Gilles Lambert

PCSK9 Association With Lipoprotein(a)

RATIONALEnLipoprotein(a) [Lp(a)] is a highly atherogenic low-density lipoprotein-like particle characterized by the presence of apoprotein(a) [apo(a)] bound to apolipoprotein B. Proprotein convertase subtilisin/kexin type 9 (PCSK9) selectively binds low-density lipoprotein; we hypothesized that it can also be associated with Lp(a) in plasma.nnnOBJECTIVEnCharacterize the association of PCSK9 and Lp(a) in 39 subjects with high Lp(a) levels (range 39-320 mg/dL) and in transgenic mice expressing either human apo(a) only or human Lp(a) (via coexpression of human apo(a) and human apolipoprotein B).nnnMETHODS AND RESULTSnWe show that PCSK9 is physically associated with Lp(a) in vivo using 3 different approaches: (1) analysis of Lp(a) fractions isolated by ultracentrifugation; (2) immunoprecipitation of plasma using antibodies to PCSK9 and immunodetection of apo(a); (3) ELISA quantification of Lp(a)-associated PCSK9. Plasma PCSK9 levels correlated with Lp(a) levels, but not with the number of kringle IV-2 repeats. PCSK9 did not bind to apo(a) only, and the association of PCSK9 with Lp(a) was not affected by the loss of the apo(a) region responsible for binding oxidized phospholipids. Preferential association of PCSK9 with Lp(a) versus low-density lipoprotein (1.7-fold increase) was seen in subjects with high Lp(a) and normal low-density lipoprotein. Finally, Lp(a)-associated PCSK9 levels directly correlated with plasma Lp(a) levels but not with total plasma PCSK9 levels.nnnCONCLUSIONSnOur results show, for the first time, that plasma PCSK9 is found in association with Lp(a) particles in humans with high Lp(a) levels and in mice carrying human Lp(a). Lp(a)-bound PCSK9 may be pursued as a biomarker for cardiovascular risk.

A MARCH6 and IDOL E3 Ubiquitin Ligase Circuit Uncouples Cholesterol Synthesis from Lipoprotein Uptake in Hepatocytes

ABSTRACT Cholesterol synthesis and lipoprotein uptake are tightly coordinated to ensure that the cellular level of cholesterol is adequately maintained. Hepatic dysregulation of these processes is associated with pathological conditions, most notably cardiovascular disease. Using a genetic approach, we have recently identified the E3 ubiquitin ligase MARCH6 as a regulator of cholesterol biosynthesis, owing to its ability to promote degradation of the rate-limiting enzymes 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGCR) and squalene epoxidase (SQLE). Here, we present evidence for MARCH6 playing a multifaceted role in the control of cholesterol homeostasis in hepatocytes. We identify MARCH6 as an endogenous inhibitor of the sterol regulatory element binding protein (SREBP) transcriptional program. Accordingly, loss of MARCH6 increases expression of SREBP-regulated genes involved in cholesterol biosynthesis and lipoprotein uptake. Unexpectedly, this is associated with a decrease in cellular lipoprotein uptake, induced by enhanced lysosomal degradation of the low-density lipoprotein receptor (LDLR). Finally, we provide evidence that induction of the E3 ubiquitin ligase IDOL represents the molecular mechanism underlying this MARCH6-induced phenotype. Our study thus highlights a MARCH6-dependent mechanism to direct cellular cholesterol accretion that relies on uncoupling of cholesterol synthesis from lipoprotein uptake.

Effect of atorvastatin, cholesterol ester transfer protein inhibition, and diabetes mellitus on circulating proprotein subtilisin kexin type 9 and lipoprotein(a) levels in patients at high cardiovascular risk

BACKGROUNDnProprotein subtilisin kexin type 9 (PCSK9) and lipoprotein (a) [Lp(a)] levels are causative risk factors for coronary heart disease.nnnOBJECTIVESnThe objective of the study was to determine the impact of lipid-lowering treatments on circulating PCSK9 and Lp(a).nnnMETHODSnWe measured PCSK9 and Lp(a) levels in plasma samples from Investigation of Lipid Level Management to Understand its Impact in Atherosclerotic Events trial patients with coronary heart disease and/or type II diabetes (T2D) mellitus. Patients received atorvastatin, which was titrated (10, 20, 40, or 80xa0mg/d) to achieve low-density lipoprotein cholesterol levels <100xa0mg/dL (baseline) and were subsequently randomized either to atorvastatinxa0+xa0torcetrapib, a cholesterol ester transfer protein inhibitor, or to atorvastatinxa0+xa0placebo.nnnRESULTSnAt baseline, both plasma PCSK9 and Lp(a) were dose-dependently increased with increasing atorvastatin doses. Compared with patients without T2D, those with T2D had higher PCSK9 (357xa0±xa0123 vs 338xa0±xa0115xa0ng/mL, Pxa0=xa0.0012) and lower Lp(a) levels (28xa0±xa032 vs 32xa0±xa033xa0mg/dL, Pxa0=xa0.0005). Plasma PCSK9 levels significantly increased in patients treated with torcetrapib (+13.1xa0±xa0125.3xa0ng/mL [+3.7%], Pxa0=xa0.005), but not in patients treated with placebo (+2.6xa0±xa0127.9xa0ng/mL [+0.7%], Pxa0=xa0.39). Plasma Lp(a) levels significantly decreased in patients treated with torcetrapib (-3.4xa0±xa010.7xa0mg/dL [-11.1%], Pxa0<xa0.0001), but not in patients treated with placebo (+0.3xa0±xa09.4xa0mg/dL [+0.1%], Pxa0=xa0.92).nnnCONCLUSIONnIn patients at high cardiovascular disease risk, PCSK9 and Lp(a) are positively and dose-dependently correlated with atorvastatin dosage, whereas the presence of T2D is associated with higher PCSK9 but lower Lp(a) levels. Cholesterol ester transfer protein inhibition with torcetrapib slightly increases PCSK9 levels and decreases Lp(a) levels.

Emerging Cardiovascular Disease Biomarkers and Incident Diabetes Mellitus Risk in Statin-Treated Patients With Coronary Artery Disease (from the Treating to New Targets [TNT] Study).

Whether biomarkers associated with cardiovascular disease risk also predict incident diabetes mellitus (DM) is unknown. Our objective was to determine if a panel of 18 biomarkers previously associated with risk of cardiovascular disease also predicts incident DM in statin-treated patients with coronary artery disease (CAD). The Treating to New Targets (TNT) study is a randomized trial that compared the efficacy of high (80xa0mg) versus low (10xa0mg) dose atorvastatin for the secondary prevention of coronary heart disease events. Fasting plasma levels of standard lipids and of 18 emerging CAD risk biomarkers were obtained after an 8-week run-in period on atorvastatin 10xa0mg in a random sample of 1,424 TNT patients. After exclusion of patients with DM at baseline (nxa0= 253), 101 patients developed DM during the median follow-up of 4.9xa0years. Patients with incident DM had lower levels of total and high-molecular weight adiponectin, lipoprotein-associated phospholipase A2 (Lp-PLA2), soluble receptor of advanced glycation end products, and vitamin D compared with patients without incident DM. In contrast, insulin, soluble CD40 ligand, and soluble intercellular adhesion molecule-1 levels were higher in patients with incident DM compared with those without. Plasma levels of C-reactive protein, cystatin C, lipoprotein(a), monocyte chemotactic protein-1, matrix metalloproteinase-9, myeloperoxidase, neopterin, N-terminal fragment of pro-B-type natriuretic peptide, osteopontin, and soluble vascular cell adhesion molecule-1 were comparable in patients with and without incident DM. After multivariate adjustment, total and high-molecular weight adiponectin as well as Lp-PLA2 were negatively associated with incident DM. Results of this study suggest that plasma lipids and some emerging CAD risk biomarkers, such as adiponectin and Lp-PLA2, may be useful for predicting incident DM in statin-treated patients with stable CAD.

PCSK9 inhibition with alirocumab reduces lipoprotein(a) levels in nonhuman primates by lowering apolipoprotein(a) production rate

Therapeutic antibodies targeting proprotein convertase subtilisin kexin type 9 (PCSK9) (e.g. alirocumab) lower low-density lipoprotein cholesterol (LDL-C) and lipoprotein (a) [Lp(a)] levels in clinical trials. We recently showed that PCSK9 enhances apolipoprotein(a) [apo(a)] secretion from primary human hepatocytes but does not affect Lp(a) cellular uptake. Here, we aimed to determine how PCSK9 neutralization modulates Lp(a) levels in vivoSix nonhuman primates (NHP) were treated with alirocumab or a control antibody (IgG1) in a crossover protocol. After the lowering of lipids reached steady state, NHP received an intravenous injection of [2H3]-leucine, and blood samples were collected sequentially over 48 h. Enrichment of apolipoproteins in [2H3]-leucine was assessed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Kinetic parameters were calculated using numerical models with the SAAMII software. Compared with IgG1, alirocumab significantly reduced total cholesterol (TC) (-28%), LDL-C (-67%), Lp(a) (-56%), apolipoprotein B100 (apoB100) (-53%), and apo(a) (-53%). Alirocumab significantly increased the fractional catabolic rate of apoB100 (+29%) but not that of apo(a). Conversely, alirocumab sharply and significantly reduced the production rate (PR) of apo(a) (-42%), but not significantly that of apoB100, compared with IgG1, respectively.In line with the observations made in human hepatocytes, the present kinetic study establishes that PCSK9 neutralization with alirocumab efficiently reduces circulating apoB100 and apo(a) levels by distinct mechanisms: apoB primarily by enhancing its catabolism and apo(a) primarily by lowering its production.

A hemorrhagic transformation model of mechanical stroke therapy with acute hyperglycemia in mice

Clinical benefit for mechanical thrombectomy (MT) in stroke was recently demonstrated in multiple large prospective studies. Acute hyperglycemia (HG) is an important risk factor of poor outcome in stroke patients, including those that underwent MT. The aim of this therapy is to achieve a complete reperfusion in a short time, given that reperfusion damage is dependent on the duration of ischemia. Here, we investigated the effects of acute HG in a mouse model of ischemic stroke induced by middle cerebral artery occlusion (MCAO). Hyperglycemic (intraperitoneal [ip] injection of glucose) and control (ip saline injection) 10‐week male C57BL6 mice were subjected to MCAO (30, 90, and 180 min) followed by reperfusion obtained by withdrawal of the monofilament. Infarct volume, hemorrhagic transformation (HT), neutrophil infiltration, and neurological scores were assessed at 24 hr by performing vital staining, ELISA immunofluorescence, and behavioral test, respectively. Glucose injection led to transient HG (blood glucoseu2009=u2009250–390 mg/dL) that significantly increased infarct volume, HT, and worsened neurological outcome. In addition, we report that HG promoted blood‐brain barrier disruption as shown by hemoglobin accumulation in the brain parenchyma and tended to increase neutrophil extravasation within the infarcted area. Acute HG increased neurovascular damage for all MCAO durations tested. HTs were observed as early as 90 min after ischemia under hyperglycemic conditions. This model mimics MT ischemia/reperfusion and allows the exploration of brain injury in hyperglycemic conditions.

Inhibiting PCSK9 — biology beyond LDL control

Clinical trials have unequivocally shown that inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9) efficaciously and safely prevents cardiovascular events by lowering levels of LDL cholesterol. PCSK9 in the circulation is derived mainly from the liver, but the protein is also expressed in the pancreas, the kidney, the intestine and the central nervous system. Although PCSK9 modulates cholesterol metabolism by regulating LDL receptor expression in the liver, in vitro and in vivo studies have suggested that PCSK9 is involved in various other physiological processes. Although therapeutic PCSK9 inhibition could theoretically have undesired effects by interfering with these non-cholesterol-related processes, studies of individuals with genetically determined reduced PCSK9 function and clinical trials of PCSK9 inhibitors have not revealed clinically meaningful adverse consequences of almost completely eradicating PCSK9 from the circulation. The clinical implications of PCSK9 functions beyond lipid metabolism in terms of wanted or unwanted effects of therapeutic PCSK9 inhibition therefore appear to be limited. The objective of this Review is to describe the physiological role of PCSK9 beyond the LDL receptor to provide a rational basis for monitoring the effects of PCSK9 inhibition as these drugs gain traction in the clinic.Proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors prevent cardiovascular events by lowering levels of LDL cholesterol derived from the liver. However, PCSK9 is expressed in many other tissues, including the pancreas and central nervous system. This Review explores the functions of PCSK9 beyond the control of cholesterol levels.Key pointsPCSK9 is expressed in several tissues other than the liver, including the pancreas, the kidney, the intestine and the brain.Although PCSK9 might be involved in various pathophysiological and physiological processes in different organ systems, the clinical implications for therapeutic PCSK9 inhibition seem to be limited.Clinical trials of PCSK9 inhibitors and studies of individuals with genetically determined reduced PCSK9 activity have provided reassurance regarding the safety of therapeutic PCSK9 inhibition.

Kinetics of plasma Apolipoprotein E isoforms by LC-MS/MS: a pilot study

Human apoE exhibits three major isoforms (apoE2, apoE3, and apoE4) corresponding to polymorphism in the APOE gene. Total plasma apoE concentrations are closely related to these isoforms, but the underlying mechanisms are unknown. We aimed to describe the kinetics of apoE individual isoforms to explore the mechanisms for variable total apoE plasma concentrations. We used LC-MS/MS to discriminate between isoforms by identifying specific peptide sequences in subjects (three E2/E3, three E3/E3, and three E3/E4 phenotypes) who received a primed constant infusion of 2H3-leucine for 14 h. apoE concentrations and leucine enrichments were measured hourly in plasma. Concentrations of apoE2 were higher than apoE3, and concentrations of apoE4 were lower than apoE3. There was no difference between apoE3 and apoE4 catabolic rates and between apoE2 and apoE3 production rates (PRs), but apoE2 catabolic rates and apoE4 PRs were lower. The mechanisms leading to the difference in total plasma apoE concentrations are therefore related to contrasted kinetics of the isoforms. Production or catabolic rates are differently affected according to the specific isoforms. On these grounds, studies on the regulation of the involved biochemical pathways and the impact of pathological environments are now warranted.

Severe decrease in high-density lipoprotein cholesterol with the combination of fibrates and ezetimibe: A case series

A sudden and severe drug-induced decrease in plasma high-density lipoprotein cholesterol (HDL-C) is a rare condition. We report 2 patients with familial hypercholesterolemia treated with statins and fibrates and 2 others with mixed dyslipidemia treated with fibrates, who presented with a sudden and severe decrease in HDL-C (from -44% to -95%, compared with baseline). Three of the patients were treated with fibrates and had a sudden decrease in HDL-C after the adjunction of ezetimibe. HDL-C returned to normal levels after discontinuation of the offending therapies. In 2 of these patients, the reintroduction of ezetimibe with no fibrates did not affect HDL-C. In conclusion, we report a new profile of patients who are at risk for a sudden drop of HDL-C related to treatment with a combination of fibrates and ezetimibe. Although a sudden drop of HDL-C is a rare event, we recommend to carefully monitor plasma HDL-C in patients submitted to both drugs.