Peripheral microvascular function was measured by peripheral arterial tonometry (PAT)(Endo-PAT2000®, Itamar Medical, software version 3.2.4, Caesarea, Israel). Relative ischemia was provoked by inflating a blood pressure cuff on the forearm to a minimum of 100 mmHg above systolic blood pressure. Following a 5-minute period, reactive hyperemia was induced by rapidly deflating the cuff. The reactive hyperemia index (RHI) is defined as the ratio of the average amplitude of the PAT signal over a one-minute period starting one minute after cuff deflation (maximum pulse amplitude) divided by the average amplitude of the PAT signal over a 3.5 minute period preceding cuff inflation (baseline pulse amplitude). RHI and Framingham modified RHI (fRHI) were calculated by dedicated software (Itamar Medical).

The carotid intima-media thickness (IMT) was measured: B-mode and color Doppler ultrasound, equipped with a 10 MHz linear array transducer (Aloka Prosound 6 Ultrasound Linear Probe LN 5413, Hitachi Aloka Medical, Japan) was used to evaluate the right common, internal and external carotid arteries. A standardized protocol was applied to evaluate carotid IMT: briefly, all subjects were examined in a supine position with their head turned 45 degrees from the site being scanned. Measurements were made using automated e-tracking of the wall along a distance of 2 cm. Three measurements were taken at the common carotid artery, approximately 2 cm proximal to the carotid bifurcation, and the values were averaged to determine the mean IMT.

Systemic large artery stiffness was measured using pulse wave velocity (PWV) and augmentation index (AIx)(Sphygmocor system®, AtCor Medical, West Ryde, Australia).

For PWV, an applanation tonometer (Millar Instruments) was positioned on both the right common carotid artery and the right femoral artery. Proximal (i.e., carotid) and distal (i.e., femoral) pulses were recorded consecutively and were averaged. PWV was calculated as the carotid-to-distal path length divided by the transit time. In order to prevent overestimation, a scaling factor of 0.8 was applied to convert PWV values obtained using the direct carotid-femoral distance to the corrected PWV (PWVc).

Systemic large artery stiffness was measured using pulse wave velocity (PWV) and augmentation index (AIx)(Sphygmocor system®, AtCor Medical, West Ryde, Australia).

For central AIx, three measurements were obtained using the applanation tonometer (Millar Instruments) at the level of the right carotid artery. AIx was determined as the ratio of augmentation (which is attributed to wave reflection) to pulse pressure (defined as the difference between systolic and diastolic pressure).

Fasting peripheral blood was collected using ethylenediaminetetraacetic acid (EDTA) tubes (BD Vacutainer®, Canada). EDTA samples were analyzed using a Sysmex XN-9100 (Sysmex, Germany) to quantify white blood cell counts.

Fasting peripheral blood was collected using serum vacuette tubes (BD Vacutainer®, Canada). Serum samples were analyzed using an Atellica® IM/CH Analyzer (Siemens Healthcare, Germany) to quantify high-sensitivity C-reactive protein (hs-CRP) concentrations.

Superoxide anion radical levels were quantified using electron paramagnetic resonance (EPR) spectroscopy.

When fasting venous blood samples from the patients were obtained in a heparin tube (BD vacutainer®, Canada), 100 µl of blood was immediately added to 100 µL of spin probe CMH. Immediately after mixing, the sample was snap frozen and stored at -80°C until analysis. For analysis conducted at the University of Maastricht (Department of Toxicogenomics), the mixture of CMH and blood was thawed and transferred into a 50 μl glass capillary (Hirschmann®, Germany). The glass capillaries were placed in the resonator of the EPR. EPR measurements were carried out on a Bruker EMX 1273 spectrometer equipped with an ER 4119HS high-sensitivity resonator and 12 kW power supply operating at X band frequencies. The data were analyzed using WinEPR (Brüker, Germany) software and radicals were identified and quantified as ESR peak amplitude arbitrary units (A.U.).

All echocardiographic examinations were performed by two experienced sonographers utilizing harmonic imaging on an EPIQ7 ultrasound system (Philips Medical Systems, Best, the Netherlands). A detailed 2D transthoracic echocardiogram was performed with the patient placed in the left lateral decubitus position.

For the assessment of left ventricular (LV)* systolic function, LV ejection fraction (LVEF) was measured using the Simpson biplane method.

* The term "LV" refers to the morphological LV

All echocardiographic examinations were performed by two experienced sonographers utilizing harmonic imaging on an EPIQ7 ultrasound system (Philips Medical Systems, Best, the Netherlands). A detailed 2D transthoracic echocardiogram was performed with the patient placed in the left lateral decubitus position.

For the assessment of left ventricular (LV)* systolic function, LV global longitudinal strain (LV GLS) was calculated by combining 3 apical longitudinal LV chamber views (2-, 3- and 4-chamber).

* The term "LV" refers to the morphological LV

The right ventricle (RV)* was comprehensively assessed using the novel two-dimensional multiplane echocardiography (2D MPE) approach revealing 4 RV views: a focused, non-foreshortened RV view (4C), coronary sinus view (CS), aortic view (Ao) and coronal view (CV).

RV fractional area change (FAC)(calculated as: (end-diastolic-end-systolic area)/end-diastolic area x100) was recorded in all 4 RV views. In addition to the values from the individual RV walls, a multiwall average was computed when measurements from at least 3 walls of one individual were feasible to obtain.

* The term "RV" refers to the morphological RV

The right ventricle (RV)* was comprehensively assessed using the novel two-dimensional multiplane echocardiography (2D MPE) approach revealing 4 RV views: a focused, non-foreshortened RV view (4C), coronary sinus view (CS), aortic view (Ao) and coronal view (CV).

RV tricuspid annular plane systolic excursion (TAPSE)(measured with 2D echocardiography-guided M-mode) was recorded in all 4 RV views. In addition to the values from the individual RV walls, a multiwall average was computed when measurements from at least 3 walls of one individual were feasible to obtain.

* The term "RV" refers to the morphological RV

The right ventricle (RV)* was comprehensively assessed using the novel two-dimensional multiplane echocardiography (2D MPE) approach revealing 4 RV views: a focused, non-foreshortened RV view (4C), coronary sinus view (CS), aortic view (Ao) and coronal view (CV).

RV tissue Doppler imaging systolic velocity (TDI S') was recorded in all 4 RV views. In addition to the values from the individual RV walls, a multiwall average was computed when measurements from at least 3 walls of one individual were feasible to obtain.

* The term "RV" refers to the morphological RV

The right ventricle (RV)* was comprehensively assessed using the novel two-dimensional multiplane echocardiography (2D MPE) approach revealing 4 RV views: a focused, non-foreshortened RV view (4C), coronary sinus view (CS), aortic view (Ao) and coronal view (CV).

Peak systolic global longitudinal RV strain (RV LS) was assessed in 3 of the 4 RV views (i.e., 4C, CS, Ao) using an automated strain software (AutoStrain; Philips Medical Systems, Best, the Netherlands). In each RV view, a single RV free wall strain value was derived from the average of 3 segments (basal, mid and apical). A multiwall average was calculated if at least 2 RV free walls were feasible to measure.

* The term "RV" refers to the morphological RV

The patients were asked to complete a quality of life questionnaire consisting of a combination of the RAND 36-Item Health Survey 1.0 and the EuroQol visual analogue scale (EQ-VAS).

The RAND 36-Item Health Survey 1.0 encompasses 8 health domains: physical functioning, bodily pain, role limitations due to physical health issues, role limitations due to personal or emotional concerns, general mental health, social functioning, energy/fatigue, and general health perception. Additionally, it incorporates a single item aimed at assessing perceived change in health.

The patients were asked to complete a quality of life questionnaire consisting of a combination of the RAND 36-Item Health Survey 1.0 and the EuroQol visual analogue scale (EQ-VAS).

The EQ-VAS is a vertical scale to self-rate the patients' current health state, ranging from 0 (worst imaginable) to 100 (best imaginable).

A cardiopulmonary exercise test was conducted until maximal exhaustion. Therefore, a continuously incrementing ramp protocol (increase of work rate, e.g., every 2-15 seconds) was implemented with the objective of achieving maximal exertion within 8-12 minutes using a Lode Corival bike ergometer. The protocol was chosen based on the Jones' predictions of maximal power output. Throughout the test, gas exchange measurements and 12-lead electrocardiogram readings were continuously recorded, while blood pressure was monitored every minute. Peak oxygen consumption (pVO2) was determined as the mean VO2 during the final 30 seconds of exercise. Subsequently, percent-predicted peak oxygen consumption (%ppVO2) was computed, using the reference values following Wasserman et al. and the LowLands registry.

The one-repetition maximum (1RM) was measured for 4 different exercises targeting major muscle groups: chest press, vertical traction, low row, and leg press (BioCircuit Series 4, TechnoGym®, Benelux).