Wilfried Le Goff

Action of Atorvastatin in Combined Hyperlipidemia: Preferential Reduction of Cholesteryl Ester Transfer From HDL to VLDL1 Particles

Combined hyperlipidemia (CHL) is characterized by a concomitant elevation of plasma levels of triglyceride-rich, very low density lipoproteins (VLDLs) and cholesterol-rich, low density lipoproteins (LDLs). The predominance of small, dense LDLs contributes significantly to the premature development of coronary artery disease in patients with this atherogenic dyslipoproteinemia. In the present study, we evaluated the impact of atorvastatin, a newly developed inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMGCoA) reductase, on the cholesteryl ester transfer protein (CETP)-mediated remodeling of apolipoprotein (apo) B-containing lipoprotein subspecies, and more specifically, the particle subpopulations of VLDL and LDL in CHL. In parallel, we evaluated the atorvastatin-induced modulation of the quantitative and qualitative features of atherogenic apo B-containing and cardioprotective apo AI-containing lipoprotein subspecies. Atorvastatin therapy (10 mg/d for a 6-week period) in patients with a lipid phenotype typical of CHL (n=18) induced reductions of 31% (P<0.0001) and 36% (P<0.0001) in plasma total cholesterol and LDL cholesterol, respectively. In addition, atorvastatin significantly reduced VLDL cholesterol, triglycerides, and apo B levels by 43% (P<0.0001), 27% (P=0.0006), and 31% (P<0.0001), respectively. The plasma concentrations of triglyceride-rich lipoproteins (VLDL1, Sf 60 to 400; VLDL2, Sf 20 to 60; and intermediate density lipoproteins, Sf 12 to 20) and of LDL, as determined by chemical analysis, were markedly diminished after drug therapy (-30% and -28%, respectively; P<0.0007). Atorvastatin significantly reduced circulating levels of all major LDL subspecies, ie, light (-28%, P<0.0008), intermediate (-27%, P<0.0008), and dense (-32%, P<0.0008) LDL; moreover, in terms of absolute lipoprotein mass, the reduction in dense LDL levels (mean -62 mg/dL) was preponderant. In addition, the reduction in plasma dense LDL concentration after therapy was significantly correlated with a reduction in plasma VLDL1 levels (r=0.429, P=0.0218). Atorvastatin induced a significant reduction (-7%, P=0.0039) in total CETP-dependent CET activity, which accurately reflects a reduction in plasma CETP mass concentration. Total CETP-mediated CET from high density lipoproteins to apo B-containing lipoproteins was significantly reduced (-26%, P<0.0001) with drug therapy. Furthermore, CETP activity was significantly correlated with the atorvastatin-induced reduction in plasma VLDL1 levels (r=0.456, P=0. 0138). Indeed, atorvastatin significantly and preferentially decreased CET from HDL to the VLDL1 subfraction (-37%, P=0.0064), thereby reducing both the levels (-37%, P=0.0001) and the CE content (-20%, P<0.005) of VLDL1. We interpret our data to indicate that 2 independent but complementary mechanisms may be operative in the atorvastatin-induced reduction of atherogenic LDL levels in CHL: first, a significant degree of normalization of both the circulating levels and the quality of their key precursors, ie, VLDL1, and second, enhanced catabolism of the major LDL particle subclasses (ie, light, intermediate, and dense LDL) due to upregulation of hepatic LDL receptors.

Cholesteryl ester transfer protein: at the heart of the action of lipid-modulating therapy with statins, fibrates, niacin, and cholesteryl ester transfer protein inhibitors

Subnormal plasma levels of high-density lipoprotein cholesterol (HDL-C) constitute a major cardiovascular risk factor; raising low HDL-C levels may therefore reduce the residual cardiovascular risk that frequently presents in dyslipidaemic subjects despite statin therapy. Cholesteryl ester transfer protein (CETP), a key modulator not only of the intravascular metabolism of HDL and apolipoprotein (apo) A-I but also of triglyceride (TG)-rich particles and low-density lipoprotein (LDL), mediates the transfer of cholesteryl esters from HDL to pro-atherogenic apoB-lipoproteins, with heterotransfer of TG mainly from very low-density lipoprotein to HDL. Cholesteryl ester transfer protein activity is elevated in the dyslipidaemias of metabolic disease involving insulin resistance and moderate to marked hypertriglyceridaemia, and is intimately associated with premature atherosclerosis and high cardiovascular risk. Cholesteryl ester transfer protein inhibition therefore presents a preferential target for elevation of HDL-C and reduction in atherosclerosis. This review appraises recent evidence for a central role of CETP in the action of current lipid-modulating agents with HDL-raising potential, i.e. statins, fibrates, and niacin, and compares their mechanisms of action with those of pharmacological agents under development which directly inhibit CETP. New CETP inhibitors, such as dalcetrapib and anacetrapib, are targeted to normalize HDL/apoA-I levels and anti-atherogenic activities of HDL particles. Further studies of these CETP inhibitors, in particular in long-term, large-scale outcome trials, will provide essential information on their safety and efficacy in reducing residual cardiovascular risk.

Proatherogenic Role of Elevated CE Transfer From HDL to VLDL1 and Dense LDL in Type 2 Diabetes

Abstract —Plasma cholesteryl ester transfer protein (CETP) facilitates intravascular lipoprotein remodeling by promoting the heteroexchange of neutral lipids. To determine whether the degree of triglyceridemia may influence the CETP-mediated redistribution of HDL CE between atherogenic plasma lipoprotein particles in type 2 diabetes, we evaluated CE mass transfer from HDL to apoB-containing lipoprotein acceptors in the plasma of type 2 diabetes subjects (n=38). In parallel, we investigated the potential relationship between CE transfer and the appearance of an atherogenic dense LDL profile. The diabetic population was divided into 3 subgroups according to fasting plasma triglyceride (TG) levels: group 1 (G1), TG<100 mg/dL; group 2 (G2), 100200 mg/dL. Type 2 diabetes patients displayed an asymmetrical LDL profile in which the dense LDL subfractions predominated. Plasma levels of dense LDL subfractions were strongly positively correlated with those of plasma triglyceride (TG) (r =0.471;P =0.0003). The rate of CE mass transfer from HDL to apoB-containing lipoproteins was significantly enhanced in G3 compared with G2 or G1 (46.2±8.1, 33.6±5.3, and 28.2±2.7 &mgr;g CE transferred · h−1 · mL−1 in G3, G2, and G1, respectively;P <0.0001 G3 versus G1, P =0.0001 G2 versus G1, and P =0.02 G2 versus G3). The relative capacities of VLDL and LDL to act as acceptors of CE from HDL were distinct between type 2 diabetes subgroups. LDL particles represented the preferential CE acceptor in G1 and accounted for 74% of total CE transferred from HDL. By contrast, in G2 and G3, TG-rich lipoprotein subfractions accounted for 47% and 72% of total CE transferred from HDL, respectively. Moreover, the relative proportion of CE transferred from HDL to VLDL1 in type 2 diabetes patients increased progressively with increase in plasma TG levels. The VLDL1 subfraction accounted for 34%, 43%, and 52% of total CE transferred from HDL to TG-rich lipoproteins in patients from G1, G2, and G3, respectively. Finally, dense LDL acquired an average of 45% of total CE transferred from HDL to LDL in type 2 diabetes patients. In conclusion, CETP contributes significantly to the formation of small dense LDL particles in type 2 diabetes by a preferential CE transfer from HDL to small dense LDL, as well as through an indirect mechanism involving an enhanced CE transfer from HDL to VLDL1, the specific precursors of small dense LDL particles in plasma.

Stimulation of Cholesterol Efflux by LXR Agonists in Cholesterol-Loaded Human Macrophages Is ABCA1-Dependent but ABCG1-Independent

Objective—Maintenance of cholesterol homeostasis in human macrophages is essential to prevent foam cell formation. We evaluated the relative contribution of the ABCA1 and ABCG1 transporters to cholesterol efflux from human macrophages, and of the capacity of LXR agonists to reduce foam cell formation by stimulating export of cellular cholesterol. Methods and Results—ABCG1 mRNA levels were strongly increased in acLDL-loaded THP-1 macrophages and in HMDM on stimulation with LXR agonists. However, silencing of ABCG1 expression using ABCG1-specific siRNA indicated that ABCG1 was not essential for cholesterol efflux to HDL in cholesterol-loaded human macrophages stimulated with LXR agonists. Indeed, ABCA1 was solely responsible for the stimulation of cholesterol efflux to HDL on LXR activation, as this effect was abolished in HMDM from Tangier patients. Furthermore, depletion of cellular ATP indicated that the LXR-induced export of cholesterol was an ATP-dependent transport mechanism in human macrophages. Finally, use of an anti–Cla-1 blocking antibody identified the Cla-1 receptor as a key component in cholesterol efflux to HDL from cholesterol-loaded human macrophages. Conclusion—Our data indicate that stimulation of cholesterol efflux to HDL by LXR agonists in human foam cells involves an ATP-dependent transport mechanism mediated by ABCA1 that it appears to be independent of ABCG1 expression.

Small, Dense High-Density Lipoprotein-3 Particles Are Enriched in Negatively Charged Phospholipids Relevance to Cellular Cholesterol Efflux, Antioxidative, Antithrombotic, Anti-Inflammatory, and Antiapoptotic Functionalities

Objective— High-density lipoprotein (HDL) displays multiple atheroprotective activities and is highly heterogeneous in structure, composition, and function; the molecular determinants of atheroprotective functions of HDL are incompletely understood. Because phospholipids represent a major bioactive lipid component of HDL, we characterized the phosphosphingolipidome of major normolipidemic HDL subpopulations and related it to HDL functionality. Approach and Results— Using an original liquid chromatography–mass spectrometry/mass spectrometry methodology for phospholipid and sphingolipid profiling, 162 individual molecular lipid species were quantified across the 9 lipid subclasses, in the order of decreasing abundance, phosphatidylcholine>sphingomyelin>lysophosphatidylcholine>phosphatidylethanolamine>phosphatidylinositol>ceramide>phosphatidylserine>phosphatidylglycerol>phosphatidic acid. When data were expressed relative to total lipid, the contents of lysophosphatidylcholine and of negatively charged phosphatidylserine and phosphatidic acid increased progressively with increase in hydrated density of HDL, whereas the proportions of sphingomyelin and ceramide decreased. Key biological activities of HDL subpopulations, notably cholesterol efflux capacity from human THP-1 macrophages, antioxidative activity toward low-density lipoprotein oxidation, antithrombotic activity in human platelets, cell-free anti-inflammatory activity, and antiapoptotic activity in endothelial cells, were predominantly associated with small, dense, protein-rich HDL3. The biological activities of HDL particles were strongly intercorrelated, exhibiting significant correlations with multiple components of the HDL phosphosphingolipidome. Specifically, the content of phosphatidylserine revealed positive correlations with all metrics of HDL functionality, reflecting enrichment of phosphatidylserine in small, dense HDL3. Conclusions— Our structure–function analysis thereby reveals that the HDL lipidome may strongly affect atheroprotective functionality. # Significance {#article-title-54}Objective—High-density lipoprotein (HDL) displays multiple atheroprotective activities and is highly heterogeneous in structure, composition, and function; the molecular determinants of atheroprotective functions of HDL are incompletely understood. Because phospholipids represent a major bioactive lipid component of HDL, we characterized the phosphosphingolipidome of major normolipidemic HDL subpopulations and related it to HDL functionality. Approach and Results—Using an original liquid chromatography–mass spectrometry/mass spectrometry methodology for phospholipid and sphingolipid profiling, 162 individual molecular lipid species were quantified across the 9 lipid subclasses, in the order of decreasing abundance, phosphatidylcholine>sphingomyelin>lysophosphatidylcholine>phosphatidylethanolamine>phosphatidylinositol>ceramide>phosphatidylserine>phosphatidylglycerol>phosphatidic acid. When data were expressed relative to total lipid, the contents of lysophosphatidylcholine and of negatively charged phosphatidylserine and phosphatidic acid increased progressively with increase in hydrated density of HDL, whereas the proportions of sphingomyelin and ceramide decreased. Key biological activities of HDL subpopulations, notably cholesterol efflux capacity from human THP-1 macrophages, antioxidative activity toward low-density lipoprotein oxidation, antithrombotic activity in human platelets, cell-free anti-inflammatory activity, and antiapoptotic activity in endothelial cells, were predominantly associated with small, dense, protein-rich HDL3. The biological activities of HDL particles were strongly intercorrelated, exhibiting significant correlations with multiple components of the HDL phosphosphingolipidome. Specifically, the content of phosphatidylserine revealed positive correlations with all metrics of HDL functionality, reflecting enrichment of phosphatidylserine in small, dense HDL3. Conclusions—Our structure–function analysis thereby reveals that the HDL lipidome may strongly affect atheroprotective functionality.

HDL Particle Size Is a Critical Determinant of ABCA1-Mediated Macrophage Cellular Cholesterol Export

RATIONALE High-density lipoprotein (HDL) is a heterogeneous population of particles. Differences in the capacities of HDL subfractions to remove cellular cholesterol may explain variable correlations between HDL-cholesterol and cardiovascular risk and inform future targets for HDL-related therapies. The ATP binding cassette transporter A1 (ABCA1) facilitates cholesterol efflux to lipid-free apolipoprotein A-I, but the majority of apolipoprotein A-I in the circulation is transported in a lipidated state and ABCA1-dependent efflux to individual HDL subfractions has not been systematically studied. OBJECTIVE Our aims were to determine which HDL particle subfractions are most efficient in mediating cellular cholesterol efflux from foam cell macrophages and to identify the cellular cholesterol transporters involved in this process. METHODS AND RESULTS We used reconstituted HDL particles of defined size and composition, isolated subfractions of human plasma HDL, cell lines stably expressing ABCA1 or ABCG1, and both mouse and human macrophages in which ABCA1 or ABCG1 expression was deleted. We show that ABCA1 is the major mediator of macrophage cholesterol efflux to HDL, demonstrating most marked efficiency with small, dense HDL subfractions (HDL3b and HDL3c). ABCG1 has a lesser role in cholesterol efflux and a negligible role in efflux to HDL3b and HDL3c subfractions. CONCLUSIONS Small, dense HDL subfractions are the most efficient mediators of cholesterol efflux, and ABCA1 mediates cholesterol efflux to small dense HDL and to lipid-free apolipoprotein A-I. HDL-directed therapies should target increasing the concentrations or the cholesterol efflux capacity of small, dense HDL species in vivo.

Dose-dependent action of atorvastatin in type IIB hyperlipidemia: preferential and progressive reduction of atherogenic apoB- containing lipoprotein subclasses (VLDL-2, IDL, small dense LDL) and stimulation of cellular cholesterol efflux

Type IIB hyperlipidemia is associated with premature vascular disease, an atherogenic lipoprotein phenotype characterised by elevated levels of triglyceride-rich VLDL and small dense LDL, together with subnormal levels of HDL. The dose-dependent and independent effects of a potent HMGCoA reductase inhibitor, Atorvastatin, at daily doses of 10 and 40 mg, were evaluated on triglyceride-rich lipoprotein subclasses (VLDL-1, VLDL-2 and IDL), on the major LDL subclasses (light LDL, LDL-1+LDL-2, D: 1.019-1.029 g/ml; intermediate LDL, LDL-3, D: 1.029-1.039 g/ml and small dense LDL, LDL-4+LDL+5, D: 1.039-1.063 g/ml), on CETP-mediated cholesteryl ester transfer from HDL to apoB-containing lipoproteins, on phospholipid transfer protein activity and on plasma-mediated cellular cholesterol efflux in patients (n=10) displaying type IIB hyperlipidemia. Plasma concentrations of triglyceride-rich lipoprotein subclasses (TRL: VLDL-1, Sf 60-400; VLDL-2, Sf 20-60 and IDL, Sf 12-20) and of LDL (D: 1.019-1.063 g/ml) were markedly diminished after 6 weeks of statin treatment at 10 mg per day (-31 and -36%, respectively; P<0.002) and by 42 and 51%, respectively at the 40 mg per day dose. Increasing doses of atorvastatin progressively normalised both the quantitative and qualitative features of the LDL subclass profile, in which dense LDL predominated at baseline. Indeed, dense LDL levels were reduced by up to 57% at the 40-mg dose, leading to a shift in the peak of the density profile towards larger, buoyant LDL particles typical of normolipidemic subjects. In addition, marked reduction in numbers of apoB100-containing particle acceptors led to a 30% decrease (P<0.02) in CETP-mediated CE transfer from HDL. Finally, a significant dose-dependent statin-mediated elevation (+15% at 10 mg; P=0.0003 and +35% at 40 mg; P<0.0001 compared to baseline) in the capacity of plasma from type IIB subjects to mediate free cholesterol efflux from Fu5AH hepatoma cells was observed. Moreover, atorvastatin (40 mg per day) significantly increased plasma apoAI levels (+24%; P<0.05), thereby suggesting that this statin enhances production of apoAI and with it, formation of nascent pre-beta HDL particles. Plasma PLTP activity was not affected by either dose of atorvastatin. We conclude that increasing the dose of atorvastatin leads to dose-dependent, preferential and progressive reduction in particle numbers of atherogenic VLDL-2, IDL and dense LDL, and concomitantly, to enhanced cellular cholesterol efflux in type IIB dyslipidemia, thereby diminishing the atherosclerotic burden in subjects characterised by high cardiovascular risk.

Coexistence of Foam Cells and Hypocholesterolemia in Mice Lacking the ABC Transporters A1 and G1

The concept that macrophages can become foam cells as a result of a disturbed balance between the uptake of cholesterol from lipoproteins and cholesterol efflux is generally accepted. ABCA1 and ABCG1 are two cholesterol transporters that may act sequentially to remove cellular cholesterol, but currently their combined role in vivo is unknown. We report here that targeted disruption of both ABCA1 and ABCG1 in mice, despite severe plasma hypocholesterolemia, leads to massive lipid accumulation and foam cell formation of tissue macrophages. A complete ablation of cellular cholesterol efflux in vitro is observed, whereas in vivo macrophage-specific reverse cholesterol transport to the feces is markedly decreased. Despite the massive foam cell formation of tissue macrophages, no lipid accumulation was observed in the vascular wall, even in mice of 1 year old, indicating that the double knockout mice, possibly because of their hypocholesterolemia, lack the trigger to attract macrophages to the vessel wall. In conclusion, even under hypocholesterolemic conditions macrophages can be converted into foam cells, and ABCA1 and ABCG1 play an essential role in the prevention of foam cell formation.

Mutations at a single codon in Mad homology 2 domain of SMAD4 cause Myhre syndrome

Myhre syndrome (MIM 139210) is a developmental disorder characterized by short stature, short hands and feet, facial dysmorphism, muscular hypertrophy, deafness and cognitive delay. Using exome sequencing of individuals with Myhre syndrome, we identified SMAD4 as a candidate gene that contributes to this syndrome on the basis of its pivotal role in the bone morphogenetic pathway (BMP) and transforming growth factor (TGF)-β signaling. We identified three distinct heterozygous missense SMAD4 mutations affecting the codon for Ile500 in 11 individuals with Myhre syndrome. All three mutations are located in the region of SMAD4 encoding the Mad homology 2 (MH2) domain near the site of monoubiquitination at Lys519, and we found a defect in SMAD4 ubiquitination in fibroblasts from affected individuals. We also observed decreased expression of downstream TGF-β target genes, supporting the idea of impaired TGF-β–mediated transcriptional control in individuals with Myhre syndrome.

Small, Dense High-Density Lipoprotein-3 Particles Are Enriched in Negatively Charged Phospholipids

Objective— High-density lipoprotein (HDL) displays multiple atheroprotective activities and is highly heterogeneous in structure, composition, and function; the molecular determinants of atheroprotective functions of HDL are incompletely understood. Because phospholipids represent a major bioactive lipid component of HDL, we characterized the phosphosphingolipidome of major normolipidemic HDL subpopulations and related it to HDL functionality. Approach and Results— Using an original liquid chromatography–mass spectrometry/mass spectrometry methodology for phospholipid and sphingolipid profiling, 162 individual molecular lipid species were quantified across the 9 lipid subclasses, in the order of decreasing abundance, phosphatidylcholine>sphingomyelin>lysophosphatidylcholine>phosphatidylethanolamine>phosphatidylinositol>ceramide>phosphatidylserine>phosphatidylglycerol>phosphatidic acid. When data were expressed relative to total lipid, the contents of lysophosphatidylcholine and of negatively charged phosphatidylserine and phosphatidic acid increased progressively with increase in hydrated density of HDL, whereas the proportions of sphingomyelin and ceramide decreased. Key biological activities of HDL subpopulations, notably cholesterol efflux capacity from human THP-1 macrophages, antioxidative activity toward low-density lipoprotein oxidation, antithrombotic activity in human platelets, cell-free anti-inflammatory activity, and antiapoptotic activity in endothelial cells, were predominantly associated with small, dense, protein-rich HDL3. The biological activities of HDL particles were strongly intercorrelated, exhibiting significant correlations with multiple components of the HDL phosphosphingolipidome. Specifically, the content of phosphatidylserine revealed positive correlations with all metrics of HDL functionality, reflecting enrichment of phosphatidylserine in small, dense HDL3. Conclusions— Our structure–function analysis thereby reveals that the HDL lipidome may strongly affect atheroprotective functionality. # Significance {#article-title-54}Objective—High-density lipoprotein (HDL) displays multiple atheroprotective activities and is highly heterogeneous in structure, composition, and function; the molecular determinants of atheroprotective functions of HDL are incompletely understood. Because phospholipids represent a major bioactive lipid component of HDL, we characterized the phosphosphingolipidome of major normolipidemic HDL subpopulations and related it to HDL functionality. Approach and Results—Using an original liquid chromatography–mass spectrometry/mass spectrometry methodology for phospholipid and sphingolipid profiling, 162 individual molecular lipid species were quantified across the 9 lipid subclasses, in the order of decreasing abundance, phosphatidylcholine>sphingomyelin>lysophosphatidylcholine>phosphatidylethanolamine>phosphatidylinositol>ceramide>phosphatidylserine>phosphatidylglycerol>phosphatidic acid. When data were expressed relative to total lipid, the contents of lysophosphatidylcholine and of negatively charged phosphatidylserine and phosphatidic acid increased progressively with increase in hydrated density of HDL, whereas the proportions of sphingomyelin and ceramide decreased. Key biological activities of HDL subpopulations, notably cholesterol efflux capacity from human THP-1 macrophages, antioxidative activity toward low-density lipoprotein oxidation, antithrombotic activity in human platelets, cell-free anti-inflammatory activity, and antiapoptotic activity in endothelial cells, were predominantly associated with small, dense, protein-rich HDL3. The biological activities of HDL particles were strongly intercorrelated, exhibiting significant correlations with multiple components of the HDL phosphosphingolipidome. Specifically, the content of phosphatidylserine revealed positive correlations with all metrics of HDL functionality, reflecting enrichment of phosphatidylserine in small, dense HDL3. Conclusions—Our structure–function analysis thereby reveals that the HDL lipidome may strongly affect atheroprotective functionality.