Niacin Increases Atherogenic Proteins in High-Density Lipoprotein of Statin-Treated Subjects.

OBJECTIVE: Niacin therapy fails to reduce cardiovascular events in statin-treated subjects even though it increases plasma HDL-C (HDL [high-density lipoprotein] cholesterol) and decreases LDL-C (LDL [low-density lipoprotein] cholesterol) and triglyceride levels. To investigate potential mechanisms for this lack of cardioprotection, we quantified the HDL proteome of subjects in 2 niacin clinical trials: the CPC study (Carotid Plaque Composition) and the HDL Proteomics substudy of the AIM-HIGH trial (Atherothrombosis Intervention in Metabolic Syndrome with Low HDL/High Triglycerides). APPROACH AND RESULTS: Using targeted proteomics, we quantified levels of 31 HDL proteins from 124 CPC subjects and 120 AIM-HIGH subjects. The samples were obtained at baseline and after 1 year of statin monotherapy or niacin-statin combination therapy. Compared with statin monotherapy, niacin-statin combination therapy did not reduce HDL-associated apolipoproteins APOC1, APOC2, APOC3, and APOC4, despite significantly lowering triglycerides. In contrast, niacin markedly elevated HDL-associated PLTP (phospholipid transfer protein), CLU (clusterin), and HP/HPR (haptoglobin/haptoglobinrelated proteins; P≤0.0001 for each) in both the CPC and AIM-HIGH cohorts. CONCLUSIONS: The addition of niacin to statin therapy resulted in elevated levels of multiple HDL proteins linked to increased atherosclerotic risk, which might have compromised the cardioprotective effects associated with higher HDL-C levels and lower levels of LDL-C and triglycerides. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique identifier: NCT00715273; NCT00880178; NCT00120289.

The Role of Phospholipid Transfer Protein in the Development of Atherosclerosis.

PURPOSE OF REVIEW: Phospholipid transfer protein (PLTP), a member of lipid transfer protein family, is an important protein involved in lipid metabolism in the circulation. This article reviews recent PLTP research progresses, involving lipoprotein metabolism and atherogenesis. RECENT FINDINGS: PLTP activity influences atherogenic and anti-atherogenic lipoprotein levels. Human serum PLTP activity is a risk factor for human cardiovascular disease and is an independent predictor of all-cause mortality. PLTP deficiency reduces VLDL and LDL levels and attenuates atherosclerosis in mouse models, while PLTP overexpression exerts an opposite effect. Both PLTP deficiency and overexpression result in reduction of HDL which has different size, inflammatory index, and lipid composition. Moreover, although both PLTP deficiency and overexpression reduce cholesterol efflux capacity, but this effect has no impact in macrophage reverse cholesterol transport in mice. Furthermore, PLTP activity is related with metabolic syndrome, thrombosis, and inflammation. PLTP could be target for the treatment of dyslipidemia and atherosclerosis, although some potential off-target effects should be noted.

Esterification of 4ß-hydroxycholesterol and other oxysterols in human plasma occurs independently of LCAT.

The acyltransferase LCAT mediates FA esterification of plasma cholesterol. In vitro studies have shown that LCAT also FA-esterifies several oxysterols, but in vivo evidence is lacking. Here, we measured both free and FA-esterified forms of sterols in 206 healthy volunteers and 8 individuals with genetic LCAT deficiency, including familial LCAT deficiency (FLD) and fish-eye disease (FED). In the healthy volunteers, the mean values of the ester-to-total molar ratios of the following sterols varied: 4ß-hydroxycholesterol (4ßHC), 0.38; 5,6α-epoxycholesterol (5,6αEC), 0.46; 5,6ß-epoxycholesterol (5,6ßEC), 0.51; cholesterol, 0.70; cholestane-3ß,5α,6ß-triol (CT), 0.70; 7-ketocholesterol (7KC), 0.75; 24S-hydroxycholesterol (24SHC), 0.80; 25-hydroxycholesterol (25HC), 0.81; 27-hydroxycholesterol (27HC), 0.86; and 7α-hydroxycholesterol (7αHC), 0.89. In the individuals with LCAT deficiency, the plasma levels of the FA-esterified forms of cholesterol, 5,6αEC, 5,6ßEC, CT, 7αHC, 7KC, 24SHC, 25HC, and 27HC, were significantly lower than those in the healthy volunteers. The individuals with FLD had significantly lower FA-esterified forms of 7αHC, 24SHC, and 27HC than those with FED. It is of note that, even in the three FLD individuals with negligible plasma cholesteryl ester, substantial amounts of the FA-esterified forms of 4ßHC, 5,6αEC, 7αHC, 7KC, and 27HC were present. We conclude that LCAT has a major role in the FA esterification of many plasma oxysterols but contributes little to the FA esterification of 4ßHC. Substantial FA esterification of 4ßHC, 5,6αEC, 7αHC, 7KC, and 27HC is independent of LCAT.

A thumbwheel mechanism for APOA1 activation of LCAT activity in HDL.

APOA1 is the most abundant protein in HDL. It modulates interactions that affect HDL's cardioprotective functions, in part via its activation of the enzyme, LCAT. On nascent discoidal HDL, APOA1 comprises 10 α-helical repeats arranged in an anti-parallel stacked-ring structure that encapsulates a lipid bilayer. Previous chemical cross-linking studies suggested that these APOA1 rings can adopt at least two different orientations, or registries, with respect to each other; however, the functional impact of these structural changes is unknown. Here, we placed cysteine residues at locations predicted to form disulfide bonds in each orientation and then measured APOA1's ability to adopt the two registries during HDL particle formation. We found that most APOA1 oriented with the fifth helix of one molecule across from fifth helix of the other (5/5 helical registry), but a fraction adopted a 5/2 registry. Engineered HDLs that were locked in 5/5 or 5/2 registries by disulfide bonds equally promoted cholesterol efflux from macrophages, indicating functional particles. However, unlike the 5/5 registry or the WT, the 5/2 registry impaired LCAT cholesteryl esterification activity (P < 0.001), despite LCAT binding equally to all particles. Chemical cross-linking studies suggest that full LCAT activity requires a hybrid epitope composed of helices 5-7 on one APOA1 molecule and helices 3-4 on the other. Thus, APOA1 may use a reciprocating thumbwheel-like mechanism to activate HDL-remodeling proteins.

Genetic and secondary causes of severe HDL deficiency and cardiovascular disease.

We assessed secondary and genetic causes of severe HDL deficiency in 258,252 subjects, of whom 370 men (0.33%) and 144 women (0.099%) had HDL cholesterol levels <20 mg/dl. We excluded 206 subjects (40.1%) with significant elevations of triglycerides, C-reactive protein, glycosylated hemoglobin, myeloperoxidase, or liver enzymes and men receiving testosterone. We sequenced 23 lipid-related genes in 201 (65.3%) of 308 eligible subjects. Mutations (23 novel) and selected variants were found at the following gene loci: 1) ABCA1 (26.9%): 2 homozygotes, 7 compound or double heterozygotes, 30 heterozygotes, and 2 homozygotes and 13 heterozygotes with variants rs9282541/p.R230C or rs111292742/c.-279C>G; 2) LCAT (12.4%): 1 homozygote, 3 compound heterozygotes, 13 heterozygotes, and 8 heterozygotes with variant rs4986970/p.S232T; 3) APOA1 (5.0%): 1 homozygote and 9 heterozygotes; and 4) LPL (4.5%): 1 heterozygote and 8 heterozygotes with variant rs268/p.N318S. In addition, 4.5% had other mutations, and 46.8% had no mutations. Atherosclerotic cardiovascular disease (ASCVD) prevalence rates in the ABCA1, LCAT, APOA1, LPL, and mutation-negative groups were 37.0%, 4.0%, 40.0%, 11.1%, and 6.4%, respectively. Severe HDL deficiency is uncommon, with 40.1% having secondary causes and 48.8% of the subjects sequenced having ABCA1, LCAT, APOA1, or LPL mutations or variants, with the highest ASCVD prevalence rates being observed in the ABCA1 and APOA1 groups.

Arginine 123 of apolipoprotein A-I is essential for lecithin:cholesterol acyltransferase activity.

ApoA-I activates LCAT that converts lipoprotein cholesterol to cholesteryl ester (CE). Molecular dynamic simulations suggested earlier that helices 5 of two antiparallel apoA-I molecules on discoidal HDL form an amphipathic tunnel for migration of acyl chains and unesterified cholesterol to the active sites of LCAT. Our recent crystal structure of Δ(185-243)apoA-I showed the tunnel formed by helices 5/5, with two positively charged residues arginine 123 positioned at the edge of the hydrophobic tunnel. We hypothesized that these uniquely positioned residues Arg123 are poised for interaction with fatty acids produced by LCAT hydrolysis of the sn-2 chains of phosphatidylcholine, thus positioning the fatty acids for esterification to cholesterol. To test the importance of Arg123 for LCAT phospholipid hydrolysis and CE formation, we generated apoA-I[R123A] and apoA-I[R123E] mutants and made discoidal HDL with the mutants and WT apoA-I. Neither mutation of Arg123 changed the particle composition or size, or the protein conformation or stability. However, both mutations of Arg123 significantly reduced LCAT catalytic efficiency and the apparent Vmax for CE formation without affecting LCAT phospholipid hydrolysis. A control mutation, apoA-I[R131A], did not affect LCAT phospholipid hydrolysis or CE formation. These data suggest that Arg123 of apoA-I on discoidal HDL participates in LCAT-mediated cholesterol esterification.

Overexpression and deletion of phospholipid transfer protein reduce HDL mass and cholesterol efflux capacity but not macrophage reverse cholesterol transport.

Phospholipid transfer protein (PLTP) may affect macrophage reverse cholesterol transport (mRCT) through its role in the metabolism of HDL. Ex vivo cholesterol efflux capacity and in vivo mRCT were assessed in PLTP deletion and PLTP overexpression mice. PLTP deletion mice had reduced HDL mass and cholesterol efflux capacity, but unchanged in vivo mRCT. To directly compare the effects of PLTP overexpression and deletion on mRCT, human PLTP was overexpressed in the liver of wild-type animals using an adeno-associated viral (AAV) vector, and control and PLTP deletion animals were injected with AAV-null. PLTP overexpression and deletion reduced plasma HDL mass and cholesterol efflux capacity. Both substantially decreased ABCA1-independent cholesterol efflux, whereas ABCA1-dependent cholesterol efflux remained the same or increased, even though preß HDL levels were lower. Neither PLTP overexpression nor deletion affected excretion of macrophage-derived radiocholesterol in the in vivo mRCT assay. The ex vivo and in vivo assays were modified to gauge the rate of cholesterol efflux from macrophages to plasma. PLTP activity did not affect this metric. Thus, deviations in PLTP activity from the wild-type level reduce HDL mass and ex vivo cholesterol efflux capacity, but not the rate of macrophage cholesterol efflux to plasma or in vivo mRCT.

An in silico model of retinal cholesterol dynamics (RCD model): insights into the pathophysiology of dry AMD.

We developed an in silico mathematical model of retinal cholesterol (Ch) dynamics (RCD) to quantify the physiological rate of Ch turnover in the rod outer segment (ROS), the lipoprotein transport mechanisms by which Ch enters and leaves the outer retina, and the rates of drusen growth and macrophage-mediated clearance in dry age-related macular degeneration. Based on existing experimental data and mechanistic hypotheses, we estimated the Ch turnover rate in the ROS to be 1-6 pg/mm2/min, dependent on the rate of Ch recycling in the outer retina, and found comparable rates for LDL receptor-mediated endocytosis of Ch by the retinal pigment epithelium (RPE), ABCA1-mediated Ch transport from the RPE to the outer retina, ABCA1-mediated Ch efflux from the RPE to the choroid, and the secretion of 70 nm ApoB-Ch particles from the RPE. The drusen growth rate is predicted to increase from 0.7 to 4.2 µm/year in proportion to the flux of ApoB-Ch particles. The rapid regression of drusen may be explained by macrophage-mediated clearance if the macrophage density reaches ∼3,500 cells/mm2 The RCD model quantifies retinal Ch dynamics and suggests that retinal Ch turnover and recycling, ApoB-Ch particle efflux, and macrophage-mediated clearance may explain the dynamics of drusen growth and regression.

Atomistic MD simulation reveals the mechanism by which CETP penetrates into HDL enabling lipid transfer from HDL to CETP.

Inhibition of cholesterol ester transfer protein (CETP), a protein mediating transfer of neutral lipids between lipoproteins, has been proposed as a means to elevate atheroprotective HDL subpopulations and thereby reduce atherosclerosis. However, off-target and adverse effects of the inhibition have raised doubts about the molecular mechanism of CETP-HDL interaction. Recent experimental findings have demonstrated the penetration of CETP into HDL. However, atomic level resolution of CETP penetration into HDL, a prerequisite for a better understanding of CETP functionality and HDL atheroprotection, is missing. We constructed an HDL particle that mimics the actual human HDL mass composition and investigated for the first time, by large-scale atomistic molecular dynamics, the interaction of an upright CETP with a human HDL-mimicking model. The results demonstrated how CETP can penetrate the HDL particle surface, with the formation of an opening in the N barrel domain end of CETP, put in evidence the major anchoring role of a tryptophan-rich region of this domain, and unveiled the presence of a phenylalanine barrier controlling further access of HDL-derived lipids to the tunnel of CETP. The findings reveal novel atomistic details of the CETP-HDL interaction mechanism and can provide new insight into therapeutic strategies.

HDL-apolipoprotein A-I exchange is independently associated with cholesterol efflux capacity.

HDL is the primary mediator of cholesterol mobilization from the periphery to the liver via reverse cholesterol transport (RCT). A critical first step in this process is the uptake of cholesterol from lipid-loaded macrophages by HDL, a function of HDL inversely associated with prevalent and incident cardiovascular disease. We hypothesized that the dynamic ability of HDL to undergo remodeling and exchange of apoA-I is an important and potentially rate-limiting aspect of RCT. In this study, we investigated the relationship between HDL-apoA-I exchange (HAE) and serum HDL cholesterol (HDL-C) efflux capacity. We compared HAE to the total and ABCA1-specific cholesterol efflux capacity of 77 subjects. We found that HAE was highly correlated with both total (r = 0.69, P < 0.0001) and ABCA1-specific (r = 0.47, P < 0.0001) efflux, and this relationship remained significant after adjustment for HDL-C or apoA-I. Multivariate models of sterol efflux capacity indicated that HAE accounted for approximately 25% of the model variance for both total and ABCA1-specific efflux. We conclude that the ability of HDL to exchange apoA-I and remodel, as measured by HAE, is a significant contributor to serum HDL efflux capacity, independent of HDL-C and apoA-I, indicating that HDL dynamics are an important factor in cholesterol efflux capacity and likely RCT.

LCAT deficiency does not impair amyloid metabolism in APP/PS1 mice.

A key step in plasma HDL maturation from discoidal to spherical particles is the esterification of cholesterol to cholesteryl ester, which is catalyzed by LCAT. HDL-like lipoproteins in cerebrospinal fluid (CSF) are also spherical, whereas nascent lipoprotein particles secreted from astrocytes are discoidal, suggesting that LCAT may play a similar role in the CNS. In plasma, apoA-I is the main LCAT activator, while in the CNS, it is believed to be apoE. apoE is directly involved in the pathological progression of Alzheimer's disease, including facilitating ß-amyloid (Aß) clearance from the brain, a function that requires its lipidation by ABCA1. However, whether apoE particle maturation by LCAT is also required for Aß clearance is unknown. Here we characterized the impact of LCAT deficiency on CNS lipoprotein metabolism and amyloid pathology. Deletion of LCAT from APP/PS1 mice resulted in a pronounced decrease of apoA-I in plasma that was paralleled by decreased apoA-I levels in CSF and brain tissue, whereas apoE levels were unaffected. Furthermore, LCAT deficiency did not increase Aß or amyloid in APP/PS1 LCAT(-/-) mice. Finally, LCAT expression and plasma activity were unaffected by age or the onset of Alzheimer's-like pathology in APP/PS1 mice. Taken together, these results suggest that apoE-containing discoidal HDLs do not require LCAT-dependent maturation to mediate efficient Aß clearance.

Identification of four novel genes contributing to familial elevated plasma HDL cholesterol in humans.

While genetic determinants strongly influence HDL cholesterol (HDLc) levels, most genetic causes underlying variation in HDLc remain unknown. We aimed to identify novel rare mutations with large effects in candidate genes contributing to extreme HDLc in humans, utilizing family-based Mendelian genetics. We performed next-generation sequencing of 456 candidate HDLc-regulating genes in 200 unrelated probands with extremely low (≤10th percentile) or high (≥90th percentile) HDLc. Probands were excluded if known mutations existed in the established HDLc-regulating genes ABCA1, APOA1, LCAT, cholesteryl ester transfer protein (CETP), endothelial lipase (LIPG), and UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 2 (GALNT2). We identified 93 novel coding or splice-site variants in 72 candidate genes. Each variant was genotyped in the proband's family. Family-based association analyses were performed for variants with sufficient power to detect significance at P < 0.05 with a total of 627 family members being assessed. Mutations in the genes glucokinase regulatory protein (GCKR), RNase L (RNASEL), leukocyte immunoglobulin-like receptor 3 (LILRA3), and dynein axonemal heavy chain 10 (DNAH10) segregated with elevated HDLc levels in families, while no mutations associated with low HDLc. Taken together, we have identified mutations in four novel genes that may play a role in regulating HDLc levels in humans.

Implications of High-Density Cholesterol Metabolism for Oocyte Biology and Female Fertility.

Cholesterol is an essential component of animal cells. Different regulatory mechanisms converge to maintain adequate levels of this lipid because both its deficiency and excess are unfavorable. Low cell cholesterol content promotes its synthesis and uptake from circulating lipoproteins. In contrast, its excess induces the efflux to high-density lipoproteins (HDL) and their transport to the liver for excretion, a process known as reverse cholesterol transport. Different studies suggest that an abnormal HDL metabolism hinders female fertility. HDL are the only lipoproteins detected in substantial amounts in follicular fluid (FF), and their size and composition correlate with embryo quality. Oocytes obtain cholesterol from cumulus cells via gap junctions because they cannot synthesize cholesterol de novo and lack HDL receptors. Recent evidence has supported the possibility that FF HDL play a major role in taking up excess unesterified cholesterol (UC) from the oocyte. Indeed, genetically modified mouse models with disruptions in reverse cholesterol transport, some of which show excessive circulating UC levels, exhibit female infertility. Cholesterol accumulation can affect the egg´s viability, as reported in other cell types, and activate the plasma membrane structure and activity of membrane proteins. Indeed, in mice deficient for the HDL receptor Scavenger Class B Type I (SR-B1), excess circulating HDL cholesterol and UC accumulation in oocytes impairs meiosis arrest and hinders the developmental capacity of the egg. In other cells, the addition of cholesterol activates calcium channels and dysregulates cell death/survival signaling pathways, suggesting that these mechanisms may link altered HDL cholesterol metabolism and infertility. Although cholesterol, and lipids in general, are usually not evaluated in infertile patients, one study reported high circulating UC levels in women showing longer time to pregnancy as an outcome of fertility. Based on the evidence described above, we propose the existence of a well-regulated and largely unexplored system of cholesterol homeostasis controlling traffic between FF HDL and oocytes, with significant implications for female fertility.

Postprandial remodeling of high-density lipoprotein following high saturated fat and high carbohydrate meals.

BACKGROUND: Humans spend most of the time in the postprandial state, yet most knowledge about high-density lipoproteins (HDL) derives from the fasted state. HDL protein and lipid cargo mediate HDL's antiatherogenic effects, but whether these HDL constituents change in the postprandial state and are affected by dietary macronutrients remains unknown. OBJECTIVES: This study aimed to assess changes in HDL protein and lipid composition after the consumption of a high-carbohydrate or high saturated fat (HSF) meal. METHODS: We isolated HDL from plasma collected during a randomized, cross-over study of metabolically healthy subjects. Subjects consumed isocaloric meals consisting predominantly of either carbohydrate or fat. At baseline and at 3 and 6 hours postprandial, we quantified HDL protein and lipid composition by liquid chromatography-mass spectrometry. RESULTS: A total of 15 subjects were included (60% female, aged 34 ± 15 years, body mass index: 24.1 ± 2.7 kg/m2). Consumption of the HSF meal led to HDL enrichment in total lipid (P = .006), triglyceride (P = .02), and phospholipid (P = .008) content and a corresponding depletion in protein content. After the HSF meal, 16 of the 25 measured phosphatidylcholine species significantly increased in abundance (P values range from .027 to <.001), along with several sphingolipids including ceramides (P < .004), lactosylceramide (P = .023), and sphingomyelin-14 (P = .013). Enrichment in apolipoprotein A-I (P = .001) was the only significant change in HDL protein composition after the HSF meal. The high-carbohydrate meal conferred only minimal changes in HDL composition. CONCLUSION: Meal macronutrient content acutely affects HDL composition in the postprandial state, with the HSF meal resulting in enrichment of HDL phospholipid content with possible consequences for HDL function.

ApoA-I-Mediated Lipoprotein Remodeling Monitored with a Fluorescent Phospholipid.

We describe simple, sensitive and robust methods to monitor lipoprotein remodeling and cholesterol and apolipoprotein exchange, using fluorescent Lissamine Rhodamine B head-group tagged phosphatidylethanolamine (*PE) as a lipoprotein reference marker. Fluorescent Bodipy cholesterol (*Chol) and *PE directly incorporated into whole plasma lipoproteins in proportion to lipoprotein cholesterol and phospholipid mass, respectively. *Chol, but not *PE, passively exchanged between isolated plasma lipoproteins. Fluorescent apoA-I (*apoA-I) specifically bound to high-density lipoprotein (HDL) and remodeled *PE- and *Chol-labeled synthetic lipoprotein-X multilamellar vesicles (MLV) into a pre-ß HDL-like particle containing *PE, *Chol, and *apoA-I. Fluorescent MLV-derived *PE specifically incorporated into plasma HDL, whereas MLV-derived *Chol incorporation into plasma lipoproteins was similar to direct *Chol incorporation, consistent with apoA-I-mediated remodeling of fluorescent MLV to HDL with concomitant exchange of *Chol between lipoproteins. Based on these findings, we developed a model system to study lipid transfer by depositing fluorescent *PE and *Chol-labeled on calcium silicate hydrate crystals, forming dense lipid-coated donor particles that are readily separated from acceptor lipoprotein particles by low-speed centrifugation. Transfer of *PE from donor particles to mouse plasma lipoproteins was shown to be HDL-specific and apoA-I-dependent. Transfer of donor particle *PE and *Chol to HDL in whole human plasma was highly correlated. Taken together, these studies suggest that cell-free *PE efflux monitors apoA-I functionality.

Diabetes alters the association between high-density lipoprotein subfractions and carotid intima-media thickness: The Brazilian Longitudinal Study of Adult Health (ELSA-Brasil).

INTRODUCTION: High-density lipoprotein cholesterol comprises a group of heterogeneous subfractions that might have differential effects on atherosclerosis. Moreover, prior investigations suggest that the presence of diabetes (T2D) modifies the impact of some subfractions on atherosclerosis. In this study, we aimed to evaluate the association between high-density lipoprotein cholesterol subfractions and carotid intima-media thickness in the baseline assessment of the Brazilian Longitudinal Study of Adult Health participants from the São Paulo investigation centre. METHODS: We evaluated 3930 individuals between 35 and 74 years without previous cardiovascular disease not using lipid-lowering drugs. High-density lipoprotein cholesterol subfractions (HDL2-C and HDL3-C) were measured by vertical ultracentrifugation (vertical auto profile). The relationship between each high-density lipoprotein cholesterol subfraction and carotid intima-media thickness was analysed by multiple linear regression models. RESULTS: Total high-density lipoprotein cholesterol, as well as HDL2-C and HDL3-C, was negatively associated with carotid intima-media thickness after adjustment for demographic data (all p < 0.001) and traditional risk factors (all p < 0.05). When stratified by T2D status, the HDL2-C/HDL3-C ratio showed a negative association with carotid intima-media thickness in participants with T2D ( p = 0.032), even after fully controlling for confounding variables, including total high-density lipoprotein cholesterol. CONCLUSION: HDL2-C, HDL3-C and HDL2/HDL3-C ratio are inversely associated with carotid intima-media thickness after adjustment for traditional risk factors. Association of the HDL2-C/HDL3-C ratio is modified by the presence of diabetes, being more pronounced in diabetic individuals.