Heme oxygenase-1-Dependent anti-inflammatory effects of atorvastatin in zymosan-injected subcutaneous air pouch in mice.

Statins exert pleiotropic and beneficial anti-inflammatory and antioxidant effects. We have previously reported that macrophages treated with statins increased the expression of heme oxygenase-1 (HO-1), an inducible anti-inflammatory and cytoprotective stress protein, responsible for the degradation of heme. In the present study, we investigated the effects of atorvastatin on inflammation in mice and analyzed its mechanism of action in vivo. Air pouches were established in 8 week-old female C57BL/6J mice. Atorvastatin (5 mg/kg, i.p.) and/or tin protoporphyrin IX (SnPPIX), a heme oxygenase inhibitor (12 mg/kg, i.p.), were administered for 10 days. Zymosan, a cell wall component of Saccharomyces cerevisiae, was injected in the air pouch to trigger inflammation. Cell number and levels of inflammatory markers were determined in exudates collected from the pouch 24 hours post zymosan injection by flow cytometry, ELISA and quantitative PCR. Analysis of the mice treated with atorvastatin alone displayed increased expression of HO-1, arginase-1, C-type lectin domain containing 7A, and mannose receptor C-type 1 in the cells of the exudate of the air pouch. Flow cytometry analysis revealed an increase in monocyte/macrophage cells expressing HO-1 and in leukocytes expressing MRC-1 in response to atorvastatin. Mice treated with atorvastatin showed a significant reduction in cell influx in response to zymosan, and in the expression of proinflammatory cytokines and chemokines such as interleukin-1α, monocyte chemoattractant protein-1 and prostaglandin E2. Co-treatment of mice with atorvastatin and tin protoporphyrin IX (SnPPIX), an inhibitor of heme oxygenase, reversed the inhibitory effect of statin on cell influx and proinflammatory markers, suggesting a protective role of HO-1. Flow cytometry analysis of air pouch cell contents revealed prevalence of neutrophils and to a lesser extent of monocytes/macrophages with no significant effect of atorvastatin treatment on the modification of their relative proportion. These findings identify HO-1 as a target for the therapeutic actions of atorvastatin and highlight its potential role as an in vivo anti-inflammatory agent.

Statins Modulate Cyclooxygenase-2 and Microsomal Prostaglandin E Synthase-1 in Human Hepatic Myofibroblasts.

Statins have been shown to exert anti-inflammatory and anti-fibrogenic properties in the liver. In the present study, we explored the mechanisms underlying anti-fibrogenic effects of statins in isolated hepatic myofibroblasts and focused on cyclooxyegnase-2, a major anti-proliferative pathway in these cells. We show that simvastatin and fluvastatin inhibit thymidine incorporation in hMF in a dose-dependent manner. Pretreatment of cells with NS398, a COX-2 inhibitor, partially blunted this effect. cAMP levels, essential to the inhibition of hMF proliferation, were increased by statins and inhibited by non-steroidal anti-inflammatory drugs. Since statins modify prenylation of some important proteins in gene expression, we investigated the targets involved using selective inhibitors of prenyltransferases. Inhibition of geranylgeranylation resulted in the induction of COX-2 and mPGES-1. Using gel retardation assays, we further demonstrated that statins potentially activated the NFκB and CRE/E-box binding for COX-2 promoter and the binding of GC-rich regions and GATA for mPGES-1. Together these data demonstrate that statin limit hepatic myofibroblasts proliferation via a COX-2 and mPGES-1 dependent pathway. These data suggest that statin-dependent increase of prostaglandin in hMF contributes to its anti-fibrogenic effect.

Role of nitric oxide and CCAAT/enhancer-binding protein transcription factor in statin-dependent induction of heme oxygenase-1 in mouse macrophages.

The effect of statins on heme oxygenase-1 (HO-1) was compared in 2 murine cell lines, RAW 264.7 and J774A.1 cell lines, and in primary peritoneal macrophages of BALB/c or C57BL/6 mice. The role of endogenous nitric oxide and the type of transcription factors involved were explored. Simvastatin and fluvastatin induced HO-1. Pretreatment of cells with l-NMMA or 1400 W, two different nitric oxide synthase inhibitors, partially blocked statin-dependent induction of HO-1 in RAW 264.7 and J774A.1 but not in primary peritoneal macrophages. Induction of HO-1 by statins was dependent on p-38 MAP kinase activation in all types of macrophages. In RAW 264.7 cells, both statins increased the activity of reporter genes linked to the proximal 1.3 kbp promoter of HO-1 (EC50 of 1.4±0.3 µM for simvastatin and 0.6±0.03 µM for fluvastatin). This effect was significantly blocked by 1400 W (80±5.2% inhibition, p<0.02) and mevalonate, the direct metabolite of HMGCoA reductase. Gel retardation experiments implicated C/EBPß, AP-1 but not USF, for both RAW 264.7 and primary peritoneal macrophages of C57BL/6 mice. Collectively we showed a differential role of endogenous nitric oxide between macrophage cell lines and primary macrophages and an effect of statins in the protection against inflammation by increasing HO-1 expression.

Statins modulate transcriptional activity of heme-oxygenase-1 promoter in NIH 3T3 Cells.

Statins, inhibitors of HMG CoA reductase, have pleiotropic effects independent of their capacity to lower cholesterol. Heme-oxygenase-1(HO-1) plays an important role as an anti-oxidant and anti-inflammatory enzyme. In the present study, we used NIH 3T3 cells which express HO-1 to investigate the molecular mechanisms of HO-1 induction by statins. Simvastatin or fluvastatin induced a significant increase in HO-1 protein expression and mRNA levels. Both statins stimulated activity of a mouse HO-1 promoter (-1,287 to +73 bp)/luciferase reporter gene, 3.25 ± 0.23 (Mean ± S.E.M., n = 15, P < 0.001, t-test) and 3.13 ± 0.33 (Mean ± S.E.M., n = 6, P < 0.001, t-test), respectively. This effect was more pronounced in the short proximal promoter than the full promoter of HO-1. Gel retardation experiments for C/EBP and upstream stimulatory factor (USF) DNA-binding activities using simvastatin- or fluvastatin-treated cells showed significant nuclear protein-DNA complexes which were supershifted with antibodies specific for C/EBP ß and δ or USF-1 and USF-2. Point mutations of the proximal HO-1 promoter (-149 to +73 bp) for the myc/max which binds USF or the C/EBP binding sequences showed a reduction in statin-induced reporter activity whereas no role of the distal C/EBP binding elements located at -4 kb was observed. Moreover, overexpression of mutated C/EBP ß and USF factor or the siRNA for both factors supported a role of these transcription factors in statin-dependent induction of HO-1, with a clearer effect for C/EBP.