investigation and its translational relevance should be viewed with some cautions. Indeed, the causes of dyslipidemia in HIV infected patients taking a PI are not completely clarified. In addition to the above described mechanisms, alternative regulatory pathways could have been activated by exposure of macrophages to ritonavir. The pregnane –x-receptor (PXR) is a nuclear receptor and xenobiotic sensor and is a ritonavir target gene. Previous studies have shown that PXR directly activates SREBP1c and CD36 by binding to PXR responsive elements in the CD36 promoter. However, we have ruled out a possible role for PXR in the negative regulatory effects exerted by FXR agonists on this scavenger receptor, because FXR is a known PXR inducer [45,46]. Thus, exposure to FXR ligand would results in the induction of PXR, that is a positive regulatory gene for CD36. In conclusion, we have provided evidence that FXR activation exerts anti-atherosclerotic effects in ApoE2/2 mice treated with an HIV PI and that these effects are independent on modulation of lipid metabolism, but requires the regulation of CD36 expression and activity. These data suggest that CDCA by modulating SREPBP1c expression specifically antagonizes the effects of ritonavir of CD36, a mechanism involved in dyslipidemia caused by HIV-PI. Materials and Methods Animal and treatments The present study involves the use of rodents. All experiments were performed in accordance to international and Italian guidelines for use of laboratory animals. The present protocol was approved by the Italian Minister Health and conforms to national guidelines. The ID for this project is #11/2010-B. The authorization was released to prof. Stefano Fiorucci, as a principal investigator, on January 25, 2010. Mice were maintained in a temperature controlled facility with a 12-hour light/dark cycle and were given free access to food and water. Eight weeks old wild type and FXR2/2 mice were randomized into four groups (N = 10): group 1, no treatment; group 2 intraperitoneally administration of ritonavir 5 mg/Kg/day in a total volume of 100 ml alone; groups 3 and 4 received ritonavir (5 mg/Kg/day) in combination with CDCA (20 mg/kg/day by gavage) and gemfibrozil (100 mg/kg/ day by gavage) respectively. While FXR mice, eight weeks old, were randomized into two groups (N = 10): group 1, no treatment and group 2 intraperitoneally administration of 100 ml ritonavir (5 mg/Kg/day) alone. All drugs were administered 14 days. During the experiment mice were weight once a week. At the end of the experiment, 12-hour fasted animals received the latest administration of the specific agent and 6 hours later were anesthetized with sodium pentobarbital and then sacrificed. Blood and livers were collected. Serum content of total cholesterol, triglyceride, HDL, LDL, glucose and alanine aminotransferase were measured by enzymatic assays (Wako Chemicals; Osaka, Japan). ApoE2/2 male mice on a C57BL6/J background and wild type C57BL6/J male mice were from the Harlan Nossan (Udine, Italy). Ten weeks old ApoE2/2 mice were randomized into four groups (n = 10 per group): group 1, no treatment; group 2 mice were administered intraperitoneally with 100 ml of a solution containing 5 mg/Kg/day ritonavir; groups 3 and 4 mice were administered with r ritonavir (5 mg/Kg/day) in combination with CDCA (15 mg/kg/day by gavage) or gemfibrozil (100 mg/kg/day by gavage) respectively. All drugs were administered five days a week for 12 weeks. During the experiment mice were weight once a week. At the end of the experiment animals were fasted for 16 hours, anesthetized with sodium pentobarbital before harvest- FXR and Anti-HIV Therapy ing blood for subsequent lipid measurements and monocytes/ macrophages isolated for the measurement of CD36 expression by flow cytometry. Liver samples for RNA isolation were immediately snap frozen. Liver samples for histology were embedded in paraffin. The aortas were processed for enface atherosclerotic lesion coloration. This protocol was repeated three fold and results on one experiment are shown. In the second set of experiments (8 mice per group), animals received the same treatments described above and at the end blood was collected for RNA isolation and monocytes/macrophages isolated for CD36 expression. Quantification of atherosclerotic plaques and liver histology The heart and upper section of the aorta were removed from animals, cleaned of peripheral fat under a dissecting microscope, and sectioned parallel to the atria leaflets. En face analysis, aortas, including the ascending arch, thoracic, and abdominal segments, were dissected, gently cleaned of the adventitia, and stained with Sudan IV. Exactly the aortic parts were then stretched between two glass plates and fixed in neutral aqueous phosphate-buffered formaldehyde solution. The fixed aortic parts were stained en face with a filtrated solution containing Sudan IV (0.5 g Sudan IV dissolved in 200 ml ethanol:acetone:H2O, 21:10:9, vol/vol/vol; Sigma Chemical, Milan, Italy) to demonstrate areas of developed atheromatous plaques. To identify early atherosclerotic lesions, the aortas were then stained with Sudan IV without removing the pins. Aortas were briefly rinsed in 70% ethanol, immersed for 6 minutes in a filtered solution containing 0–5% Sudan IV (Sigma Chemical Co), 35% ethanol, and 50% acetone for 6 minutes, and destained for 5 minutes in 80% ethanol. The mean lesion area was quantified from ten digitally captured sections per mice using a BX60 microscope (Olympus Co., Rome, Italy) and digitalized using a SPOT-2 camera (Diagnostic Instruments Inc., Sterling Heights, MI) with a resolution of 131561033 pixels. Area measurements (expressed in pixels) were done using the free software Image J 1.33u (Rasband W., National Institutes of Health; Bethesda, MD). For liver histology, samples of the right and left liver lobes (100 mg/each) taken from each animal were fixed in 10% formalin, embedded in paraffin, sectioned, and stained with H&E. Tissue triacylglycerols and FFA For determination of total triglyceride, cholesterol and FFA (N = 6-4) content about 100 mg fragments of liver or heart were homogenized with 1 ml of T-PER (Pirce). The homogenized were used for protein concentration, Bradford assay (Bio-rad), and at 100ml of lised tissue extract was added 1.6 ml CHCl3 : MeOH (2:1) for 16 h at 4uC, after which 200mL of 0.6% NaCl was added and the solution centrifuged at 2,000 g for 20 min. The organic layer was removed and dried by speed vac system (HETO- Holten). The resulting pellet was dissolved in 100mL of phosphate buffered saline containing 1% Triton X-100 and triglyceride and free fatty acids content ware determined using specific enzymatic reagents, the results were expressed for mg of protein. Flow cytometry quantification of CD36 expression on monocytes/macrophages Blood-derived macrophages were collected from wild-type and ApoE2/2 mice (n = 12/genotype) as described in the previous section. The cells were treated with ACK solution to eliminate red blood cells, stained by anti-CD14-PE antibody and anti-CD36 antibody (BD Biosciences Pharmingen, San Diego, USA) and analyzed by a flow cytometer (Beckman Coulter, Fullerton, CA). PLoS ONE | www.plosone.org 11 October 2010 | Volume 5 | Issue 10 | e13238