Hypercholesterolemia-Induced HDL Dysfunction Can Be Reversed: The Impact of Diet and Statin Treatment in a Preclinical Animal Model

Abstract

High-density lipoproteins (HDL) undergo adverse remodeling and loss of function in the presence of comorbidities. We assessed the potential of lipid-lowering approaches (diet and rosuvastatin) to rescue hypercholesterolemia-induced HDL dysfunction. Hypercholesterolemia was induced in 32 pigs for 10 days. Then, they randomly received one of the 30-day interventions: (I) hypercholesterolemic (HC) diet; (II) HC diet + rosuvastatin; (III) normocholesterolemic (NC) diet; (IV) NC diet + rosuvastatin. We determined cholesterol efflux capacity (CEC), antioxidant potential, HDL particle number, HDL apolipoprotein content, LDL oxidation, and lipid levels. Hypercholesterolemia time-dependently impaired HDL function (−62% CEC, −11% antioxidant index (AOI); p < 0.01), increased HDL particles numbers 2.8-fold (p < 0.0001), reduced HDL-bound APOM (−23%; p < 0.0001), and increased LDL oxidation 1.7-fold (p < 0.0001). These parameters remained unchanged in animals on HC diet alone up to day 40, while AOI deteriorated up to day 25 (−30%). The switch to NC diet reversed HDL dysfunction, restored apolipoprotein M content and particle numbers, and normalized cholesterol levels at day 40. Rosuvastatin improved HDL, AOI, and apolipoprotein M content. Apolipoprotein A-I and apolipoprotein C-III remained unchanged. Lowering LDL-C levels with a low-fat diet rescues HDL CEC and antioxidant potential, while the addition of rosuvastatin enhances HDL antioxidant capacity in a pig model of hypercholesterolemia. Both strategies restore HDL-bound apolipoprotein M content.

Keywords: HDL dysfunction; diet; hypercholesterolemia; pig; statin.

Figure 1

Diet-induced hypercholesterolemia onset increases lipid parameters while HDL function is impaired. Progression of plasma lipid parameters including total cholesterol (A), LDL-C (B), HDL-C (C), non-HDL-C (D), and HDL functional measures cholesterol efflux capacity (CEC) from macrophages to HDL (E), and antioxidant capacity (G) are depicted during the first 10 days of HC diet. The correlation between cholesterol efflux capacity and HDL-C levels (d0 and d10 (F) is shown with linear regressions. Individual values are depicted with bars representing the mean ± SD over time. Shapiro–Wilk test confirmed normality (alpha = 0.05), and data were analyzed by one-way ANOVA with Tukey’s multicomparison test and considered significant with a p-value < 0.05. * p < 0.05, ** p < 0.0001 in comparison to day 0; † p < 0.05, †† p < 0.0001 in comparison to day 1; # p < 0.05, ## p < 0.0001 in comparison to day 3; §§ p < 0.0001, § p < 0.05 in comparison to day 5. LDL-C: low-density lipoprotein cholesterol; HDL-C: high-density lipoprotein cholesterol; CEC: cholesterol efflux capacity; AOI: antioxidant index.

Figure 2

Effects of diet and rosuvastatin intervention on lipid parameters and CEC. Changes in plasma total cholesterol (TC, circle with continuous line), low-density lipoprotein cholesterol (LDL-C, square with dotted line), and high-density lipoprotein cholesterol (HDL-C, triangles with dashed line) levels (A,C,E,G), and percent cholesterol efflux from macrophages to HDL (% Efflux; B,D,F,H) are depicted as mean (bars or line through respective symbol) ± SD (whiskers) for all four groups over the period of 40 days. Shapiro–Wilk test confirmed normality (alpha = 0.05), and data were analyzed by unpaired t-test and considered significant with a p-value < 0.05. ** p < 0.0001 in comparison to day 0; †† p < 0.0001 in comparison to day 10. CEC: cholesterol efflux capacity; TC: total cholesterol; HDL: high-density lipoprotein cholesterol; LDL: low-density lipoprotein cholesterol; HC: hypercholesterolemic; NC: normocholesterolemic; rosu: rosuvastatin; d0–40: day 0–40.

Figure 3

Effects of diet and rosuvastatin intervention on HDL antioxidant capacity. Changes in HDL antioxidant capacity are presented as percent antioxidant index ((A), % AOI; measure for capacity to reverse LDL oxidation) and levels of conjugated dienes ((B–E) product of lipid oxidation). Data are shown as mean ± SEM ((A); for visual clarity of the graph) or SD (whiskers) over the period of 40 days. (A) includes all four groups, whereas (B–E) represent each group individually. Shapiro–Wilk test confirmed normality (alpha = 0.05), and data were analyzed by one-way ANOVA with Tukey’s multicomparison test and considered significant with a p-value < 0.05. For (A): * p < 0.05 in comparison to day 0; † p < 0.05 in comparison to day 10; # p < 0.05 compared to HC; § p < 0.05 in comparison to NC. For (B–E): * p < 0.05, ** p < 0.0001 in comparison to day 0; † p < 0.05 in comparison to day 10. d0–40: day 0–40; HC: hypercholesterolemic diet; NC: normocholesterolemic diet; +R/+rosu: + rosuvastatin; LDL: low-density lipoprotein.

Figure 4

Effects of diet and rosuvastatin intervention on HDL apolipoprotein levels. Changes in protein levels of apolipoproteins (APO) APOA-I (A), APOC-III (B), and APOM (C) in isolated HDL particles are presented as relative quantification and representative Western blots (D) at study endpoint. Western blot quantifications are shown as mean ± SD. Shapiro–Wilk test confirmed normality (alpha = 0.05), and data were analyzed by unpaired t-test and considered significant with a p-value < 0.05. ** p < 0.0001 in comparison to day 0; †† p < 0.0001 in comparison to day 10; # p < 0.05 in comparison to day 40 of animals on HC diet. d0–40: day 0–40; HC: hypercholesterolemic diet; NC: normocholesterolemic diet; +R: + rosuvastatin.

Figure 5

Study design. All animals were fed a hypercholesterolemic diet for the first 10 days of the study. From this day onwards, the animals were randomly divided into four groups to receive 30 days of (I) hypercholesterolemic diet alone (n = 9), (II) hypercholesterolemic diet with 40 mg rosuvastatin daily (n = 7), (III) normocholesterolemic diet alone (n = 8), (IV) normocholesterolemic diet with 40 mg rosuvastatin daily (n = 8). Samples were collected at indicated timepoints. HDL: high-density lipoprotein; CEC: cholesterol efflux capacity; AOI: antioxidant index; WB: Western blot. * High-fat and normal diet refer to hypercholesterolemic and normocholesterolemic diet, respectively.