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Abstract

Context

Elevated serum lipoprotein(a) [Lp(a)] levels are associated with increased cardiovascular disease risk. ABCA1-mediated cholesterol efflux from macrophages may be an antiatherogenic process. Plasminogen (PLG) is a driver of ABCA1-mediated cholesterol efflux, and its action is inhibited by purified human Lp(a).

Objective

To determine the effects of Lp(a) in human serum on ABCA1 cholesterol efflux.

Methods

Cholesterol efflux capacity (CEC) was measured with two different cell-culture models using serum from 76 patients with either low (<50 mg/dL) or high (>50 mg/dL) Lp(a) levels.

Results

Using cAMP-stimulated J774 macrophages or baby hamster kidney fibroblasts overexpressing human ABCA1, we show that CEC was lower in patients with high Lp(a) levels compared with patients with low levels (−30.6%, P = 0.002 vs −24.1%, P < 0.001, respectively). Total-serum CEC negatively correlated with Lp(a) levels (r = −0.433, P = 0.0007 vs r = −0.505, P = 0.0011, respectively). These negative associations persisted after adjusting for serum cholesterol, age, sex, and statin use in a multiple linear regression model (adjusted R2 = 0.413 or 0.405, respectively) and were strengthened when further adjusting for the interaction between Lp(a) and PLG levels (adjusted R2 = 0.465 and 0.409, respectively). Total-serum and isolated Lp(a) from patients with high Lp(a) inhibited PLG-mediated ABCA1 cholesterol efflux.

Conclusion

Total-serum CEC is reduced in patients with high Lp(a) levels. This is in part due to the inhibition of PLG-mediated ABCA1 cholesterol efflux by Lp(a). Our findings suggest an atherogenic role for Lp(a) through its ability to inhibit CEC.

Lipoprotein(a) [Lp(a)] is a low-density lipoprotein (LDL)-like particle composed of apolipoprotein(a) (LPA) bound to apolipoprotein B-100 through a single disulfide bond. LPA is very heterogeneous in size, depending on the number of Kringle IV type 2 repeats, ranging from single digits to >40 (1). Plasma concentration of Lp(a) is largely genetically determined and independently associated with risk for myocardial infarction (2, 3), stroke, peripheral arterial disease, and calcific aortic valve stenosis (4, 5). The Lp(a) levels in the population follows a non-Gaussian distribution with 70% to 80% of the population having levels <30 mg/dL. These levels are not associated with cardiovascular disease (CVD). A linear increase in Lp(a)-mediated CVD risk is found in patients with Lp(a) levels >30 mg/dL (70th percentile) or 50 mg/dL (80th percentile) (6). Physiologic, dietary, and environmental factors play relatively minor roles in modulating Lp(a) serum levels (7). In humans, circulating Lp(a) levels are determined by variation in the LPA gene through the heterogeneity in the number of Kringle IV type 2 repeats that affect LPA size and also through single nucleotide polymorphisms that affect LPA expression levels (8).

Although Lp(a) is an independent CVD risk factor, the molecular mechanisms of its atherogenicity are unclear. Lp(a) affects CVD risk by at least two mechanisms: (1) by altering the prothrombotic/antifibrolytic properties of the plasminogen (PLG)/plasmin system and (2) by promoting intimal deposition of Lp(a) cholesterol, similar to the atherogenic function of LDL (9, 10). Results from in vitro studies have also shown that Lp(a) increases the expression of adhesion molecules (11) and induces human vascular endothelial cells to produce monocyte chemotactic protein (12). Lp(a) also inhibits the generation of active TGFβ and thus enhances smooth muscle cell proliferation and migration (13, 14). Moreover, Lp(a) is a storage site for oxidized phospholipids, which may aggravate inflammatory and atherogenic pathways in the artery wall (5, 15).

The protease domain of LPA contains 88% amino acid sequence homology with the protease domain of PLG, a zymogen that plays an important role in the fibrinolytic cascade, cell migration, angiogenesis, and inflammation (16). Several independent meta-analyses have reported that polymorphisms in PLG strongly associate with atherosclerotic CVD (17, 18). In addition, serum PLG levels were independently and directly associated with risk for coronary artery disease in two prospective cohort studies and continued to predict risk even after adjustment for cholesterol and high-density lipoprotein cholesterol (HDL-C) levels (17, 18). Regarding its role in atherogenesis, the data are controversial. Low levels of PLG in intimal tissue were found associated with increased atherosclerosis in one study (19), whereas high levels of PLG in the atherosclerotic plaque where reported in another study (20).

Our previous studies using either fibroblasts or bone marrow–derived macrophages isolated from Abca1−/−, Abca1+/−,- or Abca1+/+ mice showed that PLG promotes cholesterol efflux only in the presence of ABCA1 (21). We also studied the interaction of Lp(a) and PLG using either Lp(a) isolated from human serum or a 17-kringle recombinant LPA, and showed that both inhibited PLG-mediated cholesterol efflux capacity (CEC) through ABCA1 in a concentration-dependent manner. Neither PLG nor Lp(a) affects cholesterol efflux in cells that do not express ABCA1. Removal of PLG from human plasma reduced sterol efflux by ∼30%, whereas addition of PLG to depleted human plasma reinstated sterol efflux (21). Moreover, plasma from mice deficient in Plg had ∼20% reduction in sterol efflux when compared with plasma from wild-type mice (21).

To understand the effect of Lp(a) levels on CEC, we used total-serum and isolated lipoproteins from patients with varying Lp(a) levels (range, 2 to 291 mg/dL; mean, 64 mg/dL), and LPA size isoforms. We show that whereas Lp(a) in itself serves as a cholesterol acceptor, its serum levels are inversely associated with total-serum CEC, and that Lp(a), either in serum or as isolated particles, inhibits PLG-dependent, ABCA1-mediated cholesterol efflux.

Methods

Patients and samples

Seventy-six patients from the outpatient clinic at the Oregon Health & Sciences University (OHSU) Center for Preventive Cardiology (CPC) were included. This study represents a preplanned analysis of a prospectively enrolled clinical cohort. Subjects enrolled in the CPC registry and biorepository consented to take part in an institutional review board (IRB)-approved research study [OHSU IRB approval no. 18643 (n = 45); Vanderbilt University IRB 101615 (n = 31)]. Patient characteristics are presented in an online repository (22). Samples were collected as EDTA plasma and converted to serum using 25 μM CaCl2.

Lipid measurements

Levels of cholesterol, triglycerides, HDL-C, and Lp(a) in plasma were measured at the Knight Cardiovascular Institute Lipoprotein Analytical Core at OHSU using a Hitachi 704 Chemistry Analyzer with Roche Diagnostics reagents for cholesterol, triglycerides, and HDL-C measurements or Medtest reagents for Lp(a) testing. Levels of cholesterol, triglycerides, HDL-C, low-density lipoprotein cholesterol (LDL-C; calculated) are reported as the lipid concentration. Lp(a) is reported as mass concentration. The Lp(a) assay was based on the Denka method standardized to the World Health Organization/International Federation of Clinical Chemistry reference material, SRM 2B (23) and is the closest in terms of agreement to the ELISA reference method (24). The assay is provided with a five-point calibrator with accuracy-based assigned target values, which reflect the heterogeneity of isoforms present in the general population.

Lipoproteins isolation

Lp(a) and LDL were isolated by iodixanol natural-gradient ultracentrifugation, as previously described (25). To further purify Lp(a) from HDL, an additional ultracentrifugation step was performed, after adjusting density to 1.0935 g/mL, at 100,000 RPM for 16 hours at 4°C. Levels of isolated lipoprotein levels used for cholesterol efflux were matched to their level in 2% total serum, which was used for cholesterol efflux. LDL and HDL levels were matched on the basis of their measured cholesterol levels in plasma (i.e., LDL-C and HDL-C, respectively). Lp(a) was matched on the basis of the measured mass concentration in plasma. We are able to purify Lp(a) using the method described; however, one limitation was obtaining enough material to generate a 5-point inhibition curve for both conditions (i.e., ±ABCA1). For LPA isoform studies, nine patients were screened, of whom only three had samples with enough volume to run a dose-dependent curve using five Lp(a) concentrations.

Cholesterol efflux assays

CEC was assessed using J774 macrophages labeled with [3H]-cholesterol and stimulated with a cAMP analog, as previously described (21, 26). Efflux by the ABCA1 pathway was measured in baby hamster kidney (BHK) cells expressing mifepristone-inducible human ABCA1 that was radiolabeled with [3H]cholesterol and 1 mg/mL fatty-acid free albumin (21, 27, 28). Efflux of [3H]-cholesterol (Perkin Elmer, Seattle, WA) was measured after a 4-hour incubation at 37°C in medium with either 2% total serum; apolipoprotein B (apoB)-depleted serum (serum-HDL); isolated HDL, LDL, and Lp(a) from human serum; or human PLG (R&D Systems, Minneapolis, MN). PLG concentration (range, 4 to 20 μg/mL based on 1 to 5 μg in 0.25 mL of media) used in the various assays approximated physiologic levels. The serum efflux assays were done with 2% serum per well, which corresponds to 2 ug/mL PLG, given that the mean plasma PLG concentration is ∼10 mg/dL.

The PLG concentration used in this assay matched that of the serum levels for each patient. We used store-bought, serum-isolated PLG instead of isolating it from each patient. Cholesterol efflux was calculated as the percentage of radiolabel in the medium of the cells at the end of the incubation divided by the total radioactivity in both medium and cells. J774 macrophage efflux was determined as cholesterol efflux from cAMP-stimulated cells. BHK fibroblast cholesterol efflux was determined as the difference in cholesterol efflux of cells with and without mifepristone induction of ABCA1 expression. Interplate normalization was done using a pooled control sample, and the coefficient of variation intra- and interplate was <10%.

Lp(a) inhibition of PLG-mediated, ABCA1-specific CEC was determined by subtracting baseline efflux levels (i.e., cells without ABCA1) from the values obtained from cells that express ABCA1 (after overnight stimulation with mifepristone to turn on ABCA1 transcription). Although the PLG concentration was kept constant (4 μg/mL) the isolated Lp(a) from three patients was introduced at increasing concentrations.

PLG urokinase PLG activator cleavage

Different concentrations of urokinase PLG activator were incubated in DMEM media with 2 µg/mL PLG for 2 hours at 37°C and then used for efflux and PLG ELISA.

PLG ELISA

The Human Plasminogen Total Antigen ELISA Kit was used to measure PLG levels (Molecular Innovations, Novi, MI). The test was performed according to kit protocol. Human samples were diluted 1:10,000 in kit blocking buffer.

Western blotting

For LPA size estimation, 1 μL of serum was loaded onto NuPage 3% to 8% Tris-Acetate precast gels for electrophoresis. The size-separated proteins were then transferred to nitrocellulose membranes, and a primary antibody was used to detect target LPA. Signal was detected by a LiCore imager. Size was estimated using a HiMark protein standard, which gives an accurate size evaluation up to 460 kDa.

STAT3 phosphorylation assay

Phosphorylation of signal transducer and activator of transcription 3 (pSTAT3) in BHK fibroblasts was determined as the difference in the pSTAT3-to-STAT3 ratio of cells with and without mifepristone induction of ABCA1 upon incubation with apolipoprotein A1 (APOA1) or PLG for 40 minutes. Cells were then fixed with 2% formaldehyde for 15 minutes at room temperature, then washed and blocked with Odyssey Blocking Buffer for 90 minutes at room temperature. The florescent antibodies were used and plates were imaged on the LiCor Clx and analyzed using the In-Cell Western Image Studio software.

Statistical analyses

Data are reported as mean ± SD, with median and interquartile range reported for the skewed variable [i.e., Lp(a)]. Differences among groups were assessed with ANOVA with Tukey post hoc analysis performed to correct for multiple comparisons. Linear correlations were assessed with the Pearson product-moment coefficient. Two sample t tests (for normally distributed variables) and Kruskal-Wallis tests [for non-normal variables, such as Lp(a)] were used to compare continuous characteristics between Lp(a) and LDL groups. Pearson correlation (reported as a r value), is a univariate measure of correlation between two measures. The R2 reported is the coefficient of determination, or the proportion of variance of the outcome (i.e., dependent variable) explained by all of the predictors (i.e., independent variables) in the regression model.

Linear regression models were fit with sterol efflux as the dependent variable and PLG, Lp(a), LDL, HDL, and sex as independent predictors. Linear regression models were fit with cholesterol efflux as the dependent variable and PLG, Lp(a), LDL, HDL, and statin use (yes/no) as independent predictors. Regression coefficients (β) are reported with SE, and regression results are reported with adjusted R2 and global F-statistic P values. Only patients with complete data were included in the regression models.

A sensitivity analysis to determine whether familial hypercholesterolemia status affected associations was conducted by dichotomizing LDL as LDL ≥190 mg/dL and including interactions of LDL >190 mg/dL with PLG and Lp(a). All reported P values are two-tailed, with P < 0.05 indicating statistical significance. Analyses were performed with Prism and R GNU software, version 3.4.2. Lp(a) inhibition slopes were determined with quadratic inhibition curve analysis using GraphPad Prism 8 software.

Results

Total-serum cholesterol efflux was lower in patients with high Lp(a) levels

We studied serum from 62 patients with Lp(a) levels between 2 and 291 mg/dL (mean, 64 mg/dL) to understand if total-serum CEC varies in relation to serum Lp(a) levels. A summary of the patient characteristics is provided in an online repository (22).

Total-serum CEC measured in cAMP-stimulated macrophages was 30.6% lower in patients with Lp(a) concentrations >50 mg/dL (n = 38; median Lp(a) concentration, 98 mg/dL), compared with patients with Lp(a) concentrations <50 mg/dL (n = 24; median Lp(a) concentration, 13 mg/dL), measuring 17.0% ± 8.0% vs 24.4% ± 9.1%, respectively (P = 0.002; Fig. 1A). Furthermore, total-serum CEC negatively correlated with Lp(a) levels as a continuous variable (r = −0.43, P = 0.0007; Fig. 1B). After adjusting for serum cholesterol, age, sex, and statin use, the multiple linear regression model for total-serum CEC (Table 1) demonstrated a negative correlation between total-serum CEC and Lp(a) levels (β = −0.022, P = 0.106; R2 = 0.413, P < 0.05), which was strengthened when further adjusted for the interaction between Lp(a) and PLG levels (β = −0.079, P = 0.008; R2=0.465, P < 0.05). The interaction between Lp(a) and PLG used here is a mathematical device to evaluate the association of Lp(a) with efflux for different levels of PLG and vice versa. For a fixed value of PLG (X), the estimated slope of the association between Lp(a) and efflux is equal to the Lp(a) coefficient (β) + X*Lp(a):PLG coefficient.

Total-serum cholesterol efflux in patients with low and high Lp(a) levels. (A) J774 macrophage CEC after stimulation with cAMP in patients with low Lp(a) levels (≤50 mg/dL) vs high Lp(a) levels (>50 mg/dL). (B) J774 macrophage CEC after stimulation with cAMP of the same samples analyzed in (A), shown as a function of continuous Lp(a) levels. (C) ABCA1-specific CEC after stimulation with mifepristone minus CEC of unstimulated cells in patients with high vs low Lp(a) levels. (D) ABCA1-specific CEC after stimulation with mifepristone minus CEC of unstimulated cells of the same samples analyzed in (C), shown as a function of continuous Lp(a) levels. ***P < 0.001. PLG levels are color coded along a gradient: red, low Lp(a) level; blue, high Lp(a) level. Cholesterol levels are represented by circle sizes: small, <200 mg/dL; medium, 201 to 300 mg/dL; and large, 301 to 400 mg/dL.
Figure 1.

Total-serum cholesterol efflux in patients with low and high Lp(a) levels. (A) J774 macrophage CEC after stimulation with cAMP in patients with low Lp(a) levels (≤50 mg/dL) vs high Lp(a) levels (>50 mg/dL). (B) J774 macrophage CEC after stimulation with cAMP of the same samples analyzed in (A), shown as a function of continuous Lp(a) levels. (C) ABCA1-specific CEC after stimulation with mifepristone minus CEC of unstimulated cells in patients with high vs low Lp(a) levels. (D) ABCA1-specific CEC after stimulation with mifepristone minus CEC of unstimulated cells of the same samples analyzed in (C), shown as a function of continuous Lp(a) levels. ***P < 0.001. PLG levels are color coded along a gradient: red, low Lp(a) level; blue, high Lp(a) level. Cholesterol levels are represented by circle sizes: small, <200 mg/dL; medium, 201 to 300 mg/dL; and large, 301 to 400 mg/dL.

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Table 1.
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Linear Regression Models for Macrophage and BHK Cell–Mediated Efflux

Macrophage-Mediated Efflux% Cholesterol Efflux
J774BHK Cells
With Lp(a) and PLG InteractionWithout Lp(a) and PLG InteractionWith Lp(a) and PLG InteractionWithout Lp(a) and PLG Interaction
Lp(a) level−0.079 (0.028); P = 0.008−0.022 (0.013); P = 0.106−0.049 (0.014); P = 0.002−0.027 (0.008); P = 0.004
PLG level−1.423 (0.418); P = 0.002−0.729 (0.298); P = 0.019−0.179 (0.192); P = 0.3620.066 (0.151); P = 0.667
HDL-C level0.095 (0.058); P = 0.1100.071 (0.060); P = 0.2440.031 (0.043); P = 0.4740.039 (0.045); P = 0.396
LDL-C level0.055 (0.019); P = 0.0070.037 (0.018); P = 0.0490.042 (0.009); P = 0.00020.035 (0.009); P = 0.001
Male sex3.004 (2.044); P = 0.1503.109 (2.140); P = 0.154−1.709 (0.930); P = 0.072−1. 582 (0.973); P = 0.119
Age0.012 (0.070); P = 0.869−0.041 (0.069); P = 0.5520.090 (0.031); P = 0.0080.077 (0.032); P = 0.022
Takes lipid-lowering medication−4.737 (2.074); P = 0.028−5.417 (2.149); P = 0.0160.760 (0.979); P = 0.4460.359 (1.009); P = 0.725
Lp(a) and PLG interaction0.008 (0.003); P = 0.029N/A0.004 (0.002); P = 0.068N/A
Constant14.254 (8.146); P = 0.08817.646 (8.383); P = 0.0426.304 (5.573); P = 0.1306.594 (4.221); P = 0.132
Observationsa51516.304 (5.573); P = 0.1306.594 (4.221); P = 0.132
r20.550.4950.7280.685
Adjusted r20.4650.4130.6340.593
F statistic6.427b (df = 8; 42) (P = 0.00002)6.032b (df = 7; 43) (P = 0.0001)7.700b (df = 8; 23) (P = 0.0001)4.015b (df = 7; 24) (P = 0.0001)
Macrophage-Mediated Efflux% Cholesterol Efflux
J774BHK Cells
With Lp(a) and PLG InteractionWithout Lp(a) and PLG InteractionWith Lp(a) and PLG InteractionWithout Lp(a) and PLG Interaction
Lp(a) level−0.079 (0.028); P = 0.008−0.022 (0.013); P = 0.106−0.049 (0.014); P = 0.002−0.027 (0.008); P = 0.004
PLG level−1.423 (0.418); P = 0.002−0.729 (0.298); P = 0.019−0.179 (0.192); P = 0.3620.066 (0.151); P = 0.667
HDL-C level0.095 (0.058); P = 0.1100.071 (0.060); P = 0.2440.031 (0.043); P = 0.4740.039 (0.045); P = 0.396
LDL-C level0.055 (0.019); P = 0.0070.037 (0.018); P = 0.0490.042 (0.009); P = 0.00020.035 (0.009); P = 0.001
Male sex3.004 (2.044); P = 0.1503.109 (2.140); P = 0.154−1.709 (0.930); P = 0.072−1. 582 (0.973); P = 0.119
Age0.012 (0.070); P = 0.869−0.041 (0.069); P = 0.5520.090 (0.031); P = 0.0080.077 (0.032); P = 0.022
Takes lipid-lowering medication−4.737 (2.074); P = 0.028−5.417 (2.149); P = 0.0160.760 (0.979); P = 0.4460.359 (1.009); P = 0.725
Lp(a) and PLG interaction0.008 (0.003); P = 0.029N/A0.004 (0.002); P = 0.068N/A
Constant14.254 (8.146); P = 0.08817.646 (8.383); P = 0.0426.304 (5.573); P = 0.1306.594 (4.221); P = 0.132
Observationsa51516.304 (5.573); P = 0.1306.594 (4.221); P = 0.132
r20.550.4950.7280.685
Adjusted r20.4650.4130.6340.593
F statistic6.427b (df = 8; 42) (P = 0.00002)6.032b (df = 7; 43) (P = 0.0001)7.700b (df = 8; 23) (P = 0.0001)4.015b (df = 7; 24) (P = 0.0001)

Data are reported as β coefficient (SE), unless otherwise indicated.

Abbreviations: df, degrees of freedom; N/A, not applicable.

a

Observations are considered as the number of patients with all parameters mentioned in the table (i.e., 51 of 59 patients for J774 efflux and 32 of 39 patients for BHK efflux).

b

P < 0.0001.

Table 1.
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Linear Regression Models for Macrophage and BHK Cell–Mediated Efflux

Macrophage-Mediated Efflux% Cholesterol Efflux
J774BHK Cells
With Lp(a) and PLG InteractionWithout Lp(a) and PLG InteractionWith Lp(a) and PLG InteractionWithout Lp(a) and PLG Interaction
Lp(a) level−0.079 (0.028); P = 0.008−0.022 (0.013); P = 0.106−0.049 (0.014); P = 0.002−0.027 (0.008); P = 0.004
PLG level−1.423 (0.418); P = 0.002−0.729 (0.298); P = 0.019−0.179 (0.192); P = 0.3620.066 (0.151); P = 0.667
HDL-C level0.095 (0.058); P = 0.1100.071 (0.060); P = 0.2440.031 (0.043); P = 0.4740.039 (0.045); P = 0.396
LDL-C level0.055 (0.019); P = 0.0070.037 (0.018); P = 0.0490.042 (0.009); P = 0.00020.035 (0.009); P = 0.001
Male sex3.004 (2.044); P = 0.1503.109 (2.140); P = 0.154−1.709 (0.930); P = 0.072−1. 582 (0.973); P = 0.119
Age0.012 (0.070); P = 0.869−0.041 (0.069); P = 0.5520.090 (0.031); P = 0.0080.077 (0.032); P = 0.022
Takes lipid-lowering medication−4.737 (2.074); P = 0.028−5.417 (2.149); P = 0.0160.760 (0.979); P = 0.4460.359 (1.009); P = 0.725
Lp(a) and PLG interaction0.008 (0.003); P = 0.029N/A0.004 (0.002); P = 0.068N/A
Constant14.254 (8.146); P = 0.08817.646 (8.383); P = 0.0426.304 (5.573); P = 0.1306.594 (4.221); P = 0.132
Observationsa51516.304 (5.573); P = 0.1306.594 (4.221); P = 0.132
r20.550.4950.7280.685
Adjusted r20.4650.4130.6340.593
F statistic6.427b (df = 8; 42) (P = 0.00002)6.032b (df = 7; 43) (P = 0.0001)7.700b (df = 8; 23) (P = 0.0001)4.015b (df = 7; 24) (P = 0.0001)
Macrophage-Mediated Efflux% Cholesterol Efflux
J774BHK Cells
With Lp(a) and PLG InteractionWithout Lp(a) and PLG InteractionWith Lp(a) and PLG InteractionWithout Lp(a) and PLG Interaction
Lp(a) level−0.079 (0.028); P = 0.008−0.022 (0.013); P = 0.106−0.049 (0.014); P = 0.002−0.027 (0.008); P = 0.004
PLG level−1.423 (0.418); P = 0.002−0.729 (0.298); P = 0.019−0.179 (0.192); P = 0.3620.066 (0.151); P = 0.667
HDL-C level0.095 (0.058); P = 0.1100.071 (0.060); P = 0.2440.031 (0.043); P = 0.4740.039 (0.045); P = 0.396
LDL-C level0.055 (0.019); P = 0.0070.037 (0.018); P = 0.0490.042 (0.009); P = 0.00020.035 (0.009); P = 0.001
Male sex3.004 (2.044); P = 0.1503.109 (2.140); P = 0.154−1.709 (0.930); P = 0.072−1. 582 (0.973); P = 0.119
Age0.012 (0.070); P = 0.869−0.041 (0.069); P = 0.5520.090 (0.031); P = 0.0080.077 (0.032); P = 0.022
Takes lipid-lowering medication−4.737 (2.074); P = 0.028−5.417 (2.149); P = 0.0160.760 (0.979); P = 0.4460.359 (1.009); P = 0.725
Lp(a) and PLG interaction0.008 (0.003); P = 0.029N/A0.004 (0.002); P = 0.068N/A
Constant14.254 (8.146); P = 0.08817.646 (8.383); P = 0.0426.304 (5.573); P = 0.1306.594 (4.221); P = 0.132
Observationsa51516.304 (5.573); P = 0.1306.594 (4.221); P = 0.132
r20.550.4950.7280.685
Adjusted r20.4650.4130.6340.593
F statistic6.427b (df = 8; 42) (P = 0.00002)6.032b (df = 7; 43) (P = 0.0001)7.700b (df = 8; 23) (P = 0.0001)4.015b (df = 7; 24) (P = 0.0001)

Data are reported as β coefficient (SE), unless otherwise indicated.

Abbreviations: df, degrees of freedom; N/A, not applicable.

a

Observations are considered as the number of patients with all parameters mentioned in the table (i.e., 51 of 59 patients for J774 efflux and 32 of 39 patients for BHK efflux).

b

P < 0.0001.

Experiments were repeated using ABCA1-expressing fibroblasts with samples from a subgroup of patients (n = 39, due to limited plasma volume availability from the rest of the cohort). Total-serum CEC measured in ABCA1-expressing fibroblasts was 24.1% lower in patients with Lp(a) concentrations >50 mg/dL (n = 23; median Lp(a) concentration, 84 mg/dL), compared with patients with Lp(a) concentrations <50 mg/dL (n = 16; median Lp(a) concentration, 13 mg/dL), measuring 15.3% ± 4.0% vs 20.2% ± 2.8%, respectively (P < 0.001; Fig. 1C). Total-serum CEC negatively correlated with Lp(a) as a continuous variable (r = −0.505, P = 0.0011; Fig. 1D). After adjusting for serum cholesterol, age, sex, and statin use, the multiple linear regression model for total-serum CEC (Table 1) demonstrated a negative association between total-serum CEC and Lp(a) levels (β = −0.027, P = 0.004; R2=0.593, P < 0.05), which was strengthened when further adjusted for the interaction between Lp(a) and PLG levels (β = −0.049, P = 0.002; R2=0.634, P < 0.05). Overall, efflux in patients with high Lp(a) levels was 24% to 31% lower than in patients with Lp(a) levels <50 mg/dL, a difference with physiologic relevance when considering the reported ∼9% change in serum-HDL CEC between patients undergoing cardiac catherization and control patients (29).

LDL is also a promoter of cholesterol efflux (30, 31), and LDL-C levels positively correlated with cholesterol efflux in our multiple regression model (for J774 macrophages, β = 0.055, P = 0.007; for ABCA1-expressing fibroblasts, β = 0.042, P = 0.0002). Thus, we further stratified our cohort according to LDL-C levels above and below 190 mg/dL. Our data demonstrate that although hypercholesterolemia predicted total-serum CEC, it did not influence the association between Lp(a) and CEC and did not interact with Lp(a) levels and/or PLG levels (22). Furthermore, the Pearson correlation r as univariate measure of correlation between efflux and PLG levels was not significant for the BHK model (r = 0.07; P = 0.65), and the multivariate regression model that considered all the predictors produced comparable results. One possible explanation for the difference between the two models is that BHK cells only use ABCA1 as cholesterol transporter, whereas J774 macrophages use multiple transporters, including ABCG1, SR-B1, and ABCA1 (26).

Serum determinants of cholesterol efflux

To understand the relative contributions of each lipoprotein compartment to cellular cholesterol efflux, we compared CEC measured using total serum, serum-HDL, and ultracentrifugation-isolated HDL, LDL, and Lp(a). The concentrations of serum-HDL, isolated lipoproteins, and PLG in the incubation medium represented the same amounts in the corresponding total-serum treatment (Fig. 2A). Lipoprotein isolation purity was visualized via lipid gel (22). To study ABCA1-mediated effects, we used ABCA1-expressing fibroblasts that do not express other cholesterol transporters such as SR-B1 and ABCG123. In ABCA1-expressing fibroblasts, CEC levels were similar between total serum and serum-HDL (11.0% ± 1.1% vs 9.3% ± 1.9%, respectively; Fig. 2B). ABCA1-mediated efflux for isolated HDL, LDL, Lp(a), and PLG were 4.7% ± 2.9%, 2.8% ± 1.9%, 1.0% ± 0.5%, and 4.5% ± 0.9%, respectively (Fig. 2B). Average and individual patient values for CEC in stimulated and unstimulated BHK cells are provided in an online repository, as are patients characteristics for this subgroup (22).

Serum determinants of cholesterol efflux. (A) Experimental design to measure ABCA1-specific CEC of total serum (TS), serum-HDL, ultracentrifugation-isolated lipoproteins [i.e., LDL, Lp(a), HDL], and recombinant PLG. (B) ABCA1-specific CEC after stimulation of BHK fibroblasts with mifepristone minus CEC of unstimulated cells.
Figure 2.

Serum determinants of cholesterol efflux. (A) Experimental design to measure ABCA1-specific CEC of total serum (TS), serum-HDL, ultracentrifugation-isolated lipoproteins [i.e., LDL, Lp(a), HDL], and recombinant PLG. (B) ABCA1-specific CEC after stimulation of BHK fibroblasts with mifepristone minus CEC of unstimulated cells.

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The mathematical addition of CEC values from serum-HDL, LDL, PLG, and Lp(a) resulted in a total calculated CEC (17.6% ± 5.2%) higher than that observed for total-serum CEC (11.0% ± 1.1%), thus suggesting that inhibitory interactions in serum modulate ABCA1-specific CEC.

Inhibitory effects of Lp(a) levels and LPA isoforms on PLG-mediated efflux in total serum

To study how PLG-mediated efflux is altered in response to varying levels of Lp(a), we studied a subgroup of patients with normal (<30 mg/dL; n = 5), high (30 to 80 mg/dL; n = 4), and extremely high Lp(a) (>80 mg/dL; n = 5) levels, and analyzed the ability of their total serum to inhibit PLG-mediated efflux. Patients characteristics for this subgroup are provided in an online repository (22). Confirming our hypothesis, total serum with increasing Lp(a) levels inhibited efflux mediated by 1 μg or 5 μg PLG (Fig. 3A). These levels corresponded to the levels of PLG found in plasma for the total-serum CEC assays, as explained in Methods.

Effect of Lp(a) levels and LPA isoforms on PLG-mediated efflux in total serum. (A) PLG-mediated, ABCA1-specific CEC after stimulation with mifepristone minus CEC of unstimulated cells in total serum of patients with low (<30 mg/dL; n = 5), medium (30 to 80 mg/dL; n = 4), and high (>80 mg/dL; n = 5) Lp(a) levels. Experiments were performed using two PLG concentrations (1 μg and 5 μg in 0.25 mL of media; range, 4 to 20 μg/mL). (B) LPA immunoblot for the nine patients with low-medium (30 to 80 mg/dL; n = 4) and high (>80 mg/dL; n = 5) Lp(a) levels shown in (A). (C) PLG-mediated BHK fibroblasts CEC after stimulation with mifepristone minus CEC of unstimulated cells with increasing concentration of isolated Lp(a) from patients 2, 5, and 7 [shown in (B)] with constant levels of PLG and increasing concentrations of Lp(a). Red line, efflux with varying Lp(a) levels; blue line, efflux for a constant PLG and varying Lp(a) levels.
Figure 3.

Effect of Lp(a) levels and LPA isoforms on PLG-mediated efflux in total serum. (A) PLG-mediated, ABCA1-specific CEC after stimulation with mifepristone minus CEC of unstimulated cells in total serum of patients with low (<30 mg/dL; n = 5), medium (30 to 80 mg/dL; n = 4), and high (>80 mg/dL; n = 5) Lp(a) levels. Experiments were performed using two PLG concentrations (1 μg and 5 μg in 0.25 mL of media; range, 4 to 20 μg/mL). (B) LPA immunoblot for the nine patients with low-medium (30 to 80 mg/dL; n = 4) and high (>80 mg/dL; n = 5) Lp(a) levels shown in (A). (C) PLG-mediated BHK fibroblasts CEC after stimulation with mifepristone minus CEC of unstimulated cells with increasing concentration of isolated Lp(a) from patients 2, 5, and 7 [shown in (B)] with constant levels of PLG and increasing concentrations of Lp(a). Red line, efflux with varying Lp(a) levels; blue line, efflux for a constant PLG and varying Lp(a) levels.

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A 17-kringle recombinant LPA inhibits PLG-mediated efflux (21). Given that Lp(a) exhibits size, density, and concentration heterogeneity as a result of the LPA length polymorphism (8, 32–34) we screened nine patients whose Lp(a) could be isolated without contamination and we selected three distinct molecular-weight LPA isoforms (Fig. 3B) to show that all LPA isoforms inhibited PLG-mediated efflux with varying slopes (−0.1, −0.8, and −1.2 for patients 2, 5, and 7, respectively; Fig. 3C). The number of samples (n = 3) and resolution of LPA gel separation do not allow us to make definitive claims on the LPA isoform–specific inhibition of PLG-mediated efflux.

PLG-mediated activation of ABCA1 downstream signaling

Binding of APOA1 to ABCA1 activates cAMP production, which increases APOA1 lipidation (35). Another pathway activated within minutes of APOA1 binding to ABCA1 is the phosphorylation of STAT3, which is independent of the lipid transport function of ABCA1 and suppresses proinflammatory cytokine secretion (36). To study whether PLG-mediated activation of ABCA1 results in downstream signaling events, we incubated APOA1 or PLG with fibroblasts that either express or do not express ABCA1, and we analyzed the levels of STAT3 phosphorylation. Both APOA1 and PLG activated at comparable levels the phosphorylation of STAT3 in cells that express ABCA1 (Fig. 4A).

Proposed model for Lp(a) inhibition PLG-mediated efflux through ABCA1. (A) In-Cell Western image for STAT3 phosphorylation, measured as the levels of phosphor-STAT3 (red) to total STAT3 (green) ratio in BHK fibroblasts before or after stimulation of ABCA1 expression with mifepristone. (B) PLG promotes ABCA1-specific cholesterol efflux from cells and STAT3 phosphorylation, likely through a direct interaction with the transporter. (C) Lp(a) interferes with the binding of PLG to ABCA1.
Figure 4.

Proposed model for Lp(a) inhibition PLG-mediated efflux through ABCA1. (A) In-Cell Western image for STAT3 phosphorylation, measured as the levels of phosphor-STAT3 (red) to total STAT3 (green) ratio in BHK fibroblasts before or after stimulation of ABCA1 expression with mifepristone. (B) PLG promotes ABCA1-specific cholesterol efflux from cells and STAT3 phosphorylation, likely through a direct interaction with the transporter. (C) Lp(a) interferes with the binding of PLG to ABCA1.

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Discussion

In clinical trials studying cholesterol efflux serum-HDL, CEC has become the diagnostic method of choice for prediction of cardiovascular diseases (29, 37). However, this method does not consider other plasma factors that can interact with ABCA1 and affect CEC, such as PLG (21), LDL (30, 31), and Lp(a). Total-serum CEC was previously found to predict atherosclerosis in macaques (38). It remains to be determined whether total-serum CEC adds to serum-HDL CEC in prediction of cardiovascular diseases in humans.

Serum PLG levels were previously shown to be independently associated with risk for coronary artery disease in two prospective cohort studies: the FINRISK ‘92 Hemostasis Study (39) and the Atherosclerosis Risk in Communities Study (40). PLG remained a risk factor after adjustment for cholesterol and HDL-C levels (39, 40, 30). In contrast, there was no correlation between serum and intimal concentrations of PLG in a study of normal and atherosclerotic human intimal tissue (19). We previously showed that both isolated Lp(a) and recombinant LPA inhibit PLG-mediated efflux by ABCA1 (21). Since serum levels of both PLG and Lp(a) correlate with CVD risk, the interplay between PLG, Lp(a), and ABCA1 may affect the overall atherogenicity of Lp(a). Here we suggest that the previous contradicting results about the role of PLG in CVD may be attributable to the interplay between PLG and Lp(a) and their effect on CEC.

Both Lp(a) and LPA inhibit the activation of PLG by t-PA or u-PA (16), in part by competing with PLG for binding to the endothelial cell surface. The cleavage of PLG to plasmin abolished PLG-mediated efflux (23), suggesting that Lp(a) inhibits PLG-mediated CEC by mechanisms other than the activation of PLG. Thus, the mechanisms by which the Lp(a) inhibits PLG-mediated efflux remain to be determined.

To summarize, standard efflux assays only account for the power of serum-HDL as a cholesterol acceptor. We show that in total serum, factors contributing to sterol efflux capacity can be interactive and even inhibitory. The contribution of apoB-lipoproteins and the interplay between Lp(a) and PLG on ABCA1-mediated cholesterol efflux are of physiologic significance: Patients with high Lp(a) levels have lower sterol efflux capacity. Though additional studies are needed to determine the mechanisms by which Lp(a) inhibits ABCA1-mediated sterol efflux, our model supports a scenario in which serum PLG mediates cholesterol efflux through ABCA1 (Fig. 4B), whereas Lp(a) interferes with the binding of PLG to ABCA1 (Fig. 4C). Although our data do not directly attest to the relative importance of PLG in efflux in vivo and to the fate of the cholesterol picked up by PLG, we do show that PLG is a cholesterol acceptor through ABCA1. Additional studies are needed to assess if these new metrics provide additional predictive power for CVD risk. The atherogenic function of Lp(a) can be exploited for diagnostic, predictive, and therapeutic purposes.

Acknowledgments

The authors thank all the providers enrolling patients into the CPC registry and biorepository and all patients who generously contributed blood samples to our study.

Financial Support: H.T. was partially supported by American Heart Association Grant 16SGD27520011; S.F. was partially supported by National Institutes of Health (NIH) Grant R01HL132985; and N.P. was partially supported by NIH Grant R01HL136373.

Disclosure Summary: The authors have nothing to disclose.

Abbreviations:

    Abbreviations:
     
  • ABCA1

    ATP-binding cassette A1

    •  
  • APOA1

    apolipoprotein A1

    •  
  • apoB

    apolipoprotein B

    •  
  • BHK

    baby hamster kidney

    •  
  • CEC

    cholesterol efflux capacity

    •  
  • CVD

    cardiovascular disease

    •  
  • HDL-C

    high-density lipoprotein cholesterol

    •  
  • IRB

    institutional review board

    •  
  • LDL

    low-density lipoprotein

    •  
  • Lp(a)

    lipoprotein(a)

    •  
  • LPA

    apolipoprotein(a)

    •  
  • OHSU

    Oregon Health & Sciences University

    •  
  • PLG

    plasminogen

    •  
  • pSTAT3

    phosphorylation of signal transducer and activator of transcription 3

    •  
  • serum-HDL

    apolipoprotein B–depleted serum

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Copyright © 2019 Endocrine Society
Issue Section:
Clinical Research Articles > Lipids and Cardiovascular
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