Omega-6 polyunsaturated fatty acids prevent atherosclerosis development in LDLr-KO mice, in spite of displaying a pro-inflammatory profile similar to.

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Omega-6 polyunsaturated fatty acids prevent atherosclerosis development in LDLr-KO mice, in spite of displaying a pro-inflammatory profile similar to trans fatty acids  Roberta M. Machado, Edna R. Nakandakare, Eder C.R. Quintao, Patricia M. Cazita, Marcia K. Koike, Valeria S. Nunes, Fabiana D. Ferreira, Milessa S. Afonso, Renata P.A. Bombo, Adriana Machado-Lima, Francisco G. Soriano, Sergio Catanozi, Ana Maria Lottenberg  Atherosclerosis  Volume 224, Issue 1, Pages 66-74 (September 2012) DOI: 10.1016/j.atherosclerosis.2012.06.059 Copyright © 2012 Elsevier Ireland Ltd Terms and Conditions

Fig. 1 Dietary fatty acids and the development of atherosclerosis. The atherosclerotic lesion area was determined in the aortas isolated from LDLr-KO mice fed a high fat diet for 16 weeks. Serial sections were analyzed for (A) lipid content using Oil Red-O stain; (B) macrophage infiltrate (anti-CD68); and (C) ABCA1 content using immunohistochemistry (anti-ABCA1). The following parameters were quantified: (D) the atherosclerotic lesion area (n = 5); (E) macrophage infiltrate (n = 7) and (F) ABCA1 content (n = 7); (G) determination of the ABCA1: macrophage ratio of the atherosclerotic plaque; (H) aortic cholesterol content in whole aortas (n = 8–13) isolated from LDLr-KO mice fed either a TRANS, SAFA or PUFA diet for 16 weeks; *p < 0.05, **p < 0.001. Data were checked for normality and transformation was performed prior to statistical analysis. One-way ANOVA followed by the post-hoc Newman–Keuls Multiple Comparison Test for pair-wise comparisons was performed. Results are shown as the mean ± SEM. Atherosclerosis 2012 224, 66-74DOI: (10.1016/j.atherosclerosis.2012.06.059) Copyright © 2012 Elsevier Ireland Ltd Terms and Conditions

Fig. 2 Correlations between (A) aortic total cholesterol (TC) with lesion area, plasma TC and plasma TG concentration: the aortic TC content was positively correlated with lesion area, plasma TC and plasma TG; (B) aortic cholesteryl ester (CE) and free cholesterol (FC) with lesion areas. Atherosclerosis 2012 224, 66-74DOI: (10.1016/j.atherosclerosis.2012.06.059) Copyright © 2012 Elsevier Ireland Ltd Terms and Conditions

Fig. 3 Co-localization of macrophage and ABCA1 on the atherosclerotic plaque. Dual immunofluorescent staining in serial sections did not show co-localization of ABCA1 (red) and macrophage (green) in the atherosclerotic plaques of LDLr-KO mice fed high fat diets enriched with TRANS, SAFA or PUFA for 16 weeks. DAPI (blue) (n = 3). Atherosclerosis 2012 224, 66-74DOI: (10.1016/j.atherosclerosis.2012.06.059) Copyright © 2012 Elsevier Ireland Ltd Terms and Conditions

Fig. 4 Correlations between plasma lipid concentrations (TC and TG) and aortic root ABCA1, and macrophage infiltration and lesion areas. Plasma TC and TG concentrations were positively correlated with aortic ABCA1 content, macrophage infiltration and, atherosclerotic lesion area. Atherosclerosis 2012 224, 66-74DOI: (10.1016/j.atherosclerosis.2012.06.059) Copyright © 2012 Elsevier Ireland Ltd Terms and Conditions

Fig. 5 Plasma inflammatory profile and macrophage inflammatory response. Peritoneal macrophages isolated from mice fed one of the three fatty diets for 16 weeks, and cultured in RPMI medium for 48 h. After this period the cells were washed remaining only adhered macrophages in the plaque. Cells (0.5 × 106/well) were stimulated with 1 μg/mL lipopolysaccharide (LPS) from Escherichia coli, for 24 h, and inflammatory factors (TNF-α, IL-6 and IL-10) released into the culture medium were measured using a commercial ELISA kit. (A) plasma IL-6 (n = 4); (B) plasma TNF-α (n = 4); (C) culture medium cytokines (n = 4). Atherosclerosis 2012 224, 66-74DOI: (10.1016/j.atherosclerosis.2012.06.059) Copyright © 2012 Elsevier Ireland Ltd Terms and Conditions

Fig. 6 Collagen content of the atherosclerotic lesion. Atherosclerotic plaque collagen content in LDLr-KO mice fed high fat diets enriched with TRANS, SAFA or PUFA fatty acids for 16 weeks was analyzed. (A) Picrosirius-red (PSR)-stained sections show the presence of a fibrous cap (arrows) with depleted collagen contents and necrotic cores (*) indicative of an unstable plaque. (B) Quantification of the collagen volume fraction (CVF) expressed as a percentage (%) of the total plaque area (n = 5). Atherosclerosis 2012 224, 66-74DOI: (10.1016/j.atherosclerosis.2012.06.059) Copyright © 2012 Elsevier Ireland Ltd Terms and Conditions