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. 2023 Nov;64(11):100456.
doi: 10.1016/j.jlr.2023.100456. Epub 2023 Oct 10.

Serum opacity factor normalizes erythrocyte morphology in Scarb1-/- mice in an HDL-free cholesterol-dependent way

Ziyi Wang  1 , Dedipya Yelamanchili  2 , Jing Liu  3 , Antonio M Gotto Jr  4 , Corina Rosales  4 , Baiba K Gillard  4 , Henry J Pownall  5
Affiliations

Serum opacity factor normalizes erythrocyte morphology in Scarb1-/- mice in an HDL-free cholesterol-dependent way

Ziyi Wang et al. J Lipid Res. 2023 Nov.

Abstract

Compared with WT mice, HDL receptor-deficient (Scarb1-/-) mice have higher plasma levels of free cholesterol (FC)-rich HDL and exhibit multiple pathologies associated with a high mol% FC in ovaries, platelets, and erythrocytes, which are reversed by lowering HDL. Bacterial serum opacity factor (SOF) catalyzes the opacification of plasma by targeting and quantitatively converting HDL to neo HDL (HDL remnant), a cholesterol ester-rich microemulsion, and lipid-free APOA1. SOF delivery with an adeno-associated virus (AAVSOF) constitutively lowers plasma HDL-FC and reverses female infertility in Scarb1-/- mice in an HDL-dependent way. We tested whether AAVSOF delivery to Scarb1-/- mice will normalize erythrocyte morphology in an HDL-FC-dependent way. We determined erythrocyte morphology and FC content (mol%) in three groups-WT, untreated Scarb1-/- (control), and Scarb1-/- mice receiving AAVSOF-and correlated these with their respective HDL-mol% FC. Plasma-, HDL-, and tissue-lipid compositions were also determined. Plasma- and HDL-mol% FC positively correlated across all groups. Among Scarb1-/- mice, AAVSOF treatment normalized reticulocyte number, erythrocyte morphology, and erythrocyte-mol% FC. Erythrocyte-mol% FC positively correlated with HDL-mol% FC and with both the number of reticulocytes and abnormal erythrocytes. AAVSOF treatment also reduced FC of extravascular tissues to a lesser extent. HDL-FC spontaneously transfers from plasma HDL to cell membranes. AAVSOF treatment lowers erythrocyte-FC and normalizes erythrocyte morphology and lipid composition by reducing HDL-mol% FC.

Keywords: HDLs; atherosclerosis; cholesterol; erythrocyte morphology; hyperalphalipoproteinemia; scavenger receptor class B member 1.

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Conflict of interest statement

Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article.

Figures

None
Graphical abstract
Fig. 1
Fig. 1
Plasma and HDL lipid composition. AAVSOF treatment decreases the elevated plasma and HDL TC, FC, CE, mol% FC, and FC/TC levels in Scarb1−/− mice toward WT levels. Plasma (A–F) and HDL (G–L) lipid concentrations of female (left panels) and male (right panels) WT, Scarb1−/−, and AAVSOF-treated Scarb1−/− mice. M and N: Plasma and HDL-mol% FC were highly correlated for both females (M) and males (N). HDLs were obtained from individual mouse plasma by heparin-manganese depletion of APOB lipoproteins. Data points are values for individual mice, and bars are mean ± SD. Numbers of female (-F) and male (-M) mice per group were WT-F (n = 17), Scarb1−/−F (n = 16), Scarb1−/−FAAVSOF (n = 18), WT-M (n = 15), Scarb1−/−M (n = 11), and Scarb1−/−MAAVSOF (n = 17). Group means were compared by ANOVA with Tukey comparison of means as described in the Materials and Methods section. P values for significantly different pairwise comparisons (∗P ≤ 0.05, ∗∗P ≤ 0.01, and∗∗∗P ≤ 0.001) are indicated over brackets. The slope m, R2, and P values for the linear regression line for the correlation plots (M, N) are shown on the graphs. Comparisons between male and female data within the same genotype or treatment group for plasma: CE: WT-F < WT-M, P = 0.0048; TG: AAVSOF-F < AAVSOF-M, P = 0.0005, whereas for HDL, there were no significant differences between sexes for any of the analytes.
Fig. 2
Fig. 2
Erythrocyte lipid composition. AAVSOF treatment decreases the elevated erythrocyte (red blood cell) TC, FC, and mol% FC in Scarb1−/− mice toward WT levels. Panels A–C (female) and D–F (male) provide the lipid composition relative to protein (W/W), mol% FC, and the FC/TC ratio (W/W). G and H: Correlation of erythrocyte versus HDL-mol% FC. Data points are values for individual mice, and bars are mean ± SD. Mice/group for the bar graphs were WT-F (n = 11), Scarb1−/−F (n = 11), Scarb1−/−FAAVSOF (n = 18), WT-M (n = 9), Scarb1−/−M (n = 14), and Scarb1−/−MAAVSOF (n = 17) and for the correlation plots WT-F (n = 5), Scarb1−/−F (n = 5), Scarb1−/−FAAVSOF (n = 18), WT-M (n = 5), Scarb1−/−M (n = 6), and Scarb1−/−MAAVSOF (n = 17). Statistics are as described in the legend to Figure 1. Comparisons between male and female data within the same genotype or treatment group showed no significant differences between sexes for any of the analytes. Note: Data in the correlation plots are only for those mice from which both plasma and erythrocytes were collected so there are fewer paired values for the correlations (G, H) than the total number of mice in the bar graphs.
Fig. 3
Fig. 3
Erythrocyte (red blood cell [RBC]) morphology correlates with RBC-FC. AAVSOF normalizes both the RBC-FC content and morphology. A: Micrographs of representative fields of blood smears stained with Wright/Giemsa stain from WT, Scarb1−/−, and AAVSOF-treated Scarb1−/− mice (Arrows: abnormal cells; circles: reticulocytes). B, quantitation of erythrocyte morphology based on counts of abnormal cells (acanthocytes) and reticulocytes. Data from male and female mice were not significantly different; so pooled data are shown. C: Correlation of percent abnormal cells versus percent reticulocytes. D and E: Correlation of percent abnormal and percent reticulocyte cells with erythrocyte-mol% FC. Mice/group: WT (n = 10), Scarb1−/− (n = 10), and Scarb1−/−AAVSOF (n = 12). Data plotted by sex were not different; so male and female data were pooled.
Fig. 4
Fig. 4
Heart, lung, and liver lipid composition. AAVSOF treatment has variable effects on the cholesterol content of heart, lung, and liver in Scarb1−/− mice. A–H: Heart; (I–P) Lung; and (Q–X) Liver. The respective panels provide the lipid composition relative to protein (W/W), mol% FC, and the FC/TC ratio (W/W). Data points are values for individual mice, and bars are mean ± SD. Mice/group were WT-F (n = 12), Scarb1−/−F (n = 11), Scarb1−/−FAAVSOF (n = 11), WT-M (n = 10), Scarb1−/−M (n = 5), and Scarb1−/−MAAVSOF (n = 9). Statistics are as described in the legend to Figure 1. Comparisons between male and female data within the same genotype or treatment group gave the following significant differences between sexes: heart: TC: Scarb1−/−F> Scarb1−/−M, P = 0.0137; FC: AAVSOF-F> AAVSOF-M, P = 0.0084; CE: Scarb1−/−F> Scarb1−/−M, P = 0.0015; FC/TC: Scarb1−/−M> Scarb1−/−F, P = 0.0025. Lung: TC: AAVSOF-M> AAVSOF-F, P = 0.0286; AAVSOF-M> AAVSOF-F, P = 0.0019. Liver: TC: Scarb1−/−F> Scarb1−/−M, P = 0.0085; FC: Scarb1−/−F> Scarb1−/−M, P = 0.0017; TG: Scarb1−/−F> Scarb1−/−M, P = 0.0074; mol% FC: Scarb1−/−F> Scarb1−/−M, P = 0.0587 and AAVSOF-M> AAVSOF-F, P = 0.0470. Note: In some instances, the calculated FC/TC ratio was >1 because of the imprecision of some of the analyses at concentrations near the detection limits of the assay.
Fig. 5
Fig. 5
Steroidogenic tissue lipid composition. AAVSOF treatment decreases the elevated mol% FC to WT levels in ovaries but does not restore the low CE levels of ovaries or adrenals of Scarb1−/− mice. A–C and G: ovary; D–F and H: testis, and I–P: adrenals. The respective panels provide the lipid composition relative to protein (W/W), mol% FC, and the FC/TC ratio (W/W). Data points are values for individual mice, and bars are mean ± SD. Mice/group for ovaries : WT-F (n = 17), Scarb1−/−F (n = 14), Scarb1−/−FAAVSOF (n = 18); for testis, WT-M (n = 15), Scarb1−/−M (n = 11), and Scarb1−/−MAAVSOF (n = 17); for adrenals: WT-F (n = 5–8), Scarb1−/−F (n = 5–8), Scarb1−/−FAAVSOF (n = 18), WT-M (n = 5–8), Scarb1−/−M (n = 6–13), and Scarb1−/−MAAVSOF (n = 17). Statistics are as described in the legend to Figure 1. Comparisons between male and female adrenal data within the same genotype or treatment group gave the following significant differences between sexes: CE: Scarb1−/−F> Scarb1−/−M, P = 0.0426; TG: WT-M> WT-F, P = 0.0473; FC/TC: Scarb1−/−M> Scarb1−/−F, P < 0.0001 and AAVSOF-M> AAVSOF-F, P < 0.0001.
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