Atherosclerosis Regression and Cholesterol Efflux in Hypertriglyceridemic Mice

Abstract

[Figure: see text].

Keywords: atherosclerosis; cardiovascular disease; lipoprotein lipase; lipoproteins; macrophages; triglyceride.

Figure 1.. LpL deficiency increased VLDL-TG and reduced HDL-C, which was further reduced with hCETP expression.

(A) Study Setup (B) Total plasma cholesterol levels at time of harvest (C) Total triglyceride (TG) levels in baseline and regression groups before and after the 2 weeks atherosclerosis regression period (D) CETP activity measured in circulation. Cholesterol (E) and triglyceride levels (F) of isolated lipoproteins. N= (B, C) Baseline 26, Lpl^fl/fl 19, Lpl^−/− 18, Lpl^fl/fl +hCETP 11, Lpl^−/− + hCETP 7; (D) Lpl^fl/fl 8, Lpl^−/− 4, Lpl^fl/fl +hCETP 9, Lpl^−/− + hCETP 5; (E, F) Baseline 4, Lpl^fl/fl 15, Lpl^−/− 13, Lpl^fl/fl +hCETP 10, Lpl^−/− + hCETP 6. Data represented as mean ± SEM, * P < 0.05, ** P<0.01, *** P < 0.001, **** P < 0.0001, 1-way ANOVA with Tukey’s multiple comparison test; HDL-C and HDL-TG Welch ANOVA with Games-Howell’s multiple comparison test; VLDL-TG & LDL-TG Kruskal-Wallis Test with Dunn’s multiple comparison test.

Figure 2.. HyperTG due to global LpL deficiency does not affect atherosclerosis regression in the aortic transplant mouse model.

(A) Representative stainings; CD68, MOVATs, Picrosirius Red Staining (brightfield & polarized light); scale bar = 500 μm. Quantification of (B) Lesion Area (μm²) and (C) CD68 Area (% of Lesion Area ) (D) Necrotic Core (% of Lesion Area) (E) Fibrous Cap determined by number of layers above the Necrotic Core, Thick >10layers, Thin 5–10layers, Very Thin <5 layers; scale bar = 100 μm (F) Collagen (% of Lesion Area); N= (B, C, D) Baseline 15, Lpl^fl/fl 18, Lpl^−/− 16, Lpl^fl/fl +hCETP 8, Lpl^−/− + hCETP 6; (E) Baseline 5, Lpl^fl/fl 7, Lpl^−/− 5, Lpl^fl/fl +hCETP 7, Lpl^−/− + hCETP 6 (F) Baseline 8, Lpl^fl/fl 8, Lpl^−/− 9, Lpl^fl/fl +hCETP 5, Lpl^−/− + hCETP 6. Data represented as mean ± SEM, **** P < 0.0001, 1-way ANOVA with Tukey’s multiple comparison test; (E) 2-way ANOVA with Tukey’s multiple comparison test (F) Welch ANOVA with Games-Howell’s multiple comparison test.

Figure 3.. LpL deletion does not alter atherosclerosis regression in a non-invasive model of regression.

(A) Study design: atherosclerosis was created in Lpl^fl/fl and iLpl^−/− mice with LDLR antisense oligonucleotides (ASO) and western diet feeding for 16 weeks. One set of mice were analyzed at 16 weeks as the baseline group and the rest of the mice were treated with SO to induce regression and were analyzed after 3 weeks. The mice in regression group were also treated with tamoxifen at week 13 to induce hypertriglyceridemia in iLpl^−/− mice. (B) Plasma total cholesterol (TC) and (C) triglyceride (TG) levels in baseline and regression groups. (D) Total plaque area, (E) % of macrophages (CD68+) within aortic root lesions, (F) total plaque area, (G) % of macrophage (Mac2+) in plaques within the BCA in the baseline and regression groups. N=(B,C) Baseline Lpl^fl/fl 9, Regression Lpl^fl/fl 10 and iLpl^−/− 7, (D,E) Baseline Lpl^fl/fl 10, Regression Lpl^fl/fl14 and iLpl^−/− 9 and (F,G) Baseline Lpl^fl/fl 15, Regression Lpl^fl/fl 19 and iLpl^−/− 11. Results are expressed as mean ± SEM. * P < 0.05, ** P<0.01 and **** P < 0.0001 using 1-way ANOVA with Tukey’s multiple comparison test.

Figure 4.. LpL deficiency (+/− hCETP) does not affect macrophage phenotype.

Macrophage phenotype was assessed by isolating mRNA from atherosclerotic CD68+ macrophages using Laser Capture Microdissection and mRNA expression measured for (A) Metabolism-related genes (B) Inflammatory genes (C) Anti-inflammatory genes. Additionally, immunofluorescence staining of atherosclerotic arches was performed for (D-E) Arginase-1 (%cells per total cells) and (F-G) iNOS (%cells per total cells), scale bars = 500 μm. N= (A-C) Lpl^fl/fl +hCETP 5, Lpl^−/− + hCETP 5; (E, G) Lpl^fl/fl 6, Lpl^−/− 5, Lpl^fl/fl +hCETP 6, Lpl^−/− + hCETP 5. Data represented as mean ± SEM, * P < 0.05, (A-C) unpaired t-test, (E, G) 1-way ANOVA with Tukey’s multiple comparison test.

Figure 5.. LpL deficiency-mediated HyperTG reduces HDL-C and HDL-P, but does not impair CEC.

(A) Total HDL-P and its subfractions. (B) Total and ABCA1-mediated HDL Cholesterol Efflux Capacity (CEC). (C) Total and ABCA1-mediated CEC per HDL-P. (D) Volcano-plot of HDL Proteomics in iLpl^−/− versus Lpl^f//fl. (E) Main regulated proteins are part of plasma lipoprotein remodeling. (F) Volcano plot iLpl^−/− + hCETP vs. Lpl^fl/f + hCETP. (G) Regulated proteins in the plasma lipoprotein remodeling pathway. Proteomics normalized to spiked ApoA-I; complete list of proteins can be found in Supplemental Table II–V. N= (A) Lpl^fl/fl 19, Lpl^−/− 17, Lpl^fl/fl +hCETP 9, Lpl^−/− + hCETP 5; (B,C) Lpl^fl/fl 19, Lpl^−/− 13, Lpl^fl/fl +hCETP 8, Lpl^−/− + hCETP 5; (D-G) Lpl^fl/fl n=8, iLpl^−/− n=8, Lpl^fl/f + hCETP n=17, iLpl^−/− + hCETP n=10. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, 1-way ANOVA with Tukey’s multiple comparison test; Medium HDL-P and (C) Kruskal-Wallis Test with Dunn’s multiple comparison test.

Figure 6.. Subjects with HyperTG, incl. LpL deficient subjects show decreased HDL-P, but no impairment in CEC.

(A) Plasma TG and Cholesterol levels (mg/dL). (B) total HDL-P and HDL-P subsets. (C) Total and ABCA1-mediated CEC (%). (D) CEC per HDL-P (E) Volcano-plot of HDL Proteomics showing changes with HyperTG. (F) Main regulated proteins are part of plasma lipoprotein remodeling pathway. White-filled dots refer to HyperTG patients, blue filled dots refer to LpL deficient patients. Proteomics normalized to spiked ApoA-I; complete list of proteins can be found in Supplemental Table VI. N=6 (Control), N=9 (HyperTG). * P < 0.05, ** P<0.01, **** P < 0.0001, students unpaired t-test; (C, D) Mann-Whitney test.