Specific Disruption of Abca1 Targeting Largely Mimics the Effects of miR-33 Knockout on Macrophage Cholesterol Efflux and Atherosclerotic Plaque Development - PubMed (original) (raw)
Specific Disruption of Abca1 Targeting Largely Mimics the Effects of miR-33 Knockout on Macrophage Cholesterol Efflux and Atherosclerotic Plaque Development
Nathan L Price et al. Circ Res. 2019.
Abstract
Rationale: Inhibition of miR-33 reduces atherosclerotic plaque burden, but miR-33 deficient mice are predisposed to the development of obesity and metabolic dysfunction. The proatherogenic effects of miR-33 are thought to be in large part because of its repression of macrophage cholesterol efflux, through targeting of Abca1 (ATP-binding cassette subfamily A member 1). However, targeting of other factors may also be required for the beneficial effects of miR-33, and currently available approaches have not allowed researchers to determine the specific impact of individual miRNA target interactions in vivo.
Objective: In this work, we sought to determine how specific disruption of Abca1 targeting by miR-33 impacts macrophage cholesterol efflux and atherosclerotic plaque formation in vivo.
Methods and results: We have generated a novel mouse model with specific point mutations in the miR-33 binding sites of the Abca1 3'untranslated region, which prevents targeting by miR-33. Abca1 binding site mutant ( Abca1BSM) mice had increased hepatic ABCA1 expression but did not show any differences in body weight or metabolic function after high fat diet feeding. Macrophages from Abca1BSM mice also had increased ABCA1 expression, as well as enhanced cholesterol efflux and reduced foam cell formation. Moreover, LDLR (low-density lipoprotein receptor) deficient animals transplanted with bone marrow from Abca1BSM mice had reduced atherosclerotic plaque formation, similar to mice transplanted with bone marrow from miR-33 knockout mice.
Conclusion: Although the more pronounced phenotype of miR-33 deficient animals suggests that other targets may also play an important role, our data clearly demonstrate that repression of ABCA1 is primarily responsible for the proatherogenic effects of miR-33. This work shows for the first time that disruption of a single miRNA/target interaction can be sufficient to mimic the effects of miRNA deficiency on complex physiological phenotypes in vivo and provides an approach by which to assess the impact of individual miRNA targets.
Keywords: atherosclerosis; binding sites; bone marrow; foam cells; obesity.
Figures
Figure 1.. Generation of Abca1 binding site mutant mice results in derepression of ABCA-1 in vivo.
A-B) CRISPR/Cas9 mediated mutation of the 3 binding sites (BS) for miR-33 (black bars) in the genomic region of Abca1 was performed to generate Abca1 BSM mice (mutated sequences in red). This was confirmed by direct sequencing A) and PCR amplification B) in DNA isolated from WT and Abca1 BSM mice. C-D) qPCR analysis of the expression of miR-33 normalized to U6 (n=5) C) and the mRNA expression of miR-33 target genes normalized to 18s (n=4–5) D) in liver samples isolated from WT and Abca1 BSM mice. E) Representative western blot analysis of miR-33 targets in liver samples isolated from WT and Abca1 BSM mice. Right panel shows the quantification of band densitometry values and are expressed in a.u. after correction for loading control vinculin (n=4). F) Body weight and fat mass after high fat diet (60% fat) feeding in WT, miR-33 −/−, and Abca1BSM mice (n=6–9 per group). G-H) Measurements of G) cholesterol and H) HDL-cholesterol in plasma of chow diet fed WT, miR-33 −/− and Abca1 BSM mice (n=7). I) Lipoprotein profile from FPLC fractionation of pooled plasma of WT, miR-33 −/− and Abca1 BSM mice on a chow diet. J-K) Measurements of J) cholesterol and K) HDL-cholesterol in plasma of HFD fed WT, miR-33 −/− and Abca1 BSM mice (n=7). L) Lipoprotein profile from FPLC fractionation of pooled plasma of WT, miR-33 −/− and Abca1 BSM mice on a HFD. All data are represented as the mean ± SEM. *P≤0.05 compared with WT mice under the same conditions.
Figure 2.. Macrophages from Abca1 BSM mice have increased ABCA1 expression, enhanced cholesterol efflux, and reduced foam cell formation.
A-B) Protein and mRNA expression of miR-33 target genes (Abca1, Abcg1, Hadhβ and Hmga2) from peritoneal macrophages isolated from WT and Abca1 BSM mice, untreated or stimulated with the LXR ligand TO901317 (TO90). A) Representative western blot analysis out of three with similar results. Relative protein levels were determined by band densitometry and are expressed in a.u after correction for loading control HSP90 (n=3). B) Relative mRNA expression levels normalized to 18S rRNA (n=4–5). C) Expression levels of miR-33 in PM from WT and Abca1 BSM mice normalized to U6 (n=7–8). D-E) Cholesterol efflux to APOA1 D) and HDL E) in peritoneal macrophages isolated from WT, miR-33 −/− and Abca1 BSM mice, untreated or stimulated with TO90 (n=3). F) [3H]-cholesterol in plasma (left) fecal bile acids (middle) and total fecal cholesterol (right) from WT mice injected intraperitoneally with [3H]-cholesterol-loaded bone marrow derived macrophages from WT and Abca1 BSM mice (n=11–14). G) Representative high magnification images of Oil red O (ORO) staining in foam cells generated in vivo from Ldlr−/− mice transplanted with bone marrow from WT and Abca1 BSM mice after feeding a Western diet (1.25% cholesterol). Quantification of the ORO-positive area per cell (left) and integrated optical density (IOD) (Right) is shown in the right panel. For each animal quantification was performed in at least six low magnification images from different fields (n=4) Scale bars, 100μm. All data are represented as the mean ± SEM. *P≤0.05 compared with macrophages from WT mice under the same conditions.
Figure 3.. Expression of inflammatory genes and efferocytosis capacity in macrophages from Abca1 BSM mice.
A) mRNA expression of a number of inflammatory genes in foam cells generated in vivo from Ldlr −/− mice transplanted with bone marrow from WT and Abca1 BSM mice after feeding a Western diet (1.25% cholesterol) (n=3–4). B) Representative images of the in vitro engulfment of CellTracker Red labeled apoptotic Jurkat cells by peritoneal macrophages isolated from WT and Abca1 BSM mice. Right, quantification of phagocytic index, which is the number of apoptotic cells (AC, red) ingested in 1h per F4/80-positive macrophage (green) x 100. Quantification performed in at least five images from different fields (n=3). Scale bars, 70μm. All data are represented as the mean ± SEM. *P≤0.05 compared with macrophages from WT mice under the same conditions.
Figure 4.. Disruption of Abca1 binding by miR-33 is sufficient to reduce atherosclerotic plaque size.
Representative histological analysis of cross-sections of the aortic sinus stained from Ldlr −/− mice transplanted with WT, miR-33 −/− and Abca1 BSM bone marrow after 12 months of western diet (1.25% cholesterol) feeding. A-B) Representative images of H&E stained sections with quantification of total plaque area A) and necrotic core size B). Dashed lines show the boundary of the developing necrotic core (NC) (n=9–14 animals). C) Representative images of CD68 stained sections with quantification of percent CD68 positive area (n=5–8). All data are represented as the mean ± SEM. *P≤0.05 compared with mice reconstituted with WT bone marrow. Each dot is based on quantification of ≥ 9 sections from an individual animal. Scale bars, 200μm.
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