Susceptibility of low-density lipoprotein particles to aggregate depends on particle lipidome, is modifiable, and associates with future cardiovascular deaths

Abstract

Aims: Low-density lipoprotein (LDL) particles cause atherosclerotic cardiovascular disease (ASCVD) through their retention, modification, and accumulation within the arterial intima. High plasma concentrations of LDL drive this disease, but LDL quality may also contribute. Here, we focused on the intrinsic propensity of LDL to aggregate upon modification. We examined whether inter-individual differences in this quality are linked with LDL lipid composition and coronary artery disease (CAD) death, and basic mechanisms for plaque growth and destabilization.

Methods and results: We developed a novel, reproducible method to assess the susceptibility of LDL particles to aggregate during lipolysis induced ex vivo by human recombinant secretory sphingomyelinase. Among patients with an established CAD, we found that the presence of aggregation-prone LDL was predictive of future cardiovascular deaths, independently of conventional risk factors. Aggregation-prone LDL contained more sphingolipids and less phosphatidylcholines than did aggregation-resistant LDL. Three interventions in animal models to rationally alter LDL composition lowered its susceptibility to aggregate and slowed atherosclerosis. Similar compositional changes induced in humans by PCSK9 inhibition or healthy diet also lowered LDL aggregation susceptibility. Aggregated LDL in vitro activated macrophages and T cells, two key cell types involved in plaque progression and rupture.

Conclusion: Our results identify the susceptibility of LDL to aggregate as a novel measurable and modifiable factor in the progression of human ASCVD.

Figures

Figure 1

Measurement of the susceptibility of low-density lipoprotein (LDL) from healthy human subjects and from coronary artery disease patients to aggregate ex vivo. (A) LDL is isolated from blood plasma by ultracentrifugation and aggregation was induced by incubation with human recombinant secretory sphingomyelinase (hrSMase) at pH 5.5. The size of LDL particles was measured before hrSMase treatment (time = 0 h), and formation of LDL aggregates was followed in real time by measuring their size with dynamic light scattering. (B) LDL particles were isolated from 100 plasma samples collected from the Finnish Health 2000 Health Examination Survey and the aggregation susceptibility of the particles was analysed. Based on LDL aggregate size at 2 h, the particles were divided into quartiles. (C) Size distributions of LDL aggregates at the 2 h time point. The box encompasses the middle 50% of the measured values; the horizontal line within each box shows the median of the measured values; the whiskers encompass the most extreme data point that is still no further from the margins of the box than 1.5 times the interquartile range. (D) Patients (n = 48) from the Corogene study, having >50% stenosis in their coronary arteries were divided into two groups: (i) CAD death group, in which patients died of coronary events during an average 2.5-year follow-up period and (ii) stable CAD group, having no cardiovascular events during the follow-up period. The patients were matched for the conventional cardiovascular risk factors. LDL was isolated and LDL aggregation was induced by treatment with hrSMase. The box plot diagram shows the distribution of aggregate sizes after incubation for 2 h in the two groups from Corogene study and in 100 subjects from the Health 2000 study (all quartiles from C combined). Statistical differences between the groups were determined using Kruskal–Wallis test followed by Dunn’s test. P < 0.001 by Kruskal–Wallis test; *P < 0.05, ***P <0.001 by Dunn’s test.

Figure 2

The susceptibility of low-density lipoprotein (LDL) to aggregate strongly correlates with the surface lipid composition of the particles. (A) LDL was isolated from plasma and LDL lipidome was analysed using mass spectrometry. Volcano plots showing Spearman correlation coefficients of LDL aggregate size at 2 h vs. LDL surface lipids in (B) Health 2000 samples and (C) in Corogene samples. Red circles indicate positive correlations, and blue circles indicate negative correlations. The identities of only those lipids with significance correlation values (P < 0.05) are indicated. Cer, ceramide; LPC, lysophosphatidylcholine; PC, phosphatidylcholine; SM, sphingomyelin.

Figure 3

The susceptibility of low-density lipoprotein (LDL) to aggregate strongly correlates with the core lipid composition of the particles. Volcano plots showing Spearman correlation coefficients of LDL aggregate size at 2 h vs. LDL core lipids (A) in Health 2000 samples and (B) in Corogene samples. Red circles indicate positive correlations, and blue circles indicate negative correlations. The identities of only those lipids with significance correlation values (P < 0.05) are indicated. CE, cholesteryl ester; TAG, triacylglycerol.

Figure 4

A dietary intervention and PCSK9 inhibition in human subjects improves their low-density lipoprotein (LDL) composition and renders their particles less susceptible to aggregate. Plasma samples were obtained from the SYSDIET-study, where participants were placed on either an isocaloric healthy Nordic diet (n = 33) or a control diet (n = 25) for 18 or 24 weeks and from the EQUATOR study, a randomized placebo-controlled phase II trial of a monoclonal antibody inhibiting the function of PCSK9, RG7652, (n = 25), or placebo (n = 15) for 29 days. LDL was isolated, and aggregation analysed from samples before and after the diet/treatment period. (A and B) LDL aggregate sizes at the 2-h time point are shown in the diet group and control group before and after the diet period. Each line represents one subject and blue lines show a decrease and red lines an increase in aggregate size. (D and E) LDL aggregate sizes at the 2-h time point are shown in the PSCK9 inhibitor group and placebo group before and after the treatment period. Each line represents one subject and blue lines show a decrease and red lines an increase in aggregate size. (C and F) Volcano plot showing the Spearman correlation coefficients of LDL aggregate size at 2 h vs. LDL surface lipids in the SYSDIET study and in the EQUATOR study. PC, phosphatidylcholine; PE, phosphatidylethanolamine; SM, sphingomyelin.

Figure 5

Pharmacological and genetic interventions that favourably alter low-density lipoprotein (LDL) lipid composition in vivo in hypercholesterolaemic mice render their LDL aggregation-resistant. (A) Human APOB transgenic/LDLr−/− mice were given a single intravenous injection of large ‘empty’ vesicles (LEVs) made from PC 16:0/18:1 or equivalent volume of PBS (control, n= 8 per group). After 1 h, plasma was collected, LDL isolated, and aggregation induced by treatment with hrSMase. LDL aggregation was followed by dynamic light scattering at the indicated time points. The insert shows LDL aggregation for up to 24 h. (B) LDLr−/−/Apob100/100 mice were simultaneously started on an atherogenic western-type diet and intraperitoneal injections three times per week of either myriocin, and inhibitor of SM biosynthesis, or PBS (n = 11 mice per group). Diet and injections continued for 10 weeks. LDL was isolated, then treated with hrSMase, and particle aggregation was followed by dynamic light scattering at the indicated time points. (C) Representative aortas from a control mouse and a myriocin-treated mouse stained with Sudan IV. The extent of atherosclerosis was determined by measuring the amounts of Sudan IV positive areas in the aortas. (D) LDL was isolated from the plasma of human APOB transgenic/LDLr−/−/Soat2−/− mice and human APOB transgenic/LDLr−/−/Soat2+/+ littermates. LDL was treated with hrSMase and LDL aggregation followed by dynamic light scattering at the indicated time points. The line and column graphs display averages ± standard deviations. *P <0.05, **P < 0.01, and ***P < 0.001 by Student’s t-test.

Figure 6

The effect of aggregated low-density lipoprotein (LDL) on macrophages and T-cells. (A) Oil Red O-stained human monocyte-derived macrophages incubated for 20 h with 100 µg/mL of LDL or aggregated LDL (24-h treatment with hrSMase). (B) The amounts of cholesteryl esters in monocyte-derived macrophages incubated in the presence of 100 µg/mL of the variously treated LDL preparations for 20 h. (C) The amount of MMPs secreted from the cells was determined by a Multiplex array. Only secretion of MMP-7 was induced by SMase-treated LDL. (D) Activation of human apoB-100-specific T-cell hybridoma 48-5 measured by IL-2 secretion after 24 h co-culture with antigen presenting cells and 10 µg/mL LDL with increasing amount of sphingomyelinase modification (left panel) or oxidation (right panel). The column graphs show averages ± standard deviations. Statistical differences between the groups were determined using Kruskal–Wallis test followed by post hoc pairwise comparison using Dunn’s test. *P < 0.05 and **P < 0.01.

Take home figure

Development of a measurement of LDL aggregation susceptibility revealed the importance of qualitative differences in LDL particles in ASCVD. The circulating LDL particles of patients succumbing in CAD have a high proportion of sphingomyelins (SM) and ceramides. When such aggregation-prone LDL particles enter the arterial intima, they easily aggregate upon modification. Intimal LDL aggregates promote atherogenesis, inflammation and plaque rupture by inducing foam cell formation, secretion of matrix metalloproteinase 7 (MMP7) and by activating T-cells, ultimately increasing the risk for fatal CAD. Therefore, decreasing the aggregation susceptibility of circulating LDL could reduce CAD risk. In vitro and in mouse models, aggregation-prone LDL particles can be rendered aggregation-resistant by reducing their sphingolipid content. In humans, a healthy diet or treatment with a PCSK9 inhibitor improves the quality of LDL particles resulting in aggregation-resistant phosphatidylcholine (PC)- and lysophosphatidylcholine (LPC)-rich LDL particles.