Olive oil, compared to a saturated dietary fat, has a protective role on atherosclerosis in niacin-treated mice with metabolic syndrome

! 1! Olive oil, compared to a saturated dietary fat, has a protective role on 1! atherosclerosis in niacin-treated mice with metabolic syndrome 2! 3! Sergio Montserrat-de la Paza,*, Maria C Naranjoa, Sergio Lopeza, Rocio 4! Abiaa, Francisco JG Murianaa, Beatriz Bermudezb,* 5! 6! a Laboratory of Cellular and Molecular Nutrition, Instituto de la Grasa, CSIC, 7! Ctra. de Utrera Km. 1, 41013 Seville, Spain. 8! b Department of Cell Biology, School of Biology, University of Seville, C/ 9! Profesor Garcia Gonzalez s/n, 41012 Seville, Spain. 10! 11! Running title: MUFAs vs SFAs on atherosclerosis in NA-treated mice with 12! MetS 13! 14! * Corresponding author: 15! Sergio Montserrat-de la Paz, Beatriz Bermudez 16! Laboratory of Cellular and Molecular Nutrition, Instituto de la Grasa, CSIC. 17! Ctra. de Utrera Km. 1, Campus Universitario Pablo de Olavide, Edificio 46, 18! 41013 Seville (Spain) 19! Tel: +34 954 611 550 (Ext. 360/359) 20! E-mail: [email protected], [email protected] 21! 22! 23! 24! 25! Postprint of Journal of Functional Foods 26: 557-564 (2016) http://dx.doi.org/10.1016/j.jff.2016.08.028 ! 2! ABSTRACT 26! We aimed to investigate the impact of high-fat low-cholesterol diets rich in 27! saturated fatty acids (HFLCD-SFAs), monounsaturated FAs (HFLCD-MUFAs) 28! or MUFAs + omega-3 long-chain polyunsaturated FAs (HFLCD-PUFAs) in 29! combination with niacin (NA) on atherosclerotic plaque characteristics in a 30! mouse model (Lepob/obLDLR−/−) of metabolic syndrome (MetS). Compared to 31! a low-fat low-cholesterol diet (LFLCD), HFLCDs increased body weight, 32! triglycerides, insulin, pro-inflammatory cytokines, and circulating monocytes, 33! contributing the HFLCD-SFAs to a predominance of a classical pro-34! inflammatory Ly6Chi population, whereas HFLCD-MUFAs and HFLCD-PUFAs 35! to a non-classical patrolling Ly6Clo population. HFLCDs promoted 36! atherosclerosis in the aortic roots of animals but the plaque size, collagen, 37! and macrophage content were higher with the HFLCD-SFAs than with the 38! HFLCD-MUFAs or HFLCD-PUFAs. Furthermore, HFLCD-SFAs promoted the 39! intra-plaque accumulation of M1 macrophages, whereas HFLCD-MUFAs and 40! HFLCD-PUFAs favoured the accumulation of M2 macrophages. These data 41! suggest that dietary MUFAs had advantage over SFAs to prevent 42! atherosclerotic events in the NA-treated MetS. 43! 44! Keywords: Monounsaturated fatty acids; niacin; monocytes; atherosclerosis; 45! macrophages; metabolic syndrome 46! 47! 48! 49! 50! ! 3! 1. Introduction 51! Dyslipidemia, insulin resistance, and obesity, the defining components of the 52! metabolic syndrome (MetS), are well-known risk factors implicated in the 53! aetiology and pathogenesis of certain cardiovascular diseases (CVDs) such 54! as atherosclerosis, the main cause of CVD death in developed and some 55! developing countries (Libby, 2012). Atherosclerosis is a systemic lipid-driven 56! inflammatory condition associated with endothelial dysfunction that results in 57! accumulation and subsequent oxidation of lipids in the vessel wall or plaque 58! development. These abnormalities trigger inflammatory cell infiltration and 59! macrophage foam cell formation leading to apoptosis and secondary necrosis 60! and plaque advancement (Tabas, 2010). It is important to address the factors 61! involved in the progression of atherosclerosis because advanced 62! atherosclerotic lesions are prone to rupture, leading to disability or death. The 63! monocyte-macrophage lineage is of fundamental interest in understanding 64! atherosclerotic lesion progression (Ziegler-Heitbrock et al., 2010). Monocytes 65! can be found into two distinct subsets based on the expression of specific 66! surface markers. In mice, monocytes are divided based on Ly6C expression 67! into inflammatory Ly6C high (Ly6Chi) and patrolling Ly6C low (Ly6Clo) 68! monocytes (Auffray et al., 2007; Sunderkotter et al., 2004). CC-motif 69! chemokine receptor 2 (CCR2) is expressed at high levels by inflammatory 70! monocytes (Zlotnik & Yoshie, 2000), whereas CX3C-motif chemokine receptor 71! 1 (CX3CR1) is more abundant on patrolling monocytes (Geissmann, Jung, & 72! Littman, 2003; Woollard & Geissmann, 2010). Inflammatory monocytes are 73! generally thought to be the most important subtype in early atherogenesis, 74! ! 4! being efficiently recruited to the lesion in a CCR2-dependent manner 75! (Robbins et al., 2012; Soehnlein et al., 2013). 76! 77! The atheroprotective effects of niacin (NA), also known as nicotinic acid or 78! vitamin B3, were first described in the 1950s (Carlson, 2005; Montserrat-de la 79! Paz et al., 2016a). According to a meta-analysis of 30 randomized controlled 80! trials, NA potently reduces triglycerides (TGs) by 15-30%, total cholesterol 81! (TC) by 5-15%, and LDL-cholesterol (LDL-C) by 5-20% and increases HDL-82! cholesterol (HDL-C) by 10-25% in plasma of patients with dyslipidaemia 83! and/or hypercholesterolemia (Birjmohun, Hutten, Kastelein, & Stroes, 2005). 84! Additional to the pharmacology of NA, several translational studies have 85! identified the differential role between saturated and unsaturated fats at 86! cardiovascular level. Compared to saturated fatty acids (SFAs), the 87! consumption of monounsaturated (MUFAs) and omega-3 long-chain 88! polyunsaturated (PUFAs) fatty acids have beneficial effects on lowering blood 89! lipids (Ortega et al., 2012; Vafeiadou et al., 2015) and inflammatory mediators 90! (Naranjo et al., 2016;Teng, Chang, Chang, & Nesaretnam, 2014). However, 91! the potential anti-atherogenic effects from a combination of dietary fatty acids 92! and NA have not yet been defined. Therefore, the current study aimed to 93! explore the influence of a pharmacological dose of NA and diets enriched in 94! SFAs, MUFAs or MUFAs + omega-3 long-chain PUFAs on atherosclerosis in 95! a mouse model (Lepob/obLDLR−/−) of MetS. 96! 97! 2. Materials and methods 98! 2.1. Fatty acid composition of dietary fats 99! ! 5! The fatty acid composition of dietary fats [cow's milk cream, rich in SFAs; 100! refined olive oil, rich in MUFAs; and refined olive oil plus eicosapentaenoic 101! acid (EPA) and docosahexaenoic acid (DHA), rich in MUFAs and omega-3 102! long-chain PUFAs] was determined by the method described in 103! EEC/796/2002 (Montserrat-de la Paz et al., 2016b), using a gas 104! chromatography system (HP-5890, Hewlett-Packard, Palo-Alto, USA) 105! equipped with flame ionization detector and a SP-2380 capillary column 106! (Supelco, Bellefonte, USA, 30 m x 0.32 mm) packed with cyanopropyl 107! siloxane (0.25 µm). The initial column temperature was 165 ºC, which was 108! held for 10 min, then programmed from 165 ºC to 200 ºC at 1.5 ºC/min. 109! Injector and detector temperature were 250 ºC, with the carrier gas H2. The 110! fatty acid composition of different dietary fats is detailed in Table 1. 111! 112! 2.2. Animal diets and experimental design 113! Male Lepob/obLDLR−/− mice bred onto a C57BL/6J background (B6.Cg-Lepob 114! Ldlrtm1Her/J, The Jackson Laboratory, Bar Harbor, ME, USA) was used for 115! the study. These mice are obese and develop plasma lipid alterations that 116! closely reflect MetS-related hyperlipidaemia (Kennedy et al., 2010; 117! Montserrat-de la Paz et al., 2016c). All diets were prepared by Panlab 118! Laboratoires (SAFE, Augy, France) and presented as pellets to the animals. 119! Mice received one of the following diets for 8 weeks: a standard normal-fat 120! diet (low-fat low-cholesterol diet, LFLCD) containing 3% energy as fat, used 121! as control, or high-fat low-cholesterol diets (HFLCDs), which contained 24% 122! energy as fat. All the diets were based on the standard rodent diet A04-10, 123! containing 0.01% cholesterol, 20 mg/kg BHT, and 3% binder. Three different 124! ! 6! HFLCDs were prepared by replacing the fat source from A04-10 diet by cow's 125! milk cream (21% energy) (HFLCD-SFAs), refined olive oil (21% energy) 126! (HFLCD-MUFAs) or refined olive oil (20% energy) plus EPA+DHA in the form 127! of ethyl esters (1% energy) (HFLCD-PUFAs). The cow’s milk cream provided 128! an additional amount of 0.006% by weight. All the diets contained equal 129! proportion of protein (19.5% energy) and carbohydrate was used to adjust the 130! total energy content. 131! 132! After weaning, mice were randomly allocated into 4 groups (n = 10 per group) 133! as follows: (1) group that received LFLCD; (2) group that received HFLCD-134! SFAs; (3) group that received HFLCD-MUFAs; and (4) group that received 135! HFLCD-PUFAs. The four groups received NA (1%, w/v; Twinlab, UT, USA) in 136! the drinking water. Body weight, food, and water intake were daily evaluated. 137! Sacrifice of all animals was carried out within the animal facilities (Instituto de 138! Biomedicina de Sevilla, IBiS), at the beginning of the light cycle and after 10 h 139! of food deprivation. Animals were euthanized with an overdose of 140! pentobarbital (1:10 in PBS, 150 mg/kg body weight). Cardiac puncture was 141! used to blood collection. Heart samples were collected upon sacrifice. All 142! animal protocols received appropriate institutional approval (Animal Care and 143! Use Committee of the University of Seville) and were performed according to 144! the official rules formulated in the Spanish law on the care and use of 145! experimental animals (UE Directive of 2010: 2010/63/UE; RD 53/2013). 146! 147! 2.3. Biochemical parameters 148! ! 7! Serum samples were obtained from blood by centrifugation at 1800 g for 15 149! min at 4 ºC. Fasting serum levels of glucose, TC, and TGs were assessed by 150! colorimetric assay kits (Bio-science-medical, Madrid, Spain). Serum cytokines 151! were measured using Multiplex Biomarker Immunoassays for Luminex xMAP 152! technology (Millipore, Billerica, USA). 153! 154! 2.4. Immunostaining of circulating monocytes by FACS 155! Cardiac blood samples were collected at the end of each dietary period. 156! Circulating monocyte membrane expression of CD11b (PerpCy5.5 anti-mouse 157! CD11b, Becton Dickinson, Oxford, UK), Ly6G (FITC anti-mouse Ly6G, 158! Miltenyi, Madrid, Spain), Ly6C (APC-Cy7 anti-mouse Ly6C, Miltenyi), CCR2 159! (APC anti-mouse CCR2, Miltenyi), and CX3CR1 (PE-Cy7 anti-mouse 160! CX3CR1, BD) was assessed by flow cytometry. Cells were incubated with 161! antibodies at room temperature, in the dark, for 15 min, followed by fixation 162! and lysing of erythrocytes with 20× volume of Fluorescence Activated Cell 163! Sorting (FACS) lysing solution (BD). Fluorescence intensity was measured by 164! using a FACSCanto II cytometer and calibrated using FACSCanto II Cell 165! analyser software (BD). Mean fluorescence intensity (MFI) of 104 counted 166! cells was measured in each sample. Monocytes were gated as forward 167! scatterhigh (FSChi)-side scatterhigh (SSChi)-cells. Expression levels were 168! presented as MFI corrected for nonspecific binding of isotype control 169! antibodies. 170! 171! 2.5. Immunohistochemistry 172! ! 8! Mouse heart was dissected, fixed in 1% paraformaldehyde, and embedded in 173! paraffin. Size of atherosclerotic lesions was determined as previously 174! described (Gijbels et al., 1999). Serial sections (6 µm) of the aortic root were 175! cut and stained with haematoxylin-eosin (HE) for morphometric analysis and 176! routine qualitative examination of collagen content, necrosis, and amount of 177! inflammatory B (B220) and C (CD3) cells. Sirius Red was used for the 178! detection of collagen. A polarized filter and birefringence colour discrimination 179! was used to differentiate various collagen structures [ranging from loosely 180! patched, immature, thin collagen (green) to tightly packed, mature, thick 181! collagen fibres (red)] as previously described (Mackenna, Omens, & Covell, 182! 1996). For evaluation of macrophage content were used MAC3 (an antibody 183! recognizing pan macrophages), CCR7 (antibody against M1 macrophages), 184! and CD163 (antibody against M2 macrophages). Samples were captured at 185! 20× magnifications by a Leica DM3000 light microscope (Leica, Wetzlar, 186! Germany) and an independent operator, in a blinder manner, performed 187! histological analyses using Quantimet with Qwin3 quantification software 188! (Leica). 189! 190! 2.6. Statistical analysis 191! All values in the figures and text are expressed as the arithmetic mean ± SD. 192! Experiments were carried out in triplicate. Data were evaluated with Graph 193! Pad Prism Version 5.01 software. The statistical significance of any difference 194! in each parameter among the groups was evaluated by one-way analysis of 195! variance (ANOVA), using Tukey's test for multiple comparison analysis. P 196! values of <0.05 were considered statistically significant. 197! ! 9! 198! 3. Results 199! 3.1. MUFA-rich diets reduce body weight gain and serum TG, insulin, 200! and pro-inflammatory cytokine levels compared to the SFA-rich diet in 201! NA-treated Lepob/obLDLR−/− mice 202! In the setting of NA treatment, mean daily food intake (LFLCD, 4.07 ± 0.32; 203! HFLCD-SFAs, 4.15 ± 0.61; HFLCD-MUFAs, 4.01 ± 0.47; HFLCD-PUFAs, 204! 4.17 ± 0.41 g/mouse) and water intake (LFLCD, 6.28 ± 0.83; HFLCD-SFAs, 205! 6.35 ± 1.01; HFLCD-MUFAs, 6.12 ± 0.77; HFLCD-PUFAs, 6.11 ± 0.81 206! mL/mouse) were not different among the four diet groups. Despite similarities 207! in food and drink intake, the final body weight and body weight gain of animals 208! increased but to a different degree depending on the diet (HFLCD-SFAs > 209! HFLCD-MUFAs = HFLCD-PUFAs > LFLCD, Table 2). Serum TG levels were 210! significantly higher in animals fed with HFLCDs (HFLCD-SFAs > HFLCD-211! MUFAs = HFLCD-PUFAs) than in those fed with the LFLCD. Serum TC levels 212! did not differ among the HFLCD groups but were higher than those observed 213! in the LFLCD group. Notably, serum insulin levels were significantly different 214! in all groups: HFLCD-SFAs > HFLCD-MUFAs > HFLCD-PUFAs > LFLCD. No 215! differences were observed for serum glucose levels. In relation to pro-216! inflammatory cytokines, serum TNF-α levels were higher only in animals fed 217! with HFLCD-SFAs, whereas serum IL-6 and monocyte chemoattractant 218! protein-1 (MCP-1/CCL2) levels were found to be higher in animals fed with 219! HFLCDs (for IL-6, HFLCD-SFAs > HFLCD-MUFAs = HFLCD-PUFAs; for 220! MCP-1, HFLCD-SFAs = HFLCD-MUFAs = HFLCD-PUFAs) than in those fed 221! with the LFLCD. 222! ! 16! REFERENCES 372! Ait-Oufella, H., Taleb, S., Mallat, Z., & Tedgui, A. (2011). Recent advances on 373! the role of cytokines in atherosclerosis. Arteriosclerosis, Thrombosis, and 374! Vascular Biology, 31, 969-979. 375! Ancuta, P., Rao, R., Moses, A., Mehle, A., Shaw, S.K., Luscinskas, F.W., & 376! Gabuzda, D. (2003). Fractalkine preferentially mediates arrest and migration 377! of CD16+ monocytes. Journal of Experimental Medicine, 197, 1701-1707. 378! Auffray, C., Fogg, D., Garfa, M., Elain, G., Join-Lambert, O., Kayal, S., 379! Sarnacki, S., Cumano, A., Lauvau, G., & Geissmann, F. (2007). Monitoring of 380! blood vessels and tissues by a population of monocytes with patrolling 381! behavior. Science, 317, 666-670. 382! Birjmohun, R.S., Hutten, B.A., Kastelein, J.J., & Stroes, E.S. (2005). Efficacy 383! and safety of high-density lipoprotein cholesterol-increasing compounds: a 384! meta-analysis of randomized controlled trials. Journal of American College of 385! Cardiology, 45, 185–197. 386! Carlson, L.A. (2005). Nicotinic acid: the broad-spectrum lipid drug. A 50th 387! anniversary review. Journal of Internal Medicine, 258, 94–114. 388! Deshmane, S.L., Kremlev, S., Amini, S., & Sawaya, B.E. (2009). Monocyte 389! chemoattractant protein-1 (MCP-1): an overview. Journal of Interferon & 390! Cytokine Research, 29, 313-326. 391! Finucane, O.M., Lyons, C.L., Murphy, A.M., Reynolds, C.M., Klinger, R., 392! Healy, N.P., Cooke, A.A., Coll, R.C., McAllan, L., Nilaweera, K.N., et al. 393! (2015). Monounsaturated fatty acid-enriched high-fat diets impede adipose 394! NLRP3 inflammasome-mediated IL-1β secretion and insulin resistance 395! despite obesity. Diabetes, 64, 2116-2128. 396! ! 17! Geissmann, F., Jung, S., & Littman, D.R. (2003). Blood monocytes consist of 397! two principal subsets with distinct migratory properties. Immunity, 19, 71-82. 398! Gijbels, M.J., van der Cammen, M., van der Laan, L.J., Emeis, J.J., Havekes, 399! L.M., Hofker, M.H., & Kraal, G. (1999). Progression and regression of 400! atherosclerosis in apoe3-leiden transgenic mice: An immunohistochemical 401! study. Atherosclerosis, 143, 15-25. 402! Git, K.C., & Adan R.A. (2015). Leptin resistance in diet-induced obesity: the 403! role of hypothalamic inflammation. Obesity Reviews, 16, 207-224. 404! Gui, T., Shimokado, A., Sun, Y., Akasaka, T., & Muragaki, Y. (2012). Diverse 405! roles of macrophages in atherosclerosis: From inflammatory biology to 406! biomarker discovery. Mediators of Inflammation, 2012, 693083. 407! Hilgendorf, I., Swirski, F.K., & Robbins, C.S. (2015). Monocyte fate in 408! atherosclerosis. Arteriosclerosis, Thrombosis, and Vascular Biology, 35, 272-409! 279. 410! Kennedy, A.J., Ellacott, K.L.J., King, V.L., & Hasty, A.H. (2010). Mouse 411! models of the metabolic syndrome. Disease Models & Mechanisms, 3, 156-412! 166. 413! Landsman, L., Bar-On, L., Zernecke, A., Kim, K.W., Krauthgamer, R., 414! Shagdarsuren, E., Lira, S.A., Weissman, I.L., Weber, C., & Jung, S. (2009). 415! CX3CR1 is required for monocyte homeostasis and atherogenesis by 416! promoting cell survival. Blood, 113, 963-972. 417! Libby, P. (2012). Inflammation in atherosclerosis. Arteriosclerosis, 418! Thrombosis, & Vascular Biology, 32, 2045–2051. 419! Lipszyc, P.S., Cremaschi, G.A., Zorrilla-Zubilete, M., Bertolino, M.L., Capani, 420! F., Genaro, A.M., & Wald, M.R. (2013). Niacin Modulates Pro-inflammatory 421! ! 18! Cytokine Secretion. A Potential Mechanism Involved in its Anti-atherosclerotic 422! Effect. Open Cardiovascular Medicine Journal, 7, 90-98. 423! Mackenna, D.A., Omens, J.H., & Covell, J.W. (1996). Left ventricular 424! perimysial collagen fibers uncoil rather than stretch during diastolic filling. 425! Basic Research in Cardiology, 91, 111-122. 426! Montserrat-de la Paz, S., Bermudez, B., Naranjo, M.C., Lopez, S., Abia, R., & 427! Muriana, F.J.G. (2016a). Pharmacological effects of niacin on acute 428! hyperlipemia. Current Medicinal Chemistry (Epub ahead of print) 429! Montserrat-de la Paz, S., Naranjo, M.C., Bermudez, B., Lopez, S., Moreda, 430! W., Abia, R., & Muriana, F.J. (2016b). Postprandial dietary fatty acids exert 431! divergent inflammatory responses in retinal-pigmented epithelium cells. Food 432! & Function, 7, 1345-1353. 433! Montserrat-de la Paz, S., Naranjo, M.C., Lopez, S., Abia, R., Muriana, F.J., & 434! Bermudez, B. (2016c). Niacin and olive oil promote skewing to the M2 435! phenotype in bone marrow-derived macrophages of mice with metabolic 436! syndrome. Food & Function, 7, 2233-2238. 437! Moore, K.J., Sheedy, F.J., & Fisher, E.A. (2013). Macrophages in 438! atherosclerosis: a dynamic balance. Nature Reviews in Immunology, 13, 709-439! 721. 440! Naranjo, M.C., Garcia, I., Bermudez, B., Lopez, S., Cardelo, M.P., Abia, R., 441! Muriana, F.J.G., & Montserrat-de la Paz, S. (2016). Acute effects of dietary 442! fatty acids on osteoclastogenesis via RANKL/RANK/OPG system. Molecular 443! Nutrition & Food Research doi: 10.1002/mnfr.201600303 444! Ohta, H., Wada, H., Niwa, T., Kirii, H., Iwamoto, N., Fujii, H., Saito, K., 445! Sekikawa, K., & Seishima, M. (2005). Disruption of tumor necrosis factor-446! ! 19! alpha gene diminishes the development of atherosclerosis in ApoE-deficient 447! mice. Atherosclerosis, 180, 11-17. 448! Ortega, A., Varela, L.M., Bermudez, B., Lopez, S., Abia, R., & Muriana, F.J. 449! (2012). Dietary fatty acids linking postprandial metabolic response and 450! chronic diseases. Food & Function, 2, 22-27. 451! Robbins, C.S., Chudnovskiy, A., Rauch, P., Figuereido, J.L., Iwamoto, Y., 452! Gorbatov, R., Etzrodt, M., Weber, G.F., Ueno, T., van Rooijen, N., et al. 453! (2012). Extremedullary hematopoiesis generates Ly-6C(high) monocytes that 454! infiltrate atherosclerotic lesions. Circulation, 125, 364-374. 455! Soehnlein, O., Drechsler, M., Doring, Y., Lievens, D., Hartwig, H., Kemmerich, 456! K., Ortega-Gomez, A., Mandl, M., Vijayan, S., Projahn, D., et al. (2013). 457! Distinct functions of chemokine receptor axes in the atherogenic mobilization 458! and recruitment of classical monocytes. EMBO Molecular Medicine, 5, 471–459! 481. 460! Soehnlein, O., & Swirski, F.K. (2013). Hypercholesterolemia links 461! hematopoiesis with atherosclerosis. Trends in Endocrinology and Metabolism, 462! 24, 129-136. 463! Sunderkotter, C., Nikolic, T., Dillon, M.J., Van Rooijen, N., Stehling, M., 464! Drevets, D.A., & Leenen, P.J. (2004). Subpopulations of mouse blood 465! monocytes differ maturation stage and inflammatory response. Journal of 466! Immunology, 172, 4410-4417. 467! Tabas, I. (2010) Macrophage death and defective inflammation resolution in 468! atherosclerosis. Nature Review in Immunology, 10, 36-46. 469! ! 20! Teng, K.T., Chang, C.Y., Chang, L.F., & Nesaretnam, K. (2014). Modulation 470! of obesity-induced inflammation by dietary fats: mechanisms and clinical 471! evidence. Nutrition Journal, 13, 12. 472! Thomas, G., Tacke, R., Hedrick, C.C., & Hanna, R.N. (2015). Nonclassical 473! patrolling monocyte function in the vasculature. Arteriosclerosis, Thrombosis, 474! and Vascular Biology, 35, 1306-1316. 475! Tolani, S., Pagler, T.A., Murphy, A.J., Bochem, A.E., Abramowicz, S., Welch, 476! C., Nagareddy, P.R., Holleran, S., Hovingh, G.K., Kuivenhoven, J.A., et al. 477! (2013). Hypercholesterolemia and reduced HDL-C promote hematopoietic 478! stem cell proliferation and monocytosis: studies in mice and FH children. 479! Atherosclerosis, 229, 79-85. 480! Vafeiadou, K., Weech, M., Altowaijri, H., Todd, S., Yaqoob, P., Jackson, K.G., 481! & Lovegrove, J.A. (2015). Replacement of saturated with unsaturated fats had 482! no impact on vascular function but beneficial effects on lipid biomarkers, E-483! selectin, and blood pressure: results from the randomized, controlled Dietary 484! Intervention and VAScular function (DIVAS) study. American Journal of 485! Clinical Nutrition, 102, 40-48. 486! van der Valk, F.M., Kuijk, C., Verweij, S.L., Stiekema, L.C., Kaiser, Y., 487! Zeerleder, S., Nahrendorf, M., Voermans, C., & Stroes, E.S. (2016). 488! Increased haematopoietic activity in patients with atherosclerosis. European 489! Heart Journal (Epub ahead of print) 490! Woollard, K.J., & Geissmann, F. (2010). Monocytes in aterosclerosis: subsets 491! and functions. Nature Review in Cardiology, 7, 77-86. 492! ! 21! Ziegler-Heitbrock, L., Ancuta, P., Crowe, S., Dalod, M., Grau, V., Hart, D.N., 493! Leenen, P.J., Liu, Y.J., MacPherson, G., Randolph, G.J., et al. (2010). 494! Nomenclature of monocytes and dendritic cells in blood. Blood, 116, e74-80. 495! Zlotnik, A., & Yoshie, O. (2000). Chemokines: a new classification system and 496! their role in immunity. Immunity, 12, 121-127. 497! 498! 499! 500! 501! 502! 503! 504! 505! 506! 507! 508! 509! 510! 511! 512! 513! 514! 515! 516! 517! ! 22! Figure Legends 518! Figure 1. (A) Representative FACS plots showing the gating strategy to 519! identify and quantify the circulating monocyte populations, (B) percentage of 520! Ly6Clo monocytes over total circulating monocytes, (C) CX3CR1 and (D) 521! CCR2 expression (MFI, mean fluorescence intensity) in total circulating 522! monocytes in Lepob/obLDLR−/− mice treated with NA and fed with LFLCD, 523! HFLCD-SFAs, HFLCD-MUFAs, and HFLCD-PUFAs during 8 weeks. Values 524! are presented as the mean ± SD (n = 10) and those marked with different 525! lowercase letter are statistically different (P < 0.05). 526! 527! Figure 2. (A) Representative pictures of HE stained sections (original 528! magnification ×40) and (B) quantification of aortic root plaque area in 529! Lepob/obLDLR−/− mice treated with NA and fed with LFLCD, HFLCD-SFAs, 530! HFLCD-MUFAs, and HFLCD-PUFAs during 8 weeks. Scale bar is applicable 531! to all pictures of the panel (A). Values are presented as the mean ± SD (n = 532! 10) and those marked with different lowercase letter are statistically different 533! (P < 0.05). 534! 535! Figure 3. Representative pictures of Sirius Red stained sections (original 536! magnification ×40) of aortic root plaque area examined by normal light (A) and 537! polarized (B) microscopy, (C) percentage of collagen in aortic root plaque 538! area, and (D) quantification of different collagen fibres per unit of aortic root 539! plaque area in Lepob/obLDLR−/− mice treated with NA and fed with LFLCD, 540! HFLCD-SFAs, HFLCD-MUFAs, and HFLCD-PUFAs during 8 weeks. Values 541! ! 23! are presented as the mean ± SD (n = 10) and those marked with different 542! lowercase letter are statistically different (P < 0.05). 543! 544! Figure 4. Representative pictures of immunohistochemically stained sections 545! (original magnification ×40) for (A) MAC3, (B) CCR7, and (C) CD163 positive 546! cells, and percentage of (D) total, (E) M1, and (F) M2 macrophages in aortic 547! root plaque area of Lepob/obLDLR−/− mice treated with NA and fed with LFLCD, 548! HFLCD-SFAs, HFLCD-MUFAs, and HFLCD-PUFAs during 8 weeks. Values 549! are presented as the mean ± SD (n = 10) and those marked with different 550! lowercase letter are statistically different (P < 0.05).551! ! 24! Table 1. Fatty acid composition of dietary fats. Cow’s milk cream Refined olive oil Refined olive oil plus EPA + DHA Fatty acid g/100 g of fatty acid 10:0, capric 2.5 ± 0.1 - - 12:0, lauric 3.1 ± 0.4 - - 14:0, myristic 10.9 ± 0.9 - - 16:0, palmitic 35.5 ± 0.8 20.4 ± 0.9 20.5 ± 0.6 16:1(n-7), palmitoleic 3.6 ± 0.3 1.0 ± 0.2 0.8 ± 0.1 18:0, stearic 11.5 ± 0.8 5.7 ± 0.1 4.5 ± 0.4 18:1(n-9), oleic 25.3 ± 0.7 61.9 ± 1.2 61.5 ± 1.0 18:2(n-6), linoleic 4.3 ± 0.8 8.0 ± 0.7 8.0 ± 0.5 18:3(n-3), α-linolenic 0.4 ± 0.1 1.0 ± 0.1 0.9 ± 0.0 20:5(n-3), eicosapentaenoic - - 0.9 ± 0.1 22:6(n-3), docosahexaenoic - - 0.7 ± 0.1 Others 3.0 ± 1.7 2.1 ± 1.1 2.0 ± 0.9 SFAs 63.5 ± 1.9a 26.1 ± 1.0b 25.0 ± 0.9b MUFAs 28.9 ± 0.8b 62.8 ± 1.4a 62.4 ± 1.0a PUFAs 4.7 ± 0.8c 9.0 ± 0.7b 10.6 ± 0.7a Values are expressed as the mean ± SD (n = 3) and those marked with different lowercase letter in the same row are statistically different (P < 0.05). ! 25! Table 2. Food intake, final body and relative weights, and biochemical blood analysis of Lepob/obLDLR−/− mice treated with NA and fed with LFLCD, HFLCD-SFAs, HFLCD-MUFAs, and HFLCD-PUFAs during 8 weeks. LFLCD HFLCD-SFAs HFLCD-MUFAs HFLCD-PUFAs Food intake (g/wk/animal) 26.2 ± 4.4a 32.8 ± 7.6a 29.6 ± 10.6a 31.77 ± 7.4a Final body weight (g) 28.6 ± 2.7c 38.5 ± 2.3a 32.4 ± 1.1b 31.7 ± 1.8b Body weight gain (g) 9.0 ± 0.7c 18.4 ± 1.6a 11.7 ± 0.3b 11.0 ± 1.1b Biochemical blood parameters TC (mmol/L) 3.6 ± 0.2b 4.3 ± 0.3a 4.1 ± 0.2a 4.0 ± 0.1a TGs (mmol/L) 0.4 ± 0.1c 0.8 ± 0.1a 0.5 ± 0.1b 0.5 ± 0.1b Glucose (mg/dL) 138.2 ± 6.7a 147.5 ± 4.9a 143.8 ± 7.3a 151.5 ± 4.9a Insulin (pg/mL) 412.5 ± 92.0d 1103.7 ± 105.1a 873.3 ± 82.7b 707.3 ± 92.2c TNF-α (pg/mL) 4.0 ± 0.2b 4.7 ± 0.2a 4.2 ± 0.1b 4.2 ± 0.1b IL-6 (pg/mL) 2.9 ± 0.2c 9.7 ± 1.0a 5.0 ± 0.4b 4.7 ± 0.3b MCP-1 (pg/mL) 32.9 ± 2.3b 41.0 ± 3.3a 38.2 ± 2.1a 38.4 ± 2.7a Values are expressed as the mean ± SD (n = 10) and those marked with different lowercase letter in the same row are statistically different (P < 0.05).