Inbred female C57Bl/6J mice, 4 weeks old, were obtained from the Facultad de Veterinaria, Universidad de Buenos Aires (UBA), Argentina and were housed in the animals facility of the Centro de Estudios Farmacológicos y Botánicos (CEFYBO)- Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET)- UBA. Animals were maintained on a 8:00 am to 8:00 pm light/dark cycle under controlled temperature (18-22ºC). All animal experiments and care were in accordance with the principles and guidelines of the Guide for the Care and Use of Laboratory Animals, US National Research (NIH) accredited by the Institutional Committee for use and care of Laboratory Animal (CICUAL, Facultad de Medicina, Universidad de Buenos Aires, Argentina). It is worth to note that this murine strain was previously shown to develop diet-induced fatty streak lesions [23].

A total of 125 female mice were used for atherogenic diet and other 125 animals were assigned for the chow diet group. Animals were randomly assigned to two groups: one that was fed with conventional chow diet (Control mice) and the other with the atherogenic diet (AGD mice). This diet was a pellet obtained by mixing an special argentine commercially breeding chow containing 10% fat, 26 % proteins, 34 % carbohydrate, with the addition of 15% dairy butter. As previously described this type of diet exerts the largest atherogenic action [23,24]. The standard diet contains 26% proteins, 45 % carbohydrate and 5% fat. The two diets contain AIN-93 mineral and vitamin mix.

Mice were anesthetized with 28 % (w/v) chloral hydrate, 0, 1 ml/ 100g of body, and perfused with 4% paraformaldehyde in phosphate buffer 0, 1 M, pH 7.4 through the abdominal aorta. Later this artery was dissected and the thoracic and abdominal segment selected and post fixed in the same solution during 2 hours, immersed overnight in phosphate buffer 0, 1 M, pH 7.4 and them embedded in paraffin for optical microscope evaluation. Sections of 4 µm were cut and mounted in silanized slices.

For immunohistochemical studies, artery sections (4 µm) were incubated overnight with the primary antibody (α- Actin, 1:100, monoclonal mouse antibody, 1A4, DAKO). Following several wash steps with PBS, artery sections were incubated with the proper secondary antibody (Vector Laboratories Inc., Burlingame, CA, USA) and followed by incubation with a biotin streptavidin complex (HRP Histo Mark, Caramillo, CA, USA). After washing in PBS, sections were developed and AEC substrate kit (Invitrogen Gaithersburg, MD, USA) until staining was optimal as examined by light microscopy using Leica Microscopes. Images were collected using a CCD video camera CU-M50 (Sony Inc.). Pictures were analyzed and compiled using Adobe Photoshop 8.0 CS3.

Macrophages were purified from peritoneal exudates as previously described [25]. Briefly, after sacrifice, mice were injected intraperitoneally with 10 ml of sterile PBS and after 10 min the ascitic fluid was removed. This fluid was centrifuged 10 min at 200g and the cell pellet was washed twice with PBS. Cells were seeded at a concentration of 1x10⁶cel/cm² in culture medium (RPMI 1640, supplemented with 10% FCS and antibiotics) in 60x15mm Petri Dish with Cover (Corning®) to purify adherent cells. After 2 h non-adherent cells were discarded and macrophages were removed by trypsin treatment. This procedure renders approximately 2-4 x 10⁶ macrophages per mouse.

Saturation binding assays were carried out on purified macrophages according to Lorenzen et al., 2001 [26] and

Wise et al., 2003 [8]. Briefly, 6 x 10⁶ cells per binding point in a total volume of 250 μl were incubated with increasing concentrations of 5,6-³H nicotinic acid (American Radiolabeled Chemicals Inc, 50 Ci/mmol) ranging from 10 to 200 nmol/L for 3 h at room temperature with agitation. Nonspecific binding was assessed in the presence of 1 mM nicotinic acid. The bound ligand was recovered onto presoaked GF/B filters, washed four times with 1 ml of ice-cold binding buffer, and measured by liquid scintillation counting.

1 x 10⁶ cells were incubated in 1 ml of supplemented medium (RPMI-1640 with 10% fetal calf serum and antibiotics) on 24 well-plates. These cells were incubated alone or with increasing concentrations of Niacin, ranging from 50 to 1000 μM and stimulated with lipopolysaccharide (LPS) and interferon-γ (IFN-γ) (Sigma Aldrich Co.) for 24 hours as previously described [27]. After incubation cell-free supernatants were obtained and the cytokine levels indicated in the Result section were measured by enzyme-linked immunosorbent assays (ELISA) using commercially available kits, following the manufacturer instructions. To further characterize the role of the GPR109A receptor, that is coupled to G protein, studies were also performed on macrophages pretreated with an inhibitor of G protein coupling, namely pertussis toxin (PTX), for 18 h.

An additional group of mice (n=12) was used to study the effect of Niacin on in vivo cytokine production. For this purpose animals were divided in four experimental groups: injected intraperitoneally with PBS or 500 µg (20 mg/kg) of LPS (Escherichia coli 026:B6) and simultaneously oral treated or non-treated with Niacin (250 mg/Kg)). Twelve hour after the injection, intraperitoneal macrophages were obtained and incubated in supplemented medium for additional twelve hours to collect supernatants for cytokine analysis [28]

Group differences were tested by one-way analysis of variance (ANOVA). When multiple comparisons were necessary after ANOVA, the Student-Newman-Keuls test was applied. Weight analysis was done with repeated measures ANOVA. When results showed not normally distribution the non-parametric Mann-Whitney test was performed. Differences between means were considered significant when p< 0.05.

To validate the atherogenic response in AGD mice, serum lipid levels were measured in peripheral blood samples and tissue lesions were evaluated in aortic sections by immunohistochemistry. As shown in Fig. (1), AGD, but not control mice, displayed an increase in body weight and in cholesterol levels. No changes in triglycerides, glucose, HDL, LDL or VLDL were observed in both groups. Moreover these animals had important aortic lesions that were not present in control mice. As can be seen in Fig. (2), control animals showed aortic arteries sections without morphological changes. All layers were preserved and no evidence of cell migration or endothelial activation was observed (panel A). In AGD mice the arteries wall sections showed significant alterations such as inner elastic layer disruption, disorganization of smooth muscle cells (SMC) from middle layer, vacuolization and ulterior migration of smooth muscle cells to the neointimal layer and an aortic wall intimal thickening (panel B and E). Also, in control animal’s contractile cells (differenciated) predominate over the other group; opposite what was found in AGD animals (panel F). It is worth noting that myoepithelial cells were finally confirmed by their strong positive immunohistochemical staining with anti-(-actin antibody (Panel C). Moreover, the presence of CD68+ cells (macrophages) was observed (Panel D).

Fig. (1). Characteristics of mice submitted to the atherogenic diet. The mean weights of n=30 control (circles) or 30 atherogenic diet-fed (AGD mice, squares) mice recorded at different weeks are depicted (panel A). Plasma levels of glycemia (panel B) and lipid profile: triglycerides (panel C) cholesterol (panel D), HDL (panel E), LDL (panel F) and VLDL (panel G) were determined for control and AGD mice. Results shown are the mean ± SEM of n = 30 mice from each group. Differs significantly from control animals, * p<0.05, ** p<0.01.

Fig. (2). Aortic lesions in AGD mice. Histological sections of aortic arteries, representative of n=10 sections from different animals of each group, are shown. Panel A: Control aortic wall stained with Hematoxylin-eosin (HE) shows that all layers are conserved. Panel B: HE staining of aortic wall from AGD mice: 1. rupture of the internal elastic layer; 2. migration of smooth muscle cells into the neo-intima; 3. vacuolization of sub-intimal smooth muscle cells; 4. disruption of smooth muscle. Panel C: immunohistochemical staining with anti-a-actin antibody: 1-myoepithelial cells; 2-endothelial cells. Panel D: immunohistochemical staining anti-CD68 (macrophages). 1- myoepitelial cells, 2- macrophage. Panel E: size of the intimal aortic wall. Panel F: average number of medial smooth muscle cells (SMCs). This value of intimal cells was determined by counting nuclear profiles of two types of intimal cells: “synthetic”, undifferentiated cells (gray), and “contractile”, differenciated cells: (white). Results are expressed as cells per unit area of intima (i.e., cells per square millimeter) in control and treated (AGD) animals. Data represent mean ± SEM; n=3 in each group (Panel E and F), ** p< 0.01 and *** p<0.001

Fig. (3). Niacin receptor characterization in macrophages. Panel A: Saturation Binding Assays: Increasing concentrations of [3H]- Nicotinic acid were incubated with 6 x 106 macrophages during 3 h at 25°C. Total (black) and non-specific (white, in the presence of 0.1 mM of Nicotinic acid) binding curves for [3H]-Nicotinic acid to isolated peritoneal macrophages from C57Bl/6J control mice were obtained. Panel B: Scatchard analysis of the results shown in panel A. The ratio of specific bound [3H]-Nicotinic acid to free (B/F) is plotted as a function of [3H]-Nicotinic acid bound (B). The intercept with the abscissa is the number of binding sites (Bmax) and the negative reciprocal of the slope is the dissociation constant (Kd). A representative experiment from 5 independent assays performed in duplicate is depicted in both panels. The Kd and Bmax values, mean ± S.E.M. from 5 experiments, for control and AGD mice macrophages are shown in the adjoined table. * p<0.05.

Fig. (4). Modulation of macrophage pro-inflammatory cytokine production by Niacin. Macrophages from control (white) or AGD (gray) mice were incubated in the presence or absence of increasing concentrations of Niacin (µM) and stimulated with LPS (10 mg/ml) + IFN-γ (100 U/ml) for 24 h. Cytokine levels, namely IL-1 (Panel A), TNF-α (Panel B) and IL-6 (Panel C) were measured in cell free supernatants, using commercial kits. Results shown are the mean ± SEM of 3 independent experiments performed in duplicate. + p< 0.05 Basal (B) AGD vs Control mice; * p< 0.05, ** p<0.01 vs the corresponding B value.

To evaluate if the changes compatible with atherosclerosis induction in AGD mice were accompanied by modifications in macrophages, Niacin receptor expression was studied on purified macrophages from control and AGD mice by ³H Nicotinic acid binding. As can be seen in Fig. (3), a saturable and specific (more than 80%) binding of radiolabelled Niacin was obtained in these cells. Scatchard analysis of saturation curves showed that macrophages from AGD mice have twice binding sites than control mice. No differences were found in the nicotinic acid receptor affinity, as similar Kd were observed.

As basal macrophage cytokines levels in both control and AGD mice are too low, we used two experimental approaches to evaluate the effect of Niacin on cytokine production: i. by assaying in vitro cytokine secretion from macrophages after a pro-inflammatory stimulus and ii. by evaluating the production of cytokines in macrophages from mice injected with an inflammatory dose of LPS. To induce the production of pro-inflammatory cytokines, in vitro macrophages were stimulated with LPS and IFN-γ as described [27]. To study the action of Niacin on pro- inflammatory cytokine production, cells were stimulated in the absence or presence of increasing concentrations of Niacin (ranging from 50 µM to 1000 µM) and Tumor Necrosis Factor-α (TNFα), Interleukin-1 (IL-1) and Interleukin-6 (IL-6) levels were measured in cell free supernatants of these cultures. As shown in Fig. (4) stimulated cytokine production in the absence of niacin (Basal) is higher in macrophages from AGD than those from control mice. Pro-inflammatory cytokine production was reduced in the presence of 500 to 1000 μM Niacin both in control and AGD mice, being the effect more evident in AGD than in control animals. To further characterize the role of the GPR109A receptor, macrophages were pretreated with PTX, a nonselective G protein-uncoupling agent, frequently used to examine the function of G protein-coupled receptors. As can be seen in Table 1, pretreatment with PTX blocked the Niacin-mediated effect on pro-inflammatory cytokine production.

To induce the production of pro-inflammatory cytokines in vivo, mice were injected with 500 μg of LPS. As shown in Table 2, production of IL-1, TNF-a and IL-6 for macrophages were significantly increased 12 h after LPS injection respect to PBS injected mice. Niacin treatment significantly reduced this elevated production of cytokines (Table 2).