Dietary fish oil supplementation prevents SCD1 ASO-driven atherosclerosis in LDLr^−/−, ApoB^100/100 mice. Starting at six weeks of age, male mice were fed diets containing 0.1% (w/w) cholesterol enriched in either saturated (Sat.) or long chain ω-3 fatty acids (Fish) for 20 weeks in conjunction with biweekly injections (25 mg/kg) of a non-targeting control ASO ■ or SCD1 ASO □. A. En face morphometric analysis of total aortic lesion area. Data shown in panel A represent the mean ± SEM from 6 mice per group. GLC analysis of aortic cholesteryl ester (B) and free cholesterol (C) was determined. Data in panels B and C represents the mean ± SEM from 8–15 mice per group. Values not sharing a common superscript differ significantly (p<0.05). D. Representative Verhoeff-van Giesen stained sections of proximal aortae from mice treated with diet and ASO for 20 weeks. E. Representative hematoxylin and eosin stained sections of proximal aortae from mice treated with diet and ASO for 20 weeks.

Dietary fish oil supplementation prevents SCD1 ASO-driven TLR4 hypersensitivity in macrophages. Starting at six weeks of age, male mice were fed diets containing 0.1% (w/w) cholesterol enriched in either saturated (Sat.) or long chain ω-3 fatty acids (Fish) for 6 weeks in conjunction with biweekly injections (25 mg/kg) of a non-targeting control ASO (Cont.) or SCD1 ASO (SCD1). Following six weeks of diet and ASO treatment, freshly isolated thioglycollate-elicited macrophages were pooled (n=5–7 mice per pool) and cultured as described in materials and methods. A. Phospholipid fatty acid (PL-FA) composition [16:1 to 16:0 ratio and % of total FA as long chain ω-3 (eicosapentaenoic and docosahexaenoic) fatty acids] of freshly isolated (2h culture) macrophages. B. Macrophage cholesterol efflux to apoAI (10 μg/ml) or high density lipoprotein (HDL, 50 μg/ml). C. TLR4-driven gene expression: Freshly isolated macrophages were treated with vehicle or 10ng/ml Kdo₂-Lipid A (TLR4 agonist) for 6 hours, and mRNA levels were measured for interleukins 1 beta (IL-1β), 6 (IL-6), and 12p40 (IL-12p40), tumor necrosis factor alpha (TNFα), C-X-C motif ligand 10 (IP-10), and gluocorticoid attenuated response gene 16 (Garg-16), and normalized to GAPDH. Data shown in panel C are expressed as the mean relative mRNA expression, where all values were normalized to the levels in the Sat. – Control vehicle treated group, and the standard error of the mean (SEM) was calculated from triplicate plates for each pool. D. TLR4-driven cytokine secretion: Freshly isolated macrophages were treated with vehicle or 10ng/ml Kdo₂-Lipid A (TLR4 agonist) for 6 hours, and cytokine levels (pg/ml) were measured for IL-1β, IL-6, IL-12p40, TNFα, Chemokine C-X-C motif ligand 1 (KC), Regulated upon Activation Normal T-cell Expressed and Secreted (RANTES,) monocyte chemotactic protein 1 (MCP-1), and macrophage inflammatory protein 1 alpha (MIP-1α) using conditioned media. Data shown in panel D the mean ± SEM from triplicate plates for each pool. N.D. = values not detectable

Combined therapy of dietary fish oil and SCD1 ASO synergistically improves hyperlipidemia in LDLr^−/−, ApoB^100/100 mice. Starting at six weeks of age, male mice were fed diets containing 0.1% (w/w) cholesterol enriched in either saturated (Sat.) or long chain ω-3 fatty acids (Fish) for 20 weeks in conjunction with biweekly injections (25 mg/kg) of a non-targeting control ASO (Control) or SCD1 ASO (SCD1). Plasma samples were collected at baseline (6 weeks of age), and after 4, 8, or 20 weeks of diet and ASO treatment. Plasma triglycerides (A) and total plasma cholesterol (TPC) (B) were measured enzymatically. Data shown in panels (A) and (B) represents the mean ± SEM from 5–8 mice per group, * = significantly different than the saturated diet fed control ASO treated group, within each time point (p<0.05). Panels C–E represents cholesterol levels in very-low-density lipoproteins (VLDLc), low-density lipoproteins (LDLc), and high-density lipoproteins (HDLc) in mice receiving dietary and ASO treatment for 20 weeks. Data shown in panel C–E represent the mean ± SEM from 6 mice per group, and values not sharing a common superscript differ significantly (p<0.05). F. Fatty acid (FA) composition [% of total FA as saturated or long chain ω-3 (eicosapentaenoic and docosahexaenoic) fatty acids] of LDL cholesteryl esters (LDL CE-FA). Data shown in panel (F) represents the mean ± SEM (n=5 per group), and values not sharing a common superscript differ significantly (p<0.05).