High-Sensitivity CRP is Independently Associated with HDL-Mediated Cholesterol Eux Capacity and HDL Remodeling in Patients with Coronary Artery Disease

Background: Cholesterol eux capacity (CEC), a crucial atheroprotective function of high-density lipoprotein (HDL), has proven to be a reliable predictor of cardiovascular risk. Inammation can damage CEC, but few studies have focused on the relationship between the systemic inammation marker high-sensitivity C-reactive protein (hsCRP) and CEC in patients with coronary artery disease (CAD). Methods: Thirty-six CAD patients and sixty-one non-CAD controls were enrolled in this observational, cross-sectional study. CEC was measured using a [ 3 H] cholesterol loading Raw 264.7 cell model with apolipoprotein B-depleted plasma (a surrogate for HDL). Proton nuclear magnetic resonance (NMR) spectroscopy was used to assess HDL components and subclass distribution. hsCRP was measured with a latex particle, enhanced immunoturbidimetric assay. Results: CEC was impaired in CAD patients compared to controls (11.9±2.3% vs. 13.0±2.2%, p=0.022). In the control group, CEC was positively correlated with enzymatically measured HDL cholesterol (HDL-C) levels (r=0.358, p=0.006) or NMR-determined HDL-C levels (r=0.416, p=0.001). However, in the CAD group, the signicance of correlation disappeared (enzymatic method: r=0.216, p=0.206; NMR spectroscopy: r=0.065, p=0.708). Instead, we found that the level of hsCRP was negatively correlated with CEC (r=-0.351, p=0.036), and this relationship was not modied by CAD risk factors, HDL-C, and HDL subclasses. NMR showed that HDL particles shifted to larger ones in patients with high hsCRP levels, and this phenomenon was accompanied by decreased CEC. Conclusions: NMR-HDL-C, NMR-determined high-density lipoprotein cholesterol. Boldface type emphasizes signicant changes.


Introduction
Over the last few decades, epidemiological studies have con rmed a strong and inverse relationship between the level of high-density lipoprotein (HDL) cholesterol (HDL-C) and the risk of coronary artery disease (CAD) [1]. However, the role of HDL-C has been challenged by the failure of HDL-C raising trials using niacin or cholesteryl ester transfer protein (CETP) inhibitors [2,3]. In addition, a genetically increased HDL-C does not necessarily translate to a decreased risk of myocardial infarction [4]. Even worse, higher HDL-C levels secondary to SCARB1 gene mutations lead to an increased risk of CAD [5]. A recent epidemiological study has also revealed that extremely high HDL-C levels are associated with increased CAD mortality [6]. These results highlight the potential limitations of using HDL-C levels, a static mass-based parameter, to assess the risk of CAD, and call for investigations on more robust HDL functional markers for evaluating cardiovascular risks.
HDL particle exerts favorable effects against atherosclerosis, primarily by reverse cholesterol transport (RCT) [7]. A meta-analysis of 11 studies (n = 63,064 patients) has reported that the concentration of HDL particles was inversely related to cardiovascular events [8]. Cholesterol e ux capacity (CEC), a metric re ecting the ability of HDL particles as a cellular cholesterol acceptor, has been demonstrated to be inversely associated with subclinical atherosclerosis [9,10], the incidence of cardiovascular events [11][12][13], and prognosis of CAD [14,15]. The HDL particle numbers and CEC have been considered novel and reliable CAD markers [11] and may be better targets for intervention than HDL-C.
The measurement of CEC requires radiolabeled cholesterol and cultured cells, which is time-consuming and not applicable in clinical settings. It has been consistently observed that CEC was positively correlated with HDL-C levels in healthy populations [9,12,13,16]. However, in patients with CAD, this relationship was inconsistent in different studies. Data from Khera, et al. [9] and Shao, et al. [17] showed that the correlation coe cients between HDL-C and CEC were 0.51 (p < 0.0001) and 0.31 (p < 0.05), respectively, while two other studies showed that the correlation was weak [18] or even inexistent [14].
The unstable relationship between HDL-C and CEC may be subjected to dynamic changes in the components of HDL subclasses. In ammation has been a well-established factor that affects HDL components and subclass distribution [19,20], and patients with in ammatory connective tissue disease always present a decreased CEC [21], suggesting in ammatory markers may serve as surrogate parameters for HDL dysfunction.
Proton nuclear magnetic resonance (NMR) spectroscopy is an emerging technique that can provide a ne-grained snapshot of a person's lipid metabolism. Using NMR-determined HDL subclasses can improve the mortality risk discrimination in the cardiac catheterization cohort [22] and predict the prognosis of patients with pulmonary arterial hypertension [23]. This study used NMR spectroscopy to provide more detailed information about the components of HDL and HDL subclasses. By simultaneously examining the level of high-sensitivity C-reactive protein (hsCRP), a sensitive marker of systemic in ammation, and HDL functional marker CEC, we intended to clarify the relationship between HDL-C and CEC and between hsCRP and CEC in patients with CAD.  [24,25], the diagnosis of MI was based on a combination criteria, namely the increased level of serum high-sensitivity troponin T (hsTnT), with at least one value above the 99th percentile of the upper reference limit (0.0140 µg/L) and at least one of the following: (1) symptoms of ischemia; (2) new or presumably new signi cant ST-T wave changes or left bundle branch block on the 12-lead electrocardiogram (ECG); (3) development of pathological Q waves on the ECG; and (4) Imaging evidence of new or presumably new loss of viable myocardium or regional wall motion abnormality. MI with persistent (> 20 min) ST-segment elevation was diagnosed as ST-segment elevated MI; otherwise, it was diagnosed as non-ST-segment elevated MI. In patients with normal hsTnT levels that presented evidence of cardiac ischemia, the diagnosis was unstable angina. All patients with suspected CAD underwent coronary angiography, and the diagnosis was con rmed by a coronary angiography showing ≥ 50% stenosis in at least one main coronary artery. Sixty-one healthy controls (non-CAD) free from atherosclerotic disorders con rmed by coronary angiography or coronary computed tomography were recruited in the same period to our department. Patients during the gestational phase, under hormone therapy, taking corticosteroid or anti-in ammatory drugs were not included. Patients with human immunode ciency virus (HIV) were not included either, because HIV infection has been reported to damage CEC [26]. Exclusion criteria included hemodynamic instability, a history of renal failure, hepatic function insu ciency, severe infections, autoimmunity disease, cancer, severe medical illnesses, heavy alcohol use (the average daily alcohol intake ≥ 40 g (for Female) or ≥ 80 g (for male) ), or intensive exercise (comparable to a basketball competition) within one week before admission. Written informed consent was obtained from all the individuals included in this study. The research related to human use complied with all the relevant national regulations, institutional policies, and followed the tenets of the Helsinki Declaration, and has been approved by the Medical Ethics Committee of the Second Xiangya

Subjects And
Hospital of Central South University. This trial was registered at the Chinese Clinical Trial Registry as ChiCTR1900020873.

Clinical and biochemical measurements
Demographic information, such as height, weight, blood pressure, and heart rate were measured and recorded. Fasting blood samples were collected the morning after admission. Routine blood, urine, and lipid pro les, including triglycerides (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and HDL-C, were analyzed via the enzymatic method. hsCRP was measured with a latex particle, enhanced immunoturbidimetric assay. Cardiac troponin T was measured using a high-sensitivity assay. For the subsequent experiments, fresh plasma was aliquoted and stored at − 80 °C.

ApoB-depleted plasma preparation
The plasma samples were thawed in a refrigerator at 4 °C before the experiment. According to the protocol of the previous experiment [27], 540 µL of heparin sodium solution (280 mg/mL, Aladdin, China) and 10 mL of a manganese chloride solution (1.06 mol/L, Aladdin, China) were mixed. Plasma was incubated for 30 min at 4 °C with a mixed solution (10:1 vol/vol) and then centrifuged at 1500 × g for 30 min. The supernatant was collected and if it was still turbid (especially samples with a high concentration of triglycerides), plasma was centrifuged again at 12000 × g for 10 min, and the lower liquid fraction was recovered for the next procedure. Compared to conventional ultracentrifugation, this precipitation method is simpler and more e cient for HDL isolation [9]. A previous study revealed that heparin sodium/manganese chloride precipitation had a minor effect on HDL distribution [28].

Measurement of Cholesterol e ux capacity
The cholesterol e ux assay was performed according to established procedures [9,13]. In brief, murine

Nuclear Magnetic Resonance Spectroscopy
The total plasma apolipoprotein A-I (apoA-I)-rich lipoprotein and 30 discrete HDL-related lipoproteins were measured by NMR spectroscopy at ProteinT Biotechnology Co., Ltd (Tianjin, China) by Bruker 600 MHz NMR spectrometer. In the context of the result, the HDL-C measured enzymatically was designated as ENZ-HDL-C, and the HDL-C measured by NMR was described as NMR-HDL-C. The lipoprotein-distributionprediction method selected for the analysis was the commercial Bruker IVDr Lipoprotein Subclass Analysis (B.I.-LISA) method as previously described methods [23,29]. HDL-related lipoproteins were classi ed into four subclasses, labeled numerically according to decreasing size and increasing density.

Patient characteristics
The demographic and biochemical characteristics of the subjects have been shown in Table 1. The subjects in the CAD group were older, with a higher percentage of the male sex, diabetes, hypertension, statin use, and current smoking than subjects in the non-CAD group. Concentrations of TC, ENZ-HDL-C, and LDL-C were lower, but serum hsCRP was signi cantly higher in CAD patients than in the non- patients with CAD were admitted to the hospital more than 7 days after event onset, and 15 (41.7%) patients were admitted after more than 1 month of event onset, suggesting that most of our patients with CAD were beyond the acute stage (usually de ned as within 7 days of event onset).

Correlation between cholesterol e ux capacity and HDL-C levels
The CEC of the standard sample on each 48-well plate has been shown in Supplementary Fig. 1. The intra-and inter-assay coe cients of variation were 5.7% and 4.8%, respectively, which was comparable to previous studies [9,31]. CEC in CAD group was signi cantly lower compared to the non-CAD group (11.9 ± 2.3% vs. 13.0 ± 2.2%, p = 0.022, Fig. 1). Correlation analysis showed that ENZ-HDL-C was positively correlated with CEC in the non-CAD group (r = 0.358, p = 0.006, Fig. 2A ), while there was no signi cant correlation in the CAD group (r = 0.216, p = 0.206, Fig. 2B).
We also measured the level of HDL-C and other HDL-related lipoproteins using NMR spectroscopy (see Supplementary Table 2). Consistent with the results of enzymatic methods, NMR-HDL-C was positively correlated with CEC in non-CAD controls (r = 0.416, p = 0.001, Fig. 2C). However, in CAD patients, there was no correlation between NMR-HDL-C and CEC (r = 0.065, p = 0.708, Fig. 2D).

In the CAD group, cholesterol e ux capacity was negatively correlated with the hsCRP level
To investigate which factor correlated with HDL-mediated CEC in CAD patients, univariate analysis was performed. There was no correlation between CEC and age, body mass index, severity of coronary stenosis (expressed as the Gensini score), hsTnT, or the serum levels of TG, TC, LDL-C, fasting glucose (see Supplementary Table 3). However, we found that CEC was negatively correlated with the hsCRP level (r=-0.351, p = 0.036, Fig. 2E). Then, we divided 36 CAD patients into hsCRP-low (n = 18) and hsCRP-high (n = 18) groups by using the median hsCRP level value (1.75 mg/L) as the criterion (the values of hsCRP in all patients have been shown in Supplementary Fig. 2). The baseline characteristics, the concentrations of total HDL lipids and apolipoproteins were comparable in two groups, except for the signi cantly higher level of hsTnT in the hsCRP-high group (see Supplementary Table 4). Nonetheless, CEC was signi cantly lower in the hsCRP-high group than in the hsCRP-low group (11.3 ± 2.2% vs. 12.6 ± 2.1%, p = 0.038, Fig. 2F).

In CAD patients, HDL particles underwent extensive remodeling with a high level of hsCRP
To explore why CEC reduced despite the level of total HDL lipids and major apolipoproteins remained unchanged between the hsCRP-high and the hsCRP-low group, we compared HDL subclass distribution in the two groups. In the hsCRP-high group, the concentrations of lipids and major apolipoproteins in the largest HDL subclass (HDL1) were signi cantly higher, while those in the smallest HDL subclass (HDL4) were signi cantly lower than those in the hsCRP-low group (Fig. 3). Further analysis showed that the levels of lipids and major apolipoproteins in HDL1 were positively correlated with the level of hsCRP, while in HDL4 there was a negative correlation (see Supplementary Table 5).

Multiple linear regression analysis of cholesterol e ux capacity in patients with CAD
To clarify whether the relationship between hsCRP and CEC was independent, multiple linear regression analysis was performed. Cardiovascular risk factors including age, sex, LDL-C, diabetes, current smoking, body mass index, TG (log-transformed), and hsTnT (log-transformed) were included as covariates. Adjustments were made for ENZ-HDL-C, NMR-HDL-C, HDL1-cholesterol, and HDL4-cholesterol. Results showed that hsCRP had an independent relationship with HDL-mediated CEC, regardless of conventional CAD risk factors, hsTnT, HDL-C, and HDL subclasses (Table 3). Table 3 Multiple linear regression analysis between hsCRP and cholesterol e ux capacity in CAD patients (n = 36).

Discussion
HDL has been recognized as a traditional protective factor against atherosclerosis. Early epidemiological studies have consistently shown that HDL-C is inversely correlated with cardiovascular risk [1]. However, subsequent attempts for drug therapies that aim to raise HDL-C levels with niacin [3] or CETP inhibitor [2] have both been in vain. HDL-mediated RCT is the key protective function of HDL. CEC, a metric de ned by ex vivo experiments which re ects the rst and rate-limiting step of RCT, has been demonstrated to be more valuable than HDL-C in predicting CAD risks [9-12, 14, 15]. However, CEC is susceptible to impairment in many disease states.
Our results showed that compared to non-CAD controls, CEC was lower in CAD patients and was not signi cantly related to HDL-C. The reduced CEC in CAD patients has already been well described in a high-quality study [9] but the correlation between HDL-C and CEC was inconsistent in previous reports.
For example, the correlation coe cient between HDL-C and CEC was 0.51 (p < 0.0001) in the study by Khera et al. [9] (combining 442 CAD patients and 351 controls) but it was − 0.09 in the study by Zhang et al. (313 CAD patients) [14]. The discordance could result from the different study populations, as most of the patients in Zhang et al.'s study and our study had ACS, while Khera et al.'s study excluded ACS patients. In addition to CAD, the CEC of HDL is also impaired in other diseases. Our previous observation found that patients with end-stage renal disease exhibited signi cantly decreased CEC compared to controls [10]. HIV infection has been reported to induce structural and functional changes in HDL particles [26]. A similar impairment of CEC was also found in patients with type 1 [32] or type 2 diabetes [12] and autoimmune diseases [21].
A common feature of the above diseases is that in ammation is a major part of their pathophysiological mechanism. The in ammatory process can in uence HDL components, shifting HDL proteome to in ammatory pro le [33], and some of these uninvited guests can be detrimental to CEC. For example, the enrichment of serum amyloid A (SAA) or apolipoprotein C-III (ApoC-III) in HDL can impair HDL-mediated CEC [19,31]. In addition to the change of HDL components, in ammation can also lead to modi cation of apoA-I, the major protein of HDL. Our early experimental study has found that myeloperoxidase (MPO)mediated tryptophan oxidation of apoA-I was associated with decreased cholesterol e ux [34]. Modi ed apoA-I was also abundant in the plasma recovered from patients with CAD [17]. Although we did not measure the level of serum MPO or modi ed apoA-I in this study, correlation analysis revealed that the level of apoA-I (determined by NMR) was not correlated with CEC in CAD patients, suggesting that apoA-I modi cation could be a reason for HDL dysfunction in this study.
Our study showed that hsCRP was inversely correlated with CEC in CAD patients. The correlation remained signi cant after adjusting other conventional risk factors, HDL-C levels, and HDL subclasses.
This independent role of hsCRP to CEC has not been reported in patients with CAD, although univariate negative correlation has been noted before [17]. In ammation in patients with CAD mainly derives from the activation of in ammatory pathways by various atherogenic risk factors. IL-1-IL-6-CRP axis is one of the most critical in ammatory pathways, and its activation can explain our ndings. Cells in the atheroma (e.g., endothelial cells, macrophages, smooth muscle cells, etc.) produce IL-1β when exposed to stimuli (e.g., oxidized lipoproteins, disturbed blood ow, etc.). IL-1β strongly augments the production of IL-6 by various cell types. IL-6 induces liver to produce acute-phase proteins, such as CRP and SAA [35,36]. We speculated that the activation of the IL-1-IL-6-CRP axis increased the level of hsCRP, and at the same time, led to the SAA-mediated CEC impairment. In ammation was the possible reason behind the negative correlation between hsCRP and CEC.
hsCRP is a valuable marker of CAD. In people without a history of vascular disease, the risk ratios for CAD per 1-SD higher of log e CRP concentration were 1.37 (1.27-1.48) after adjustment for conventional risk factors [37]. In patients with CAD, it has been shown that patients with higher hsCRP baseline level (usually ≥ 2 mg/L) have signi cantly higher risk of major adverse cardiovascular events (MACE) compared to those with lower hsCRP baseline level (usually < 2 mg/L) [38,39]. hsCRP is not only a risk marker but can also predict the clinical bene t of antiatherogenic agents. Results from the JUPITER trial showed that participants without dyslipidemia but who had hsCRP levels higher than 2 mg/L can bene t more from rosuvastatin treatment [40]. In the CANTOS trial, participants with canakinumab, a monoclonal antibody targeting IL-1β, who achieved an on-treatment hsCRP level < 2 mg/L had a 25% reduction in the risk of MACE, while no signi cant bene t was observed in participants whose on-treatment hsCRP concentration was 2 mg/L or above [41]. Recent results from the COLCOT trial also showed that the nonspeci c anti-in ammation agent colchicine reduced stroke and coronary revascularization in patients with myocardial infarction, accompanied by a large reduction (> 65%) of the hsCRP level [42]. On the other hand, treatment with another anti-in ammation medicine, methotrexate, did not reduce the risk of MACE, as there was no decrease in the hsCRP levels following treatment [43]. Results from these highquality studies and the current study consistently revealed the potential role of IL-1-IL-6-CRP axis in CAD and underscored the importance of hsCRP testing in patients with CAD.
In our study, the HDL subclass distribution was also determined by NMR spectroscopy. Our results showed that CAD patients with above median hsCRP levels exhibited a different HDL subclass distribution compared to patients with hsCRP levels below the median, namely the increased large HDL particles (HDL1) and the decreased small HDL particles (HDL4). Partial correlation analysis revealed that even after controlling for hsTnT levels, age, LDL-C levels, and diabetes, hsCRP levels remained positively correlated with HDL1-cholesterol levels, suggesting that the systematic in ammatory status of CAD was the underlying reason for HDL subfraction-distribution abnormality. HDL is a heterogeneous population of particles with sizes ranging from 7 nm-14 nm in diameter [7], and HDL particles are known to shift toward larger ones as a result of in ammation [20,46]. The mechanism by which in ammation affects HDL remodeling is still not fully understood. In our study, the underlying reason, as mentioned before, could be the increased SAA levels [47] or the altered activity of plasma enzymes related to HDL metabolism, such as CETP [48].
In conclusion, we found that HDL-C levels could not re ect the functional status of HDL in patients with CAD, and the impaired correlation between HDL-C levels and CEC was possibly due to in ammationinduced HDL-subclass remodeling. The in ammatory marker, hsCRP, had an independent and negative relationship with CEC in CAD patients. As a relatively small group of patients was taken into account, further studies with larger cohorts including stable CAD patients, ACS patients, and mechanistic studies will need to be undertaken to further verify our conclusions and to elucidate the underlying mechanism for CEC impairment and HDL remodeling. Comparison of CEC between non-CAD controls (n=61) and CAD patients (n=36). CEC, cholesterol e ux capacity; CAD, coronary artery disease; * p <0.05. density lipoprotein cholesterol; NMR-HDL-C, NMR measured high-density lipoprotein cholesterol; hsCRP, high-sensitivity C-reactive protein; CEC, cholesterol e ux capacity; CAD, coronary artery disease; * p <0.05.

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