Mathieu R. Brodeur

Genotype-Dependent Effects of Dalcetrapib on Cholesterol Efflux and Inflammation: Concordance with Clinical Outcomes

Background—Dalcetrapib effects on cardiovascular outcomes are determined by adenylate cyclase 9 gene polymorphisms. Our aim was to determine whether these clinical end point results are also associated with changes in reverse cholesterol transport and inflammation. Methods and Results—Participants of the dal-OUTCOMES and dal-PLAQUE-2 trials were randomly assigned to receive dalcetrapib or placebo in addition to standard care. High-sensitivity C-reactive protein was measured at baseline and at end of study in 5243 patients from dal-OUTCOMES also genotyped for the rs1967309 polymorphism in adenylate cyclase 9. Cholesterol efflux capacity of high-density lipoproteins from J774 macrophages after cAMP stimulation was determined at baseline and 12 months in 171 genotyped patients from dal-PLAQUE-2. Treatment with dalcetrapib resulted in placebo-adjusted geometric mean percent increases in high-sensitivity C-reactive protein from baseline to end of trial of 18.1% (P=0.0009) and 18.7% (P=0.00001) in participants with the GG and AG genotypes, respectively, but the change was −1.0% (P=0.89) in those with the protective AA genotype. There was an interaction between the treatment arm and the genotype groups (P=0.02). Although the mean change in cholesterol efflux was similar among study arms in patients with GG genotype (mean: 7.8% and 7.4%), increases were 22.3% and 3.5% with dalcetrapib and placebo for those with AA genotype (P=0.005). There was a significant genetic effect for change in efflux for dalcetrapib (P=0.02), but not with placebo. Conclusions—Genotype-dependent effects on C-reactive protein and cholesterol efflux are supportive of dalcetrapib benefits on atherosclerotic cardiovascular outcomes in patients with the AA genotype at polymorphism rs1967309. Clinical Trials Registration—ClinicalTrials.gov; Unique Identifiers: NCT00658515 and NCT01059682.

Evaluation of Links Between High-Density Lipoprotein Genetics, Functionality, and Aortic Valve Stenosis Risk in Humans

Objective—Studies have shown that high-density lipoprotein (HDL)–raising compounds induce regression of aortic valve stenosis (AVS) in animal models. However, whether patients with AVS have an impaired HDL metabolism is unknown. Approach and Results—A total of 1435 single nucleotide polymorphisms in genes associated with HDL cholesterol levels (in or around GALNT2, LPL, ABCA1, APOA5, SCARB1, LIPC, CETP, LCAT, LIPG, APOC4, and PLTP) were genotyped in 382 patients with echocardiography-confirmed AVS (aortic jet velocity ≥2.5 m/s) and 401 controls. After control for multiple testing, none of the genetic variants showed a positive association with case/control status (adjusted P≥0.05 for all single nucleotide polymorphisms tested). In a subsample of this cohort, HDL cholesterol levels, apolipoprotein AI levels, lecithin-cholesterol acyltransferase activity, pre–&bgr;-HDL, HDL size, and 4 parameters of cholesterol efflux capacity were measured in apolipoprotein B–depleted serum samples from 86 patients with and 86 patients without AVS. Cholesterol efflux capacity was measured using J774 macrophages with and without stimulation of ATP-binding cassette A-1 expression by cAMP, and HepG2 hepatocytes for scavenger receptor class B type 1–mediated efflux. None of these parameters were different between cases and controls. However, compared with patients without coronary artery disease, sera from patients with coronary artery disease had lower HDL cholesterol levels, scavenger receptor class B type 1–mediated efflux, and HDL size (P⩽0.003), independently of the presence or absence of AVS. Conclusions—Results of the present study suggest that, based on HDL genetics and HDL functionality, HDL metabolism does not seem to predict the risk of AVS. Because of our limited sample size, additional studies are needed to confirm these findings.

An update on the clinical development of dalcetrapib (RO4607381), a cholesteryl ester transfer protein modulator that increases HDL cholesterol levels

CETP is the target of CETP inhibitors such as anacetrapib and the modulator dalcetrapib. Both molecules have entered Phase III clinical trials, with the ultimate goal of reducing cardiovascular events by raising HDL cholesterol. At the 600-mg dose selected for the dal-OUTCOMES study, dalcetrapib is expected to inhibit CETP activity by approximately 30% and raise HDL-C by approximately 30% with limited effects on LDL cholesterol. Importantly, dalcetrapib does not raise blood pressure or aldosterone levels, two effects previously associated with the CETP inhibitor torcetrapib. Dalcetrapib has been well tolerated at the 600-mg dose. In the dal-PLAQUE atherosclerosis imaging study, dalcetrapib reduced the enlargement of total vessel area over time. In May 2012, following the results of the second interim analysis of dal-OUTCOMES, the Data and Safety Monitoring Board recommended stopping the study owing to a lack of clinically significant benefit, which was followed by Roches (Basel, Switzerland) decision to terminate the study and the dalcetrapib program (dal-HEART). Contrary to anacetrapib, a potent CETP inhibitor that markedly increases HDL cholesterol and significantly reduces LDL cholesterol, dalcetrapib has allowed us to test the hypothesis that an isolated, moderate elevation in HDL cholesterol prevents cardiovascular events.

Lipids, Apolipoproteins, and Inflammatory Biomarkers of Cardiovascular Risk: What Have We Learned?

Cardiovascular diseases (CVD) are the first cause of death in the world. CVD risk is influenced by multiple factors, some nonmodifiable such as age, sex, and genetic background, and others modifiable. Great progress has been made over the last decades in the identification of biomarkers of incident or recurrent CV risk and surrogate endpoints of CV outcomes. We present the current state of knowledge for CV biomarkers in plasma including lipids, apolipoproteins, inflammation‐related, and emerging omics‐based biomarkers. Clinically validated surrogate endpoints for CV outcomes include plasma low‐density lipoprotein‐cholesterol reduction, and plasma triglyceride reduction is a likely relevant surrogate endpoint. High‐density lipoprotein‐cholesterol is not a validated surrogate endpoint, but is a useful biomarker of CV risk. CV risk biomarkers of interest include apolipoprotein B and non‐HDL‐cholesterol, lipoprotein (a), C‐reactive protein, and recently, genetic and protein‐based risk scores and gut microbiota‐derived trimethylamine oxide levels.

HDL mimetic peptide CER-522 treatment regresses left ventricular diastolic dysfunction in cholesterol-fed rabbits

OBJECTIVES High-density lipoprotein (HDL) infusions induce rapid improvement of experimental atherosclerosis in rabbits but their effect on ventricular function remains unknown. We aimed to evaluate the effects of the HDL mimetic peptide CER-522 on left ventricular diastolic dysfunction (LVDD). METHODS Rabbits were fed with a cholesterol- and vitamin D2-enriched diet until mild aortic valve stenosis and hypercholesterolemia-induced LV hypertrophy and LVDD developed. Animals then received saline or 10 or 30mg/kg CER-522 infusions 6 times over 2weeks. We performed serial echocardiograms and LV histology to evaluate the effects of CER-522 therapy on LVDD. RESULTS LVDD was reduced by CER-522 as shown by multiple parameters including early filling mitral deceleration time, deceleration rate, Em/Am ratio, E/Em ratio, pulmonary venous velocities, and LVDD score. These findings were associated with reduced macrophages (RAM-11 positive cells) in the pericoronary area and LV, and decreased levels of apoptotic cardiomyocytes in CER-522-treated rabbits. CER-522 treatment also resulted in decreased atheromatous plaques and internal elastic lamina area in coronary arteries. CONCLUSIONS CER-522 improves LVDD in rabbits, with reductions of LV macrophage accumulation, cardiomyocyte apoptosis, coronary atherosclerosis and remodelling.

HDL biogenesis revisited: how desmocollin-1 could sabotage reverse cholesterol transport in the arterial wall

High-density lipoproteins (HDLs) are highly heterogeneous complexes of hundreds of lipids and 100 proteins that can be classified according to their composition, intrinsic density, size, and functions. They are often defined as lipoproteins containing apolipoprotein (apo) A-I, the most abundant protein in HDL. HDL from healthy people exerts multiple antiatherogenic effects including cellular cholesterol efflux, anti-inflammatory, antithrombotic, and vasculoprotective activities. These normal functions of HDL may explain the established inverse relationship of HDL-cholesterol levels and incident cardiovascular disease. However, simply raising HDL-cholesterol levels is not a successful strategy to prevent cardiovascular events: disappointing results of phase III studies conducted with niacin or cholesterylester transfer protein (CETP) inhibitors in patients with high cardiovascular risk suggest that enhancing HDL functionality is rather the path to explore. Indeed, beneficial effects of HDL could not exist without HDL biogenesis, the molecular events that leads to lipidation of apo A-I with cellular membrane lipids by the key transporter ABCA1 and the subsequent esterification of cholesterol by lecithin:cholesterol acyltransferase (LCAT). In the arterial wall, the majority of apo A-I can be found in the lipid-poor state rather than in mature particles. In atherosclerotic plaques, lipid-poor apo A-I molecules undergo oxidative modifications, lose the capability to interact with the ATP-binding cassette A1 (ABCA1) transporter, and accumulate through mechanisms that remain to be defined. These observations support the concept that HDL biogenesis is impaired in the context of atherosclerotic plaques. In this issue of the journal, Choi and colleagues provide a seminal contribution to the understanding of how HDL biogenesis is prevented by the interaction of apo A-I with a subtype of cholesteroland sphingomyelin-rich membrane microdomain, enriched in desmocollin-1 (Dsc1) and other desmosomal cadherins such as desmoglein 1 and 3. They isolated and characterized these microdomains from human fibroblasts with a new method involving: (i) detergent-free isolation of membranes on sucrose gradients in the presence of apo A-I; (ii) immunoprecipitation of apo A-I-associated microdomains after sonication in the absence of cross-linking agents; and (iii) lipidomic and proteomic analyses of the composition of microdomains. Co-immunoprecipitation of apo A-I with Dsc1 was also obtained in the absence of ABCA1 (in fibroblasts from Tangier disease patients) and was confirmed in human embryonic kidney (HEK) cells overexpressing human Dsc1. In both cell types, apo A-I co-localized with Dsc1 as shown by confocal microscopy. Using transient overexpression of Dsc1 or ABCA1 in HEK cells, the authors concluded that both proteins are able to increase apo A-I binding independently, but that cholesterol efflux was uniquely stimulated through ABCA1 expression and not by Dsc1 overexpression. While Dsc1 overexpression per se did not reduce ABCA1-mediated cholesterol efflux to apo A-I, interference with endogenous Dsc1 expression by stable transfection of short hairpin RNA or CRISPR/Cas9 constructs increased ABCA1-mediated cholesterol efflux. This was paralleled by an increase in ABCA1 protein, which was apparently stabilized by the increased availability of apo A-I for binding to ABCA1 or increased availability of membrane cholesterol following reduced Dsc1 expression. Both mechanisms have been shown to extend the half-life of this rapid turnover protein. The concept that Dsc1-containing microdomains sequester apo A-I and/or membrane lipids efficiently and impact ACBA1-mediated biogenesis is novel (Figure 1). As the authors justly point out, the balance between Dsc1 and ACBA1 expression at the cell surface could regulate the intensity of HDL biogenesis. One limitation of this study is that HDL biogenesis was described essentially by cellular cholesterol efflux assays, but the process of apo A-I lipidation also involves

Adenylate Cyclase Type 9 (ADCY9) Inactivation Protects from Atherosclerosis Only in the Absence of Cholesteryl Ester Transfer Protein (CETP)

Background: Pharmacogenomic studies have shown that ADCY9 genotype determines the effects of the CETP (cholesteryl ester transfer protein) inhibitor dalcetrapib on cardiovascular events and atherosclerosis imaging. The underlying mechanisms responsible for the interactions between ADCY9 and CETP activity have not yet been determined. Methods: Adcy9-inactivated (Adcy9Gt/Gt) and wild-type (WT) mice, that were or not transgenic for the CETP gene (CETPtgAdcy9Gt/Gt and CETPtgAdcy9WT), were submitted to an atherogenic protocol (injection of an AAV8 [adeno-associated virus serotype 8] expressing a PCSK9 [proprotein convertase subtilisin/kexin type 9] gain-of-function variant and 0.75% cholesterol diet for 16 weeks). Atherosclerosis, vasorelaxation, telemetry, and adipose tissue magnetic resonance imaging were evaluated. Results: Adcy9Gt/Gt mice had a 65% reduction in aortic atherosclerosis compared to WT (P<0.01). CD68 (cluster of differentiation 68)-positive macrophage accumulation and proliferation in plaques were reduced in Adcy9Gt/Gt mice compared to WT animals (P<0.05 for both). Femoral artery endothelial-dependent vasorelaxation was improved in Adcy9Gt/Gt mice (versus WT, P<0.01). Selective pharmacological blockade showed that the nitric oxide, cyclooxygenase, and endothelial-dependent hyperpolarization pathways were all responsible for the improvement of vasodilatation in Adcy9Gt/Gt (P<0.01 for all). Aortic endothelium from Adcy9Gt/Gt mice allowed significantly less adhesion of splenocytes compared to WT (P<0.05). Adcy9Gt/Gt mice gained more weight than WT with the atherogenic diet; this was associated with an increase in whole body adipose tissue volume (P<0.01 for both). Feed efficiency was increased in Adcy9Gt/Gt compared to WT mice (P<0.01), which was accompanied by prolonged cardiac RR interval (P<0.05) and improved nocturnal heart rate variability (P=0.0572). Adcy9 inactivation–induced effects on atherosclerosis, endothelial function, weight gain, adipose tissue volume, and feed efficiency were lost in CETPtgAdcy9Gt/Gt mice (P>0.05 versus CETPtgAdcy9WT). Conclusions: Adcy9 inactivation protects against atherosclerosis, but only in the absence of CETP activity. This atheroprotection may be explained by decreased macrophage accumulation and proliferation in the arterial wall, and improved endothelial function and autonomic tone.

Genotype-Dependent Effects of Dalcetrapib on Cholesterol Efflux and InflammationCLINICAL PERSPECTIVE

Background—Dalcetrapib effects on cardiovascular outcomes are determined by adenylate cyclase 9 gene polymorphisms. Our aim was to determine whether these clinical end point results are also associated with changes in reverse cholesterol transport and inflammation. Methods and Results—Participants of the dal-OUTCOMES and dal-PLAQUE-2 trials were randomly assigned to receive dalcetrapib or placebo in addition to standard care. High-sensitivity C-reactive protein was measured at baseline and at end of study in 5243 patients from dal-OUTCOMES also genotyped for the rs1967309 polymorphism in adenylate cyclase 9. Cholesterol efflux capacity of high-density lipoproteins from J774 macrophages after cAMP stimulation was determined at baseline and 12 months in 171 genotyped patients from dal-PLAQUE-2. Treatment with dalcetrapib resulted in placebo-adjusted geometric mean percent increases in high-sensitivity C-reactive protein from baseline to end of trial of 18.1% (P=0.0009) and 18.7% (P=0.00001) in participants with the GG and AG genotypes, respectively, but the change was −1.0% (P=0.89) in those with the protective AA genotype. There was an interaction between the treatment arm and the genotype groups (P=0.02). Although the mean change in cholesterol efflux was similar among study arms in patients with GG genotype (mean: 7.8% and 7.4%), increases were 22.3% and 3.5% with dalcetrapib and placebo for those with AA genotype (P=0.005). There was a significant genetic effect for change in efflux for dalcetrapib (P=0.02), but not with placebo. Conclusions—Genotype-dependent effects on C-reactive protein and cholesterol efflux are supportive of dalcetrapib benefits on atherosclerotic cardiovascular outcomes in patients with the AA genotype at polymorphism rs1967309. Clinical Trials Registration—ClinicalTrials.gov; Unique Identifiers: NCT00658515 and NCT01059682.

Genotype-Dependent Effects of Dalcetrapib on Cholesterol Efflux and InflammationCLINICAL PERSPECTIVE: Concordance With Clinical Outcomes

Background—Dalcetrapib effects on cardiovascular outcomes are determined by adenylate cyclase 9 gene polymorphisms. Our aim was to determine whether these clinical end point results are also associated with changes in reverse cholesterol transport and inflammation. Methods and Results—Participants of the dal-OUTCOMES and dal-PLAQUE-2 trials were randomly assigned to receive dalcetrapib or placebo in addition to standard care. High-sensitivity C-reactive protein was measured at baseline and at end of study in 5243 patients from dal-OUTCOMES also genotyped for the rs1967309 polymorphism in adenylate cyclase 9. Cholesterol efflux capacity of high-density lipoproteins from J774 macrophages after cAMP stimulation was determined at baseline and 12 months in 171 genotyped patients from dal-PLAQUE-2. Treatment with dalcetrapib resulted in placebo-adjusted geometric mean percent increases in high-sensitivity C-reactive protein from baseline to end of trial of 18.1% (P=0.0009) and 18.7% (P=0.00001) in participants with the GG and AG genotypes, respectively, but the change was −1.0% (P=0.89) in those with the protective AA genotype. There was an interaction between the treatment arm and the genotype groups (P=0.02). Although the mean change in cholesterol efflux was similar among study arms in patients with GG genotype (mean: 7.8% and 7.4%), increases were 22.3% and 3.5% with dalcetrapib and placebo for those with AA genotype (P=0.005). There was a significant genetic effect for change in efflux for dalcetrapib (P=0.02), but not with placebo. Conclusions—Genotype-dependent effects on C-reactive protein and cholesterol efflux are supportive of dalcetrapib benefits on atherosclerotic cardiovascular outcomes in patients with the AA genotype at polymorphism rs1967309. Clinical Trials Registration—ClinicalTrials.gov; Unique Identifiers: NCT00658515 and NCT01059682.