antioxidants Article Nicotinamide Prevents Apolipoprotein B-Containing Lipoprotein Oxidation, Inflammation and Atherosclerosis in Apolipoprotein E-Deficient Mice Karen Alejandra Méndez-Lara 1,2 , Nicole Letelier 3 , Nuria Farre 4 , Elena M. G. Diarte-Añazco 1,2 , Núria Nieto-Nicolau 1,5 , Elisabeth Rodríguez-Millán 1 , David Santos 6 , Victor Pallarès 1 , Joan Carles Escolà-Gil 1,2,6 , Tania Vázquez del Olmo 7 , Enrique Lerma 7 , Mercedes Camacho 1,8 , Ricardo P Casaroli-Marano 1,5,9 , Annabel F. Valledor 3 , Francisco Blanco-Vaca 2,4,6, * and Josep Julve 1,2,6, * 1 Institut de Recerca de l’Hospital de la Santa Creu i Sant Pau i Institut d’Investigació Biomèdica de l’Hospital de la Santa Creu i Sant Pau (IIB-Sant Pau), 08041 Barcelona, Spain; kmendez1101@gmail.com (K.A.M.-L.); ediarte@santpau.cat (E.M.G.D.-A.); nurianieton@gmail.com (N.N.-N.); elisabethrodriguez78@gmail.com (E.R.-M.); mpallaresl@santpau.cat (V.P.); jescola@santpau.cat (J.C.E.-G.); mcamacho@santpau.cat (M.C.); rcasaroli@ub.edu (R.P.C.-M.) 2 Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain 3 Departament de Biologia Cel·lular, Fisiologia i Immunologia. Facultat de Biologia, Universitat de Barcelona i Institut de Biomedicina de la Universitat de Barcelona (IBUB), 08028 Barcelona, Spain; nicole.letelier.t@gmail.com (N.L.); afernandezvalledor@ub.edu (A.F.V.) 4 Servei de Bioquímica, Hospital de la Santa Creu i Sant Pau, IIB-Sant Pau, 08041 Barcelona, Spain; farre.nuria88@gmail.com 5 Barcelona Tissue Bank (BTB) & Donor Center, Banc de Sang i Teixits (BST), 08005 Barcelona, Spain 6 CIBER de Diabetes y Enfermedades Metabólicas Asociadas, CIBERDEM, 28029 Madrid, Spain; daymer11@hotmail.com 7 Servei d’Anatomia Patològica, Hospital de la Santa Creu i Sant Pau, IIB-Sant Pau, 08041 Barcelona, Spain; tvazquez@santpau.cat (T.V.d.O.); elerma@santpau.cat (E.L.) 8 CIBER de Enfermedades Cardiovasculares (CIBERCV), 28029 Madrid, Spain 9 Departament de Cirurgia, Facultat de Medicina & Hospital Clínic de Barcelona, Universitat de Barcelona, 08036 Barcelona, Spain * Correspondence: fblancova@santpau.cat (F.B.-V.); jjulve@santpau.cat (J.J.); Tel.: +34-935537377 (F.B.-V.); +34-935537586 (J.J.)  Received: 30 October 2020; Accepted: 18 November 2020; Published: 21 November 2020  Abstract: The potential of nicotinamide (NAM) to prevent atherosclerosis has not yet been examined. This study investigated the effect of NAM supplementation on the development of atherosclerosis in a mouse model of the disease. The development of aortic atherosclerosis was significantly reduced (NAM low dose: 45%; NAM high dose: 55%) in NAM-treated, apolipoprotein (Apo)E-deficient mice challenged with a Western diet for 4 weeks. NAM administration significantly increased (1.8-fold) the plasma concentration of proatherogenic ApoB-containing lipoproteins in NAM high-dose (HD)-treated mice compared with untreated mice. However, isolated ApoB-containing lipoproteins from NAM HD mice were less prone to oxidation than those of untreated mice. This result was consistent with the decreased (1.5-fold) concentration of oxidized low-density lipoproteins in this group. Immunohistochemical staining of aortas from NAM-treated mice showed significantly increased levels of IL-10 (NAM low-dose (LD): 1.3-fold; NAM HD: 1.2-fold), concomitant with a significant decrease in the relative expression of TNFα (NAM LD: −44%; NAM HD: −57%). An improved anti-inflammatory pattern was reproduced in macrophages cultured in the presence of NAM. Thus, dietary NAM supplementation in ApoE-deficient mice prevented the development of atherosclerosis and improved protection against ApoB-containing lipoprotein oxidation and aortic inflammation. Antioxidants 2020, 9, 1162; doi:10.3390/antiox9111162 www.mdpi.com/journal/antioxidants Antioxidants 2020, 9, 1162 2 of 15 Keywords: macrophage; cytokine; ATP-binding cassette (ABC) transporters; niacinamide; vitamin B3; cardiovascular disease 1. Introduction Coronary artery disease (CAD) represents one of the main outcomes of cardiovascular disease [1]. Atherosclerosis is an inflammatory process that is characterized by the infiltration of macrophages and other inflammatory cell subsets in the arterial wall and subsequently contributes to cardiovascular disease [2–4]. Statins have been proven to be effective at reducing cardiovascular disease-related mortality and morbidity [5]. However, a substantial risk of adverse cardiovascular outcomes persists [6, 7]. Therefore, over the last few decades, researchers have expressed increasing interest in investigating other pharmacological agents to further decrease the residual cardiovascular disease risk [8]. Nicotinic acid (NA) administration produces favorable antiatherogenic effects in vivo [9–18] and on humans [19]. NA significantly reduced CAD progression or clinical cardiovascular events in several studies [20]. However, the results from these earlier clinical trials have not been replicated in more recent trials that also included statins [20]. The occurrence of adverse side effects of NA therapy has also limited its use in clinical practice. Dietary supplementation with other vitamin B3 derivatives, such as nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR), confer favorable age-related vasoprotective effects by reversing endothelial dysfunction, attenuating oxidative stress, or rescuing age-related changes in gene expression [21–25]. However, these favorable effects have not been linked to changes in the inflammatory status of target tissues in these studies. Moreover, atherosclerosis was not directly assessed in these studies. Nicotinamide (NAM), the amide form of vitamin B3 , has also been shown to exert potent anti-inflammatory effects on chronic inflammatory diseases, including intestinal [26] or dermatological diseases [27–30]. Consistent with these findings, a dietary deficiency of vitamin B3 forms has been associated with different inflammatory processes, including dermatitis, irritation, inflammation of mucous membranes and tubular inflammation [31–35]. The anti-inflammatory effects of NAM have also been explored in vitro in immune cell types [36–45], which are involved in chronic inflammatory processes such as atherosclerosis. Notably, in one of these studies [35], the authors proposed that NAM may promote monocyte differentiation into macrophages with restricted inflammatory traits. Increased oxidation often underlies inflammation and triggers cardiovascular disease [46]. Importantly, NAM also exerts a beneficial effect on oxidative stress [47]. Indeed, this vitamin B3 form prevents both protein and lipid oxidation [48], at least in part through its ability to scavenge reactive oxidative species [47]. Antioxidant effects of other vitamin B3 derivatives, such as NR [49] and NMN [18], on vascular cells have also been reported. Based on experimental evidence, NAM might also protect against atherogenesis in vitro [36–45]; however, the effect of NAM has not yet been assessed in vivo. We therefore tested the hypothesis that the administration of NAM in vivo prevents the development of atherosclerosis in a murine model of substantial hypercholesterolemia and atherosclerosis. 2. Materials and Methods All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee of the Institut de Recerca de l’Hospital de la Santa Creu i Sant Pau (Procedure ID 10434). The methods were conducted in accordance with the approved guidelines. The effect of NAM supplementation on male apolipoprotein (Apo)E-deficient mice on a C57BL/6J genetic background was examined. Two doses of NAM (high dose: 1%; low dose: 0.25%) (cat#N0636, Merck KGaA, Darmstadt, Germany) were administered to mice in the drinking water, starting at the same time as the high-fat diet was initiated. The design of the intervention is shown in Figure S1. Antioxidants 2020, 9, 1162 3 of 15 The biochemical parameters were analyzed using commercial kits and adapted to a COBAS 6000/c501 autoanalyzer (Roche Diagnostics, Basel, Switzerland), as described in a previous study [50]. NAM and me-NAM levels were analyzed using high-performance liquid chromatography (HPLC) with mass spectrometry (MS). Plasma concentrations of cytokines (IL-10, IL-6, IL-4 and TNFα) were analyzed using Luminex xMAP® technology (Millipore Corporation, Billerica, MA, USA). The susceptibility of mouse lipoproteins to copper-induced lipid oxidation and the capacity of NAM to inhibit the oxidative modification of human low-density lipoproteins (LDL) were measured by monitoring the formation of conjugated dienes at a λ of 234 nm at 37 ◦ C for 6 h using a BioTek Synergy HT spectrophotometer (BioTek Synergy, Winooski, VT, USA) [51]. Serum oxidized LDL (OxLDL) levels were determined using a murine OxLDL sandwich ELISA (cat# SEA527Mu-96T, Cloud-Clone Corp, Houston, TX, USA). Cellular cholesterol efflux induced by human high-density lipoprotein (HDL) was determined in vitro using [3 H]-cholesterol-labeled J774A.1 mouse macrophages (ATCC® TIB67™, Manassas, VA, USA), as previously described [52]. Radioactivity counts were measured in both the cell culture medium and cell extract, and the percentage of cholesterol efflux was calculated. Proximal aortic atherosclerotic lesions in mice were evaluated in serial cryosections of optimal cutting temperature (OCT) compound-embedded preparations. Cryosections were stained with Oil Red O for lipids and the lesion area (surface area stained with Oil Red O) was quantified using AxioVision image analysis software (Zeiss, Jena, Germany). For immunohistochemical staining, thoracic aorta segments were fixed with a 10% neutral buffered formalin solution (cat# HT501128, Merck KGaA, Darmstadt, Germany). Seven-micrometer sections of paraffin-embedded tissue samples were incubated with mouse monoclonal antibodies against IL-10 (cat# GTX632359, diluted 1:50, v:v) and rabbit polyclonal antibodies against TNFα (cat# GTX110520, diluted 1:200, v:v) from GeneTex and stained with diaminobenzidine (DAB) in a Dako Autostainer Link 48 using the Dako EnVision+System-HRP-DAB-kit, according to the manufacturer’s protocol. Slides were then dehydrated and coverslipped and images were obtained using a BX61 Olympus bright-field microscope. Images were quantified using ImageJ-Fiji software. RNA was extracted from tissues and reverse transcribed, and the relative mRNA levels of each gene were determined using fluorescence-based quantitative real time PCR (qPCR) (Supplementary Methods (Table S1–S4)). The data are presented as medians (interquartile ranges). Statistical analyses were performed using GraphPad Prism software (GPAD, version 5.0, San Diego, CA, USA). The effects of NAM administration on gross and biochemical parameters, histological traits, and gene expression levels were determined using a nonparametric Kruskal–Wallis test followed by the Dunn multiple comparison test. Spearman’s rho correlation coefficients were calculated to determine the correlations between atherosclerosis and parameters of lipoprotein function, with all parameters considered as nonparametric variables. Differences between groups were considered statistically significant when the p value was <0.05. Additional details about the methods are available in the online version of the paper (Supplementary Methods (Table S1–S4)). 3. Results 3.1. Effect of NAM on Gross Parameters and Systemic Phenotype Plasma NAM concentrations were increased in a dose-dependent manner (NAM low-dose (LD): ~48-fold, p < 0.05; NAM high-dose (HD): ~145-fold, p < 0.05) in NAM-treated mice (Table 1). The plasma level of the methylated form of NAM (me-NAM) was also increased (~6-fold, p < 0.05) in mice receiving the maximal dose. Body weight gain was significantly reduced only in mice receiving the highest dose of NAM (p < 0.05) (Table 1). The latter was not accompanied by changes in daily food intake (Table 1). Supplementation with NAM did not affect the liver size (Table 1). Plasma levels of alanine aminotransferase were reduced (NAM LD: 40%, NAM HD: 56%; p < 0.05) in NAM-treated mice compared to untreated mice, while plasma aspartate aminotransferase levels did not differ among groups. Plasma creatinine concentrations did not change with NAM treatment (Table 1). Plasma Antioxidants 2020, 9, 1162 4 of 15 glucose and insulin levels in NAM HD-treated mice did not significantly differ from the levels in untreated mice. However, total cholesterol levels were significantly increased in NAM HD-treated mice (~1.8-fold, p < 0.05) compared to untreated mice, exclusively due to increased levels of the non-HDL cholesterol fraction (Table 1). Notably, plasma cholesterol and non-HDL cholesterol levels were approximately 0.7-fold lower in NAM LD-treated mice than in NAM HD-treated mice, which should be considered in the interpretation of the analysis of atherosclerosis. Table 1. Effect of NAM administration on gross parameters and plasma biochemical parameters of male ApoE-deficient mice. Parameters Untreated NAM LD NAM HD p Gross parameters Body weight (g) 28.7 (27.0; 29.9) 27.4 (23.3; 28.1) 24.6 (22.3; 26.0) * <0.05 Liver weight (g) 1.3 (1.2; 1.4) 1.3 (1.2;1.4) 1.2 (1.1;1.4) 0.20 Diet intake (g/day) 2.5 (2.4; 2.8) 2.7 (2.6; 2.8) 2.4 (2.1; 2.5) † <0.05 Water intake (g/day) 3.9 (3.2; 5.1) 4.6 (4.4; 4.8) 4.6 (3.8; 5.4) 0.20 Calculated dose of NAM - 0.5 (0.4; 0.7) 1.9 (1.6; 2.2) † <0.05 (g/kg/day) Plasma biochemistry NAM (µM) 4.0 (3.3; 5.5) 193.5 (90.5, 248.5) 580.0 (526.0; 605.0) * <0.05 me-NAM (relative values) 0.15 (0.13; 0.16) 0.28 (0.19; 0.31) 0.85 (0.45; 0.89) * <0.05 (×10−3 ) a Glucose (mM) 12.3 (10.6; 15.3) 11.1 (9.4; 11.8) 10.9 (8.4; 12.9) 0.17 Insulin (µg/L) 0.7 (0.6; 0.8) n. d. 0.7 (0.6; 0.7) 0.32 Triglycerides (mM) 0.5 (0.4; 1.0) 1.8 (1.3; 2.5) * 1.5 (1.1; 2.3) * <0.05 Total cholesterol (mM) 43.3 (33.7; 47.4) 53.0 (44.2; 55.2) 77.6 (71.6, 82.3) * † <0.05 Non-HDL cholesterol (mM) 43.1 (33.7; 47.0) 52.6 (43.8; 54.9) 77.3 (71.2, 82.0) * † <0.05 HDL cholesterol (mM) 0.2 (0.1; 0.4) 0.4 (0.3; 0.4) 0.4 (0.1; 0.5) 0.45 AST (U/L) 61 (24;128) 25 (19; 39) 63 (27; 117) 0.33 ALT (U/L) 16 (7; 48) 5 (3; 6) * 6 (2; 11) * <0.05 Creatinine (mM) 0.02 (0.01; 0.03) 0.01 (0.01; 0.02) 0.01 (0.01; 0.02) 0.16 Results are reported as medians (interquartile ranges) (n = 8 mice per group). All analyses were conducted in three-month-old mice. At two months of age, the mice were challenged with a Western diet, and treated with or without NAM for 1 month. Food and water intake was measured at the end of the study as described in the Materials and Methods section (Supplementary Methods (Table S1–S4)). Plasma concentration of NAM was expressed in µM, whereas that of me-NAM were shown in relative values (n = 5–6 mice per group). Plasma levels of the HDL fractions were determined in the plasma supernatants after precipitating with phosphotungstic acid (Roche); the non-HDL fraction was calculated by subtracting the HDL moiety from the total plasma. Statistically significant differences among groups for each variable were determined using a nonparametric a Kruskal–Wallis test followed by Dunn’s posttest; differences were considered significant when p < 0.05. Specifically, * p < 0.05 vs. Untreated group; † p < 0.05 vs. NAM LD group. Abbreviations used: ALT, alanine aminotransferase; AST, aspartate aminotransferase; NAM, nicotinamide, me-NAM, methylated form of nicotinamide; NAM LD, low-dose, NAM-treated mice; NAM HD, high-dose, NAM-treated mice, HDL, high-density lipoprotein; n. d., not determined. a relative units. 3.2. NAM Administration Prevents the Development of Aortic Atherosclerosis NAM supplementation significantly decreased the areas of aortic atherosclerotic lesions in treated male mice (NAM LD: 0.55-fold, −45%, p < 0.05; NAM HD: 0.45-fold, −55%, p < 0.05) compared with untreated mice (Figure 1 and Figure S2, panel (a)). Representative images of the lesions observed in each group are shown. Mice treated with NAM developed less advanced atherosclerotic lesions that were mainly restricted to aortic valves compared with the larger lesions that extended to the free aortic wall in untreated mice. The areas of aortic atherosclerotic lesions were also significantly reduced in female NAM-treated mice (NAM LD: 0.6-fold, p < 0.05; NAM HD: 0.3-fold, p < 0.05) compared with untreated mice (Figure S2, panels (b)–(d)). Antioxidants 2020, 12, x FOR PEER REVIEW 5 of 15 The Antioxidants 2020,area of atherosclerotic plaques was inversely correlated with the plasma NAM 9, 1162 5 of 15 concentration (Spearman’s r = −0.45, p ˂ 0.05, n = 24). Figure 1. Analysis Figure of of 1. Analysis proximal proximalaortic aorticlesions lesions in apolipoprotein (Apo)E-deficient apolipoprotein (Apo)E-deficient mice mice treated treated withwith different doses different of NAM. doses of NAM.(a) Representative (a) Representative images images of aortic atherosclerotic of aortic lesion atherosclerotic lesioninin 12-week-old 12-week-oldmice mice challenged challenged to a western to a western diet and diet NAMand over NAM1overmonth 1 month at 2 months at 2 months of age. of age. (b) Area (b) Area of proximal of proximal aortic aortic lesion lesion quantified quantified from 8 mice from per8group. mice per Data group. Data are as are expressed expressed as the the median median (interquartile (interquartile range) of four range) of sections consecutive four consecutive sections throughout thethroughout aortic sinus thethat aortic weresinus that were obtained obtained every 20 µmevery when20 µm when aortic valves aortic valves became visible. Statistically significant differences among groups for each became visible. Statistically significant differences among groups for each variable were determined variable were determined using a nonparametric a Kruskal–Wallis test followed by Dunn’s posttest. Differences using a nonparametric a Kruskal–Wallis test followed by Dunn’s posttest. Differences were considered were considered significant when p < 0.05. Specifically, * p < 0.05 vs. Untreated group. Abbreviations significant when p < 0.05. Specifically, * p < 0.05 vs. Untreated group. Abbreviations used: NAM LD, used: NAM LD, low-dose, NAM-treated mice; NAM HD, high-dose, NAM-treated mice. low-dose, NAM-treated mice; NAM HD, high-dose, NAM-treated mice. 3.3. NAM Administration Directly Protects against the Oxidation of Non-HDL Lipoproteins The area of atherosclerotic plaques was inversely correlated with the plasma NAM concentration (Spearman’s r = −0.45,modification The oxidative p < 0.05, n = of 24). LDL leads to cholesterol accumulation in macrophages and foam cell formation during atherosclerosis [53]. Notably, the susceptibility of non-HDL particles to 3.3. oxidation, NAM Administration as measuredDirectly Protectsdiene by conjugated against the Oxidation formation, of Non-HDLdelayed was significantly Lipoproteins (2-fold, p < 0.05) in NAM HD-treated mice (Figure 2, panels (a) and (b)). The oxidative modification of LDL leads to cholesterol accumulation in macrophages and foam Serum oxLDL concentrations were lower (35%, p < 0.05) in the NAM HD mice than in cell formation during untreated mice, atherosclerosis concomitant [53]. with the Notably, elevated theofsusceptibility levels of non-HDL non-HDL cholesterol particles in these to oxidation, mice (Figure 2, as measured by conjugated diene formation, was significantly delayed (~2-fold, p < 0.05) panel (c)). However, serum oxLDL levels were not associated with the area of atherosclerotic lesions in NAM HD-treated mice (Figure S3, (Figure panel (a)). 2, panels (a) and (b)). Serum TheoxLDL concentrations LDL fraction were and was isolated, lower effect pof