Use of Lipid Parameters to Identify Healthy Men at a Higher Risk of Arterial Stiffness Progression


 Background: Dyslipidemia contributes to the development and progression of arterial stiffness. We aimed to confirm the superiority of the serum lipids and their calculated ratios in predicting arterial stiffness progression.Methods: Total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and brachial-ankle pulse wave velocity (baPWV) of 612 healthy males (47.0 ± 10.5 years) were measured at baseline. Values for non-HDL-C, TC/HDL-C, TG/HDL-C, LDL-C/HDL-C, and non-HDL-C/HDL-C were calculated. BaPWV was re-performed after 4.1 years follow-up. A baPWV cutoff of 1400 cm/s was used to diagnose arterial stiffness.Results: Over the follow-up period, the mean baPWV value increased from 1330 cm/s to 1400 cm/s, and 309 individuals increased/persisted with high baPWV (outcome 1). Among the 430 subjects who were free of arterial stiffness at baseline, 90 arterial stiffness events occurred (outcome 2). Only logTG (OR 1.65 [95% CI: 1.14-2.40] for outcome 1; 2.08 [1.24-3.52] for outcome 2) and logTG/HDL-C (1.56 [1.15-2.13] for outcome 1; 1.69 [1.10-2.62] for outcome 2) were significantly associated with arterial stiffness progression after adjusting for confounding factors. Adding logTG or logTG/HDL-C to age and blood pressure improved the accuracy of risk predictions for arterial stiffness progression. These associations remained significant when lipids were analyzed as categorical variables.Conclusions: Baseline serum TG and TG/HDL-C were independently associated with increases in/persistently high baPWV and incident arterial stiffness, and they performed more effectively than other lipid variables in identifying healthy men at a higher risk of arterial stiffness progression.


Background
Cardiovascular disease (CVD) has become the leading cause of death in China [1]. The increase in CVD risk is largely driven by adverse changes of the vasculature, including arterial stiffening. Brachial-ankle pulse wave velocity (baPWV) is the most widely used routine clinical practice in Asia to assess arterial stiffness and is being increasingly incorporated into studies in the US and Europe [2,3], since the measurement is valid, reproducible, minimal-risk, convenient, and cost-saving. Accumulating evidence demonstrates that arterial stiffness, evaluated by baPWV, is an independent predictor for CVD event and mortality in the general population and various patient populations [4][5][6]. Arterial stiffness is also one of the major age-related arterial phenotypes [7], and the 'Vascular Aging Continuum' has regarded increased arterial stiffness as the fundamental and vital link [8]. Pulse wave velocity is considered a physiological method for quantifying vascular aging [9,10]. Therefore, a relatively simple means to identify individuals who are at higher risk of arterial stiffness progression would be clinically useful.
Dyslipidemia characterized by the increase of total cholesterol (TC), triglyceride (TG), or low-density lipoprotein cholesterol (LDL-C), or the decrease of high-density lipoprotein cholesterol (HDL-C) contributes to arterial stiffness and has been proven to be associated with baPWV in extensive cross-sectional studies [7,11]. However, associations observed in cross-sectional analyses limit inferences about temporality and longitudinal studies regarding the role of serum lipids on arterial stiffness progression are limited and far from conclusive. In addition, arterial stiffness progresses at different rates and can be accelerated by several long-standing cardiovascular risk factors [12], and participants enrolled in previous longitudinal studies were often affected by a variety of chronic diseases. Therefore we selected healthy individuals according to strict criteria in this study to reduce the potential bias from other diseases and capture the dyslipidemia-related atrial stiffness progression difference. We aimed to determine the association of baPWV progression with baseline serum lipids, including TC, TG, LDL-C, HDL-C, non-HDL-C, TC/HDL-C, TG/HDL-C, LDL-C/HDL-C, and non-HDL-C/HDL-C, and to con rm the superiority of those lipid parameters in identifying healthy men at a higher risk of arterial stiffness progression.

Subjects
This retrospective longitudinal study was conducted at the physical examination center of the geriatric department of Tongji Hospital and was approved by the medical ethics committee of Tongji Hospital (TJ-IRB20190410). The study protocol conforms to the Declaration of Helsinki. We collected physical examination records from March 2011 to July 2019. In total, 612 healthy males aged 18 years or older were selected. All of them had measurements of baPWV and a complete lipid panel (including TC, TG, LDL-C, and HDL-C) at baseline and had their second baPWV measurement after a delay of more than three years. The exclusion criteria were: inheritable dyslipidemia, use of lipid-lowering medications, hypertension [de ned as systolic blood pressure (SBP) ≥ 140 mm Hg, diastolic blood pressure (DBP) ≥ 90 mm Hg, or use of antihypertensive drugs], diabetes [de ned as fasting blood glucose (FBG) of ≥ 7.0 mmol/L, HbA1c ≥ 6.5%, or use of hypoglycemic drugs], coronary disease, stroke, obvious arrhythmia (persistent atrial brillation, frequent premature beats, or wearing a pacemaker), cardiomyopathy, valvular heart disease, chronic liver or kidney disease, cancer, ankle-brachial index (ABI) less than 0.9, and missing data.

Clinical characteristics
Every time the patients visited the physical examination center, trained personnel conducted standardized in-person interviews with the patients to collect information regarding age, sex, current cigarette smoking, medical history, and medication use. Anthropometric indexes including height and weight were measured.
Body mass index (BMI) was computed as the weight in kilograms divided by the square of the height in meters. Blood pressure and heart rate were measured using an OMRON sphygmomanometer (OMRON Corporation, Japan). The blood pressure and heart rate used in the analysis were calculated as the average of three measured values. Mean arterial pressure (MAP) was calculated from the standard equation MAP = (2/3)DBP + (1/3)SBP (in mm Hg). Fasting venous blood samples were collected and sent to the hospital's clinical chemistry laboratory. TC, TG, LDL-C, HDL-C, FBG, HbA1c, creatinine, and uric acid were measured using standard certi ed assays. We also calculated values for non-HDL-C (TC minus HDL-C), TC/HDL-C (TC divided by HDL-C), TG/HDL-C (TG divided by HDL-C), LDL-C/HDL-C (LDL-C divided by HDL-C), and non-HDL-C/HDL-C (non-HDL-C divided by HDL-C).
Arterial stiffness measurements and de nition of the outcomes BaPWV and ABI were measured using the Vascular Pro ler BP-203RPE system (Omron, Kyoto, Japan).
Trained technicians placed the pressure cuffs on the subjects, i.e., one on the upper part of each arm and one on each ankle. Then, the subjects were examined after ten minutes of rest in the supine position. The device simultaneously recorded the bilateral pulse waves of the brachial and posterior tibial arteries using an oscillometric method. BaPWV was calculated as the ratio of the traveled distance (which was automatically estimated from the body height) divided by the transit time of the pulse wave between the brachial and posterior tibial arteries. We classi ed the outcome in 2 different manners. Outcome 1: the baseline and follow-up baPWV were both divided into quartiles, respectively. Then we classi ed subjects into two subgroups: those who decreased their quartile distribution or persisted within the two lower quartile groups, and those who increased their quartile distribution or persisted within the two higher quartile groups [13]. Outcome 2: a cutoff value of more than 1400 cm/s for baPWV was used to diagnose arterial stiffness [4,14]. Individuals without arterial stiffness at baseline were divided into "non-arterial stiffness" and "incident arterial stiffness" groups based on their follow-up baPWV levels.

Statistical analysis
Data were analyzed using R and RStudio 3.6.2. Continuous variables were presented as the means ± standard deviation or medians (interquartile range), as appropriate for the distribution. Categorical variables were shown as counts and proportions. Paired t-tests were used to determine if the baPWV levels changed over the follow-up period. We compared the baseline variables between groups using unpaired t-test, Mann-Whitney U test, and Chi-squared test accordingly. Crude and multivariable-adjusted binary logistic regression models were developed to estimate odd ratios (ORs) and 95% con dence intervals (CIs) for the two outcomes associated with each serum lipid, respectively. Prior to regression analysis, TG and TG/HDL-C were log-transformed to achieve normality. Age and MAP, the most important determinants for baPWV, were controlled for in the rst model. Further adjustments were made for BMI, smoking (current smoker and non-current smoker), heart rate, FBG, HbA1c, creatinine, and uric acid in the second model. The improvement in the ability of each serum lipid to predict outcomes was summarized using area under receiver operating characteristic (ROC) curves (AUC), positive net reclassi cation improvement (NRI), and integrated discrimination improvement (IDI) [15]. In sensitivity analyses, TC, TG, HDL-C, LDL-C, and non-HDL were divided into three or two levels by clinical cut-points [16]; TC/HDL-C, TG/HDL-C, LDL-C/HDL-C, and non-HDL-C/HDL-C ratios were divided into three levels by tertile cut-points.
ORs and 95% CIs for the outcomes across categories of each serum lipid were also calculated, using the most favorable category as the reference. Trends in ORs across categories of each lipid were calculated by modeling the lipid categories as an ordinal variable. Two-tailed p-values < 0.05 were considered signi cant.

General characteristics
The clinical characteristics of the study subjects are shown in Table 1. The population had a mean baseline age of 47.0 ± 10.5 years and the median duration of the follow-up was 4.1 years. The mean baPWV value increased from 1330 ± 204 cm/s to 1400 ± 240 cm/s over the follow-up period, with a mean annual baPWV change of 16 cm/s and the difference showed a statistical signi cance (p < 0.001).
Of all the study participants, 309 (50.5%) individuals had increased/persisted with high baPWV (outcome 1), and they had higher levels of age, blood pressure, heart rate, ABI, TG, TC/HDL-C, TG/HDL-C, and non-HDL-C/HDL-C, compared with those who presented a reduction or persisted with low baPWV. There were no statistical differences in other lipids, including TC, LDL-C, non-HDL-C, HDL-C, and LDL-C/HDL-C.
A total of 430 subjects were free of arterial stiffness at baseline, and 90 (20.9%) of them developed arterial stiffness during the follow-up period (outcome 2). Individuals with incident arterial stiffness had higher levels of age, blood pressure, ABI, FBG, TG, and TG/HDL-C, compared with those who stayed arterial health. Other lipids, including TC, LDL-C, non-HDL-C, HDL-C, TC/HDL-C, LDL-C/HDL-C, and non-HDL-C/HDL-C had no statistical difference.

Arterial stiffness progression and lipid parameters as continuous variables
Only logTG and logTG/HDL-C were signi cantly associated with increased risk of arterial stiffness progression in both crude and adjusted logistic regression models (all p <0.05; Figure 1). After adjusting for potential confounding factors, a unit increase in logTG and logTG/HDL-C resulted in ORs for outcome 1 of 1.65 (95% CI: 1.14-2.40) and 1.56 (95% CI: 1.15-2.13), respectively. Meanwhile, the ORs for outcome 2 per unit increase in logTG and logTG/HDL-C was 2.08 (95% CI: 1.24-3.52) and 1.69 (95% CI: 1.10-2.62), respectively. TC/HDL-C and non-HDL-C/HDL-C were not signi cantly associated with arterial stiffness progression in the adjusted models, while the ORs of TC, LDL-C, non-HDL-C, HDL-C, and LDL-C/HDL-C did not reach statistical signi cance in all the models.
We further performed ROC analyses to study the predictive power of logTG and logTG/HDL-C ( Figure 2 and Table 2). When adding logTG or logTG/HDL-C to the basic model, the prediction of the outcomes showed an increase in AUC. For example, AUC for outcome 2 increased from 0.72 to 0.74 when logTG was added. Positive NRI and IDI indicated that adding logTG or logTG/HDL-C signi cantly improved risk reclassi cation for both outcomes (p < 0.05).

Arterial stiffness progression and lipid parameters as categorical variables
The association between each serum lipid and arterial stiffness was also present when serum lipids were analyzed as categorical variables (Figure 3 and Figure 4). TG and TG/HDL-C were signi cantly associated with both outcome 1 and outcome 2 (all p-trend < 0.05). The ORs associated with the increased versus appropriate level of TG (≥ 2.30 versus ≤ 1.69 mmol/L) was 2.00 (95% CI: 1.20-3.38) for outcome 1 and 2.86 (95% CI: 1.35-6.04) for outcome 2. The ORs associated with the highest versus lowest quartile of TG/HDL was 2.03 (95% CI: 1.24-3.37; ≥ 1.58 versus ≤ 0.91) for outcome 1 and 2.98 (95% CI: 1.44-6.36; ≥ 1.47 versus ≤ 0.86) for outcome 2. ORs for outcome 1 among participants with the highest tertile compared with those with the lowest tertile of TC/HDL-C or non-HDL-C/HDL-C also had statistical signi cance.

Discussion
In this study, we compared the predictive power of serum lipid parameters on arterial stiffness progression in a sample of apparently healthy Chinese men. We found that even after adjusting for the confounding factors, high TG and TG/HDL-C were correlated with a higher risk of increases in/persistently high baPWV and incident arterial stiffness. In addition, we also found that serum TG was positively associated with baPWV and low TG was a protective factor for vascular aging in healthy elderly subjects in a previous study [10]. Hence we con rmed the superiority of TG and TG/HDL-C in midand late-life.
High TG is a traditional risk factor for CVD [17]. However, previous investigations, which have included TG in multivariable regression analyses for arterial stiffness progression, were con icting. Some studies showed that baseline TG levels and changes of TG levels over time independently predicted arterial stiffness progression after adjusting for other cardiovascular risk factors [18][19][20][21], while others have not [22][23][24][25]. TG/HDL-C has been considered as an independent predictor of insulin resistance [26,27], diabetes mellitus [28], and CVD [29]. High-level TG/HDL-C ratio was also proved to be associated with higher arterial stiffness in cross-sectional studies [30][31][32], and the relationship might be non-linear [30]. TG/HDL-C was also found to be an independent determinant of arterial stiffness in adolescents and young adults [33]. Consistent with our study, subjects with high TG/HDL-C ratio also had a higher risk of carotidfemoral pulse wave velocity (cfPWV) progression in healthy individuals [34]. However, the result of another investigation revealed that higher cfPWV in late-life was not related to faster annual rates of change in TG/HDL-C from mid-life [32]. The con icting results of these investigations were likely based on the differences in the study populations and the evaluation indexes of arterial stiffness and we provided evidence of the independent predictive value of baseline TG and TG/HDL for baPWV progression in Chinese healthy males.
It has long been thought that arterial stiffness is only a feature of hypertension-mediated organ damage, while more recent studies show that arterial stiffness precedes hypertension [35,36]. Arterial stiffness re ects the cumulative damage of cardiovascular risk factors on the vascular wall and is even better than the blood pressure in re ecting the risk of CVD [37]. Arterial stiffness begins in early childhood and shows increasing progression in adults [7]. Our study population displayed a mean baPWV increase of 16 cm/s per year. The progression rate was quite similar to that of another group of Chinese males, in which the 5year change was 70 cm/s for the healthy men [19]. The average annual change of baPWV of a Japanese/American population-based cohort study was 9 cm/s, but this study merely focused on men aged 40 to 49 years [38]. Valid baPWV progression rates have not been established so far.
We have some limitations to consider. First, although the results were adjusted for multiple covariates that may be associated with baPWV, the possibility of residual confounds remains. Second, we referred to some articles about cfPWV, because data regarding baPWV progression is limited. Although baPWV is highly correlated with cfPWV, we do not think the results are interchangeable. Moreover, whether controlling for TG and HDL-C can attenuate progression of arterial stiffness or whether any long-term reduction in arterial stiffness can translate into a reduction in cardiovascular events has not been directly demonstrated in randomized controlled trials.

Conclusions
In conclusion, we provided evidence in this longitudinal study that baseline TG and TG/HDL-C which could be obtained from routine serum lipids independently predicted increases in/persistently high baPWV and incident arterial stiffness, and they were superior to other traditional lipid variables in identifying healthy men who are at an increased risk of arterial stiffness progression. This positive association between TG, TG/HDL-C, and baPWV progression should be considered in the management of vascular health.

Consent for publication
Not applicable.

Availability of data and materials
The datasets analyzed during the current study are available from the corresponding author on reasonable request.  Figure 1 Forest plot of ORs of lipids for arterial stiffness progression. Left: outcome 1 (increases in/persistently high baPWV). Right: outcome 2 (incident arterial stiffness). Model 1: adjustmet for age and mean arterial pressure. Model 2: adjustment for age, mean arterial pressure, body mass index, smoking, heart rate, fasting blood glucose, HbA1c, creatinine, and uric acid. BaPWV indicates brachial-ankle pulse wave veloctiy; TC, total cholesterol; TG, triglyceride; LDL-C, low-density lipoprotein cholesterol; HDL-C, highdensity lipoprotein cholesterol.  Forest plot of ORs for outcome 1 (increases in/persistently high baPWV). All ORs were adjusted for age, mean arterial pressure, body mass index, smoking, heart rate, fasting blood glucose, HbA1c, creatinine, and uric acid. BaPWV indicates brachial-ankle pulse wave veloctiy; TC, total cholesterol; TG, triglyceride;