Replacing Saturated Fat With Unsaturated Fat in Western Diet Reduces Foamy Monocytes and Atherosclerosis in Male Ldlr-/- Mice

Abstract

Objective: A Mediterranean diet supplemented with olive oil and nuts prevents cardiovascular disease in clinical studies, but the underlying mechanisms are incompletely understood. We investigated whether the preventive effect of the diet could be due to inhibition of atherosclerosis and foamy monocyte formation in Ldlr^-/- mice fed with a diet in which milkfat in a Western diet (WD) was replaced with extra-virgin olive oil and nuts (EVOND). Approach and Results: Ldlr^-/- mice were fed EVOND or a Western diet for 3 (or 6) months. Compared with the Western diet, EVOND decreased triglyceride and cholesterol levels but increased unsaturated fatty acid concentrations in plasma. EVOND also lowered intracellular lipid accumulation in circulating monocytes, indicating less formation of foamy monocytes, compared with the Western diet. In addition, compared with the Western diet, EVOND reduced monocyte expression of inflammatory cytokines, CD36, and CD11c, with decreased monocyte uptake of oxLDL (oxidized LDL [low-density lipoprotein]) ex vivo and reduced CD11c⁺ foamy monocyte firm arrest on vascular cell adhesion molecule-1 and E-selectin-coated slides in an ex vivo shear flow assay. Along with these changes, EVOND compared with the Western diet reduced the number of CD11c⁺ macrophages in atherosclerotic lesions and lowered atherosclerotic lesion area of the whole aorta and aortic sinus.

Conclusions: A diet enriched in extra-virgin olive oil and nuts, compared with a Western diet high in saturated fat, lowered plasma cholesterol and triglyceride levels, inhibited foamy monocyte formation, inflammation, and adhesion, and reduced atherosclerosis in Ldlr^-/- mice.

Keywords: atherosclerosis; cardiovascular diseases; cholesterol; cytokines; monocyte.

Figure 1.

In low-density lipoprotein receptor–knockout (Ldlr^–/–) mice, extra-virgin olive oil and nuts diet (EVOND) improved lipid profiles compared to western diet (WD). Eight-week-old male Ldlr^–/– mice were fed normal laboratory diet (ND), WD, or EVOND for 3 months. A, Body weight (n=12–20/group) and average food intake (n=5/group). B. Liver and epididymal fat pad weight (n=12–20/group). C, Fasting plasma total cholesterol and triglyceride (TG) levels (n=12–20/group). D, Plasma lipoprotein profile from mice on WD or EVOND showing cholesterol and TG distribution in each lipoprotein fraction (n=7/group). E, Hepatic TG and cholesterol content (n=9–13/group). F, mRNA levels of lipogenic genes in the liver (n=9–12/group). Data are presented as mean±SEM. *p<0.05, **p<0.01, ***p<0.001; ns, not significant.

Figure 2.

Plasma fatty acid (FA) profiles showed higher unsaturated fatty acid (UFA) and lower saturated fatty acid (SFA) concentrations in mice on EVOND than on WD. A, Concentrations of each SFA and UFA in total hydrolyzed FA profiles or nonesterified fatty acid (NEFA) profiles. B, Percentages of each SFA and UFA in total hydrolyzed FA profiles or NEFA profiles. C, UFA/SFA ratio in total hydrolyzed FA profiles or NEFA profiles. Data are shown as mean±SEM (n=9–12/group); *p<0.05, **p<0.01, ***p<0.001.

Figure 3.

EVOND compared to WD reduced foamy monocyte formation and inflammation in Ldlr^–/– mice. A, Monocyte frequency in total leukocytes of mice on ND, WD, and EVOND (n=12–20/group). B, Side scatter (SSC) value and Nile red mean fluorescence intensity (MFI) of circulating monocytes of mice on different diets (3 months on diets for Nile red staining). C, Representative fluorescence-activated cell sorter (FACS) examples showing foamy monocytes in blood of Ldlr^–/– mice on different diets (left panel). Monocytes (CD115⁺) were divided into two subsets based on CD36. Elevations in SSC indicated lipid accumulation and foamy monocyte formation; quantification of SSC values of CD36^– and CD36⁺ monocytes in Ldlr^–/– mice on diets (n=9–18/group; right panel). D, CD11c expression on CD36^– and CD36⁺ monocytes in Ldlr^–/– mice on diets (n=9–18/group). E, Expression of TNFα and IL-1β in monocytes of Ldlr^–/– mice on diets (n=4/group). Data are shown as mean±SEM. *p<0.05, **p<0.01, ***p<0.001.

Figure 4.

EVOND reduced monocyte CD36 expression and oxidized LDL (oxLDL) uptake. A, SSC value of monocytes (from ND-fed Ldlr^–/– mice) after incubation with triglyceride-rich lipoprotein (TGRL) fraction from mice on WD or EVOND (n=6/group). B, Expression level of CD36 on circulating monocytes from mice on different diets (left panel; n=9–15 mice/group) or on monocytes from ND-fed Ldlr^–/– mice after incubation with TGRL fraction from mice on WD or EVOND (right panel; n=6/group). C, Monocyte uptake of DiI-oxLDL. Total leukocytes from Ldlr^–/– mice on WD or EVOND were incubated ex vivo with DiI-oxLDL in the absence or presence of anti-mouse CD36 antibody for 1 h, and DiI signals representing monocyte uptake of oxLDL were examined by FACS (n=4/group). Data are shown as mean±SEM. *p<0.05, ***p<0.001.

Figure 5.

Reduced on-chip adhesion of foamy monocytes from mice on EVOND vs. WD. Circulating monocytes were stained with CD115 and CD11c and perfused through VCAM-1– and E-selectin–coated chip. The number of arrested CD11c⁺ monocytes were counted and normalized by total infused monocyte number. A, Frequency of CD11c⁺ monocytes arrested on chips in the total infused monocyte. Data are shown as mean±SEM, *p<0.05, n=4/group. B, Representative images showing arrested cells on chips under flow with FITC-anti-CD11c (Green), PE-anti-CD115 (Red), and DAPI (Blue) staining. See “Ex vivo micro–flow adhesion assay” under Materials and Methods for experimental procedures. Foamy monocytes marked with white arrows were identified by CD115 and CD11c double staining.

Figure 6.

EVOND reduced atherosclerosis compared to WD in Ldlr^–/– mice on diets for 3 months. A, Representative en face oil red O staining of whole aorta and aortic sinus (10x original magnification). B, Statistics of plaque area of whole aorta (upper panel) and aortic sinus (lower panel) (n=8/group). C, Representative immunofluorescence staining of Mac3 (green) and CD11c (red), with DAPI staining (blue) for nuclei, in aortic sinus lesions. D, CD11c fluorescent intensity (relative fluorescent unit [RFU]) in plaque of mice from WD and EVOND groups (n=6/group). Data are shown as mean±SEM. *p<0.05, **p<0.01.