Inducible phospholipid transfer protein deficiency ameliorates atherosclerosis

Abstract

Background and aims: Atherosclerosis progression and regression studies are related to its prevention and treatment. Although we have gained extensive knowledge on germline phospholipid transfer protein (PLTP) deficiency, the effect of inducible PLTP deficiency in atherosclerosis remains unexplored.

Methods: We generated inducible PLTP (iPLTP)-knockout (KO) mice and measured their plasma lipid levels after feeding a normal chow or a Western-type diet. Adenovirus associated virus-proprotein convertase subtilisin/kexin type 9 (AAV-PCSK9) was used to induce hypercholesterolemia in the mice. Collars were placed around the common carotid arteries, and atherosclerosis progression and regression in the carotid arteries and aortic roots were evaluated.

Results: On a normal chow diet, iPLTP-KO mice exhibited decreased cholesterol, phospholipid, apoA-I, and apoB levels compared with control mice. Furthermore, the overall amount of high-density lipoprotein (HDL) particles was reduced in these mice, but this effect was more profound for larger HDL particles. On a Western-type diet, iPLTP-KO mice again exhibited reduced levels of all tested lipids, even though the basal lipid levels were increased. Additionally, these mice displayed significantly reduced atherosclerotic plaque sizes with increased plaque stability. Importantly, inducible PLTP deficiency significantly ameliorated atherosclerosis by reducing the size of established plaques and the number of macrophages in the plaques without causing lipid accumulation in the liver.

Conclusions: Induced PLTP deficiency in adult mice reduces plasma total cholesterol and triglycerides, prevents atherosclerosis progression, and promotes atherosclerosis regression. Thus, PLTP inhibition is a promising therapeutic approach for atherosclerosis.

Keywords: Atherosclerosis progression and regression; Inducible PLTP gene knockout mice; Lipoprotein metabolism; Stability of atherosclerotic plaque.

Fig. 1.. Plasma lipoprotein analysis of iPLTP-KO and control mice on chow diet.

(A) Plasma cholesterol, phospholipid, and triglyceride measurements in male and female iPLTP-KO and control mice. (B) Fast protein liquid chromatography analysis of plasma lipid distribution using pooled plasma samples from male and female iPLTP-KO or control mice. (C) Western blot of apoA-I, apoB (top band, apoB100; bottom band, apoB48), and apoE levels. (D) Native polyacrylamide gel electrophoresis for lipoprotein separation. Mouse and human plasma (50 μL) samples were pre-stained with Sudan Black and then run on our system (Materials and Methods). The two large HDLs (yellow arrows) not existing in human plasma were detected in mouse plasma samples. Inducible PLTP deficiency resulted in a new HDL particle formation (red arrow). Addition of rPLTP into iPLTP-KO mouse plasma resulted in the formation of human-like LDL and a preβ1-HDL. M-Con, male controls; M-KO, male iPLTP-KO mice; F-Con, female controls; F–KO, female iPLTP-KO mice. Values the are mean ± standard deviation (n = 5, *p < 0.01).

Fig. 2.. Plasma lipoprotein analysis of iPLTP-KO and control mice on the Western-type diet.

(A) Plasma cholesterol, phospholipid, and triglyceride measurements in male and female iPLTP-KO and control mice. (B) Fast protein liquid chromatography analysis of plasma lipid distribution using pooled plasma samples from male and female iPLTP-KO or control mice. (C) Western blot of apoA-I, apoB (top band, apoB100; bottom band, apoB48), and apoE levels. (D) VLDL production measurements of mice. Male iPLTP-KO and control mice fed the Western-type diet for 3 weeks. Mice were fasted for 16 h and then intraperitoneally injected with Poloxamer 407 (1 mg/g) to block VLDL clearance. Plasma triglyceride tri was measured at 0, 1, 2, and 4 hour. Con, controls; KO, PLTP KO mice. h, hour. Values are the mean ± standard deviation (n = 5–6), *p < 0.01).

Fig. 3.. Atherosclerosis progression in male iPLTP-KO and control mice.

We used the strategy described in Supplementary Figure IA. (A) Illustration of carotid collar surgery. (B) Collared carotid artery with atherosclerotic plaques (red arrows). (C) Hematoxylin and eosin staining of collared carotid artery, 2 and 4 weeks after carotid collar surgery. At 2 weeks, all control mice exhibited lesions (8/8), whereas iPLTP-KO mice did not (0/8). At 4 weeks, both mice exhibited lesions, but iPLTP-KO mice had smaller-sized lesions than controls. (D) Quantification of lesion size 4 weeks after surgery. (E) En face aortic plaque analysis after Oil Red O staining. (F) Quantification of whole-aortic lesion area (en face). Con, controls; KO, iPLTP-KO mice. Values are the mean ± standard deviation (n = 6, *p < 0.01).

Fig. 4.. Atherosclerotic plaque lipid, macrophage, collagen, and smooth muscle cell (SMC) staining and plaque stability analysis.

(A) Frozen sections were used. Lipids were stained with Oil Red O; macrophages were immunostained with rat anti-mouse-monocyte/macrophage antibody (MOMA2); collagen was stained with Masson’s trichrome; and SMCs were immunostained with rabbit anti-mouse oc-SMC antibody. (B) Staining quantification. (C) Vulnerability index calculation. Con, controls; KO, iPLTP-KO mice. Coll, collage; Mϕ, macrophage. Values are the mean ± standard deviation (n = 6–7, *p < 0.01).

Fig. 5.. Atherosclerosis regression in male iPLTP-KO and control mice.

We used the strategy described in Supplementary Fig. 1B. (A) Hematoxylin and eosin staining of collared carotid artery and aortic root with atherosclerotic plaques (lesion areas highlighted by blue lines). (B and C) Quantification of lesion area of collared carotid artery and aortic root. D. Whole aorta (en face) with atherosclerotic plaques stained with Oil Red O. (E) Quantification of whole-aortic lesion area (en face). (F) Frozen aortic root sections were immunostained with rat anti-mouse monocyte/macrophage antibody (MOMA-2). (G) Quantification of macrophages in carotid plaques. (H) Quantification of macrophages in aortic root plaques. Con, controls; KO, iPLTP-KO mice. Values are the mean ± standard deviation (n = 9–10, *p < 0.01).