Rosuvastatin Alters the Proteome of High Density Lipoproteins: Generation of alpha-1-antitrypsin Enriched Particles with Anti-inflammatory Properties

Abstract

Statins lower plasma cholesterol by as much as 50%, thus reducing future cardiovascular events. However, the physiological effects of statins are diverse and not all are related to low density lipoprotein cholesterol (LDL-C) lowering. We performed a small clinical pilot study to assess the impact of statins on lipoprotein-associated proteins in healthy individuals (n = 10) with normal LDL-C (<130 mg/dL), who were treated with rosuvastatin (20 mg/day) for 28 days. Proteomic analysis of size-exclusion chromatography isolated LDL, large high density lipoprotein (HDL-L), and small HDL (HDL-S) fractions and spectral counting was used to compare relative protein detection before and after statin therapy. Significant protein changes were found in each lipoprotein pool and included both increases and decreases in several proteins involved in lipoprotein metabolism, complement regulation and acute phase response. The most dramatic effect of the rosuvastatin treatment was an increase in α-1-antirypsin (A1AT) spectral counts associated with HDL-L particles. Quantitative measurement by ELISA confirmed an average 5.7-fold increase in HDL-L associated A1AT. Molecular modeling predictions indicated that the hydrophobic reactive center loop of A1AT, the functional domain responsible for its protease inhibitor activity, is likely involved in lipid binding and association with HDL was found to protect A1AT against oxidative inactivation. Cell culture experiments, using J774 macrophages, demonstrated that the association of A1AT with HDL enhances its antiprotease activity, preventing elastase induced production of tumor necrosis factor α. In conclusion, we show that statins can significantly alter the protein composition of both LDL and HDL and our studies reveal a novel functional relationship between A1AT and HDL. The up-regulation of A1AT on HDL enhances its anti-inflammatory functionality, which may contribute to the non-lipid lowering beneficial effects of statins.

Fig. 1.

Effect of rosuvastatin on plasma lipids and lipoprotein particle numbers. A, Rosuvastatin effects on total plasma lipid levels (TC = total cholesterol; HDL-C = HDL cholesterol; LDL-C = LDL cholesterol; TG = triglyceride). The effect of rosuvastatin on LDL particle number (B) and HDL particle number (C) were measured by nuclear magnetic resonance. T0 and T28 are timepoints indicating baseline and after 28 days of rosuvastatin treatment, respectively. Data are mean ± standard deviation. * indicates p < 0.01.

Fig. 2.

Effect of rosuvastatin on plasma lipid distributions by size-exclusion chromatography. Plasma from patients at baseline (T0) and after 28 days (T28) of rosuvastatin treatment was separated on two Superose 6 columns arranged in series. Collected fractions were analyzed for total cholesterol, free cholesterol, phosphatidylcholine and triglyceride. Data are mean ± standard deviation.

Fig. 3.

ApoB spectral counts correlate with LDL particle number. As validation of the semi-quantitative potential of spectral counting under our experimental conditions we compared spectral counts for apolipoprotein B (apoB) versus LDL particle number. ApoB is a core protein of LDL and has a well-established 1:1 (mol apoB/mol LDL) stoichiometry.

Fig. 4.

Rosuvastatin alters the lipoprotein proteome. Statistically significant changes to the LDL (A) and HDL (B) proteomes resulting from rosuvastatin treatment are displayed as percent change compared with baseline. HDL-L = large HDL; HDL-S = small HDL; PGRP-L = N-acetylmuramoyl-l-alanine amidase. Statistical comparisons were made using student's t test. All displayed data are p < 0.05.

Fig. 5.

Quantitative measurement of α-1-antitrypsin on HDL and in plasma. A, Individual patient spectral counts for α-1-antitrypsin (A1AT) in large HDL at baseline (T0) and after 28 days (T28) of rosuvastatin treatment, n = 10 for each time point. The “n = 5” indicator points to data from 5 subjects with a high degree of overlap. B, Quantitative measurement of A1AT in large HDL by ELISA assay. C, Time course of plasma A1AT concentrations during rosuvastatin treatment and after two-week washout period (Day 42 time point). * indicates p < 0.05 and ** indicates p < 0.01 compared with T0.

Fig. 6.

Structural prediction of lipid binding by α-1-antitrypsin. A, Predicted binding of α-1-antitrypsin (A1AT) to a lipid surface (red spheres). Inset demonstrates that methionine residues (Met³⁵¹ and Met³⁵⁸) are embedded in the lipid (white arrows) and indicates the cut site for neutrophil elastase (red arrow). B, Predicted lipid binding of A1AT structure with the reactive center loop removed (A1AT Δ 346).

Fig. 7.

α-1-antitrypsin has reduced anti-elastase activity when bound to reconstituted HDL. Reconstituted HDL (rHDL) were prepared from apoA-I and phospholipids by cholate dialysis and then co-incubated with α-1-antitrypsin (A1AT) to generate A1AT enriched rHDL. A, Size exclusion chromatography on tandem Superdex 200 columns was used to isolate HDL bound A1AT from lipid free protein. B, The ability of lipid free and rHDL bound A1AT to inhibit neutrophil elastase (NE) activity was measured by fluorometric assay.

Fig. 8.

Binding to HDL protects α-1-antitrypsin anti-elastase activity from oxidation by H₂O₂. HDL isolated from healthy human donors was co-incubated with α-1-antitrypsin (A1AT) to generate A1AT enriched nHDL. Lipid free A1AT and A1AT nHDL were exposed to varying concentrations of H₂O₂ for 30 min before measurement of anti-elastase activity by fluorometric assay. Nonlinear regression analysis was used for comparison of curve fits and found the two curves to be significantly different (p < 0.0001).

Fig. 9.

α-1-antitrypsin enriched HDL prevents elastase induced TNF-α production by macrophages. A, J774 mouse macrophages treated with increasing amounts of porcine pancreatic elastase (PPE) or heat inactivated elastase for 4 h, TNF-α in the culture media was measured by ELISA. B, J774 cells pretreated with PBS, isolated human HDL (nHDL), the same HDL enriched with α-1-antitrypsin (A1AT nHDL), or lipid free A1AT for 1 h prior to PPE addition. C, The ability of each of the cell treatments to inhibit elastase activity was measured in a cell-free assay. D, Treatments were pre-incubated with PPE prior to addition to cells and TNF- α was measured in the culture media after 4 h. E, J774 cells were pre-incubated with each treatment for 1 h; cells were then washed twice with PBS and placed in fresh media containing PPE and TNF- α was measured in the culture media after 4 h. All experiments were repeated at least 3 times and were done in triplicates. Treatments were compared using one-way ANOVA and Tukey's multiple comparisons test, p < 0.05 was considered significant. The letters above each treatment indicate statistical significance; within each graph, bars bearing different letters were statistically different from each other.