EP3365463A1

EP3365463A1 - Biomarker for predicting coronary artery disease in smokers

Info

Publication number: EP3365463A1
Application number: EP16787799.2A
Authority: EP
Inventors: Sara Johanna Pinto-Sietsma; Josephus Cornelis Maria Meijers; Maayke Guda Maria KOK; Perry MOERLAND; Esther Elisa Johanna Maria Creemers
Original assignee: Academisch Medisch Centrum
Current assignee: Academisch Medisch Centrum
Priority date: 2015-10-23
Filing date: 2016-10-24
Publication date: 2018-08-29
Also published as: WO2017068198A1

Abstract

The invention relates to a method for determining the risk of suffering from or developing a coronary artery disease (CAD), atherosclerosis or a cardiovascular disease of a smoker, comprising: determining in a biological sample from the smoker the expression level of mi R-124-3p, comparing said expression level with a reference level, and determining said risk based on said comparison.

Description

Biomarker for predicting coronary artery disease in smokers

Technical Field The present invention relates to the field of diagnostics. In particular, the invention relates to a method for determining whether a smoker is at risk of developing coronary artery disease (CAD), atherosclerosis or cardiovascular disease.

Background

Tobacco use is the most important avoidable cause of cardiovascular disease in the world. The risk of developing myocardial infarction (MI) is twice as high among smokers compared to non-smoking individuals. However, numerous smokers never develop cardiovascular complaints at all.

There are biomarkers known in the art for determining the risk of developing cardiovascular complaints in smokers. Serum adiponectin level was an independent predictor of early atherosclerosis in smokers (see Fan LH et al., Nutrition. 2015 Jul-Aug;31(7-8):955-8. doi: 10.1016/j.nut.2015.01.010. Epub 2015 Feb 26). There remains a need for further biomarkers for determining the risk of cardiovascular diseases in smokers.

It is therefore an object of the invention to provide an alternative biomarker for determining the risk of cardiovascular diseases in smokers.

Summary

The invention is based on the find that elevated levels of miR-124-3p are associated with subclinical atherosclerosis in smoking individuals. The inventors found that miR-124-3p was differentially expressed in smokers compared to non-smoking individuals. All non-smoking individuals had a low miR-124-3p expression level, whereas miR-124-3p expression was quite heterogeneous in smoking individuals (Fig 1A).

The inventors observed that in a cohort of smoking and former smoking individuals an increase in miR-124-3p expression level was related to a higher risk of subclinical atherosclerosis (OR 2.36; 95%

CI 1.23-4.50; p=0.009). They further found that currently smoking individuals have an even higher risk of subclinical atherosclerosis which is related with increased miR-124-3p levels, whereas in individuals that stopped smoking less than 5 years ago, no increased expression of miR-124-3p levels was observed. They further performed a flow cytometry analysis of monocyte surface markers which revealed that high miR-124-3p expression levels in smoking individuals were correlated to phenotypic changes in monocytes. Additional qPCR analyses showed that high miR-124-3p levels were associated with subclinical atherosclerosis in smoking individuals. It has been reported that smoking alters plasma miRNA profiles in healthy subjects¹⁰'¹¹. Additionally, it was disclosed that the plasma miRNAs miR-124 and let-7a showed increased expression in smoking individuals compared to controls¹². However, it was unknown that miR-124-3p expression levels are related to increased risk of subclinical atherosclerosis in smokers.

The invention provides a method for determining the risk of suffering from or developing coronary artery disease (CAD), atherosclerosis or a cardiovascular disease of a smoker, comprising:

determining in a biological sample from the smoker the expression level of miR-124-3p, comparing said expression level with a reference level, and determining said risk based on said comparison.

Preferably, said biological sample comprises whole blood, isolated monocytes.

In a preferred embodiment, said CAD is subclinical atherosclerosis. Preferably, said method further comprising assessing the expression level of a further expression marker in the smoker; and determining said risk based on the expression level of miR-124-3p and said further expression marker. Preferably, said expression marker is selected from the group consisting of CD206, CD29 and CD45RA.

Preferably, said method further comprising assessing a clinical factor of the smoker, wherein said clinical factor is associated with an increased risk of suffering from or developing CAD,

atherosclerosis or cardiovascular disease; and determining said risk based on the expression level of miR-124-3p and said clinical factor. Preferably, said clinical factor is selected from the group consisting of: age, gender, chest pain type, neutrophil count, ethnicity, disease duration, diastolic blood pressure, systolic blood pressure, a family history parameter, cholesterol level, diabetes, use of medication, a medical history parameter, a medical symptom parameter, height, weight, a body-mass index, CAC score, and resting heart rate.

Preferably, said expression level of said miRNA is normalized using one or more reference miRNAs. Preferably, said one or more reference miRNAs is selected from the group consisting of consisting of miR-130b and miR-342-3p. Preferably, the method comprising a step of amplifying miR-124-3p using reverse amplification and determining the amount of amplified products. The invention further provides a kit of parts, comprising at least one molecule capable of specifically binding to at least one miRNA selected from the group consisting of miR-124-3p, miR-130b and miR- 342-3p, and optionally one or more controls and/or one or more standards.

Brief description of the drawings

Figure 1 shows microarray miR-124-3p expression in monocytes of smoking and non-smoking individuals. It shows MiR-124-3p expression in non-smokers, individuals that stopped smoking <5 years ago and current smokers in (Fig. 1A) Cohort I and (Fig. IB) Cohort II. Triangles represent patients with atherosclerosis, whereas hexagons represent healthy controls. Figure 2 shows the correlation between miR-124-3p expression and monocyte surface markers.

Correlations between miRNA-124-3p and FACS markers (Fig 2A: CD45RA, classical monocytes; Fig. 2B: CD29, non-classical monocytes; Fig. 2C: CD206, classical monocytes; Fig. 2D: CD29, intermediate monocytes). Pearson correlation coefficient (r) and p-value of the log transformed monocyte surface marker and miRNA expression (log transformed) are displayed as well as the log- regression line.

Figure 3 shows the expression of monocyte surface markers after transfection with a miR-124-3p mimic. Expression of monocyte function markers after human monocyte induction with IL-4 and transfection with a miR-124-3p mimic. (Fig. 3A: CD206; Fig. 3B: CD64; Fig. 3C: CCR7; Fig. 3D: CD200R). * =P<0.05

Detailed description

Definitions

In accordance with the invention and as used herein, the following terms are defined with the following meanings, unless explicitly stated otherwise. The article "a" and "an" as used herein refers to one or to more than one (i.e., at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

The term "coronary artery disease" or "CAD" encompasses all forms of atherosclerotic disease affecting the coronary arteries. The term "atherosclerosis" refers to a form of arteriosclerosis in which deposits of yellowish plaques containing cholesterol, lipoid material , and lipophages are formed within the intima and innner media of large and medium-sized arteries.

As used herein the term "cardiovascular disease" is a disease of the blood vessels of the circulation system caused by abnormally high concentrations of lipids in the vessels.

The term "subclinical atherosclerosis" as used herein refers to an illness of an individual not exhibiting clinical symptoms, most preferably chest pain or ECG changes, indicative of stable angina, unstable angina, and/or myocardial infarction.

The term "smoker" as used herein means a human that has smoked in the past 5 years. In a preferred embodiment, said smoker is a current smoker. Preferably, said smoker has smoked at least 100 smokable tobacco products in his/her lifetime and preferably currently smokes tobacco every day (daily) or some days (nondaily).

The term "non-smoker" as used herein means a human that has not smoked in the past 5 years.

The term "biological sample", as used herein, refers to a sample obtained from an individual. The sample may be of any biological tissue, cells or fluid. Such samples include, but are not limited to, sputum, blood, serum, plasma, blood cells (e.g., white cells), tissue, nipple aspirate, core or fine needle biopsy samples, cell-containing body fluids, free floating nucleic acids, urine, peritoneal fluid, and pleural fluid, or cells there from. Biological samples may also include sections of tissues such as frozen or fixed sections taken for histological purposes or microdissected cells or extracellular parts thereof.

As used herein "determining the level of a certain miRNA in a sample" means assaying a test sample, e.g. a biological sample from a patient, in vitro to determine the concentration or amount of the miRNA in the sample. Any convenient qualitative, semi-quantitative or, preferably, quantitative detection method for determining nucleic acids can be used to determine the concentration or amount of the miRNA in the sample. A variety of methods for determining nucleic acids are well known to those of skill in the art, e.g. determination by nucleic acid hybridization and/or nucleic acid amplification. Exemplary methods to determine the concentration or amount of the miRNA in the sample are provided below.

The term "expression level" refers to a value that represents a direct, indirect, or comparative measurement of the level of expression of a nucleotide (e.g., RNA or DNA) or polypeptide. For example, "expression level" can refer to a value that represents a direct, indirect, or comparative measurement of the RNA expression level of a miRNA marker of interest. The term "miR-124-3p" or "hsa-miR-124-3p" as used herein refers to a miRNA having the nucleic acid sequence of UAAGGCACGCGGUGAAUGCC (SEQ ID NO: l).

The term "reference level" or "reference value" as used herein refers to the level of expression of miR- 124-3p which is indicative for the risk a smoker of suffering from coronary artery disease (CAD), atherosclerosis or cardiovascular disease.

The term "reference miRNA" as used herein refers to a miRNA which is stably expressed in the cells of the biological sample.

Embodiments

The concentration or amount of the miRNAs in the sample may be directly determined in the sample, that is, without an RNA extraction step. Alternatively, RNA may be extracted from the sample prior to miRNA processing for detection. RNA may be purified using a variety of standard procedures as described, for example, in RNA Methodologies, A laboratory guide for isolation and characterization, 2nd edition; 1998, Robert E. Farrell, Jr., Ed., Academic Press. In addition, there are various processes as well as products commercially available for isolation of small molecular weight RNAs, including miRNeasy™ kit (Qiagen), MagMAX™ kit (Life Technologies), Pure Link™ kit (Life Technologies), and mirVANA™ miRNA Isolation Kit (Ambion). For example, small molecular weight RNAs may be isolated by organic extraction followed by purification on a glass fiber filter. Alternative methods for isolating miRNAs include hybridization to magnetic beads.

The determination of the risk of coronary artery disease (CAD), atherosclerosis or cardiovascular disease is based on comparing the expression level(s) of the miRNAs in the smoker's biological sample with those obtained using relevant controls. A person skilled in the art may choose a control suitable for this purpose, including but not limited to internal standards, samples of purified monocytes from subjects known to be suffering from coronary artery disease (CAD), atherosclerosis or cardiovascular disease. It is not necessary to determine both the reference value and the test value at the same time. It is for instance possible to determine a reference value, preferably using a sample from a subject not suffering from, or at risk of suffering from, a coronary artery disease (CAD), atherosclerosis or cardiovascular disease, and use said reference value over and over again to determine whether or not the relative expression of miR-124-3p in a test sample is indicative for a smoker at risk of suffering from coronary artery disease (CAD), atherosclerosis or cardiovascular disease. It is possible to use as a reference value a level of expression that has been shown to be indicative for a low risk of suffering from a coronary artery disease (CAD), atherosclerosis or cardiovascular disease, for instance the mean level of miRNA in a number of samples from healthy individuals. On the other hand it is also possible to use as a reference value a level of expression that has been shown to be indicative for cardiovascular disease or for a high risk of suffering from a cardiovascular disease. Such reference value is for instance obtained using a sample of an individual suffering from a coronary artery disease (CAD), atherosclerosis or cardiovascular disease. It is thus possible to determine whether a subject is at risk of suffering from a coronary artery disease (CAD), atherosclerosis or cardiovascular disease, by comparing the level of expression of any of the above mentioned miRNA in a sample of the smoker with the level of expression in a sample of a healthy individual or with the level of expression in a sample of an individual suffering from a cardiovascular disease. With a "individual suffering from, or at risk of suffering from, a coronary artery disease (CAD), atherosclerosis or cardiovascular disease " is meant a subject which is already diagnosed with cardiovascular disease and/or has an increased risk relative to the normal population of suffering from cardiovascular disease. A level of expression which is comparable with that of a healthy individual is indicative for said subject being not at risk of a coronary artery disease (CAD), atherosclerosis or cardiovascular disease. A level of expression which is comparable with that of an individual suffering from a coronary artery disease (CAD),

atherosclerosis or cardiovascular disease is indicative for said subject being at risk of suffering from cardiovascular disease. With comparable is meant that the levels of expression are preferably less than 1.8 fold different, more preferably less than 1.5 fold different, more preferably less than 1.3 fold different, most preferably less than 1.2 fold different.

The step of determining whether a smoker is at risk of developing coronary artery disease (CAD), atherosclerosis or cardiovascular disease may suitably be based on the information obtained by comparison between the expression level with a reference level. The expression level of the analysed miRNA when statistically analysed will have a threshold whereby expression levels of the individual miRNAs below or above the threshold are indicative for respectively the presence or absence of CAD, atherosclerosis or cardiovascular disease. Threshold miRNA levels for each of the analysed miRNAs can be determined by any suitable algorithm. Such an algorithm may involve classifying a sample between smokers who suffer from a coronary artery disease (CAD), atherosclerosis or cardiovascular disease and healthy control group. For example, samples may be classified on the basis of threshold values, or based upon Mean and/or Median miRNA levels in smokers suffering from CAD, atherosclerosis or cardiovascular disease versus non-smokers (e.g., a cohort from the general population or a patient cohort with diseases unrelated to CAD, atherosclerosis or cardiovascular disease). Various classification schemes are known for classifying samples between two or more groups, including Decision Trees, Logistic Regression, Principal Components Analysis, Naive Bayes model, Support Vector Machine model, and Nearest Neighbour model. In addition, the predictions from multiple models can be combined to generate an overall prediction. The miRNA expression level (miRNA signature, level, or miRNA concentration) is generated (determined) from (in) the biological-sample using any of various methods known in the art for quantifying miRNA levels. Such methods include polymerase-based assays, such as Real-Time PCR (e.g., Taqman™), hybridization-based assays, for example using microarrays (e.g. miRNome microRNA Profilers QuantiMir Human PCR array (Biocat)), nucleic acid sequence based

amplification (NASBA), flap endonuclease -based assays, as well as direct RNA capture with branched DNA (QuantiGene™), Hybrid Capture™ (Digene), or nCounter™ miRNA detection (nanostring). The assay format, in addition to determining the miRNA levels will also allow for the control of, inter alia, intrinsic signal intensity variation. Such controls may include, for example, controls for background signal intensity and/or sample processing, and/or hybridization efficiency, as well as other desirable controls for quantifying miRNA levels across samples (e.g., collectively referred to as "controls"). Many of the assay formats for amplifying and quantitating miRNA sequences, and thus for generating miRNA levels are commercially available and/or have been described, e.g. in WO 2008/153692, WO 2010/139810, and WO 2011/163214, or references cited therein. Although the miRNA tested for is indicated as RNA sequence, it will be understood that, when referring to hybridizations or other assays, corresponding DNA sequences can be used as well. For example, RNA sequences may be reverse transcribed and amplified using the polymerase chain reaction (PCR) in order to facilitate detection. In these cases, it will actually be DNA and not RNA that is directly quantitated. It will also be understood that the complement of the reverse transcribed DNA sequences can be analysed instead of the sequence itself. In this context, the term "complement" refers to an oligonucleotide that has an exactly complementary sequence, i.e. for each adenine there is a thymine, etc.

Also preferred, the method of the invention can further comprise determining the level of one or more normalization control(s) in the sample. Preferably, the sample is spiked with the normalization control(s).

In a preferred embodiment, the normalization control is a non-endogenous RNA or miRNA, or a miRNA not expressed in the sample. For example, the normalization control may be one or more exogenously added RNA(s) or miRNA(s) that are not naturally present in the biological sample, e.g. an RNA or miRNA from another organism, and/or one or more human miRNAs not expressed in the sample-sample undergoing analysis. In a highly preferred embodiment, said level of the miRNA of the invention is normalized using one or more reference miRNAs which are stably expressed in whole blood. Preferably, said one or more reference miRNAs is selected from the group consisting of miR- miR-130b and miR-342-3p. The normalization may suitably be performed as described in European patent application EP15166257. In the method of the invention, the miRNA level (or miRNA concentration) is preferably determined by an amplification-and/or hybridization-based assay. The amplification- and/or hybridization-based assay can be quantitative miRNA real-time polymerase chain reaction (RT-PCR), e.g. TaqMan. The miRNA level may also be determined by preparing cDNA, followed by RT-PCR. In addition to the miRNA 124-3p biomarker, all variant sequences having at least 70, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or greater identity to said marker sequence. The percentage of sequence identity may be determined using algorithms well known to those of ordinary skill in the art, including, e.g., BLASTn, and BLASTp, as described in Stephen F. Altschul et al, J. Mol. Biol. 215:403-410 (1990) and available at the National Center for

Biotechnology Information website maintained by the National Institutes of Health. In accordance with an embodiment of the invention, all operable markers and methods for their use in identifying subjects at risk of CAD now known or later discovered to be highly correlated with the expression of an exemplary marker can be used in addition to that exemplary marker. For the purposes of the invention, such highly correlated markers are contemplated to be within the literal scope of the claimed inventions or alternatively encompassed as equivalents to the 124-3p biomarker. Identification of markers having expression values that are highly correlated to those ofl24-3p biomarker, and their use in identifying a subject at risk of CAD is well within the level of ordinary skill in the art.

In a preferred embodiment, a method according to the invention is provided, comprising determining whether the level of expression of said miRNA in said biological sample is at least 1.50 fold increased relative to the level of expression of said miRNA in a sample of a healthy individual not at risk of cardiovascular disease. In a more preferred embodiment, it is determined whether said level of expression is at least 1.55, more preferably at least 1.60, more preferably at least 1.65, more preferably at least 1.75, most preferably at least 1.85 fold increased.

In an embodiment, a condition can include one clinical factor or a plurality of clinical factors. In an embodiment, the invention can include assessing a clinical factor in a subject and combining the assessment with an analysis of the first dataset (see above) to identify risk of CAD atherosclerosis or cardiovascular disease in the smoker. In an embodiment, a clinical factor can be included within a dataset, e.g., the first dataset. A dataset can include one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or more, sixteen or more, seventeen or more, eighteen or more, nineteen or more, twenty or more, twenty-one or more, twenty-two or more, twenty-three or more, twenty-four or more, twenty-five or more, twenty-six or more, twenty- seven or more, twenty-eight or more, twenty-nine or more, or thirty or more overlapping or distinct clinical factor(s). A clinical factor can be, for example, the condition of a subject in the presence of a disease or in the absence of a disease. Alternatively, or in addition, a clinical factor can be the health status of a smoker. Alternatively, or in addition, a clinical factor can be age, gender, chest pain type, neutrophil count, ethnicity, disease duration, diastolic blood pressure, systolic blood pressure, a family history parameter, a medical history parameter, a medical symptom parameter, height, weight, a body- mass index, and resting heart rate. Clinical factors can include whether the subject has stable chest pain, whether the subject has typical angina, whether the subject has atypical angina, whether the subject has an anginal equivalent, whether the subject has been previously diagnosed with MI, whether the subject has had a revascularization procedure, whether the subject has diabetes, whether the subject has an inflammatory condition, whether the subject has an infectious condition, whether the subject is taking a steroid, whether the subject is taking an immunosuppressive agent, and/or whether the subject is taking a chemo therapeutic agent.

The invention further provides a kit for quantifying the amount of miR-124-3p in a biological sample comprising an amplification primer set, comprising at least one primer comprising a sequence that is complementary to a portion of miR-124-3p. In the method of the invention, the miRNA level (or miRNA concentration) is preferably determined by an amplification-and/or hybridization-based assay. The amplification- and/or hybridization-based assay can be quantitative miRNA real-time polymerase chain reaction (RT-PCR), e.g. TaqMan. The miRNA level may also be determined by preparing cDNA, followed by RT-PCR.

The kit for determining the risk of suffering from or developing a coronary artery disease (CAD), atherosclerosis or a cardiovascular disease of a smoker according to the invention may comprise means for determining the concentration (expression level) of miR-124-3p in a biological sample from a subject. The means for determining the concentration of these miRNAs; or miRNA-specific primers for reverse transcribing or amplifying of miR-124-3p. For example, the means for determining the concentration of miR-124-3p may be TaqMan probes. Further, the kit may comprise an enzyme for cDNA preparation (e.g. , reverse transcriptase) and/or PCR amplification (e.g., Taq polymerase), and/or a reagent for detecting and/or quantifying miRNA. Additionally, the kit may further comprise include a reagent for miRNA isolation from samples. The kit can also comprise one or more normalization control(s). The normalization control(s) can, for example, be provided as one or more separate reagent(s) for spiking samples or reactions. Preferably, the normalization control(s) is/are selected from non-endogenous RNA or miRNA, or a miRNA not expressed in the sample. In a preferred embodiment, said kit comprises a specific primer for reverse transcribing or amplifying one or more reference miRNAs is selected from the group consisting of miR-130b and miR-342-3p. The design of oligonucleotide probes specific for miR-124-3p, miR-130b and miR-342-3p; or miRNA-specific primers for reverse transcribing or amplifying each of miR-124-3p, miR-130b and miR-342-3p to detect their expression levels (concentrations) in accordance with suitable assay formats is well known to those of skill in the art, and appropriate probes and/or primers can be commercially purchased.

It is especially preferred to combine the preferred embodiments of the present invention in any possible manner.

Example

We performed a miRNA microarray experiment on isolated monocytes of 2 independent cohorts of patients with premature CAD and healthy controls and found that miR-124-3p was heterogeneously expressed among smoking individuals, whereas expression levels were low in non-smokers.

Subsequent flow cytometry analysis showed that high miR-124-3p levels were associated with up- regulation of the monocyte surface markers CD45RA, CD29 and CD206, suggesting an altered function of these monocytes, which may contribute to the development of atherosclerosis. Next, we performed a RT-qPCR experiment on whole blood from 2 new independent cohorts and showed that increased miR-124-3p levels were associated with subclinical atherosclerosis in smoking individuals, but not in non-smoking individuals.

We showed that elevated levels of miR-124-3p are predictive for subclinical CAD in smoking individuals and that this miRNA is therefore a candidate biomarker for CAD in smoking individuals.

Cohorts Cohort I consisted of 40 male premature coronary artery disease (CAD) patients and 40 age- matched male controls. Premature CAD was defined as a first cardiovascular event before the age of 51. These individuals might have a genetic predisposition for atherosclerosis, which may be accelerated by smoking. Patients between the age of 35 and 65 years who had a premature cardiovascular event and were willing to participate were included from the outpatient clinic for premature CAD in the Academic Medical Center in Amsterdam between December 2009 and June 2010. Sample collection was performed on average 5.6 ± 3.5 years after the first cardiovascular event. Control individuals were recruited by advertisement and were matched for age. Controls were eligible for participation if they did not have a personal or family history for CAD and did not use any medication. To study the effect of medication on miRNA expression levels, we administered 100 mg of acetyl salicylic acid, once daily, for two weeks, and simvastatin 40 mg, once daily, for 6 weeks, of which the last 2 weeks in combination with the acetyl salicylic acid. Blood samples were collected at baseline in the absence of aspirin and statins and after six weeks of medication use.

Cohort II consisted of 4 families with a high prevalence of premature CAD, of which CAD patients were treated at the outpatient clinic for premature CAD. From these families we included 21 patients with established CAD and 46 apparently healthy family members. All family members were screened at the outpatient clinic and underwent a coronary CT-scan to assess the coronary calcification score (CAC) as a marker for premature CAD. CAD cases were defined as patients with established atherosclerosis (n=21) and family members with a CAC score > 80^th percentile corrected for age and gender (n=6). Controls were defined as healthy family members without any signs of complaints of CAD and a CAC score < 80^th percentile (n=41). This group also included subjects under the age of 30 years, in whom no CAC score was measured. Since atherosclerosis is usually not as pronounced at a young age, CAC score determination is not sensitive enough in those individuals and therefore a low CAC score will not rule out the presence of advanced atherosclerosis.

Cohort III To investigate the effect of smoking on monocyte function we performed fluorescence- activated cell sorting (FACS) on isolated monocytes of 22 healthy smoking first degree relatives (FDRs) of patients with premature CAD that visited the outpatient clinic of premature CAD at the Academic Medical Center (AMC) in Amsterdam. All selected subjects were current smokers or had stopped smoking <5 years ago, according to the information provided by a questionnaire. We choose to include subjects that quit smoking<5 years ago, since the adverse effect of smoking on CAD is known to last during this period. To exclude the effect of statins on the monocytes, subjects that used statins were asked to discontinue statin therapy 6 weeks prior to their visit. This was only asked if discontinuation of therapy for a short period of time was medically justified, which was the case in all individuals. Due to technical reasons, two individuals were excluded from this cohort.

Cohort TV consisted of 71 FDRs of subjects with premature CAD that visited the outpatient clinic for risk assessment between April 2010 and May 2013. We only selected smoking FDRs or FDRs that quit smoking <5 years ago. Of these FDRs, 39 individuals had a CAC score >80^ώ percentile corrected for age and gender and were considered as having subclinical atherosclerosis. The remaining 32 FDRs had a CAC score of zero and were considered healthy controls.

Cohort V consisted of 65 non-smoking FDRs of subjects with premature CAD. Selection criteria were similar to those in cohort IV. Of these FDRs, 28 individuals had a CAC score >80^th percentile corrected for age and gender and were considered as having subclinical atherosclerosis. The remaining 37 FDRs had a CAC score of zero and were considered healthy controls. Sample collection and processing

In the microarray Cohorts I and II, circulating monocytes were isolated from peripheral blood samples. Non-fasting venous blood was drawn in CTAD tubes (Becton Dickinson, Alphen aan de Rijn, the Netherlands) and centrifuged for 20 minutes at 163 g at 20°C. The buffy coat was collected and monocytes were positively selected with CD14+ Dynal beads (Invitrogen, Dynal Biotech, Oslo, Norway) according to the manufacturer's instructions. Monocytes were incubated in RNA later (Ambion) for 45 minutes. Subsequently, ice-cold PBS was added in a 1: 1 volume ratio and the samples were centrifuged for one minute at 5000 g, after which monocytes were obtained and snap- frozen in liquid nitrogen. Monocyte RNA was isolated using the ra rVana PARIS kit (Ambion, Inc.), according to the manufacturer's protocol.

For the flow cytometry analysis in Cohort III monocytes were isolated from fresh whole blood samples through density centrifugation using Lymphoprep™ (Axis-Shield). CD 14+ monocytes were isolated using human CD 14 magnetic beads (Miltenyi) and MACS® cell separation columns

(Miltenyi) according to the manufacturer' s instruction.

RNA was isolated from CD 14+ monocytes using TRIzol® Reagent (Life Technologies) according to the manufacturer' s instruction.

In Cohorts IV and V whole blood samples were collected in EDTA tubes (Becton Dickinson, Alphen aan de Rijn, the Netherlands) and directly stored at -80°C. Whole blood RNA was isolated using the miREasy Mini kit (Qiagen), according to the manufacturer' s protocol.

MiRNA microarray

The integrity of total RNA including miRNAs from monocytes was investigated with the BioAnalyzer (Agilent Technologies) using the RNA 6000 Pico kit (Agilent Technologies) and Small RNA kit (Agilent Technologies) according to the manufacturer's instructions.

100 ng of total RNA including microRNAs was dried down in a Centrivap concentrator (Labconco) and dissolved in 2 μΐ RNase-free water. Sample labeling with Cy3 was performed as described in the miRNA Microarray System with miRNA Complete Labeling and Hyb Kit manual version 2.2 (Agilent Technologies) with the inclusion of spike -ins and the optional desalting step with spin columns (Micro Bio-Spin 6, Bio-Rad). Labeled samples were hybridized on Human 8x15k miRNA microarrays based on Sanger miRBase release 19.0 containing 866 human and 89 human viral miRNAs (G4470C, Agilent Technologies) at 55°C and 20 rpm for 20 hours. After washing, the arrays were scanned using the Agilent DNA microarray scanner (G2565CA, Agilent Technologies). Data were extracted with Feature Extraction software (vlO.7.3.1 , Agilent Technologies) with the miRNA_107_Sep09 protocol for miRNA microarrays. miRNA microarray pre-processing and analysis

Microarrays for cohort I and II were pre-processed and analysed separately. A two-step normalisation approach was taken. In the first step, we corrected for systematic technical effects in the raw probe- level data as extracted via the Agilent Feature Extraction software. For this purpose, we fitted a linear mixed-effects model with coefficients for two technical effects (slide and slide position, that is, upper or lower half of a slide), and patient status using the R/MAANOVA package. Residuals after correcting for the two technical effects were further pre-processed and summarized using a modified version of the robust multi-array average (RMA) method with background correction, as implemented in the AgiMicroRna R package¹⁴. Quality control was performed using the arrayQualityMetrics R package. Based on arrayQualityMetrics outlier detection and visual inspection of heatmaps, MA-plots, and intensity distributions, seven arrays were excluded from further analysis for cohort I and one array for cohort II. Data from the remaining arrays were renormalized using the two-stage procedure described above. Only non-control miRNAs detected on at least one array according to Agilent Feature Extraction software were included in the further analysis. Normalized expression values of technical replicate arrays were averaged. To detect miRNAs differentially expressed between smokers and non-smokers, gene-wise linear models were fit with smoking status as explanatory variable corrected for patient status (case/control), BMI and age followed by a moderated t-test (limma R package). Resulting p-values were adjusted to correct for multiple hypothesis testing using the Benjamin-Hochberg false discovery rate.

Flow cytometry analyses

Blood samples were obtained by peripheral venipuncture and collected into K3EDTA BD Vacutainer (BD Biosciences, San Jose, CA) tubes. After red blood cell lysis (eBioscience lx RBC lysis buffer), cells were washed and incubated with fluorescently labeled antibodies for 20 minutes at room temperature in the dark. We used antibodies against the following surface markers: CDl lb, CDl lc, CD18, CD29, CD32, CD36, CD45RA, CD49d, CD80, CD86, CD163, CD206, CCR5, CCR7, CX3CR1, SRA and TLR4 (Supplementary table 1). After an additional wash, fluorescence was measured with a BD Canto II and analyzed with FlowJo. CountBright Absolute Counting Beads (Life technologies) were used to determine absolute cell counts as described by the manufacturer.

Compensation was performed with Ebioscience OneComp eBeads. Monocytes were gated using a protocol that was adapted from a previous study¹⁵. Briefly, cells were first plotted on a FCS/SSC plot and a first gate (A) was drawn to exclude the majority of debris, residual red blood cells and granulocytes. These cells were next viewed on a CD14/CD16 plot to gate CD 14+ and/or CD 16+ cells (B). When cells from gate B were viewed on a CD16/HLA-DR plot, monocytes (gate C) were easily distinguished from contaminating cells ('not monocytes'). The final monocyte population was viewed again on a CD14/CD16 plot to gate CD14++/CD16-, CD14++/CD16+ and CD14dim/CD16+ monocyte subsets. In addition to the percentage of each subset and their absolute cell count, the expression of various surface markers was assessed and calculated as AMFI = [median fluorescence intensity] _{positive staining} - [median fluorescence intensity]i_{SOtype∞ntrol}.

MiR-124-3p mimic experiment

Human monocytes were isolated from Buffy coats (Sanquin) via density centrifugation and CD 14+ beads isolation as described above, cultured in IMDM complete (25mM HEPES + 10% Fetal Calf Serum, +1% L-glutamine, +1% penicillin streptomycin) and stimulated half of the cells with 50ng/ml MCSF (Miltenyi). On day 6, cells were transferred to a 12-wells plate in a density of 1 · 10⁶ cells/ml. Hereafter, monocytes and macrophages were transfected with 20 μΜ miRIDIAN hsa-miR-124-3p mimic (Dharmacon, cat# C-300592-05) or 20 μΜ negative control #1 mimic (Dharmacon, cat# CN- 001000-01-05) using the Lipofectamine 2000 transfection reagent (Invitrogen) in triplicate.

Additionally, a subset of cells were induced with IL-4 (50ng/ml) for 24 hours after transfection with the negative control mimic. 24 hours after transfection FACS analyses of the cells was performed as described above. We determined expression of CD64, CCR7, CD200R, CD206, CD29 and CD45RA).

Quantitative PCR MiR-124-3p specific reverse transcription was performed on lOOng of purified total RNA, including miRNAs, using the TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems, Gent, Belgium). RT-qPCR reactions were carried out in duplicate, on a LightCycler 480 system II (Roche, Basel, Switzerland). Data were analysed using LinRegPCR quantitative PCR data analysis software, version 11¹⁶ MiR-124-3p expression was normalized to the geometric mean of a previously established miRNA normalization panel for whole blood samples consisting of miR-130b and miR-342-3p¹⁷.

Additionally, we determined CD29 and CD45RA mRNA expression in monocytes and macrophages from the mimic experiment. qPCR was performed using SYBR Green according to the manufacture's instructions. Statistical analysis

Results are expressed as mean ± standard deviation, except when indicated otherwise. Student's t-tests and Chi-square tests were used to calculate differences in baseline characteristics. Variables with a skewed distribution, e.g. the miRNA expression levels, were log transformed before they were analysed. We used Pearson correlation to analyze the correlation between miRNA expression levels and monocyte surface markers.

To analyze differences in monocyte surface marker expression between the miR-124-3p mimic and the negative control treated monocytes, we used one-way ANOVA and Tukey post-hoc tests.

We used a logistic regression model to analyse the relation between miRNA expression levels and the presence of subclinical atherosclerosis in FDRs. This model was adjusted for age, gender and medication use. All analyses were performed using SPSS for Windows 19.0. A p-value < 0.05 was considered statistically significant.

Results

MiRNA microarrays show elevated miR-124-3p levels in smoking individuals Clinical characteristics of the individuals included in Cohort I are displayed in Table 1. In this cohort 17.5% of the included individuals were current smokers. Most of these smoking individuals were CAD patients (n=10, 71.4%). Furthermore, hypercholesterolemia and hypertension were significantly more often present in CAD patients compared to healthy controls.

MiRNA microarray analysis of isolated monocytes in Cohort I showed that miR-124-3p was differentially expressed in smokers compared to non-smoking individuals (adjusted Ρ=1.22·10^~12). All non-smoking individuals had a low miR-124-3p expression level, whereas miR-124-3p expression was quite heterogeneous in smoking individuals (Fig 1A). In this analysis we did not observe any differences in monocyte miR-124-3p expression between CAD patients and healthy controls.

Interestingly, individuals who quit smoking less than 5 years ago (n=13, 16.3%) also had a low miR- 124-3p expression level, suggesting that the effect of smoking on miRNA 124-3p is only relatively short. There was no difference in miR-124-3p expression before and after medication use.

Additionally, we analysed the miRNA microarray data for isolated monocytes of a second, independent cohort. Clinical characteristics of Cohort II are displayed in Table 1. In this cohort 19% (n=13) of the individuals were current smokers. 40% of the included subjects were CAD patients, of which 44% (n=12) were smokers. In the group of healthy controls 27% of the individuals were smokers (n=l l). Furthermore, CAD patients were significantly older, had more often hypertension and did more often use medication compared to healthy controls (Table 1).

We observed exactly the same effect as for the micro-array data of Cohort I. MiR-124-3p expression was low in all non-smokers, whereas it was heterogeneously expressed among smokers (adjusted P=0.0007). No differences were seen in monocyte miR-12-3p expression between CAD patients and healthy controls. Again, individuals who had stopped smoking less than 5 years ago showed low miR- 124-3p expression levels (fig. IB).

Flow cytometry analysis reveals a correlation between miR-124-3p expression and monocyte surface markers Since miR-124-3p was heterogeneously expressed within the group of smoking individuals in both independent microarray cohorts, we tested whether miRNA 124-3p expression in smoking individuals was associated with the expression of monocyte surface markers. To investigate this, we performed flow cytometry on whole blood of subjects from Cohort III and gated for monocytes. We correlated the expression of the monocyte surface markers to the miR-124- 3p expression in isolated monocytes as determined by RT-qPCR. Baseline characteristics of Cohort III are displayed in Table 1. In individuals with a high miR-124-3p expression we observed an increase in the monocyte surface marker CD45RA, CD29 and CD206. CD45RA, which is present on activated monocytes¹⁸, was increased in the classical monocyte subtype (p=0.57; p=0.008). ( 1)29 was increased in the non- classical monocyte subtype (p=0.59, p=0.006). ( 1)29 is part of the LH integrin very late antigen-4 (VLA-4) which is also present on activated monocytes¹⁹. ( 1)206 was increased in both the classical and intermediate monocyte subtype (p=0.47, p=0.038 and p=0.53, p=0.017 respectively) (Figure 2). CD206 or the mannose receptor, is a marker of the alternatively activated (M2) macrophages²⁰.

CD206 and CD64 are up regulated after stimulation of human macrophages with a miR-124-3p mimic

To investigate whether up regulation of miR-124-3p indeed leads to an upregulation of the markers CD45RA, CD29 and CD206, we overexpressed miR-124-3p using miRNA mimic transfections in human monocytes and macrophages. Transfection with the miR-124-3p mimic resulted in a significant increase of CD206 expression (p=0.01) (Figure 3A), which is in line with the flow cytometry results, where high CD206 levels correlated with high miR-124-3p levels. Additionally, we observed a significant increase in CD64 expression after miR-124-3p overexpression (p=0.004) (Figure 3B). Expression of the Ml marker CCR7 and the M2 marker CD200R were not altered after miR-124-3p overexpression (Figure 3C and 3D). The monocyte activation markers CD29 and CD45RA were not expressed on these macrophages.

Similar results were observed when human monocytes were not stimulated using MCFS to become macrophages upon transfection with a miR-124-3p mimic. Since it has been reported that IL-4 stimulation of macrophages leads to increased miR-124 expression we investigated whether the monocyte surface markers are upregulated after stimulation with IL-4. As expected, IL-4-induced macrophage polarization resulted in a significant down regulation of the Ml marker CD64 (p=0.026) and a significant increase in the expression of M2 markers CD206 (p<0.001) and CD200R (p<0.001). Expression of CCR7, also a Ml macrophage marker, was not altered by IL-4 induction (Figure 3). Furthermore, expression of miR-124-3p, did not change after incubation with IL-4. This implies that in human monocytes and macrophages, miR-124-3p results in altered surface marker expression through a pathway independent of IL-4. CD29 and CD45RA mRNA expression is not altered after stimulation of human macrophages with a miR-124-3p mimic

CD29 and CD45RA expression was upregulated in monocytes of individuals with high miR-124-3p expression of Cohort III, but not after monocyte and macrophage transfection with a miR-124-3p mimic. We hypothesized that this could be due to the short duration of incubation with the mimic. Alterations in mRNA expression might be ahead of surface marker expression. Therefore, we determined CD29 and CD45RA mRNA expression in human monocytes and macrophages transfected with a miR-124-3p mimic. In this experiment we did not observe differences in mRNA expression between miR-124-3p transefected cells and controls. MiR-124-3p expression levels in whole blood predict risk of subclinical atherosclerosis in smokers

Since we showed that miR-124-3p was heterogeneously expressed in circulating monocytes of smoking individuals and that high levels of miR-124-3p correlated with changes in monocyte surface markers, we hypothesized that an increase in miR-124-3p expression is related to subclinical atherosclerosis. Therefore, we related whole blood miR-124-3p expression levels to coronary calcium abnormalities as measured by coronary CT scanning. We choose to analyse this in whole blood samples for practical reasons, since isolated monocytes are not easily obtained in clinical daily practice.

We determined miR-124-3p expression levels by RT-qPCR in Cohort IV, which consisted of 44 smoking FDRs and 27 FDRs that stopped smoking <5 years ago. Of these, 39 had subclinical atherosclerosis and 32 did not. Characteristics of Cohort IV are shown in Table 1. Among the individuals with subclinical atherosclerosis there were significantly more diabetes patients and they more often used medication compared to individuals without subclinical atherosclerosis (Table 1).

Logistic regression analysis showed that, in a cohort of smoking and former smoking individuals an increase in miR-124-3p expression level was related to a higher risk of subclinical atherosclerosis (OR 2.36; 95% CI 1.23-4.50; p=0.009). Since individuals that stopped smoking < 5 years ago from Cohort I and II showed low miR-124-3p expression levels on the miRNA microarray, thereby resembling non-smoking individuals from these cohorts, we repeated the analyses in smokers and individuals that stopped smoking <5 years ago from Cohort IV separately. We found that in currently smoking individuals an even higher risk of subclinical atherosclerosis could be observed with increasing miR- 124-3p levels (OR 3.24; 95% CI 1.23-8.46; p=0.016), whereas in individuals that stopped smoking <5 years ago this association could not be observed (OR 1.36; 95% CI 0.42-4.43; p=0.61). This observation underlines the short-lasting effect of smoking on monocytes. We repeated the analysis in Cohort V. This cohort consisted of 65 FDRs of subjects with premature CAD that had never smoked, of which 31 had subclinical atherosclerosis and 38 did not.

Characteristics of Cohort V are shown in Table 1. Individuals with subclinical atherosclerosis had significantly more often hypertension and hypercholesterolemia compared to controls for which they significantly more often used medication (Table 1).

We did not observe a relation between higher miR-124-3p expression levels and subclinical atherosclerosis in non-smoking individuals (OR 1.35; 95% CI 0.43-4.30; p=0.61). This indicates that the observed association between miR-124-3p expression levels and the risk of subclinical atherosclerosis among smoking individuals is likely to be related to smoking, and not to CAD itself. Discussion

In this study we showed that miR-124-3p expression is heterogeneously expressed in monocytes of smoking individuals in two independent cohorts and that expression of miR-124-3p positively correlates with the expression of the surface markers CD29, CD45RA and CD206, suggesting a potential role for miR-124-3p in the development of atherosclerosis. Finally, we showed that elevated levels of miR-124-3p are associated with subclinical atherosclerosis in smoking individuals.

In two independent miRNA microarray experiments we found that miR-124-3p was heterogeneously expressed in monocytes of smoking individuals, whereas in non-smokers miR-124-3p expression was low.

To investigate a possible relation between elevated miR-124-3p levels and atherosclerosis we performed a flow cytometry analysis of monocyte surface markers which revealed that high miR-124- 3p expression levels in smoking individuals were correlated to phenotypic changes in monocytes. Additional qPCR analyses showed that high miR-124-3p levels were associated with subclinical atherosclerosis in smoking individuals.

MiR-124 levels were previously reported to be elevated in plasma of smoking individuals¹¹. In this study by Banerjee and colleagues, a dose -dependent effect on miR-124 expression was reported, with an increase in miR-124-3p expression with each additionally smoked cigarette. Here, we report a heterogeneous expression of miR-124-3p in smoking individuals, whereas miR-124-3p expression was low in non-smokers. A dose-dependent effect of cigarette smoke was not specifically analysed, but it could very well be that the heterogeneous expression reflects the dose-dependent effect that was reported by Banerjee. Interestingly, analogous to Banerjee's study, we also observed that subjects who quit smoking showed low miR-124-3p levels, suggesting a short, direct effect of smoking on monocyte miR-124-3p expression. Flow cytometry analyses of monocytes showed that an increase in miR-124-3p expression is associated with an increase of several monocyte surface markers, CD45RA, CD 29 and CD206.

CD45RA (or protein tyrosine phosphate receptor C (PTPRC)), is considered a pro-inflammatory surface marker. This marker is mainly present on naive T-cells, however, heterogeneous populations of monocytes also express CD45RA as a marker of monocyte activation¹⁸. In vitro studies showed that CD45RA is co-expressed with Bl integrin very late antigen -4 (VLA-4) and that these cells are most likely to migrate over inflamed endothelial cells. Furthermore, this population of monocytes has been correlated to an atherogenic lipid profile, which in turn forms a risk factor for endothelial

inflammation²². It has been reported that cigarette smoking contributes to the oxidation of low density lipoprotein, contributing to an atherogenic lipid profile²³. This might eventually lead to an up regulation of CD45RA expression, which is correlated with increase miR-124-3p expression as seen in smoking individuals.

Together with CD49d, ( 1)29 forms the Bl integrin very late antigen-4 (VLA-4), which binds to its iigand vascular cell adhesion molecule 1 (VCAM-1) on the endothelium¹⁹. VLA-4 displays a particular high affinity for inflamed endothelial ceils and facilitates monocyte adhesion to and migration over the endothelium²⁴. In vitro studies have shown that due to cigarette smoke, VCAM-1 expression on endothelial cells is increased²⁵. Together with the increase in ( 1)29 expression on monocytes with high miR-124-3p expression reported here, this may have consequences for monocyte migration into the vessel wall, where they reside and transform into either activated macrophages or iipid-iaden foam ceils, accelerating the process of atherosclerotic plaque formation.

We also found a positive correlation between the expression of CD206 and miR-124-3p levels.

CD206, or the mannose receptor, is a marker present on the alternatively activated (M2) macrophages. Although M2 macrophages are generally known as anti-inflammatory, their presence has been described in various stages of the atherosclerotic plaque⁵'²⁶. It was previously shown that miR-124 up regulation in bone-marrow derived mouse macrophages was associated with a shift from the Ml to the M2 macrophage phenotype²⁷. Subsequently, monocytes stimulation with IL-4 and IL-13 to obtain M2 macrophages, resulted in high miR-124 expression and up regulation of CD206 expression¹³.

Interestingly, after blocking miR-124 using an antimiR, IL-4 stimulation did not result in an increase in CD206. The results of our mimic experiment confirmed the causal relation between miR-124-3p and CD206 by showing that transfection with a miR-124-3p mimic induces an increase in CD206 expression. However, we did not observe an increase in miR-124-3p expression after stimulation of human monocytes and macrophages with IL-4. This implies that in humans, miR-124-3p results in CD206 upregulation independent of the IL-4 pathway. Besides upregulation of the M2 marker CD206, our miR-124-3p mimic experiment also showed up- regulation of CD64, a marker of the classical activated (Ml) macrophages²⁸. These inflammatory macrophages produce reactive oxygen species that induce and exacerbate oxidative stress in the atherosclerotic plaque . Furthermore, they secrete the cytokine IL-6 that enhances atherogenesis . The regulation of both Ml and M2 markers on macrophages transfected with a miR-124-3p mimic indicates that up regulation of miR-124-3p does not result in a polarization towards a single macrophage subtype, but rather identifies miR-124-3p as a complex regulator of

monocytes/macrophages.

Since we showed that miR-124-3p is elevated in monocytes of smoking individuals and that increased miR-124-3p levels are associated with pro-atherogenic changes in monocyte phenotype, we studied whether miR-124-3p expression levels could be used as a biomarker for subclinical atherosclerosis in smoking individuals. For this purpose, we determined miR-124-3p levels in two independent cohorts of smoking and non-smoking individuals. The analysis revealed that in smoking individuals an increase in miR-124-3p levels was associated with a 2.36-fold increased risk of having subclinical atherosclerosis, whereas no such association was observed in non-smoking individuals.

Thus, we showed that miR-124-3p was heterogeneously expressed among smoking individuals, whereas high miR-124-3p was associated with an increased risk of having subclinical atherosclerosis in Cohort IV. This suggests a susceptibility for the adverse effects of smoking and may explain why several smoking individuals never develop cardiovascular complaints. Therefore, miR-124-3p is a suitable whole blood biomarker for subclinical atherosclerosis in smoking individuals.

Conclusion

We showed that miR-124-3p expression is heterogeneously expressed in monocytes of smoking individuals in two independent cohorts. Phenotypical analyses of these monocytes revealed that elevated miR-124-3p levels are associated with the expression of the pro-atherogenic surface markers CD29 and CD45RA, suggesting a potential role for miR-124-3p in the development of atherosclerosis. Moreover, we showed that an increase in miR-124-3p levels in whole blood is associated with subclinical atherosclerosis in smoking individuals and could therefore be used as a suitable biomarker in these individuals, thus identifying individuals with a susceptibility for the adverse effects of smoking.

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Table 1. B seline characteristics

Cohort I Cohort II Cohort III Cohort IV Cohort V

Subclinical Subclinical

Premature Premature atherosclerosis atherosclerosis

CAD cases Controls CAD cases Controls cases Controls cases Contro

N 40 40 27 40 22 39 32 31 38

Age at visit, years + SD 51.4 ±4.7 51.0+4.6 59.4 + 9.8 37.7 + 11.2* 50.4 + 8.7 48.7 + 9.9 48.7 + 6.0 48.8 + 7.9 46.9+

Gender, male (%) 40 (100) 40 (100) 8 (29.6) 20 (50.0) 9(41) 16(41) 11 (34) 15 (48.4) 12 (31.

BMI, kg/m², ± SD 28.0 ±3.1 25.2 ±2.6* 29.6 ±4.4 26.6 ±4.6 * 26.8 ±5.0 27.5 ±26.7 26.7 ±4.6 27.5 ± 4.4 28.2 ±

Hypercholesterolemia, n (%) 5 (12.5) 0(0) * 1(3) 4(15) 9(41) 12(31) 9 (28) 18 (58.1) 11 (28.9

Hypertension, n (%) 6(15) 0(0) * 10 (37) 2(5) * 6(27) 15 (36) 7 (22) 12 (38.7) 6(15.8)

Diabetes, n (%) 0(0) 0(0) 6(22) 1 (3)* 1(5) 6(15) 0(0) * 4(12.9) 1 (2.6

Medication use, n (%) 40 (100) 0(0) 21 (78) 4 (10) * 10 (46) 14 (35.9) 6(19) 16(51.6) 10 (26.3

Current smoking, n (%) 11 (28) 5 (13) * 12 (44) 11 (28) 13 (59) 26 (67) 18 (56) 0(0) 0(0)

Smoking quitted <5 years ago, n (%) 9 (22.5) 4 (10) * 2(7) 4(10) 9(41) 13 (33) 14 (44) 0(0) 0(0)

Systolic blood pressure, mmHG ± SD 136.8 ±19.6 128.7 ±13.4 137.2 ±22.8 124.4 ± 10.1 * 133.0 ±23.2 131.1 ± 19.0 124.4 ±9.7 131.9 ± 18.9 126.4 ±1

Diastolic blood pressure, mmHG + SD 86.7 + 12.0 84.0 + 10.1 80.4 + 8.6 77.3 + 8.3 78.7+10.1 77.6 + 10.5 78.7 + 8.2 78.7 + 9.1 77.3+

Continuous data are expressed as mean ± SD, categorical data as absolute number with (percentages).N, number, SD standard deviation, * p<0.05 compared to cases.

Claims

1. A method for determining the risk of suffering from or developing a coronary artery disease

(CAD), atherosclerosis or a cardiovascular disease of a smoker, comprising: a. determining in a biological sample from the smoker the expression level of miR-124-3p, b. comparing said expression level with a reference level, and c. determining said risk based on the comparison of step b.

2. The method according to claim 1, wherein the expression level of miR-124-3p positively

correlates with an increased risk of suffering from or developing CAD, atherosclerosis or cardiovascular disease in the smoker.

3. The method of claims 1-2, wherein said biological sample comprises whole blood, isolated or monocytes.

4. The method of claims 1-3, wherein said CAD is subclinical atherosclerosis.

5. The method of claims 1-4, said method further comprising assessing the expression level of a further expression marker in the smoker; and determining said risk based on the expression level of miR-124-3p and said further expression marker.

6. The method according to claim 5, wherein said expression marker is selected from the group consisting of CD206, CD29 and CD45RA.

7. The method of claims 1-6, said method further comprising assessing a clinical factor of the

smoker, wherein said clinical factor is associated with an increased risk of suffering from or developing CAD, atherosclerosis or cardiovascular disease; and determining said risk based on the expression level of miR-124-3p and said clinical factor.

8. The method of claims 1-7, wherein said clinical factor is selected from the group consisting of: age, gender, chest pain type, neutrophil count, ethnicity, disease duration, diastolic blood pressure, systolic blood pressure, a family history parameter, cholesterol level, diabetes, use of medication, a medical history parameter, a medical symptom parameter, height, weight, a body-mass index, CAC score, and resting heart rate.

9. The method according to any of the previous claims, wherein said expression level of said miRNA is normalized using one or more reference miRNAs.

10. The method according to any of the previous claims, wherein said one or more reference miRNAs is selected from the group consisting of consisting of miR-130b and miR-342-3p.

11. The method according to any of previous claims, the method comprising a step of amplifying miR-124-3p using reverse amplification and determining the amount of amplified products.

12. A kit of parts, comprising at least one molecule capable of specifically binding to at least one miRNA selected from the group consisting of miR-124-3p, miR-130b and miR-342-3p, and optionally one or more controls and/or one or more standards.