Cholesteryl Ester Transfer Protein Activity Research Articles

Cholesteryl ester transfer protein: ace of spades, queen of hearts, or the joker?

Cholesteryl ester transfer protein: ace of spades, queen of hearts, or the joker?

Increasing HDL levels by inhibiting cholesteryl ester transfer protein activity in rabbits with hindlimb ischemia is associated with increased angiogenesis

BackgroundHigh density lipoprotein (HDL) infusions increase new blood vessel formation (angiogenesis) in rodents with ischemic injury. This study asks if increasing HDL levels by inhibiting cholesteryl ester transfer protein (CETP) activity increases angiogenesis in New Zealand White (NZW) rabbits with hindlimb ischemia. Methods and resultsNZW rabbits were maintained for 6weeks on chow or chow supplemented with 0.07% or 0.14% (wt/wt) of the CETP inhibitor, des-fluoro-anacetrapib. The left femoral artery was ligated after 2weeks of des-fluoro-anacetrapib treatment. The animals were sacrificed 4weeks after femoral artery ligation. Treatment with 0.07% and 0.14% (wt/wt) des-fluoro-anacetrapib reduced CETP activity by 63±12% and 81±8.6%, increased plasma apoA-I levels by 1.3±0.1- and 1.4±0.1-fold, and increased plasma HDL-cholesterol levels by 1.4±0.1- and 1.7±0.2-fold, respectively. Treatment with 0.07% and 0.14% (wt/wt) des-fluoro-anacetrapib increased the number of collateral arteries by 60±16% and 84±27%, and arteriole wall area in the ischemic hindlimbs by 84±16% and 94±13%, respectively. Capillary density in the ischemic hindlimb adductor muscle increased from 1.1±0.2 (control) to 2.1±0.3 and 2.2±0.4 in the 0.07% and 0.14% (wt/wt) des-fluoro-anacetrapib-treated animals, respectively. Incubation of HDLs from des-fluoro-anacetrapib-treated animals with human coronary artery endothelial cells at apoA-I concentrations comparable with their plasma levels increased tubule network formation. These effects were abolished by knockdown of scavenger receptor-B1 (SR-B1) and PDZK1, and pharmacological inhibition of PI3K/Akt. ConclusionIncreasing HDL levels by inhibiting CETP activity is associated with increased collateral blood vessel formation in NZW rabbits with hindlimb ischemia in an SR-B1- and PI3K/Akt-dependent manner.

Lack of Atorvastatin Protective Effect Against Atrial Fibrillation in CETP TaqIB2B2 Genotype.

There has been some evidence for a role of statins in reducing the risk of atrial fibrillation, but the response to statin treatment varies considerably due to environmental and genetic factors. One of these is related to CETP expression. So we assessed whether CETP TaqIB polymorphism influences atrial fibrillation occurrence after treatment with statins. 200 unrelated dyslipidemic Caucasian patients (146 men and 54 women; mean age 75±8) from Salento (Southern Italy), assigned to atorvastatin treatment, and 158 normolipidemic subjects (119 men and 39 women; mean age 75±11), selected from the same ward, were enrolled. All patients were followed at six-month intervals. CETP TaqIB polymorphism was genotyped by RFLP-PCR. During a mean follow-up time of 71±6 months, 64 patients (32%) of the group treated with atorvastatin and 70 subjects (44%) of the group without atorvastatin experienced at least one episode of AF, with a statistically significant difference (p = 0,0208) between the two groups. No significant differences were observed between the two groups with regard to demographic and echocardiographic data, to clinical history and pharmacological treatment. While in patients not assuming atorvastatin there was no significant difference (p = 1) between TaqIB genotype and atrial fibrillation occurence, in subjects treated with atorvastatin B2B2 genotype was more frequent in patients with atrial fibrillation (p = 0,0001). According to these data the subjects with the B2B2 genotype seem to be more susceptible to atrial fibrillation development (RR 2,74; IC 95% 1,92-3,90; p<0.025). Our data seem to provide a further evidence for the hypothesis that statins may have adverse effect in subjects with genetically low CETP levels. Because statins reduce CETP activity up to 30%, we hypothesize that such CETP activity reduction by statins, in patients with low CETP levels induced by polymorphism, may counteract the beneficial effect of statins on atrial fibrillation.

In-silico approach towards identification of inhibitor for cholesteryl ester transfer protein (CETP): A novel strategy

Atherosclerosis is a multifactorial disease which caused by a long term process of an accumulation of lipids combines with an inflammatory response which forms plaques or atheroma. The current focus in preventing atherosclerosis is by raising the HDL-C level. One novel approach is by inhibition of Cholesterol Ester Transfer Protein (CETP) function. Aim: The aim of this study is to see whether HCA does bind to the same active site in the CETP tunnel and to see the stability of the ligand-protein binding. Method: Docking studies are carried out by using X-ray crystallography structure (PDB ID: 20BD and 4EWS) by using Glides software from Schrodinger Inc and validated results using GOLD. The molecular dynamic simulation are being carried out by Desmond (Schrodinger inc) by using the ligand-protein complexes from the docking results. Results: Based on the molecular docking studies, HCA does bind to the active sites of torcetrapib is and based on the MD simulation analysis, the binding of the ligand-protein complexes are stable throughout the simulation. Further verification on in vitro experimentation of HCA does shows the potency against CETP activity and the results are in consonance with the in silico studies. Conclusion: This study prepares a solid foundation on how the binding mode of HCA against CETP and the study of mechanism of actions should be carry forward in order to see the real interaction between CETP protein residue and HCA in increasing the HDL-C level

New CETP inhibitor K-312 reduces PCSK9 expression: a potential effect on LDL cholesterol metabolism.

Despite significant reduction of cardiovascular events by statin treatment, substantial residual risk persists, driving emerging needs for the development of new therapies. We identified a novel cholesteryl ester transfer protein (CETP) inhibitor, K-312, that raises HDL and lowers LDL cholesterol levels in animals. K-312 also suppresses hepatocyte expression of proprotein convertase subtilisin/kexin 9 (PCSK9), a molecule that increases LDL cholesterol. We explored the underlying mechanism for the reduction of PCSK9 expression by K-312. K-312 inhibited in vitro human plasma CETP activity (IC50; 0.06 μM). Administration of K-312 to cholesterol-fed New Zealand White rabbits for 18 wk raised HDL cholesterol, decreased LDL cholesterol, and attenuated aortic atherosclerosis. Our search for additional beneficial characteristics of this compound revealed that K-312 decreases PCSK9 expression in human primary hepatocytes and in the human hepatoma cell line HepG2. siRNA silencing of CETP in HepG2 did not compromise the suppression of PCSK9 by K-312, suggesting a mechanism independent of CETP. In HepG2 cells, K-312 treatment decreased the active forms of sterol regulatory element-binding proteins (SREBP-1 and -2) that regulate promoter activity of PCSK9. Chromatin immunoprecipitation assays demonstrated that K-312 decreased the occupancy of SREBP-1 and SREBP-2 on the sterol regulatory element of the PCSK9 promoter. PCSK9 protein levels decreased by K-312 treatment in the circulating blood of cholesterol-fed rabbits, as determined by two independent mass spectrometry approaches, including the recently developed, highly sensitive parallel reaction monitoring method. New CETP inhibitor K-312 decreases LDL cholesterol and PCSK9 levels, serving as a new therapy for dyslipidemia and cardiovascular disease.

Abstract 34: Elevated Plasma CETP Activity and Hyper-procoagulant R451Q-CETP Mutation Linked to Venous Thrombosis Risk

Cholesteryl ester transfer protein (CETP) is an important lipid transfer factor in plasma that enhances prothrombinase activity in purified reaction mixtures. To test the hypothesis that CETP activity is linked to venous thrombosis (VTE), we measured CETP lipid transfer activity in plasmas of 49 male VTE patients (age &lt; 55 yrs old) and matched controls. CETP activity levels were significantly higher for VTE patients than controls (p= 0.0008) (see Figure panel A). Elevated CETP activity (&gt; 90th percentile of controls) was significantly associated with VTE risk (odds ratio= 4.1 (95%CI, 1.4-13)). A subset of patients carrying CETP mutations A373P and R451Q, which were also linked to VTE risk, showed significantly higher plasma CETP activity compared with non-carriers (110 % (105-115 %) vs 80 % (66-92 %) (p=0.0004)). To test the hypothesis that these mutations enhance procoagulant activity of CETP, we made recombinant CETPs in a stable HEK293 cell expression system and measured the procoagulant activity of recombinant wt-CETP, A373P-CETP, and R451Q-CETP that were added to reaction mixtures containing factors Xa, Va, prothrombin and lipids. Quite remarkably, the R451Q-CETP mutant had five-fold higher thrombin generation activity compared to wt-CETP or A373P-CETP (see Figure panel B). In summary, our data show that (1) elevated CETP lipid transfer activity in plasma was associated with the risk of VTE and (2) recombinant R451Q-CETP which is linked to VTE has much higher procoagulant activity than wt-CETP. These clinical and basic findings indicate that the lipid transfer activity of CETP may contribute to risk for thrombosis and that further studies of this hypothesis are merited.

Abstract 132: CETP Deficiency in Rabbits Protects High Fat High Cholesterol Diet Induced Atherosclerosis

CETP has been suggested as an effective and promising target for further reducing cardiovascular events through elevation of the HDL-C levels. The failure of Pfizer’s torcetrapib and Roche’s dalcetrapib raised the question whether increased HDL from pharmacological inhibition of CETP are cardioprotective. Because mice lack CETP naturally, we generated CETP knockout (KO) rabbits by Zinc-finger nucleases (ZFN) to gain insights onto CETP and HDL biology in CVD. The ZFN pairs target on exon 3 of rabbit CETP gene. By microinjection of ZFN mRNA into rabbit embryos, we generated 5 CETP knockout (CETPko) founder rabbits. The founder used for this study contained 4 nucleotides deletion in exon 3 that results in a premature stop codon. Western blot confirms that there is no detectable CETP protein in the homozygous CETPko rabbit plasma and liver. No CETP activity was detected in the CETPko rabbit plasma, consistent with their genotype. At the normal chow fed conditions, there is no significant difference between wild type and CETPko male rabbits in plasma HDLc and total cholesterol (TC) level; whereas female CETPko rabbits show 40 to 100% increase in both HDLc and TC levels compared to wild type ones. By sequential ultracentrifugation method to separate plasma lipoproteins, we found that there is much higher HDL3 component in female CETPko rabbits, but not in male CETPko ones, comparing to their WT age matched control. At the HFHC (3% soy bean oil and 0.3% cholesterol) diet fed conditions, both male and female CETPko rabbits show significantly lower level of TC and LDLc compared to wild type rabbits. At the same time, the HDLc levels are significantly higher in the CETPko rabbits. In addition, apoB depleted plasma from CETPko rabbit showed 2-fold increase in cholesterol efflux tested in human THP1 cells. Furthermore, we measured the atherosclerotic plaque in the rabbit aorta after 16 weeks HFHC diet treatment. The total gross lesion areas in CETPko rabbits were only one-third of those in the wild type rabbits. In conclusion, our study shows that genetic deficiency of CETP in rabbits has beneficial effects on enhancing HDL function and reducing atherosclerosis. Our work sheds light on CETP and HDL biology, and provides valuable information to the development of CETP drugs.

Abstract 113: Phospholipid Physical State Determines the Efficiency of Cholesteryl Ester Transfer by Cholesteryl Ester Transfer Protein between Discoidal High Density Lipoproteins

The transfer of cholesteryl ester by recombinant cholesteryl ester transfer protein (CETP) between reconstituted discoidal HDL was studied. Particles contained apolipoprotein A-I, unsaturated palmitoyllinoleoylphosphatidylcholine (PLPC), palmitoyloleoylphosphatidylcholine (POPC) or saturated dipalmitoylphosphatidylcholine (DPPC) and cholesteryl ester up to 3 mol% as cholesteryl 1-pyrenedecanoate (CPD) or cholesteryl laurate (CL) in donor and acceptor rHDL, respectively. The concentration dependencies of excimerization upward deviated from a linear dependence, being more curvilinear for DPPC. Separately for DPPC complexes, monomer fluorescence compared to excimer was more efficiently quenched by acrylamide. The nonradiative energy transfer between apoA-I tryptophan residues and CPD molecules was more efficient for PLPC compared to DPPC complexes. Data fulfilled the quenching sphere-of-action model, the sphere with adjacent probe molecules being larger and more distant from apoA-I tryptophan residues in DPPC complexes. CETP-catalyzed cholesteryl ester exchange between donor and acceptor HDL followed by probe excimerization was characterized by a heterogeneous kinetics; the fast exchanging CPD pool was much higher in a case of POPC compared to DPPC complexes. Probe fraction accessible to CETP increased with temperature, suggesting a more homogeneous probe distribution. The noncompetitive inhibition of probe transfer by acceptor particles seems to satisfy the zero off-rate compatible with probe tunneling. The values of V max (0.063 μM·min -1 ) and k cat (0.42 s -1 ) together with a similarity of K m (0.9 μM CPD) and K I (2.8 μM CL) values for POPC-containing rHDL suggest the efficient cholesteryl ester transfer between nascent HDL with unsaturated phosphatidylcholine in vivo opposite to much less efficiency with HDL with saturated phosphatidylcholine. The physical state of phospholipid matrix may underlie CETP activity with discoidal HDL through self-association and location of cholesteryl ester in the bilayer, the availability of cholesteryl ester to cholesterol-binding site in apoA-I structure and the binding of apoA-I-interacting cholesteryl ester to CETP. We are grateful to Dr. A. Tall for providing the recombinant CETP.

Analysis of "On/Off" Kinetics of a CETP Inhibitor Using a Mechanistic Model of Lipoprotein Metabolism and Kinetics.

RG7232 is a potent inhibitor of cholesteryl-ester transfer protein (CETP). Daily oral administration of RG7232 produces a dose- and time-dependent increase in high-density lipoprotein-cholesterol (HDL-C) and apolipoproteinA-I (ApoA-I) levels and a corresponding decrease in low-density lipoprotein-cholesterol (LDL-C) and apolipoproteinB (ApoB) levels. Due to its short plasma half-life (∼3 hours), RG7232 transiently inhibits CETP activity during each dosing interval (“on/off” kinetics), as reflected by the temporal effects on HDL-C and LDL-C. The influence of RG7232 on lipid-poor ApoA-I (i.e., pre-β1) levels and reverse cholesterol transport rates is unclear. To investigate this, a published model of lipoprotein metabolism and kinetics was combined with a pharmacokinetic model of RG7232. After calibration and validation of the combined model, the effect of RG7232 on pre-β1 levels was simulated. A dose-dependent oscillation of pre-β1, driven by the “on/off” kinetics of RG7232 was observed. The possible implications of these findings are discussed.

A Multidose Study to Examine the Effect of Food on Evacetrapib Exposure at Steady State.

Purpose:To determine the effect of a high-fat meal on evacetrapib exposure at steady state in healthy participants.Methods:This was a randomized, 2-period, 2-sequence, open-label, crossover study. Patients were randomly assigned to 1 of the 2 treatment sequences in which they received evacetrapib 130 mg/d for 10 days following a 10-hour fast each day or following a high-fat breakfast each day. Plasma samples collected through 24 hours were analyzed for evacetrapib concentrations and pharmacokinetic parameter estimates including area under the concentration–time curve during a dosing interval (AUCτ), maximum observed concentration (Cmax), and time of Cmax (tmax) were calculated. Pharmacodynamic parameters, including cholesteryl ester transfer protein (CETP) activity, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), total cholesterol, and triglycerides, were also assessed.Results:A total of 34 males and 6 females, mean age 41.5 years and mean body mass index 26.6 kg/m2, were enrolled. Statistical analysis showed AUCτ was 44% higher (90% confidence interval [CI]: 29%-62%) and Cmax was 51% higher (90% CI: 28%-79%) in the fed state than in the fasted state, indicating an effect of food. Consistent with higher evacetrapib exposure, changes in HDL-C, LDL-C, and CETP activity appeared to be greater in the fed state than in the fasted state. There were no notable changes in total cholesterol or triglycerides following administration in the fed and fasted states. The 130-mg doses of evacetrapib were well tolerated with and without food.Conclusion:A high-fat meal increased evacetrapib mean exposure at steady state by 44% in healthy participants.

Modification of CETP function by changing its substrate preference: a new paradigm for CETP drug design

We previously determined that hamster cholesteryl ester transfer protein (CETP), unlike human CETP, promotes a novel one-way transfer of TG from VLDL to HDL, causing HDL to gain lipid. We hypothesize that this nonreciprocal lipid transfer activity arises from the usually high TG/cholesteryl ester (CE) substrate preference of hamster CETP. Consistent with this, we report here that ∼25% of the total lipid transfer promoted by the human Q199A CETP mutant, which prefers TG as substrate, is nonreciprocal transfer. Other human CETP mutants with TG/CE substrate preferences higher or lower than wild-type also possess nonreciprocal lipid transfer activity. Mutants with high TG/CE substrate preference promote the nonreciprocal lipid transfer of TG from VLDL to HDL, but mutants with low TG/CE substrate preference promote the nonreciprocal lipid transfer of CE, not TG, and this lipid flow is in the reverse direction (from HDL to VLDL). Anti-CETP TP2 antibody alters the TG/CE substrate preference of CETP and also changes the extent of nonreciprocal lipid transfer, showing the potential for externally acting agents to modify the transfer properties of CETP. Overall, these data show that the lipid transfer properties of CETP can be manipulated. Function-altering pharmaceuticals may offer a novel approach to modify CETP activity and achieve specific modifications in lipoprotein metabolism.

Transcriptional regulation of the apolipoprotein F (ApoF) gene by ETS and C/EBPα in hepatoma cells.

Apolipoprotein F (ApoF) inhibits cholesteryl ester transfer protein (CETP) activity and plays an important role in lipid metabolism. In the present study, the full-length human ApoF promoter was cloned, and the molecular mechanism of the regulation of ApoF was investigated. The ApoF promoter displayed higher activities in hepatoma cell lines, and the-198nt to+79nt promoter region contained the maximum promoter activity. In the promoter region of-198nt to-2nt there were four putative binding sites for transcription factors ETS-1/ETS-2 (named EBS-1 to EBS-4) and one for C/EBP. Mutation of EBS-2, EBS4 and the C/EBP binding site abolished the promoter activity, and ETS-1/ETS-2 and C/EBPα could interact with corresponding binding sites. In addition, overexpression of ETS-1/2 or C/EBPα enhanced, while dominant-negative mutants of ETS-1/2 and knockdown of C/EBPα decreased, ApoF promoter activities. ETS-1 and C/EBPα associated physically, and acted synergistically to activate ApoF transcription. These results demonstrated combined activation of the ApoF promoter by liver-enriched and ubiquitous transcription factors. Direct interactions between C/EBPα and ETS-1 were important for high liver-specific expression of ApoF.

Cholesteryl ester transfer protein inhibition enhances endothelial repair and improves endothelial function in the rabbit.

High-density lipoproteins (HDLs) can potentially protect against atherosclerosis by multiple mechanisms, including enhancement of endothelial repair and improvement of endothelial function. This study asks if increasing HDL levels by inhibiting cholesteryl ester transfer protein activity with the anacetrapib analog, des-fluoro-anacetrapib, enhances endothelial repair and improves endothelial function in New Zealand White rabbits with balloon injury of the abdominal aorta. New Zealand White rabbits received chow or chow supplemented with 0.07% or 0.14% (wt/wt) des-fluoro-anacetrapib for 8 weeks. Endothelial denudation of the abdominal aorta was carried out after 2 weeks. The animals were euthanized 6 weeks postinjury. Treatment with 0.07% and 0.14% des-fluoro-anacetrapib reduced cholesteryl ester transfer protein activity by 81±4.9% and 92±12%, increased plasma apolipoprotein A-I levels by 1.4±0.1-fold and 1.5±0.1-fold, increased plasma HDL-cholesterol levels by 1.8±0.2-fold and 1.9±0.1-fold, reduced intimal hyperplasia by 37±11% and 51±10%, and inhibited vascular cell proliferation by 25±6.1% and 35±6.7%, respectively. Re-endothelialization of the injured aorta increased from 43±6.7% (control) to 69±6.6% and 76±7.7% in the 0.07% and 0.14% des-fluoro-anacetrapib-treated animals, respectively. Aortic ring relaxation and guanosine 3',5'-cyclic monophosphate production in response to acetylcholine were also improved. Incubation of HDLs from the des-fluoro-anacetrapib-treated animals with human coronary artery endothelial cells increased cell proliferation and migration relative to control. These effects were abolished by knockdown of scavenger receptor-B1 and PDZ domain-containing protein 1 and by pharmacological inhibition of phosphatidylinositol-4,5-bisphosphate 3-kinase/Akt. Increasing HDL levels by inhibiting cholesteryl ester transfer protein reduces intimal thickening and regenerates functional endothelium in damaged New Zealand White rabbit aortas in an scavenger receptor-B1-dependent and phosphatidylinositol-4,5-bisphosphate 3-kinase/Akt-dependent manner.

Remarkable quantitative and qualitative differences in HDL after niacin or fenofibrate therapy in type 2 diabetic patients

HDL-increasing drugs such as fenofibrate and niacin have failed to decrease the cardiovascular risk in patients with type 2 diabetes. Drug-mediated quantitative and qualitative HDL modifications could be involved in these negative results. To evaluate the quantitative and qualitative effects of niacin and fenofibrate on HDL in patients with type 2 diabetes, a prospective, randomised controlled intervention trial was conducted. Thirty type 2 diabetic patients with low HDL were randomised to receive either fenofibrate (FFB) or niacin + laropiprant (ERN/LPR) as an add-on to simvastatin treatment for 12 weeks according to a crossover design. At the basal point and after each intervention period, physical examinations and comprehensive standard biochemical determinations and HDL metabolomics were performed. Thirty nondiabetic patients with normal HDL were used as a basal control group. ERN/LRP, but not FFB, significantly increased HDL cholesterol. Neither ERN/LRP nor FFB reversed the HDL particle size or particle number to normal. ERN/LRP increased apoA-I but not apoA-II, whereas FFB produced the opposite effect. FFB significantly increased Preβ1-HDL, whereas ERN/LRP tended to lower Preβ1-HDL. CETP and LCAT activities were significantly decreased only by ERN/LRP. PAF-AH activity in HDL and plasma decreased with the use of both agents. Despite their different actions on antioxidant parameters, none of the treatments induced detectable antioxidant improvements.ERN/LRP and FFB had strikingly different effects on HDL quantity and quality, as well as on HDL cholesterol concentrations. When prescribing HDL cholesterol increasing drugs, this differential action should be considered.

A vaccine targeted at CETP alleviates high fat and high cholesterol diet-induced atherosclerosis and non-alcoholic steatohepatitis in rabbit.

Low HDL-C levels are associated with atherosclerosis and non-alcoholic steatohepatitis, and increased levels may reduce the risk of these diseases. Inhibition of cholesteryl ester transfer protein (CETP) activity is considered a promising strategy for increasing HDL-C levels. Since CETP is a self-antigen with low immunogenicity, we developed a novel CETP vaccine (Fc-CETP6) to overcome the low immunogenicity of CETP and for long-term inhibition of CETP activity. The vaccine consists of a rabbit IgG Fc domain for antigen delivery to antigen-presenting cells fused to a linear array of 6 repeats of a CETP epitope to efficiently activate B cells. Rabbits were fed a high fat/cholesterol (HFC) diet to induce atherosclerosis and NASH, and immunized with Fc-CETP6 vaccine. The Fc-CETP6 vaccine successfully elicited anti-CETP antibodies and lowered plasma CETP activity. The levels of plasma HDL-C and ApoA-I were higher, and plasma ox-LDL lower, in the Fc-CETP6-immunized rabbits as compared to the unimmunized HFC diet-fed rabbits. Pathological analyses revealed less lipid accumulation and inflammation in the aorta and liver of the Fc-CETP6-immunized rabbits. These results show that the Fc-CETP6 vaccine efficiently elicited antibodies against CETP and reduced susceptibility to both atherosclerosis and steatohepatitis induced by the HFC diet. Our findings suggest that the Fc-CETP6 vaccine may improve atherosclerosis and NASH and has high potential for clinical use.

Abstract 16404: CETP Inhibitor K-312 Lowers PCSK9 Expression and LDL Cholesterol Levels in vivo

Background: Despite potent LDL-cholesterol (LDL-C) therapies, substantial residual cardiovascular risk remains. Proprotein convertase subtilisin/kexin 9 (PCSK9) is a promising target for lowering LDL-C. We recently demonstrated that K-312, originally found as a cholesteryl ester transfer protein (CETP) inhibitor, decreased PCSK9 expression in vitro through the reduction of its promoter activity. The present study has further examined the underlying molecular mechanisms and in vivo effects of K-312 on PCSK9 levels using mass spectrometry. Methods and Results: CETP silencing by siRNA in the human hepatocyte cell line HepG2 did not compromise suppression of PCSK9 expression by K-312, indicating a mechanism independent of CETP inhibition. Transcription factors sterol-regulatory element binding protein (SREBP)-1 and SREBP-2 bind to the sterol regulatory element (SRE) of PCSK9 promoter when their precursor forms are cleaved into active forms. K-312 treatment decreased binding of SREBP-1 and SREBP-2 to SRE of the PCSK9 promoter (chromatin immunoprecipitation) and active forms of SREBP-1 and SREBP-2 (western blotting), suggesting the suppression of PCSK9 expression by K-312 involves SREBP-1 and SREBP-2. We then examined the effects of K-312 in cholesterol-fed New Zealand White rabbits administered either vehicle (N=7) or K-312 30 mg/kg (N=7) for 2 weeks. K-312 treatment decreased LDL-C (vehicle: 272.5 ± 42.2 mg/dl; K-312: 193.0 ± 36.5 mg/dl), increased HDL cholesterol (vehicle: 29.9 ± 3.7 mg/dl; K-312: 93.3 ± 14.7 mg/dl) and decreased PCSK9 mRNA levels in the liver by 63%. We further used two mass spectrometry-based approaches to measure PCSK9 levels in rabbit plasma: spectral counting and a novel high resolution (HR)-MS/MS quantification method. Immunoprecipitation of PCSK9 successfully enriched PCSK9 signal. Spectral counting and HR-MS/MS of PCSK9 peptides both demonstrated that K-312 treatment significantly decreased plasma PCSK9 levels by 76% (p&lt;0.01) and 60% (p&lt;0.05), respectively. Conclusion: K-312 may lower LDL cholesterol levels by suppressing CETP activity and PCSK9 expression, serving as a novel therapy for dyslipidemia and cardiovascular disease.

Abstract 17913: Inhibition of CETP by Dalcetrapib Results in a Modest Increase in Cholesterol Efflux Capacity Associated With an Increase in Large HDL Particles, but Does Not Impact Carotid Intima-Media Thickness in a dal-PLAQUE-2 Substudy

Inhibition of cholesteryl ester transfer protein (CETP) is an approach aiming at raising HDL-cholesterol levels and reducing cardiovascular risk. The dal-PLAQUE-2 phase III study recruited 988 subjects with stable coronary artery disease. Its primary objective was to evaluate the effect of dalcetrapib on the progression of carotid atherosclerotic disease measured by intima-media thickness after one year of therapy. As dalcetrapib showed a neutral effect on cardiovascular risk in the dal-OUTCOMES study, our objective was to evaluate its effect on cIMT and markers of HDL mass, lipoprotein subclass distribution and HDL function. All subjects from dal-PLAQUE-2 who had provided serum samples at baseline and one year were included in our substudy of 193 subjects on placebo and 186 subjects on dalcetrapib. Comparisons between groups at one year were made by ANCOVA after adjustment for baseline levels. No significant differences between groups for all variables considered were observed at baseline. Dalcetrapib reduced CETP activity as measured by a fluorescent method by 30% after 1 year (p&lt;0.001), which resulted in increases in HDL-cholesterol and apoA-I levels by 32% and 11%, respectively (both p&lt;0.001). Dalcetrapib markedly increased the concentration of large HDL particles measured by NMR profiling (+59%, p&lt;0.001), at the expense of small HDL particles (-9.5%, p&lt;0.001). Cholesterol efflux capacity of serum was measured from J774 macrophages under basal conditions and after cAMP stimulation. Dalcetrapib increased basal and stimulated efflux by 7.1% and 5.5% (both p&lt;0.001), but was without effect on the ABCA1-dependent component (p=0.26). Despite these effects, dalcetrapib had no impact on mean and maximal cIMT (p=0.98 and p=0.85). While the change in cIMT was inversely correlated with basal cholesterol efflux in the placebo group (Spearman r=-0.163, p&lt;0.05), such a relationship was not found in the dalcetrapib group. Moreover, the change in total HDL particles concentration was inversely correlated with change in cIMT (r=0.181, p&lt;0.05) in the placebo arm only. In conclusion, dalcetrapib raised HDL-C and larger HDL, but this had minimal effects on cholesterol efflux capacity of patients’ serum and had a neutral effect on carotid artery plaque burden.

How anacetrapib inhibits the activity of the cholesteryl ester transfer protein? Perspective through atomistic simulations.

Cholesteryl ester transfer protein (CETP) mediates the reciprocal transfer of neutral lipids (cholesteryl esters, triglycerides) and phospholipids between different lipoprotein fractions in human blood plasma. A novel molecular agent known as anacetrapib has been shown to inhibit CETP activity and thereby raise high density lipoprotein (HDL)-cholesterol and decrease low density lipoprotein (LDL)-cholesterol, thus rendering CETP inhibition an attractive target to prevent and treat the development of various cardiovascular diseases. Our objective in this work is to use atomistic molecular dynamics simulations to shed light on the inhibitory mechanism of anacetrapib and unlock the interactions between the drug and CETP. The results show an evident affinity of anacetrapib towards the concave surface of CETP, and especially towards the region of the N-terminal tunnel opening. The primary binding site of anacetrapib turns out to reside in the tunnel inside CETP, near the residues surrounding the N-terminal opening. Free energy calculations show that when anacetrapib resides in this area, it hinders the ability of cholesteryl ester to diffuse out from CETP. The simulations further bring out the ability of anacetrapib to regulate the structure-function relationships of phospholipids and helix X, the latter representing the structural region of CETP important to the process of neutral lipid exchange with lipoproteins. Altogether, the simulations propose CETP inhibition to be realized when anacetrapib is transferred into the lipid binding pocket. The novel insight gained in this study has potential use in the development of new molecular agents capable of preventing the progression of cardiovascular diseases.

Elevated CETP activity during acute phase of myocardial infarction is independently associated with endothelial dysfunction and adverse clinical outcome

Objective: Recent data suggests that cholesteryl ester transfer protein (CETP) activity may interact with acute stress conditions via inflammatory-oxidative response and thrombogenesis. We investigated this assumption in patients with ST-elevation myocardial infarction (STEMI). Methods: Consecutive patients with STEMI (n = 116) were enrolled <24-h of symptoms onset and were followed for 180 days. Plasma levels of C-reactive protein (CRP), interleukin-2 (IL-2), tumor necrosis factor (TNFα), 8-isoprostane, nitric oxide (NOx) and CETP activity were measured at enrollment (D1) and at fifth day (D5). Flow-mediated dilation (FMD) was assessed by ultrasound and coronary thrombus burden (CTB) was evaluated by angiography. Results: Neither baseline nor the change of CETP activity from D1 to D5 was associated with CRP, IL-2, TNFα, 8-isoprostane levels or CTB. The rise in NOx from D1 to D5 was inferior [3.5(−1; 10) vs. 5.5(−1; 12); p < 0.001] and FMD was lower [5.9(5.5) vs. 9.6(6.6); p = 0.047] in patients with baseline CETP activity above the median value than in their counterparts. Oxidized HDL was measured by thiobarbituric acid reactive substances (TBARS) in isolated HDL particles and increased from D1 to D5, and remaining elevated at D30. The change in TBARS content in HDL was associated with CETP activity (r = 0.72; p = 0.014) and FMD (r = −0.61; p = 0.046). High CETP activity at admission was associated with the incidence of sudden death and recurrent MI at 30 days (OR 12.8; 95% CI 1.25–132; p = 0.032) and 180 days (OR 3.3; 95% CI 1.03–10.7; p = 0.044). Conclusions: An enhanced CETP activity during acute phase of STEMI is independently associated with endothelial dysfunction and adverse clinical outcome.

Cholesteryl ester diffusion, location and self-association constraints determine CETP activity with discoidal HDL: Excimer probe study

The transfer of cholesteryl ester by recombinant cholesteryl ester transfer protein (CETP) between reconstituted discoidal high-density lipoprotein (rHDL) was studied. Particles contained apolipoprotein A-I, unsaturated POPC or saturated DPPC and cholesteryl ester as cholesteryl 1-pyrenedecanoate (CPD) or cholesteryl laurate (CL) in donor and acceptor rHDL, respectively. Probe dynamics fulfilled the quenching sphere-of-action model. The cholesteryl ester exchange between donor and acceptor particles was characterized by a heterogeneous kinetics; the fast exchanging CPD pool was much higher in a case of POPC compared to DPPC complexes. Probe fraction accessible to CETP increased with temperature, suggesting a more homogeneous probe distribution. Noncompetitive inhibition of probe transfer by acceptor particles was observed. The values of Vmax (0.063μMmin−1) and catalytic rate constant kcat (0.42s−1) together with a similarity of Km (0.9μM CPD) and KI (2.8μM CL) values for POPC-containing rHDL suggest the efficient cholesteryl ester transfer between nascent HDL with unsaturated phosphatidylcholine in vivo. The phospholipid matrix in discoidal HDL may underlie CETP activity through the self-association, diffusivity and location of cholesteryl ester in the bilayer, the accessibility of cholesteryl ester to cholesterol-binding site in apoA-I structure and the binding of cholesteryl ester, positionable by apoA-I, to CETP.