ABSTRACT: HMG-CoA reductase inhibitors, statins, have beneficial vascular effects beyond their cholesterol-lowering action. These pleiotropic effects include an anti-inflammatory effect on macrophages. Since macrophages play a central role in atherogenesis, we further characterized the effects on peripheral blood monocyte-macrophages (HPBM). Using Affymetrix gene chip analysis of simvastatin-treated HPBM, we found that simvastatin treatment lead to the downregulation of the expression of many proinflammatory genes including several chemokines (e.g. MCP-1, MIP-1 alpha and β, RANTES, several other CC and CXC chemokines, IL-2 receptor-β, and leukemia inhibitory factor), members of the tumor necrosis factor family (e.g. lymphotoxin beta and TRAIL), VCAM-1, ICAM-3, and tissue factor (TF). Simvastatin also modulated the expression of several transcription factors essential for the inflammatory response: simvastatin downregulated the expression of NF-kappaB relA/p65 subunit and ets-1 transcription factor, and upregulated the expression of a novel atheroprotective transcription factor, Kruppel-like factor 2 (KLF-2). The effects of simvastatin on KLF-2 and its target genes were dependent on protein prenylation, since inhibitors of protein prenylation had a similar inhibitory effect in THP-1 derived macrophages. Additionally, by lentiviral overexpression KLF-2 we showed that the effect of simvastatin on MCP-1 and TF were dependent on KLF-2. We concluded that simvastatin had a strong anti-inflammatory effect on macrophages, which includes upregulation of the atheroprotective transcription factor KLF-2. These findings further explain the beneficial pleiotropic effects of statins on cardiovascular diseases. Experiment Overall Design: Cell culture studies. Experiment Overall Design: Human peripheral blood monocytes (HPBM) were isolated from buffy coats from healthy blood-donor volunteers (Finnish Red Cross, Helsinki, Finland) using Ficoll-Paque gradient centrifugation. None of the blood donors were on statin therapy. During isolation, monocytes from 3 individuals were pooled. Adherent cells were cultured in standard medium (20% human serum, Cambrex for differentiation into macrophages. The macrophage-phenotype at day 7 after isolation was confirmed with the typical shape of macrophages and also by macrophage-immunostaining (mAB CD68, DAKO, Denmark, dilution 1:200), where macrophages presented >95% of the cell population. The study protocol has been accepted by the Ethical Committee of the University of Kuopio. Experiment Overall Design: Simvastatin treatment. Experiment Overall Design: Simvastatin was a generous gift from Merck & Co. The inactive lactone form of simvastatin was hydrolyzed to the corresponding β-hydroxy acid. The HPBM-macrophages were treated with statin at day 7 after the isolation. 12 hours prior to statin treatment the cell growth media were changed to serum-free media. The statin-treated (10 uM simvastatin in serum-free media) cells were collected at 12 h and 24 h for Affymetrix analyses. The toxicity of simvastatin was assessed in cell culture: 5x concentration of simvastatin (50 uM) had no effect on cell viability. Experiment Overall Design: RNA isolation. Experiment Overall Design: Total RNA was isolated from the cells using Trizol reagent (Gibco BRL, USA) according to manufacturersâ?? instructions. The quality and amount of RNA was assessed by spectrophotometry (NanoDrop, USA) and by agarose gel electrophoresis. Experiment Overall Design: Microarray analyses. Experiment Overall Design: For Affymetrix analyses three separate HPBM cell isolation and simvastatin experiments were performed (HPBMs from 3 individuals at each time, total n=9). Cells were collected at 12 h and 24 h after the statin treatment. Total RNA was isolated as above. cDNAs were prepared from RNAs (5 ug of RNA) with reverse transcriptase (Superscript II primed by a poly (T) oligomer/T7 promoter). cDNAs were subsequently used as a template to make biotin-labeled cRNA with an in vitro transcription reaction. cRNAs (15 ug) were hybridized to Affymetrix HGU133 Plus 2.0 oligonucleotide arrays, which was processed and scanned according to manufacturerâ??s instructions. Each array quantifies the expression of over 47 000 transcripts (including full-length mRNA sequences and expressed sequence tags) derived from build 133 of the UniGene database (available at www.affymetrix.com). Experiment Overall Design: Microarray data analysis. Experiment Overall Design: Affymetrix GeneChip® Operating Software (GCOS) was used to generate .CEL files which were then converted into .DCP files using dChip (http://www.dchip.org) V1.3 software 28. The arrays were normalized to baseline array with median probe intensity, and gene expression data were generated calculating model-based expression values. In this study, genes were considered differentially expressed if they changed more than 1.5-fold (90% confidence bound of the fold change), absolute difference of signals was >100, at least 40% of the samples were called present in both groups and False Discovery Rate (FDR) was less than 1%. Hierarchical clustering was performed by dChip using Pearson correlation with a centroid-linkage method. Gene function analysis was performed by using the gene ontology mining tool GoSurfer incorporated in dChip program.
