**2. Methods**

## *2.1. Study Population*

In brief, ATTICA is a prospective, population-based, cohort study performed in Attica (Athens metropolitan region, Greece), which recruited 3042 non-institutionalized adults (Caucasians; women/men: 1528/1514; age: ≥18 years) without previous CVD. The enrolment of the participants was carried out during 2001–2002, and after that, two follow-up waves followed 5 (in 2006) and 10 years (in 2012) later. Random, multistage sampling based on the age and gender distribution of the reference population, as defined by the Hellenic National Statistical Service Census Survey of 2001, was applied. Sampling procedures anticipated enrolling only one participant per household, while

institutionalized individuals were excluded from study participation. All participants underwent detailed baseline assessments which included medical history, physical examination, and blood sampling for biochemical measurements. Baseline CVD was excluded in all participants by the study physicians [24]. For the purposes of the present work, only baseline data for the study's sample of over 50 years old (i.e., *n* = 1128) men and women were used (there was no psychological assessment at follow-up examinations).

#### *2.2. Sociodemographic and Life-Style Variables*

The study questionnaire included demographic information such as age, gender, family status (married, divorced, widowed), financial status, and education level. The level of education (as a proxy of social status) was determined by years of schooling and classified into 3 groups: (1) <9 years; (2) up to high school or technical college (10–14 years); (3) university. Mean annual income during the past 3 years was also recorded.

Current smokers were defined as those who smoked at least one cigarette/day, never smokers as those who had never tried a cigarette in their life, and former smokers as those who had stopped smoking for at least one year. Occasional smokers (7 cigarettes/week) were recorded and combined with current smokers because of their small sample size. In order to evaluate more accurately the smoking habits, we calculated the pack-years (cigarette packs/day × years of smoking), adjusted for a nicotine content of 0.8 mg/cigarette.

The physical activity level of each participant was also assessed at baseline using the International Physical Activity Questionnaire (IPAQ; participants reporting no physical activities or exercise on the IPAQ were classified as physically inactive) [25].

#### *2.3. Clinical and Biochemical Assessments*

Standardized measurements of anthropometric parameters were performed by trained study researchers, including body weight and height, BMI (kg/m2), and WC (cm). Resting arterial blood pressure (BP, average of 3 recordings in sitting position) was also measured, and participants exhibiting average BP ≥ 140/90 mmHg or taking antihypertensive medication(s) were categorized as hypertensive.

At baseline, fasting blood samples were obtained from all participants after overnight fasting. Triglyceride (TG), total cholesterol (TC) and high-density lipoprotein-cholesterol (HDL-C) levels were measured by a chromatographic enzymatic method using a Technicon automatic analyzer RA-1000 (Dade Behring, Marburg, Germany; corresponding intra- and inter-assay coefficients of variation (CV) were <4%, <9%, and <4%, respectively). Low-density lipoprotein-cholesterol (LDL-C) was calculated by the Friedewald formula [26]. Hypercholesterolemia was defined as TC > 200 mg/dL or treatment with lipid-lowering drug(s). Moreover, fasting blood glucose (FBG) levels were measured by a Beckman glucose analyzer (Beckman Instruments, Fullerton, CA, USA) and subjects with FBG >125 mg/dL or on antidiabetic treatment were classified as having diabetes.

## *2.4. Dietary Assessment*

All participants underwent a detailed baseline dietary evaluation through the EPIC-Greek questionnaire [27], which is a validated semi-quantitative food-frequency questionnaire that was kindly provided by the Unit of Nutrition of Athens Medical School. The energy intake in kcal/day was also calculated based on the participants' responses in this questionnaire. Energy intake tertiles were also created, those in the 1st energy tertile were the participants consuming <1840 kcals/day.

All participants were asked to report the average intake (per week or day) of several food items that they consumed (during the last 12 months). Then, the frequency of consumption was quantified approximately in terms of the number of times a month this food was consumed. Thus, daily consumption multiplied by 30 and weekly consumption multiplied by 4 and a value of 0 was assigned to food items rarely or never consumed. Alcohol consumption was measured in wineglasses

(100 ml) and quantified by ethanol intake (grams per drink). One wineglass was considered equal to 12% ethanol concentration.

A dietary pyramid was developed to describe the Mediterranean dietary pattern [28]. This dietary pattern consist of: (a) daily consumption of non-refined cereals and products (whole grain bread, pasta, brown rice, etc.), vegetables (2–3 servings/day), fruits (6 servings/day), olive oil (as the main added lipid) and dairy products (1–2 servings/day); (b) weekly consumption of fish (4–5 servings/week), poultry (3–4 servings/week), olives, pulses, and nuts (3 servings/week), potatoes, eggs and sweets (3–4 servings/week) and monthly consumption of red meat and meat products (4–5 servings/month). TheMediterranean diet (MedDiet) is also characterized by moderate consumption of wine (1–2 wineglasses/day) and high monounsaturated to saturated fat ratio (>2).

A special diet score that assessed adherence to MedDiet was calculated. In particular, we assigned a score of 0 for rare or no consumption of food items that are close to this dietary pattern, 1 for 1 to 4 times/month, 2 for 5 to 8 times/month, 3 for 9 to 12 times/month, 4 for 13 to 18 times/month and 5 for almost daily consumption. On the other hand, for the consumption of foods that are away from this traditional diet, like meat and meat products, we assigned the opposite scores (i.e. 0 for almost daily consumption to 5 for rare or no consumption). For alcohol, we assigned a score of 5 for consumption of less than 3 wineglasses per day, a score of 0 for consumption of more than 7 wineglasses/day and scores of 1 to 4 for consumption of 3, 4–5, 6 and 7 wine glasses per day.

The calculation of the dietary inflammatory load of the participants' diet was according to the methodology and the rationale of the Dietary Inflammation Index (DII) that has been previously proposed by Shivappa and colleagues [29]. Thus, a Dietary Anti-Inflammation Index (D-AII) was developed based on participants' dietary habits. Detailed information on the D-AII has been reported in a previous ATTICA publication, by Georgousopoulou et al [30].

Aside from the classic indexes of MedDiet and D-AII, additional dietary patterns were defined for the daily dietary intake of the participants. Three main components were extracted using the principal components (PC) methods (Table 1). These were: (A) The lacto-fish-vegetarian (LFV) dietary pattern including the food components of vegetables, cereals, fruits, legumes, fish, and dairy products; (B) The meat-eaters dietary pattern including food components of all kinds of meat, read meat, and poultry; (C) The saturated fat and added sugars (SFAS) dietary pattern including the food components of sweets, soft drinks, nuts, and potatoes.

## *2.5. Psychological Assessments*

Anxiety levels were assessed using the validated Greek translation of the 20 item, self-report State-Trait Anxiety Inventory (STAI) [31], the total score of which ranges from 20 to 80. Higher scores in this scale are indicative of more severe anxiety, and according to Spielberger's criteria, a score of 40 or higher reflects clinically relevant symptoms of anxiety.

Depressive symptomatology was assessed using a translated and validated version of the Zung Self-Rating Depression Scale (ZDRS) [32]. The ZDRS consists of 20 items that cover affective, psychological, and somatic symptoms, and ranges from 20 to 80. A subject with a ZDRS score below 50 is considered normal but with a score of 50 to 59, 60 to 69, or 70 or above is considered to suffer from mild, moderate, or severe depression, respectively.

Cognitive distortion symptomatology was assessed using a validated version of the cognitive distortions scale (CDS) [33]. This tool captures cognitive symptoms or distortions among individuals who have experienced inter-personal victimization: self-criticism, self-blame, helplessness, hopelessness, and preoccupation with danger.
