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Delayed graft function (DGF) following kidney transplantation affects long-term graft function and survival and is considered a manifestation of ischemia reperfusion injury. Preclinical studies characterize metabolic defects resulting from mitochondrial damage as primary driver of ischemia reperfusion injury. In a comprehensive approach that included sequential establishment of postreperfusion arteriovenous concentration differences over the human graft, metabolomic and genomic analysis in tissue biopsies taken before and after reperfusion, we tested whether the preclinical observations translate to the context of clinical DGF. This report is based on sequential studies of 66 eligible patients of which 22 experienced DGF. Grafts with no DGF immediately recovered aerobic respiration as indicated by prompt cessation of lactate release following reperfusion. In contrast, grafts with DGF failed to recover aerobic respiration and showed persistent adenosine triphosphate catabolism indicated by a significant persistently low post reperfusion tissue glucose-lactate ratio and continued significant post-reperfusion lactate and hypoxanthine release (net arteriovenous difference for lactate and hypoxanthine at 30 minutes). The metabolic data for the group with DGF point to a persistent post reperfusion mitochondrial defect, confirmed by functional (respirometry) and morphological analyses. The archetypical mitochondrial stabilizing peptide SS-31 significantly preserved mitochondrial function in human kidney biopsies following simulated ischemia reperfusion. Thus, development of DGF is preceded by a profound post-reperfusion metabolic deficit resulting from severe mitochondrial damage. Strategies aimed at preventing DGF should be focused on safeguarding a minimally required post-reperfusion metabolic competence., (Copyright © 2016 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Background and Aims: Besides LDL-cholesterol, local vascular inflammation plays a key role in atherogenesis. Efficient therapies to treat the inflammatory component of the disease have not been established. The discovery of specialized inflammation-resolving mediators, such as resolvins may provide new opportunities for treatment. This study examines whether the ω-3 fatty acid eicosapentaenoic acid-derived resolvin E1 (RvE1), can reduce atherosclerosis, when administered alone or in combination with a cholesterol-lowering statin., Methods: ApoE*3Leiden mice were fed a hypercholesterolemic diet for 9 weeks and subsequently treated with RvE1-low (1 mg/kg/day), RvE1-high (5 mg/kg/day), atorvastatin (1.5 mg/kg/day) or the combination of atorvastatin and RvE1-low for the following 16 weeks., Results: RvE1-low and RvE1-high reduced atherosclerotic lesion size to the same extent (-35%; p < 0.05), attenuated the formation of severe lesions, also seen as a proportional increase in the presence of mild lesions, but did not alter plasma cholesterol levels. Cholesterol-lowering atorvastatin reduced atherosclerosis (-27%, p < 0.05), and the combination of RvE1 and atorvastatin further attenuated lesion size (-51%, p < 0.01) and increased the content of mild lesions. RvE1 did not affect plasma SAA, E-selectin, VCAM-1 or MCP-1 but did reduce plasma EPHX4 and down-regulated the local expression of pro-atherogenic genes in the aortae, (e.g. Cd74, Cd44, Ccl2, Ccr5 and Adam17) and significantly inactivated IFN-γ (p < 0.001) and TNF-α (p < 0.001) signalling pathways., Conclusions: RvE1 attenuates atherogenesis both alone and on top of a statin. The local effects of RvE1 are demonstrated by the modulated aortic expression of genes involved in inflammatory and immune responses, without altering plasma cholesterol or circulating SAA., (Copyright © 2016 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.)

Background & Aims: Anthocyanins may have beneficial effects on lipid metabolism and inflammation and are demonstrated to have hepatoprotective properties in models of restraint-stress- and chemically-induced liver damage. However, their potential to protect against non-alcoholic steatohepatitis (NASH) under conditions relevant for human pathogenesis remains unclear. Therefore, we studied the effects of the standardised anthocyanin-rich extract Mirtoselect on diet-induced NASH in a translational model of disease., Methods: ApoE(∗)3Leiden mice were fed a Western-type cholesterol-containing diet without (HC) or with 0.1% (w/w) Mirtoselect (HCM) for 20weeks to study the effects on diet-induced NASH., Results: Mirtoselect attenuated HC-induced hepatic steatosis, as observed by decreased macro- and microvesicular hepatocellular lipid accumulation and reduced hepatic cholesteryl ester content. This anti-steatotic effect was accompanied by local anti-inflammatory effects in liver, as demonstrated by reduced inflammatory cell clusters and reduced neutrophil infiltration in HCM. On a molecular level, HC diet significantly induced hepatic expression of pro-inflammatory genes Tnf, Emr1, Ccl2, Mpo, Cxcl1, and Cxcl2 while this induction was less pronounced or significantly decreased in HCM. A similar quenching effect was observed for HC-induced pro-fibrotic genes, Acta2 and Col1a1 and this anti-fibrotic effect of Mirtoselect was confirmed histologically. Many of the pro-inflammatory and pro-fibrotic parameters positively correlated with intrahepatic free cholesterol levels. Mirtoselect significantly reduced accumulation and crystallisation of intrahepatic free cholesterol, providing a possible mechanism for the observed hepatoprotective effects., Conclusions: Mirtoselect attenuates development of NASH, reducing hepatic lipid accumulation, inflammation and fibrosis, possibly mediated by local anti-inflammatory effects associated with reduced accumulation and crystallisation of intrahepatic free cholesterol., (Copyright © 2014 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Objective: Previous studies investigating flavanol-rich foods provide indications for potential cardioprotective effects of these foods, but the effects of individual flavanols remain unclear. We investigated whether the flavanol epicatechin can reduce diet-induced atherosclerosis, with particular emphasis on the cardiovascular risk factors dyslipidaemia and inflammation., Methods: ApoE*3-Leiden mice were fed a cholesterol-containing atherogenic diet with or without epicatechin (0.1% w/w) to study effects on early- and late-stage atherosclerosis (8 w and 20 w). In vivo effects of epicatechin on diet-induced inflammation were studied in human-CRP transgenic mice and NFκB-luciferase reporter mice., Results: Epicatechin attenuated atherosclerotic lesion area in ApoE*3-Leiden mice by 27%, without affecting plasma lipids. This anti-atherogenic effect of epicatechin was specific to the severe lesion types, with no effect on mild lesions. Epicatechin mitigated diet-induced increases in plasma SAA (in ApoE*3-Leiden mice) and plasma human-CRP (in human-CRP transgenic mice). Microarray analysis of aortic gene expression revealed an attenuating effect of epicatechin on several diet-induced pro-atherogenic inflammatory processes in the aorta (e.g. chemotaxis of cells, matrix remodelling), regulated by NFκB. These findings were confirmed immunohistochemically by reduced lesional neutrophil content in HCE, and by inhibition of diet-induced NFκB activity in epicatechin-treated NFκB-luciferase reporter mice., Conclusion: Epicatechin attenuates development of atherosclerosis and impairs lesion progression from mild to severe lesions in absence of an effect on dyslipidaemia. The observed reduction of circulating inflammatory risk factors by epicatechin (e.g. SAA, human-CRP), as well as its local anti-inflammatory activity in the vessel wall, provide a rationale for epicatechin's anti-atherogenic effects., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Objective: Polyphenols such as quercetin may exert several beneficial effects, including those resulting from anti-inflammatory activities, but their impact on cardiovascular health is debated. We investigated the effect of quercetin on cardiovascular risk markers including human C-reactive protein (CRP) and on atherosclerosis using transgenic humanized models of cardiovascular disease., Methods: After evaluating its anti-oxidative and anti-inflammatory effects in cultured human cells, quercetin (0.1%, w/w in diet) was given to human CRP transgenic mice, a humanized inflammation model, and ApoE*3Leiden transgenic mice, a humanized atherosclerosis model. Sodium salicylate was used as an anti-inflammatory reference., Results: In cultured human endothelial cells, quercetin protected against H(2)O(2)-induced lipid peroxidation and reduced the cytokine-induced cell-surface expression of VCAM-1 and E-selectin. Quercetin also reduced the transcriptional activity of NFκB in human hepatocytes. In human CRP transgenic mice (quercetin plasma concentration: 12.9 ± 1.3 μM), quercetin quenched IL1β-induced CRP expression, as did sodium salicylate. In ApoE*3Leiden mice, quercetin (plasma concentration: 19.3 ± 8.3 μM) significantly attenuated atherosclerosis by 40% (sodium salicylate by 86%). Quercetin did not affect atherogenic plasma lipids or lipoproteins but it significantly lowered the circulating inflammatory risk factors SAA and fibrinogen. Combined histological and microarray analysis of aortas revealed that quercetin affected vascular cell proliferation thereby reducing atherosclerotic lesion growth. Quercetin also reduced the gene expression of specific factors implicated in local vascular inflammation including IL-1R, Ccl8, IKK, and STAT3., Conclusion: Quercetin reduces the expression of human CRP and cardiovascular risk factors (SAA, fibrinogen) in mice in vivo. These systemic effects together with local anti-proliferative and anti-inflammatory effects in the aorta may contribute to the attenuation of atherosclerosis., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Chronic inflammation and proatherogenic lipids are important risk factors of cardiovascular disease (CVD). Specific dietary constituents such as polyphenols and fish oils may improve cardiovascular risk factors and may have a beneficial effect on disease outcomes. We hypothesized that the intake of an antiinflammatory dietary mixture (AIDM) containing resveratrol, lycopene, catechin, vitamins E and C, and fish oil would reduce inflammatory risk factors, proatherogenic lipids, and endpoint atherosclerosis. AIDM was evaluated in an inflammation model, male human C-reactive protein (CRP) transgenic mice, and an atherosclerosis model, female ApoE*3Leiden transgenic mice. Two groups of male human-CRP transgenic mice were fed AIDM [0.567% (wt:wt) powder and 0.933% (wt:wt oil)] or placebo for 6 wk. The effects of AIDM on basal and IL-1β-stimulated CRP expression were investigated. AIDM reduced cytokine-induced human CRP and fibrinogen expression in human-CRP transgenic mice. In the atherosclerosis study, 2 groups of female ApoE*3Leiden transgenic mice were fed an atherogenic diet supplemented with AIDM [0.567% (wt:wt) powder and 0.933% (wt:wt oil)] or placebo for 16 wk. AIDM strongly reduced plasma cholesterol, TG, and serum amyloid A concentrations compared with placebo. Importantly, long-term treatment of ApoE*3Leiden mice with AIDM markedly reduced the development of atherosclerosis by 96% compared with placebo. The effect on atherosclerosis was paralleled by a reduced expression of the vascular inflammation markers and adhesion molecules inter-cellular adhesion molecule-1 and E-selectin. Dietary supplementation of AIDM improves lipid and inflammatory risk factors of CVD and strongly reduces atherosclerotic lesion development in female transgenic mice.

Objective: Inflammation plays an important role in all stages of atherosclerosis, but little is known about the therapeutic effects of quenching inflammation in already existing atherosclerotic lesions. Putative beneficial effects of salicylate, an inhibitor of NF-κB activation, were studied in mice with established lesions., Methods: ApoE*3-Leiden mice received a high-cholesterol diet (HC) to establish atherosclerotic lesions. Reference mice (REF) were sacrificed to determine the lesion area at the start of two interventions. In one intervention group HC diet feeding was continued, but the diet contained salicylate (HC+SAL). As salicylate not only quenches inflammation but also reduces plasma cholesterol, a second intervention group was fed a low-cholesterol diet (LC) resulting in cholesterol levels comparable to HC+SAL. The effects of these interventions on lesion area and composition were assessed after 8 and 16 weeks., Results: HC+SAL markedly reduced hepatic NF-κB activity compared to REF, and was significantly more effective than LC diet feeding. HC+SAL and LC also quenched aortic NF-κB activity. While continuing HC diet typically further increases total lesion area, 16 weeks of intervention with HC+SAL and LC halted further disease progression and resulted in lesion sizes comparable to that of REF. At the same time, lesion composition was significantly improved, particularly with salicylate. Strikingly, HC+SAL resulted in a lower lesional macrophage content and a greater plaque stability index (ratio of collagen to macrophage area) than LC., Conclusion: Anti-inflammatory salicylate reduces atherosclerotic macrophage content and increases lesion stability of pre-existing plaques through quenching of NF-κB activity and reducing plasma cholesterol., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

Objective: The use of the HIV-protease inhibitor ritonavir (RTV) is associated with induction of hypertriglyceridemia, which is a cardiovascular risk factor. Therefore, we investigated the effect of RTV on atherosclerosis development in APOE*3-Leiden transgenic mice, a model for human-like lipoprotein metabolism and atherosclerosis., Methods and Results: APOE*3-Leiden mice were fed a Western-type diet without or with RTV (35 mg/kg/day) for 19 weeks. RTV increased plasma TG levels throughout the study (approximately 2-fold; P<0.05). Despite these increased TG levels, RTV decreased the atherosclerotic lesion area in the aortic root (-57%; P<0.05), concomitant with reduced macrophage area (-72%; P<0.01) and decreased lesion severity. This could not be explained by reduced inflammatory markers in plasma (i.e. serum amyloid A, E-selectin and fibrinogen), nor by decreased lipid accumulation in macrophages or increased cholesterol efflux from macrophages, as assessed using peritoneal macrophages in vitro. Rather, whereas RTV did not affect plasma total cholesterol levels, RTV decreased (V)LDL-cholesterol and increased cholesterol in apoE-rich large HDL., Conclusion: Despite inducing hypertriglyceridemia, RTV decreases atherosclerotic lesion area and severity, associated with decreased (V)LDL-cholesterol and increased atheroprotective apoE-rich large HDL., (Copyright (c) 2009 Elsevier Ireland Ltd. All rights reserved.)

Background: Low-grade chronic inflammation in overweight subjects is thought to play an important role in disease development., Objective: It was hypothesized that specific dietary components are able to reduce low-grade inflammation as well as metabolic and oxidative stress., Design: Dietary products [resveratrol, green tea extract, alpha-tocopherol, vitamin C, n-3 (omega-3) polyunsaturated fatty acids, and tomato extract] selected for their evidence-based antiinflammatory properties were combined and given as supplements to 36 healthy overweight men with mildly elevated plasma C-reactive protein concentrations in a double-blind, placebo-controlled, crossover study with treatment periods of 5 wk. Inflammatory and oxidative stress defense markers were quantified in plasma and urine. Furthermore, 120 plasma proteins, 274 plasma metabolites (lipids, free fatty acids, and polar compounds), and the transcriptomes of peripheral blood mononuclear cells and adipose tissue were quantified., Results: Plasma adiponectin concentrations increased by 7%, whereas C-reactive protein (principal inflammation marker) was unchanged. However, a multitude of subtle changes were detected by an integrated analysis of the "omics" data, which indicated modulated inflammation of adipose tissue, improved endothelial function, affected oxidative stress, and increased liver fatty acid oxidation., Conclusion: An intervention with selected dietary products affected inflammatory processes, oxidative stress, and metabolism in humans, as shown by large-scale profiling of genes, proteins, and metabolites in plasma, urine, and adipose tissue. This trial was registered at clinical trials.gov as NCT00655798.

Objective: Cholesteryl ester transfer protein (CETP) adversely affects the plasma lipoprotein profile by increasing VLDL-cholesterol and decreasing HDL-cholesterol. The relative contribution of either of these changes to atherosclerosis development is not known. We investigated to what extent the increase in VLDL-cholesterol can explain the atherogenic action of human CETP expression in APOE*3-Leiden (E3L) mice, a model for human-like lipoprotein metabolism., Methods and Results: E3L mice and E3L.CETP mice were fed a low cholesterol (LC) diet, resulting in a 4-fold increased VLDL-cholesterol level as well as a 9-fold increased atherosclerotic lesion area in the aortic root in E3L.CETP mice compared to E3L-LC mice. E3L mice fed a high cholesterol (HC) diet to match the increased VLDL-cholesterol levels in E3L.CETP mice, displayed a similar atherosclerotic lesion area as observed in E3L.CETP mice. Hence, the CETP-induced raise in atherosclerosis can largely be explained by increased VLDL-cholesterol. Despite similar atherosclerosis development, E3L.CETP mice had lower HDL-cholesterol as compared to E3L-HC mice (-49%) indicating that the HDL-cholesterol lowering effect of CETP is unlikely to contribute to atherosclerosis development in this experimental setting. Remarkably, atherosclerotic lesions in CETP-expressing mice were enriched in collagen, suggesting a role of CETP or the diet in modifying lesion collagen content., Conclusions: In this experimental setting, the proatherogenic effect of CETP is largely explained by increased VLDL-cholesterol.

The aim of this study was to define the anti-atherosclerotic role of liver-X-receptors (LXRs) under lesion progressive and lesion regressive conditions, to establish a temporal line of events, and to gain insights into the mechanisms underlying the anti-atherogenic potency of LXRs. We used apoE*3Leiden (E3L) mice to comprehensively and time-dependently dissect how T0901317, an LXR-agonist, inhibits initiation and progression of atherosclerotic lesions and regresses existing lipid- and macrophage-rich lesions. T0901317 suppresses lesion evolution and promotes lesion regression regarding lesion number, area, and severity. Quantitative plasma and vessel wall analyses corroborated by immunohistochemical evaluation of the aortic lesions revealed that under progressive (high-cholesterol diet) as well as regressive (cholesterol-free diet) conditions T0901317: i) significantly increases plasma triglyceride and total cholesterol levels; ii) does not affect the systemic inflammation marker, Serum amyloid A (SAA); iii) suppresses endothelial monocyte adhesion; and iv) induces the expression of the cholesterol efflux-related genes apolipoprotein E (apoE), ATP binding cassette (ABC) transporters ABCA1 and ABCG1. Furthermore, under progressive conditions, T0901317 suppresses the vascular inflammatory status (NF-kappaB) and the vascular expression of adhesion molecules [E-selectin, intercellular adhesion molecule (ICAM)-1, and CD44], lowers lesional macrophage accumulation, and blocks lesion evolution at the transition from lesional stage II to III. Under regressive conditions, T0901317 induces lesional macrophage disappearance and increases the expression of the chemokine receptor CCR7, a factor functionally required for regression. The LXR-agonist T0901317 retards vascular lesion development and promotes lesion regression at several levels. The findings support that vascular LXR is a potential anti-atherosclerotic target.

We report a sensitive, generic method for quantitative profiling of bile acids and other endogenous metabolites in small quantities of various biological fluids and tissues. The method is based on a straightforward sample preparation, separation by reversed-phase high performance liquid-chromatography mass spectrometry (HPLC-MS) and electrospray ionisation in the negative ionisation mode (ESI-). Detection is performed in full scan using the linear ion trap Fourier transform mass spectrometer (LTQ-FTMS) generating data for many (endogenous) metabolites, not only bile acids. A validation of the method in urine, plasma and liver was performed for 17 bile acids including their taurine, sulfate and glycine conjugates. The method is linear in the 0.01-1 microM range. The accuracy in human plasma ranges from 74 to 113%, in human urine 77 to 104% and in mouse liver 79 to 140%. The precision ranges from 2 to 20% for pooled samples even in studies with large number of samples (n>250). The method was successfully applied to a multi-compartmental APOE*3-Leiden mouse study, the main goal of which was to analyze the effect of increasing dietary cholesterol concentrations on hepatic cholesterol homeostasis and bile acid synthesis. Serum and liver samples from different treatment groups were profiled with the new method. Statistically significant differences between the diet groups were observed regarding total as well as individual bile acid concentrations.

The increased consumption of fruits and vegetables is associated with reduced cardiovascular disease. The molecular basis of this health effect is not fully understood, yet dietary flavonoids are thought to play an important role. Genetic engineering has enabled us to overexpress specific flavonoids (flavones and flavonols) in tomato fruit. Human C-reactive protein transgenic (CRPtg) mice express markers of cardiovascular risk that allow us to study of the putative health effects of wild-type tomato (wtTom) and flavonoid-enriched tomato (flTom). In this study, we analyzed whether consumption of wtTom, at a dose achievable with a human diet, has beneficial effects on cardiovascular risk markers and whether flTom may enhance such effects. CRPtg mice were fed a diet containing 4 g/kg wtTom, flTom peel, vehicle, or 1 g/kg fenofibrate, which reportedly reduces cardiovascular risk, for 7 wk. Markers of general health (bodyweight, food intake, and plasma alanine aminotransferase activities) and of cardiovascular risk (plasma CRP, fibrinogen, E-selectin, and cholesterol levels) were analyzed. All groups had comparable food intakes and body-weight gains. Plasma alanine aminotransferase activities increased significantly in vehicle and fenofibrate-treated mice. Compared with baseline, wtTom and flTom significantly reduced basal human CRP concentrations by 43 and 56%, respectively. The CRP-lowering effect of flTom significantly exceeded that of wtTom. The effects of flTom on CRP were reversed within a 2-wk washout period. WtTom and flTom did not affect fibrinogen, but comparably repressed E-selectin expression and upregulated HDL cholesterol. Tomato peel consumption improved cardiovascular risk factors in CRPtg mice, a beneficial effect that was further enhanced by enrichment of the flavonoid content.

Inflammatory processes, aside from cholesterol, play a central role in atherogenesis. Human C-reactive protein (huCRP) signals systemic inflammation and independently predicts future cardiovascular risk. Cholesterol-lowering statins reduce atherosclerosis and plasma huCRP levels. Evidence is sought for a direct anti-inflammatory statin effect in vivo, independent of effects on plasma cholesterol and atherogenesis. The effect of atorvastatin and simvastatin on huCRP expression was studied in nonatherosclerotic huCRP transgenic mice and compared with another class of hypolipidemic drugs, peroxisome proliferator-activated receptor-alpha (PPARalpha) activators, notably fenofibrate and Wy14643. Like statins, PPARalpha activators combine antiatherosclerotic properties with huCRP-lowering effects. Dietary treatment with statins or PPARalpha activators decreased basal and interleukin-1beta (IL-1beta)-induced plasma huCRP levels independently of cholesterol lowering. These direct anti-inflammatory in vivo effects occurred at the transcriptional level and could be confirmed in cultured human liver slices and in human hepatoma cells transiently transfected with a huCRP promoter-driven luciferase reporter. A molecular rationale for the suppression of IL-1-induced huCRP transcription is provided by showing that statins and PPARalpha activators up-regulate IkappaBalpha protein expression. This results in a reduced nuclear translocation of p50-nuclear factor kappa B (NFkappaB) and thereby decreased amounts of nuclear p50-NFkappaB approximately CCAAT/enhancer binding protein beta (C/EBPbeta) complexes, which determine the huCRP transcription rate. Our results provide conclusive evidence for a direct suppressive effect of statins and PPARalpha activators on huCRP expression independent of cholesterol lowering and atherogenesis.

Background: Patients treated with peritoneal dialysis frequently suffer from recurrent peritonitis episodes. During peritonitis, inflammatory mediators are released and a serofibrinous exudate is formed in the peritoneal cavity, which promotes fibrosis and abdominal adhesion development. Human peritoneal mesothelial cells (HMC) play a critical role in maintaining the intraperitoneal balance between fibrinolysis and coagulation by expressing the fibrinolytic enzyme tissue-type plasminogen activator (t-PA) and its specific inhibitor, plasminogen activator inhibitor-1 (PAI-1) as well as the procoagulant protein, tissue factor., Methods: Cultured HMC were used to examine the effect of a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, simvastatin, on the expression of t-PA, PAI-1 and tissue factor after activation of the cells with tumor necrosis factor-alpha (TNF-alpha). Antigen concentrations in the cell supernatants were measured by enzyme-linked immunosorbent assay (ELISA). Northern blot analysis was conducted for mRNA expression. Luciferase reporter gene assays and Western blot analysis in human fibrosarcoma HT1080 cells and HMC were performed to analyze the effect of simvastatin on the transcription factors nuclear factor kappa B (NF-kappa B) and activator protein-1 (AP-1), which regulate tissue factor gene expression., Results: Incubation of HMC with TNF-alpha resulted in significantly decreased t-PA and increased PAI-1 synthesis. In the presence of simvastatin t-PA synthesis in control and TNF-alpha-treated cells dose-dependently increased, reaching 5.8-fold and 7.7-fold higher t-PA levels, respectively, at 5 micromol/L simvastatin after 48 hours. Simvastatin dose-dependently suppressed PAI-1 production in both control and TNF-alpha-treated cells. At 5 micromol/L, simvastatin lowered PAI-1 synthesis 3.4-fold and 4.0-fold, respectively, thereby also completely suppressing the TNF-alpha effect itself. Similarly, simvastatin down-regulated the expression of tissue factor and also completely opposed the TNF-alpha-induced tissue factor expression. The effects of simvastatin on t-PA, PAI-1 and tissue factor expression were prevented by mevalonate and geranylgeraniol (GG), suggesting the involvement of geranylgeranyl-modified intermediates in simvastatin's mode of action. Also, simvastatin reduced NF-kappa B- and AP-1-dependent reporter gene activity in TNF-alpha-treated HT-1080 fibrosarcoma cells and reduced the nuclear levels of p50-NF-kappa B, p65-NF-kappa B, and the AP-1 components c-fos and c-jun in HMC., Conclusion: The HMG-CoA reductase inhibitor simvastatin is an effective stimulator of the mesothelial fibrinolytic capacity and suppresses the procoagulant activity both under normal and inflammatory conditions. Our findings provide a molecular explanation for the anti-inflammatory properties of statins in HMC and a rationale for the use of these drugs to protect peritoneal dialysis patients from peritoneal fibrosis and adhesion development during bacterial peritonitis.

C-reactive protein (CRP) is a major acute-phase protein in humans. Elevated plasma CRP levels are a risk factor for cardiovascular disease. CRP is predominantly expressed in hepatocytes and is induced by interleukin-1 (IL-1) and IL-6 under inflammatory situations, such as the acute phase. Fibrates are hypolipidemic drugs that act through the nuclear receptor peroxisome proliferator-activated receptor-alpha (PPAR-alpha). Fibrates have been shown to reduce elevated CRP levels in humans, but the molecular mechanism is unknown. In this study, we demonstrate that different PPAR-alpha activators suppress IL-1-induced, but not IL-6-induced, expression of CRP in primary human hepatocytes and HuH7 hepatoma cells. Induction of CRP expression by IL-1 occurs at the transcriptional level. Site-directed mutagenesis experiments show that IL-1 induces CRP expression through 2 overlapping response elements, the binding sites for CCAAT-box/enhancer-binding protein-beta (C/EBP-beta) and p50-nuclear factor-kappaB (p50-NFkappaB). Cotransfection of C/EBP-beta and p50-NFkappaB enhances CRP promoter activity, and coimmunoprecipitation experiments indicate that the increase in CRP promoter activity by IL-1 is related to the generation and nuclear accumulation of C/EBP-beta-p50-NFkappaB complexes. Interestingly, PPAR-alpha activators reduce the formation of nuclear C/EBP-beta-p50-NFkappaB complexes, and thereby CRP promoter activity, by 2 mechanisms. First, PPAR-alpha increases IkappaB-alpha expression and thus prevents p50-NFkappaB translocation to the nucleus. Second, fibrates decrease hepatic C/EBP-beta and p50-NFkappaB protein levels in mice in a PPAR-alpha-dependent way. Our findings identify C/EBP-beta and p50-NFkappaB as novel targets for PPAR-alpha and provide a molecular explanation for the reduction of plasma CRP levels by fibrates.

Background: The continuous physical and chemical irritation of the peritoneum in peritoneal dialysis patients can result in a nonbacterial serositis with increased fibrin deposition, thus promoting peritoneal fibrosis and adhesion development. By expressing the fibrinolytic enzyme tissue-type plasminogen activator (t-PA) and its specific inhibitor, plasminogen activator inhibitor-1 (PAI-1), human peritoneal mesothelial cells (HMC) play an important role in regulating peritoneal fibrinolysis., Methods: Cultured HMC were used to examine the effect of a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, simvastatin, on the expression of t-PA and PAI-1. Antigen concentrations in the cell supernatants were measured by ELISA and Northern blot analysis was conducted for mRNA expression., Results: Simvastatin time- and concentration-dependently increased t-PA and decreased PAI-1 synthesis, reaching maximal effects after 48 hours, when simvastatin (1 micromol/L) increased t-PA levels 5.1 +/- 0.1-fold and suppressed PAI-1 levels 2.6 +/- 0.2-fold. This was accompanied by a twofold increase in mesothelial cell-associated t-PA activity. Qualitatively similar results were obtained in cultured human endothelial cells, but the effects were less pronounced and required higher simvastatin concentrations. Northern blot analysis revealed that the action of simvastatin on t-PA and PAI-1 expression in HMC can be explained by parallel changes in t-PA and PAI-1 mRNA. The effects of simvastatin were prevented in the presence of mevalonate and geranylgeraniol, suggesting that the effect of simvastatin on t-PA and PAI-1 synthesis is mediated through geranylgeranyl-modified intermediates. Experiments using specific inhibitors of geranylgeranylated Rho GTPases excluded a role of members of this family of small GTP-binding proteins in simvastatin action in HMC. The effects of simvastatin on t-PA and PAI-1 expression as well as on cell shape were completely mimicked by cytochalasin D, a disrupter of cellular actin filaments, but not by colchicine, a disrupter of microtubules., Conclusions: In conclusion, the cholesterol-lowering drug simvastatin is an effective stimulator of local peritoneal fibrinolytic activity, as it increases t-PA and decreases PAI-1 production in mesothelial cells by a mechanism involving geranylgeranyl-modified intermediates and actin skeleton perturbation. These results provide a new rationale to prevent peritoneal fibrin deposition and adhesion development in peritoneal dialysis patients.