Vascular target organ damage (TOD), defined as structural or functional deleterious changes in large and small arteries, is related to unfavorable arterial biomechanics, atherosclerosis and arteriosclerosis. Endothelial dysfunction due to unfavorable redox and glycation states on the bases of these phenomena. However, little is known about the role of glycoxidation on arterial biomechanics and TOD in apparently healthy individuals. The main hypothesis is that glycation and glycoxidation status are associated with arterial biomechanical abnormalities and TOD in patients with moderate to high cardiovascular risk. This is an observational, ambispective, and multicenter project that will include non-smoking patients over 18 years, without diabetes mellitus or established cardiovascular disease. Demographic, epidemiological, and clinical-anthropometric variables will be collected, including data from ambulatory blood pressure monitoring. The investigators will measure the serum percentage of glycated hemoglobin, glycated albumin, and fructosamine levels; along with quantification of skin advanced glycation and glycoxidation end productos (AGEs). Plasma concentration, activity, and structure of catalase, glutathione peroxidase, and superoxide dismutase in relation to the patient's glycation and glycoxidation status will be also evaluated. Concurrently, several biomechanical parameters will be assessed in the Common, Internal Carotid Artery, and distal limb arteries using ultrasound exploration. Incipient microvasculature damage will be also evaluated by retinal image. Patients will be followed up for the development of arterial biomechanical abnormalities and TOD, along with cardiovascular events.

INTRODUCTION

Atherosclerotic cardiovascular disease (ASCVD) represents one of the leading causes of morbidity and mortality in the population. Arterial hypertension (AHT), along with other cardiovascular risk (CVR) factors, predisposes to abnormalities in the structure and function of large and small arteries, leading to premature vascular aging and culminating in the development of target organ damage (TOD),which reresents a clear but underdiagnosed precursor of ASCVD. However, not all patients develop TOD, nor do they do so over the same time span or with the same intensity. Additionally, patients who initially present with low to moderate CVR are already considered to be at moderate to high CVR in the presence of TOD, with more stringent therapeutic objectives.

Vascular biomechanics primarily focuses on the study of mechanical properties and behavior of arteries. Unfavorable arterial biomechanics are associated with the development and progression of atherosclerosis and arteriosclerosis. These processes represent a continuum of vascular damage mediated by the accumulation of oxidized lipoproteins in the subendothelial space, activation and amplification of inflammatory pathways, and development of arterial fibrosis and calcification. At the core of these phenomena lies the occurrence of endothelial dysfunction, characterized by increased permeability and loss of the vascular endothelium's filtering capacity for deleterious molecules to the arterial wall.

Oxidative stress, defined as an imbalance between reactive species production and antioxidant systems in favor of the former, plays a crucial role in the development of endothelial dysfunction, deposition of oxidized lipoproteins, and proinflammatory interaction between these and the mononuclear-phagocytic system. The accumulation of reactive oxygen species (ROS) and others induces lipid peroxidation and glycoxidation phenomena culminating in the formation of advanced lipoxidation and glycation end products (ALEs and AGEs, respectively). ALEs and AGEs can bind to proteins, causing functional and structural abnormalities, and also influence cellular signaling by activating, among others, death pathways.

Unfavorable redox status and glycation levels are linked to aging, degenerative diseases, and accumulated vascular damage in certain subpopulations, particularly patients with diabetes mellitus (DM). However, little is known about the influence of antioxidant system glycation and glycoxidation on arterial biomechanics and the development of target organ damage in apparently healthy patients with moderate to high CVR. The investigators aim to study the specific relationship between arterial biomechanical abnormalities and TOD with serum glycation levels, glycoxidation status, and function-structure balance of some antioxidant system elements, highlighting catalase, superoxide dismutase, and glutathione peroxidase.

MATERIALS AND METHODS

Project Design and Setup

This is an observational, ambispective, and multicenter project promoted by the Unit of Hypertension and Cardiovascular risk belonging to the Internal Medicine Department at the University Hospital of Santiago de Compostela. Participants will be individuals belonging to the Organizational Structure of Integrated Management (EOXI) of Santiago de Compostela and Barbanza who seek consultation for CVR diagnosis, management, and follow-up.

Participants. Inclusion and Exclusion Criteria

From the total pool of consulted patients, only individuals over 18 years with a moderate to high CVR profile based on guideline reference scores will be preselected. Exclusion criteria will include the presence of DM according to current clinical practice guidelines, smoking habit (defined as current consumption and within the 6 months prior to recruitment), risky alcohol consumption (defined as consumption exceeding 10 g and 20 g daily for women and men, respectively), and established cardiovascular disease.

Assessment of Arterial Biomechanical Markers and TOD

The investigators will perform bilateral ultrasound exploration of the Common Carotid Artery (CCA), Internal Carotid Artery (ICA) and distal arteries of the lower limbs using a Mindray Z60 device (Mindray®, Guangdong, China) with high-frequency probe. The following measurements and calculations will be extracted:

1. In motion mode (M-mode), concomitant measurement of arterial radius, arterial pressures, and total wall thickness for the calculation of distensibility and tension parameters in the middle segment of the CCA.
2. In two-dimensional mode (B-mode), automated measurement of intima-media thickness (IMT) in the distal region of the CCA, along with detection and characterization of cholesterol plaque.
3. In spectral Doppler mode, morphological description of volume-time (flow) function in terms of flow velocities and vascular resistance indices in the middle segment of the CCA and ICA.
4. In B-mode with elastography module, direct evaluation of local stiffness degree in a middle segment of the CCA.
5. In spectral Doppler mode with ECG synchronization, assessment of carotid-femoral pulse wave velocity (c-f PWV).
6. In spectral Doppler mode, assessment of the ankle-brachial index (ABI).

The investigators will also assess incipient damage in the retinal microvasculature using retinography with a Topcon TRC-NW6S device (Electronics®, Tokyo, Japan).

Assessment of Oxidative Stress markers and Antioxidant System Activity-Structure

To assess the redox status, plasma and urine thiobarbituric acid reactive substances (TBARS) and reduced thiols will be assessed according to the referenced protocols via spectrophotometric quantification. Enzymatic function of glutathione peroxidase, catalase, and superoxide dismutase will be measured as enzymatic activity according to the referenced protocols. These analyses will be conducted using an Asys UVM-340 analyzer (Biochrom®, Cambridge, UK). Structure features of antioxidant enzymes based on glycation and glycoxidation patterns will be assessed using proteomics.

Determination of Glycation, Glycoxidation, and Lipoxidation status

The percentage of glycated hemoglobin will be assessed using high-performance liquid chromatography (HPLC) with the Hemoglobin next analyzer (A. Menarini Diagnostics®, Florence, Italy). Fructosamine levels will be determined by the diazyme glycated serum protein enzymatic method (Diazyme, Kent, UK) on an Advia 2400 analyser (Siemens Healthcare Diagnostics Inc.®, Tarrytown, USA). The percentage of glycated albumin will be estimated from fructosamine and albumin levels. This laboratory assessment will be performed by the Central Hospital Laboratory department.

General levels of glycoxidation and lipoxidation will be estimated through quantification of dicarbonyl levels using the TBARS spectrophotometric approach. Global glycoxidation status will be also estimated through assessment of skin advanced glycation end products (AGEs) using an AGE Reader (DiagnOptics BV®, Groningen, Netherlands). AGEs mainly represent glycated adducts bound to proteins that arise during glycoxidation processes. The AGE Reader has been developed to assess skin autofluorescence (SAF) non-invasively using the fluorescent properties of various AGEs.

Outcomes in Cardiovascular Risk

The investigators will measure the number needed to treat (NNT) of consultations and exploration time to detect abnormalities in arterial biomechanics and target organ damage (TOD), and thus determine an increased CVR warranting therapeutic intensification. Cardiovascular and cerebrovascular events, hospital admissions, consultations, death and disability will be also assessed.

Ethical Considerations and Good Clinical Practice

This study will be conducted in accordance with the ethical principles of the Declaration of Helsinki and the good practice standards in the research of the Galician (Spain) Health Service (SERGAS). Written informed consent will be obtained from all patients who agreed to participate. The protocols were approved by the Research Ethics Committee of Santiago-Lugo (codes 2021/401 and 2023/302).

Work Plan and Project Flow

In a first phase, the investigators plan to include patients meeting the criteria and consenting to participate. Clinical-anthropometric evaluation, arterial biomechanics, TOD detection, and sample collection will be conducted in a single medical act. Biomechanical, oxidative stress, glycation and glycoxidation markers will be quantified in both patients with and without biomechanical abnormalities and arterial TOD. The aim of this phase is to evaluate the differences between patient groups based on biomechanical parameters and arterial TOD. In a subgroup of patients, differences in the structure-function of antioxidant enzymes in terms of glycation and glycoxidation levels, and their relationship with arterial biomechanical abnormalities and TOD will also be evaluated. This phase will include the extraction, collection, processing, and analysis of preliminary data, along with the attainment of the project's initial results.

In a second phase,

1. Patients without biomechanical abnormalities or TOD will undergo a 5-year follow-up, with anual and final evaluations of biomechanical abnormalities, TOD, and cardiovascular outcomes. The aim of this phase is to evaluate differences in the previous markers between patient groups based on biomechanical parameters and arterial TOD. In a subgroup of patients, differences in the structure-function of antioxidant enzymes in terms of glycation and glycoxidation levels, and their relationship with arterial biomechanical abnormalities and TOD will be also assessed. This phase will include the extraction, collection, processing, and analysis of preliminary data, along with the attainment of the project's final results.
2. Patients with biomechanical abnormalities and/or TOD will undergo a 5-year follow-up, with annual and final evaluations of TOD progression and cardiovascular outcomes. The aim of this phase is to evaluate differences in the previous markers between patient groups based arterial TOD and cardiovascular outcomes. In a subgroup of patients, differences in the structure-function of antioxidant enzymes in terms of glycation and glycoxidation levels, and their relationship with TOD and cardiovascular outcomes will be assessed. This phase will include the extraction, collection, processing, and analysis of preliminary data, along with the attainment of the project's final results.

Mathematical analysis and modeling

The SPSS 22.0 statistical software package (SPSS Inc.®, Chicago, IL, EE.UU) will be employed. A descriptive statistic approach will be performed, including assessment of normality for quantitative variables. Univariate statistics will be used to compare groups with specific tests depending on the variable type. If relevant results are found, linear correlation studies will be conducted. After univariate analysis, multivariate models will be constructed, both explanatory for the association of predictors with the outcomes, and predictive for the risk of biomechanical abnormalities, TOD and cardiovascular results. To assess factors that may influence biomechanical parameters, TOD and cardiovascular results over time, specific mathematical procedures related to joint modeling approach and time series analysis will be applied. Sample size calculations will be based on specific statistical tests according to the variable type and distribution, always considering a moderate effect size, 95% confidence interval (95%CI), and at least 80% power.

Inclusion Criteria:

• Patients aged 18 years or older.
• Moderate to high Cardiovascular Risk estimated by SCORE2OP.
• Signed written consent for participation in the study.

Exclusion Criteria:

• Absence of current smoking habit and in the last 6 months.
• High-risk alcohol consumption (More than 10 and 20 g/day in women and men, respectively).
• Presence of Diabetes mellitus.
• Established cardiovascular disease, including heart failure, ischemic heart disease, valvular heart disease, atrial fibrillation, peripheral artery disease, and cerebrovascular disease.

Patients belonging to the health area of Santiago de Compostela and Barbanza (A Coruña, Spain), referred from primary care and hospital services to the Cardiovascular Risk and Hypertension Unit of the Internal Medicine Department at the reference hospital.