Docetaxel as a Model Compound to Promote HDL (High-Density Lipoprotein) Biogenesis and Reduce Atherosclerosis.

The recent identification of the cell-surface protein DSC1 (desmocollin 1) as a negative regulator of HDL (high-density lipoprotein) biogenesis has attracted us to revisit the old HDL biogenesis hypothesis: HDL biogenesis reduces atherosclerosis. The location and function of DSC1 suggest that DSC1 is a druggable target for the promotion of HDL biogenesis, and the discovery of docetaxel as a potent inhibitor of the DSC1 sequestration of apolipoprotein A-I has provided us with new opportunities to test this hypothesis. The FDA-approved chemotherapy drug docetaxel promotes HDL biogenesis at low-nanomolar concentrations that are far lower than used in chemotherapy. Docetaxel has also been shown to inhibit atherogenic proliferation of vascular smooth muscle cells. In accordance with these atheroprotective effects of docetaxel, animal studies have shown that docetaxel reduces dyslipidemia-induced atherosclerosis. In the absence of HDL-directed therapies for atherosclerosis, DSC1 constitutes an important new target for the promotion of HDL biogenesis, and the DSC1-targeting compound docetaxel serves as a model compound to prove the hypothesis. In this brief review, we discuss opportunities, challenges, and future directions for using docetaxel in the prevention and treatment of atherosclerosis.

Sex differences in the perception of cardiovascular risk in familial hypercholesterolemia.

BACKGROUND: Familial hypercholesterolemia (FH), a common genetic condition, is characterized by elevated low-density lipoprotein cholesterol (LDL-C) and premature atherosclerotic cardiovascular disease (ASCVD). Recent data indicate an undertreatment of females with FH. OBJECTIVE: To characterize the role of sex in the perception of FH, its associated ASCVD risk and treatment. METHODS: A survey investigating for sex differences in the perception of FH was sent to 1073 patients with FH using a cross sectional study design. RESULTS: A total of 412 patients (51.9 % male) responded to the survey; mean age was 56.2 ± 14.4 years. There was a higher proportion of males with ASCVD than females (41.5 % vs. 16.5 %, respectively, p<0.001). Analyses of the survey responses showed that a majority of both males and females agreed that their risk of ASCVD is higher than healthy individuals of same age (70.8 % vs. 74.7 %, respectively, p = 0.434). Females were more concerned about having high LDL-C levels (67.5 % vs. 56.5 % in males, p = 0.024), especially those in secondary prevention programs. As for treatment of FH, approximately 75 % of both sex groups considered statins to be efficient in reducing the risk of myocardial infarction, but less than half of the females considered statins to be safe (44.8 % vs. 60.0 % in males, p = 0.003). No major sex differences were noted regarding the influence of the doctor in their understanding of FH as a disease. CONCLUSION: Overall, both males and females with FH were well informed about FH, although females were more concerned about having high LDL-C levels and they feared the safety of statins.

Sex differences in the presentation, treatment and outcomes of patients with homozygous familial hypercholesterolemia.

BACKGROUND: Homozygous familial hypercholesterolemia (HoFH) is a rare, autosomal semi-dominant lipid metabolism disorder characterized by extremely high low-density lipoprotein cholesterol (LDL-C) levels and premature cardiovascular disease. The objective of this study was to investigate sex-differences in the treatment and outcomes of patients with HoFH. METHODS: We examined clinical characteristics, lipid-lowering therapy (LLT), and cardiovascular events using descriptive statistics of patients in the Canadian HoFH registry. Major adverse cardiovascular events (MACE) were defined as the composite of cardiovascular death, non-fatal myocardial infarction, and stroke. Sex differences between continuous and categorical variables were analyzed using Mann-Whitney U test and Fisher's Exact test, respectively. RESULTS: This study included 48 patients (27 (56%) female). The median age at diagnosis in females was 14.0 (interquartile range (IQR) 9.0-30.0) and in males was 8.0 (IQR 2.0-23.0) (p = 0.07). Baseline clinical characteristics were comparable between both sexes. The median baseline LDL-C was 12.7 mmol/L (10.0-18.3) in females and 15.3 (10.5-20.0) in males (p = 0.51). Follow up LDL-C levels were 7.6 mmol/L (IQR 4.8-11.0) in females and 6.3 (IQR 4.6-7.5) in males (p = 0.1). Most patients were taking 3 or more LLTs, with comparable proportions in both sexes (p = 0.26). Apheresis was similar in both sexes, 14 (51.8%) vs. 10 (47.6%) (p = 0.2). Over a mean of 10 years of follow-up, MACE occurred in 3 females (11.1%) and 4 males (19.1%) (p = 0.2). CONCLUSION: Lipid levels and treatment were similar between sexes. MACE occurred in similar proportions between sexes, indicating that HoFH offsets the inherently lower cardiovascular risk in pre-menopausal females. Further investigation into sex-differences in HoFH in larger sample sizes is warranted.

Biomedical Advances in ABCA1 Transporter: From Bench to Bedside.

ATP-binding cassette transporter A1 (ABCA1) has been identified as the molecular defect in Tangier disease. It is biochemically characterized by absence of high-density lipoprotein cholesterol (HDL-C) in the circulation, resulting in the accumulation of cholesterol in lymphoid tissues. Accumulation of cholesterol in arteries is an underlying cause of atherosclerosis, and HDL-C levels are inversely associated with the presence of atherosclerotic cardiovascular disease (ASCVD). ABCA1 increases HDL-C levels by driving the generation of new HDL particles in cells, and cellular cholesterol is removed in the process of HDL generation. Therefore, pharmacological strategies that promote the HDL biogenic process by increasing ABCA1 expression and activity have been intensively studied to reduce ASCVD. Many ABCA1-upregulating agents have been developed, and some have shown promising effects in pre-clinical studies, but no clinical trials have met success yet. ABCA1 has long been an attractive drug target, but the failed clinical trials have indicated the difficulty of therapeutic upregulation of ABCA1, as well as driving us to: improve our understanding of the ABCA1 regulatory system; to develop more specific and sophisticated strategies to upregulate ABCA1 expression; and to search for novel druggable targets in the ABCA1-dependent HDL biogenic process. In this review, we discuss the beginning, recent advances, challenges and future directions in ABCA1 research aimed at developing ABCA1-directed therapies for ASCVD.

Statin associated muscle symptoms (SAMS): strategies for prevention, assessment and management.

INTRODUCTION: Statins are the cornerstone for atherosclerotic cardiovascular disease risk reduction with recognized efficacy in primary and secondary prevention. Despite this, they remain underutilized due to concerns regarding adverse effects. Statin-associated muscle symptoms (SAMS) are the most frequent cause of medication intolerance and discontinuation with a prevalence estimated at 10%, regardless of causality, with the consequence of increased risk of adverse cardiovascular outcomes. AREAS COVERED: This clinical perspective reviews recent developments in mechanisms underlying the pathogenesis of statin myopathy, the role of the nocebo effect in perception of statin intolerance, and explores diverse components endorsed by international societies in establishing a statin intolerance syndrome. Non-statin drug alternatives that reduce low-density lipoprotein-cholesterol are also discussed, with emphasis on therapies with established effects on cardiovascular outcomes. EXPERT OPINION: Ultimately, a patient-centered clinical approach to managing SAMS is proposed to optimize statin tolerability, achieve guideline-recommended therapeutic goals and improve cardiovascular outcomes.

Genetic testing for familial hypercholesterolemia in Quebec, Canada: a single-centre retrospective cohort study.

BACKGROUND: Familial hypercholesterolemia (FH) is associated with premature atherosclerotic cardiovascular disease caused by elevated low-density lipoprotein cholesterol (LDL-C) levels. We determined the impact of a full next-generation sequencing (NGS) genetic panel on reclassification of patients with a clinical diagnosis of FH in Quebec compared to the partial genetic panel currently offered by the Quebec Ministère de la Santé et des Services sociaux (Ministry of Health and Social Services) (MSSS), which includes 11 variants that are common in French Canadians. METHODS: We conducted a retrospective cohort study in a subgroup of patients in the Canadian FH Registry seen at the McGill University Health Centre Preventive Cardiology/Lipid Clinic, Montréal, between September 2017 and September 2021 who were clinically diagnosed with severe hypercholesterolemia, probable FH or definite FH according to the Canadian definition of FH. Next-generation sequencing of the LDLR, APOB and PCSK9 genes, and multiplex ligation-dependent probe amplification of the LDLR gene to detect genetic variants, were performed. RESULTS: Among 335 consecutive patients with heterozygous FH (184 men [54.9%] and 151 women [45.1%]), the baseline LDL-C level was 6.96 (standard deviation 1.79) mmol/L. Patients identified through cascade screening were 11 years younger on average than index patients, and smaller proportions presented to the clinic with cardiovascular risk factors. A pathogenic FH variant was identified in 169 (73.8%) of the 229 patients who underwent genetic testing; the majority had variants in the LDLR (146 [86.4%]) or APOB (24 [14.2%]) gene. The genetic panel offered by the MSSS accounted for only 48% of the variants identified with the full NGS panel. Of the 229 patients, 90 (39.3%, 95% confidence interval 32.9%-46.0%) were reclassified from a clinical diagnosis of probable FH to definite FH after genetic screening with a full FH panel. INTERPRETATION: Genetic testing in patients suspected of having FH provided diagnostic certainty and permitted many patients with a clinical diagnosis of probable FH to be reclassified as having definite FH. Genetic screening allows for increased identification of patients with FH and may therefore help reduce the burden of cardiovascular disease and mortality rates among Canadians with FH. Trial registration: ClinicalTrials.gov, no. NCT02009345.

Atherosclerotic Coronary Plaque Regression and Risk of Adverse Cardiovascular Events: A Systematic Review and Updated Meta-Regression Analysis.

Importance: The association between changes in atherosclerotic plaque induced by lipid-lowering therapies (LLTs) and reduction in major adverse cardiovascular events (MACEs) remains controversial. Objective: To evaluate the association between coronary plaque regression assessed by intravascular ultrasound (IVUS) and MACEs. Data Sources: A comprehensive, systematic search of publications in PubMed, Embase, Cochrane Central Register of Controlled Trials, and Web of Science was performed. Study Selection: Clinical prospective studies of LLTs reporting change in percent atheroma volume (PAV) assessed by IVUS and describing MACE components were selected. Data Extraction and Synthesis: Reporting was performed in compliance with Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. The association between mean change in PAV and MACEs was analyzed by meta-regression using mixed-effects, 2-level binomial logistic regression models, unadjusted and adjusted for clinical covariates, including mean age, baseline PAV, baseline low-density lipoprotein cholesterol level, and study duration. Main Outcome and Measures: Mean PAV change and MACE in intervention and comparator arms were assessed in an updated systematic review and meta-regression analysis of IVUS trials of LLTs that also reported MACEs. Results: This meta-analysis included 23 studies published between July 2001 and July 2022, including 7407 patients and trial durations ranging from 11 to 104 weeks. Mean (SD) patient age ranged from 55.8 (9.8) to 70.2 (7.6) years, and the number of male patients from 245 of 507 (48.3%) to 24 of 26 (92.3%). Change in PAV across 46 study arms ranged from -5.6% to 3.1%. The number of MACEs ranged from 0 to 72 per study arm (17 groups [37%] reported no events, 9 [20%] reported 1-2 events, and 20 [43%] reported ≥3 events). In unadjusted analysis, a 1% decrease in mean PAV was associated with 17% reduced odds of MACEs (unadjusted OR, 0.83; 95% CI, 0.71-0.98; P = .03), and with a 14% reduction in MACEs in adjusted analysis (adjusted OR, 0.86; 95% CI, 0.75-1.00; P = .050). Further adjustment for cardiovascular risk factors showed a 19% reduced risk (adjusted OR, 0.81; 95% CI, 0.68-0.96; P = .01) per 1% decrease in PAV. A 1% reduction of PAV change between intervention and comparator arms within studies was also associated with a significant 25% reduction in MACEs (OR, 0.75; 95% CI, 0.56-1.00; P = .046). Conclusions and Relevance: In this meta-analysis, regression of atherosclerotic plaque by 1% was associated with a 25% reduction in the odds of MACEs. These findings suggest that change in PAV could be a surrogate marker for MACEs, but given the heterogeneity in the outcomes, additional data are needed.

Homozygous Familial Hypercholesterolemia in Canada: An Observational Study.

Background: Homozygous familial hypercholesterolemia (HoFH) is a rare genetic disease characterized by very high levels of low-density lipoprotein cholesterol (LDL-C). Untreated patients present with extensive xanthomas and premature atherosclerosis. Lipid-lowering therapy is highly efficacious and has dramatically increased life expectancy of patients with HoFH. Objectives: The aim of the study was to obtain a comprehensive registry of HoFH in Canada, known to have several founder effect regions, and describe the clinical characteristics and cardiovascular outcomes of this population over time. Methods: Clinical and genetic data on patients with HoFH were collected via a standardized questionnaire sent to academic sites participating in the Familial Hypercholesterolemia Canada network. Results: A total of 48 patients with HoFH were enrolled. The median age at diagnosis was 12 years (interquartile range [IQR]: 5-24) and untreated LDL-C levels were 15.0 mmol/L (IQR: 10.5-18.6 mmol/L; 580 mg/dL IQR: 404-717 mg/dL). At last follow-up visit, median age was 40 years (IQR: 26-54 years). Treated LDL-C levels were 6.75 mmol/L (IQR: 4.73-9.51 mmol/L; 261 mg/dL IQR: 183-368 mg/dL) with 95.5% of patients on statins, 88.6% on ezetimibe, 34.1% on proprotein convertase subtilisin/kexin type 9 inhibitors, 27.3% on lomitapide, 13.6% on evinacumab, and 56.8% were treated with low-density lipoprotein apheresis or plasmapheresis. Deaths were reported in 7 (14.5%) and major adverse cardiovascular events were observed in 14.6% of patients with the average onset at 30 years (IQR: 20-36 years). Aortic stenosis was reported in one-half the patients (47.9%) and 10 (20.8%) underwent aortic valve replacement. Conclusions: This HoFH patient registry in Canada will provide important new health-related knowledge about the phenotypic manifestations and determinants of cardiovascular risk in this population, allowing for closer examination of quality of life and burden to the health care system.

Genetics of cholesterol efflux.

Plasma levels of high-density lipoprotein cholesterol (HDL-C) show an inverse association with coronary heart disease (CHD). As a biological trait, HDL-C is strongly genetically determined, with a heritability index ranging from 40 % to 60 %. HDL represents an appealing therapeutic target due to its beneficial pleiotropic effects in preventing CHD. This review focuses on the genetic basis of cellular cholesterol efflux, the rate-limiting step in HDL biogenesis. There are several monogenic disorders (e.g., Tangier disease, caused by mutations within ABCA1) affecting HDL biogenesis. Importantly, many disorders of cellular cholesterol homeostasis cause a reduced HDL-C. We integrate information from family studies and linkage analyses with that derived from genome-wide association studies (GWAS) and review the recent identification of micro-RNAs (miRNA) involved in cellular cholesterol metabolism. The identification of genomic pathways related to HDL may help pave the way for novel therapeutic approaches to promote cellular cholesterol efflux as a therapeutic modality to prevent atherosclerosis.

High-Density Lipoprotein and Cardiovascular Disease-Where do We Stand?

Decades of research have shown that high-density lipoprotein cholesterol (HDL-C) levels in humans are associated with atherosclerotic cardiovascular disease (ASCVD). This association is strong and coherent across populations and remains after the elimination of covariates. Animal studies show that increasing HDL particles prevent atherosclerosis, and basic work on the biology of HDL supports a strong biological plausibility for a therapeutic target. This enthusiasm is dampened by Mendelian randomization data showing that HDL-C may not be causal in ASCVD. Furthermore, drugs that increase HDL-C have largely failed to prevent or treat ASCVD.

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-ß-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.

Tenofovir-induced osteomalacia with hypophosphataemia.

Tenofovir disoproxil fumarate (TDF) is an antiretroviral drug widely used as a first-line treatment of hepatitis B virus (HBV) and HIV. Increasing evidence has emerged associating its use with the development of Fanconi syndrome, renal insufficiency and bone disease. We report a case of a 61-year-old woman with a remote history of liver transplant for cirrhosis due to HBV. Over 1 year, the patient had recurrent falls, generalised myalgias and arthralgias, misdiagnosed as fibromyalgia. We discuss a complication of her transplant treatment regimen with the drug TDF leading to a rare but reversible disorder: tenofovir-induced Fanconi osteomalacia with renal phosphate wasting. Though recognised, this rare disorder was initially likely missed due to clinical unfamiliarity with the diagnosis, concomitant psychiatric symptoms and premature diagnostic closure.

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 alpha-(125)I-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 alpha-(125)I-LpA-I to prebeta-HDL. To further investigate the dynamics of lipid transfer, nascent LpA-I were labeled with cell-derived [(3 )H]cholesterol (UC) or [(3)H]phosphatidylcholine (PC) and incubated with plasma at 37 degrees 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 (125)I-LpA-I into rabbits resulted in a rapid size redistribution of (125)I-LpA-I. The majority of [(3)H]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.

Effect of ABCA1 mutations on risk for myocardial infarction.

The adenosine triphosphate-binding cassette A1 (ABCA1) gene codes for a cellular phospholipid and cholesterol transporter that mediates the initial and essential step in high-density lipoprotein (HDL) biogenesis: the formation of nascent HDL particles. Mutations at the ABCA1 gene locus cause severe familial HDL deficiency and, in the homozygous form, cause Tangier disease. Several studies have investigated the influence of ABCA1 variation on lipid metabolism and coronary heart disease, but they have resulted in controversial and inconsistent results. Genetic variability at the ABCA1 gene has also been associated with increased risk of myocardial infarction. In one study, this association was independent of HDL cholesterol levels, raising the possibility that the measurement of HDL cholesterol levels may not provide adequate information on the functional roles of HDL particles. Nevertheless, genomic screening for complex diseases, such as coronary heart disease, and HDL deficiency in particular, may not add additional information to that gained from conventional global cardiovascular risk stratification.

The WWOX gene modulates high-density lipoprotein and lipid metabolism.

BACKGROUND: Low levels of high-density lipoprotein (HDL) cholesterol constitutes a major risk factor for atherosclerosis. Recent studies from our group reported a genetic association between the WW domain-containing oxidoreductase (WWOX) gene and HDL cholesterol levels. Here, through next-generation resequencing, in vivo functional studies and gene microarray analyses, we investigated the role of WWOX in HDL and lipid metabolism. METHODS AND RESULTS: Using next-generation resequencing of the WWOX region, we first identified 8 variants significantly associated and perfectly segregating with the low-HDL trait in 2 multigenerational French Canadian dyslipidemic families. To understand in vivo functions of WWOX, we used liver-specific Wwox(hep-/-) and total Wwox(-/-) mice models, where we found decreased ApoA-I and Abca1 levels in hepatic tissues. Analyses of lipoprotein profiles in Wwox(-/-), but not Wwox(hep-/-) littermates, also showed marked reductions in serum HDL cholesterol concentrations, concordant with the low-HDL findings observed in families. We next obtained evidence of a sex-specific effect in female Wwox(hep-/-) mice, where microarray analyses revealed an increase in plasma triglycerides and altered lipid metabolic pathways. We further identified a significant reduction in ApoA-I and Lpl and an upregulation in Fas, Angptl4, and Lipg, suggesting that the effects of Wwox involve multiple pathways, including cholesterol homeostasis, ApoA-I/ABCA1 pathway, and fatty acid biosynthesis/triglyceride metabolism. CONCLUSIONS: Our data indicate that WWOX disruption alters HDL and lipoprotein metabolism through several mechanisms and may account for the low-HDL phenotype observed in families expressing the WWOX variants. These findings thus describe a novel gene involved in cellular lipid homeostasis, which effects may impact atherosclerotic disease development.

Exome sequencing identifies 2 rare variants for low high-density lipoprotein cholesterol in an extended family.

BACKGROUND: Exome sequencing is a recently implemented method to discover rare mutations for Mendelian disorders. Less is known about its feasibility to identify genes for complex traits. We used exome sequencing to search for rare variants responsible for a complex trait, low levels of serum high-density lipoprotein cholesterol (HDL-C). METHODS AND RESULTS: We conducted exome sequencing in a large French-Canadian family with 75 subjects available for study, of which 27 had HDL-C values less than the fifth age-sex-specific population percentile. We captured ≈50 Mb of exonic and transcribed sequences of 3 closely related family members with HDL-C levels less than the fifth age-sex percentile and sequenced the captured DNA. Approximately 82,000 variants were detected in each individual, of which 41 rare nonsynonymous variants were shared by the sequenced affected individuals after filtering steps. Two rare nonsynonymous variants in the ATP-binding cassette, subfamily A (ABC1), member 1 (ABCA1), and lipoprotein lipase genes predicted to be damaging were investigated for cosegregation with the low HDL-C trait in the entire extended family. The carriers of either variant had low HDL-C levels, and the individuals carrying both variants had the lowest HDL-C values. Interestingly, the ABCA1 variant exhibited a sex effect which was first functionally identified, and, subsequently, statistically demonstrated using additional French-Canadian families with ABCA1 mutations. CONCLUSIONS: This complex combination of 2 rare variants causing low HDL-C in the extended family would not have been identified using traditional linkage analysis, emphasizing the need for exome sequencing of complex lipid traits in unexplained familial cases.

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=1.86x10(-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.

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 (125)I-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 Delta(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 (125)I-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 (125)I-apoA-I from the PM was 4-fold slower than that from ICCs at 37 degrees 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.

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 (125)I-apoA-I binding to the HCBS, whereas (125)I-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 (Delta187-243) 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 (125)I-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.