Isabelle Ruel

The HDL proteome in acute coronary syndromes shifts to an inflammatory profile

Inflammation is a major factor underlying acute coronary syndromes (ACS). HDL particles may be remodeled, becoming functionally defective, under the inflammatory conditions seen in ACS. Shotgun proteomics was used to monitor changes in the HDL proteome between male age-matched control, stable CAD, and ACS subjects (n=10/group). HDL was isolated by ultracentrifugation and separated by 1D-gel followed by LC-MS/MS. We identified 67 HDL-associated proteins, 20 of which validated recently identified proteins including vitronectin and complement C4B, and 5 of which were novel. Using gene ontology analysis, we found that the HDL-proteome consisted of proteins involved in cholesterol homeostasis (~50%), with significant contributions by proteins involved in lipid binding, antioxidant, acute-phase response, immune response, and endopeptidase/protease inhibition. Importantly, levels of apoA-IV were significantly reduced in ACS patients, whereas levels of serum amyloid A (SAA) and complement C3 (C3) were significantly increased (spectral counting; t-test p≤0.05), as confirmed by immunoblot or ELISA. Despite differences in protein composition, ABCA1, ABCG1, and SR-BI mediated cholesterol efflux assays did not indicate that HDL from ACS patients is functionally deficient as compared to controls, when corrected for apoA-I mass. Our results support that the HDL proteome differs between control, CAD and ACS patients. Increased abundance of SAA, C3, and other inflammatory proteins in HDL from ACS patients suggests that HDL reflects a shift to an inflammatory profile which, in turn, might alter the protective effects of HDL on the atherosclerotic plaque. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).

Identification of an ABCA1-dependent phospholipid-rich plasma membrane apolipoprotein A-I binding site for nascent HDL formation: implications for current models of HDL biogenesis

It is well accepted that both apolipoprotein A-I (apoA-I) and ABCA1 play crucial roles in HDL biogenesis and in the human atheroprotective system. However, the nature and specifics of apoA-I/ABCA1 interactions remain poorly understood. Here, we present evidence for a new cellular apoA-I binding site having a 9-fold higher capacity to bind apoA-I compared with the ABCA1 site in fibroblasts stimulated with 22-(R)-hydroxycholesterol/9-cis-retinoic acid. This new cellular apoA-I binding site was designated “high-capacity binding site” (HCBS). Glyburide drastically reduced 125I-apoA-I binding to the HCBS, whereas 125I-apoA-I showed no significant binding to the HCBS in ABCA1 mutant (Q597R) fibroblasts. Furthermore, reconstituted HDL exhibited reduced affinity for the HCBS. Deletion of the C-terminal region of apoA-I (Δ187-243) drastically reduced the binding of apoA-I to the HCBS. Interestingly, overexpressing various levels of ABCA1 in BHK cells promoted the formation of the HCBS. The majority of the HCBS was localized to the plasma membrane (PM) and was not associated with membrane raft domains. Importantly, treatment of cells with phosphatidylcholine-specific phospholipase C, but not sphingomyelinase, concomitantly reduced the binding of 125I-apoA-I to the HCBS, apoA-I-mediated cholesterol efflux, and the formation of nascent apoA-I-containing particles. Together, these data suggest that a functional ABCA1 leads to the formation of a major lipid-containing site for the binding and the lipidation of apoA-I at the PM. Our results provide a biochemical basis for the HDL biogenesis pathway that involves both ABCA1 and the HCBS, supporting a two binding site model for ABCA1-mediated nascent HDL genesis.

Canadian Cardiovascular Society Position Statement on Familial Hypercholesterolemia

Familial hypercholesterolemia (FH) is the most common genetic disorder causing premature cardiovascular disease and death. Heterozygous FH conservatively affects approximately 1:500 Canadians, and the more serious homozygous form affects approximately 1:1,000,000 Canadians, although these numbers might be underestimated. Of approximately 83,500 Canadians estimated to have FH, most are undiagnosed, which represents a simultaneous public health deficit and opportunity, because early treatment of heterozygous FH can normalize life expectancy. Diagnostic algorithms for FH incorporate increased plasma low-density lipoprotein cholesterol, pathognomonic clinical features, and family history of early cardiovascular disease and hyperlipidemia. DNA-based detection of causative mutations in FH-related genes can help with diagnosis. Maximizing diagnosis and treatment of FH in Canada will involve a multipronged approach, including: (1) increasing awareness of FH among health care providers and patients; (2) creating a national registry for FH individuals; (3) setting standards for screening, including cascade screening in affected families; (4) ensuring availability of standard-of-care therapies, in particular optimization of plasma low-density lipoprotein cholesterol levels and timely access to future validated therapies; (5) promoting patient-based support and advocacy groups; and (6) forming alliances with international colleagues, resources, and initiatives that focus on FH. This document aims to raise awareness of FH nationally, and to mobilize knowledge translation, patient support, and availability of treatment and health care resources for this underrecognized, but important medical condition.

Quantitative analysis of ABCA1-dependent compartmentalization and trafficking of apolipoprotein A-I: implications for determining cellular kinetics of nascent high density lipoprotein biogenesis.

The molecular mechanisms underlying the apoA-I/ABCA1 endocytic trafficking pathway in relation to high density lipoprotein (HDL) formation remain poorly understood. We have developed a quantitative cell surface biotinylation assay to determine the compartmentalization and trafficking of apoA-I between the plasma membrane (PM) and intracellular compartments (ICCs). Here we report that 125I-apoA-I exhibited saturable association with the PM and ICCs in baby hamster kidney cells stably overexpressing ABCA1 and in fibroblasts. The PM was found to have a 2-fold higher capacity to accommodate apoA-I as compared with ICCs. Overexpressing various levels of ABCA1 in baby hamster kidney cells promoted the association of apoA-I with PM and ICCs compartments. The C-terminal deletion of apoA-I Δ(187–243) and reconstituted HDL particles exhibited reduced association of apoA-I with both the PM and ICCs. Interestingly, cell surface biotinylation with a cleavable biotin revealed that apoA-I induces ABCA1 endocytosis. Such endocytosis was impaired by naturally occurring mutations of ABCA1 (Q597R and C1477R). To better understand the role of the endocytotic pathway in the dynamics of the lipidation of apoA-I, a pulse-chase experiment was performed, and the dissociation (re-secretion) of 125I-apoA-I from both PM and ICCs was monitored over a 6-h period. Unexpectedly, we found that the time required for 50% dissociation of 125I-apoA-I from the PM was 4-fold slower than that from ICCs at 37 °C. Finally, treatment of the cells with phosphatidylcholine-specific phospholipase C, increased the dissociation of apoA-I from the PM. This study provides evidence that the lipidation of apoA-I occurs in two kinetically distinguishable compartments. The finding that apoA-I specifically mediates the continuous endocytic recycling of ABCA1, together with the kinetic data showing that apoA-I associated with ICCs is rapidly re-secreted, suggests that the endocytotic pathway plays a central role in the genesis of nascent HDL.

APOE p.Leu167del mutation in familial hypercholesterolemia.

BACKGROUND Autosomal dominant hypercholesterolemia (ADH) is caused by mutations in the low density lipoprotein receptor (LDLR), its ligand apoB (APOB) or proprotein convertase subtilisin/kexin type 9 (PCSK9) genes. Yet DNA sequencing does not identify mutations in these genes in a significant number of cases, suggesting that ADH has multiple genetic etiologies. METHODS Through a combination of clinical examination, biochemical analysis, candidate gene approach and next-generation exome sequencing we investigated the genetic basis of an ADH phenotype in a proband of an Italian origin. RESULTS The proband presented with an acute myocardial infarction at age 43. He had tendinous xanthomas, xanthelasmas and elevated levels of total and LDL cholesterol, at 11.2 and 9.69 mmol/L, respectively, with normal levels of HDL cholesterol and triglycerides at 1.62 and 1.13 mmol/L, respectively. HPLC lipoprotein profile showed selective increase in LDL-C. DNA sequencing did not identify any mutation in the LDLR, PCSK9, LDLRAP1 and APOB gene. We then performed exome sequencing on three individuals from the family. The strongest evidence of association was found for the previously identified apolipoprotein E mutation (APOE, chromosome 19:45412053-55) known as APOE Leu167del, an in-frame three base-pair deletion. Computational biology confirmed the deleterious nature of this mutation. The Leu167del mutation is predicted to alter the protein structure of apoE near the α-helix within the receptor binding domain. CONCLUSIONS This report confirms a previous report that ADH can be caused by mutations within the APOE gene and represents the 4th loci causing ADH. Standard screening for ADH should include APOE gene.

Membrane microdomains modulate oligomeric ABCA1 function: impact on apoAI-mediated lipid removal and phosphatidylcholine biosynthesis

Recent studies have identified an ABCA1-dependent, phosphatidylcholine-rich microdomain, called the “high-capacity binding site” (HCBS), that binds apoA-I and plays a pivotal role in apoA-I lipidation. Here, using sucrose gradient fractionation, we obtained evidence that both ABCA1 and [125I]apoA-I associated with the HCBS were found localized to nonraft microdomains. Interestingly, phosphatidylcholine (PtdCho) was selectively removed from nonraft domains by apoA-I, whereas sphingomyelin and cholesterol were desorbed from both detergent-resistant membranes and nonraft domains. The modulatory role of cholesterol on apoA-I binding to ABCA1/HCBS was also examined. Loading cells with cholesterol resulted in a drastic reduction in apoA-I binding. Conversely, depletion of membrane cholesterol by methyl-β-cyclodextrin treatment resulted in a significant increase in apoA-I binding. Finally, we obtained evidence that apoA-I interaction with ABCA1 promoted the activation and gene expression of key enzymes in the PtdCho biosynthesis pathway. Taken together, these results provide strong evidence that the partitioning of ABCA1/HCBS to nonraft domains plays a pivotal role in the selective desorption of PtdCho molecules by apoA-I, allowing an optimal environment for cholesterol release and regeneration of the PtdCho-containing HCBS. This process may have important implications in preventing and treating atherosclerotic cardiovascular disease.

Analysis of lipid transfer activity between model nascent HDL particles and plasma lipoproteins: implications for current concepts of nascent HDL maturation and genesis.

The specifics of nascent HDL remodeling within the plasma compartment remain poorly understood. We developed an in vitro assay to monitor the lipid transfer between model nascent HDL (LpA-I) and plasma lipoproteins. Incubation of α-125I-LpA-I with plasma resulted in association of LpA-I with existing plasma HDL, whereas incubation with TD plasma or LDL resulted in conversion of α-125I-LpA-I to preβ-HDL. To further investigate the dynamics of lipid transfer, nascent LpA-I were labeled with cell-derived [3 H]cholesterol (UC) or [3H]phosphatidylcholine (PC) and incubated with plasma at 37°C. The majority of UC and PC were rapidly transferred to apolipoprotein B (apoB). Subsequently, UC was redistributed to HDL for esterification before being returned to apoB. The presence of a phospholipid transfer protein (PLTP) stimulator or purified PLTP promoted PC transfer to apoB. Conversely, PC transfer was abolished in plasma from PLTP−/− mice. Injection of 125I-LpA-I into rabbits resulted in a rapid size redistribution of 125I-LpA-I. The majority of [3H]UC from labeled r(HDL) was esterified in vivo within HDL, whereas a minority was found in LDL. These data suggest that apoB plays a major role in nascent HDL remodeling by accepting their lipids and donating UC to the LCAT reaction. The finding that nascent particles were depleted of their lipids and remodeled in the presence of plasma lipoproteins raises questions about their stability and subsequent interaction with LCAT.

Exome Sequencing in Suspected Monogenic Dyslipidemias

Background—Exome sequencing is a promising tool for gene mapping in Mendelian disorders. We used this technique in an attempt to identify novel genes underlying monogenic dyslipidemias. Methods and Results—We performed exome sequencing on 213 selected family members from 41 kindreds with suspected Mendelian inheritance of extreme levels of low-density lipoprotein cholesterol (after candidate gene sequencing excluded known genetic causes for high low-density lipoprotein cholesterol families) or high-density lipoprotein cholesterol. We used standard analytic approaches to identify candidate variants and also assigned a polygenic score to each individual to account for their burden of common genetic variants known to influence lipid levels. In 9 families, we identified likely pathogenic variants in known lipid genes (ABCA1, APOB, APOE, LDLR, LIPA, and PCSK9); however, we were unable to identify obvious genetic etiologies in the remaining 32 families, despite follow-up analyses. We identified 3 factors that limited novel gene discovery: (1) imperfect sequencing coverage across the exome hid potentially causal variants; (2) large numbers of shared rare alleles within families obfuscated causal variant identification; and (3) individuals from 15% of families carried a significant burden of common lipid-related alleles, suggesting complex inheritance can masquerade as monogenic disease. Conclusions—We identified the genetic basis of disease in 9 of 41 families; however, none of these represented novel gene discoveries. Our results highlight the promise and limitations of exome sequencing as a discovery technique in suspected monogenic dyslipidemias. Considering the confounders identified may inform the design of future exome sequencing studies.

Comparison of Treatment of Severe High-Density Lipoprotein Cholesterol Deficiency in Men With Daily Atorvastatin (20 mg) Versus Fenofibrate (200 mg) Versus Extended-Release Niacin (2 g)

To determine whether available lipid-modifying medication can increase high-density lipoprotein (HDL) cholesterol in well-defined genetic or familial HDL-deficiency states, we studied 19 men with HDL deficiency (HDL cholesterol <5th percentile for age and gender) 55 +/- 10 years of age. Concomitant risk factors included diabetes (n = 3) and hypertension (n = 7) and 8 patients had coronary artery disease. Molecular analysis revealed that 4 patients had a mutation in the ABCA1 gene. Patients were assigned to sequentially receive atorvastatin 20 mg/day, fenofibrate 200 mg/day, and extended-release niacin 2 g/day for 8 weeks, with a 4-week washout period between each treatment. Patients in whom a statin was required, according to current treatment guidelines, were kept on atorvastatin throughout the study. Baseline HDL cholesterol level was 0.63 +/- 0.12 mmol/L (24 +/- 5 mg/dl), triglycerides 2.01 +/- 0.98 mmol/L (180 +/- 86 mg/dl), and low-density lipoprotein (LDL) cholesterol 2.29 +/- 0.95 mmol/L (94 +/- 39 mg/dl). Mean percent changes in HDL cholesterol on atorvastatin, fenofibrate, and niacin were -6% (p = NS), +6% (p = NS), and +22% (p <0.05), respectively. Furthermore, niacin significantly increased the large alpha-1 apolipoprotein A-I-containing HDL subspecies (12 to 17 nm). In conclusion, niacin was the only effective drug to increase HDL cholesterol. The absolute increase in HDL cholesterol, approximately 0.10 mmol/L (3.9 mg/dl), is of uncertain clinical significance. Biomarkers of HDL-mediated cellular cholesterol efflux were not changed by niacin therapy. Atorvastatin or fenofibrate had little effect on HDL cholesterol; atorvastatin decreased the total cholesterol/HDL cholesterol ratio by 26%. Fenofibrate did not change HDL cholesterol levels and caused an increase in LDL cholesterol. Aggressive LDL cholesterol lowering may be the strategy of choice in such patients.

Genetic Variation at the Proprotein Convertase Subtilisin/Kexin Type 5 Gene Modulates High-Density Lipoprotein Cholesterol Levels

Background— A low level of plasma high-density lipoprotein cholesterol (HDL-C) is a risk factor for cardiovascular disease. HDL particles are modulated by a variety of lipases, including endothelial lipase, a phospholipase present on vascular endothelial cells. The proprotein convertase subtilisin/kexin type 5 (PCSK5) gene product is known to directly inactivate endothelial lipase and indirectly cleave and activate angiopoetin-like protein 3, a natural inhibitor of endothelial lipase. We therefore investigated the effect of human PCSK5 genetic variants on plasma HDL-C levels. Methods and Results— Haplotypes at the PCSK5 locus were examined in 9 multigenerational families that included 60 individuals with HDL-C <10th percentile. Segregation with low HDL-C in 1 family was found. Sequencing of the PCSK5 gene in 12 probands with HDL-C <5th percentile identified 7 novel variants. Using a 2-stage design, we first genotyped these single-nucleotide polymorphisms (SNPs) along with 163 tagSNPs and 12 additional SNPs (n=182 total) in 457 individuals with documented coronary artery disease. We identified 9 SNPs associated with HDL-C (P<0.05), with the strongest results for rs11144782 and rs11144766 (P=0.002 and P=0.005, respectively). The SNP rs11144782 was also associated with very low-density lipoprotein (P=0.039), triglycerides (P=0.049), and total apolipoprotein levels (P=0.022). In stage 2, we replicated the association of rs11144766 with HDL-C (P=0.014) in an independent sample of Finnish low HDL-C families. In a combined analysis of both stages (n=883), region-wide significance of rs11144766 and low HDL-C was observed (unadjusted P=1.86×10−4 and Bonferroni-adjusted P=0.031). Conclusions— We conclude that variability at the PCSK5 locus influences HDL-C levels, possibly through the inactivation of endothelial lipase activity, and, consequently, atherosclerotic cardiovascular disease risk.