Anti-inflammatory salicylate beneficially modulates pre-existing atherosclerosis through quenching of NF-κB activity and lowering of cholesterol.

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.

Ritonavir protects against the development of atherosclerosis in APOE*3-Leiden mice.

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.

MIF deficiency reduces chronic inflammation in white adipose tissue and impairs the development of insulin resistance, glucose intolerance, and associated atherosclerotic disease.

Chronic inflammation in white adipose tissue (WAT) is positively associated with obesity, insulin resistance (IR) and the development of type 2 diabetes. The proinflammatory cytokine MIF (macrophage migration inhibitory factor) is an essential, upstream component of the inflammatory cascade. This study examines whether MIF is required for the development of obesity, IR, glucose intolerance, and atherosclerosis in the LDL receptor-deficient (Ldlr(-/-)) mouse model of disease. Ldlr(-/-) mice develop IR and glucose intolerance within 15 weeks, whereas Mif(-/-)Ldlr(-/-) littermates are protected. MIF deficiency does not affect obesity and lipid risk factors but specifically reduces inflammation in WAT and liver, as reflected by lower plasma serum amyloid A and fibrinogen levels at baseline and under inflammatory conditions. Conversely, MIF stimulates the in vivo expression of human C-reactive protein, an inflammation marker and risk factor of IR and cardiovascular disease. In WAT, MIF deficiency reduces nuclear c-Jun levels and improves insulin sensitivity; MIF deficiency also reduces macrophage accumulation in WAT and blunts the expression of two proteins that regulate macrophage infiltration (intercellular adhesion molecule-1, CD44). Mechanistic parallels to WAT were observed in aorta, where the absence of MIF reduces monocyte adhesion, macrophage lesion content, and atherosclerotic lesion size. These data highlight the physiological importance of chronic inflammation in development of IR and atherosclerosis and suggest that MIF is a potential therapeutic target for reducing the inflammatory component of metabolic and cardiovascular disorders.

Human CETP aggravates atherosclerosis by increasing VLDL-cholesterol rather than by decreasing HDL-cholesterol in APOE*3-Leiden mice.

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.

Bexarotene induces dyslipidemia by increased very low-density lipoprotein production and cholesteryl ester transfer protein-mediated reduction of high-density lipoprotein.

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.

PXR agonism decreases plasma HDL levels in ApoE3-Leiden.CETP mice.

Pregnane X receptor (PXR) agonism has been shown to affect multiple steps in both the synthesis and catabolism of HDL, but its integrated effect on HDL metabolism in vivo remains unclear. The aim of this study was to evaluate the net effect of PXR agonism on HDL metabolism in ApoE3-Leiden (E3L) and E3L.CETP mice, well-established models for human-like lipoprotein metabolism. Female mice were fed a diet with increasing amounts of the potent PXR agonist 5-pregnen-3beta-ol-20-one-16alpha-carbonitrile (PCN). In E3L and E3L.CETP mice, PCN increased liver lipids as well as plasma cholesterol and triglycerides. However, whereas PCN increased cholesterol contained in large HDL-1 particles in E3L mice, it dose-dependently decreased HDL-cholesterol in E3L.CETP mice, indicating that CETP expression dominates the effect of PCN on HDL metabolism. Analysis of the hepatic expression of genes involved in HDL metabolism showed that PCN decreased expression of genes involved in HDL synthesis (Abca1, Apoa1), maturation (Lcat, Pltp) and clearance (Sr-b1). The HDL-increasing effect of PCN, observed in E3L mice, is likely caused by a marked decrease in hepatic SR-BI protein expression, and completely reversed by CETP expression. We conclude that chronic PXR agonism dose-dependently reduces plasma HDL-cholesterol in the presence of CETP.

LXR agonist suppresses atherosclerotic lesion growth and promotes lesion regression in apoE*3Leiden mice: time course and mechanisms.

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.

Quantitative profiling of bile acids in biofluids and tissues based on accurate mass high resolution LC-FT-MS: compound class targeting in a metabolomics workflow.

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.

Torcetrapib does not reduce atherosclerosis beyond atorvastatin and induces more proinflammatory lesions than atorvastatin.

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.

Mouse models for atherosclerosis and pharmaceutical modifiers.

Atherosclerosis is a multifactorial highly-complex disease with numerous etiologies that work synergistically to promote lesion development. The ability to develop preventive and ameliorative treatments will depend on animal models that mimic the human subject metabolically and pathophysiologically and will develop lesions comparable to those in humans. The mouse is the most useful, economic, and valid model for studying atherosclerosis and exploring effective therapeutic approaches. Among the most widely used mouse models for atherosclerosis are apolipoprotein E-deficient (ApoE-/-) and LDL receptor-deficient (LDLr-/-) mice. An up-and-coming model is the ApoE*3Leiden (E3L) transgenic mouse. Here, we review studies that have explored how and to what extent these mice respond to compounds directed at treatment of the risk factors hypercholesterolemia, hypertriglyceridemia, hypertension, and inflammation. An important outcome of this survey is that the different models used may differ markedly from one another in their response to a specific experimental manipulation. The choice of a model is therefore of critical importance and should take into account the risk factor to be studied and the working spectrum of the compounds tested.