Acute coronary syndrome remodels the protein cargo and functions of high-density lipoprotein subfractions

Abstract

Objectives: This study examined alterations in the functions and proteome of high-density lipoprotein (HDL) subfractions (HDL2 and HDL3) isolated from patients with acute coronary syndrome (ACS) compared with control subjects.

Methods: We measured HDL subfraction cholesterol efflux capacity, inflammatory index (HII), paraoxonase-1 (PON1) activity, and lipid hydroperoxide (LOOH) levels in both male age-matched controls and the ACS group (n = 40/group). Additionally, proteomic analysis was used to monitor changes in the HDL subfraction proteome between controls and ACS subjects.

Results: Both HDL2 and HDL3 from ACS patients had greater HII and LOOH levels compared with controls (P<0.001); PON1 activity and cholesterol efflux capacity in both HDL2 and HDL3 from the ACS group were significantly less than those of controls (P<0.001). Using proteomic analysis, we demonstrated that, compared with the control group, nine proteins were selectively enriched in HDL3 from subjects with ACS, and ras-related protein Rab-7b was decreased in HDL3. Additionally, in the ACS subjects, 12 proteins were decreased in HDL2 and 4 proteins were increased in HDL2.

Conclusions: Functional HDL subfractions shifted to dysfunctional HDL subfractions during ACS, and the functional impairment was linked to remodeled protein cargo in HDL subfractions from ACS patients.

Figure 1. Comparison of HDL subfraction functions between acute coronary syndrome and control groups.

The high-density lipoprotein subfraction cholesterol efflux capacity, inflammatory index (HII), paraoxonase-1 (PON1) activity, and LOOH levels were measured in both male age-matched controls and ACS subjects (n = 40/group). (A), HDL subfractions, HDL2 and HDL3, mediated reverse cholesterol transport capacity; (B), anti-inflammatory capacity; (C) and (D) anti-oxidant capacity. PON1 activity was normalized to mg of protein; *P<0.001 compared with the control group.

Figure 2. Forty-eight differentially expressed protein spots in HDL subfractions between the ACS group and controls analyzed by DeCyder 2D software.

Proteins were extracted as described and separated in pH–10 NL IPG strips for the first dimension and 12.5% polyacrylamide for the second dimension. The image was acquired on a Typhoon 9400 scanner at 633/670 nm excitation/emission wavelengths. Spots detected by the analysis software are indicated.

Figure 3. Overlapped sections of the 2D-DIGE proteome map of the acute coronary syndrome group and controls in HDL2.

A total of 50 µg of each type of HDL subfraction sample was labeled with CyDye 3 (control group) and CyDye 5 (ACS group), respectively. An internal standard (50 µg) comprised of an equal amount of proteins from all samples (control and ACS group) was labeled with CyDye 2 and included in all gels. Cy2, Cy3, and Cy5 images appeared as blue, green, and red using the ImageQuant TL software. The green spots indicate downregulated proteins, while the red spots indicate upregulated proteins. Compared with the controls, 12 proteins were downregulated in HDL2 from ACS subjects, while 4 proteins were upregulated.

Figure 4. Overlapped sections of the 2D-DIGE proteome map of the acute coronary syndrome group and controls in HDL3.

The green spots indicate downregulated proteins, while the red spots indicate upregulated proteins. Compared with the controls, nine proteins were selectively enriched in HDL3 from ACS subjects, while ras-related protein Rab-7b was decreased in HDL3.

Figure 5. Validation of HDL-associated proteins by western blot and ELISA.

(A) and (B), to confirm the results obtained by MS+MS/MS, HDL subfractions isolated from ACS patients and control subjects were subjected to immunoblot analysis. HDL subfraction-associated proteins were separated by SDS-PAGE, transferred to PVDF membranes, and probed using specific antibodies. Molecular mass is indicated on the right. (C), comparison of serum amyloid P-component concentrations between acute coronary syndrome and control groups. SAP denotes serum amyloid P-component. *P<0.001 compared with controls. Data are presented here after log transformation of SAP levels (for nonparametric results).