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Comparative Study
. 2021 Oct;41(10):e453-e467.
doi: 10.1161/ATVBAHA.121.316535. Epub 2021 Aug 12.

High Free Cholesterol Bioavailability Drives the Tissue Pathologies in Scarb1-/- Mice

Jing Liu  1   2   3 , Baiba K Gillard  3   4 , Dedipya Yelamanchili  3 , Antonio M Gotto Jr  3   4 , Corina Rosales  3   4 , Henry J Pownall  3   4
Affiliations
Comparative Study

High Free Cholesterol Bioavailability Drives the Tissue Pathologies in Scarb1-/- Mice

Jing Liu et al. Arterioscler Thromb Vasc Biol. 2021 Oct.

Abstract

Objective: Overall and atherosclerosis-associated mortality is elevated in humans with very high HDL (high-density lipoprotein) cholesterol concentrations. Mice with a deficiency of the HDL receptor, Scarb1 (scavenger receptor class B type 1), are a robust model of this phenotype and exhibit several additional pathologies. We hypothesized that the previously reported high plasma concentration of free cholesterol (FC)-rich HDL in Scarb1-/- mice produces a state of high HDL-FC bioavailability that increases whole-body FC and dysfunction in multiple tissue sites. Approach and Results: The higher mol% FC in Scarb1-/- versus WT (wild type) HDL (41.1 versus 16.0 mol%) affords greater FC bioavailability for transfer to multiple sites. Plasma clearance of autologous HDL-FC mass was faster in WT versus Scarb1-/- mice. FC influx from Scarb1-/- HDL to LDL (low-density lipoprotein) and J774 macrophages was greater ([almost equal to]4x) than that from WT HDL, whereas FC efflux capacity was similar. The higher mol% FC of ovaries, erythrocytes, heart, and macrophages of Scarb1-/- versus WT mice is associated with previously reported female infertility, impaired cell maturation, cardiac dysfunction, and atherosclerosis. The FC contents of other tissues were similar in the two genotypes, and these tissues were not associated with any overt pathology. In addition to the differences between WT versus Scarb1-/- mice, there were many sex-dependent differences in tissue-lipid composition and plasma FC clearance rates. Conclusions: Higher HDL-FC bioavailability among Scarb1-/- versus WT mice drives increased FC content of multiple cell sites and is a potential biomarker that is mechanistically linked to multiple pathologies.

Keywords: atherosclerosis; cholesterol; lipoproteins, HDL; macrophages; phenotype.

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Figures

Figure 1:
Figure 1:
Plasma (A–C) and HDL (D–F) Lipid Concentrations of Male (M) and Female (F) WT and Scarb1−/− Mice. HDL were obtained from individual mouse plasma by heparin-manganese depletion of APOB lipoproteins. Bars are mean ± SEM. Numbers of mice per group are: WT-F (n = 12), WT-M (n = 10), Scarb1−/−-F (n = 11) and Scarb1−/−-M (n = 5). ANOVA and pairwise comparisons were calculated as described in Methods. P-values for significantly different pairwise comparisons (p <0.05) are indicated over brackets.
Figure 2:
Figure 2:
HDL-FC Transfer to human LDL and J774 Macrophages. A, B: Equilibrium distribution of [3H]FC between various HDL and human LDL according to HDL-protein (A) and HDL-FC (B). The specific activities of WT mouse, Scarb1−/− mouse and human HDL-FC were 21,488, 7,942, and 38,233 dpm/nmol FC respectively. In A, m, the slopes of the linear regression lines, are different, p ≤ 0.0001 for all three curves. In B, where transfer is plotted vs. starting HDL-FC, data for the three HDL fall on a single line. C–E: Kinetics of HDL-[3H]FC Transfer to human LDL as labeled. The red and black symbols are from two independent experiments, and the lines are exponential decay fits of the combined data, as described in Methods. F: Extent of FC transfer from HDL to LDL at the asymptote, i.e., kinetic equilibrium, calculated from C–E. FC transferred from Scarb1−/− HDL > WT HDL (p < 0.0001). G–J: FC Flux between HDL and J774 Macrophages. G, H: FC influx from HDL to macrophages according to time (G) at 20 μg HDL-protein/mL and dose at 2 h (H). [3H]FC specific radioactivities were 40,767, 9,242 and 15,247 dpm/nmol FC respectively for human, WT, and Scarb1−/− HDL. At all times and doses, FC influx from SCARB1−/− HDL > WT HDL (p<0.0001). I, J: FC efflux from macrophages to HDL, time course (I; 20 μg HDL-protein/mL) and dose response (2 h; J). The plots are representative of two to three independent experiments, each done in triplicate. The points are mean ± SD with error bars sometimes smaller than the symbols.
Figure 3:
Figure 3:
FC Accretion and Lipid Composition of Erythrocytes, Heart, Lung, and Liver. Within each group—A–C (erythrocytes), D–F (heart), G–I (lung), and J–L (liver)—the respective panels provide the lipid composition relative to protein (W/W), mol% FC, and the FC/total cholesterol (TC) ratio (W/W). Bars are mean ± SEM. Statistics are as described in Figure 1 legend. Mice/group are as follows: Erythrocytes, WT-F (n = 6), WT-M (n = 4), Scarb1−/−-F (n = 6) and Scarb1−/−-M (n = 8); for heart, lung and liver WT-F (n = 12), WT-M (n = 10), Scarb1−/−-F (n = 11) and Scarb1−/−-M (n = 5).
Figure 4:
Figure 4:
FC Accretion and Lipid Compositions in Abdominal-, Ovary-, and Testes-Fat. TG values are at 1/20 scale vs. other lipids. Within each group—A–C (abdominal fat), E–F (ovary fat), and G–I (testes fat)—the respective panels provide lipid composition vs. protein (W/W), mol% FC, and FC/TC ratio (W/W). Bars are mean ± SEM. Mice/group are as follows: Abdominal fat, WT-F (n = 4), WT-M (n = 5), Scarb1−/−-F (n = 4) and Scarb1−/−-M (n = 5); ovary fat, WT-F (n = 5) and Scarb1−/−-F (n = 4); testis fat, WT-M (n = 5), and Scarb1−/−-M (n = 5). Statistics are as described in Figure 1 legend. In Panels A and D, some values for CE that were at the detection limit are nevertheless shown with their SEM for completeness.
Figure 5:
Figure 5:
FC Accretion and Lipid Compositions in Ovaries (A–C), Testes (D–F), and Adrenals (G–I). Bars are mean ± SEM. Numbers of mice per group for ovaries were as follows: WT-F (n = 12), Scarb1−/−-F (n = 10). For testis: WT-M (n = 10), Scarb1−/−-M (n = 5). For adrenals: WT-F (n = 3); WT-M (n = 3); Scarb1−/−-F (n = 3); Scarb1−/− M (n = 7). Statistics as described in Figure 1 legend.
Figure 6:
Figure 6:
In vivo HDL-FC Kinetics. A) Plasma decay kinetics of autologous HDL-[3H]FC in female and male WT and Scarb1−/− mice. B) In the same experiments, erythrocyte-[3H]FC. Mice of each genotype/sex (n = 3–4) were injected and blood collected at each time point; symbols are mean ± SD. The respective specific activities of the injected WT and Scarb1−/− mouse HDL (50 μg protein/mouse) were 6,160 and 5,110 dpm/μg. Injected HDL-FC < 1% of endogenous FC. Rate constants and statistical comparisons are given in Supplementary Table IV.
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