Results Plaque burden was higher in men (63.4 mm 3 [interquartile range (IQR): 23.8 to 144.8 mm 3 ] vs. 25.7 mm 3 [IQR: 11.5 to 61.6 mm 3 ] in women; p < 0.001), in the femoral territory (64 mm 3 [IQR: 27.6 to 140.5 mm 3 ] vs. 23.1 mm 3 [IQR: 9.9 to 48.7 mm 3 ] in the carotid territory; p < 0.001), and with increasing age (p < 0.001). Age, sex, smoking, and dyslipidemia were more strongly associated with femoral than with carotid disease burden, whereas hypertension and diabetes showed no territorial differences. Plaque burden was directly associated with estimated cardiovascular risk independently of the number of plaques or territories affected (p < 0.01).

Key Words

Detection of subclinical atherosclerosis improves cardiovascular risk stratification (1,2). A direct relationship between atherosclerotic lesions and conventional cardiovascular risk factors (CVRFs) has been previously established, first with necropsy findings (3), and subsequently using different noninvasive imaging techniques. Coronary artery calcium score (CACS) and intima-media thickness (IMT) have been more widely explored in their relationship with CVRFs and cardiovascular risk than direct evaluation of atheromas. Nonetheless, increasing evidence reinforces the potential value for risk assessment of detecting atherosclerotic lesions (4–6), including multi-territorial evaluation since atherosclerosis is a systemic process (7,8). Two-dimensional vascular ultrasound (2DVUS) is well suited, not only for detecting, but also for grading, the extent of atherosclerosis in different territories. However, methods for quantifying atherosclerotic plaque burden with 2DVUS have differed between studies, and no consensus exists on which measurement should be used or which arterial territory is more suitable for cardiovascular risk prediction (9).

Recently introduced semiautomated 3-dimensional vascular ultrasound (3DVUS) has been proposed as a better method for quantifying peripheral atherosclerotic burden (10). Plaque volume may be a more comprehensive measure that incorporates disease presence and amount, and is thus more likely to reflect the cumulative effect of chronic CVRF exposure and individual susceptibility. However, 3DVUS has not been used to quantify multiterritorial plaque burden in large population studies, and the associations of plaque volume with CVRFs have not been explored. Our goals were to: 1) evaluate feasibility and reproducibility, and provide reference values of carotid and femoral plaque burden distribution by 3DVUS in asymptomatic middle-aged individuals; 2) determine the relationship between CVRFs and plaque burden; and 3) explore the potential additive value of quantifying plaque volume versus plaque detection alone.

Global plaque burden by 3DVUS is a right-skewed variable. The relationship between CVRFs and global plaque burden was explored by ordinal logistic regression, with plaque volume tertiles (outcome) used to classify atherosclerotic burden as none, mild, moderate, or severe. The candidate variables in the multivariable model were sex, age, smoking in pack-years, dyslipidemia, diabetes, hypertension, obesity, and family history of cardiovascular disease. To compare the effects of different CVRFs on plaque burden in our cohort, we calculated the “adequacy” index (15) . When excluding participants without plaques, linear regression on log-transformed plaque burden was used to investigate sex- and age-related changes in the extent of carotid and femoral subclinical atherosclerosis. We used linear regression on log-transformed data to explore the association between global plaque burden and ASCVD risk score stratified by number of plaques and affected territories. A p for trend <0.05 was considered significant. Statistical analyses were conducted with Stata version 12 (Stata Corp., College Station, Texas).

Baseline characteristics are presented as mean ± SD or median and interquartile range (IQR) for continuous variables, and counts and proportions for categorical variables. Differences between continuous variables and categorical variables were tested with unpaired Student t tests and chi-square tests, respectively. Variables with non-normal distribution were log-transformed before comparison. For reproducibility analyses, the intraclass correlation coefficient (ICC) and Bland-Altman plots were employed. Kappa coefficients were used to assess agreement of plaque detection between techniques. For ICC and kappa, good agreement was defined as >0.70 and excellent agreement as >0.90.

The 3DVUS examinations were performed with a Philips iU22 ultrasound system equipped with a VL13-5 3D volume-linear array transducer (Philips Healthcare, Andover, Massachusetts). Detailed 3DVUS acquisition and analysis methodology have been reported previously, together with experimental validation of this novel 3D vascular technique (13) . The PESA study 3DVUS methodology included evaluation of carotid and femoral arterial segments adjacent to the bifurcation in a standardized fashion. The protocol for the carotid arteries consisted of a 30° automatic sweep (explored vessel segment ≈6 cm long) centered at the carotid bulb to include the distal common carotid artery, the bulb, the bifurcation, and the proximal internal and external carotid artery segments. For the femoral arteries, the 30° and ≈6 cm acquisition was centered at the bifurcation and included the mid-distal common femoral artery, the bifurcation, and the proximal superficial and deep femoral artery segments ( Online Figure 1 ). The acquired images were analyzed off-line using the Vascular Plaque Quantification (VPQ) tool in QLAB version 10.2 (Philips Healthcare) at the Centro Nacional de Investigaciones Cardiovasculares (CNIC) Imaging Core Laboratory. For analysis, 3 contours were defined: the media–adventitia boundary, the lumen–intima boundary, and the plaque–lumen boundary ( Online Figure 1 ). Ultrasound readers can then review and manually correct the contours that the software semiautomatically propagates throughout the imaged vessel. In this study, 3DVUS acquisitions were analyzed by 4 trained technicians blinded to other test results. There is currently no standard definition of atherosclerosis plaque using 3DVUS, and plaque was therefore defined using the Mannheim criteria for 2DVUS as a focal protrusion into the arterial lumen of thickness >0.5 mm or >50% of the IMT or IMT >1.5 mm (14) . Plaque burden was quantified by measuring the volumes of all atherosclerotic plaques visualized within the standardized 6-cm acquisition. Global plaque burden was defined as the sum of plaque volumes in the right and left carotid and femoral arteries. We also recorded plaque presence (yes/no), the number of arteries affected, and the total number of plaques present in the 3DVUS acquisitions. Participants without plaques in the analyzed segment were assigned a plaque burden of 0 mm 3 . Plaque volume was not adjusted for artery size because we analyzed a fixed 6-cm segment regardless of total vessel size.

The PESA-CNIC-Santander (Progression of Early Subclinical Atherosclerosis) study is an ongoing observational prospective cohort study characterizing early subclinical atherosclerotic burden and determinants of atherosclerosis presence and progression using different noninvasive image techniques. The study rationale and design have been previously reported (11) . Briefly, between June 2010 and February 2014, PESA enrolled 4,184 volunteers between 40 and 54 years of age without prior cardiovascular disease, who were employees at the Banco de Santander headquarters in Madrid (Spain). A baseline visit included 3DVUS in the carotid and femoral territories, clinical interviews, standardized lifestyle questionnaires, physical examination, fasting blood draw, urine sample collection, and 12-lead electrocardiogram. Participants are being prospectively followed-up with repeat visits at 3 and 6 years. The study protocol has been approved by the Instituto de Salud Carlos III Ethics Committee and complies with the Declaration of Helsinki. All participants provided written informed consent. Definitions of CVRFs, including dyslipidemia, smoking, hypertension, diabetes, obesity, and family history of premature cardiovascular disease, are as reported previously (7) . Cumulative smoking exposure was determined in pack-years by multiplying the number of years smoked by the average number of packs per day. The American College of Cardiology/American Heart Association atherosclerotic cardiovascular disease (ASCVD) risk score was quantified, and classified according to 10-year risk as low (<5%), intermediate (5% to <7.5%), or high (≥7.5%) (12) .

A and C show the distribution of disease among ASCVD risk categories stratified by the number of territories affected and the number of plaques (excluding no disease). B and D show mean global plaque burden across risk strata adjusted by the number of territories affected and number of plaques. The p values for the trend between plaque burden and ASCVD risk are shown. 3DVUS = 3-dimensional vascular ultrasound; ACC/AHA = American College of Cardiology/American Heart Association; ASCVD = atherosclerotic cardiovascular disease.

Most participants were disease-free (53.6%). Involvement of 1, 2, 3, or 4 territories was noted in 22.9%, 14.2%, 5.6%, and 3.6% participants, and 1, 2, 3, or ≥4 plaques were detected in 19.8%, 11.6%, 6.3%, and 8.7% participants, respectively ( Table 5 ). Panels A and C in the Central Illustration display the distribution of disease presence across ASCVD risk categories. Plaque presence increased across risk categories whether assessed by the number of territories affected or the number of plaques. However, a non-negligible proportion of participants classified at high risk had only localized disease (19.1% and 15.4% of participants with 1 territory or plaque, respectively), and participants with extensive disease were found among the low-risk subgroup (1.8% and 4.6% of participants with 4 territories or ≥4 plaques, respectively). Detailed data on the number of plaques and affected territories, as well as plaque burden across ASCVD risk categories, are shown in Table 5 .

The cardiovascular risk profile of participants without plaque and across tertiles of global plaque burden is shown in Table 3 , and separately for carotid and femoral arteries in Online Tables 5 and 6 . As expected, participants in the highest tertile were older and had a higher prevalence of all CVRFs except for family history of premature cardiovascular disease. The univariate and multivariate predictors of plaque burden are summarized in Table 4 . Age, male sex, and all CVRFs demonstrated independent associations with plaque burden, but not family history and obesity. The strongest associations were noted for age, sex, and smoking exposure, followed by dyslipidemia and hypertension, whereas diabetes was the weakest independent predictor in our cohort, and was not significant for carotid plaque burden. When comparing the association of risk factors with either carotid or femoral plaque burden, the multivariate associations of age, male sex, smoking, and dyslipidemia were stronger for the femoral than the carotid territory, whereas diabetes and hypertension showed no significant territorial differences. The magnitudes of these differences are shown in Figure 4 .

Plaque detection and plaque burden quantification by age, sex, and territory are shown in Table 2 . Median global plaque burden was 50.8 mm 3 (IQR: 18.7 to 121.5 mm 3 ), being more pronounced in men than in women (63.4 mm 3 [IQR: 23.8 to 144.8 mm 3 ] vs. 25.7 mm 3 [IQR: 11.5 to 61.6 mm 3 ]; p < 0.001). For both sexes, plaque burden was higher in the femoral than the carotid territory (p < 0.001) and increased significantly with age ( Table 2 ). Men tended to have a higher age-related plaque burden increase for the femoral territory ( Figure 2 ); nevertheless, this did not reach statistical significance when compared with the carotid territory or with women (p = 0.10 for both). Plaque burden values by percentiles are shown in Online Table 4 , and percentile curves for each sex as a function of age are shown in Figure 3 .

A total of 15,936 arteries (99% of the 3DVUS studies) were successfully analyzed, with good image quality in 93% ( Online Table 1 ). Acceptable quality window was slightly more frequent in the carotid territory than in the femoral territory (7.6% vs. 6.2%). Low-echogenic plaques (4.4%) or plaques with complex morphologies (2.3%) did not generally prevent adequate plaque volume analysis; and severe calcification was rare (0.03%). Interobserver and intraobserver agreement was good for the detection of plaque (kappa ranging from 0.87 to 0.97 and 0.94 to 1, respectively) and for global plaque burden quantification (ICC 0.89 [95% confidence interval 0.86 to 0.91] and 0.87 [95% confidence interval 0.83 to 0.90], respectively), with similar good results for carotid and femoral territories when analyzed separately ( Online Tables 2 and 3 ). Bland-Altman plots for global and regional plaque volume quantification are shown in Online Figure 2 .

The PESA cohort consists of 4,184 participants. Thirty-three participants withdrew, and 167 were excluded due to incomplete 3DVUS studies in at least 1 of the 4 territories. A further 8 participants were excluded due to inadequate image quality in 1 or more territories, and 116 were excluded due to missing information about CVRFs. The final study population thus comprised 3,860 (92.2%) of PESA participants ( Figure 1 ). Baseline demographic, clinical, and laboratory characteristics are summarized in Table 1 . There was a relatively low prevalence of diabetes, hypertension, obesity, and family history of premature cardiovascular disease; by contrast, there was a non-negligible prevalence of dyslipidemia and cigarette smoking. Overall, the PESA participants are a low-risk population (median ASCVD risk 2.17% [IQR: 0.95% to 4.37%]), with most (79.4%) in the low-risk category.

Discussion

In the present study, we evaluated 3DVUS in a large population. We provide evidence that 3DVUS is a feasible and reproducible approach to not only detect, but also quantify, early atherosclerotic burden in the carotid and femoral arteries. In addition, we report the reference values of atherosclerotic plaque volume for a middle-aged, asymptomatic population. The key messages derived from our observations are: 1) carotid and femoral 3DVUS identifies higher plaque burden in men, in the femoral arteries, and with increasing age; moreover, the pattern of disease changes with age is sex- and artery-dependent; 2) 3DVUS-detected plaque burden is strongly associated with CVRFs, and this association is more significant for the femoral than for the carotid territory; and 3) the evaluation of plaque burden in addition to plaque presence provides a closer match with global cardiovascular risk.

Prevalence and distribution of plaque burden by 3DVUS A “pseudo-3DVUS” (freehand-2D-sweep/3D-like reconstruction) method was used previously for the carotid territory in the BioImage study (10); however, the present study is the first study to evaluate carotid and femoral atherosclerosis with a true 3DVUS approach in a large cohort. In agreement with previous 2DVUS studies, we found that the plaque burden in men is almost double that in women within the middle-age range (16,17). However, we also found a higher plaque volume in the femoral territory in both sexes, whereas plaque presence was more frequent in the femoral arteries in men but in the carotid arteries in women. Furthermore, age-related changes in plaque volume tended to be more rapid in the femoral arteries in men. Thus, compared with plaque detection, plaque volume quantification unveils novel sex- and artery-related differences in the early development of atherosclerotic disease. Prevalence of carotid atherosclerosis has been reported to be low in women before menopause (18,19), subsequently becoming similar to the prevalence in men (20). Our data suggest that the same pattern may occur with femoral atherosclerosis evaluated by 3DVUS. These observations are in accordance with the later development of cardiovascular disease in women until menopause worsens their cardiovascular risk profile. In addition, there is an increasing awareness of the importance of femoral artery evaluation for the detection of early subclinical atherosclerosis (7,8,21–23). Our results confirm that this territory is more extensively diseased in both sexes, suggesting a potential to improve early disease evaluation in the young. The availability of reference values in the population has been extremely valuable for image-based strategies for cardiovascular risk stratification. In the literature on CACS and carotid IMT, values above the 75th percentile are considered pathological in the general population (24,25). There are no previous reports on true 3DVUS-volumetric data in population-based cohorts, and this study provides a large dataset for future reference. Ongoing long-term PESA study follow-up will help to identify associations with cardiovascular events.

Relationship between plaque burden and CVRFs Many previous reports have associated CVRFs with carotid IMT and CACS (26,27). More recently, the AWHS (Aragon Workers’ Heart Study) assessed the association between CVRFs and carotid and femoral atherosclerosis in 1,423 asymptomatic men between 40 and 59 years of age (22). However, this study only evaluated the presence of plaque with 2DVUS and not its burden. Only a few groups have used quantitative 2DVUS measurements of peripheral atherosclerosis, such as plaque area or plaque thickness, in the general population (28,29). Our study confirms the strong association between multiple CVRFs and atherosclerotic plaque burden as measured with 3DVUS. Age was the strongest predictor of plaque burden, followed by sex and the modifiable risk factors, as demonstrated by the adequacy index. Among the modifiable risk factors, smoking exposure in pack-years was strongly associated with atheroma burden, followed by dyslipidemia, hypertension, and to a lesser extent, diabetes. Although this is in apparent contradiction with the well-documented relation between diabetes and increased atherosclerosis, it probably reflects the low prevalence of diabetes in our cohort and the likely short disease exposure among those who are affected. In a further analysis (data not shown), being a smoker also showed an independent association with plaque burden (adequacy index = 0.16; p < 0.001). However, pack-years of smoking was more strongly related than the simple smoking status to baseline plaque burden. Contrasting with our results, the AWHS did not find dyslipidemia and diabetes to be significant predictors of the presence of carotid plaques even though these risk factors were more prevalent than in our study, highlighting the difference between simple plaque detection versus burden quantification. Yerly et al. (29) evaluated the link between CVRFs and 2DVUS-quantified carotid and femoral atherosclerosis in a middle-aged cohort (N = 496, age 45 to 64 years) and found that CVRFs were more strongly associated with quantitative plaque measures than IMT. Notably, the relationship between CVRFs and carotid plaque thickness was inconsistent and become clearer when plaque area was measured. The authors attributed this to high variability in 2DVUS measurements, something that 3DVUS has partially overcome (30). Together, the results of these studies and ours suggest that more comprehensive measures of atherosclerotic burden such as plaque volume may better reflect long-term CVRF exposure.

Territorial differences in the relationship between plaque burden and CVRFs Beyond the age- and sex-based differences discussed in the preceding text, the associations between CVRFs and plaque burden tended to be stronger for the femoral than the carotid territory. Similar observations were reported in the AWHS (22) and by Yerly et al. (29), both using 2DVUS, although these studies did not make formal comparisons. Our analyses confirm these observations, showing statistical significant differences for sex, age, smoking, and dyslipidemia. Smoking has been linked to peripheral artery disease and intermittent claudication (31), and early atherosclerotic changes are more significant in the femoral compared with the carotid territory in patients with familial hypercholesterolemia (32). The apparent lack of territorial differences in hypertension contrasts with previous reports that have tended to link high blood pressure with carotid atherosclerosis and stroke (16,33). However, these studies did not include assessment of the femoral territory and usually enrolled older populations. In accordance with our results, the few available reports in middle-aged participants show a relationship between hypertension and both carotid and femoral subclinical atherosclerosis (22,29,34). The mechanisms of these artery-related differences are unclear, and are likely related to both histological and hemodynamic factors (35). Overall, our findings support multiterritorial imaging for comprehensive evaluation of subclinical atherosclerosis and the combined effect of CVRFs in early atheroma formation.

Relationship of plaque volume and plaque presence with cardiovascular risk Our results confirm the well-established mismatch between cardiovascular risk profile determined by risk scales and the presence of subclinical atherosclerosis (7,36), which has been attributed to variable individual susceptibility. However, adding plaque burden quantification to the detection of plaque presence provides a clearer picture of the relationship between risk and subclinical disease: among individuals with the same number of plaques or affected territories, global plaque burden was independently and positively associated with estimated ASCVD risk. Global plaque volume, by integrating plaque presence, number, and plaque size, is a more comprehensive index of disease burden that may better reflect individual susceptibility, and thus partially explain the mismatch between risk profile and plaque presence. The difference between disease presence and volume is also illustrated by the discrepancy between these 2 methods in comparing disease severity between the femoral and carotid arteries in women; the number of plaques was similar or even lower in the femoral territory, but femoral plaque volume was almost double that in the carotid territory. The potential of plaque burden quantification to improve cardiovascular risk stratification is suggested by the BioImage study, which showed that quantifying carotid burden as the sum of consecutive plaque areas in mm2 improves risk prediction, risk reclassification, and statin eligibility for primary prevention strategies with comparable results to CACS (37,38). Also, previous studies using 2D plaque area measurements demonstrated significant improvement in risk prediction of myocardial infarction, stroke, and even cardiovascular death (39–41). Our results suggest that the ability of global plaque burden to predict cardiovascular risk is likely to be improved by adding evaluation of the femoral arteries at early stages of cardiovascular disease.

Feasibility and reproducibility of 3DVUS PESA study protocol Assessment of the 3DVUS method in the PESA study demonstrated good feasibility for screening early atherosclerosis, providing a unique opportunity to study subclinical disease in large populations. Previous 3DVUS approaches reported dropout rates that varied depending on the clinical context, ranging from 23% in patients undergoing revascularization of peripheral artery disease (42) to 33% in patients with recent ischemic stroke or transient ischemic attack (43). The main reported cause of dropout in these studies was a high percentage of severely calcified plaques, suggesting that 3DVUS performs worse at advanced stages of atherosclerosis compared with our sample of individuals in the early stages of disease. Also, the 3DVUS protocol developed in the PESA study, by centering the acquisition at bifurcations without probe displacement, is simple and has good reproducibility, ensuring the re-evaluation of the same segment during follow-up to detect small changes and facilitating the generalizability of our results across sites.