The Effect of Lipoprotein (a) on Arterial Stiffness, Endothelial Function and Myocardial Deformation

1. Introduction Lipoprotein(a), or Lp(a), is a type of lipoprotein that is structurally similar to LDL (low-density lipoprotein) but carries an additional protein called apolipoprotein (a). Recent data establishes Lp(a) as a predisposing risk factor for cardiovascular diseases, such as coronary artery disease, stroke, and aortic valve stenosis. Elevated levels of Lp(a) contribute to these conditions by promoting atherosclerosis. Its levels are primarily determined genetically, with minimal influence from diet and lifestyle. This makes it a significant factor for assessing cardiovascular risk, particularly in individuals with a family history of coronary artery disease or patients who experience early cardiovascular problems despite having normal lipid levels.

   While Lp(a) levels can vary greatly, the generally accepted threshold for increased cardiovascular risk is 50 mg/dL (or 125 nmol/L). Individuals with levels above this limit are at a higher risk for cardiovascular events. It is estimated that 20-30% of the general population has elevated Lp(a) levels, making it a common risk factor. Currently, there are no widely available treatments specifically targeting Lp(a) levels, though PCSK9 inhibitors and inclisiran have shown promising results in clinical trials.

2. Purpose of the Study There is currently insufficient bibliographic documentation regarding the effect of Lp(a) on arterial stiffness, endothelial function, and the deformation of the left atrium and left ventricle.

   The primary purpose of this study is to investigate the effect of Lp(a) levels on arterial stiffness, endothelial function, and left atrial (LA) and left ventricular (LV) deformation over a 6-month follow-up period.

   Secondarily, the study will investigate:

   • a) The incidence of major adverse cardiovascular events (MACE), including cardiovascular death, acute myocardial infarction, and acute stroke.
   • b) The correlation between MACE incidence and parameters of arterial stiffness, endothelial function, and LA/LV deformation.
   • c) The levels of oxidative load markers.
3. Materials and Methods

This observational study will include adults aged 18-75 years (regardless of gender) who visit the outpatient clinics of the 2nd University Cardiology Clinic at "Attikon" General Hospital. All participants will sign a consent form. A full medical history, clinical examination, and blood collection will be performed to determine levels of Total Cholesterol, LDL-C, HDL-C, triglycerides, and Lp(a) at each visit. Participants will be divided into three groups:

• Group A: Lp(a) ≥50 mg/dL with Total Cholesterol\<200 mg/dl
• Group B : Lp(a) \<50 mg/dL. with Total Cholesterol>200 mg/dl
• Group C (Control): Lp(a) \<50 mg/dL. with Total Cholesterol\<200 mg/dl The exclusion criteria encompass: 1. History of autoimmune/autoinflammatory disease, 2. Severe valvular heart disease, 3. severe chronic kidney disease(eGFR\<60 ml/min/1.73 m2), 4. Active Pregnancy and 5. Severe hepatic impairment.

At each group n ≥ 100 participants are anticipated.

The following measurements will be conducted for each group at baseline and after 6 and 12 months post enrollment:

• Arterial Stiffness: Determination of carotid-femoral pulse wave velocity (cf-PWV) using the Complior SP device and 24-hour pulse wave analysis with the Mobil-O-Graph device. Pulse wave velocity is the gold standard for the evaluation of arterial stiffness [21,22]. The investigators will use two non-invasive pressure sensors to record the carotid and femoral waveforms. The investigators will measure the distance between the two arterial sites with a tape measure. PWV is calculated by dividing the distance by transit time between waves (m/s). The appropriate corrections of the PWV were applied, after multiplying the default PWV output values by 0.8 [22]. Central systolic blood pressure (cSBP, mmHg) and central diastolic blood pressure (cDBP, mm Hg) will be measured using Complior.
• Endothelial Function: The investigators will measure the perfused boundary region (PBR) of the sublingual arterial microvessels (ranged from 4 to 25μm) using Sidestream Darkfield (SDF) imaging (Microscan, Glycocheck, Microvascular Health Solutions Inc., Salt Lake City, Utah, United States) [18]. This technique enables the assessment of the endothelial glycocalyx, using a non-invasive method. The PBR is the cell poor layer, which results from the phase separation between the flowing RBC's and plasma on the surface of the microvessel lumen. The PBR includes the most luminal part of glycocalyx that does allow cell penetration. Thus, an increased PBR is consistent with deeper penetration of erythrocytes into glycocalyx, indicating a loss of glycocalyx barrier properties and is a marker of reduced glycocalyx thickness. The measurement of endothelial glycocalyx thickness using SDF imaging is easy to perform (duration of 3 minutes) and is not biased by operator skills. Moreover, it has a standardized methodology, provides measurements of multiple sample sites (>3,000 vascular segments of sublingual microvasculature), has very good reproducibility and thus is proposed as a valid method to assess endothelial integrity by the European Society of Cardiology Working Group on Peripheral Circulation. The PBR measurements are independent of RBC filling of the vessels segments (hematocrit), because the software only includes vessel segments that have a filling percentage more than 50% [9,18]. Hence, vessel segments are only selected when at least 11 of the 21-line markers have a positive signal for the presence of an erythrocyte. Thus, PBR values are independent of hematocrit, reflecting a damaged glycocalyx that is more accessible for circulating erythrocytes.
• Cardiac Deformation: In all participants the investigators will measure longitudinal systolic strain from standard two dimensional (2D) acquisitions (frame rate: 70-80/second) with the use of a dedicated software (EchoPac 206, GE Healthcare). Global longitudinal strain (GLS) is calculated using the 17 LV segment model imaged from apical chamber views (4,2 and 3 chamber view). The myocardial deformations at the basal, mid-ventricular, and apical segments were averaged to GLS. All variables represent the mean value measurements taken in three consecutive cardiac cycles. Inter- and intra- observer variability of GLS was 8 and 5%, respectively. LA function will be evaluated using transthoracic echocardiographic speckle -tracking strain imaging. A speckle -tracking algorithm was applied to the LA myocardium in apical 4- and 2- chamber views. The region of interest width was adjusted to the thickness of the LA wall, and the cardiac cycle was demarcated by indicating QRS onset. LA reservoir strain, a metric of LA compliance, was measured as peak longitudinal strain (average of all 12 segments in the apical 4- and 2- chamber views) during ventricular systole, taking the QRS onset as the reference point. After having defined by anatomical M-mode mitral and aortic valve opening and closure as well as atrial contraction, LA longitudinal strain, obtained by 2D strain, will be measured in all three atrial phases (reservoir, conduit, and atrial contraction) as follows : reservoir period was defined as the interval between mitral valve closure and mitral valve opening, conduit period as the interval between mitral valve opening and the onset of atrial contraction, as assessed by anatomical M- mode, and contractile period as the interval between the onset of atrial contraction, as assessed by anatomical M-mode, and mitral valve closure. Subsequently, the atrial deformation of each period was calculated by measuring the respective difference of strain (values at the end minus values at the beginning).
• Oxidative Load: Determination of malondialdehyde (MDA) and protein carbonyls (PCs) levels as markers of oxidative stress. MDA will be estimated spectrophotometrically with a commercial kit (Oxford Biomedical Research, Rochester Hills, MI) of colorimetric assay for lipid peroxidation (measurements range, 1-20 nmol/l). For the determination of PCs (nmol/mL), the investigators used the spectrophotometric assessment of 2,4-dinitrophenylhydrazine derivatives of PCs.
• Statistical Analysis: Statistical analysis will be conducted by employing SPSS version 29 (IBM SPSS Statistics, Inc., Chicago, IL). All continuous variables variables will be presented as mean ±SD if they are distributed normally, as indicated by the Kolmogorov-Smirnov and Shapiro-Wilk normality tests. In case of non-normal distribution, transformation into ranks will be performed. Nominal variables are presented as percentages. Correlation analysis will be performed via Spearman or Pearson correlation tests, based upon data distribution. Categorical variables are analyzed via either Chi-square tests or Fisher's exact tests, as appropriate. ANOVA for repeated measurements will be used for (1) measurements of the aforementioned at the beginning, at 6 and at 12 months (which is considered a within-subject factor) and (2) also the differenced of different groups, as a between-subject factor). Post-hoc comparisons will be conducted with Bonferroni's correction. The F and the respective P values between the different time periods of measurements will be calculated. Furthermore the F and P values between time of measurement of the biomarkers and the examined groups will be estimated. The Greenhouse-Geisser, Huynh-Feldt or Lower-Bound corrections will be used when the sphericity assumption, as evaluated by Mauchly's test, will not be met. The percent changes of the examined variables after intervention will also be compared by one way ANOVA. All statistical tests are two-tailed and a p-value \< 0.05 is considered statistically significant.