Mipu1 overexpression protects macrophages from oxLDL-induced foam cell formation and cell apoptosis

Abstract

Mipu1 (myocardial ischemic preconditioning upregulated protein 1) is a novel N-terminal Kruppel-associated box (KRAB)/C2H2 zinc finger superfamily protein, that displays a powerful effect in protecting H9c2 cells from oxidative stress-induced cell apoptosis. The present study aims to investigate the effect of Mipu1 overexpression on oxidized low-density lipoprotein (oxLDL)-induced foam cell formation, cell apoptosis, and its possible mechanisms. New Zealand healthy rabbits were used to establish atherosclerosis model, and serum levels of triglycerides, total cholesterol, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol were detected by an automatic biochemical analyzer. Sudan IV staining was used to detect atherosclerotic lesions. The RAW264.7 macrophage cell line was selected as the experimental material. Oil red O staining, high-performance liquid chromatography, and Dil-labeled lipoprotein were used to detect cholesterol accumulation qualitatively and quantitatively, respectively. Flow cytometry was used to determine cell apoptosis. Real-time quantitative polymerase chain reaction (PCR) was used to detect the mRNA expression of the main proteins that are associated with the transport of cholesterol, such as ABCA1, ABCG1, SR-BI, and CD36. Western blot analysis was used to detect the protein expression of Mipu1. There were atherosclerotic lesions in the high-fat diet group with Sudan IV staining. High-fat diet decreased Mipu1 expression and increased CD36 expression significantly at the 10th week compared with standard-diet rabbits. Mipu1 overexpression decreased oxLDL-induced cholesterol accumulation, oxLDL uptake, cell apoptosis, and cleaved caspase-3. Mipu1 overexpression inhibited the oxLDL-induced CD36 mRNA and protein expression, but it did not significantly inhibit the mRNA expression of ABCA1, ABCG1, and SR-BI. Mipu1 overexpression inhibits oxLDL-induced foam cell formation and cell apoptosis. Mipu1 overexpression reduces the lipid intake of macrophages and might be associated with the downregulation of CD36 expression in the presence of oxLDL.

FIG. 1.

Change of Mipu1 (myocardial ischemic preconditioning upregulated protein 1) and CD36 expression in high-fat diet induced atherosclerosis in rabbits. (A) Plasma levels of total triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) after 10 weeks of high-fat diet feeding. (B) Plaque quantification of thoracic aorta stained with Sudan IV. Protein expression of Mipu1 (C) and CD36 (D) in the aortic tissue (n=4 each), values are mean±SEM, *p<0.05 versus control group.

FIG. 2.

Mipu1 overexpression inhibits oxLDL-induced lipid accumulation in macrophages. (A) LDH release in oxidized low-density lipoprotein (oxLDL)-treated group (75 μg/mL of oxLDL for 24 h) was 23.4%±3.1% (relative to total LDH release), and control group (no oxLDL treatment) was 5.0%±0.7%. (B) Expression of Mipu1 protein in RAW264.7 cells transfected with pcDNA3.1-Mipu1 was analyzed by western blot. (C) RAW264.7-vector and RAW264.7-Mipu1 cells were treated with 75 μg/mL oxLDL for 24 h, and the extent of lipid loading was assessed by Oil red O staining. (D) Under the same conditions as in (C), the intracellular cholesterol content was measured through high-performance liquid chromatography. (E) Dil-oxLDL fluorescence intensity in cells incubated with Dil-labeled oxLDL (75 μg/mL) for 24 h. Data are presented as the mean±SEM of at least four independent experiments. ^#p<0.05 versus control group; *p<0.05 versus pcDNA3.1 group.

FIG. 3.

Mipu1 overexpression inhibited apoptosis of RAW264.7 cells. RAW264.7-vector and RAW264.7-Mipu1 cells were treated with 75 μg/mL oxLDL for 24 h. (A) FACS analysis with PE-Annexin-V staining revealed that Mipu1 overexpression inhibited apoptosis. (B) Caspase-3 activities were measured as described in methods. Data are presented as the mean±SEM of at least four independent experiments. *p<0.05 versus pcDNA3.1 group.

FIG. 4.

Mipu1 overexpression inhibited CD36 expression in the presence of oxLDL. RAW264.7-vector and RAW264.7-Mipu1 cells were incubated with 75 μg/mL oxLDL for 24 h and the amount of mRNA encoding ABCA1, ABCG1, SR-BI, and CD36 was measured by quantitative PCR (A–D) (data show mean fold change relative to untreated RAW264.7-vector±SEM from four independent experiments). (E) Protein expression of CD36 was detected by western blot after oxLDL treatment. ^#p<0.05 versus untreated group, ^$p<0.05 versus RAW264.7-vector group.