Perilipin1 deficiency in whole body or bone marrow-derived cells attenuates lesions in atherosclerosis-prone mice

Abstract

Aims: The objective of this study is to determine the role of perilipin 1 (Plin1) in whole body or bone marrow-derived cells on atherogenesis.

Methods and results: Accumulated evidence have indicated the role of Plin1 in atherosclerosis, however, these findings are controversial. In this study, we showed that Plin1 was assembled and colocalized with CD68 in macrophages in atherosclerotic plaques of ApoE-/- mice. We further found 39% reduction of plaque size in the aortic roots of Plin1 and ApoE double knockout (Plin1-/-ApoE-/-) females compared with ApoE-/- female littermates. In order to verify whether this reduction was macrophage-specific, the bone marrow cells from wild-type or Plin1 deficient mice (Plin1-/-) were transplanted into LDL receptor deficient mice (LDLR-/-). Mice receiving Plin1-/- bone marrow cells showed also 49% reduction in aortic atherosclerotic lesions compared with LDLR-/- mice received wild-type bone marrow cells. In vitro experiments showed that Plin1-/- macrophages had decreased protein expression of CD36 translocase and an enhanced cholesterol ester hydrolysis upon aggregated-LDL loading, with unaltered expression of many other regulators of cholesterol metabolism, such as cellular lipases, and Plin2 and 3. Given the fundamental role of Plin1 in protecting LD lipids from lipase hydrolysis, it is reasonably speculated that the assembly of Plin1 in microphages might function to reduce lipolysis and hence increase lipid retention in ApoE-/- plaques, but this pro-atherosclerotic property would be abrogated on inactivation of Plin1.

Conclusion: Plin1 deficiency in bone marrow-derived cells may be responsible for reduced atherosclerotic lesions in the mice.

Fig 1. Plin1 expresses in atherosclerotic lesions.

(A) Immunoblotting analysis of Plin1 expression in perivascular adipose tissue and in peritoneal macrophages isolated from Plin1-/- and wild-type mice. Incubation with modified LDL increases Plin1 expression in the macrophages. Anti-Plin1 antiserum from (Abcam, #ab3526) recognizes the C-terminal amino-acid residues of Plin1. (B) Unstained frozen section of arotic root of ApoE-/- mice. (C) The frozen section of arotic root in ApoE-/- mice was immunostained (Green) with the primary antibody against CD68, a macrophagemarker. (D) Immunostaining of Plin1 (Red). (E and F) Merged image shows colocalization of Plin1 and CD68 in atheroma. Boxed area in panel E (a-c) were magnified to show colocalization of Plin1 and CD68 (F, a-c) in macrophages in atherosclerotic lesion in ApoE-/- mice.

Fig 2. Lipid content is decreased in peritoneal macrophages and foam cells derived from Plin1-/- mice.

Peritoneal macrophages from Plin1+/+ and Plin1-/- mice were incubated for 48 hours in the absence or presence of 50 μg/ml aggLDL. (A and B) The contents of free cholesterol (FC), cholesterol ester (CE) and triglyceride (TG) were measured. n = 4,*P < 0.05. (C) Nile red staining of LDs in peritoneal macrophages from Plin1+/+ and Plin1-/- mice with aggLDL (50μg/ml) (n = 6).

Fig 3. Ablation of Plin1 restricts As in female ApoE-/- mice.

(A and B) The mean lesion area (±SEM) in the aortic roots of male (A) and female (B) ApoE-/- and Plin1-/-ApoE-/- mice, *** p < 0.001. (C and D) Representative photographs of aortic roots from male (C) and female (D) ApoE-/- and Plin1-/-ApoE-/- mice stained with oil red O. (E) Representative photographs of the aorta en face from female ApoE-/- and Plin1-/-ApoE-/- mice. (F) Quantification of atheroma of total aortic lesion area. n = 15. (G) Regional analysis of lesion distribution in the aorta.

Fig 4. The bone marrow cells from male Plin1-/- mice transplanted to female LDLR-/- mice reduce atherosclerotic lesion.

(A) Representative photographs of aortic roots from LDLR-/- mice received bone marrow cells from Plin1+/+ and Plin1-/- mice respectively. (B) The mean lesion area (±SEM) in the aortic roots in mice transplanted with bone marrow cells from Plin1+/+ or Plin1-/- mice. ** p < 0.01 for Plin-/- vs. Plin+/+.

Fig 5. The cholesterol metabolism in macrophages.

(A) Immunoblotting of CD36 and ABCA1 in peritoneal macrophages and foam cells and (B) The density of the protein band was quantitated. (C) Immunoblotting of HSL and ATGL in peritoneal macrophages and foam cells and (D) the density of the protein bands was quantitated. (E) The rate of cholesterol ester hydrolysis in peritoneal macrophages from Plin1+/+ and Plin1-/- mice. (F) Time-course of cholesterol efflux to ApoA-I in peritoneal macrophages from Plin1+/+ and Plin1-/- mice. * p < 0.05, ** p < 0.01 for Plin-/- vs. Plin+/+.

Fig 6. The expressions of Plin2 and Plin3 in Plin1+/+ and Plin1-/- macrophages and foam cells.

(A) The expression of Plin2 protein and (B) Plin2 mRNA in the macrophages and foam cells of Plin1+/+ and Plin1-/- mice. (C) Protein band density of Plin2 shown in panel A. (D) Immunoblotting of Plin3 from the macrophages and foam cells of Plin1+/+ and Plin1-/- mice. (E) Protein band density of Plin3 shown in panel D. ** p < 0.01 for Plin-/- vs. Plin+/+.