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Proprotein convertase subtilisin kexin type 9 (PCSK9) inhibits the clearance of low-density lipoprotein (LDL) cholesterol (LDL-C) from plasma by directly binding with the LDL receptor (LDLR) and sending the receptor for lysosomal degradation. As the interaction promotes elevated plasma LDL-C levels, and therefore a predisposition to cardiovascular disease, PCSK9 has attracted intense interest as a therapeutic target. Despite this interest, an orally bioavailable small-molecule inhibitor of PCSK9 with extensive lipid-lowering activity is yet to enter the clinic. We report herein the discovery of NYX-PCSK9i, an orally bioavailable small-molecule inhibitor of PCSK9 with significant cholesterol-lowering activity in hyperlipidemic APOE∗3-Leiden.CETP mice. NYX-PCSK9i emerged from a medicinal chemistry campaign demonstrating potent disruption of the PCSK9-LDLR interaction in vitro and functional protection of the LDLR of human lymphocytes from PCSK9-directed degradation ex vivo. APOE∗3-Leiden.CETP mice orally treated with NYX-PCSK9i demonstrated a dose-dependent decrease in plasma total cholesterol of up to 57%, while its combination with atorvastatin additively suppressed plasma total cholesterol levels. Importantly, the majority of cholesterol lowering by NYX-PCSK9i was in non-HDL fractions. A concomitant increase in total plasma PCSK9 levels and significant increase in hepatic LDLR protein expression strongly indicated on-target function by NYX-PCSK9i. Determinations of hepatic lipid and fecal cholesterol content demonstrated depletion of liver cholesteryl esters and promotion of fecal cholesterol elimination with NYX-PCSK9i treatment. All measured in vivo biomarkers of health indicate that NYX-PCSK9i has a good safety profile. NYX-PCSK9i is a potential new therapy for hypercholesterolemia with the capacity to further enhance the lipid-lowering activities of statins., Competing Interests: Conflict of Interest A. K. S., J. P., J. B., and B. J. E. are employees of Nyrada Inc. J. T. P. and G. L. are members of the Scientific Advisory Board of Nyrada Inc. J. T. P., G. L., J. B., and B. J. E. have share/stock options in Nyrada Inc. A. K. S., P. A. C., J. T. P., J. B., and B. J. E. are listed as inventors on the patent that discloses NYX-PCSK9i and related compounds. G. S., P. A. C., E. J. P., N. K., G. L., K. C., A. K. J., and H. M. G. P. are employees or members of their respective companies or laboratories who were contracted and/or consulted by Nyrada Inc. to perform specific research activities., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Atherosclerosis-related CVD causes nearly 20 million deaths annually. Most patients are treated after plaques develop, so therapies must regress existing lesions. Current therapies reduce plaque volume, but targeting all apoB-containing lipoproteins with intensive combinations that include alirocumab or evinacumab, monoclonal antibodies against cholesterol-regulating proprotein convertase subtilisin/kexin type 9 and angiopoietin-like protein 3, may provide more benefit. We investigated the effect of such lipid-lowering interventions on atherosclerosis in APOE*3-Leiden.CETP mice, a well-established model for hyperlipidemia. Mice were fed a Western-type diet for 13 weeks and thereafter matched into a baseline group (euthanized at 13 weeks) and five groups that received diet alone (control) or with treatment [atorvastatin; atorvastatin and alirocumab; atorvastatin and evinacumab; or atorvastatin, alirocumab, and evinacumab (triple therapy)] for 25 weeks. We measured effects on cholesterol levels, plaque composition and morphology, monocyte adherence, and macrophage proliferation. All interventions reduced plasma total cholesterol (37% with atorvastatin to 80% with triple treatment; all P < 0.001). Triple treatment decreased non-HDL-C to 1.0 mmol/l (91% difference from control; P < 0.001). Atorvastatin reduced atherosclerosis progression by 28% versus control ( P < 0.001); double treatment completely blocked progression and diminished lesion severity. Triple treatment regressed lesion size versus baseline in the thoracic aorta by 50% and the aortic root by 36% (both P < 0.05 vs. baseline), decreased macrophage accumulation through reduced proliferation, and abated lesion severity. Thus, high-intensive cholesterol-lowering triple treatment targeting all apoB-containing lipoproteins regresses atherosclerotic lesion area and improves lesion composition in mice, making it a promising potential approach for treating atherosclerosis., (Copyright © 2020 Pouwer et al.)

Background and Aims: Activation of brown adipose tissue (BAT) reduces both hyperlipidemia and atherosclerosis by increasing the uptake of triglyceride-derived fatty acids by BAT, accompanied by formation and clearance of lipoprotein remnants. We tested the hypothesis that the hepatic uptake of lipoprotein remnants generated by BAT activation would be accelerated by concomitant statin treatment, thereby further reducing hypercholesterolemia and atherosclerosis., Methods: APOE*3-Leiden.CETP mice were fed a Western-type diet and treated without or with the selective β3-adrenergic receptor (AR) agonist CL316,243 that activates BAT, atorvastatin (statin) or both., Results: β3-AR agonism increased energy expenditure as a result of an increased fat oxidation by activated BAT, which was not further enhanced by statin addition. Accordingly, statin treatment neither influenced the increased uptake of triglyceride-derived fatty acids from triglyceride-rich lipoprotein-like particles by BAT nor further lowered plasma triglyceride levels induced by β3-AR agonism. Statin treatment increased the hepatic uptake of the formed cholesterol-enriched remnants generated by β3-AR agonism. Consequently, statin treatment further lowered plasma cholesterol levels. Importantly, statin, in addition to β3-AR agonism, also further reduced the atherosclerotic lesion size as compared to β3-AR agonism alone, without altering lesion severity and composition., Conclusions: Statin treatment accelerates the hepatic uptake of remnants generated by BAT activation, thereby increasing the lipid-lowering and anti-atherogenic effects of BAT activation in an additive fashion. We postulate that, in clinical practice, combining statin treatment with BAT activation is a promising new avenue to combat hyperlipidemia and cardiovascular disease., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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.)

The present study describes the effects of atorvastatin on whole body synthesis of nitric oxide (NO), prostacyclin (PGI2), and thromboxane A2 (TxA2), on oxidative stress and nitrite/nitrate-related renal carbonic anhydrase (CA) activity in patients with type 2 diabetes mellitus (T2DM). A double-blind, randomized, placebo-controlled parallel-group trial (the DALI study group) on 217 patients with T2DM and dyslipidemia was performed. Urinary samples were collected before and after administration of a standard dose (10 mg/d, n=73), a maximal dose atorvastatin (80 mg/d, n=72) or placebo (n=72) for 30 weeks. Urinary nitrite and nitrate were measured to assess whole body NO synthesis. The urinary molar ratio of nitrate to nitrite (UNOxR) served as a measure of renal CA activity. Free radical- and cyclooxygenase (COX)-catalyzed lipid peroxidation was estimated by measuring urinary 8-iso-prostaglandin F2α (8-iso-PGF2α). In subgroups, systemic PGI2 and TxA2 synthesis was assessed by measuring their major urinary metabolites 2,3-dinor-6-keto-prostaglandin F1α and 2,3-dinor-thromboxane B2, respectively. All biochemical parameters were measured by GC-MS and GC-MS/MS methods. T2DM patients had elevated levels of nitrate, nitrite, UNOxR, and 8-iso-PGF2α compared to healthy non-diabetic and normolipidemic subjects. Thirty-week treatment with atorvastatin (10 or 80 mg/d) did not significantly alter NO, PGI2, TxA2 and 8-iso-PGF2α synthesis and did not improve the renal reabsorption of nitrite which is considered an important reservoir of NO. Our study suggests that atorvastatin (10 or 80 mg/d) does not provide cardiovascular benefit beyond its cholesterol lowering effect in patients with T2DM., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

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.)

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.)

Some cross-sectional epidemiological studies have reported positive associations of serum concentrations of non-high density lipoprotein cholesterol with serum perfluorooctanesulfonate (PFOS) and perfluorooctanoate (PFOA). However, the strength of the reported associations is inconsistent for exposure-response across three orders of magnitude of serum PFOS and/or PFOA concentrations. These positive associations are unexpected based on toxicological/mechanistic studies, suggesting that the associations may have a biological, rather than a causal, basis. This study tested the hypothesis that PFOS and PFOA distribute into serum lipoprotein fractions such that increases in serum lipoproteins would result in corresponding increases in serum concentrations of PFOS and PFOA. Based on observed binding of PFOS and PFOA to isolated β-lipoproteins in physiological saline (96% and 40% bound, respectively) in preliminary experiments using ultrafiltration and LC-MS/MS methods, binding to human donor plasma lipoprotein fractions was investigated by two density gradient methods. The majority of PFOS and PFOA recovered masses were found in lipoprotein-depleted plasma. Plasma density gradient fractionation data suggested that maximally 9% of PFOS distributes to lipoprotein-containing fractions, yet only 1% or less of PFOA is so distributed. These data do not support a strong role for plasma lipoprotein fractions in explaining the inconsistent dose-response associations reported in cross-sectional epidemiological studies., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

The cholesteryl ester transfer protein (CETP) facilitates the bidirectional transfer of cholesteryl esters and triglycerides (TG) between HDL and (V)LDL. By shifting cholesterol in plasma from HDL to (V)LDL in exchange for VLDL-TG, CETP aggravates atherosclerosis in hyperlipidemic APOE*3-Leiden (E3L) mice. The aim of this study was to investigate the role of CETP in TG metabolism and high-fat diet-induced obesity by using E3L mice with and without the expression of the human CETP gene. On chow, plasma lipid levels were comparable between both male and female E3L and E3L.CETP mice. Further mechanistic studies were performed using male mice. CETP expression increased the level of TG in HDL. CETP did not affect the postprandial plasma TG response or the hepatic VLDL-TG and VLDL-apolipoprotein B production rate. Moreover, CETP did not affect the plasma TG clearance rate or organ-specific TG uptake after infusion of VLDL-like emulsion particles. In line with the absence of an effect of CETP on tissue-specific TG uptake, CETP also did not affect weight gain in response to a high-fat diet. In conclusion, the CETP-induced increase of TG in the HDL fraction of E3L mice is not associated with changes in the production of TG or with tissue-specific clearance of TG from the plasma.

Objective: In addition to lowering low-density lipoprotein (LDL)-cholesterol, statins modestly increase high-density lipoprotein (HDL)-cholesterol in humans and decrease cholesteryl ester transfer protein (CETP) mass and activity. Our aim was to determine whether the increase in HDL depends on CETP expression., Methods and Results: APOE*3-Leiden (E3L) mice, with a human-like lipoprotein profile and a human-like responsiveness to statin treatment, were crossbred with mice expressing human CETP under control of its natural flanking regions resulting in E3L.CETP mice. E3L and E3L.CETP mice were fed a Western-type diet with or without atorvastatin. Atorvastatin (0.01% in the diet) reduced plasma cholesterol in both E3L and E3L.CETP mice (-26 and -33%, P<0.05), mainly in VLDL, but increased HDL-cholesterol only in E3L.CETP mice (+52%). Hepatic mRNA expression levels of genes involved in HDL metabolism, such as phospholipid transfer protein (Pltp), ATP-binding cassette transporter A1 (Abca1), scavenger receptor class B type I (Sr-b1), and apolipoprotein AI (Apoa1), were not differently affected by atorvastatin in E3L.CETP mice as compared to E3L mice. However, in E3L.CETP mice, atorvastatin down-regulated the hepatic CETP mRNA expression (-57%; P<0.01) as well as the total CETP level (-29%) and cholesteryl esters (CE) transfer activity (-36%; P<0.05) in plasma., Conclusions: Atorvastatin increases HDL-cholesterol in E3L.CETP mice by reducing the CETP-dependent transfer of cholesterol from HDL to (V)LDL, as related to lower hepatic CETP expression and a reduced plasma (V)LDL pool.

In addition to efficiently decreasing VLDL-triglycerides (TGs), fenofibrate increases HDL-cholesterol levels in humans. We investigated whether the fenofibrate-induced increase in HDL-cholesterol is dependent on the expression of the cholesteryl ester transfer protein (CETP). To this end, APOE*3-Leiden (E3L) transgenic mice without and with the human CETP transgene, under the control of its natural regulatory flanking regions, were fed a Western-type diet with or without fenofibrate. Fenofibrate (0.04% in the diet) decreased plasma TG in E3L and E3L.CETP mice (-59% and -60%; P < 0.001), caused by a strong reduction in VLDL. Whereas fenofibrate did not affect HDL-cholesterol in E3L mice, fenofibrate dose-dependently increased HDL-cholesterol in E3L.CETP mice (up to +91%). Fenofibrate did not affect the turnover of HDL-cholesteryl ester (CE), indicating that fenofibrate causes a higher steady-state HDL-cholesterol level without altering the HDL-cholesterol flux through plasma. Analysis of the hepatic gene expression profile showed that fenofibrate did not differentially affect the main players in HDL metabolism in E3L.CETP mice compared with E3L mice. However, in E3L.CETP mice, fenofibrate reduced hepatic CETP mRNA (-72%; P < 0.01) as well as the CE transfer activity in plasma (-73%; P < 0.01). We conclude that fenofibrate increases HDL-cholesterol by reducing the CETP-dependent transfer of cholesterol from HDL to (V)LDL, as related to lower hepatic CETP expression and a reduced plasma (V)LDL pool.

Fibrates are drugs of choice in patients with hypertriglyceridemia (HTG), but may increase the risk for gallstones by decreasing bile acid synthesis. Fish oil might be a therapeutic alternative, but its effect on bile acid metabolism in humans is unknown. We compared the effects of triglyceride-lowering therapy by fish oil or bezafibrate on cholesterol synthesis and bile acid metabolism in HTG. Cholesterol synthesis, bile acid pool sizes, and synthesis rates were compared between 9 male HTG patients and 10 normolipidemic controls matched for age, sex, and BMI. Effects of bezafibrate or fish oil were studied only in HTG patients in a randomized crossover trial. Patients had 14-fold higher serum triglyceride concentrations and greater cholesterol synthesis, as indicated by a 107% higher ratio of serum lathosterol to cholesterol (P < 0.01) than controls. The groups did not differ in bile acid metabolism. Both bezafibrate and fish oil reduced serum TG concentration (-68 and -51% vs. baseline, respectively). Compared with baseline, bezafibrate therapy was associated with reduced cholesterol synthesis (-25%, P = 0.009) without changes in bile acid synthesis rate and pool size. In contrast, fish oil increased bile acid synthesis (+31% vs. baseline, P = 0.07 and +53% vs. bezafibrate, P = 0.02) and altered bile acid distribution, as reflected by an increased ratio of the cholic acid (CA) synthesis rate to the chenodeoxycholic acid (CDCA) synthesis rate (+35% vs baseline, P = 0.05 and + 32% vs bezafibrate, P = 0.07) without effects on bile acid pool size or cholesterol synthesis. In conclusion, cholesterol synthesis is greater in HTG patients than in controls, whereas bile acid synthesis does not differ. Bezafibrate and fish oil have similar triglyceride-lowering capacities, but distinct effects on cholesterol synthesis. Bile acid synthesis is increased by fish oil, but not by bezafibrate therapy.

Unfiltered coffee brews such as French press and espresso contain a lipid from coffee beans named cafestol that raises serum cholesterol in humans. Cafestol decreases the expression and activity of cholesterol 7alpha-hydroxylase, the rate-limiting enzyme in the classical pathway of bile acid synthesis, in cultured rat hepatocytes and livers of APOE3Leiden mice. Inhibition of bile acid synthesis has been suggested to be responsible for the cholesterol-raising effect of cafestol. Therefore, we assessed whether cafestol decreases the activity of cholesterol 7alpha-hydroxylase in humans. Because liver biopsies were not feasible, we measured plasma levels of 7alpha-hydroxy-4-cholesten-3-one, a marker for the activity of cholesterol 7alpha-hydroxylase in the liver. Plasma 7alpha-hydroxy-4-cholesten-3-one was measured in 2 separate periods in which healthy volunteers consumed coffee oil containing cafestol (69 mg/d) for 5 wk. Plasma levels of 7alpha-hydroxy-4-cholesten-3-one increased by 47 +/- 13% (mean +/- SEM, n = 38, P = 0.001) in the first period and by 23 +/- 10% (n = 31, P = 0.03) in the second treatment period. Serum cholesterol was raised by 23 +/- 2% (P < 0.001) in the first period and by 18 +/- 2% (P < 0.001) in the second period. We corrected individual 7alpha-hydroxy-4-cholesten-3-one levels for serum cholesterol levels, because coffee oil increases serum cholesterol and 7alpha-hydroxy-4-cholesten-3-one is probably present in the lipoprotein fraction of serum. After correction, the increase in 7alpha-hydroxy-4-cholesten-3-one was 24 +/- 11% (P = 0.04) in the first period and there was no effect in period 2. Our study showed that coffee oil did not decrease, and actually increased, plasma levels of 7alpha-hydroxy-4-cholesten-3-one in humans in 2 separate treatment periods. Therefore, this study does not support the hypothesis that cafestol decreases bile acid synthesis in humans.

Numerous animal studies have reported that garlic can protect against atherosclerosis. However, a comparable number of studies do not support this observation. This contradiction may result from differences in study design, use of different animal models, and use of different garlic formulations and preparations. Here, we investigated the effect of the chemically well-characterized and production-controlled garlic powder printanor on atherosclerosis in the APOE*3-Leiden transgenic mouse, a mouse model well suited for evaluating anti-atherosclerotic properties of drugs and food components under human-like conditions. APOE*3-Leiden mice were fed a Western diet supplemented with either 5 or 50 g kg(-1) printanor. As a reference, the commercially available fermented garlic kyolic was included (1.6 g kg(-1) diet). Treatment with printanor demonstrated reduced body weight, coinciding with increased feces production and fecal fatty acids excretion. Printanor and kyolic treatment did not affect plasma lipids, markers of inflammation (serum amyloid A, serum-soluble intercellular adhesion molecule-1, and blood-leukocytes tumor necrosis factor-alpha (TNFalpha) production) and vascular activation (plasma von Willebrand factor (vWF)). As analyzed after 28 weeks of treatment, printanor and kyolic did not affect atherosclerotic lesion type, area or composition. Under conditions relevant to the human situation, the well-characterized and production-controlled garlic powder printanor does not display hypolipidemic, anti-inflammatory or anti-atherosclerotic properties.

The response of plasma lipids to dietary cholesterol and fat varies among individuals. Variations in genes involved in cholesterol metabolism can be important in these interindividual differences. The rate-limiting enzyme in the conversion of cholesterol into bile acids is cholesterol 7alpha-hydroxylase (CYP7A1). We investigated the effect of the A278-C promoter polymorphism in the CYP7A1 gene on responses of plasma lipids to an increased intake in dietary cholesterol (742 +/- 114 mg/d), cafestol (57 +/- 6 mg/d), saturated fat [change of 8-9 energy percent/d (en%/d)] and trans fat (change of 10-11 en%/d) in 496 normolipidemic subjects. These responses were measured in 26 previously published dietary trials. After adjustment for the apolipoprotein E genotype effect, AA-subjects consuming a cholesterol-rich diet had a smaller increase in plasma HDL cholesterol than CC-subjects (0.00 +/- 0.02 vs. 0.17 +/- 0.04 mmol/L; P < 0.001). Upon intake of cafestol, AA-subjects had a smaller increase in plasma total cholesterol than CC-subjects (0.69 +/- 0.10 vs. 1.01 +/- 0.10 mmol/L; P = 0.028). No effects of the polymorphism were found in the saturated and trans fat interventions. In conclusion, the CYP7A1 polymorphism has a small but significant effect on the increase in plasma HDL cholesterol and plasma total cholesterol after an increased intake of dietary cholesterol and cafestol, respectively.

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.

Garlic is reported to have beneficial effects on risk factors associated with cardiovascular disease, including normalization of plasma lipid levels. However, numerous studies do not support this beneficial effect of garlic on plasma lipids. This contradiction may result from the use of different garlic-derived materials, experimental designs, and/or animal models. The present study investigated the hypolipidemic effect of garlic-derived materials in APOE*3-Leiden mice, a model well suited for drug and dietary intervention studies of hyperlipidemia. APOE*3-Leiden mice were fed a garlic-derived sulfur-rich compound, either allicin (0.29 g.L drinking water(-1)) or diallyldisulfide (0.27 g.kg diet(-1)), or powdered garlic, of either the kwai (42 g.kg diet(-1)) or morado (42 g.kg diet(-1)) variety. The amounts of garlic-derived materials supplied allowed free intake of allicin or allicin equivalents (diallyldisulfide, kwai, or morado) at 44 mg.kg body wt(-1).d(-1). Mice were fed a nonpurified diet for 4 wk, followed by a Western diet for 8 wk, both supplemented with the garlic-derived materials. These diets had no consistent effect on plasma lipids and did not affect lipoprotein profiles, which are markers for whole-body cholesterol synthesis and intestinal sterol absorption. The current data indicate that the postulated effects of garlic on cardiovascular disease are not caused via modulation of plasma lipid levels.

The low-density lipoprotein (LDL) receptor-related protein (LRP) has a well-established role in the hepatic removal of atherogenic apolipoprotein E (APOE)-rich remnant lipoproteins from plasma. In addition, LRP recognizes multiple distinct pro- and antiatherogenic ligands in vitro. Here, we investigated the role of hepatic LRP in atherogenesis independent of its role in removal of APOE-rich remnant lipoproteins. Mice that allow inducible inactivation of hepatic LRP were combined with LDL receptor and APOE double-deficient mice (MX1Cre(+)LRP(flox/flox)LDLR(-/-)APOE(-/-)). On an LDLR(-/-)APOE(-/-) background, hepatic LRP deficiency resulted in decreased plasma cholesterol and triglycerides (cholesterol: 17.1 +/- 5.2 vs 23.4 +/- 6.3 mM, P =.025; triglycerides: 1.1 +/- 0.5 vs 2.2 +/- 0.8 mM, P =.002, for MX1Cre(+)LRP(flox/flox)-LDLR(-/-)APOE(-/-) and control LRP(flox/flox)-LDLR(-/-)APOE(-/-) mice, respectively). Lower plasma cholesterol in MX1Cre(+)LRP(flox/flox)-LDLR(-/-)APOE(-/-) mice coincided with increased plasma lipoprotein lipase (71.2 +/- 7.5 vs 19.1 +/- 2.4 ng/ml, P =.002), coagulation factor VIII (4.4 +/- 1.1 vs 1.9 +/- 0.5 U/mL, P =.001), von Willebrand factor (2.8 +/- 0.6 vs 1.4 +/- 0.3 U/mL, P =.001), and tissue-type plasminogen activator (1.7 +/- 0.7 vs 0.9 +/- 0.5 ng/ml, P =.008) compared with controls. Strikingly, MX1Cre(+)LRP(flox/flox)LDLR(-/-)APOE(-/-) mice showed a 2-fold higher atherosclerotic lesion area compared with controls (408.5 +/- 115.1 vs 219.1 +/- 86.0 10(3)microm(2), P =.003). Our data indicate that hepatic LRP plays a clear protective role in atherogenesis independent of plasma cholesterol, possibly due to maintaining low levels of its proatherogenic ligands.

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.

Raman spectroscopy allows quantitative, non-destructive evaluation of entire, intact atherosclerotic plaques. We quantified the anti-atherosclerotic effects of atorvastatin and amlodipine on progression of atherosclerosis using post-mortem Raman spectroscopic plaque imaging in 28 APOE*3 Leiden transgenic mice who were fed a high fat/high cholesterol diet for 28 weeks. Mice were assigned to a control group receiving the diet alone or to groups that received the diet with either 0.01% w/w atorvastatin, 0.002% w/w amlodipine, or the combination. The entire excised aortic arch was scanned with Raman microspectroscopy for quantitation of the distribution of cholesterol and calcification content. When mice had been treated with atorvastatin, cholesterol accumulation and calcification in the aortic arch was reduced by 91 and 98%, respectively, (both P<0.001). Amlodipine did not reduce the cholesterol content but reduced calcification of the aorta by 69% (P<0.05). The combination of amlodipine and atorvastatin was as effective as atorvastatin alone. This study demonstrates the strong atheroprotective potential of atorvastatin. In addition it is demonstrated that amlodipine reduces mineralization of atherosclerotic plaque. No synergistic effect of the combination of amlodipine and atorvastatin on plaque development is demonstrated. This study encourages Raman spectroscopic evaluations of anti-atherosclerotic drugs in larger animals and humans in vivo.