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OBJECTIVE:: Selective cyclooxygenase-2 inhibition by rofecoxib was associated with increased risk of cardiovascular events. We hypothesized that concomitant treatment with thromboxane prostanoid receptor antagonist S18886 might ameliorate possible negative effects. We evaluated the effects of S18886, rofecoxib, and the interaction of both compounds in a combined treatment on myocardial infarct (MI) size and cardiac function after experimental ischemia/reperfusion injury in hyperlipidemic APOE*3Leiden transgenic mice. DESIGN:: Prospective, randomized, control trial. SETTING:: Research laboratory. SUBJECTS:: Hyperlipidemic APOE*3Leiden transgenic mice. INTERVENTIONS:: After four weeks of feeding an atherogenic diet, MI was induced by a 30-min ligation of the left anterior descending coronary artery, followed by reperfusion. Oral compound treatment was initiated 90 mins before MI, and continued daily by gavage for seven days. Four treatment groups (n = 12, each) were studied: solvent (Control), S18886, rofecoxib, and S18886 plus rofecoxib. MEASUREMENTS AND MAIN RESULTS:: One week after MI, the mice were anesthetized and cardiac function was quantified by left ventricular (LV) pressure-volume relationships obtained by miniature pressure-conductance catheters. The ischemic area was measured by morphometry and expressed as percentage of LV area. No significant differences in infarct size were found between groups. Compared with control, treatment with S18886 did not affect heart function whereas the rofecoxib group had significantly lower cardiac output (4.5 +/- 0.8 vs. 3.2 +/- 1.1 mL/min, p < 0.01), lower ejection fraction (40 +/- 8 vs. 27 +/- 11%, p < 0.005), and increased end-systolic volume (18.6 +/- 5.7 vs. 28.6 +/- 9.0 muL, p < 0.05). The group with combined (S18886+rofecoxib) treatment was not different from control. Statistical analysis showed significant interactive effects between S18886 and rofecoxib indicating that negative effects of rofecoxib on cardiac function were prevented by S18886 treatment. CONCLUSIONS:: Rofecoxib treatment reduced global and systolic LV function after ischemia-reperfusion injury in APOE*3Leiden mice. These negative effects are prevented by combined treatment with thromboxane prostanoid-receptor antagonist S18886 (terutroban). [ABSTRACT FROM AUTHOR]

Background & Aims: While fibrosis stage predicts liver-associated mortality, cardiovascular disease (CVD) is still the major overall cause of mortality in patients with NASH. Novel NASH drugs should thus ideally reduce both liver fibrosis and CVD. Icosabutate is a semi-synthetic, liver-targeted eicosapentaenoic acid (EPA) derivative in clinical development for NASH. The primary aims of the current studies were to establish both the anti-fibrotic and anti-atherogenic efficacy of icosabutate in conjunction with changes in lipotoxic and atherogenic lipids in liver and plasma respectively., Methods: The effects of icosabutate on fibrosis progression and lipotoxicity were investigated in amylin liver NASH (AMLN) diet (high fat, cholesterol and fructose) fed ob/ob mice with biopsy-confirmed steatohepatitis and fibrosis and compared with the activity of obeticholic acid. APOE*3Leiden.CETP mice, a translational model for hyperlipidaemia and atherosclerosis, were used to evaluate the mechanisms underlying the lipid-lowering effect of icosabutate and its effect on atherosclerosis., Results: In AMLN ob/ob mice, icosabutate significantly reduced hepatic fibrosis and myofibroblast content in association with downregulation of the arachidonic acid cascade and a reduction in both hepatic oxidised phospholipids and apoptosis. In APOE*3Leiden.CETP mice, icosabutate reduced plasma cholesterol and TAG levels via increased hepatic uptake, upregulated hepatic lipid metabolism and downregulated inflammation pathways, and effectively decreased atherosclerosis development., Conclusions: Icosabutate, a structurally engineered EPA derivative, effectively attenuates both hepatic fibrosis and atherogenesis and offers an attractive therapeutic approach to both liver- and CV-related morbidity and mortality in NASH patients., (© 2020 The Authors. Liver International published by John Wiley & Sons Ltd.)

Icosabutate is a structurally engineered eicosapentaenoic acid derivative under development for nonalcoholic steatohepatitis (NASH). In this study, we investigated the absorption and distribution properties of icosabutate in relation to liver targeting and used rodents to evaluate the effects of icosabutate on glucose metabolism, insulin resistance, as well as hepatic steatosis, inflammation, lipotoxicity, and fibrosis. The absorption, tissue distribution, and excretion of icosabutate was investigated in rats along with its effects in mouse models of insulin resistance ( ob/ob ) and metabolic inflammation/NASH (high-fat/cholesterol-fed APOE*3Leiden.CETP mice) and efficacy was compared with synthetic peroxisome proliferator-activated receptor α (PPAR-α) (fenofibrate) and/or PPAR-γ/(α) (pioglitazone and rosiglitazone) agonists. Icosabutate was absorbed almost entirely through the portal vein, resulting in rapid hepatic accumulation. Icosabutate demonstrated potent insulin-sensitizing effects in ob/ob mice, and unlike fenofibrate or pioglitazone, it significantly reduced plasma alanine aminotransferase. In high-fat/cholesterol-fed APOE*3Leiden.CETP mice, icosabutate, but not rosiglitazone, reduced microvesicular steatosis and hepatocellular hypertrophy. Although both rosiglitazone and icosabutate reduced hepatic inflammation, only icosabutate elicited antifibrotic effects in association with decreased hepatic concentrations of multiple lipotoxic lipid species and an oxidative stress marker. Hepatic gene-expression analysis confirmed the changes in lipid metabolism, inflammatory and fibrogenic response, and energy metabolism, and revealed the involved upstream regulators. In conclusion, icosabutate selectively targets the liver through the portal vein and demonstrates broad beneficial effects following insulin sensitivity, hepatic microvesicular steatosis, inflammation, lipotoxicity, oxidative stress, and fibrosis. Icosabutate therefore offers a promising approach to the treatment of both dysregulated glucose/lipid metabolism and inflammatory disorders of the liver, including NASH., (© 2019 The Authors. Hepatology Communications published by Wiley Periodicals, Inc., on behalf of the American Association for the Study of Liver Diseases.)

Antisense oligonucleotides (AONs) are promising candidates for treatment of Duchenne muscular dystrophy (DMD), a severe and progressive disease resulting in premature death. However, more knowledge on the pharmacokinetics of new AON drug candidates is desired for effective application in the clinic. We assessed the feasibility of using noninvasive single-photon emission computed tomography-computed tomography (SPECT-CT) imaging to determine AON pharmacokinetics in vivo. To this end, a 2'-O-methyl phosphorothioate AON was radiolabeled with 123 I or 111 In, and administered to mdx mice, a rodent model of DMD. SPECT-CT imaging was performed to determine AON tissue levels, and the results were compared to data obtained with invasive analysis methods (scintillation counting and a ligation-hybridization assay). We found that SPECT-CT data obtained with 123 I-AON and 111 In-AON were qualitatively comparable to data derived from invasive analytical methods, confirming the feasibility of using SPECT-CT analysis to study AON pharmacokinetics. Notably, also AON uptake in skeletal muscle, the target tissue in DMD, could be readily quantified using SPECT-CT imaging, which was considered a particular challenge in mice, due to their small size. In conclusion, our results demonstrate that SPECT-CT imaging allows for noninvasive characterization of biodistribution and pharmacokinetics of AONs, thereby enabling quantitative comparisons between different radiolabeled AON drug candidates and qualitative conclusions about the corresponding unmodified parent AONs. This technology may contribute to improved (pre)clinical drug development, leading to drug candidates with optimized characteristics in vivo.

Background and Aims: High-density lipoprotein cholesterol (HDL-C) is inversely related to cardiovascular risk. HDL-C raising ester transfer protein (CETP) inhibitors, are novel therapeutics. We studied the effects of CETP inhibitors anacetrapib and evacetrapib on triglycerides, cholesterol and lipoproteins, cholesterol efflux, paraoxonase activity (PON-1), reactive oxygen species (ROS), and endothelial function in E3L and E3L.CETP mice., Methods: Triglycerides and cholesterol were measured at weeks 5, 14 and 21 in E3L.CETP mice on high cholesterol diet and treated with anacetrapib (3 mg/kg/day), evacetrapib (3 mg/kg/day) or placebo. Cholesterol efflux was assessed ex-vivo in mice treated with CETP inhibitors for 3 weeks on a normal chow diet. Endothelial function was analyzed at week 21 in isolated aortic rings, and serum lipoproteins assessed by fast-performance liquid chromatography., Results: Anacetrapib and evacetrapib increased HDL-C levels (5- and 3.4-fold, resp.) and reduced triglycerides (-39% vs. placebo, p = 0.0174). Total cholesterol levels were reduced only in anacetrapib-treated mice (-32%, p = 0.0386). Cholesterol efflux and PON-1 activity (+45% and +35% vs. control, p < 0.005, resp.) were increased, while aortic ROS production was reduced with evacetrapib (-49% vs. control, p = 0.020). Anacetrapib, but not evacetrapib, impaired endothelium dependent vasorelaxation (p < 0.05). In contrast, no such effects were observed in E3L mice for all parameters tested., Conclusions: Notwithstanding a marked rise in HDL-C, evacetrapib did not improve endothelial function, while anacetrapib impaired it, suggesting that CETP inhibition does not provide vascular protection. Anacetrapib exerts unfavorable endothelial effects beyond CETP inhibition, which may explain the neutral results of large clinical trials in spite of increased HDL-C., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background and Purpose: Salsalate (salicylsalicylic acid) is an anti-inflammatory drug that was recently found to exert beneficial metabolic effects on glucose and lipid metabolism. Although its utility in the prevention and management of a wide range of vascular disorders, including type 2 diabetes and metabolic syndrome has been suggested before, the potential of salsalate to protect against non-alcoholic steatohepatitis (NASH) remains unclear. The aim of the present study was therefore to ascertain the effects of salsalate on the development of NASH., Experimental Approach: Transgenic APOE*3Leiden.CETP mice were fed a high-fat and high-cholesterol diet with or without salsalate for 12 and 20 weeks. The effects on body weight, plasma biochemical variables, liver histology and hepatic gene expression were assessed., Key Results: Salsalate prevented weight gain, improved dyslipidemia and insulin resistance and ameliorated diet-induced NASH, as shown by decreased hepatic microvesicular and macrovesicular steatosis, reduced hepatic inflammation and reduced development of fibrosis. Salsalate affected lipid metabolism by increasing β-oxidation and decreasing lipogenesis, as shown by the activation of PPAR-α, PPAR-γ co-activator 1β, RXR-α and inhibition of genes controlled by the transcription factor MLXIPL/ChREBP. Inflammation was reduced by down-regulation of the NF-κB pathway, and fibrosis development was prevented by down-regulation of TGF-β signalling., Conclusions and Implications: Salsalate exerted a preventive effect on the development of NASH and progression to fibrosis. These data suggest a clinical application of salsalate in preventing NASH., (© 2015 The British Pharmacological Society.)

Recently, we showed in APOE*3-Leiden cholesteryl ester transfer protein (E3L.CETP) mice that anacetrapib attenuated atherosclerosis development by reducing (V)LDL cholesterol [(V)LDL-C] rather than by raising HDL cholesterol. Here, we investigated the mechanism by which anacetrapib reduces (V)LDL-C and whether this effect was dependent on the inhibition of CETP. E3L.CETP mice were fed a Western-type diet alone or supplemented with anacetrapib (30 mg/kg body weight per day). Microarray analyses of livers revealed downregulation of the cholesterol biosynthesis pathway (P < 0.001) and predicted downregulation of pathways controlled by sterol regulatory element-binding proteins 1 and 2 (z-scores -2.56 and -2.90, respectively; both P < 0.001). These data suggest increased supply of cholesterol to the liver. We found that hepatic proprotein convertase subtilisin/kexin type 9 (Pcsk9) expression was decreased (-28%, P < 0.01), accompanied by decreased plasma PCSK9 levels (-47%, P < 0.001) and increased hepatic LDL receptor (LDLr) content (+64%, P < 0.01). Consistent with this, anacetrapib increased the clearance and hepatic uptake (+25%, P < 0.001) of [(14)C]cholesteryl oleate-labeled VLDL-mimicking particles. In E3L mice that do not express CETP, anacetrapib still decreased (V)LDL-C and plasma PCSK9 levels, indicating that these effects were independent of CETP inhibition. We conclude that anacetrapib reduces (V)LDL-C by two mechanisms: 1) inhibition of CETP activity, resulting in remodeled VLDL particles that are more susceptible to hepatic uptake; and 2) a CETP-independent reduction of plasma PCSK9 levels that has the potential to increase LDLr-mediated hepatic remnant clearance., (Copyright © 2015 by the American Society for Biochemistry and Molecular Biology, Inc.)

Introduction: The efflux transporters P-glycoprotein (P-gp, ABCB1) and breast cancer resistance protein (BCRP, ABCG2) are expressed at the blood-brain barrier (BBB), and can limit the access of a wide range of drugs to the brain. In this study we developed a PET-CT imaging method for non-invasive, quantitative analysis of the effect of ABCB1 and ABCG2 on brain penetration of the anti-cancer drug gefitinib, and demonstrated the applicability of this method for identification and quantification of potential modulators of ABCB1 and ABCB2 using the dual inhibitor elacridar., Methods: In vitro cellular accumulation studies with [(14)C]-gefitinib were conducted in LLC-PK1, MDCKII, and the corresponding ABCB1/Abcb1a and ABCG2/Abcg2 overexpressing cell lines. Subsequently, in vivo brain penetration of [(18)F]-gefitinib was quantified by PET-CT imaging studies in wild-type, Abcg2(-/-), Abcb1a/1b(-/-), and Abcb1a/1b;Abcg2(-/-) mice., Results: In vitro studies showed that [(14)C]-gefitinib is a substrate of the human ABCB1 and ABCG2 transporters. After i.v. administration of [(18)F]-gefitinib (1mg/kg), PET-CT imaging showed 2.3-fold increased brain levels of [(18)F]-gefitinib in Abcb1a/1b;Abcg2(-/-) mice, compared to wild-type. Levels in single knockout animals were not different from wild-type, showing that Abcb1a/1b and Abcg2 together limit access of [(18)F]-gefitinib to the brain. Furthermore, enhanced brain accumulation of [(18)F]-gefitinib after administration of the ABCB1 and ABCG2 inhibitor elacridar (10 mg/kg) could be quantified with PET-CT imaging., Conclusions: PET-CT imaging with [(18)F]-gefitinib is a powerful tool to non-invasively assess potential ABCB1- and ABCG2-mediated drug-drug interactions (DDIs) in vivo., Advances in Knowledge and Implications for Patient Care: This minimally-invasive, [(18)F]-based PET-CT imaging method shows the interplay of ABCB1 and ABCG2 at the BBB in vivo. The method may be applied in the future to assess ABCB1 and ABCG2 activity at the BBB in humans, and for personalized treatment with drugs that are substrates of ABCB1 and/or ABCG2., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Non-HDL-cholesterol is well recognised as a primary causal risk factor in cardiovascular disease. However, despite consistent epidemiological evidence for an inverse association between HDL-C and coronary heart disease, clinical trials aimed at raising HDL-C (AIM-HIGH, HPS2-THRIVE, dal-OUTCOMES) failed to meet their primary goals. This systematic review and meta-analysis investigated the effects of established and novel treatment strategies, specifically targeting HDL, on inhibition of atherosclerosis in cholesteryl ester transfer protein-expressing animals, and the prevention of clinical events in randomised controlled trials. Linear regression analyses using data from preclinical studies revealed associations for TC and non-HDL-C and lesion area (R(2)=0.258, P=0.045; R(2)=0.760, P<0.001), but not for HDL-C (R(2)=0.030, P=0.556). In clinical trials, non-fatal myocardial infarction risk was significantly less in the treatment group with pooled odd ratios of 0.87 [0.81; 0.94] for all trials and 0.85 [0.78; 0.93] after excluding some trials due to off-target adverse events, whereas all-cause mortality was not affected (OR 1.05 [0.99-1.10]). Meta-regression analyses revealed a trend towards an association between between-group differences in absolute change from baseline in LDL-C and non-fatal myocardial infarction (P=0.066), whereas no correlation was found for HDL-C (P=0.955). We conclude that the protective role of lowering LDL-C and non-HDL-C is well-established. The contribution of raising HDL-C on inhibition of atherosclerosis and the prevention of cardiovascular disease remains undefined and may be dependent on the mode of action of HDL-C-modification. Nonetheless, treatment strategies aimed at improving HDL function and raising apolipoprotein A-I may be worth exploring., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background: The residual risk that remains after statin treatment supports the addition of other LDL-C-lowering agents and has stimulated the search for secondary treatment targets. Epidemiological studies propose HDL-C as a possible candidate. Cholesteryl ester transfer protein (CETP) transfers cholesteryl esters from atheroprotective HDL to atherogenic (V)LDL. The CETP inhibitor anacetrapib decreases (V)LDL-C by ∼15-40% and increases HDL-C by ∼40-140% in clinical trials. We evaluated the effects of a broad dose range of anacetrapib on atherosclerosis and HDL function, and examined possible additive/synergistic effects of anacetrapib on top of atorvastatin in APOE*3Leiden.CETP mice., Methods and Results: Mice were fed a diet without or with ascending dosages of anacetrapib (0.03; 0.3; 3; 30 mg/kg/day), atorvastatin (2.4 mg/kg/day) alone or in combination with anacetrapib (0.3 mg/kg/day) for 21 weeks. Anacetrapib dose-dependently reduced CETP activity (-59 to -100%, P < 0.001), thereby decreasing non-HDL-C (-24 to -45%, P < 0.001) and increasing HDL-C (+30 to +86%, P < 0.001). Anacetrapib dose-dependently reduced the atherosclerotic lesion area (-41 to -92%, P < 0.01) and severity, increased plaque stability index and added to the effects of atorvastatin by further decreasing lesion size (-95%, P < 0.001) and severity. Analysis of covariance showed that both anacetrapib (P < 0.05) and non-HDL-C (P < 0.001), but not HDL-C (P = 0.76), independently determined lesion size., Conclusion: Anacetrapib dose-dependently reduces atherosclerosis, and adds to the anti-atherogenic effects of atorvastatin, which is mainly ascribed to a reduction in non-HDL-C. In addition, anacetrapib improves lesion stability., (© The Author 2014. Published by Oxford University Press on behalf of the European Society of Cardiology.)

LDL cholesterol (LDL-C) contributes to coronary heart disease. Proprotein convertase subtilisin/kexin type 9 (PCSK9) increases LDL-C by inhibiting LDL-C clearance. The therapeutic potential for PCSK9 inhibitors is highlighted by the fact that PCSK9 loss-of-function carriers exhibit 15-30% lower circulating LDL-C and a disproportionately lower risk (47-88%) of experiencing a cardiovascular event. Here, we utilized pcsk9(-/-) mice and an anti-PCSK9 antibody to study the role of the LDL receptor (LDLR) and ApoE in PCSK9-mediated regulation of plasma cholesterol and atherosclerotic lesion development. We found that circulating cholesterol and atherosclerotic lesions were minimally modified in pcsk9(-/-) mice on either an LDLR- or ApoE-deficient background. Acute administration of an anti-PCSK9 antibody did not reduce circulating cholesterol in an ApoE-deficient background, but did reduce circulating cholesterol (-45%) and TGs (-36%) in APOE*3Leiden.cholesteryl ester transfer protein (CETP) mice, which contain mouse ApoE, human mutant APOE3*Leiden, and a functional LDLR. Chronic anti-PCSK9 antibody treatment in APOE*3Leiden.CETP mice resulted in a significant reduction in atherosclerotic lesion area (-91%) and reduced lesion complexity. Taken together, these results indicate that both LDLR and ApoE are required for PCSK9 inhibitor-mediated reductions in atherosclerosis, as both are needed to increase hepatic LDLR expression., (Copyright © 2014 by the American Society for Biochemistry and Molecular Biology, Inc.)

Background: Toll-like receptor-4 (TLR4), a receptor of the innate immune system, is suggested to have detrimental effects on cardiac function after myocardial infarction (MI). RP105 (CD180) is a TLR4 homolog lacking the intracellular signaling domain that competitively inhibits TLR4-signaling. Thus, we hypothesized that RP105 deficiency, by amplifying TLR4 signaling, would lead to aggravated cardiac dysfunction after MI., Methods and Results: First, whole blood from RP105-/- and wild-type (WT) male C57Bl/6N mice was stimulated with LPS, which induced a strong inflammatory TNFα response in RP105-/- mice. Then, baseline heart function was assessed by left ventricular pressure-volume relationships which were not different between RP105-/- and WT mice. Permanent ligation of the left anterior descending coronary artery was performed to induce MI. Infarct sizes were analyzed by (immuno)histology and did not differ. Fifteen days post MI heart function was assessed and RP105-/- mice had significantly higher heart rate (+21%, P<0.01), end systolic volume index (+57%, P<0.05), end systolic pressure (+22%, P<0.05) and lower relaxation time constant tau (-12%, P<0.05), and a tendency for increased end diastolic volume index (+42%, P<0.06), compared to WT mice. In the area adjacent to the infarct zone, compared to the healthy myocardium, levels of RP105, TLR4 and the endogenous TLR4 ligand fibronectin-EDA were increased as well as the number of macrophages, however this was not different between both groups., Conclusion: Deficiency of the endogenous TLR4 inhibitor RP105 leads to an enhanced inflammatory status and more pronounced cardiac dilatation after induction of MI, underscoring the role of the TLR4 pathway in post-infarction remodeling., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibition is a potential novel strategy for treatment of CVD. Alirocumab is a fully human PCSK9 monoclonal antibody in phase 3 clinical development. We evaluated the antiatherogenic potential of alirocumab in APOE*3Leiden.CETP mice. Mice received a Western-type diet and were treated with alirocumab (3 or 10 mg/kg, weekly subcutaneous dosing) alone and in combination with atorvastatin (3.6 mg/kg/d) for 18 weeks. Alirocumab alone dose-dependently decreased total cholesterol (-37%; -46%, P < 0.001) and TGs (-36%; -39%, P < 0.001) and further decreased cholesterol in combination with atorvastatin (-48%; -58%, P < 0.001). Alirocumab increased hepatic LDL receptor protein levels but did not affect hepatic cholesterol and TG content. Fecal output of bile acids and neutral sterols was not changed. Alirocumab dose-dependently decreased atherosclerotic lesion size (-71%; -88%, P < 0.001) and severity and enhanced these effects when added to atorvastatin (-89%; -98%, P < 0.001). Alirocumab reduced monocyte recruitment and improved the lesion composition by increasing the smooth muscle cell and collagen content and decreasing the macrophage and necrotic core content. Alirocumab dose-dependently decreases plasma lipids and, as a result, atherosclerosis development, and it enhances the beneficial effects of atorvastatin in APOE*3Leiden.CETP mice. In addition, alirocumab improves plaque morphology., (Copyright © 2014 by the American Society for Biochemistry and Molecular Biology, Inc.)

Unlabelled: The experimental autoimmune encephalomyelitis model is a model of multiple sclerosis that closely mimics the disease characteristics in humans. The main hallmarks of multiple sclerosis are neuroinflammation (microglia activation, monocyte invasion, and T-cell infiltration) and demyelination. PET imaging may be a useful noninvasive technique for monitoring disease progression and drug treatment efficacy in vivo., Methods: Experimental autoimmune encephalomyelitis was induced by myelin-oligodendrocyte glycoprotein immunization in female Dark Agouti rats. Experimental autoimmune encephalomyelitis rats were imaged at baseline and at days 6, 11, 15, and 19 after immunization to monitor monocyte and microglia activation ((11)C-PK11195) and demyelination ((11)C-MeDAS) during normal disease progression and during treatment with dexamethasone., Results: (11)C-PK11195 PET detected activation of microglia and monocytes in the brain stem and spinal cord during disease progression. The uptake of (11)C-PK11195 was elevated in dexamethasone-treated animals that had shown mild clinical symptoms that had resolved at the time of imaging. Demyelination was not detected by (11)C-MeDAS PET, probably because of the small size of the lesions (average, 0.13 mm)., Conclusion: PET imaging of neuroinflammation can be used to monitor disease progression and the consequences of treatment in the experimental autoimmune encephalomyelitis rat model. PET imaging was more sensitive than clinical symptoms for detecting inflammatory changes in the central nervous system., (© 2014 by the Society of Nuclear Medicine and Molecular Imaging, Inc.)

Aims: This study aimed to investigate systematically (i) the appropriate dietary conditions to induce the features of the MetS in APOE*3Leiden.humanCholesteryl Ester Transfer Protein (E3L.CETP) mice and (ii) whether the response of this model to different antidiabetic and hypolipidemic drugs is similar as in humans., Methods: Male obese, IR and dyslipidemic E3L.CETP mice were treated with antidiabetic drugs rosiglitazone, liraglutide or an experimental 11β-hydroxysteroid-dehydrogenase-1 (HSD-1) inhibitor, or with hypolipidemic drugs atorvastatin, fenofibrate or niacin for 4-6 weeks. The effects on bw, IR and plasma and liver lipids were assessed., Results: Rosiglitazone, liraglutide and HSD-1 inhibitor significantly decreased glucose and insulin levels or IR. Liraglutide and HSD-1 inhibitor also decreased bw. Atorvastatin, fenofibrate and niacin improved the dyslipidemia and fenofibrate and niacin increased high-density lipoprotein (HDL) cholesterol. In addition, hepatic triglycerides were significantly decreased by treatment with rosiglitazone and liraglutide, while hepatic cholesterol esters were significantly decreased by rosiglitazone and atorvastatin., Conclusions: We conclude that the E3L.CETP mouse is a promising novel translational model to investigate the effects of new drugs, alone or in combination, that affect IR, diabetic dyslipidemia and non-alcoholic fatty liver disease (NAFLD)., (© 2013 John Wiley & Sons Ltd.)

Resveratrol is a major constituent of traditional Asian medicinal herbs and red wine and is suggested to be a potential antiatherosclerotic drug due to its proposed hypolipidemic, anti-inflammatory and antioxidative properties. The aim of this study was to evaluate whether resveratrol protects against atherosclerosis development in APOE*3-Leiden.CETP (E3L.CETP) mice and adds to the antiatherogenic effect of mild statin treatment, currently the most widely used antiatherogenic therapy. E3L.CETP mice were fed a cholesterol-rich diet without (control) or with resveratrol (0.01% w/w), atorvastatin (0.0027% w/w) or both for 14 weeks. During the study plasma lipid, inflammatory and oxidative stress parameters were determined. Resveratrol reduced atherosclerotic lesion area (-52%) in the aortic root, comparable to atorvastatin (-40%) and the combination of both drugs (-47%). The collagen/macrophage ratio in the atherosclerotic lesion, a marker of plaque stability, was increased by resveratrol (+108%), atorvastatin (+124%) and the combination (+154%). Resveratrol decreased plasma cholesterol levels (-19%) comparable to atorvastatin (-19%) and the combination (-22%), which was completely confined to (very)low-density lipoprotein cholesterol levels in all groups. Post hoc analyses showed that the antiatherogenic effect of atorvastatin could be explained by cholesterol lowering, while the antiatherosclerotic effect of resveratrol could be attributed to factors additional to cholesterol lowering. Markers of inflammation and oxidative stress were not different, but resveratrol improved macrophage function. We conclude that resveratrol potently reduces atherosclerosis development and induces a more stable lesion phenotype in E3L.CETP mice. However, under the experimental conditions tested, resveratrol does not add to the antiatherogenic effect of atorvastatin., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Objective: Niacin potently lowers triglycerides, mildly decreases LDL-cholesterol, and largely increases HDL-cholesterol. Despite evidence for an atheroprotective effect of niacin from previous small clinical studies, the large outcome trials, AIM-HIGH and HPS2-THRIVE did not reveal additional beneficial effects of niacin (alone or in combination with laropiprant) on top of statin treatment. We aimed to address this apparent discrepancy by investigating the effects of niacin without and with simvastatin on atherosclerosis development and determine the underlying mechanisms, in APOE*3Leiden.CETP mice, a model for familial dysbetalipoproteinemia (FD)., Approach and Results: Mice were fed a western-type diet containing cholesterol without or with niacin (120 mg/kg/day), simvastatin (36 mg/kg/day) or their combination for 18 weeks. Similarly as in FD patients, niacin reduced total cholesterol by -39% and triglycerides by -50%, (both P<0.001). Simvastatin and the combination reduced total cholesterol (-30%; -55%, P<0.001) where the combination revealed a greater reduction compared to simvastatin (-36%, P<0.001). Niacin decreased total cholesterol and triglycerides primarily by increasing VLDL clearance. Niacin increased HDL-cholesterol (+28%, P<0.01) and mildly increased reverse cholesterol transport. All treatments reduced monocyte adhesion to the endothelium (-46%; -47%, P<0.01; -53%, P<0.001), atherosclerotic lesion area (-78%; -49%, P<0.01; -87%, P<0.001) and severity. Compared to simvastatin, the combination increased plaque stability index [(SMC+collagen)/macrophages] (3-fold, P<0.01). Niacin and the combination reduced T cells in the aortic root (-71%, P<0.01; -81%, P<0.001). Lesion area was strongly predicted by nonHDL-cholesterol (R(2) = 0.69, P<0.001) and to a much lesser extent by HDL-cholesterol (R(2) = 0.20, P<0.001)., Conclusion: Niacin decreases atherosclerosis development mainly by reducing nonHDL-cholesterol with modest HDL-cholesterol-raising and additional anti-inflammatory effects. The additive effect of niacin on top of simvastatin is mostly dependent on its nonHDL-cholesterol-lowering capacities. These data suggest that clinical beneficial effects of niacin are largely dependent on its ability to lower LDL-cholesterol on top of concomitant lipid-lowering therapy.

The anti-dyslipidemic drug niacin has recently been shown to reduce the hepatic expression and plasma levels of CETP. Since liver macrophages contribute to hepatic CETP expression, we investigated the role of macrophages in the CETP-lowering effect of niacin in mice. In vitro studies showed that niacin does not directly attenuate CETP expression in macrophages. Treatment of normolipidemic human CETP transgenic mice, fed a Western-type diet with niacin for 4 weeks, significantly reduced the hepatic cholesterol concentration (-20%), hepatic CETP gene expression (-20%), and plasma CETP mass (-30%). Concomitantly, niacin decreased the hepatic expression of CD68 (-44%) and ABCG1 (-32%), both of which are specific markers for the hepatic macrophage content. The decrease in hepatic CETP expression was significantly correlated with the reduction of hepatic macrophage markers. Furthermore, niacin attenuated atherogenic diet-induced inflammation in liver, as evident from decreased expression of TNF-alpha (-43%). Niacin similarly decreased the macrophage markers and absolute macrophage content in hyperlipidemic APOE*3-Leiden.CETP transgenic mice on a Western-type diet. In conclusion, niacin decreases hepatic CETP expression and plasma CETP mass by attenuating liver inflammation and macrophage content in response to its primary lipid-lowering effect, rather than by attenuating the macrophage CETP expression level., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Increased availability of fatty acids released from insulin-resistant adipose tissue may lead to excess fatty acid uptake in nonadipose organs, including the heart. Accumulation of toxic fatty acid intermediates may affect cardiac function. Our aim was to identify to which extent high-fat diet feeding leads to alterations in cardiac function and whether this depends on gender and (or) duration of high-fat diet feeding. Male and female C57Bl/6J mice (n = 8 per group) of 12 to 16 weeks old were fed a low-fat (10% energy) or high-fat (45% energy) lard diet for 6 or 12 weeks. Plasma lipid levels, echocardiography, and left ventricular pressure-volume relationships were obtained at 2, 1, and 0 weeks before termination, respectively. In both male and female mice, the high-fat diet increased body weight and plasma lipid content. At 10 weeks, significant increases were observed for plasma total cholesterol (males: +44%; females: +86%), phospholipids (+16% and +34%), and triglycerides (+27% and +53%) (all p < 0.001). In male mice, but not in female mice, the high-fat diet significantly affected cardiac function at 12 weeks with increased end-systolic volume (25.4 ± 6.2 vs. 17.0 ± 6.7 µL, p < 0.05), increased end-systolic pressure (72.1 ± 6.9 vs. 63.6 ± 6.9 mm Hg, p < 0.01), and decreased ejection fraction (61.2% ± 4.5% vs. 68.1% ± 3.7%, p < 0.01), indicating reduced systolic function. Multiple linear regression analysis indicated a significant diet-gender interaction for end-systolic volume and ejection fraction. In conclusion, high-fat diet feeding increased body weight and plasma lipid levels in male and in female mice, but resulted in impairment of cardiac function only in males.

Objectives: To validate near-infrared (NIR)-based optical spectroscopy measurements of hepatic fat content using a minimally invasive needle-like probe with integrated optical fibers, enabling real-time feedback during percutaneous interventions. The results were compared with magnetic resonance spectroscopy (MRS) as validation and with histopathology, being the clinical gold standard. Additionally, ex vivo magic angle spinning nuclear magnetic resonance spectroscopy and high-performance thin-layer chromatography were performed for comparison., Materials and Methods: Ten mice were used for the study, of which half received a regular chow diet and the other half received a high-fat diet to induce obesity and hepatosteatosis. The mice were imaged with a clinical 3-Tesla MR to select a region of interest within the right and left lobes of the liver, where MRS measurements were acquired in vivo. Subsequently, optical spectra were measured ex vivo at the surface of the liver at 6 different positions immediately after resection. Additionally, hepatic fat was determined by magic angle spinning nuclear magnetic resonance spectroscopy and high-performance thin-layer chromatography. Histopathologic analyses were performed and used as the reference standard. Pearson correlation and linear regression analyses were performed to assess the correlation of the various techniques with NIR. A 1-way analysis of variance including post hoc Tukey multiple comparison tests was used to study the difference in fat estimation between the various techniques., Results: For both the mice groups, the estimated fat fractions by the various techniques were significantly similar (P = 0.072 and 0.627 for chow diet and high-fat diet group, respectively). The Pearson correlation value between NIR and the other techniques for fat determination showed the same strong linear correlation (P above 0.990; P < 0.001), whereas for histopathologic analyses, which is a rather qualitative measure, the Pearson correlation value was slightly lower (P = 0.925, P < 0.001) . Linear regression coefficient computed to compare NIR with the other techniques resulted in values close to unity with MRS having the narrowest confidence interval (r = 0.935, 95% confidence interval: 0.860-1.009), demonstrating highly correlating results between NIR and MRS., Conclusions: NIR spectroscopy measurements from a needle-like probe with integrated optical fibers for sensing at the tip of the needle can quickly and accurately determine hepatic fat content during an interventional procedure and might therefore be a promising novel diagnosing tool in the clinic.

Background: Remote ischaemic preconditioning (RIPC) is a strategy to protect a target organ against ischaemia-reperfusion injury (IRI) by inducing short-term ischaemia/reperfusion (I/R) in a remote organ. RIPC of the kidney by temporary limb occlusion would be a safe, inexpensive and noninvasive method to prevent renal damage in, e.g., transplantation and aortic surgery. We investigated whether brief hind limb occlusion can protect against renal IRI and whether this protection is adenosine dependent., Methods: Rats underwent either no RIPC, unilateral RIPC or bilateral RIPC. The preconditioning stimulus was either continuous (12'/12' I/R) or fractionated (three times 4'/4' I/R). After the last reperfusion period, we induced 25' ischaemia in the right kidney., Results: After 24 h of reperfusion, renal function was improved by 30-60% in both bilateral RIPC groups and in the fractionated unilateral group. Renal tubule damage and kidney injury molecule-1 expression were reduced in three of four RIPC groups. Treatment with the adenosine receptor blocker 8-(p-sulfophenyl)theophylline had no effect on fractionated or continuous RIPC., Conclusions: Brief hind limb ischaemia induces protection against renal IRI, which makes this a promising strategy to prevent renal IRI in a clinical setting. Bilateral RIPC was more effective than unilateral RIPC, and this protection occurs via an adenosine-independent mechanism.

Perfluorobutane sulfonate (PFBS), perfluorohexane sulfonate (PFHxS), and perfluorooctane sulfonate (PFOS) are stable perfluoroalkyl sulfonate (PFAS) surfactants, and PFHxS and PFOS are frequently detected in human biomonitoring studies. Some epidemiological studies have shown modest positive correlations of serum PFOS with non-high-density lipoprotein (HDL)-cholesterol (C). This study investigated the mechanism underlying the effect of PFAS surfactants on lipoprotein metabolism. APOE*3-Leiden.CETP mice were fed a Western-type diet with PFBS, PFHxS, or PFOS (30, 6, and 3 mg/kg/day, respectively) for 4-6 weeks. Whereas PFBS modestly reduced only plasma triglycerides (TG), PFHxS and PFOS markedly reduced TG, non-HDL-C, and HDL-C. The decrease in very low-density lipoprotein (VLDL) was caused by enhanced lipoprotein lipase-mediated VLDL-TG clearance and by decreased production of VLDL-TG and VLDL-apolipoprotein B. Reduced HDL production, related to decreased apolipoprotein AI synthesis, resulted in decreased HDL. PFHxS and PFOS increased liver weight and hepatic TG content. Hepatic gene expression profiling data indicated that these effects were the combined result of peroxisome proliferator-activated receptor alpha and pregnane X receptor activation. In conclusion, the potency of PFAS to affect lipoprotein metabolism increased with increasing alkyl chain length. PFHxS and PFOS reduce plasma TG and total cholesterol mainly by impairing lipoprotein production, implying that the reported positive correlations of serum PFOS and non-HDL-C are associative rather than causal.

The peroxisome proliferator-activated receptor alpha (PPARalpha) activator fenofibrate efficiently decreases plasma triglycerides (TG), which is generally attributed to enhanced very low density lipoprotein (VLDL)-TG clearance and decreased VLDL-TG production. However, because data on the effect of fenofibrate on VLDL production are controversial, we aimed to investigate in (more) detail the mechanism underlying the TG-lowering effect by studying VLDL-TG production and clearance using APOE*3-Leiden.CETP mice, a unique mouse model for human-like lipoprotein metabolism. Male mice were fed a Western-type diet for 4 weeks, followed by the same diet without or with fenofibrate (30 mg/kg bodyweight/day) for 4 weeks. Fenofibrate strongly lowered plasma cholesterol (-38%) and TG (-60%) caused by reduction of VLDL. Fenofibrate markedly accelerated VLDL-TG clearance, as judged from a reduced plasma half-life of glycerol tri[(3)H]oleate-labeled VLDL-like emulsion particles (-68%). This was associated with an increased post-heparin lipoprotein lipase (LPL) activity (+110%) and an increased uptake of VLDL-derived fatty acids by skeletal muscle, white adipose tissue, and liver. Concomitantly, fenofibrate markedly increased the VLDL-TG production rate (+73%) but not the VLDL-apolipoprotein B (apoB) production rate. Kinetic studies using [(3)H]palmitic acid showed that fenofibrate increased VLDL-TG production by equally increasing incorporation of re-esterified plasma fatty acids and liver TG into VLDL, which was supported by hepatic gene expression profiling data. We conclude that fenofibrate decreases plasma TG by enhancing LPL-mediated VLDL-TG clearance, which results in a compensatory increase in VLDL-TG production by the liver.

A common dose-limiting side effect of treatment with the retinoid X receptor agonist bexarotene is dyslipidemia. We evaluated the effects of bexarotene on plasma lipid metabolism in patients with metastatic differentiated thyroid carcinoma and investigated the underlying mechanism(s) in apolipoprotein (APO) E*3-Leiden mice without (E3L) and with human cholesteryl ester transfer protein (CETP; E3L.CETP). To this end, 10 patients with metastatic differentiated thyroid carcinoma were treated with bexarotene (300 mg/d) for 6 wk. Bexarotene increased plasma triglyceride (TG; +150%), primarily associated with very low-density lipoprotein (VLDL), and raised plasma total cholesterol (+50%). However, whereas bexarotene increased VLDL-cholesterol (C) and low-density lipoprotein (LDL)-C (+63%), it decreased high-density lipoprotein (HDL)-C (-30%) and tended to decrease apoAI (-18%) concomitant with an increase in endogenous CETP activity (+44%). To evaluate the cause of the bexarotene-induced hypertriglyceridemia and the role of CETP in the bexarotene-induced shift in cholesterol distribution, E3L and E3L.CETP mice were treated with bexarotene through dietary supplementation [0.03% (wt/wt)]. Bexarotene increased VLDL-associated TG in both E3L (+47%) and E3L.CETP (+29%) mice by increasing VLDL-TG production (+68%). Bexarotene did not affect the total cholesterol levels or distribution in E3L mice but increased VLDL-C (+11%) and decreased HDL-C (-56%) as well as apoAI (-31%) in E3L.CETP mice, concomitant with increased endogenous CETP activity (+41%). This increased CETP activity by bexarotene-treatment is likely due to the increase in VLDL-TG, a CETP substrate that drives CETP activity. In conclusion, bexarotene causes combined dyslipidemia as reflected by increased TG, VLDL-C, and LDL-C and decreased HDL-C, which is the result of an increased VLDL-TG production that causes an increase of the endogenous CETP activity.

Background and Purpose: We have evaluated the effects of a peroxisome proliferator-activated receptor (PPAR)alpha/gamma agonist on the progression of pre-existing atherosclerotic lesions in APOE*3Leiden.cholesteryl ester transfer protein (E3L.CETP) transgenic mice., Experimental Approach: E3L.CETP mice were fed a high-cholesterol diet for 11 weeks to induce atherosclerosis, followed by a low-cholesterol diet for 4 weeks to obtain a lower plasma total cholesterol level of approximately 10 mmol.L(-1). Mice were divided into three groups, which were either killed before (baseline) or after an 8 week treatment period with low-cholesterol diet without (control) or with the PPARalpha/gamma agonist tesaglitazar (10 microg.kg(-1).day(-1)). Atherosclerosis was assessed in the aortic root., Key Results: Treatment with tesaglitazar significantly reduced plasma triglycerides, total cholesterol, CETP mass and CETP activity, and increased high-density lipoprotein-cholesterol. At baseline, substantial atherosclerosis had developed. During the 8 week low-cholesterol diet, atherosclerosis progressed in the control group with respect to lesion area and severity, whereas tesaglitazar inhibited lesion progression during this period. Tesaglitazar reduced vessel wall inflammation, as reflected by decreased monocyte adhesion and macrophage area, and modified lesions to a more stabilized phenotype, with increased smooth muscle cell content in the cap and collagen content., Conclusions and Implications: Dual PPARalpha/gamma agonism with tesaglitazar markedly improved the atherogenic triad by reducing triglycerides and very low-density lipoprotein-cholesterol and increasing high-density lipoprotein-cholesterol and additionally reduced cholesterol-induced vessel wall activation. These actions resulted in complete inhibition of progression and stabilization of pre-existing atherosclerotic lesions in E3L.CETP mice.

Objective: Niacin potently decreases plasma triglycerides and LDL-cholesterol. In addition, niacin is the most potent HDL-cholesterol-increasing drug used in the clinic. In the present study, we aimed at elucidation of the mechanism underlying its HDL-raising effect., Methods and Results: In APOE*3Leiden transgenic mice expressing the human CETP transgene, niacin dose-dependently decreased plasma triglycerides (up to -77%, P<0.001) and total cholesterol (up to -66%, P<0.001). Concomitantly, niacin dose-dependently increased HDL-cholesterol (up to +87%, P<0.001), plasma apoAI (up to +72%, P<0.001), as well as the HDL particle size. In contrast, in APOE*3Leiden mice, not expressing CETP, niacin also decreased total cholesterol and triglycerides but did not increase HDL-cholesterol. In fact, in APOE*3Leiden.CETP mice, niacin dose-dependently decreased the hepatic expression of CETP (up to -88%; P<0.01) as well as plasma CETP mass (up to -45%, P<0.001) and CETP activity (up to -52%, P<0.001). Additionally, niacin dose-dependently decreased the clearance of apoAI from plasma and reduced the uptake of apoAI by the kidneys (up to -90%, P<0.01)., Conclusions: Niacin markedly increases HDL-cholesterol in APOE*3Leiden.CETP mice by reducing CETP activity, as related to lower hepatic CETP expression and a reduced plasma (V)LDL pool, and increases HDL-apoAI by decreasing the clearance of apoAI from plasma.

Background: Although cholesteryl ester transfer protein (CETP) inhibition is regarded as a promising strategy to reduce atherosclerosis by increasing high-density lipoprotein cholesterol, the CETP inhibitor torcetrapib given in addition to atorvastatin had no effect on atherosclerosis and even increased cardiovascular death in the recent Investigation of Lipid Level Management to Understand its Impact in Atherosclerotic Events trial. Therefore, we evaluated the antiatherogenic potential and adverse effects of torcetrapib in humanized APOE*3-Leiden.CETP (E3L.CETP) mice., Methods and Results: E3L.CETP mice were fed a cholesterol-rich diet without drugs or with torcetrapib (12 mg x kg(-1) x d(-1)), atorvastatin (2.8 mg x kg(-1) x d(-1)), or both for 14 weeks. Torcetrapib decreased CETP activity in both the absence and presence of atorvastatin (-74% and -73%, respectively; P<0.001). Torcetrapib decreased plasma cholesterol (-20%; P<0.01), albeit to a lesser extent than atorvastatin (-42%; P<0.001) or the combination of torcetrapib and atorvastatin (-40%; P<0.001). Torcetrapib increased high-density lipoprotein cholesterol in the absence (30%) and presence (34%) of atorvastatin. Torcetrapib and atorvastatin alone reduced atherosclerotic lesion size (-43% and -46%; P<0.05), but combination therapy did not reduce atherosclerosis compared with atorvastatin alone. Remarkably, compared with atorvastatin, torcetrapib enhanced monocyte recruitment and expression of monocyte chemoattractant protein-1 and resulted in lesions of a more inflammatory phenotype, as reflected by an increased macrophage content and reduced collagen content., Conclusions: CETP inhibition by torcetrapib per se reduces atherosclerotic lesion size but does not enhance the antiatherogenic potential of atorvastatin. However, compared with atorvastatin, torcetrapib introduces lesions of a less stable phenotype.

Aim: This study was designed to investigate the effect of the angiotensin II receptor blocker olmesartan alone, or in combination with standard treatment with a statin, pravastatin, on atherosclerosis development in APOE*3Leiden transgenic mice., Methods and Results: Four groups of 15 mice received an atherogenic diet alone (plasma cholesterol 17.4 +/- 2.7 mmol/l) or supplemented with either 0.008% (w/w) olmesartan (9.3 mg/kg per day) (plasma cholesterol 16.4 +/- 3.9 mmol/l), 0.03% (w/w) pravastatin (35 mg/kg per day) (plasma cholesterol 14.6 +/- 2.6 mmol/l), or the combination of both (plasma cholesterol 14.5 +/- 2.9 mmol/l) for 6 months. Treatment with olmesartan or pravastatin reduced the development of atherosclerosis as compared to the control group (-46 and -39%, respectively). Pravastatin also reduced the severity of the lesions. As compared to control the combination of both treatments almost fully prevented atherosclerosis (-91%, P < 0.001) and strongly reduced lesion number (-69%), lesion severity (-79%), number of macrophages (-89%) and T lymphocytes (-86%) per cross-section. Treatment with olmesartan alone and in combination with pravastatin inhibited the adhesion of monocytes to the vessel wall (-22%; P < 0.05 and -25%; P < 0.01, respectively), and reduced the relative quantity of macrophages in the lesions (-38%; P < 0.05 and -26%; NS, respectively) as compared to control., Conclusion: Olmesartan reduced atherosclerosis development mainly by decreasing monocyte adhesion and the relative amount of macrophages, whereas pravastatin inhibited the progression of atherosclerosis to more advanced lesions, reflecting different anti-atherosclerotic modes of action of the two drugs. Combination therapy with olmesartan and pravastatin additively reduced atherosclerosis development, resulting in less and less severe lesions.

Cytochrome P450 3A (CYP3A) enzymes constitute an important detoxification system that contributes to primary metabolism of more than half of all prescribed medications. To investigate the physiological and pharmacological roles of CYP3A, we generated Cyp3a-knockout (Cyp3a-/-) mice lacking all functional Cyp3a genes. Cyp3a-/- mice were viable, fertile, and without marked physiological abnormalities. However, these mice exhibited severely impaired detoxification capacity when exposed to the chemotherapeutic agent docetaxel, displaying higher exposure levels in response to both oral and intravenous administration. These mice also demonstrated increased sensitivity to docetaxel toxicity, suggesting a primary role for Cyp3a in xenobiotic detoxification. To determine the relative importance of intestinal versus hepatic Cyp3a in first-pass metabolism, we generated transgenic Cyp3a-/- mice expressing human CYP3A4 in either the intestine or the liver. Expression of CYP3A4 in the intestine dramatically decreased absorption of docetaxel into the bloodstream, while hepatic expression aided systemic docetaxel clearance. These results suggest that CYP3A expression determines impairment of drug absorption and efficient systemic clearance in a tissue-specific manner. The genetic models used in this study provide powerful tools to further study CYP3A-mediated xenobiotic metabolism, as well as interactions between CYP3A and other detoxification systems.

The multidrug resistance gene 1 product, P-glycoprotein (P-gp), is expressed in several excretory organs, including the apical membrane of proximal tubules. After inducing acute renal failure, P-gp expression is upregulated and this might be a protective function by pumping out toxicants and harmful products of oxidative stress. We characterized renal function of P-gp knockout mice and studied its consequences in renal ischemic damage. Compared with wild-type mice, knockout mice have a lower glomerular filtration rate and renal plasma flow. An augmented urinary excretion of sodium, numerous amino acids, calcium, glucose, and low molecular weight proteins was observed along with an increased diuresis. A higher lithium plasma clearance in the knockout mice suggested proximal tubular dysfunction. Electron microscopy showed mitochondrial abnormalities in proximal tubular cells that could account for decreased adenosine triphosphate levels in the cortex. After inducing ischemia, wild-type mice showed a decrease in creatinine clearance and severe proximal tubular necrosis. In contrast, knockout mice had no signs of tubular damage. Our data indicate that P-gp knockout mice have impaired renal function but are protected against ischemic renal injury.

Hypertension and hyperlipidaemia are major risk factors for the development of atherosclerosis. Calcium channel blockers (CCBs) have been used for decades and have established antihypertensive effects. Statins have been extensively used because of their potent lipid lowering properties. Amongst other factors, inflammation and oxidation are involved in enhanced progression of atherosclerosis and new lesion development. Therefore, research has been initiated focusing on the antioxidant and anti-inflammatory properties of CCBs and statins, beyond their primary effect, in order to evaluate the possible additive effects of combined treatment of CCBs with statins as antiatherosclerotic therapy. Clinical studies (e.g., the International Nifedipine Trial on Antiatherosclerotic Therapy [INTACT]) have demonstrated that the antiatherosclerotic action of CCBs is limited to the attenuation of the first stage of atherosclerogenesis (fatty streak formation or new lesion growth). The lesions that pre-existed at the start of CCB therapy did not demonstrate progression or regression on angiography. However, because the mechanisms of action of lipid-lowering drugs and CCBs, and their role in preventing the progression of atherosclerosis differ, it is conceivable to conclude that these two classes may have an additive or synergic effect, not only on new lesion formation but also on inhibiting the progression of established coronary atherosclerosis. Indeed, this combined effect of lipid-lowering therapy and CCBs on human coronary atherosclerosis has been reported in the Regression Growth Evaluation Statin Study (REGRESS) trial. This beneficial effect of combining CCBs with statins has now been replicated in transgenic atherosclerotic mice, where the combination of amlodipine and atorvastatin produced an additional 60% reduction of atherosclerosis compared with that observed with the statin alone. Serum markers of atherosclerosis and vascular integrity also improved most in the combination group. Synergistic effects of the combination of atorvastatin and amlodipine on acute nitric oxide release/endothelial function, and additive effects of the combination of amlodipine and atorvastatin in the improvement of arterial compliance in hypertensive hyperlipidaemic patients has been demonstrated. Collectively, these studies support the clinical antiatherosclerotic advantages of combination of CCBs and statins and in particular, of atorvastatin with amlodipine beyond their established antihyperlipidaemic and antihypertensive modes of action.

In systemic lupus erythematosus (SLE), autoantibodies directed against complement components of the classical pathway, especially against C1q, are associated with severe disease and are of prognostic value for flares of lupus nephritis. Mannose-binding lectin (MBL), the recognition unit of the MBL pathway of complement activation, has structural similarities to C1q. Deficiencies of MBL have been shown to predispose to the development of SLE and to influence the course of the disease. We hypothesized that the presence of autoantibodies to MBL, analogous to autoantibodies to C1q in patients with SLE, may contribute to disease development. The occurrence of anti-MBL autoantibodies was assessed by enzyme-linked immunosorbent assay (ELISA) of 68 serum samples from 20 patients with SLE and in serum from 70 healthy controls. Levels of antibodies directed against MBL were significantly higher in patients with SLE compared to healthy subjects. No significant difference was found between patients with active disease compared to those with inactive disease. While the occurrence of anti-C1q autoantibodies was associated with renal involvement, no such relationship was found for anti-MBL autoantibodies. A significant correlation was found between anti-MBL and anti-C1q antibody levels. The level of anti-MBL antibodies was negatively correlated with MBL-complex activity of circulating MBL. Anti-MBL autoantibodies were of the immunoglobulin G (IgG) isotype and the binding site of IgG anti-MBL was located in the F(ab')2 portion. We conclude that anti-MBL are present in sera from SLE patients and influence the functional activity of MBL.