Fabiana Rached

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.

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.

An Overview of the New Frontiers in the Treatment of Atherogenic Dyslipidemias

Cardiovascular diseases (CVDs) are the leading cause of morbidity/mortality worldwide. Dyslipidemia is a major risk factor for premature atherosclerosis and CVD. Lowering low‐density‐lipoprotein cholesterol (LDL‐C) levels is well established as an intervention for the reduction of CVDs. Statins are the first‐line drugs for treatment of dyslipidemia, but they do not address all CVD risk. Development of novel therapies is ongoing and includes the following: (i) reduction of LDL‐C concentrations using antibodies to proprotein convertase subtilisin/kexin‐9, antisense oligonucleotide inhibitors of apolipoprotein B production, microsomal transfer protein (MTP) inhibitors, and acyl‐coenzyme A cholesterol acyl transferase inhibitors; (ii) reduction in levels of triglyceride‐rich lipoproteins with ω‐3 fatty acids, MTP inhibitors, and diacylglycerol acyl transferase‐1 inhibitors; and (iii) increase of high‐density‐lipoprotein (HDL) cholesterol levels, HDL particle numbers, and/or HDL functionality using cholesteryl ester transfer protein inhibitors, HDL‐derived agents, apolipoprotein AI mimetic peptides, and microRNAs. Large prospective outcome trials of several of these emerging therapies are under way, and thrilling progress in the field of lipid management is anticipated.

Defective functionality of small, dense HDL3 subpopulations in ST segment elevation myocardial infarction: Relevance of enrichment in lysophosphatidylcholine, phosphatidic acid and serum amyloid A

BACKGROUND Low plasma levels of high-density lipoprotein-cholesterol (HDL-C) are typical of acute myocardial infarction (MI) and predict risk of recurrent cardiovascular events. The potential relationships between modifications in the molecular composition and the functionality of HDL subpopulations in acute MI however remain indeterminate. METHODS AND RESULTS ST segment elevation MI (STEMI) patients were recruited within 24h after diagnosis (n=16) and featured low HDL-C (-31%, p<0.05) and acute-phase inflammation (determined as marked elevations in C-reactive protein, serum amyloid A (SAA) and interleukin-6) as compared to age- and sex-matched controls (n=10). STEMI plasma HDL and its subpopulations (HDL2b, 2a, 3a, 3b, 3c) displayed attenuated cholesterol efflux capacity from THP-1 cells (up to -32%, p<0.01, on a unit phospholipid mass basis) vs. CONTROLS Plasma HDL and small, dense HDL3b and 3c subpopulations from STEMI patients exhibited reduced anti-oxidative activity (up to -68%, p<0.05, on a unit HDL mass basis). HDL subpopulations in STEMI were enriched in two proinflammatory bioactive lipids, lysophosphatidylcholine (up to 3.0-fold, p<0.05) and phosphatidic acid (up to 8.4-fold, p<0.05), depleted in apolipoprotein A-I (up to -23%, p<0.05) and enriched in SAA (up to +10.2-fold, p<0.05); such changes were most marked in the HDL3b subfraction. In vitro HDL enrichment in both lysophosphatidylcholine and phosphatidic acid exerted deleterious effects on HDL functionality. CONCLUSIONS In the early phase of STEMI, HDL particle subpopulations display marked, concomitant alterations in both lipidome and proteome which are implicated in impaired HDL functionality. Such modifications may act synergistically to confer novel deleterious biological activities to STEMI HDL. SIGNIFICANCE Our present data highlight complex changes in the molecular composition and functionality of HDL particle subpopulations in the acute phase of STEMI, and for the first time, reveal that concomitant modifications in both the lipidome and proteome contribute to functional deficiencies in cholesterol efflux and antioxidative activities of HDL particles. These findings may provide new biomarkers and new insights in therapeutic strategy to reduce cardiovascular risk in this clinical setting where such net deficiency in HDL function, multiplied by low circulating HDL concentrations, can be expected to contribute to accelerated atherogenesis.

Radial augmentation index is a surrogate marker of atherosclerotic burden in a primary prevention cohort

Arterial stiffness is linked to cardiovascular risk and predicts clinical events independently of peripheral blood pressure. The potential relationship between the augmentation index measured at the radial artery and asymptomatic atherosclerosis remains unclear however. In order to assess relationship between the peripheral augmentation index and traditional risk factors, we estimated cardiovascular risk and presence of subclinical atherosclerosis in a large asymptomatic population in primary prevention. Patients in primary prevention (n = 1007) with at least 1 cardiovascular risk factor were included and radial augmentation index was measured. Maximum common carotid intima-media thickness, the presence of plaque and Framingham 10 year cardiovascular risk score were assessed. The mean augmentation index was 81 ± 13% in a population composed of 55% males (mean age 56 years). The augmentation index differed significantly between men (77 ± 12%) and women (86 ± 12%). In the global population, augmentation index was negatively correlated to height and weight, and positively correlated to cardiovascular risk, age, systolic blood pressure, pulse pressure, diabetes, HDL-Cholesterol, fasting glucose, intima-media thickness and to the presence of plaques. Multivariate analysis in the global and in the male population revealed an independent and positive relationship between augmentation index and intima-media thickness on the one hand, and between augmentation index and the presence of plaque on the other. Our results confirm that there are significant relationships between a surrogate marker of arterial stiffness and subclinical atherosclerosis in a large primary prevention population.

HDL particle subpopulations: Focus on biological function.

Low levels of high‐density lipoprotein‐cholesterol (HDL‐C) constitute an independent biomarker of cardiovascular morbi‐mortality. However, recent advances have drastically modified the classical and limited view of HDL as a carrier of ‘good cholesterol’, and have revealed unexpected levels of complexity in the circulating HDL particle pool. HDL particles are indeed highly heterogeneous in structure, intravascular metabolism and biological activity. This review describes recent progress in our understanding of HDL subpopulations and their biological activities, and focuses on relationships between the structural, compositional and functional heterogeneity of HDL particles.

Lack of clopidogrel-statin interaction in patients undergoing coronary stent implantation

BACKGROUND Some studies have suggested reduced activity of clopidogrel on platelet activation and adherence in patients using statins. OBJECTIVE To assess whether platelet activation and aggregation decrease with clopidogrel, and whether there is a reduction of the action of clopidogrel when associated with atorvastatin or simvastatin. METHODS This prospective study included 68 patients with stable angina with previous use of simvastatin, atorvastatin, or no statin (control group), with previous elective indication of percutaneous coronary intervention (PCI). Platelet activation was analyzed by means of platelet count, levels of P-selectin and glycoprotein IIb/IIIa (with and without ADP stimulation) by flow cytometry. The findings were analyzed before and after percutaneous coronary intervention and the administration of clopidogrel. RESULTS We observed reduction in platelet activity with use of clopidogrel. Furthermore, no differences were found between the variables analyzed to prove reduced activity of clopidogrel when combined with statins. We observed levels of p-selectin (pre-angioplasty: 14.23 ± 7.52 x 8.83 x 11.45 ± 7.65 ± 7.09; after angioplasty: 21.49 ± 23.82 x 4 37 ± 2.71 x 4.82 ± 4.47, ρ < 0.01) and glycoprotein IIb/IIIa (pre-angioplasty: 98.97 ± 0.43 ± 1.25 x 98.79 x 99.21 ± 0.40 after angioplasty: 99.37 ± 0.29 ± 1.47 x 98.50 x 98.92 ± 0.88, ρ = 0.52), respectively, in the control, atorvastatin and simvastatin groups. CONCLUSION We concluded that platelet activation decreases with administration of clopidogrel, and clopidogrel has no antiplatelet effect reduced in the presence of simvastatin or atorvastatin.FUNDAMENTO: Alguns estudos tem sugerido reducao da atividade do clopidogrel sobre a ativacao e adesao plaquetarias em pacientes em uso de estatinas. OBJETIVO: Avaliar se a ativacao e agregacao plaquetarias diminuem com clopidogrel, e se ocorre reducao da acao do clopidogrel quando associado a atorvastatina ou a sinvastatina. METODOS: Estudo prospectivo que incluiu 68 pacientes com angina estavel em uso previo de sinvastatina, atorvastatina, ou nenhuma estatina (grupo controle), com indicacao previa eletiva de realizacao de intervencao coronaria percutânea. Foi analisada a ativacao plaquetaria atraves do numero de plaquetas, niveis de P-selectina e glucoproteina IIb/IIIa (com e sem estimulo de ADP) atraves de citometria de fluxo. Os resultados foram analisados antes e apos a intervencao coronaria percutânea e da administracao de clopidogrel. RESULTADOS: Observamos reducao da atividade plaquetaria com uso de clopidogrel. Alem disso, nao houve diferencas entre as variaveis analisadas que comprovassem reducao da atividade do clopidogrel quando associado a estatinas. Observou-se niveis de p-selectina (pre-angioplastia: 14,23±7,52 x 11,45±8,83 x 7,65±7,09; pos angioplastia: 21,49±23,82 x 4,37±2,71 x 4,82±4,47, ρ<0,01) e glicoproteina IIb/IIIa (pre-angioplastia: 98,97±0,43 x 98,79±1,25 x 99,21±0,40; pos angioplastia: 99,37±0,29 x 98,50±1,47 x 98,92±0,88, ρ=0,52), respectivamente nos grupos controle, atorvastatina e sinvastatina. CONCLUSAO: Concluimos que a ativacao plaquetaria diminui com a administracao de clopidogrel, e que o clopidogrel nao tem seu efeito antiplaquetario reduzido na presenca de sinvastatina ou atorvastatina.

Early Saphenous Vein Graft In-Stent Neoatherosclerosis by Optical Coherence Tomography

In-stent lipid-rich neoatherosclerosis has been recently described as a mechanism of late stent failure in native coronary arteries. Previous studies have shown that this condition occurs more commonly and much earlier after drug-eluting stent than bare-metal stent implantation. This process is related to insudation of lipids superficial to the stent strut level in the early phase after percutaneous coronary intervention. Subsequently, the final transformation to lipid-laden neointima and neoatherosclerosis progress during the extended follow-up period. Enhanced neoatherosclerosis in drug-eluting stents may be explained by delayed arterial healing and endothelial cell dysfunction, which are natural barriers inhibiting lipid infiltration and migration of inflammatory cells. By optical coherence tomography (OCT), early in-stent restenosis ( 5 years) presents several different image patterns, including heterogeneous tissue with a low-intensity area of lipid pools and calcification, suggesting a prominent role of atherosclerosis progression as a pathogenic mechanism. However, the

Impact of Lipoproteins on Atherobiology: Emerging Insights

Apolipoprotein B-containing lipoproteins and low-density lipoprotein play a key role in atherosclerotic vascular disease. Modified forms of low-density lipoprotein drive inflammation, an integral aspect of plaque progression. High-density lipoprotein particles are equipped to protect low-density lipoprotein from enzymatic and nonenzymatic modification. Under normal conditions, high-density lipoproteins facilitate cholesterol efflux from tissues, preventing its accumulation with deleterious consequences. However, the high-density lipoprotein particles characteristic of dyslipidemic states associated with premature atherosclerosis are typically dysfunctional as a result of alteration in their metabolism and consequently their structure and composition. Such an effect indirectly enhances low-density lipoprotein atherogenicity.

Validating the benefits of lipid modifying therapies upon atherosclerotic plaque by computed tomography angiography: should we trust our eyes? Commentary on the study of Zeb et al.

Statin treatment is considered as one of the most effective therapies for the stabilization of vulnerable atherosclerotic plaques, and have been shown to reduce overall morbidity and mortality when used for either primary [1,2] or secondary prevention [3,4] of cardiovascular disease (CVD). The mechanism by which statins provide cardiovascular benefit in these clinical settings, however, is not fully elucidated. Statins are known to have a wide range of biological effects, including decrease in pro-atherogenic plasma lipoproteins, as well as a modest increase of high-density lipoprotein-cholesterol (HDLC) levels [5]. However, the favorable outcomes of these drugs have been assigned thus far to changes in LDL-C and HDL-C only. Indeed, statins have important non-lipid effects that include antiinflammatory, antioxidant and, antithrombotic actions as well as mobilization of endothelial progenitor cells [6] and modification of plaque cholesterol crystallization [7]. These combined effects lead to atherosclerotic plaque stabilization in both quantitative (slowing