Acyl-CoA:Diacylglycerol Acyltransferase 1 Expression Level in the Hematopoietic Compartment Impacts Inflammation in the Vascular Plaques of Atherosclerotic Mice

Abstract

The final step of triacylglycerol synthesis is catalyzed by acyl-CoA:diacylglycerol acyltransferases (DGATs). We have previously shown that ApoE-/-Dgat1-/- mice are protected from developing atherosclerosis in association with reduced foam cell formation. However, the role of DGAT1, specifically in myeloid and other hematopoietic cell types, in determining this protective phenotype is unknown. To address this question, we reconstituted the bone marrow of irradiated Ldlr-/-mice with that from wild-type (WT→ Ldlr-/-) and Dgat1-/-(Dgat1-/-→ Ldlr-/-) donor mice. We noted that DGAT1 in the hematopoietic compartment exerts a sex-specific effect on systemic cholesterol homeostasis. However, both male and female Dgat1-/-→ Ldlr-/-mice had higher circulating neutrophil and lower lymphocyte counts than control mice, suggestive of a classical inflammatory phenotype. Moreover, specifically examining the aortae of these mice revealed that Dgat1-/-→ Ldlr-/-mice have atherosclerotic plaques with increased macrophage content. This increase was coupled to a reduced plaque collagen content, leading to a reduced collagen-to-macrophage ratio. Together, these findings point to a difference in the inflammatory contribution to plaque composition between Dgat1-/-→ Ldlr-/-and control mice. By contrast, DGAT1 deficiency did not affect the transcriptional responses of cultured macrophages to lipoprotein treatment in vitro, suggesting that the alterations seen in the plaques of Dgat1-/-→ Ldlr-/-mice in vivo do not reflect a cell intrinsic effect of DGAT1 in macrophages. We conclude that although DGAT1 in the hematopoietic compartment does not impact the overall lipid content of atherosclerotic plaques, it exerts reciprocal effects on inflammation and fibrosis, two processes that control plaque vulnerability.

Fig 1. Dgat1^–/–→ Ldlr^–/–and WT→ Ldlr^–/–mice have comparable body weight gain and adiposity.

Weight gain of (A) female mice (n = 9) and (B) male mice (n = 10–11) fed a WTD. (C) Total body fat, (D) relative adiposity, (E) lean mass, (F) gonadal fat pad weights, and (G) liver weights of WT→ Ldlr^-/- and Dgat1^-/-→ Ldlr^-/- mice after 13 (n = 9 female mice, ♀) and 19 weeks (n = 10–11 male mice, ♂) of WTD, respectively. For all panels, data are presented as means ± SEM.

Fig 2. Altered circulating immune cell numbers in Dgat1^–/–→ Ldlr^–/–mice fed a WTD.

(A) Total leukocyte counts, and relative (B) monocyte, (C) eosinophil, (D) basophil, (E) lymphocyte, and (F) neutrophil counts in both WT→ Ldlr^–/–and Dgat1^–/–→ Ldlr^–/–after 13 weeks (females, ♀) and 19 weeks (males, ♂) of WTD feeding (n = 9 per group), showing reduced lymphocyte and increased neutrophil counts in male mice, with a similar trend in female mice. Data are presented as mean ± SEM. **, p ≤ 0.01; ***, p ≤ 0.001.

Fig 3. Comparable atherosclerotic plaque areas in the aortas of WT→ Ldlr^–/–and Dgat1^–/–→ Ldlr^–/–mice.

(A) Aortae stained en face with ORO after 13 and 19 weeks of WTD feeding. (B) Quantification of plaque size in the thoracic aortae and (C) aortic arches of WT→ Ldlr^–/–(black squares) and Dgat1^–/–→ Ldlr^–/–(white circles) mice fed a WTD as in A (n = 9 females, ♀, and 10–11 males, ♂). Horizontal lines represent mean values through data groupings.

Fig 4. Increased plaque inflammation in Dgat1^–/–→ Ldlr^–/–mice.

(A-C, left) Representative images of aortic root sections stained with ORO, MoMa-2, and Masson's Trichrome. (A-C, right) Quantification of these images to measure plaque size, macrophage content, and collagen deposition, respectively. (D-F) Integrated quantification of data from A-C to determine collagen-to-macrophage ratios, necrotic core per plaque area, and collagen-to-necrotic core ratios, respectively. (G) Representative images of Masson’s Trichrome-stained aortic root sections focused on plaque fibrotic caps, with quantification of minimal fibrotic cap thickness. All data represent means ± SEM of 12 aortic root sections for ORO and three aortic root sections in the area of maximal plaque size for MoMa-2- and Trichrome-staining per mouse after 13 (n = 9 females, ♀) and 19 weeks (n = 10–11 males, ♂) of WTD feeding. *, p < 0.05.

Fig 5. M₁ and M₂ gene expression in lipoprotein-treated WT and Dgat1^–/–macrophages.

qPCR analysis of control macrophages vs. those treated with 100 μg/ml VLDL, 100 μg/ml acLDL or 100 ng/ml LPS (positive control for M₁ activation). Shown are mRNA levels of (A) M₁ marker and (B) M₂ marker genes analyzed in duplicate. Data are means (n = 3–6) ± SEM. *, p < 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.