Lack of myeloid Fatp1 increases atherosclerotic lesion size in Ldlr-/- mice

Abstract

Background and aims: Altered metabolism is an important regulator of macrophage (MΦ) phenotype, which contributes to inflammatory diseases such as atherosclerosis. Broadly, pro-inflammatory, classically-activated MΦs (CAM) are glycolytic while alternatively-activated MΦs (AAM) oxidize fatty acids, although overlap exists. We previously demonstrated that MΦ fatty acid transport protein 1 (FATP1, Slc27a1) was necessary to maintain the oxidative and anti-inflammatory AAM phenotype in vivo in a model of diet-induced obesity. The aim of this study was to examine how MΦ metabolic reprogramming through FATP1 ablation affects the process of atherogenesis. We hypothesized that FATP1 limits MΦ-mediated inflammation during atherogenesis. Thus, mice lacking MΦ Fatp1 would display elevated formation of atherosclerotic lesions in a mouse model lacking the low-density lipoprotein (LDL) receptor (Ldlr^-/-).

Methods: We transplanted bone marrow collected from Fatp1^+/+ or Fatp1^-/- mice into Ldlr^-/- mice and fed chimeric mice a Western diet for 12 weeks. Body weight, blood glucose, and plasma lipids were measured. Aortic sinus and aorta lesions were quantified. Atherosclerotic plaque composition, oxidative stress, and inflammation were analyzed histologically.

Results: Compared to Fatp1^+/+Ldlr^-/- mice, Fatp1^-/-Ldlr^-/- mice exhibited significantly larger lesion area and elevated oxidative stress and inflammation in the atherosclerotic plaque. Macrophage and smooth muscle cell content did not differ by Fatp1 genotype. There were no significant systemic alterations in LDL, high-density lipoprotein (HDL), total cholesterol, or triacylglyceride, suggesting that the effect was local to the cells of the vessel microenvironment in a Fatp1-dependent manner.

Conclusions: MΦ Fatp1 limits atherogenesis and may be a viable target to metabolically reprogram MΦs.

Keywords: FATP1; Fatty acid transport protein; LDL receptor; Macrophage; Metabolic reprogram.

Fig. 1. Fatp1^−/−Ldlr^−/− mice displayed increased atherosclerotic lesion size compared to Fatp1^+/+Ldlr^−/− mice as determined by en face analysis

(A) Fatp1^+/+ Ldlr^−/− or Fatp1^−/−Ldlr^−/− aortas were isolated and stained with Oil red O. Representative images are shown. (B) Atherosclerotic lesion size was quantified using Image J software. Data are presented as means ± SEM, n =9. **p=0.001.

Fig. 2. Deletion of Fatp1 increased atherosclerotic lesions in the aorta sinus but not necrosis or collagen deposition

(A) 20× representative photomicrographs of Oil Red O staining for Fatp1^+/+Ldlr^−/− or Fatp1^−/−Ldlr^−/− aortic sinus. (B) Atherosclerotic lesion size was quantified using Aperio ImageScope Software. (C) 20× representative photomicrographs of Masson’s trichrome staining for Fatp1^+/+ Ldlr^−/− or Fatp1^−/−Ldlr^−/− aortic sinus. (D) %Necrotic area/total area in lesions was quantified using Aperio. (E) Collagen was quantified. (F) % Subendothelial cell numbers/total area in subendothelial space were quantified. Data are presented as means ± SEM, n = 8 or 9. *p=0.015

Fig. 3. Fatp1^−/−Ldlr^−/− mice did not display alterations in plasma total cholesterol, LDL cholesterol, HDL cholesterol, and triacylglycerol concentrations compared to Fatp1^+/+Ldlr^−/− mice

(A) Total cholesterol. (B) LDL cholesterol. (C) HDL cholesterol and (D) triacylglyceride. Data are presented as means ± SEM, n = 18 or 16.

Fig. 4. Deletion of Fatp1 increased atherosclerotic plaque oxidative stress without changing plaque cell composition

(A) 200 µm representative images of immunostaining macrophage marker MOMA2. (B) Quantification using OD × % MOMA2 positive staining. (C) 200 µm representative images of immunostaining SMC marker α-SMA. (D) Quantification using OD × %α-SMA positive staining. (E) 200 µm representative images of immunostaining oxidative stress marker 4HNE. (F) Quantification using OD × %4HNE positive staining. *p=0.013. (G) 200 µm representative images of immunostaining inflammation marker IL6. (H) Quantification using OD × %IL6 positive staining. *p=0.014. Data are presented as means ± SEM, n = 7 or 9.