Serum level of High-density lipoprotein particles are independently associated with long-term prognosis in patients with coronary artery disease: The GENES study

Background HDL-Cholesterol (HDL-C) is not a right marker to measure the cardioprotective functions of HDL in coronary artery diseases (CAD) patients. Hence, measurement of other HDL-related parameters may have prognostic superiority over HDL-C. This work aimed to examine the predictive value of HDL particles profile for long-term mortality in CAD patients. Its informative value was compared to that of HDL-C and apoA-I. Method NMR spectroscopy HDL particles profile were measured by nuclear magnetic resonance (NMR) spectroscopy in 214 male participants with stable CAD (age 45-74 years). Vital status was yearly assessed, with a median follow up of 12.5 years and a 36.4% mortality rate. Cardiovascular mortality accounted for the majority (64.5 %) of deaths. Results Mean concentrations of HDL particles (HDL-P), small and medium-sized HDL (MS-HDL) and apoA-I were lower in deceased than in surviving patients whereas no difference was observed according to HDL-C and large HDL particles. All NMR-HDL measures were correlated between themselves and with other HDL markers (HDL-C, apoA-I and LpA-I). In a multivariate model adjusted for 14 cardiovascular risk factors and bioclinical variables, HDL-P and MS-HDL-P displayed the strongest inverse association with all-cause and cardiovascular mortality. Weaker associations were recorded for apoA-I. Conclusions HDL particle profile measured by NMR spectroscopy should be considered to better stratify risk in population at high risk or in the setting of pharmacotherapy.


Introduction
HDL-Cholesterol (HDL-C) has been repeatedly inversely related to cardiovascular risk in all epidemiological studies. However, pharmacological trials aimed at increasing HDL-C have failed to demonstrate a beneficial effect on clinical outcomes [1]. Similarly, genetic variants associated to increased HDL-C have not been found associated to a decreased cardiovascular risk [2]. This has led to the concept that a single measurement of HDL-C does not necessarily reflect the functional properties of HDL particles and their effects against atherosclerosis. Indeed, HDL particles are heterogeneous in size and biochemical composition, and HDL subpopulations might have different functional properties [3]. NMR-spectroscopy has been recently proposed as a reference tool to quantify HDL particles and HDL subpopulations [3]. This technology enables to measure the total concentration of HDL particles and their size distribution. Numerous recent studies have shown that the atheroprotective properties of HDL are supported by small and medium-sized HDL particles [4], which were inversely related to cardiovascular risk in various clinical settings [5,6].
In the present study, we have evaluated HDL particles concentration and distribution in a cohort of patients with established, angiographically documented, coronary artery disease [7,8].
Patients' data at inclusion included life style parameters and clinical and biological variables documenting cardiovascular risk factors, inflammatory status, renal function and heart condition. The patients' vital status was yearly assessed and mortality was recorded, distinguishing all-cause mortality, cardiovascular mortality and other causes of death, during a 12.5-year median follow-up.
Moreover, one objective of the study was to compare HDL particles measurements to routinely available HDL markers, HDL-C and apoA-I, as predictors of mortality in CAD patients. Indeed, apoA-I constitutes 70% of HDL protein, its immunoassay is today referred to international standards and now available on automated analyzers. Moreover apoA-I is much less influenced than HDL-C by other components of the lipoprotein profile, like VLDL or LDL, which may have an impact on HDL lipid composition through action of lipid transfer proteins.
In this study, multivariate analyses demonstrate that total HDL particles (HDL-P), small and medium-sized HDL (MS-HDL-P) and apoA-I are predictors of all-cause and cardiovascular mortality in coronary patients.

Characteristics of CAD patients according to vital status.
The present cohort was constituted of 214 CAD patients. After inclusion, the vital patients' status was yearly assessed. The median follow-up period was 12.5 years (mean: 10.7 years). During followup, 78 deaths had been recorded giving a death rate of 36.4 % and a mean annual rate of 3.4%. Cardiovascular mortality accounted for the majority (64.5 %) of deaths recorded and cancers accounted for 16.5 %.
Comparison of patients' data when they were included in the cohort is given in Table 1. Patients further deceased during the follow-up period had a longer duration of CAD, a decreased left ventricle ejection volume (LVEF), a higher heart rate and a more severe angiographic lesion score (Gensini).
Regarding cardiovascular risk factors, smoking habits and treatment for diabetes were more frequent in the deceased group, whereas lipid-lowering therapy was less frequent. Among lipoprotein parameters, only apoA-I, a major HDL marker, was significantly lower in further deceased patients.
Hs-CRP, an inflammatory marker, was lower in surviving than in deceasing patients.

HDL particles according to vital status.
HDL particles' profile was determined by NMR spectroscopy, enabling to distinguish large HDL (L-HDL-P) and small and medium-sized HDL (MS-HDL-P) particles. The latter accounted for about ∼ 85 % of total HDL particles (HDL-P). HDL-P was ∼ 10% lower in deceased than in surviving patients (24.6 μmol/L [SD, 6.0] vs. 27.5 μmol/L [SD, 4.9], p = 0.001, Table 2). This difference was entirely due to a decreased number of MS-HDL-P, whereas number L-HDL-P was not different according to the vital status ( Table 2). The average size of HDL particles (HDL size) was found higher in deceased patients (8.94 nm versus 8.82 nm, p = 0.014), which is concordant with a relative higher contribution of large HDL to the total HDL particles number.

Correlations between HDL particles measures and clinical and biological parameters.
Correlations were investigated between markers of HDL particles and other clinical or biological parameters in the study population (Table 3). All NMR-HDL measures were correlated between themselves and with other HDL markers: HDL-C, apoA-I and lipoprotein A-I (LpA-I). Logically, the average HDL size was correlated positively with the number of L-HDL-P, and negatively, with the number of MS-HDL-P. Triglycerides were associated positively with HDL-P and MS-HDL-P, but negatively with L-HDL-P, mean HDL size and HDL-C. These correlations might reflect the effects of the cholesterol ester transfer protein (CETP) acting between HDL particles and triglyceride-rich lipoproteins. Alcohol consumption positively correlated with HDL-C and HDL-P, and more specifically with MS-HDL-P. Inflammation, as documented by plasma hs-CRP, was inversely associated with HDL-P and MS-HDL-P, but not with L-HDL-P. The severity of coronary lesions, as illustrated by the Gensini score, was inversely related to HDL-P. Strong positive associations were observed between LVEF and both HDL-P and MS-HDL-P. Hence, numbers of total HDL particles and, more specifically of small and medium-sized HDL seem to be associated with a better clinical condition. No relationship was recorded between HDL-C and either LVEF or the Gensini score.

Total and cardiovascular mortality according to tertiles of HDL markers.
Each one HDL marker was considered according to tertiles of its distribution in the whole study population (Table 4). Death rates during follow-up were determined across the different tertiles and associations were determined after adjustment on classical risk factors. The strongest association to total and cardiovascular mortality was observed for HDL-P distribution. A 45% reduction in death rates was recorded in tertiles 2 and 3, as compared to tertile 1. Each 1 SD increase in HDL particles number was found associated with ∼ 42 % reduction in total or cardiovascular mortality (HR = 0.58 [95%CI, 0.45-0.75] and 0.59 [95%CI, 0.44-0.80], respectively). Results were almost identical considering MS-HDL-P. By contrast, no association between L-HDL-P and mortality was observed, except for an almost significant positive trend (p = 0.07) between L-HDL-P and death rates. Concordantly, death rates were significantly different across HDL size distribution, an increase in particles size being associated with highest death rates. Considering the "classical" HDL markers, HDL-C tertiles did not display different death rates; on the other hand, apoA-I distribution was associated to total and cardiovascular mortality; each 1 standard deviation increase of apoA-I was associated to a ∼ 31% risk Associations between HDL markers and mortality are illustrated in the survival curves established during the whole follow-up period for the different tertiles ( Figure 2). For both HDL-P and MS-HDL-P, patients in the first tertile had a poorer survival than patients in tertiles 2 and 3. A comparable trend was observed for apoA-I distribution yet survival differences during the whole period did not reach statistical significance.

Discussion
In the present study, levels of total HDL particle number and of small-medium sized HDL particles were inversely related to all-cause as well as to specific cardiovascular mortality in CAD patients.
Every 1-SD increase of HDL particle number was associated to a 44% decrease in cardiovascular mortality, after multiple adjustments on cardiovascular risk factors and on clinical markers of heart condition. Among other HDL markers, apoA-I was also inversely related, though to a lesser extent, to total and cardiovascular mortality. Conversely, HDL-C or large HDL particles were not associated with mortality. However, higher death rates were recorded as average HDL particle size increased.
This prospective study carried out in CAD patients confirms numerous other ones demonstrating that small and medium-sized HDL particles are inversely related to cardiovascular risk [3]. This is already evident as regards pre-clinical atherosclerosis, as documented by carotid intima-media thickness [5,9], or coronary calcifications [10]. HDL-P were inversely related to incident coronary events in the Multi-Ethnic Study of Atherosclerosis [5]. More recently, in a large study carried out in CAD patients undergoing coronary catheterization, followed-up during 8 years, HDL-P and MS-HDL-P were independent predictors of all-cause mortality; inclusion of MS-HDL-P as a marker improved risk prediction and stratification [6]. In a different context, in patients suffering from acute heart failure, concentrations of both total and small HDL particles were inversely related to short-term (3-month) mortality, after multiple adjustments on confounding variables, including NT-proBNP, a classical marker of heart failure [11]. Altogether the studies suggest that small and medium-sized HDL particles are probably protective against atherosclerosis and its clinical consequences, and may have also positive effects on cardiomyocyte functions. HDL particles, and most particularly small-sized HDL, may act against atherosclerosis through different mechanisms. Small HDL behave as the best acceptors of ABCA1-mediated cholesterol efflux from macrophages, leading subsequently to the mobilization of intracellular cholesterol to the plasma membrane [12,13]. Small and dense HDL particles also protect LDL from oxidation. HDL particles act through removing phospholipid hydroperoxides from LDL and by inactivating oxidized lipids by specific enzymes like paraoxonase-1 (PON-1) and PAF-acétylhydrolase [14,15]. Moreover small protein-rich HDL exert anti-inflammatory properties by depressing expression of VCAM-1 at the surface of endothelial cells [16]. On these cells, HDL particles appear to be cytoprotective by inhibiting apoptosis induced by oxidized LDL, and small HDL 3 would be the most effective in this function [17]. Altogether those observations suggest that the proteome associated to small HDL particles support various biological activities, which impair atherosclerosis development.
Moreover, HDL particles may exert beneficial effects on myocardial functions. Indeed, in different experimental contexts, it was demonstrated that HDL particles protect against ischemia reperfusion injury [18], leading to a reduction in infarct size. HDL may also improve myocardial function by reducing ventricular remodelling following infarction [19]. In isolated cardiomyocytes, HDL particles were shown to prevent apoptosis through an AMP-kinase dependent mechanism [20]. These experimental observations on a direct impact of HDL on myocardial functions might translate into clinical impacts. In support of this concept is the positive correlation observed here between HDL-P, small HDL-P and the left ventricular ejection fraction, concordant with the negative association between small HDL and NT-proBNP previously reported [11].
In this study, concentrations of large HDL particles were not associated to mortality. However, higher death rates were recorded as HDL size increased (p < 0.01); following multiple adjustments, association to total mortality for the upper tertile of HDL size was close to statistical significance (p = 0.06). Similar observations regarding all-cause mortality in a cohort of coronary patients have been previously reported [6]. Large HDL-P might be less effective than MS-HDL-P regarding various atheroprotective functions, like cholesterol efflux, anti-oxidative and anti-inflammatory properties, and cytoprotective effects on endothelium [4,21]. Moreover, accumulation of large HDL might reflect a defect in HDL catabolism, and particularly in HDL liver uptake, which constitutes the last step of reverse cholesterol transport [22]. Similarly, we did not observe any association of HDL-C with mortality. This is concordant with the lack of association between L-HDL-P and mortality, since HDL-C mainly reflects cholesterol associated with large, lipid rich, HDL particles.
ApoA-I was inversely related to mortality: for each 1-SD increase of apoA-I, a 31% decrease in both all-cause and cardiovascular mortality was recorded. So far, apoA-I has been little used in epidemiological studies. However, calibration on reference international standards has made the immunoassay of apoA-I robust and comparable between studies. Furthermore, apoA-I measurement is much less influenced than HDL-C by intravascular enzymes and lipid transfer proteins, which participate in HDL remodelling. Thus, apoA-I measurement may improve assessment of cardiovascular risk [23]. Association to mortality was somewhat weaker for apoA-I than for HDL-P or MS-HDL-P. This might be explained by the fact that the apoA-I content per particle varies on average from 2 to 4, between small HDL 3 and large HDL 2 [24], so that large HDL particles are somewhat overrepresented in apoA-I quantification.
Studies on HDL metabolism had progressively led to the schematic view of an interconversion cycle of HDL particles in the plasma compartment, driven by cell cholesterol efflux, enzymes like LCAT, lipases and lipid transfer proteins [25,26]. More recently the concept has emerged that HDL particles of different geometry and chemical composition have distinct metabolic fate and display specific functional properties [3,4,27]. This supports the idea that HDL functionality might be more precisely assessed by the quantification of specific HDL particles with high atheroprotective effects.
The present study is clearly in line with this concept demonstrating that concentration of small-sized HDL particles is predictive of cardiovascular mortality in coronary patients.

Measured parameters.
Age, environmental characteristics and information on cardiovascular risk factors were collected through standardized face-to-face interviews, performed by a single physician. Smoking status was classified as current smokers, smokers having quitted for more than 3 years and non-smokers. Among current smokers, cigarette consumption was estimated with the pack-year quantification and recorded

Assessment of CAD severity and extension and estimation of cardiac function.
Coronary artery stenoses of ≥ 50% luminal narrowing were considered significant. Diffusion of coronary artery disease lesions was assessed by calculating the Gensini Score, based on data from coronary angiography [30-32]. Left Ventricular Ejection Fraction (LVEF) was assessed by contrast ventriculography using an isotopic method, and/or by echocardiography.

Laboratory assays.
Blood was collected after an overnight fast. Serum sample aliquots were subsequently stored at -

HDL measurement by Nuclear Magnetic Resonance (NMR) spectroscopy.
HDL particle concentration and size were measured by NMR spectroscopy using the AXINON ® lipoFIT ® -S100 test system (Numares AG, Regensburg, Germany). 630 μ L of serum were gently 1 1 mixed with 70 μ L of an additives solution containing reference substances, NaN3 and D2O, and 600 μ L of the mixture were transferred into 5 mm NMR tubes with barcode-labeled caps.
Briefly, 1 H NMR spectra were recorded at a temperature of 310 K on a shielded 600 MHz Avance III HD NMR spectrometer (Bruker Biospin) with a 5 mm triple resonance TXI probe head including deuterium lock channel, a z-gradient coil and automatic frequency tuning and matching.
Prior to each analytical run, calibration was performed using a calibration sample comprising an aqueous solution of various calibration substances with different molecular masses, 0.01% (w/v) NaN3,

Statistical analyses.
Continuous variables are displayed as means and standard deviations (SD Cumulative survival of patients were determined by the Kaplan-Meier method and compared, using the Log-rank test for the individual endpoints of all-cause mortality. The relation between baseline variables and mortality was assessed using Cox proportional hazards regression analysis. We tested the proportionality assumption using cumulative sums of martingale-based residuals. We performed regression analyses with polynomial models (quadratic and cubic) to examine for possible non-linear relations between continuous variables and mortality. Cox regression analyses were performed first without any adjustment for co-variables and, second, with adjustment on classical cardiovascular risk factors (age, smoking, treatments for dyslipidemia, hypertension and diabetes). Further adjustments were successively performed on extended cardiovascular risk factors (alcohol consumption, physical activity, BMI, eGFR, hs-CRP and duration of CAD) and clinical parameters related to the severity, extension of the disease and cardiac function (heart rate, LVEF and Gensini score). All statistical analyses were carried out using the SAS statistical software package 9.4 (SAS Institute, Cary, NC). All tests were considered significant at a p value < 0.05.  Graphic represents hazard ratios (dots) and corresponding 95% confidence interval (95%CI) for risk of all-cause and cardiovascular mortality per 1 standard deviation increase of apoA-I, HDL-P or MS-HDL-P. * Analyses were adjusted for age, smoking, alcohol consumption, physical activity, BMI, treatments for dyslipidemia, hypertension and diabetes, hs-CRP, eGFR, heart rate, LVEF, duration of CAD and Gensini score.  Data are expressed in mean (SD). *Student's t-test. HDL-P: HDL particle. L-HDL-P: Large-sized HDL-particle. MS-HDL-P: Small and Medium-sized HDL-particle. HDL size: average HDL particle size.   Log-rank test: CHI2=4.72, p=0.094