Healthy controls: 170 Healthy controls matched with group 1 and group 2 for age, sex, CVD presence, propensity score, DM2, renal function, BMI and SBP, retrospectively recruited from the Athens Angiometabolic Cohort

20 patients starting treatment with palopegteriparatide, previously on conventional treatment

Non-invasive estimation of aortic pressure waveforms and reflected waves by pulse wave analysis (PWA) will be performed in the Angiology laboratory by the SphygmoCor System (AtCor Medical Pty Ltd, Sydney, Australia). The radial artery is gently and steadily compressed against the underlying bone, thus flattening it and equalizing circumferential pressures, allowing radial pressure waves to be recorded by a high fidelity micromanometer placed on the tip of a hand - held tonometer the size of a pen (Millar). Optimal recording is obtained if the wrist is bent outward and supported by using a small cushion or the operator's hand.

The following indices are measured:

1. augmentation index (AI, percentage) normalized for the heart rate of 75 bpm, expressed as a percentage of the aortic pulse pressure,
2. central systolic and diastolic pressures (cBP),
3. time to the beginning of the reflected wave (in milliseconds) and
4. blood pressure amplification calculated as the ratio of peripheral

B-mode ultrasound examination will also be performed, using a 14.0 MHz multi-frequency linear array probe attached to a high-resolution ultrasound machine (Vivid 7 Pro, GE Healthcare, USA). All scans are going to be performed by the same operator. Carotid intima-media thickness (ccIMT) will be measured at the distal 1.0 cm of the common carotid proximal to the bifurcation as previously described (23) . In each segment 3 measurements of the maximal IMT in the far wall will be averaged, after excluding plaque thickness. The average of the maximal IMT will be used in the analyses. Femoral IMT (fIMT) will be measured on each side, scanning a 1cm-long arterial segment proximal to the femoral bifurcation, defined as the common femoral artery segment and the average value of IMT of the far wall will be estimated. A cutoff value of >0.9 mm for mean ccIMT or fIMT will be considered increased. Plaques to carotid and femoral arteries are defined as a focal structure that protrudes into the arteri

The measurement procedure and the calculation of the sublingual microvascular parameters is performed as follows: the probe of a hand-held side-stream darkfield (SDF) videomicroscope (CapiScope HVCS, KK Technology, Honiton, UK) is placed on the sublingual mucosa of the subject, to obtain video recordings of the sublingual microvasculature. Subsequently, an analysis software (the GlycoCheck Measurement System Software Version 5.3.3) selects and analyses SDF images that are of sufficient quality (adequate focus, adequate contrast and limited movement) and automatically calculates the sublingual microcirculation parameters, such as vascular density (VD), red blood cell filling (RBCF), and perfused boundary region (PBR).

The primary non-invasive test for the diagnosis of lower extremity artery disease (LEAD) is the ABI. A cut-off value of <0.9 will be used for the diagnosis of LEAD and as predictive index of atherosclerosis, associated with increased risk of cardiovascular morbidity and mortality. Also, a cut-off value of >1.40 will be used as an index of stiffened arteries, also associated with increased mortality. For its measurement, a 10-12 cm sphygmomanometer cuff will be placed just above the ankle and a (handheld) Doppler instrument (5-10 MHz) will measure the pressure of the posterior and anterior tibial arteries of each foot. The highest ankle systolic pressure will be divided by the highest brachial systolic pressure, resulting in an ABI per leg.

A set of colour and black-and-white fundus images photograph per eye will be taken by well-trained ophthalmic photographers: macula-centered images using the handheld Optomed Aurora fundus cameras without pupil dilation. The images obtained with the handheld fundus camera had a field of view of 50° and 5 mega-pixel resolution

Participants will undergo a baseline echocardiographic examination. Standard protocol will be used and standard measurements from 2-D and Doppler echocardiography will be made. LV end-diastolic and end-systolic volumes, as well as ejection fraction, will be derived from the apical 4- and 2-chamber views using the biplane Simpson's rule. Left ventricular mass will be calculated according to Devereux's formula. Doppler examination will include interrogation of mitral inflow, and early (E) and late (A) peak diastolic velocities and deceleration time will be measured. Tissue Doppler analysis will include pulse wave interrogation of the medial and lateral mitral annulus, peak diastolic early E΄ annular velocities will be obtained and the mean value and E/E' will be calculated. In addition, Speckle-tracking analysis will be applied to estimate LV rotational mechanics, and longitudinal strain parameters. Parasternal short-axis views at the level of the mitral valve and apex, and standard apic

In order to calculate local shear stress in brachial and carotid artery, measurements of mean flow velocity in the lumen of these arteries will be needed. These measurements will be obtained, while performing carotid ultrasonography and FMD in brachial artery, as previously described. In detail, the form to calculate shear stress is: shear stress (in dyn/cm2 ) = 8×μ× mean flow velocity/resting diameter, where μ is the viscosity of blood which was assumed to be 0.035 dyne s/cm2

The peripheral and aortic office blood pressure will be assessed using specialized equipment (certified blood pressure monitor). 24-hour Ambulatory Blood pressure monitoring for measurement of 24 hour peripheral and aortic

Fasting venous blood samples will be collected for evaluation of myocardial biomarkers (hs-troponin I, NT-proBNP). Additional samples will be obtained for isolation of PBMCs.

Fasting venous blood samples will be collected for evaluation of calcium metabolism (PTH, calcium, albumin, magnesium, phosphate, 25-hydroxyvitamin D) .

Assessment of differences in central blood pressure between patients treated with long-acting PTH analogue, disease controls on conventional therapy, and healthy controls. 24-hour Ambulatory Blood pressure monitoring for measurement of 24 hour peripheral blood pressure will be performed.

B-mode ultrasound examination will be performed, using a 14.0 MHz multi-frequency linear array probe attached to a high-resolution ultrasound machine (Vivid 7 Pro, GE Healthcare, USA). All scans are going to be performed by the same operator. Carotid intima-media thickness (ccIMT) will be measured at the distal 1.0 cm of the common carotid proximal to the bifurcation. In each segment 3 measurements of the maximal IMT in the far wall will be averaged, after excluding plaque thickness. The average of the maximal IMT will be used in the analyses. A cutoff value of >0.9 mm for mean ccIMT will be considered increased. Plaques to carotid are defined as a focal structure that protrudes into the arterial lumen of at least 0.5 mm or 50

The probe of a hand-held side-stream darkfield (SDF) videomicroscope (CapiScope HVCS, KK Technology, Honiton, UK) is placed on the sublingual mucosa of the subject, to obtain video recordings of the sublingual microvasculature. Subsequently, an analysis software (the GlycoCheck Measurement System Software Version 5.3.3) selects and analyses SDF images that are of sufficient quality (adequate focus, adequate contrast and limited movement) and automatically calculates the sublingual microcirculation parameters, (vascular density (VD). Measurements will be performed by one user, experienced in performing sublingual measurements with this tool. Measurements will be taken with subjects in supine position with the researcher standing behind the headboard. Volunteers will be asked to swallow any saliva prior to measuring after which the camera will be manually placed and held still in the sublingual region.

A set of colour and black-and-white fundus images photograph per eye will be taken by well-trained ophthalmic photographers: macula-centered images using the handheld Optomed Aurora fundus cameras without pupil dilation. The images obtained with the handheld fundus camera had a field of view of 50° and 5 mega-pixel resolution.

In order to calculate local shear stress in brachial artery, measurements of mean flow velocity in the lumen of these arteries will be needed. These measurements will be obtained, while performing carotid ultrasonography in brachial artery, as previously described. In detail, the form to calculate shear stress is: shear stress (in dyn/cm2 ) = 8×μ× mean flow velocity/resting diameter, where μ is the viscosity of blood which was assumed to be 0.035 dyne s/cm2

Circulating biomarkers of myocardial stress and injury will include N-terminal pro-B-type natriuretic peptide (NT-proBNP). Venous blood samples will be collected after standard clinical preparation, processed according to local laboratory procedures, and analyzed in the hospital's certified biochemistry laboratory using automated immunoassays.

NT-proBNP will be measured as a marker of myocardial wall stress and reported in pg/mL. Values will be interpreted according to established clinical thresholds. In the non-acute setting, NT-proBNP <125 pg/mL will be considered within the normal range, whereas values ≥125 pg/mL will be considered elevated and suggestive of increased myocardial stress. For descriptive analyses, higher-risk thresholds may also be reported, including ≥300 pg/mL and age-adjusted categories when appropriate.

High-sensitivity cardiac troponin will be measured as a marker of myocardial injury and reported in ng/L. Venous blood samples will be collected after standard clinical preparation, processed according to local laboratory procedures, and analyzed in the hospital's certified biochemistry laboratory using automated immunoassays.

Serum phosphate will be measured in certified clinical laboratories using standard clinical procedures and reported in mg/dL.

Albumin-adjusted serum calcium will be measured in certified clinical laboratories using standard clinical procedures and reported in mg/dl

Serum magnesium will be measured in certified clinical laboratories using standard clinical procedures and reported in mg/dL.

Serum intact parathyroid hormone (PTH) will be measured in certified clinical laboratories using standard clinical procedures and reported in pg/mL.

Serum 25-hydroxyvitamin D [25(OH)D] will be measured in certified clinical laboratories using standard clinical procedures and reported in ng/mL.

Left ventricular global longitudinal strain (GLS) will be assessed by speckle-tracking transthoracic echocardiography using standard apical views according to current echocardiographic recommendations. GLS will be reported as percentage (%).

Left ventricular ejection fraction (LVEF) will be measured by transthoracic echocardiography using the biplane Simpson's method and reported as percentage (%).

Left ventricular diastolic function will be evaluated by Doppler and tissue Doppler echocardiography. The E/E' ratio will be calculated using transmitral early diastolic inflow velocity (E) and the mean early diastolic mitral annular velocity (E').

Non-invasive estimation of aortic pressure waveforms and reflected waves by pulse wave analysis (PWA) will be performed in the Angiology laboratory by the SphygmoCor System (AtCor Medical Pty Ltd, Sydney, Australia). The radial artery is gently and steadily compressed against the underlying bone, thus flattening it and equalizing circumferential pressures, allowing radial pressure waves to be recorded by a high fidelity micromanometer placed on the tip of a hand - held tonometer the size of a pen (Millar). Optimal recording is obtained if the wrist is bent outward and supported by using a small cushion or the operator's hand. Non-invasive estimation of augmentation index (AIx) normalized to a heart rate of 75 bpm using pulse wave analysis (SphygmoCor System). AIx is expressed as a percentage of central aortic pulse pressure.

Non-invasive estimation of aortic pressure waveforms and reflected waves by pulse wave analysis (PWA) will be performed in the Angiology laboratory by the SphygmoCor System (AtCor Medical Pty Ltd, Sydney, Australia). The radial artery is gently and steadily compressed against the underlying bone, thus flattening it and equalizing circumferential pressures, allowing radial pressure waves to be recorded by a high fidelity micromanometer placed on the tip of a hand - held tonometer the size of a pen (Millar). Optimal recording is obtained if the wrist is bent outward and supported by using a small cushion or the operator's hand. Central systolic blood pressure measured non-invasively by pulse wave analysis using the SphygmoCor System.

Non-invasive estimation of aortic pressure waveforms and reflected waves by pulse wave analysis (PWA) will be performed in the Angiology laboratory by the SphygmoCor System (AtCor Medical Pty Ltd, Sydney, Australia). The radial artery is gently and steadily compressed against the underlying bone, thus flattening it and equalizing circumferential pressures, allowing radial pressure waves to be recorded by a high fidelity micromanometer placed on the tip of a hand - held tonometer the size of a pen (Millar). Optimal recording is obtained if the wrist is bent outward and supported by using a small cushion or the operator's hand. Central diastolic blood pressure measured non-invasively by pulse wave analysis using the SphygmoCor System.

Non-invasive estimation of aortic pressure waveforms and reflected waves by pulse wave analysis (PWA) will be performed in the Angiology laboratory by the SphygmoCor System (AtCor Medical Pty Ltd, Sydney, Australia). The radial artery is gently and steadily compressed against the underlying bone, thus flattening it and equalizing circumferential pressures, allowing radial pressure waves to be recorded by a high fidelity micromanometer placed on the tip of a hand - held tonometer the size of a pen (Millar). Optimal recording is obtained if the wrist is bent outward and supported by using a small cushion or the operator's hand. Time to the beginning of the reflected wave, measured in milliseconds by pulse wave analysis using the SphygmoCor System.

Non-invasive estimation of aortic pressure waveforms and reflected waves by pulse wave analysis (PWA) will be performed in the Angiology laboratory by the SphygmoCor System (AtCor Medical Pty Ltd, Sydney, Australia). The radial artery is gently and steadily compressed against the underlying bone, thus flattening it and equalizing circumferential pressures, allowing radial pressure waves to be recorded by a high fidelity micromanometer placed on the tip of a hand - held tonometer the size of a pen (Millar). Optimal recording is obtained if the wrist is bent outward and supported by using a small cushion or the operator's hand. Blood pressure amplification, calculated as the ratio of peripheral pulse pressure to central pulse pressure using pulse wave analysis.

The primary non-invasive test for the diagnosis of lower extremity artery disease (LEAD) is the ABI. Furthermore, The ABI is a strong marker of CVD and is predictive of cardiovascular events and mortality (28-30). A cut-off value of <0.9 will be used for the diagnosis of LEAD and as predictive index of atherosclerosis, associated with increased risk of cardiovascular morbidity and mortality as previously described (28, 29). Also, a cut-off value of >1.40 will be used as an index of stiffened arteries, also associated with increased mortality, as previously described (30). For its measurement, a 10-12 cm sphygmomanometer cuff will be placed just above the ankle and a (handheld) Doppler instrument (5-10 MHz) will measure the pressure of the posterior and anterior tibial arteries of each foot. The highest ankle systolic pressure will be divided by the highest brachial systolic pressure, resulting in an ABI per leg.