SPA Promotes Atherosclerosis Through Mediating Macrophage Foam Cell Formation-Brief Report

Abstract

Background: Atherosclerosis is a progressive inflammatory disease in which macrophage foam cells play a central role in disease pathogenesis. SPA (surfactant protein A) is a lipid-associating protein involved with regulating macrophage function in various inflammatory diseases. However, the role of SPA in atherosclerosis and macrophage foam cell formation has not been investigated.

Methods: SPA expression was assessed in healthy and atherosclerotic human coronary arteries and the brachiocephalic arteries of wild-type or ApoE-deficient mice fed high-fat diets for 4 weeks. Hypercholesteremic wild-type and SPA-deficient mice fed a high-fat diet for 6 weeks were investigated for atherosclerotic lesions in vivo. In vitro experiments using RAW264.7 macrophages, primary resident peritoneal macrophages extracted from wild-type or SPA-deficient mice, and human monocyte-derived macrophages from the peripheral blood of healthy donors determined the functional effects of SPA in macrophage foam cell formation.

Results: SPA expression was increased in atherosclerotic lesions in humans and ApoE-deficient mice and in response to a proatherosclerotic stimulus in vitro. SPA deficiency reduced the lipid profiles induced by hypercholesterolemia, attenuated atherosclerosis, and reduced the number of lesion-associated macrophage foam cells. In vitro studies revealed that SPA deficiency reduced intracellular cholesterol accumulation and macrophage foam cell formation. Mechanistically, SPA deficiency dramatically downregulated the expression of scavenger receptor CD36 (cluster of differentiation antigen 36) cellular and lesional expression. Importantly, SPA also increased CD36 expression in human monocyte-derived macrophages.

Conclusions: Our results elucidate that SPA is a novel factor promoting atherosclerosis development. SPA enhances macrophage foam cell formation and atherosclerosis by increasing scavenger receptor CD36 expression, leading to increasing cellular OxLDL influx.

Keywords: atherosclerosis; cholesterol; lipoproteins, LDL; macrophages; receptors, scavenger.

Figure 1.. SPA deficiency attenuates atherosclerosis and macrophage (MΦ) foam cell formation in vivo.

(A-B) SPA expression in coronary arteries from human patients with cardiovascular disease (CVD) or healthy controls (Ctrl) detected by Western blotting (A) and normalized to alpha-Tubulin (α-Tub) (B). *p=0.0001 vs. Ctrl (n=6); One sample t test. (C) Co-immunostaining of SPA with macrophage marker CD68 in coronary arteries from human Ctrl or CVD patients. DAPI stains nuclei. Scale bar = 200μm. L: Lumen. White dashed boxes in the middle panels are enlarged in the lower panels. White arrows indicate the co-staining of CD68 with SPA. (D-E) SPA expression was induced in the brachiocephalic artery (BCA) of ApoE^−/− mice fed a high fat diet for 4 weeks as detected by Western blotting (D) and normalized to α-Tub (E). *p=0.0004 vs. WT (n=3-4); One sample t test. (F) Co-immunostaining of SPA with macrophage F4/80 in mouse BCA atherosclerotic lesions. DAPI stains nuclei. Scale bar = 100μm. L: Lumen. White dashed line outlines the developing lesion. Yellow dashed boxes in the middle panels are enlarged in the lower panels. White arrows indicate the co-staining of F4/80 with SPA. (G) WT and SPA^−/− mice were injected with PCSK9-AAV followed by high fat diet feeding for 6 weeks. Atherosclerotic lesions in serial BCA sections were observed by H&E staining. Scale = 100μm. (H) Quantification of the total plaque area in serial sections from WT or SPA^−/− mice. Each dot represents the quantification from one animal. *p=0.029 vs. WT, for each individual location (n=4); Nonparametric Mann Whitney test. (I) Representative images of foamy macrophages within BCA atherosclerotic lesions stained with BODIPY and F4/80. DAPI stains nuclei. Scale bar = 100μm. Dashed white boxes in the top panels for each group are enlarged in the lower panels. Yellow dashed lines show the boundary between lesion and media. (J) Quantification of the macrophage foam cells (BODIPY+F4/80+) in atherosclerotic lesions (number/per section). Each dot represents the quantification from one animal. *p=0.0079 vs. WT (n=5); Nonparametric Mann Whitney test.

Figure 2.. SPA deficiency mitigates macrophage (MΦ) foam cell formation through inhibiting CD36 expression.

(A) Representative images of wild-type (WT) primary resident peritoneal MΦs (pMΦs) treated with vehicle (Ctrl) or OxLDL for 24 h and stained with anti-SPA antibody. DAPI stains nuclei. Scale bar = 5μm. White arrow indicates the increased SPA expression. (B) SPA mRNA expression in WT pMΦs treated with Ctrl or OxLDL for 24 h, as detected by qRT-PCR and normalized to CYP. *p=0.0012 vs. Ctrl (n=9); One sample t test. (C) Representative immunostaining of SPA in WT or SPA^−/− pMΦs maintained in normal culture condition, confirming SPA deficiency in SPA^−/− pMΦs. DAPI stains nuclei. Scale bar = 5μm. (D) Biochemical lipid analyses quantifying non-esterified fatty acids (NEFA) in WT or SPA^−/− pMΦs treated with Ctrl or OxLDL for 24 h. WT Ctrl vs. WT OxLDL (*p<0.0001); SPA^−/− Ctrl vs. WT OxLDL (^#p<0.0001); SPA^−/− Ctrl vs. SPA^−/− OxLDL (^#p=0.047); and WT OxLDL vs. SPA^−/− OxLDL (^p <0.0001); n=6; 2-way ANOVA followed by Tukey’s multiple comparisons test. (E) Biochemical lipid analyses quantifying total cholesterol contents in WT or SPA^−/− pMΦs treated with Ctrl or OxLDL for 24 h. WT Ctrl vs. SPA^−/− Ctrl (*p<0.0001); WT Ctrl vs. WT OxLDL (*p=0.0005); WT Ctrl vs. SPA^−/− OxLDL (*p=0.0024); SPA^−/− Ctrl vs. WT OxLDL (^#p<0.0001); SPA^−/− Ctrl vs. SPA^−/− OxLDL (^#p=0.046); and WT OxLDL vs. SPA^−/− OxLDL (^p<0.0001); n=6; 2-way ANOVA followed by Tukey’s multiple comparisons test. (F) OxLDL uptake by WT and SPA^−/− pMΦs, as shown by the presence of Dil-conjugated OxLDL (Dil-OxLDL). DAPI stains nuclei. Scale bar=5μm. (G) Quantification of OxLDL uptake by measuring the fluorescent signal intensity of Dil-OxLDL. *p=0.0001 vs. WT pMΦs; n=36 for WT and n=40 for SPA^−/− groups; Mann Whitney test. (H) Quantification of foam cell formation as % cells containing Dil-conjugated OxLDL relative to the total MΦ number. *p=0.0001 vs. WT (n=9); Unpaired two-tailed t test with Welch's correction. (I) Representative immunostaining of CD36 expression in WT or SPA−_/− pMΦs treated with Ctrl or OxLDL for 24 h. DAPI stains nuclei. Scale bar=5μm. (J) Quantification of CD36 expression by measuring the green, fluorescent signal intensity. WT Ctrl vs. WT OxLDL (*p<0.0001); WT Ctrl vs. SPA^−/− OxLDL (*p<0.0001); SPA^−/− Ctrl vs. WT OxLDL (^#p<0.0001); SPA^−/− Ctrl vs. SPA^−/− OxLDL (^#p<0.0001); WT OxLDL vs. SPA^−/− OxLDL (^p<0.0001); n=60 for WT Ctrl, n=67 for WT OxLDL, n=62 for SPA^−/− Ctrl, and n=49 for SPA^−/− OxLDL; 2-way ANOVA followed by Tukey’s multiple comparisons test. (K) CD36 mRNA expression in WT or SPA^−/− pMΦs treated with Ctrl or OxLDL for 24 h, as detected by qRT-PCR and normalized to CYP. WT Ctrl vs. SPA^−/− Ctrl (*p=0.032); WT Ctrl vs. WT OxLDL (*p<0.0001); WT Ctrl vs. SPA^−/− OxLDL (*p=0.0019); SPA^−/− Ctrl vs. WT OxLDL (^#p<0.0001); and WT OxLDL vs. SPA^−/− OxLDL (^p<0.0001); n=7-8; 2-way ANOVA followed by Tukey’s multiple comparisons test. (L) Representative immunostaining of CD36 expression in lesions in brachiocephalic arteries of WT and SPA^−/− atherosclerotic mice (n=3). DAPI stains nuclei. Scale bar=100μm. (M) Representative immunostaining of CD36 expression in human monocyte-derived-MΦs treated with vehicle (Ctrl or SPA for 24 h. DAPI stains nuclei. Scale bar=5μm. (N) Quantification of CD36 expression by measuring the green, fluorescent intensity. *p<0.0001 vs Ctrl; n=97 for Ctrl and n=95 for SPA groups; Ordinary one-way ANOVA followed by Tukey’s multiple comparisons test. (O) CD36 mRNA expression in human monocyte-derived-MΦs treated with Ctrl or SPA for 8 h, as detected by qRT-PCR and normalized to CYP expression. *p<0.0001 (n=18); One sample t test. For all data, each dot represents the intensity of a single cell, at least 3 independent experiments were conducted with at least 2 technical repeats. n indicates sample sizes in different experiments, and values are not averaged.