The Role of Glycocalyx Integrity in the Primary Prevention of Major Cardiovascular Events
The Role of Glycocalyx Integrity in the Primary Prevention of Major Cardiovascular Events
We intend to recruit individuals who are referred for a routine screening in the primary
 prevention outpatient clinic of Attikon University hospital, Athens According to
 institutional protocols, a detailed medical history for atherosclerotic risk factors and
 current medication is recorded and a baseline clinical examination is performed. Moreover
 fasting blood samples are drawn to diagnose the presence of diabetes mellitus and
 hyperlipidemia. Glycocalyx examination is performed at the same visit. The subjects are
 revaluated at one month and then followed up according to the attending physician's
 instructions. All subjects are followed up for adverse events (death, stroke, myocardial
 infarction, hospitalization for heart failure) for 6 years after enrollment via telephonic
 contact and planned appointment at the outpatient clinic.
Blood pressure is measured in each subject with the aid of a digital oscillometric
 sphygmomanometer (TensioMed, Budapest Hungary, Ltd). Each subject rests in a quiet room at
 25°C for 30 minutes before the examination. The cuff is placed in the right arm, while the
 subject is seated. The diagnosis of hypertension is made on the basis of:1) systolic blood
 pressure ≥140 mmHg/diastolic blood pressure ≥90 mmHg after 3 consecutive measurements or 2)
 the participant is receiving antihypertensive treatment. Moreover fasting blood samples are
 drawn to identify the presence of diabetes mellitus and hyperlipidemia in our sample.
 Specifically, DM is determined by either plasma glucose ≥ 126 mg/dl measured by the enzymatic
 in vitro test (Roche, automatic chemistry clinical analyzer) or treatment with antidiabetic
 agents . Similarly, hyperlipidemia was defined by either Total Cholesterol ≥230 mg/dl, and/or
 LDL ≥160 mg/dl, and /or TGs ≥200 mg /dl measured by the colorimetric spectrophotometry (Roche
 Diagnostics) or the use of lipid lowering agents according to the running guidelines held
 during the initial examination. Additionally, smokers are considered only those participants
 who are actively smoking at the time of enrollment . Also, the presence of positive family
 history of coronary artery disease is defined as coronary disease in a first degree female
 relative before 65 years or a male relative before 55 years of age. BMI is calculated as the
 ratio of weight (kg) to the square of height (m2). Furthermore, we use the SCORE estimation
 system to calculate the 10-year cardiovascular risk for each participant.

 As far as ethnicity is concerned, an individual is not considered Greek in case he/she was
 born abroad and also has at least a foreigner parent or he/she is born in Greece but both
 parents were foreigners. Inversely, a subject was considered Greek if they and simultaneously
 both parents were born in Greece.

 Exclusion criteria are the history of Coronary artery disease (CAD), Peripheral Arterial
 Disease (PAD), Heart failure (HF), Stroke, hepatic or renal failure, active neoplasia and
 poorly controlled DM, defined as Hba1C>7%.

 ENDOTHELIAL GLYCOCALYX EVALUATION We quantify the perfused boundary region (PBR, measured in
 μm) of the sublingual microvasculature by using Sideview Darkfield Imaging (Microscan,
 Glycocheck, Microvascular Health Solutions Inc., Salt Lake City, UT, USA) . Briefly, SDF
 camera utilizes green reflected light emitting diode (LED) light (540 nm) from hemoglobin
 molecules to depict the radial displacements of RBCs within the microvessels . The camera is
 inserted under the tongue and captures more than 3000 vascular segments of vessels with
 diameter ranging from 5 μm to 25 μm. The images are obtained automatically by the Glycocheck
 software . Afterwards the system checks the quality of the recordings and selects the valid
 frames (contrast enhancement greater than 60%), which are going to be further analyzed . The
 RBC column width, as well as the total perfused diameter of microvessels is estimated by the
 software with high accuracy. Then PBR is calculated by the formula [Perfused Diameter- RBC
 column width]/2.The software also provides the PBR of vessels with diameter 5-9μm, 10-19μm,
 20-25μm and the mean 5-25μm As a result, PBR expresses the outer area of the vessels' lumen
 that is vulnerable to RBC penetration and is consistent with the integrity of glycocalyx
 layer. Thus, high PBR values are considered as indicator of damaged endothelial glycocalyx
 with affected barrier properties, which allows high spatio-temporal distribution of RBCs. On
 the other hand Conversely, low PBR values are associated with robust thick glycocalyx layer.
 The whole procedure lasts for approximately 3 minutes, does not require high expertise, is
 operator independent, has satisfactory reproducibility and thus is highly recommended as a
 validated technique for the assessment of endothelial glycocalyx function . The PBR
 measurements are independent of red blood cell filling of the vessels segments (hematocrit),
 because the software only includes vessel segments that have a filling percentage of more
 than 50%. 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 .

 In our study the evaluation of PBR takes place between 9:00 and 11:00, after 8 hours fasting
 and abstinence from alcohol and smoking and temporary discontinuation of medications for 48 h
 before the examination as well.

 FOLLOW UP We perform a follow up of our study cohort 6 years after the recruitment via
 telephonic contact and planned appointment at the outpatient clinic. Briefly, at the
 reevaluation the participants or their legal representatives - in case of inability or death-
 are asked for the incidence of major cardiovascular events or death attributed to any cause.
 The primary composite outcome of major adverse events consists of death, myocardial
 infarction (ICD-10: DI20-22), stroke (ICD-10: DI60-68; DG45) and hospitalization for heart
 failure (ICD-10: DI50-51; DI42; DI11). Each of the forenamed events is documented by hospital
 medical records or attending phycians' letters and confirmed by the national security
 electronic records using the appropriate ICD10.

 STATISTICAL ANALYSIS

 Statistical analysis is performed by SPSS version 25 (IBM SPSS Statistics, Inc., Chicago, IL)
 and Stata version 16 (StataCorp LP, College Station, TX). Scale variables are presented as
 mean ±SD in case of normal distribution, as determined by the normality tests
 Kolmogorov-Smirnov test and Shapiro-Wilk, or as medians and interquartile ranges in case of
 non-normal distribution. Nominal variables are expressed as frequencies and percentages.
 Scale variables are compared using independent samples Student's t tests or Mann-Whitney
 tests as indicated, whereas categorical variables are compared using Chi-square tests or
 Fisher's exact tests, as appropriate. In addition, patients are dichotomized based on the
 mean values of PBR for the sublingual microvessel diameter 5-25μm, 5-9 μm, 10-19 μm, and
 20-25 μm . Cox proportional hazard models are built for each variable to decide whether any
 of mean values of the above PBR categories can serve as an independent predictor of major
 cardiovascular events or death at a significance level of p value<0.05. Cumulative event
 rates are calculated using the Kaplan-Meier survival analysis for subjects with lower vs
 higher mean PBR value. The log-rank test for time-to-event data with respect to the total
 events is used for comparison of these two groups. Cox proportional hazard models are built
 for each possible risk factor in relation to the study endpoints. Estimated hazard ratios and
 their respective 95% confidence intervals are obtained. P value<0.05 are considered
 significant. Multivariate analysis include factors with proven clinical value or p value ≤0.1
 at univariate analysis. Moreover, we compare chi-square values of multivariate models before
 and after adjusting for PBR. Chi square change with p value<0.05 is considered significant.
 Furthermore, we calculate the net reclassification index (NRI) and Harrell's C to evaluate
 improvement in risk prediction by PBR and p value<0.05 is considered significant.
Inclusion Criteria:

 We recruit individuals who were referred for a routine screening in the primary prevention
 outpatient clinic of Attikon University hospital.

 Exclusion Criteria:

 -  History of Coronary artery disease,

 -  History of Peripheral Arterial Disease,

 -  History of Heart failure

 -  History of Stroke

 -  hepatic failure

 -  renal failure,

 -  active neoplasia

 -  poorly controlled DM, defined as Hba1C>7%