1. Introduction
During the past few years, evidence has accumulated that autoantibodies against high-density lipoproteins (HDL) and its components may impact on atherothrombotic processes that play a role in the pathogenesis of cardiovascular disease (CVD) [
1]. Immunological assays to detect such antibodies have been recently developed in several laboratories and are currently available for clinical use [
2,
3]. Autoantibodies of IgG subclass against apolipoprotein A-1 (AAA1), the major apolipoprotein of HDL, have been shown to be elevated in subjects with established CVD, to be associated with worse outcome after acute myocardial infarction and stroke, and to predict incident atherosclerotic CVD and overall mortality in the general population [
4,
5,
6,
7,
8,
9]. Furthermore, AAA1 were shown to promote sterile inflammation in vitro and in vivo, accelerating the development of atherosclerosis and atherothrombosis in mice [
10,
11,
12,
13]. In humans, the polyclonal AAA1 response has been shown to be oriented against the last alpha helix of the c-terminus part of apolipoprotein A-1 (apoA-1) (amino acids: 220–242) [
14,
15], and the corresponding mimetic peptide can used for both the detection of anti-C-terminus apoA-1 (Ac-terAA1) and the neutralization of AAA1 deleterious effect in vitro [
15].
Inverse associations have been observed between AAA1 levels and total cholesterol, low-density lipoprotein cholesterol and HDL cholesterol [
7,
13]. These autoantibodies could, therefore, interfere with cholesterol metabolism on top of their established proinflammatory and prothrombotic properties. Cellular efflux of cholesterol to extracellular acceptors (cholesterol efflux capacity (CEC)) provides the initial step in the reverse cholesterol transport (RCT) pathway, whereby cholesterol is transported back from the arterial wall to the liver for metabolism and excretion in the bile [
16,
17,
18,
19,
20]. CEC represents a key metric of HDL function, and it has been suggested that impaired CEC represents an important predictor of CVD [
19,
20]. HDL metabolism is a complex process regulated by a number of interdependent pathways, starting with the generation of small lipid-poor HDL particles, i.e., pre-β-HDL, followed by esterification of free cholesterol (EST), which results in HDL maturation, and subsequent cholesteryl ester transfer (CET) to apolipoprotein B (apoB)-containing lipoproteins [
21,
22]. AAA1 and anti-HDL antibodies have been shown to impair the antioxidant function of HDL through paraoxonase-1 inhibition [
3,
23,
24,
25]. Such an effect on HDL function underscores the relevance of testing the hypothesis that AAA1 and Ac-terAA1 may interfere with CEC and HDL metabolism. Anti-apoA-1 autoantibodies have been found to be elevated in patients with Type 2 diabetes mellitus (T2D), though particularly only in those with CVD [
26]. T2D is characterized by increased plasma EST and CET [
27,
28,
29], which makes the diabetic state a relevant condition for which to interrogate the impact of AAA1 and Ac-terAA1 on plasma EST and CET. No data are currently available concerning the association of apoA1 autoantibodies with CEC and other metrics of HDL metabolism.
The present study was, therefore, initiated to delineate relationships of CEC, plasma EST and CET with AAA1 and Ac-terAA1. Furthermore, we explored the possible impact of T2D on such associations in view of abnormalities in HDL metabolism in this condition [
27,
28,
29].
2. Results
The study population consisted of 75 control subjects and 75 T2D patients (
Table 1). Seventeen diabetic patients were taking sulfonylurea alone and 15 were taking metformin alone, whereas both drugs were used by 21 patients. Antihypertensive medication (in most cases, angiotensin-converting enzyme inhibitors, angiotensin II receptor antagonists and diuretics, alone or in combination) was used by 29 T2D patients, but not in control subjects (
p < 0.001). Oral contraceptives were taken by 4 nondiabetic women. Diabetic patients were older, more obese, had higher blood pressure and higher fasting glucose and HbA1c levels than control subjects (
Table 1). HDL cholesterol and apoA-1 were lower in diabetic patients, coinciding with higher triglycerides. Pre-β-HDL formation was not different between the groups, but phospholipid transfer protein (PLTP) activity, lecithin–cholesterol acylesterase (LCAT) activity, cholesteryl ester transfer protein (CETP) mass, EST and CET were increased in T2D patients. CEC was not different between diabetic and control subjects (
Table 1). Median AAA1 and Ac-terAA1 levels were similar between diabetic and control subjects (
Table 1). As expected [
15], AAA1 and Ac-terAA1 were closely correlated with each other in all subjects combined (
r = 0.550,
p < 0.001), as well as in T2D patients (
r = 0.549,
p < 0.001) and control subjects separately (
r = 0.542,
p < 0.001).
Univariate regression analysis showed that in all subjects combined, Ac-terAA1 levels were inversely correlated with total cholesterol, non-HDL cholesterol, triglycerides, apoB, LCAT activity, EST, CET and CEC (
Table 2). Similar inverse correlations between these variables and Ac-terAA1 were found in control subjects separately, except for the correlation with CEC, which was not significant (
Table 2). These relationships did not reach significance in T2D patients separately. Notably, except for plasma CET, for which the correlation with Ac-terAA1 levels was stronger in control subjects than in T2D patients (
p for interaction) = 0.025), there were no differences in the strength of the correlations between T2D and control subjects (
p (interaction) > 0.10 for each). No correlations were observed between both these autoantibodies and glucose, HbA1c, HDL cholesterol, apoA-1, pre-β-HDL formation or CETP mass (
Table 2). Of further note, neither in all subjects combined nor in T2D patients and control subjects separately were significant correlations of any of the variables listed in
Table 2 with AAA1 demonstrated (
Table 2).
In all subjects combined, CEC was positively correlated with pre-β-HDL formation, PLTP and LCAT, as well as with plasma EST and CET in univariate regression analysis (
Table 3). Similar relationships were found in control subjects and T2D patients separately. CEC was unrelated to glucose and HbA1c (
Table 3). In addition, CEC was positively correlated with non-HDL cholesterol (
r = 0.450,
p < 0.001), LDL cholesterol (
r = 0.286,
p < 0.001), apoB (
r = 0.407,
p < 0.001) and triglycerides (
r = 0.429,
p < 0.001). Similar relationships of CE with apoB lipoproteins were found in both groups separately (data not shown).
We next performed multivariable linear regression analysis to disclose the independent associations of CEC with Ac-terAA1 antibodies. This analysis included age, sex, Ac-terAA1 antibodies, and in separate models, also pre-β-HDL formation, PLTP activity, LCAT activity and plasma EST, i.e., variables with which CEC was significantly correlated in univariate analysis. As shown in
Table 4, the inverse association between Ac-terAA1 and CEC was unchanged after adjusting for age, sex, diabetes status (model 1) and additionally for the use of glucose-lowering drugs and antihypertensive medication (model 2), as well as after additional adjustment for pre-β-HDL formation and PLTP activity (model 3). However, this association was lost after additional adjustment for plasma EST (
Table 4, model 4). These analyses also demonstrated independent and positive associations of CEC with pre-β-HDL formation, PLTP activity and EST.
Model 1: adjusted for age, sex and diabetes status.
Model 2: adjusted for age, sex, diabetes status, and use of metformin, sulfonylurea and antihypertensive medication.
Model 3: adjusted for age; sex; diabetes status; use of metformin, sulfonylurea and antihypertensive medication; and pre-β-HDL formation
Model 4: adjusted for age; sex; diabetes status; use of metformin, sulfonylurea and antihypertensive medication; pre-β-HDL formation; and plasma EST
In univariate analysis, there was a strong relationship of plasma EST with CET in all subjects combined (
r = 0.675,
p < 0.001), as well as in T2D patients (
r = 0.695,
p < 0.001) and control subjects separately (
r = 0.695,
p < 0.001). In all subjects combined, there remained a strong relationship of plasma EST with CET after adjustment for age, sex, diabetes status, the use of glucose-lowering drugs and antihypertensive medication, and Ac-terAA1 (β = 0.622,
p < 0.001; data not shown). Subsequently, we determined the extent to which plasma EST and CET were independently associated with Ac-terAA1. When taking account of age, sex and diabetes status, plasma EST and CET were still inversely and independently associated with Ac-terAA1 levels (
Table 5, models 1A and B, respectively). These inverse relationships remained significant after further adjustment for the use of glucose-lowering drugs and antihypertensive medication (
Table 5, models 2A and B), and additionally for LCAT activity or CETP mass (
Table 5, models 3A and B, respectively). Notably, the associations of plasma EST and CET with Ac-terAA1 were lost after further adjustment for non-HDL cholesterol and triglycerides (
Table 5, models 4A and B, respectively).
Model 1: adjusted for age, sex and diabetes status.
Model 2: adjusted for age, sex, diabetes status, and use of metformin, sulfonylurea and antihypertensive medication.
Model 3: adjusted for age; sex; diabetes status; use of metformin, sulfonylurea and antihypertensive medication; and LCAT activity (A) or CETP mass (B).
Model 4: adjusted for age; sex; diabetes status; use of metformin, sulfonylurea and antihypertensive medication; LCAT activity (A) or CETP mass (B); non-HDL cholesterol; and triglycerides.
3. Discussion
The present study is, to our knowledge, the first comprehensive exploration of possible AAA1 and Ac-terAA1 associations with key features of HDL metabolism in T2D patients and control subjects. In line with other reports, T2D patients had unaltered CEC and pre-β-HDL formation, but increased plasma PLTP activity, LCAT activity, EST, CETP mass, CET and triglycerides, together with decreased HDL cholesterol and apoA-1 [
27,
28,
29]. The first remarkable finding of our current study is that Ac-terAA1 and AAA1 do not appear to associate in a similar fashion with parameters of HDL metabolism. In all subjects combined, inverse associations were observed between Ac-terAA1 and total cholesterol, non-HDL cholesterol, triglycerides, apoB, CEC, plasma EST and CET, but these associations were not observed for AAA1. Of note, Ac-terAA1 and AAA1 were not elevated in the diabetic group, and the presence of T2D did in general not significantly modify the associations of CEC and HDL variables with Ac-terAA1. Furthermore, as the opposite of what has been reported before for AAA1 [
7,
13], no associations between Ac-terAA1, HDL cholesterol and apoA-1 levels were retrieved, a finding which would suggest that Ac-terAA1 may impact on HDL metabolism in a different manner compared to AAA1.
The inverse association between CEC and Ac-terAA1 levels was found to be independent of pre-β-HDL formation, plasma PLTP and LCAT activity, but was lost when taking account of EST. In turn, plasma EST and CET were each inversely correlated with Ac-terAA1, but these autoantibodies were not associated with pre-β-HDL formation and PLTP activity. Remarkably, the associations of Ac-terAA1 with EST and CET were no longer present when adjusting for plasma apoB lipoproteins, particularly triglycerides. Furthermore, CEC was correlated positively with apoB lipoproteins. These results are in accordance with the proposition that CEC, EST and CET are intricately coupled processes, and reiterate the importance of apoB lipoproteins in accepting cholesteryl esters from HDL [
30]. Collectively, these results would suggest that Ac-terAA1 may impact on HDL metabolism by influencing plasma cholesterol esterification and cholesteryl ester transfer from HDL towards apoB-containing lipoproteins, without a major effect on ABCA1-mediated efflux via pre-β-HDL and PLTP.
It should be noted that at present there is no gold standard to measure CEC with respect to both the preferred cell system and the acceptor medium. In the current study, we used cholesterol-loaded human cultured fibroblasts as the cholesterol donor and diluted plasma from individual subjects as the cholesterol acceptor medium. Under the experimental conditions employed, these cells abundantly express ABCA1 [
31,
32]. Lipid-poor HDL particles, i.e., pre-β-HDL, and PLTP are known to stimulate cholesterol efflux by interacting with ABCA1 [
31,
33,
34]. In line with this, CEC was positively related to pre-β-HDL, assayed as the formation of these particles under in vitro conditions of LCAT inhibition, as well as to PLTP activity. Fibroblast also express the ATP-binding cassette transporter G1 (ABCG1), but hardly any scavenger receptor class B type 1 (SR-BI), both of which are reported to interact with mature, spherical HDL particles, although SR-BI is unlikely to mediate net cholesterol mass efflux [
33,
34,
35]. The lack of correlation of CEC with HDL cholesterol and total plasma apoA-1, as shown in the present study, suggests that the CEC assay used here is largely independent of ABCG1. However, the relative contribution of ABCA1, ABCG1 and aqueous diffusion to the efflux process with human cultured fibroblasts is not well known. In addition, though still unsettled, it is relevant to mention that CEC to diluted plasma, which contains pre-β-HDL and other relevant factors involved in cholesterol efflux, could conceivably reflect the interaction of interstitial fluid with cells in a different way compared to isolated HDL. In comparison, using various macrophage cell lines and isolated patient HDL as cholesterol acceptor, CEC did correlate with HDL cholesterol and total plasma apoA-1 [
36,
37,
38,
39]. Notably, neither in the present study including diabetic patients with predominantly moderate hyperglycaemia, nor in our recent study evaluating CEC, determined using a macrophage cell line and isolated HDL as cholesterol acceptor, in subjects with various degrees of glucose tolerance, was CEC associated with glycemia. More severely hyperglycaemic circumstances could be required to affect the cholesterol efflux process [
40,
41].
Although not unequivocally reported, impaired CEC may be associated with accelerated risk of atherosclerosis development, in particular in studies in which macrophage cell lines were used as the cholesterol donor [
37,
38,
42]. Although the use of cultured fibroblasts to determine the association of CEC with incident CVD is unsettled, the inverse relationship of CEC with Ac-terAA1 could imply a proatherogenic role elicited by the presence of these autoantibodies. On the other hand, plasma CET elevations have been shown to predict greater carotid artery intima media thickness and incident CVD [
43,
44]. Increased plasma EST was also found to confer increased CVD risk, but the role of LCAT per se in atherogenesis is still uncertain [
45,
46,
47,
48]. Translating the presently shown relationships between Ac-terAA1 and CEC and EST and CET into CVD risk suggests a mixed picture with possible adverse effects on CEC and beneficial effects on EST and CET.
Several other methodological issues of our study need to be discussed. We excluded subjects using lipid-lowering drugs, since statins may affect HDL metabolism [
36]. We also excluded T2D subjects using insulin. This was done to avoid effects of insulin on CEC [
49]. As a result, it is likely that diabetic subjects with mild hyperglycaemia and mild dyslipidaemia preferentially participated, explaining modestly elevated fasting glucose and HbA1c, as well as the lack of increase in plasma total cholesterol and non-HDL cholesterol in the participating diabetic patients. This selection may limit the generalizability of our findings. Furthermore, because seropositivity cutoffs for both AAA1 and Ac-terAA1 have not been validated on ethylenediaminetetraacetic acid (EDTA) plasma, we did not perform the analyses according to this strict criterion. Nonetheless, because AAA1 and Ac-terAA1 levels were similar between healthy controls and T2D patients, we would have expected antibody seropositivities close to what have been retrieved in the general population. Furthermore, it is possible that in a larger group of participants, statistically significant relationships of AAA1 and Ac-terAA1 in T2D subjects separately to CEC and HDL metabolism could have been detected. Finally, as a consequence of case-control design of our study, no causality link can be inferred to the associations between Ac-terAA1 and HDL metabolism.
In conclusion, cholesterol efflux capacity, plasma cholesterol esterification and cholesteryl ester transfer are inversely associated with Ac-terAA1, in a way that appears at least in part to be dependent on apoB-containing lipoproteins. The mechanisms responsible for these hitherto unreported associations await further evaluation.