT pro-BNP levels have been shown to be higher in women
N-terminal pro B-type natriuretic peptide (NT pro-BNP) is a predictor of long-term survival in male patients of 75 years and older with high-grade asymptomatic internal carotid artery stenosis.
In this context, it should be noted that our results were adjusted for both age and gender. Subjects in our study were also younger than in previously reported studies
N-terminal pro B-type natriuretic peptide (NT pro-BNP) is a predictor of long-term survival in male patients of 75 years and older with high-grade asymptomatic internal carotid artery stenosis.
on relationships between NT pro-BNP and carotid atherosclerosis.
When assessing our biomarkers as predictors of carotid artery stenosis necessitating open or endovascular surgical treatment, NT pro-BNP narrowly failed to reach statistical significance (p = 0.052) in the adjusted analysis. A significant result might well have emerged in a larger material. Carotid artery stenosis of such a magnitude that surgery was considered indicated could instead be predicted by MR-proADM, for which on the other hand only a trend (p = 0.061) towards significance had been detected in the entire group of patients diagnosed with carotid artery stenosis. MR-proADM levels are associated with progression of cerebral white matter lesions and cognitive decline,
whereas relationships between MR-proADM and carotid artery disease to the best of our knowledge have not been previously demonstrated. When MR-proADM was evaluated together with renin, aldosterone, and carotid IMT,
it was related to the former variables in non-diabetic subjects but no formal analysis was made concerning its potential relationships with carotid IMT. To compare the demonstrated relationship with biomarkers in all patients with carotid artery stenosis with those demonstrated in the subgroup of 55 patients that were operated for their stenosis is difficult, however, as the respective size and composition of the latter group is highly dependent upon both indications and contraindications for surgery and upon clinical practice in the study setting. The proportion of asymptomatic cases among patients undergoing carotid surgery in our country
et al; Society for vascular surgery vascular quality initiative. Regional variation in patient selection and treatment for carotid artery disease in the vascular quality initiative.
meaning that several patients with high degree asymptomatic stenosis that were not included in the surgery group in our study would perhaps have been operated in another health care system.
As expected, most of the conventional risk markers
such as age, male gender, smoking, hypertension, and cholesterol were also independently associated with incident carotid artery stenosis, whereas diabetes mellitus, triglycerides, and HbA1c were not. CRP, a well-established cardiovascular risk marker for stroke
Association of high sensitivity C-reactive protein with coronary heart disease prediction, but not with carotid atherosclerosis, in patients with hypertension.
been suggested that CRP is less useful for prediction of carotid atherosclerosis than for prediction of coronary heart disease in hypertensive subjects.
The long follow up is the main strength of our analysis, which of course also has several limitations. Subjects were invited in 1991–1994, without prior formal power calculation concerning detection of carotid artery stenosis during long-term follow-up. Furthermore, despite the fact that all subjects with an established diagnosis of carotid artery stenosis at study start were excluded from the study, asymptomatic carotid artery plaques and stenosis already at baseline cannot be excluded, as far from all carotid plaques cause embolic symptoms, leading to subsequent investigation and an established diagnosis. As there was no systematic bilateral assessment of carotid stenosis, neither at inclusion in the study, nor at follow-up, both presence of left-side asymptomatic carotid artery stenosis at study start and development of asymptomatic carotid artery stenosis during follow-up might therefore have remained undetected. The approach of using Cox regression analysis for first diagnosis of carotid artery stenosis, including a minor proportion of asymptomatic patients, can also be discussed. Therefore, a separate Cox regression analysis was performed on the operated patients, which in fact resulted in a strengthened association between MR-proADM and incident carotid artery stenosis. Ninety-nine percent of the cases of carotid stenosis could be validated in patient files, but we cannot exclude that some patients might have undergone ultrasound or radiographic imaging documenting a stenosis which was not coded as a diagnosis. Omission of correct coding is highly unlikely among operated cases, however.
Biomarkers analyzed in this material have previously been related to incident symptomatic arterial disease in the lower extremities.
In conclusion, levels of biomarkers such as NT pro-BNP and MR-proADM can be used as independent predictors for clinically detected carotid artery stenosis during long-term follow-up of subjects from the general population.
Grant support
The Malmö Diet and Cancer study was made possible by grants from the Swedish Cancer Society, the Swedish Medical Research Council, the Swedish Dairy Association, the Albert Påhlsson and Gunnar Nilsson Foundations and the Malmö city council. G.E. was supported by the Swedish Heart-Lung Foundation (2016–0315) and the Medical Faculty of Lund University. SA and AG were supported by grants from Research Funds at Skåne University Hospital, Region Skåne (430751), the Hulda Ahlmroth Foundation, and from the Swedish Government under the LUA/ALF agreement. The funding sources had no involvement in study design, analysis, interpretation, writing, or submission of the manuscript.
Declaration of Competing Interest
None.
Acknowledgements
The authors wish to thank all participants in the Malmö Diet and Cancer Study for making this study possible.
References
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Carotid artery stenosis: medical therapy, surgery, and stenting.
Interpretation of lipoprotein-associated phospholipase A2 levels is influenced by cardiac disease, comorbidities, extension of atherosclerosis and treatments.
et al; ESC study group on biomarkers in cardiology of the acute cardiovascular care association. use of copeptin for rapid rule-out of acute myocardial infarction.
Relation between insulin resistance and carotid intima-media thickness and stenosis in non-diabetic subjects. results from a cross-sectional study in Malmo, Sweden.
The plasma protein profile and cardiovascular risk differ between intima-media thickness of the common carotid artery and the bulb: a meta-analysis and a longitudinal evaluation.
N-terminal pro B-type natriuretic peptide (NT pro-BNP) is a predictor of long-term survival in male patients of 75 years and older with high-grade asymptomatic internal carotid artery stenosis.
The influence of age, sex and other variables on the plasma level of N-terminal pro brain natriuretic peptide in a large sample of the general population.
et al; Society for vascular surgery vascular quality initiative. Regional variation in patient selection and treatment for carotid artery disease in the vascular quality initiative.
Association of high sensitivity C-reactive protein with coronary heart disease prediction, but not with carotid atherosclerosis, in patients with hypertension.
Hazard ratios (HR) were expressed per one SD increment of each respective log transformed plasma biomarker biomarker entered one at a time in the analysis.
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