Dysfunctional high-density lipoproteins have distinct composition, diminished anti-inflammatory potential and discriminate acute coronary syndrome from stable coronary artery disease patients

Abstract

There is a stringent need to find means for risk stratification of coronary artery diseases (CAD) patients. We aimed at identifying alterations of plasma high-density lipoproteins (HDL) components and their validation as dysfunctional HDL that could discriminate between acute coronary syndrome (ACS) and stable angina (SA) patients. HDL₂ and HDL₃ were isolated from CAD patients' plasma and healthy subjects. ApolipoproteinAI (apoAI), apoAII, apoCIII, malondialdehyde (MDA), myeloperoxidase (MPO), ceruloplasmin and paraoxonase1 (PON1) were assessed. The anti-inflammatory potential of HDL subfractions was tested by evaluating the secreted inflammatory molecules of tumor necrosis factor α-activated endothelial cells (EC) upon co-incubation with HDL₂ or HDL_3. We found in ACS versus SA patients: 40% increased MPO, MDA, apoCIII in HDL₂ and HDL₃, 35% augmented apoAII in HDL₂, and in HDL₃ increased ceruloplasmin, decreased apoAII (40%) and PON1 protein and activity (15% and 25%). Co-incubation of activated EC with HDL₂ or HDL₃ from CAD patients induced significantly increased levels of secreted inflammatory molecules, 15-20% more for ACS versus SA. In conclusion, the assessed panel of markers correlates with the reduced anti-inflammatory potential of HDL subfractions isolated from ACS and SA patients (mostly for HDL₃ from ACS) and can discriminate between these two groups of CAD patients.

Figure 1

Biochemical parameters measured in sera/plasma of healthy subjects (N), stable angina patients (SA) and acute coronary syndrome patients (ACS). Levels of serum paraoxonase 1 (PON1) protein (a) and paraoxonase activity (b), plasma myeloperoxidase (MPO) protein (c), thiobarbituric reactive substances (TBARS) (d), free 4-hydroxy-2-nonenal (4-HNE) (e), and MPO protein/PON1 activity (MPOp/PON1a) and MPO protein/PON1 protein (MPOp/PON1p) ratios (f). Data are expressed as means ± SEM and analyzed with two-tailed Oneway ANOVA with Least Significant Difference (LSD) Post-hoc test; *p < 0.05, ***p < 0.001 vs. N; ^#p < 0.05, ^##p < 0.01 vs SA.

Figure 2

Inflammatory parameters measured in plasma of healthy subjects (N), stable angina patients (SA) and acute coronary syndrome patients (ACS). Levels of soluble vascular cell adhesion molecule 1 (sVCAM-1) (a), monocyte chemoattractant protein 1 (MCP-1) (b) and C reactive protein (CRP) (c). Data are expressed as means ± SEM and analyzed with two-tailed Oneway ANOVA with Least Significant Difference (LSD) Post-hoc test; *p < 0.05, **p < 0.01 vs. N; ^#p < 0.05 vs. SA.

Figure 3

Characterization of HDL₂ and HDL₃ subfractions isolated by density gradient ultracentrifugation from seven independent pools of sera collected from healthy subjects (N), stable angina patients (SA) and acute coronary syndrome (ACS) patients. Levels of apolipoprotein AI (ApoAI) (a), ApoAII (b), ApoCIII (c), paraoxonase 1 (PON1) protein (d) and paraoxonase activity (e), and myeloperoxidase (MPO) protein (f). Data are expressed as means ± SD and analyzed with Independent Student’s T-test; *p < 0.05, **p < 0.01, ***p < 0.001 vs. N, ^#p < 0.05, ^##p < 0.01 vs. SA.

Figure 4

Characterization of oxidative markers in HDL₂ and HDL₃ subfractions isolated by density gradient ultracentrifugation from seven independent pools of sera from healthy subjects (N), stable angina patients (SA) and acute coronary syndrome (ACS) patients. Levels of 4-hydroxy-2-nonenal associated to apolipoprotein AI (4-HNE-apoAI) (a), malondialdehyde associated to apolipoprotein AI (MDA-apoAI) (b), conjugated dienes (c) and ceruloplasmin (CP) (d). The western blot images were processed by cropping the corresponding parts from the blots. The full-length blots are supplied in the Supplementary information. Data are expressed as means ± SD and analyzed with Independent Student’s T-test; *p < 0.05, **p < 0.01, ***p < 0.001 vs. N, ^#p < 0.05, ^##p < 0.01 vs. SA.

Figure 5

HDL₂ and HDL₃ functionality evaluated by their anti-inflammatory effects on endothelial cells (EC) activated with tumor necrosis factor α (TNFα). The vascular cell adhesion molecule 1 (VCAM-1) protein relative to β-actin (a) and soluble VCAM-1 (sVCAM-1) in the cultured media relative to cellular protein (b); protein expression of disintegrin and metalloproteinase domain-containing protein 17 (ADAM17) pro-form (c) and active form (d) relative to β-actin. Levels of monocyte chemoattractant protein 1 (MCP-1) (e) and C reactive protein (CRP) (f) secreted in the culture medium relative to cellular protein. All data come from four different experiments and are expressed as fold change versus TNFα-activated EC. C - cells unstimulated with TNFα, N - healthy subjects, SA - stable angina patients, ACS - acute coronary syndrome patients, TNFα - tumor necrosis factor α. The western blot (wb) images for (b), (e) and (f) were processed by cropping the lanes from the same blot and marked by dividing white lines where the order of the samples on wb was different from the one in the graph. The full-length blots are supplied in the Supplementary information. Data are presented as means ± SD and analyzed with Independent Student’s T-test. ^#p < 0.05, ^##p < 0.01 vs. SA, ^$p < 0.05, ^$$p < 0.01 vs. TNFα.