Apolipoprotein Signature of HDL and LDL from Atherosclerotic Patients in Relation with Carotid Plaque Typology: A Preliminary Report

Abstract

In the past years, it has become increasingly clear that the protein cargo of the different lipoprotein classes is largely responsible for carrying out their various functions, also in relation to pathological conditions, including atherosclerosis. Accordingly, detailed information about their apolipoprotein composition and structure may contribute to the revelation of their role in atherogenesis and the understanding of the mechanisms that lead to atherosclerotic degeneration and toward vulnerable plaque formation. With this aim, shotgun proteomics was applied to identify the apolipoprotein signatures of both high-density and low-density lipoproteins (HDL and LDL) plasma fractions purified from healthy volunteers and atherosclerotic patients with different plaque typologies who underwent carotid endarterectomy. By this approach, two proteins with potential implications in inflammatory, immune, and hemostatic pathways, namely, integrin beta-2 (P05107) and secretoglobin family 3A member 2 (Q96PL1), have been confirmed to belong to the HDL proteome. Similarly, the list of LDL-associated proteins has been enriched with 21 proteins involved in complement and coagulation cascades and the acute-phase response, which potentially double the protein species of LDL cargo. Moreover, differential expression analysis has shown protein signatures specific for patients with “hard” or “soft” plaques.

Molecular Characterization of Plasma HDL, LDL, and VLDL Lipids Cargos from Atherosclerotic Patients with Advanced Carotid Lesions: A Preliminary Report

“…Besides some alternative purification methods such as free solution isotachophoresis and immunoaffinity or size exclusion chromatography, ultracentrifugation in high-salt media represents the most widely used approach [ 3 ]. Indeed, the different lipoprotein classes have been historically classified according to their buoyant density.…”

“…Indeed, the different lipoprotein classes have been historically classified according to their buoyant density. We performed isopycnic salt gradient ultracentrifugation followed by a further step of fraction flotation by high centrifugal fields to obtain highly purified HDL, LDL, and VLDL fractions, as assessed by SDS-PAGE analysis [ 3 , 4 ]. In particular, the electrophoretic profiles of each fraction did not show any gross contamination by plasma proteins, particularly albumin.…”

“…Although high LDL-cholesterol levels and low HDL-cholesterol levels are well-established risk factors for cardiovascular disease (CVD), several lines of evidence indicate that a residual risk exists in patients who do not fully benefit from statin treatment, suggesting the need to identify novel markers of plaque development and evolution toward instability, useful for selecting the most appropriate patient-centered therapy [ 1 , 2 ]. In the past twenty years, large-scale MS/MS-based technologies have been applied to purified lipoprotein fractions for unravelling their specific protein cargos, in association with CVD and some CVD-associated pathological conditions including kidney disease and type 1 and 2 diabetes mellitus (an updated list of references with the main results can be found in Supplementary Material 1 of [ 3 ]). As far as our research group is concerned, we applied shotgun proteomics to the analysis of plasma LDL and HDL, identifying some novel lipoprotein-associated proteins and showing specific signatures for atherosclerotic patients with different types of carotid plaque [ 3 , 4 ], sorted by ultrasonography into hypoechoic (types 1 and 2) or “soft” and hyperechoic (types 3, 4, and 5) or “hard”, according to Gray–Weale classification [ 5 ].…”

“…In the past twenty years, large-scale MS/MS-based technologies have been applied to purified lipoprotein fractions for unravelling their specific protein cargos, in association with CVD and some CVD-associated pathological conditions including kidney disease and type 1 and 2 diabetes mellitus (an updated list of references with the main results can be found in Supplementary Material 1 of [ 3 ]). As far as our research group is concerned, we applied shotgun proteomics to the analysis of plasma LDL and HDL, identifying some novel lipoprotein-associated proteins and showing specific signatures for atherosclerotic patients with different types of carotid plaque [ 3 , 4 ], sorted by ultrasonography into hypoechoic (types 1 and 2) or “soft” and hyperechoic (types 3, 4, and 5) or “hard”, according to Gray–Weale classification [ 5 ]. Indeed, the improvement of imaging techniques has allowed routine characterization and detection of the features of carotid plaque vulnerability, also providing predictive information in both symptomatic and asymptomatic carotid artery stenosis [ 6 , 7 , 8 , 9 ].…”

High-Density Lipoproteins and Cardiovascular Disease: The Good, the Bad, and the Future II

“…In total, 84 and 16 proteins were found to be differentially expressed in HDL and LDL. These findings widen the HDL and LDL proteome with two and twenty-one additional proteins involved in the inflammatory and immune and coagulation pathways and allow protein signatures to be identified specifically for patients with "hard" or "soft" plaques [16].…”