FXR and Anti-HIV Therapy Figure 6. FXR and PPAR a ligands revert the dsysregulation of CD36 expression caused by administration of the HIV-PI ritonavir to ApoE2/2 mice. Panel A. CD36 expression on circulating was induced by administering ApoE2/2 mice with ritonavir 5 mg/kg/day for 12 week (n = 6 P,0.05) s. Co-treatment with gemfibrozil or CDCA abrobated this effect. Data are mean6 SE of 12 animal per group. *P,0.05 versus wild type naive; **P,0.05 versus ApoE2/2; # P,0.05 treatments versus ritonavir alone. Panel B–E. Ritonavir, gemfibrozil and CDCA exert opposite effects on CD36 expression/function in cultured macrophages (RAW264.7 cells). Ritonavir treatment increased the cell surface expression of CD36 on cultured macrophages in a concentration-dependent manner (Figure 6B, n = 5; P,0.05) and co-treatment of the cells with gemfibrozil and CDCA (Figure 6 C,D n = 5; p,0.05) reduced CD36 expression caused by 10 mM ritonavir by approximately 75% (Figure C,D; n = 5; P,0.05). Exposure of RAW264.7 cells to 10 mM ritonavir increased CD36-mediated uptake of acetylated LDL (ac-LDL). This effect was abrogated by gemfibrozil, 250 mM, and CDCA, 50 mM (n = 5; P,0.05 versus ritonavir alone). doi:10.1371/journal.pone.0013238.g006 The two agents exerted a comparable FFA-, triacylglycerols- and LDL-lowering effect. Both agents had minor effect on cholesterol plasma levels, confirming that ritonavir increases LDL levels primarily by inducing FFA and triacylglycerols synthesis. A further confirmation of this view comes from results of experiments carried out in FXR2/2 mice. Indeed, challenging FXR2/2 mice with ritonavir exacerbated the severity of dyslipidemia induced by ritonavir causing a two-fold increase in FFA, triacylglycerols and LDL plasma levels in comparison to wild type mice administered the HIP-PI. Administering FXR2/2 mice with ritonavir also increased cholesterol and reduced HDL levels. The latter effect is commonly observed in response to FXR activation, and it is still unclear whether it holds any potential for detrimental side effects in humans. Analysis of the expression of regulatory genes in the liver revealed that ritonavir had a minor effects on expression of SREBP1. However, expression of this gene was reduced in mice administered CDCA. Our results are consistent with previous observations indicating that ritonavir increases SREBP1c by reducing its degradation and by causing its stabilization at the promoter of target genes [9,10]. In summary these results suggest that activation of FXR reverts lipid abnormalities caused by ritonavir and that FXR gene ablation exacerbates the dyslipidemia caused by this HIV PI. FXR activation protects against atherosclerosis development in ApoE2/2 mice fed ritonavir An important observation we made is that, in contrast to wild type mice, 12-weeks administration of ritonavir (5 mg/kg/day) to ApoE2/2 mice failed to exacerbate the dyslipidemic phenotype of these mice and that, while pharmacological treatments were effective in protecting against accelerated atherosclerosis, both the FXR and PPARa ligand had no effect on serum lipoprotein profile. The lack of the effect on lipid metabolism was confirmed by the analysis of the expression of regulatory genes in the liver. Thus, in striking contrast to what observed in wild type mice, both CDCA and gemfibrozil failed to modulate the liver expression of SREBP1c, SREBP2and FAS. In addition, both treatments had no effects on liver expression of PPARa and FXR. Thought that CDCA increased the liver expression of SHP. In a previous study we have demonstrated that activation of FXR, by a semi-synthetic derivative of CDCA and PPARc reduces the liver expression of SREBP1c and its target genes PLoS ONE | www.plosone.org 8 October 2010 | Volume 5 | Issue 10 | e13238