Macrophage adipose triglyceride lipase deficiency attenuates atherosclerotic lesion development in low-density lipoprotein receptor knockout mice

Abstract

Objective: The consequences of macrophage triglyceride (TG) accumulation on atherosclerosis have not been studied in detail so far. Adipose triglyceride lipase (ATGL) is the rate-limiting enzyme for the initial step in TG hydrolysis. Because ATGL knockout (KO) mice exhibit massive TG accumulation in macrophages, we used ATGL KO mice to study the effects of macrophage TG accumulation on atherogenesis.

Methods and results: Low-density lipoprotein receptor (LDLr) KO mice were transplanted with bone marrow from ATGL KO (ATGL KO→LDLr KO) or wild-type (WT→LDLr KO) mice and challenged with a Western-type diet for 9 weeks. Despite TG accumulation in ATGL KO macrophages, atherosclerosis in ATGL KO→LDLr KO mice was 43% reduced associated with decreased plasma monocyte chemoattractant protein-1 (MCP-1) and macrophage interleukin-6 concentrations. This coincided with a reduced amount of macrophages, possibly because of a 39% increase in intraplaque apoptosis and a decreased migratory capacity of ATGL KO macrophages. The reduced number of white blood cells might be due to a 36% decreased Lin(-)Sca-1(+)cKit(+) hematopoietic stem cell population.

Conclusions: We conclude that the attenuation of atherogenesis in ATGL KO→LDLr KO mice is due to decreased infiltration of less inflammatory macrophages into the arterial wall and increased macrophage apoptosis.

Figure 1

ATGL deficiency induces lipid droplet accumulation in macrophages. A, Electron micrographs (scale bar, 2 μm) and lipid parameters of isolated peritoneal macrophages. B, BMDM from transplanted mice. Images show representative Oil Red O–stained BMDM. Magnification, ×40. ***P<0.001.

Figure 2

Decreased lesion formation and increased apoptosis in aortic root sections of ATGL KO→LDLr KO mice. Representative slides were stained with Oil Red O, MoMa-2, Masson’s trichrome, and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) for the detection of lipids, macrophages, collagen, and apoptosis, respectively. Arrows indicate apoptotic nuclei. Oil Red O and lesion composition data represent the means of 10 or 5 aortic root sections of n≥9 or n≥5 animals±SEM, respectively. Magnification, ×5 (apoptosis: ×40). *P<0.05; ***P<0.001.

Figure 3

Reduced WBC and MCP-1 concentration in ATGL KO→LDLr KO mice. A, Macrophage migration assays were performed using transwell plates. Values represent the means of n=3±SEM. WBC (B), neutrophils (C), monocytes (D), and lymphocytes (E) in plasma were determined with a hematology analyzer. F, MCP-1 concentrations were determined by ELISA. Values represent the means of n≥9±SEM. *P<0.05; **P<0.01.

Figure 4

Decreased IL-6 production in ATGL KO macrophages. A, PARP protein expression in peritoneal macrophages of ATGL KO and WT mice. B, IL-6 concentrations were determined by ELISA in supernatants of peritoneal macrophages. Values represent the means of n≥3±SEM. *P<0.05; **P<0.01.

Figure 5

Decreased LSK population in ATGL KO→LDLr KO mice. Dot plots show representative examples of the LSK population within the bone marrow (Lin⁻ cells, double positive for anti-LY6A/E [Sca-1] and anti-CD117 [cKit]). The right panel shows the percentage of LSK cells within the Lin⁻ population. Values represent the means of n=5±SEM. *P<0.05.