Study Protocol

Ninety-two consecutive Caucasian volunteers without known cardiovascular risk factors and/or overt cardiovascular disease (58 men, 34 women; mean age, 46.0 ± 13.3 years) were enrolled. The study population, referred to the local hospital, had undergone voluntary screening for cardiovascular disease at our outpatient clinic, which included a questionnaire about medical history, the use of drugs in the previous 5 years, cardiovascular risk factors, and lifestyle habits (alcohol intake, smoking, and physical activity). The questionnaire was completed with data from hospital charts and/or personal records when available. None of the subjects were taking drugs chronically, and the clinical histories of these healthy volunteers were uneventful. To generate a healthy sample, participants were excluded for the following reasons: hypertension (systolic blood pressure [BP] ≥ 140 mm Hg and diastolic BP ≥ 90 mm Hg confirmed by home BP measurements or drug treatment for hypertension), diabetes (treatment with insulin or an oral hyperglycemic agent), treatment for dyslipidemia, and history of cardiovascular disease (coronary artery disease, congestive heart failure, stroke, transient ischemic attack or intermittent claudication).

Oscillometric BP and heart rate measurements were taken twice in the right arm after 10 min in the supine position in a quiet room using a mercury sphygmomanometer, just before the transthoracic echocardiographic exam and once before the carotid study was begun (≥30 min after the first measurement). Phase V Korotkoff sounds were considered as diastolic BP except in subjects with sounds tending toward zero, in whom phase IV was taken. For the calculation of the carotid stiffness parameters, the BP taken before the carotid exam was entered into the program. The BP measurements taken before echocardiography were considered for the calculation of pulse pressure [PP] (systolic BP − diastolic BP). Weight (in kilograms) and height (in meters) were measured using standard techniques, and body mass index (BMI) was calculated as body weight divided by height squared. Body surface area was calculated using the DuBois formula (0.20247 × height ^0.725 × weight ^0.425 ).

The study was approved by the institutional ethics board, and informed consent was obtained from the participants.

Echocardiographic Measurements

Standardized transthoracic and Doppler echocardiographic examinations were performed using commercially available equipment in all subjects (Alpha 10; Aloka, Tokyo, Japan) in accordance with the American Society of Echocardiography’s guidelines under continuous electrocardiographic recording. Specific views included the parasternal long-axis and short-axis views (at the mitral valve and papillary muscle levels) and apical four-chamber, two-chamber, and three-chamber views. Pulsed-wave and continuous-wave Doppler interrogation was performed on all four cardiac valves. All studies were reviewed and analyzed offline by two independent observers blinded to the clinical characteristics of the study population. Each parameter was assessed in three to five consecutive cardiac cycles, and the mean values were obtained for the purposes of our study.

Carotid Artery Stiffness Parameters

Measurements of local arterial stiffness were obtained at the level of the left common carotid artery 1 to 2 cm below the bifurcation (using a high-definition echo-tracking system implemented in the Alpha 10). The examination was performed immediately at the end of the echocardiographic study using a 7.5-MHz linear probe. Echo-tracking uses the raw radiofrequency signals that are based on the video signals. For measurements of diameter change by echo tracking, the optimal angle between the ultrasound beam and the vessel wall is 90°. However, if the blood flow is perpendicular to the beam, it is not detected. To overcome this problem, a different ultrasound beam for measurements of diameter change and blood velocity was used, and blood velocity measurements were independently steerable. Figure 1 shows a long-axis view of the common carotid artery and the ultrasound beam configuration with the independent beam steering function. The solid line shows the ultrasound beam direction for velocity measurements, while the dotted line shows the beam direction for measurements of diameter change. These beams were steered to intersect at the center of the range gate. The steering angle of each ultrasound beam can be changed in 5° increments from −30° to +30°. The echo-tracking gates are manually set at the boundaries between the intima and media of the anterior and posterior walls. The rate gate for velocity measurements was automatically positioned at the center of the diameter using echo-tracking gates. Flow velocity is corrected for the angle between ultrasound beam direction and flow velocity vector. The right section of Figure 1 represents the M-mode for the carotid diameter measurements. During systole, the pressure and diameter change waveforms were very similar. During diastole, the carotid arterial pressure-diameter relationship showed slight nonlinearity and hysteresis. The maximal and minimal values of a diameter change waveform were calibrated according to the systolic and diastolic relationship. The relationship of pressure to diameter is thought to be linear. Brachial cuff BP was measured just before starting the carotid study and was entered into the system for the calculation of carotid stiffness parameters. Three to five beats were averaged to obtain a representative waveform.

Examination of common carotid artery with simultaneous display of waveforms derived from instantaneous change in diameter using echo-tracking technique. ( Left ) Long-axis view, B-mode: the arrow indicates the gate for velocity measurements, the orange line the beam for the carotid vessel diameter variations, and the green dotted line the beam for the blood velocity. ( Right ) Long-axis view, M-mode: carotid diameter change ( top ) and BP wave ( pink ) and electrocardiographic ( green ) trace ( bottom ).

The following indices were automatically calculated: β index, ln[(Ps/Pd)/(Ds − Dd)/Dd)], Ep (pressure-strain elasticity modulus), and (Ps − Pd)/(Ds − Dd)/Dd ( Figure 2 ), where Ps is systolic BP, Pd is diastolic BP, Ds is arterial systolic diameter, and Dd is arterial diastolic diameter.

Echo-tracking assessment of parameters of carotid stiffness. ( Top and middle ) Carotid diameter variations and electrocardiogram. ( Bottom ) Carotid variables (β index and Ep). D_max , Carotid diameter in systole; D_min , carotid diameter in diastole; HR , heart rate; P_max , systolic BP; P_min , diastolic BP.

Intima-Media Thickness (IMT) Measurement

In 76 subjects from the original cohort of 92 normal subjects, IMT was measured at the far wall of the left common carotid artery 10 to 15 mm proximal to the carotid bulb by an investigator who was blinded to measures of carotid stiffness.

An additional group of 53 subjects with the same clinical characteristics as the subjects of the original cohort were studied. They underwent echocardiography and arterial stiffness and IMT determination. In this group, DTI measurements were done at the septal and lateral mitral annulus, and the mean between the two was considered.

Statistical Analysis

Data are presented as mean ± SD for variables with normal distribution and as median (interquartile range) for skewed variables. The Shapiro-Wilk test ( P < .05 for rejecting the null hypothesis) was used to test the normality of the distribution. We used Pearson’s correlation analysis to assess univariate correlations between arterial stiffness parameters, IMT, and LV structure and functional variables. Pearson’s correlation was also used to evaluate whether age, PP, sex, and BMI were associated with arterial stiffness, systolic and diastolic function, and LV structure. Covariate analysis was then applied to adjust first for the effect of age and second for the effects of age, PP, sex, and BMI combined. The level of statistical significance was set at .05. Data analysis was performed using Stata version 10.0 (StataCorp LP, College Station, TX).