Reverse Cholesterol Transport Function Research Articles

Zebrafish as outgroup model to study evolution of scavenger receptor class B type I functions

Background and aimsScavenger receptor class B1 (SCARB1) - also known as the high-density lipoprotein (HDL) receptor - is a multi-ligand scavenger receptor that is primarily expressed in liver and steroidogenic organs. This receptor is known for its function in reverse cholesterol transport (RCT) in mammals and hence disruption leads to a massive increase in HDL cholesterol in these species. The extracellular domain of SCARB1 - which is important for cholesterol handling - is highly conserved across multiple vertebrates, except in zebrafish. Methods: To examine the functional conservation of SCARB1 among vertebrates, two stable scarb1 knockout zebrafish lines, scarb1 715delA (scarb1 −1 nt) and scarb1 715_716insGG (scarb1 +2 nt), were created using CRISPR-Cas9 technology. ResultsWe demonstrate that, in zebrafish, SCARB1 deficiency leads to disruption of carotenoid-based pigmentation, reduced fertility, and a decreased larvae survival rate, whereas steroidogenesis was unaltered. The observed reduced fertility is driven by defects in female fertility (−50 %, p < 0.001). Importantly, these alterations were independent of changes in free (wild-type 2.4 ± 0.2 μg/μl versus scarb1−/− 2.0 ± 0.1 μg/μl) as well as total (wild-type 4.2 ± 0.4 μg/μl versus scarb1−/− 4.0 ± 0.3 μg/μl) plasma cholesterol levels. Uptake of HDL in the liver of scarb1−/− zebrafish larvae was reduced (−86.7 %, p < 0.001), but this coincided with reduced perfusion of the liver. No effect was observed on lipoprotein uptake in the caudal vein. SCARB1 deficient canaries, which also lack carotenoids in their plumage, similarly as scarb1−/− zebrafish, failed to show an increase in plasma free- and total cholesterol levels. ConclusionOur findings suggest that the specific function of SCARB1 in maintaining plasma cholesterol could be an evolutionary novelty that became prominent in mammals, while other known functions were already present earlier during vertebrate evolution.

Editorial introductions.

Current Opinion in Lipidology was launched in 1990. It is part of a successful series of review journals whose unique format is designed to provide a systematic and critical assessment of the literature as presented in the many primary journals. The field of lipidology is divided into six sections that are reviewed once a year. Each section is assigned a Section Editor, a leading authority in the area, who identifies the most important topics at that time. Here we are pleased to introduce the Section Editors for this issue. SECTION EDITORS Frank M. SacksFrank M. SacksDr Sacks is Professor of Cardiovascular Disease Prevention in the Nutrition Department of Harvard Chan School of Public Health; Professor of Genetics & Complexes Diseases; and Professor of Medicine at Harvard Medical School. Dr Sacks led the panel designing the DASH Study, which crafted a healthful eating pattern and demonstrated that it lowered blood pressure more effectively than any previous dietary treatment. Subsequently Dr Sacks led the DASH-Sodium study, which determined the dose-response effect of dietary sodium on BP. These multi-center National Heart Lung and Blood Institute trials found major beneficial additive effects of low salt and a dietary pattern rich in fruits and vegetables on blood pressure. He also led the seminal PoundsLost trial which showed the percentage of calories from fat or carbohydrate had no effect on long term weight loss. Currently, he is co-investigator of the MIND study, a randomized trial of a diet that combines features of the Mediterranean and DASH diets to preserve cognition in older people, funded by the National Institute on Aging. Dr Sacks's laboratory has studied the role of apolipoproteins in atherogenic lipoprotein metabolism in obesity and hypertriglyceridemia. He authored over 30 research papers on apoC-3 and/or apoE. Recently he discovered that a type of HDL that contains apolipoprotein C-III predicted higher rates of heart disease and diabetes, the opposite to the protective relation for the total HDL; and impaired HDL function in reverse cholesterol transport. Dr Sacks teaches at Harvard School of Public Health as course director for nutritional metabolism and biochemistry. Dr Sacks received the 2011 Research Achievement Award of the American Heart Association for lifetime research accomplishment. Majken K. JensenMajken K. JensenMajken K. Jensen, PhD, is Associate Professor of Nutrition and Epidemiology at Harvard T. H. Chan School of Public Health. Dr Jensen's primary research interests include novel metabolic markers, nutrition, and genetics, in relation to chronic lifestyle diseases. In ongoing collaborations with Dr Frank Sacks, they investigate how the presence of a small proinflammatory protein (apolipoprotein C-III) on HDL particles marks a potentially dysfunctional type of HDL that is not inversely associated with risk of cardiometabolic health, such as IMT, diabetes and cardiovascular disease. Other currently funded work is focused on lipoprotein metabolism and Alzheimer's disease and stroke. Her research predominantly utilizes large-scale observational studies (including the Health Professionals Follow-Up Study, the Nurses’ Health Study, the Danish Diet, Cancer and Health study, the Multi-Ethnic Study of Atherosclerosis, and the Cardiovascular Health Study) where cost-efficient prospective case-control studies are nested within for purpose of measuring novel biomarkers or genome-wide markers. Dr Jensen's research has been published in leading scientific journals and has been recognized with numerous awards. She received the Trudy Bush Fellowship for Cardiovascular Disease Research in Women's Health from the American Heart Association in 2011 and originally came for research studies at Harvard T. H. Chan School of Public Health as a Fulbright scholar. Dr Jensen has published over 80 original research articles and 5 reviews, editorials, and letters.

HDLandKidney Diseases.

Serum lipid profiles, as well as HDL can be altered in patients with kidney diseases. There are various types of kidney diseases, including nephrotic syndrome and chronic kidney disease. In patients with nephrotic syndrome, plasma levels of HDL cholesterol and ApoA-I were within or below the normal limits. The HDL cholesterol: total cholesterol ratio decreased compared to healthy individuals. In patients with chronic kidney disease (CKD), reverse cholesterol transport function of HDL is impaired, and CKD also affects the composition and function of HDL. Cardiovascular disease (CVD) is the severe complication of CKD. Furthermore, HDL might also be a potential target for the prevention of cardiovascular complications associated with CKD.

Relationship between lipoprotein concentrations and short-term and 1-year mortality in intensive care unit septic patients: results from the HIGHSEPS study

BackgroundHigh-density lipoproteins (HDLs), particles characterized by their reverse cholesterol transport function, display pleiotropic properties, including anti-inflammatory and antioxidant functions. Moreover, all lipoproteins (HDLs but also low-density lipoproteins (LDLs)) neutralize lipopolysaccharides, leading to increased bacterial clearance. These two lipoproteins decrease during sepsis, and an association between low lipoprotein levels and poor outcome was reported. The goals of this study were to characterize the lipid profile of septic patients hospitalized in our intensive care unit (ICU) and to determine the relationship with the outcome.MethodsA prospective observational study was conducted in a university hospital ICU. All consecutive patients admitted for septic shock or sepsis were included. Total cholesterol, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and triglyceride levels were assessed at admission (day 1), at day 3, and at ICU discharge. When available, a prehospitalization lipid profile collected prior to the patient’s hospitalization was compiled. Short-term and 1-year prognostic outcomes were prospectively assessed.ResultsA total of 205 patients were included. We found a decrease in HDL-C concentration between previous values and those at admission, followed by an additional decrease at day 3. At ICU discharge, the concentration was higher than that at day 3 but did not reach the concentration measured prior to hospitalization (prior HDL-C = 1.22 (1.04–1.57) mmol/l; day 1 HDL-C = 0.44 (0.29–0.70) mmol/l; day 3 HDL-C = 0.30 (0.25–0.48) mmol/l; and HDL-C at discharge = 0.65 (0.42–0.82) mmol/l). A similar trend was found for LDL-C (prior LDL-C = 2.7 (1.91–3.33) mmol/l; day 1 LDL-C = 1.0 (0.58–1.50) mmol/l; day 3 LDL-C = 1.04 (0.64–1.54) mmol/l; and LDL-C at discharge = 1.69 (1.26–2.21) mmol/l). Mixed models for repeated measures of lipoprotein concentrations showed a significant difference in HDL-C and LDL-C concentrations over time between survivors and nonsurvivors at day 28. An HDL-C concentration at admission of less than 0.4 mmol/l was associated with increased mortality at day 28 (log-rank test, p = 0.034) but not at 1 year (log-rank test, p = 0.24). An LDL-C concentration at admission of less than 0.72 mmol/l was associated with increased mortality at day 28 and at 1 year (log-rank test, p < 0.001 and p = 0.007, respectively). No link was found between prior lipid profile and mortality.ConclusionsWe showed no relationship between the prehospitalization lipid profile and patient outcome, but low lipoprotein levels in the ICU were strongly associated with short-term mortality.

Lipoprotein concentrations over time in the intensive care unit COVID-19 patients: Results from the ApoCOVID study.

Severe acute respiratory syndrome coronavirus2 has caused a global pandemic of coronavirus disease 2019 (COVID-19). High-density lipoproteins (HDLs), particles chiefly known for their reverse cholesterol transport function, also display pleiotropic properties, including anti-inflammatory or antioxidant functions. HDLs and low-density lipoproteins (LDLs) can neutralize lipopolysaccharides and increase bacterial clearance. HDL cholesterol (HDL-C) and LDL cholesterol (LDL-C) decrease during bacterial sepsis, and an association has been reported between low lipoprotein levels and poor patient outcomes. The goal of this study was to characterize the lipoprotein profiles of severe ICU patients hospitalized for COVID-19 pneumonia and to assess their changes during bacterial ventilator-associated pneumonia (VAP) superinfection. A prospective study was conducted in a university hospital ICU. All consecutive patients admitted for COVID-19 pneumonia were included. Lipoprotein levels were assessed at admission and daily thereafter. The assessed outcomes were survival at 28 days and the incidence of VAP. A total of 48 patients were included. Upon admission, lipoprotein concentrations were low, typically under the reference values ([HDL-C] = 0.7[0.5-0.9] mmol/L; [LDL-C] = 1.8[1.3-2.3] mmol/L). A statistically significant increase in HDL-C and LDL-C over time during the ICU stay was found. There was no relationship between HDL-C and LDL-C concentrations and mortality on day 28 (log-rank p = 0.554 and p = 0.083, respectively). A comparison of alive and dead patients on day 28 did not reveal any differences in HDL-C and LDL-C concentrations over time. Bacterial VAP was frequent (64%). An association was observed between HDL-C and LDL-C concentrations on the day of the first VAP diagnosis and mortality ([HDL-C] = 0.6[0.5-0.9] mmol/L in survivors vs. [HDL-C] = 0.5[0.3-0.6] mmol/L in nonsurvivors, p = 0.036; [LDL-C] = 2.2[1.9-3.0] mmol/L in survivors vs. [LDL-C] = 1.3[0.9-2.0] mmol/L in nonsurvivors, p = 0.006). HDL-C and LDL-C concentrations upon ICU admission are low in severe COVID-19 pneumonia patients but are not associated with poor outcomes. However, low lipoprotein concentrations in the case of bacterial superinfection during ICU hospitalization are associated with mortality, which reinforces the potential role of these particles during bacterial sepsis.

Editorial introductions

Editorial introductions

Editorial introductions

Editorial introductions

Abstract 541: The Molecular Interaction of Apolipoprotein A-I Containing High Density Lipoproteins with Lecithin: Cholesterol Acyl Transferase

The structure of apolipoprotein (apo)A-I on a high density lipoprotein (HDL) particle can elucidate the protein interactions and cardioprotective functions of HDL. This includes critical interactions with HDL modifying proteins like lecithin:cholesterol acyl transferase (LCAT) which performs a key function in reverse cholesterol transport by using apoA-I as a cofactor to esterify cholesterol. Data from our lab and others demonstrate that apoA-I molecules dimerize into an antiparallel stacked ring structure that encapsulates lipid in reconstituted (r)HDL particles. Cross-linking analysis of rHDL particles imply two possible registries: one with the fifth helical repeat of an apoA-I molecule adjacent to the fifth helical repeat of its antiparallel partner (5/5), and the other with the fifth helical repeat adjacent to the second helical repeat of its antiparallel partner (5/2). We hypothesized that apoA-I registry on rHDL can modulate LCAT activity. Site-directed cysteine mutagenesis was used to lock two apoA-I molecules into each of the aforementioned helical registries, and an intermediate registry created as a negative control. rHDL particles were generated using these dimerized mutants, and their ability to activate LCAT was determined. The 5/5 mutant demonstrated higher LCAT activity than wild-type rHDL particles while the 5/2 and intermediate mutants showed dramatically lower LCAT activity (p&lt;0.001). To determine where LCAT interacts with apoA-I, rHDL containing wild-type apoA-I was cross-linked to LCAT. The majority of the cross-links were concentrated between S240 of LCAT and a region encompassing helices 4-6 of apoA-I. We propose that LCAT binds to a discontinuous epitope comprised of two apoA-I molecules in rHDL and that changes in this registry can alter LCAT function. These studies provide a basis for understanding how apoA-I structure may modulate the association and activity of a multitude of different HDL protein partners. This implicates apoA-I conformation as a target for altering HDL function in order to enhance the cardioprotective properties of HDL.

Speciated High-Density Lipoprotein Biogenesis and Functionality.

Plasma high-density lipoprotein cholesterol (HDL-C) concentration is a negative risk factor for atherosclerotic cardiovascular disease (CVD). Despite this, most attempts to raise plasma HDL-C concentrations in a cardioprotective way have failed. Recently, hypotheses about the atheroprotective effects of HDL have shifted away from quantity to quality, mostly HDL function in reverse cholesterol transport. Plasma HDL from CVD patients is a poorer acceptor of cellular cholesterol than plasma from healthy controls, independent of plasma HDL-C concentrations. The function of HDL is likely determined by two other factors, stability and composition. The kinetic instability of HDL, which varies according to subclass, is a likely determinant of its reactivity in response to many HDL-modifying activities. HDL composition is also heterogeneous and variable; all HDL particles contain apo AI but only about two-thirds contain apo AII. This occurs despite the fact that apo AI and apo AII are hepatically secreted on separate HDL that later fuse in plasma. HDL also contains traces of other proteins, some of which have not yet been associated with HDL function. One minor HDL species are those that are secreted with intact signal peptides, which enhances their binding to HDL; these HDL have special properties that are independent of cholesterol transport. Here, we review and provide a perspective about what is currently known about speciated HDL biogenesis in the context of health and disease.

Abstract 534: Abca1 Mediates Macrophage Deposition of Cholesterol Into the Extracellular Matrix

ABCG1 and ABCA1 both function in reverse cholesterol transport. We previously reported that cholesterol-enriched macrophages deposit cholesterol into the extracellular space and this process depends in part on ABCG1. The extracellular cholesterol deposits can be mobilized by HDL. The objective of the current study was to determine whether ABCA1 also contributes to macrophage deposition of extracellular cholesterol. A monoclonal antibody that detects cholesterol microdomains, and not isolated cholesterol molecules, labels these extracellular deposits. First, to confirm the specificity of the cholesterol labeling, we treated cholesterol-enriched macrophages cultures with cholesterol oxidase and found that antibody labeling of deposits was eliminated. Many biochemical studies assume that cholesterol oxidase susceptible cholesterol indicates this cholesterol is within the plasma membrane. However, as we show here, the extracellular pool of cholesterol also contributes to cholesterol oxidase susceptible cholesterol. Second, concerning ABCA1 function in cholesterol deposition, we observed that the LXR agonist, TO901317, an ABCA1-inducing agent, restored cholesterol deposition that was absent in ABCG1-/- mouse macrophages. In addition, probucol, an agent that inhibits ABCA1 function, partially inhibited cholesterol deposited by wild-type mouse macrophages, and completely inhibited deposition from TO901317-treated ABCG1-/- mouse macrophages. These results suggest that ABCA1 contributes to macrophage cholesterol deposition. We confirmed this conclusion by testing cholesterol deposition in ABCA1-/- mouse macrophages. Extracellular cholesterol deposition by these macrophages was substantially reduced compared with wild-type mouse macrophages. Thus, our findings demonstrate a novel function of ABCA1 in contributing to macrophage export of cholesterol into the extracellular space where this cholesterol can be stored until mobilized by HDL.

Our panel of experts highlight the most important research articles across the spectrum of topics relevant to the field of clinical practice

Clin. Pract. (2014) 11(2), 139–143 ISSN 2044-9038 part of Parsa A, Kao WH, Xie D et al.; AASK Study Investigators; CRIC Study Investigators. APOL1 risk variants, race and progression of chronic kidney disease. N. Engl. J. Med. 369(23), 2183–2196 (2013). For decades, it has been observed that African–Americans experience a more rapid progression of chronic kidney disease (CKD) in virtually every etiology of kidney disease. This has been ascribed to more severe hypertension, poor risk factor control and noncompliance. Heretofore, a search for genetic determinants among African–Americans has been unrevealing. High-density lipoprotein cholesterol particles contain major apolipoproteins A-I and A-II, and minor apolipoproteins, including A-IV, A-V, C-I, C-II, C-III, D, E, F, H, J, L, M, O and P. Polymorphisms of these apolipoproteins have been associated with changes in reverse cholesterol transport function, oxidation and now, most recently, altered catabolism. High-density lipoprotein is filtered in the glomerulus and then catabolized by the proximal tubular cells. A mutant form of apo-L has been described in African–Americans that may have a protective effect against trypanosomes. APOL1 polymorphisms have a prevalence of approximately 30 and 2% among African–Americans and Caucasians, respectively. In this paper, data from two clinical studies, one exclusively in nondiabetic African–Americans with hypertension (AASK) and the other in patients screened for CKD (CRIC), variants in the gene for APOL1 were assessed according to whether they had two high-risk APOL1 variants (APOL1 high-risk group) or 0 or 1 copy (APOL1 low-risk group) [1]. In both studies, among those with and without diabetes, there was a more rapid progression of CKD, including the need for dialysis, in African–Americans with two alleles containing mutant variants of the APOL1 gene. This and other studies suggest that APOL1 variants are associated with a 50–100% increased risk of rapid progression of CKD and may be related to a toxic or adverse process invoked by proximal tubular cell catabolism of this apoprotein. So, in summary, APOL1 genetic abnormalities appear to be a major genetic basis for the difference between rates of progression of CKD between African–Americans and Caucasians.

ABCG1-mediated generation of extracellular cholesterol microdomains

Previous studies have demonstrated that the ATP-binding cassette transporters (ABC)A1 and ABCG1 function in many aspects of cholesterol efflux from macrophages. In this current study, we continued our investigation of extracellular cholesterol microdomains that form during enrichment of macrophages with cholesterol. Human monocyte-derived macrophages and mouse bone marrow-derived macrophages, differentiated with macrophage colony-stimulating factor (M-CSF) or granulocyte macrophage colony-stimulation factor (GM-CSF), were incubated with acetylated LDL (AcLDL) to allow for cholesterol enrichment and processing. We utilized an anti-cholesterol microdomain monoclonal antibody to reveal pools of unesterified cholesterol, which were found both in the extracellular matrix and associated with the cell surface, that we show function in reverse cholesterol transport. Coincubation of AcLDL with 50 μg/ml apoA-I eliminated all extracellular and cell surface-associated cholesterol microdomains, while coincubation with the same concentration of HDL only removed extracellular matrix-associated cholesterol microdomains. Only at an HDL concentration of 200 µg/ml did HDL eliminate the cholesterol microdomains that were cell-surface associated. The deposition of cholesterol microdomains was inhibited by probucol, but it was increased by the liver X receptor (LXR) agonist TO901317, which upregulates ABCA1 and ABCG1. Extracellular cholesterol microdomains did not develop when ABCG1-deficient mouse bone marrow-derived macrophages were enriched with cholesterol. Our findings show that generation of extracellular cholesterol microdomains is mediated by ABCG1 and that reverse cholesterol transport occurs not only at the cell surface but also within the extracellular space.

Lack of P2Y13 in mice fed a high cholesterol diet results in decreased hepatic cholesterol content, biliary lipid secretion and reverse cholesterol transport

BackgroundThe protective effect of HDL is mostly attributed to their metabolic function in reverse cholesterol transport (RCT), a process whereby excess cellular cholesterol is taken up from peripheral cells, processed in HDL particles, and later delivered to the liver for further metabolism and biliary secretion. Mechanistically, the purinergic P2Y13 ADP-receptor is involved in hepatic HDL endocytosis (i.e., uptake of both HDL protein + lipid moieties), which is considered an important step of RCT. Accordingly, chow-fed P2Y13 knockout (P2Y13-/-) mice exhibit lower hepatic HDL uptake, which translates into a decrease of hepatic free cholesterol content and biliary cholesterol and phospholipid secretion.FindingsThe aim of this study was to determine the effect of high cholesterol diet (HCD) in P2Y13-/- mice, in order to mimic high dietary cholesterol intake, which is a major cause of dyslipidemia in humans. As previously reported with chow-diet, HCD did not affect plasma lipid levels in P2Y13-/- compared with control mice but decreased hepatic free and esterified cholesterol content (p < 0.05, P2Y13-/- versus control). Interestingly, biliary lipid secretion and macrophages-to-feces RCT were more dramatically impaired in P2Y13-/- mice fed a HCD than chow-diet. HCD did not enhance atherosclerosis in P2Y13-/- compared with control mice.ConclusionThis study demonstrates that high dietary cholesterol intake accentuated the metabolic phenotype of P2Y13-/- mice, with impaired hepatobiliary RCT. Although other animal models might be required to further evaluate the role of P2Y13 receptor in atherosclerosis, P2Y13 appears a promising target for therapeutic intervention aiming to stimulate RCT, particularly in individuals with lipid-rich diet.

High density lipoprotein/sphingosine-1-phosphate-induced cardioprotection

High density lipoprotein (HDL) cholesterol has beneficial effects beyond its atheroprotective function in reverse cholesterol transport, including cardioprotection against ischemia reperfusion (IR) injuries. Two major constituents of HDL, namely the structural protein apolipoprotein AI (apoAI) and the sphingolipid sphingosine-1-phosphate (S1P) appear to contribute to this cardioprotective effect via the activation of intrinsic prosurvival signaling pathways that still remain to be clarified. Recently, a powerful prosurvival signaling pathway, termed the survivor activating factor enhancement (SAFE) pathway, which involves the activation of signal transducer and activator of transcription 3 (STAT3) and tumor necrosis factor α (TNF), has been shown to protect against ischemia-reperfusion injuries. The present review summarizes the evidence for the roles of HDL and S1P in cardioprotection and discusses the signaling pathways that have been implicated. It thus provides support for our contention that S1P should be considered in potential formulations of reconstituted HDL (reHDL) that may be tested for cardioprotection against coronary artery disease via the activation of the SAFE pathway.

Extracellular cholesterol-rich microdomains generated by human macrophages and their potential function in reverse cholesterol transport

Previous studies have shown that cholesterol in atherosclerotic plaques is present in both intracellular and extracellular forms. In the current study, we investigated a mechanism for extracellular cholesterol accumulation and examined the capacity of this pool of cholesterol to be removed by cholesterol acceptors, a step in reverse cholesterol transport. Human monocyte-derived macrophages differentiated with macrophage-colony stimulating factor were incubated with acetylated LDL to allow cholesterol enrichment and processing. These macrophages were subsequently labeled with a monoclonal antibody that specifically detects ordered cholesterol arrays, revealing the presence of unesterified cholesterol-rich microdomains on the cell surfaces and in the extracellular matrix. Similar unesterified cholesterol-rich microdomains were present in human atherosclerotic plaques. Actin microfilaments functioned in microdomain deposition or maintenance, and Src family kinases regulated transfer of these microdomains from the cell surface onto the extracellular matrix. Mediators of reverse cholesterol transport, apolipoprotein A-I (apoA-I), and HDL were capable of removing these extracellular un-esterified cholesterol-rich microdomains. However, apoA-I removed the microdomains only when macrophages were present. ApoA-I removal of microdomains was blocked by glyburide and inhibitor of ATP-binding cassette transporter A1 (ABCA1) function. In summary, cultures of cholesterol-enriched human monocyte-derived macrophages generate extracellular unesterified cholesterol-rich microdomains, which can subsequently be removed by cholesterol acceptors and therefore potentially function in reverse cholesterol transport.

High-density lipoprotein heterogeneity and function in reverse cholesterol transport

HDL is a cardioprotective lipoprotein, at least in part, because of its ability to mediate reverse cholesterol transport (RCT). It is becoming increasingly clear that the antiatherogenic effects of HDL are not only dependent on its concentration in circulating blood but also on its biological 'quality'. This review summarizes our current understanding of how the biological activities of individual subclasses of HDL particles contribute to overall HDL performance in RCT. Recent work indicates that apolipoprotein A-I-containing nascent HDL particles are heterogeneous and that such particles exert different effects on the RCT pathway. RCT from macrophages has been examined in detail in mice and the roles of plasma factors (lecithin-cholesterol acyltransferase, cholesterol ester transfer protein, phospholipid transfer protein) and cell factors (ATP-binding cassette transporter A1, ATP-binding cassette transporter G1, scavenger receptor class B type 1) have been evaluated. Manipulation of such factors has consistent effects on RCT and atherosclerosis, but the level of plasma HDL does not reliably predict the degree of RCT. Furthermore, HDL cholesterol or apolipoprotein A-I levels do not necessarily correlate with the magnitude of cholesterol efflux from macrophages; more understanding of the contributions of specific HDL subspecies is required. The antiatherogenic quality of HDL is defined by the functionality of HDL subspecies. In the case of RCT, the rate of cholesterol movement through the pathway is critical and the contributions of particular types of HDL particles to this process are becoming better defined.

The role of dysfunctional HDL in atherosclerosis

This review focuses on HDL function in modulating LDL oxidation and LDL-induced inflammation. Dysfunctional HDL has been identified in animal models and humans with chronic inflammatory diseases including atherosclerosis. The loss of antiinflammatory function correlated with a loss of function in reverse cholesterol transport. In animal models and perhaps in humans, dysfunctional HDL can be improved by apoA-I mimetic peptides that bind oxidized lipids with high affinity.

High glucose, unsaturated and saturated fatty acids differentially regulate expression of ATP-binding cassette transporters ABCA1 and ABCG1 in human macrophages

The ATP-binding cassette transporters ABCA1 and ABCG1 are highly expressed in macrophage-derived foam cells and promote reverse cholesterol efflux via biogenesis of high-density lipoproteins. The aim of this study was to analyze the direct effects of bioactive factors related to the metabolic syndrome on macrophage transcript levels of all 47 human ABC transporters. Using in vitro M-CSF predifferentiated macrophages and TaqMan low density arrays we could show that linoleic acid, palmitic acid, and high glucose levels have a major impact on ABCA1 and ABCG1 expression but do not strongly affect most other human ABC transporters. In Western blot experiments we demonstrate that ABCA1 and ABCG1 protein levels are synchronously suppressed by high glucose levels and the w6-unsaturated fatty acid linoleic acid. We conclude that metabolites associated with the metabolic syndrome enhance the formation of atherosclerotic lesions by diminishing the reverse cholesterol transport function of ABCA1 and ABCG1.

Long-term ethanol consumption impairs reverse cholesterol transport function of high-density lipoproteins by depleting high-density lipoprotein sphingomyelin both in rats and in humans

Moderate alcohol consumption has been linked to lower incidence of coronary artery disease due to increased plasma high-density lipoprotein (HDL), whereas heavy drinking has the opposite effect. Because of the crucial role of HDL in reverse cholesterol transport and positive correlation of HDL sphingomyelin (SM) content with cholesterol efflux, we have compared HDL SM content with its reverse cholesterol transport capacity both in rats fed ethanol on long-term basis and alcoholic individuals. In rats, SM HDL content was decreased in the ethanol group (−15.4%, P < .01) with a concomitant efflux decrease (−21.0%, P < .01) compared to that in controls. Similarly, HDL from the ethanol group, when compared with HDL from the control group, exhibited 13.8% ( P < .05) less cholesterol uptake with control-group hepatocytes and 35.0% ( P < .05) less cholesterol uptake with ethanol-group hepatocytes. Conversely, hepatocytes from the ethanol group, when compared with hepatocytes from the control group, exhibited 31.0% ( P < .01) less cholesterol uptake with control-group HDL and 48.0% ( P < .01) less with ethanol-group HDL. In humans, SM content in plasma HDL was also decreased in chronically alcoholic individuals without liver disease (−51.5%, P < .01) and in chronically alcoholic individuals with liver disease (−51.3%, P < .01), compared with nondrinkers. Concomitantly, in alcoholic individuals without liver disease, both efflux and uptake were decreased by 83.0% and 54.0% ( P < .01), respectively, and in chronically alcoholic individuals with liver disease by 84.0% and 61.0% ( P < .01), respectively, compared with nondrinkers. Based on these findings, we conclude that long-term ethanol consumption significantly impairs not only cholesterol efflux function of HDL by decreasing its SM content but also cholesterol uptake by affecting presumably hepatocyte receptors for HDL.

Compositional Protein Analysis of High Density Lipoproteins in Hypercholesterolemia by Shotgun LC-MS/MS and Probabilistic Peptide Scoring

A protein of a biological sample is usually quantified by immunological techniques based on antibodies. Mass spectrometry offers alternative approaches that are not dependent on antibody affinity and avidity, protein isoforms, quaternary structures, or steric hindrance of antibody-antigen recognition in case of multiprotein complexes. One approach is the use of stable isotope-labeled internal standards; another is the direct exploitation of mass spectrometric signals recorded by LC-MS/MS analysis of protein digests. Here we assessed the peptide match score summation index based on probabilistic peptide scores calculated by the PHENYX protein identification engine for absolute protein quantification in accordance with the protein abundance index as proposed by Mann and co-workers (Rappsilber, J., Ryder, U., Lamond, A. I., and Mann, M. (2002) Large-scale proteomic analysis of the human spliceosome. Genome Res. 12, 1231-1245). Using synthetic protein mixtures, we demonstrated that this approach works well, although proteins can have different response factors. Applied to high density lipoproteins (HDLs), this new approach compared favorably to alternative protein quantitation methods like UV detection of protein peaks separated by capillary electrophoresis or quantitation of protein spots on SDS-PAGE. We compared the protein composition of a well defined HDL density class isolated from plasma of seven hypercholesterolemia subjects having low or high HDL cholesterol with HDL from nine normolipidemia subjects. The quantitative protein patterns distinguished individuals according to the corresponding concentration and distribution of cholesterol from serum lipid measurements of the same samples and revealed that hypercholesterolemia in unrelated individuals is the result of different deficiencies. The presented approach is complementary to HDL lipid analysis; does not rely on complicated sample treatment, e.g. chemical reactions, or antibodies; and can be used for projective clinical studies of larger patient groups.