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Abstract Objective The purpose of this study was to evaluate cholesterol esterification and HDL subclasses in plasma and cerebrospinal fluid (CSF) of Alzheimer’s disease (AD) patients. Methods The study enrolled 70 AD patients and 74 cognitively normal controls comparable for age and sex. Lipoprotein profile, cholesterol esterification, and cholesterol efflux capacity (CEC) were evaluated in plasma and CSF. Results AD patients have normal plasma lipids but significantly reduced unesterified cholesterol and unesterified/total cholesterol ratio. Lecithin:cholesterol acyltransferase (LCAT) activity and cholesterol esterification rate (CER), two measures of the efficiency of the esterification process, were reduced by 29% and 16%, respectively, in the plasma of AD patients. Plasma HDL subclass distribution in AD patients was comparable to that of controls but the content of small discoidal preβ-HDL particles was significantly reduced. In agreement with the reduced preβ-HDL particles, cholesterol efflux capacity mediated by the transporters ABCA1 and ABCG1 was reduced in AD patients’ plasma. The CSF unesterified to total cholesterol ratio was increased in AD patients, and CSF CER and CEC from astrocytes were significantly reduced in AD patients. In the AD group, a significant positive correlation was observed between plasma unesterified cholesterol and unesterified/total cholesterol ratio with Aβ1-42 CSF content. Conclusion Taken together our data indicate that cholesterol esterification is hampered in plasma and CSF of AD patients and that plasma cholesterol esterification biomarkers (unesterified cholesterol and unesterified/total cholesterol ratio) are significantly associated to disease biomarkers (i.e., CSF Aβ1-42).

Objectives: High-density lipoprotein (HDL) removes cholesterol from cells in atherosclerotic lesions, a function known as cholesterol efflux capacity (CEC). ATP-binding-cassette A1 (ABCA1) membrane transporter starts cholesterol transfer from macrophages to HDL particles. In rheumatoid arthritis (RA), methotrexate and biologic disease modifying drugs (bDMARDs) are atheroprotective whereas corticosteroids and C-reactive protein (CRP) are proatherogenic. We evaluated the influence of these factors on the relationship of ABCA1-CEC with atherosclerosis and cardiovascular events. Methods: Atherosclerosis was evaluated with computed tomography angiography in 140 patients with RA and repeated in 99 after 6.9 ± 0.3 years. Events including acute coronary syndromes, stroke, cardiovascular death, claudication, revascularization, and heart failure were recorded. ABCA1-CEC was quantified in J774A.1 murine macrophages and reported as percentage of effluxed over intracellular cholesterol. Results: Higher ABCA1-CEC associated with (i) more calcified plaques at baseline only in patients with CRP>7 mg/L (median) (p-interaction = 0.001) and methotrexate nonusers (p-interaction = 0.037), and more partially-calcified plaques only in bDMARD nonusers (p-interaction = 0.029); (ii) fewer new calcified plaques in patients with below-median but not higher time-averaged CRP (p-interaction = 0.028); (iii) fewer new total and calcified plaques in prednisone unexposed but not patients exposed to prednisone during follow-up (p-interaction = 0.034 and 0.004) and (iv) more new plaques in baseline bDMARD nonusers and fewer in bDMARD users (p-interaction ≤ 0.001). Also, ABCA1-CEC associated with greater cardiovascular risk only in baseline prednisone users (p-interaction = 0.027). Conclusion: ABCA1-CEC associated with decreased atherosclerosis in patients with below-median baseline and time-averaged CRP and bDMARD use. Conversely, ABCA1-CEC associated with increased plaque in those with higher CRP, corticosteroid users, methotrexate nonusers, and bDMARD nonusers. While in well-treated and controlled disease ABCA1-CEC appears atheroprotective, in uncontrolled RA its action may be masked or fail to counteract the inflammation-driven proatherogenic state.

Objectives: Cholesterol efflux capacity (CEC) is the main antiatherogenic function of high-density lipoprotein (HDL). ATP-binding-cassette A1 (ABCA1) membrane transporter initiates cholesterol export from arterial macrophages to pre-β HDL particles fostering their maturation; in turn, those accept cholesterol through ABCG1-mediated export. Impaired pre-β HDL maturation may disrupt the collaborative function of the two transporters and adversely affect atherosclerosis. Statins exert atheroprotective functions systemically and locally on plaque. We here evaluated associations between ABCA1-CEC, coronary atherosclerosis and cardiovascular risk and the influence of statins on those relationships in rheumatoid arthritis (RA). Methods: Evaluation with computed tomography angiography was undertaken in 140 patients and repeated in 99 after 6.9 ± 0.3 years. Events comprising cardiovascular death, acute coronary syndromes, stroke, claudication, revascularization and heart failure were recorded. ABCA1-CEC and ABCG1-CEC were evaluated in J774A.1 macrophages and Chinese hamster ovary (CHO) cells respectively and expressed as percentage of effluxed over total intracellular cholesterol. Covariates in all cardiovascular event risk and plaque outcome models included atherosclerotic cardiovascular disease (ASCVD) risk score and high-density lipoprotein cholesterol. Results: ABCA1-CEC negatively correlated with ABCG1-CEC (r = −0.167, p = 0.049). ABCA1-CEC associated with cardiovascular risk (adjusted hazard ratio 2.05 [95%CI 1.20–3.48] per standard deviation [SD] increment). There was an interaction of ABCA1-CEC with time-varying statin use (p = 0.038) such that current statin use inversely associated with risk only in patients with ABCA1-CEC below the upper tertile. ABCA1-CEC had no main effect on plaque or plaque progression; instead, ABCA1-CEC (per SD) associated with fewer baseline total plaques (adjusted rate ratio [aRR] 0.81, [95%CI 0.65–1.00]), noncalcified plaques (aRR 0.78 [95%CI 0.61–0.98]), and vulnerable low-attenuation plaques (aRR 0.41 [95%CI 0.23–0.74]) in statin users, and more low-attenuation plaques (aRR 1.91 [95%CI 1.18–3.08]) in nonusers (p-for-interaction = 0.018, 0.011, 0.025 and

Background and aims: Cholesterol efflux capacity is a functional property of high-density lipoproteins (HDL) reflecting the efficiency of the atheroprotective reverse cholesterol transport process in humans. Its relationship with calcific aortic valve stenosis (CAVS) has not been fully assessed yet. Methods: We evaluated HDL-CEC in a patient population with varying degrees of aortic valvular calcific disease, assessed using echocardiography and cardiac computed tomography. Measurement of biomarkers that reflect osteogenic and tissue remodeling, along with dietary and gut microbiota-derived metabolites were performed. Results: Patients with moderate-severe CAVS had significantly lower HDL-CEC compared to both control and aortic sclerosis subjects (mean: 6.09%, 7.32% and 7.26%, respectively). HDL-CEC displayed negative correlations with peak aortic jet velocity and aortic valve calcium score, indexes of CAVS severity (ρ = -0.298, p = 0.002 and ρ = -0.358, p = 0.005, respectively). In multivariable regression model, HDL-CEC had independent association with aortic valve calcium score (B: -0.053, SE: 0.014, p

Objectives Cholesterol loading capacity (CLC) describes the ability of serum to deliver cholesterol to cells. It is linked to foam cell formation, a pivotal step in atherosclerotic plaque development. We evaluate the associations of CLC with coronary atherosclerosis presence, burden and cardiovascular risk in patients with rheumatoid arthritis (RA).Methods Coronary atherosclerosis (any, high-risk low-attenuation plaque and obstructive plaque) was evaluated with CT angiography in 141 patients. Participants were prospectively followed for 6.0±2.4 years and cardiovascular events including cardiac death, myocardial infarction, unstable angina, stroke, claudication, revascularisation and hospitalised heart failure were recorded. CLC was quantified as intracellular cholesterol in human macrophages after incubation with patient serum.Results CLC was not linked to overall plaque presence or burden after adjustments for atherosclerotic cardiovascular disease (ASCVD) score, statin use and low-density lipoprotein cholesterol. However, CLC associated with presence and numbers of any, low-attenuation and obstructive plaques exclusively in biologic disease-modifying antirheumatic drugs (bDMARD) non-users (p for interaction ≤0.018). CLC associated with cardiovascular event risk overall after adjustments for ASCVD and number of segments with plaque (HR=1.76 (95% CI 1.16 to 2.67) per 1 SD increase in CLC, p=0.008). Additionally, bDMARD use modified the impact of CLC on event risk; CLC associated with events in bDMARD non-users (HR=2.52 (95% CI 1.36 to 4.65) per 1SD increase in CLC, p=0.003) but not users.Conclusion CLC was linked to long-term cardiovascular event risk in RA and associated with high-risk low attenuation and obstructive coronary plaque presence and burden in bDMARD non-users. Its prospective validation as a predictive biomarker may be, therefore, warranted.

BackgroundThe etiopathogenesis of abdominal aortic aneurysm (AAA) is still unclarified, but vascular inflammation and matrix metalloproteases activation have a recognized role in AAA development and progression. Circulating lipoproteins are involved in tissue inflammation and repair, particularly through the regulation of intracellular cholesterol, whose excess is associated to cell damage and proinflammatory activation. We analyzed lipoprotein metabolism and function in AAA and in control vasculopathic patients, to highlight possible non-atherosclerosis-related, specific abnormalities.MethodsWe measured fluorometrically serum esterified/total cholesterol ratio, as an index of lecithin-cholesterol acyltransferase (LCAT) activity, and cholesteryl ester transfer protein (CETP) activity in patients referred to vascular surgery either for AAA (n=30) or stenotic aortic/peripheral atherosclerosis (n=21) having similar burden of cardiovascular risk factors and disease. We measured high-density lipoprotein (HDL)-cholesterol efflux capacity (CEC), through the ATP-binding cassette G1 (ABCG1) and A1 (ABCA1) pathways and serum cell cholesterol loading capacity (CLC), by radioisotopic and fluorimetric methods, respectively.ResultsWe found higher LCAT (+23%; p < 0.0001) and CETP (+49%; p < 0.0001) activity in AAA sera. HDL ABCG1-CEC was lower (−16%; p < 0.001) and ABCA1-CEC was higher (+31.7%; p < 0.0001) in AAA. Stratification suggests that smoking may partly contribute to these modifications. CEC and CETP activity correlated with CLC only in AAA.ConclusionsWe demonstrated that compared to patients with stenotic atherosclerosis, patients with AAA had altered HDL metabolism and functions involved in their anti-inflammatory and tissue repair activity, particularly through the ABCG1-related intracellular signaling. Clarifying the relevance of this mechanism for AAA evolution might help in developing new diagnostic parameters and therapeutic targets for the early management of this condition.

Proprotein convertase subtilisin/kexin type 9 (PCSK9), beyond regulating LDL cholesterol (LDL-c) plasma levels, exerts several pleiotropic effects by modulating lipid metabolism in extrahepatic cells such as macrophages. Macrophage cholesterol homeostasis depends on serum lipoprotein functions, including the HDL capacity to promote cell cholesterol efflux (CEC) and the serum capacity to promote cell cholesterol loading (CLC). The aim of this observational study was to investigate the effect of PCSK9 inhibitors (PCSK9-i) treatment on HDL-CEC and serum CLC in patients with familial hypercholesterolemia (FH). 31 genetically confirmed FH patients were recruited. Blood was collected and serum isolated at baseline and after 6 months of PCSK9-i treatment. HDL-CEC was evaluated through the main pathways with a radioisotopic cell-based assay. Serum CLC was assessed fluorimetrically in human THP-1 monocyte-derived macrophages. After treatment with PCSK9-i, total cholesterol and LDL-c significantly decreased (−41.6%, p < 0.0001 and −56.7%, p < 0.0001, respectively). Total HDL-CEC was not different between patients before and after treatment. Conversely, despite no changes in HDL-c levels between the groups, ABCG1 HDL-CEC significantly increased after treatment (+22.2%, p < 0.0001) as well as HDL-CEC by aqueous diffusion (+7.8%, p = 0.0008). Only a trend towards reduction of ABCA1 HDL-CEC was observed after treatment. PCSK9-i significantly decreased serum CLC (−6.6%, p = 0.0272). This effect was only partly related to the reduction of LDL-c levels. In conclusion, PCSK9-i treatment significantly increased HDL-CEC through ABCG1 and aqueous diffusion pathways and reduced the serum CLC in FH patients. The favorable effect of PCSK9-i on functional lipid profile could contribute to the cardiovascular benefit of these drugs in FH patients.

Vaccination has drastically decreased mortality due to coronavirus disease 19 (COVID-19), but not the rate of acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Alternative strategies such as inhibition of virus entry by interference with angiotensin-I-converting enzyme 2 (ACE2) receptors could be warranted. Cyclodextrins (CDs) are cyclic oligosaccharides that are able to deplete cholesterol from membrane lipid rafts, causing ACE2 receptors to relocate to areas devoid of lipid rafts. To explore the possibility of reducing SARS-CoV-2 entry, we tested hydroxypropyl-β-cyclodextrin (HPβCD) in a HEK293T-ACE2hi cell line stably overexpressing human ACE2 and Spike-pseudotyped SARS-CoV-2 lentiviral particles. We showed that HPβCD is not toxic to the cells at concentrations up to 5 mM, and that this concentration had no significant effect on cell cycle parameters in any experimental condition tested. Exposure of HEK293T-ACEhi cells to concentrations of HPβCD starting from 2.5 mM to 10 mM showed a concentration-dependent reduction of approximately 50% of the membrane cholesterol content. In addition, incubation of HEK293T-ACEhi cells with HIV-S-CoV-2 pseudotyped particles in the presence of increasing concentrations of HPβCD (from 0.1 to 10 mM) displayed a concentration-dependent effect on SARS-CoV-2 entry efficiency. Significant effects were detected at concentrations at least one order of magnitude lower than the lowest concentration showing toxic effects. These data indicate that HPβCD is a candidate for use as a SARS-CoV-2 prophylactic agent.

Nasal delivery has been indicated as one of the most interesting alternative routes for the brain delivery of neuroprotective drugs. Nanocarriers have emerged as a promising strategy for the delivery of neurotherapeutics across the nasal epithelia. In this work, hybrid lecithin/chitosan nanoparticles (LCNs) were proposed as a drug delivery platform for the nasal administration of simvastatin (SVT) for the treatment of neuroinflammatory diseases. The impact of SVT nanoencapsulation on its transport across the nasal epithelium was investigated, as well as the efficacy of SVT-LCNs in suppressing cytokines release in a cellular model of neuroinflammation. Drug release studies were performed in simulated nasal fluids to investigate SVT release from the nanoparticles under conditions mimicking the physiological environment present in the nasal cavity. It was observed that interaction of nanoparticles with a simulated nasal mucus decreased nanoparticle drug release and/or slowed drug diffusion. On the other hand, it was demonstrated that two antibacterial enzymes commonly present in the nasal secretions, lysozyme and phospholipase A2, promoted drug release from the nanocarrier. Indeed, an enzyme-triggered drug release was observed even in the presence of mucus, with a 5-fold increase in drug release from LCNs. Moreover, chitosan-coated nanoparticles enhanced SVT permeation across a human cell model of the nasal epithelium (×11). The nanoformulation pharmacological activity was assessed using an accepted model of microglia, obtained by activating the human macrophage cell line THP-1 with the Escherichia coli–derived lipopolysaccharide (LPS) as the pro-inflammatory stimulus. SVT-LCNs were demonstrated to suppress the pro-inflammatory signaling more efficiently than the simple drug solution (−75% for IL-6 and −27% for TNF-α vs. −47% and −15% at 10 µM concentration for SVT-LCNs and SVT solution, respectively). Moreover, neither cellular toxicity nor pro-inflammatory responses were evidenced for the treatment with the blank nanoparticles even after 36 h of incubation, indicating a good biocompatibility of the nanomedicine components in vitro. Due to their biocompatibility and ability to promote drug release and absorption at the biointerface, hybrid LCNs appear to be an ideal carrier for achieving nose-to-brain delivery of poorly water-soluble drugs such as SVT.

HDL-like particles in human cerebrospinal fluid (CSF) promote the efflux of cholesterol from astrocytes toward the neurons that rely on this supply for their functions. We evaluated whether cell cholesterol efflux capacity of CSF (CSF-CEC) is impaired in Alzheimer's disease (AD) by analyzing AD (n = 37) patients, non-AD dementia (non-AD DEM; n = 16) patients, and control subjects (n = 39). As expected, AD patients showed reduced CSF Aβ 1-42, increased total and phosphorylated tau, and a higher frequency of the apoε4 genotype. ABCA1- and ABCG1-mediated CSF-CEC was markedly reduced in AD (−73% and −33%, respectively) but not in non-AD DEM patients, in which a reduced passive diffusion CEC (−40%) was observed. Non-AD DEM patients displayed lower CSF apoE concentrations (−24%) compared with controls, while apoA-I levels were similar among groups. No differences in CSF-CEC were found by stratifying subjects for apoε4 status. ABCG1 CSF-CEC positively correlated with Aβ 1-42 (r = 0.305, P = 0.025), while ABCA1 CSF-CEC inversely correlated with total and phosphorylated tau (r = −0.348, P = 0.018 and r = −0.294, P = 0.048, respectively). The CSF-CEC impairment and the correlation with the neurobiochemical markers suggest a pathophysiological link between CSF HDL-like particle dysfunction and neurodegeneration in AD.

Background: Patients with type 2 diabetes mellitus (T2DM) remain at increased cardiovascular residual risk and endothelial dysfunction, even after optimizing metabolic control and treatment by sodium-glucose-2 transporter inhibitors (SGLT2-is). The present study was based on the hypothesis that proprotein convertase subtilisin/kexin 9 inhibitor (PCSK9i) therapy may mitigate endothelial dysfunction in T2DM patients who are on regular treatment by SGLT2-i. Methods: The EXCEED-BHS3 is a prospective, single-center, investigator-blinded, open-label, randomized clinical trial. Participants ( n = 110) will be randomized (1:1) to either empagliflozin 25 mg/day alone or empagliflozin 25 mg/day plus evolocumab 140 mg every 2 weeks in addition to optimal medical care. The primary endpoint was defined as the change in the 1-min flow-mediated dilation (FMD) after 16 weeks of treatment. The secondary endpoint is the FMD change after ischemia/reperfusion injury protocol (reserve FMD) after 16 weeks of treatment. Exploratory outcomes comprise the change in FMD and reserve FMD after 8 weeks of treatment and the change after 16 weeks of treatment in the following parameters: plasma levels of nitric oxide, vascular cell adhesion molecule-1 and isoprostane, high-density lipoprotein (HDL) and low-density lipoprotein subfractions profile, HDL function, blood pressure, body mass index, waist circumference and adipokines. Conclusion: This will be the first study to evaluate the add-on effect of PCSK9i on endothelial function of T2DM patients under regular use of empagliflozin. Trial registration: ClinicalTrials.gov identifier: NCT03932721

Several studies have revealed that traditional risk factors are less effective in predicting CVD risk in the elderly, suggesting the need to identify new biomarkers. Here, we evaluated the association between serum cholesterol efflux capacity (CEC), an atheroprotective property of HDL recently identified as a novel marker of CVD risk, and atherosclerotic burden in a cohort of very old, healthy individuals. Serum CEC values were not significantly correlated either with calcium score or with markers of vulnerable plaque, such as positive remodeling, hypodensity, spotty calcification, or napking-ring sign. In addition, no association was detected between CEC and telomere length, a marker of biological aging that has been linked to atherosclerosis extent. Interestingly, elderly subjects presented a remarkably higher CEC (+30.2%; P < 0.0001) compared with values obtained from a cohort of sex-matched, cardiovascular event-free, middle-aged individuals. In conclusion, serum CEC is not related to traditional risk factors in very old, cardiovascular event-free subjects, but has significantly higher values compared with a healthy, younger population. Whether this improved HDL functionality may represent a protective factor in CVD onset must be established in future studies.

Active dressings acting on multiple fronts are requested in the field of care for chronic skin ulcers in order to ameliorate patient compliance and tissue restoration. Currently, three-dimensional polymeric hydrogels are widely investigated; however, no prototypes aiming to control oxidative stress and bacterial proliferation in the wound bed have been developed up until now. The present work describes the formulation of a novel chitosan-based printable material containing α-tocopherol at stable dosages to obtain reproducible 3D scaffolds possessing antioxidant and antimicrobial activity without the use of organic solvents. Stability assays mimicking the manufacturing process and storage conditions reveal no significant drug loss. Chemico-physical characterizations including porosity and behavior after dehydration/hydration demonstrate that the dressings are highly porous, can be dehydrated up to 80%, and can recover more than 90% of water upon 1 h of rehydration. Elasticity determined by stress/strain tests was higher than human skin and was sufficiently resistant for potential clinical manipulation. Footage of fibroblasts in in vitro cultures demonstrated the biocompatibility of the constructs over 28 days. Finally, scaffolds loaded with α-tocopherol showed dose-dependent antioxidant activity (up to 80% in less than 1 h), while antimicrobial action versus multi-drug resistant strains of Pseudomonas aeruginosa and Staphilococcus aureus was assessed by inhibition rings obtained through the Kirby–Bauer technique. The proposed hydrogels can be useful as dressings for the treatment of chronically infected wounds.

Acute phase reaction (APR) is a systemic inflammation triggered by several conditions associated with lipid profile alterations. We evaluated whether APR also associates with changes in cholesterol synthesis and absorption, HDL structure, composition, and cholesterol efflux capacity (CEC). We analyzed 59 subjects with APR related to infections, oncologic causes, or autoimmune diseases and 39 controls. We detected no difference in markers of cholesterol synthesis and absorption. Conversely, a significant reduction of LpA-I- and LpAI:AII-containing HDL (−28% and −44.8%, respectively) and of medium-sized HDL (−10.5%) occurred in APR. Total HDL CEC was impaired in APR subjects (−18%). Evaluating specific CEC pathways, we found significant reductions in CEC by aqueous diffusion and by the transporters scavenger receptor B-I and ABCG1 (−25.5, −41.1 and −30.4%, respectively). ABCA1-mediated CEC was not affected. Analyses adjusted for age and gender provided similar results. In addition, correcting for HDL-cholesterol (HDL-C) levels, the differences in aqueous diffusion total and ABCG1-CEC remained significant. APR subjects displayed higher levels of HDL serum amyloid A (+20-folds; P = 0.003). In conclusion, APR does not associate with cholesterol synthesis and absorption changes but with alterations of HDL composition and a marked impairment of HDL CEC, partly independent of HDL-C serum level reduction.

Alzheimer’s disease (AD) has been associated with dysregulation of brain cholesterol homeostasis. Proprotein convertase subtilisin/kexin type 9 (PCSK9), beyond the known role in the regulation of plasma low-density lipoprotein cholesterol, was first identified in the brain with a potential involvement in brain development and apoptosis. However, its role in the central nervous system (CNS) and in AD pathogenesis is still far from being understood. While in vitro and in vivo evidence led to controversial results, genetic studies apparently did not find an association between PCSK9 loss of function mutations and AD risk or prevalence. In addition, a potential impairment of cognitive performances by the treatment with the PCSK9 inhibitors, alirocumab and evolocumab, have been excluded, although ongoing studies with longer follow-up will provide further insights. PCSK9 is able to affect the expression of neuronal receptors involved in cholesterol homeostasis and neuroinflammation, and higher PCSK9 concentrations have been found in the cerebrospinal fluid (CSF) of AD patients. In this review article, we critically examined the science of PCSK9 with respect to its modulatory role of the mechanisms underlying the pathogenesis of AD. In addition, based on literature data, we made the hypothesis to consider brain PCSK9 as a negative modulator of brain cholesterol homeostasis and neuroinflammation and a potential pharmacological target for treatment.

Background: A wealth of experimental and epidemiological evidence suggest that Apolipoprotein A-I (ApoA-I), the main protein constituent of high-density lipoprotein (HDL), may protect against Alzheimer disease (AD). To investigate this potential role, we conducted a meta-analysis of the published studies on the relationship between serum ApoA-I and AD occurrence. Methods: We screened MEDLINE, EMBASE, Web of Science, and Scopus, for cross-sectional studies published from inception to 1 March 2021, comparing the ApoA-I serum levels between patients with AD and cognitively normal controls. Results: From an initial screening of 245 articles, 5 studies, including 397 AD patients (mean age 75.0 years, 234 females) and 367 controls (mean age 69.2 years, 182 females), met the inclusion criteria. Compared to healthy controls, AD subjects had a lower ApoA-I serum level. The pooled weighted mean difference from a random-effects model was −0.31 g/L (p < 0.0001) (95% Confidence Interval: [−0.62–0.01], with high heterogeneity (I2 = 100%). The Egger’s test confirmed an absence of publication bias (t = 0.62, p = 0.576). Conclusions: Our study showed that AD patients present lower serum levels of ApoA-I compared to cognitively normal individuals. Further studies on large population samples are required to support this finding.

Over the years, the relationship between high-density lipoprotein (HDL) and atherosclerosis, initially highlighted by the Framingham study, has been revealed to be extremely complex, due to the multiple HDL functions involved in atheroprotection. Among them, HDL cholesterol efflux capacity (CEC), the ability of HDL to promote cell cholesterol efflux from cells, has emerged as a better predictor of cardiovascular (CV) risk compared to merely plasma HDL-cholesterol (HDL-C) levels. HDL CEC is impaired in many genetic and pathological conditions associated to high CV risk such as dyslipidemia, chronic kidney disease, diabetes, inflammatory and autoimmune diseases, endocrine disorders, etc. The present review describes the current knowledge on HDL CEC modifications in these conditions, focusing on the most recent human studies and on genetic and pathophysiologic aspects. In addition, the most relevant strategies possibly modulating HDL CEC, including lifestyle modifications, as well as nutraceutical and pharmacological interventions, will be discussed. The objective of this review is to help understanding whether, from the current evidence, HDL CEC may be considered as a valid biomarker of CV risk and a potential pharmacological target for novel therapeutic approaches.

Several lines of epidemiological evidence link increased levels of high-density lipoprotein-cholesterol (HDL-C) with lower risk of Alzheimer’s disease (AD). This observed relationship might reflect the beneficial effects of HDL on the cardiovascular system, likely due to the implication of vascular dysregulation in AD development. The atheroprotective properties of this lipoprotein are mostly due to its proteome. In particular, apolipoprotein (Apo) A-I, E, and J and the antioxidant accessory protein paraoxonase 1 (PON1), are the main determinants of the biological function of HDL. Intriguingly, these HDL constituent proteins are also present in the brain, either from in situ expression, or derived from the periphery. Growing preclinical evidence suggests that these HDL proteins may prevent the aberrant changes in the brain that characterize AD pathogenesis. In the present review, we summarize and critically examine the current state of knowledge on the role of these atheroprotective HDL-associated proteins in AD pathogenesis and physiopathology.

The capacity of HDL to induce cell cholesterol efflux is considered one of its main antiatherogenic properties. Little is known about the impact of such HDL function on vascular physiology. We investigated the relationship between ABCA1-dependent serum cholesterol efflux capacity (CEC), an HDL functionality indicator, and pulse wave velocity (PWV), an indicator of arterial stiffness. Serum of 167 healthy subjects was used to conduct CEC measurement, and carotid-femoral PWV was measured with a high-fidelity tonometer. J774 macrophages, labeled with [3H]cholesterol and stimulated to express ABCA1, were exposed to sera; the difference between cholesterol efflux from stimulated and unstimulated cells provided specific ABCA1-mediated CEC. PWV is inversely correlated with ABCA1-dependent CEC (r =−0.183; P = 0.018). Moreover, controlling for age, sex, body mass index, mean arterial pressure, serum LDL, HDL-cholesterol, and fasting plasma glucose, PWV displays a significant negative regression on ABCA1-dependent CEC (β=−0.204; 95% confidence interval, −0.371 to −0.037). The finding that ABCA1-dependent CEC, but not serum HDL cholesterol level (r =−0.002; P = 0.985), is a significant predictor of PWV in healthy subjects points to the relevance of HDL function in vascular physiology and arterial stiffness prevention.

Cholesterol efflux occurs by different pathways, including transport mediated by specific proteins. We determined the effect of enriching cells with free cholesterol (FC) on the release of FC to human serum. Loading Fu5AH cells with FC had no effect on fractional efflux, whereas enriching mouse peritoneal macrophages (MPMs) resulted in a doubling of fractional efflux. Efflux from cholesterol-normal MPM and Fu5AH cells to 15 human sera correlated well with HDL parameters. However, these relationships were reduced or lost with cholesterol-loaded MPMs. Using macrophages from scavenger receptor class B type I (SR-BI)-, ABCA1-, and ABCG1-knockout mice, together with inhibitors of SR-BI- and ABCA1-mediated efflux, we were able to quantitate efflux upon loading macrophages with excess cholesterol and to establish the contributions of the various efflux pathways in cholesterol-normal and -enriched cells. The removal of ABCA1 had essentially no effect on the total efflux when cell cholesterol levels were normal. However, in cholesterol-enriched cells, the removal of ABCA1 reduced efflux by 50%. Approximately 20% of the efflux stimulated by FC-loading MPM is attributable to ABCG1. The SR-BI contribution to efflux was small. Another pathway that is present in all cells is aqueous diffusion. Our studies demonstrate that this mechanism is one of the major contributors to efflux, particularly in cholesterol-normal cells.

Cholesteryl ester transfer protein (CETP) inhibitors increase high density lipoprotein-cholesterol (HDL-C) in animals and humans, but whether CETP inhibition will be antiatherogenic is still uncertain. We tested the CETP inhibitor torcetrapib in rabbits fed an atherogenic diet at a dose sufficient to increase HDL-C by at least 3-fold (207 ± 32 vs. 57 ± 6 mg/dl in controls at 16 weeks). CETP activity was inhibited by 70–80% throughout the study. Non-HDL-C increased in both groups, but there was no difference apparent by the study's end. At 16 weeks, aortic atherosclerosis was 60% lower in torcetrapib-treated animals (16.4 ± 3.4% vs. 39.8 ± 5.4% in controls) and aortic cholesterol content was reduced proportionally. Sera from a separate group of rabbits administered torcetrapib effluxed 48% more cholesterol from Fu5AH cells than did sera from control animals, possibly explaining the reduced aortic cholesterol content. Regression analyses indicated that lesion area in the torcetrapib-treated group was strongly correlated with the ratio of total plasma cholesterol to HDL-C but not with changes in other lipid or lipoprotein levels. CETP inhibition with torcetrapib retards atherosclerosis in rabbits, and the reduced lesion area is associated with increased levels of HDL-C.

We developed an assay that quantitates bidirectional cholesterol flux between cells and lipoproteins. Incubating Fu5AH cells with increasing concentrations of human serum resulted in increased influx and efflux; however, influx was 2- to 3-fold greater at all serum concentrations. With apolipoprotein B (apoB)-depleted serum, the ratio of influx to efflux (I/E) was close to 1, indicating cholesterol exchange. The apoB fraction of serum induced influx and little efflux, with I/E > 1. Using block lipid transport-1 to block scavenger receptor class B type I (SR-BI)-mediated flux with different acceptors, we determined that 50% to 70% of efflux was via SR-BI. With HDL, 90% of influx was via SR-BI, whereas with LDL or serum, 20% of influx was SR-BI-mediated. Cholesterol-enriched hepatoma cells produced increased efflux without a change in influx, resulting in reduced I/E. The assay was applied to cholesterol-normal and -enriched mouse peritoneal macrophages exposed to serum or LDL. The enrichment enhanced efflux without shifts in influx. With cholesterol-enriched macrophages, HDL efflux was enhanced and influx was greatly reduced. With all lipoproteins, cholesterol enrichment of murine peritoneal macrophages led to a reduced I/E. We conclude that this assay can simultaneously and accurately quantitate cholesterol bidirectional flux and can be applied to a variety of cells exposed to isolated lipoproteins or serum.

Despite the efficacy in reducing acute rejection events in organ transplanted subjects, long term therapy with cyclosporine A is associated with increased atherosclerotic cardiovascular morbidity. We studied whether this drug affects the antiatherogenic process of the reverse cholesterol transport from macrophages in vivo. Cyclosporine A 50 mg/kg/d was administered to C57BL/6 mice by subcutaneous injection for 14 days. Macrophage reverse cholesterol transport was assessed by following [(3)H]-cholesterol mobilization from pre-labeled intraperitoneally injected macrophages, expressing or not apolipoprotein E, to plasma, liver and feces. The pharmacological treatment significantly reduced the amount of radioactive sterols in the feces, independently on the expression of apolipoprotein E in the macrophages injected into recipient mice and in absence of changes of plasma levels of high density lipoprotein-cholesterol. Gene expression analysis revealed that cyclosporine A inhibited the hepatic levels of cholesterol 7-alpha-hydroxylase, concomitantly with the increase in hepatic and intestinal expression of ATP Binding Cassette G5. However, the in vivo relevance of the last observation was challenged by the demonstration that mice treated or not with cyclosporine A showed the same levels of circulating beta-sitosterol. These results indicate that treatment of mice with cyclosporine A impaired the macrophage reverse cholesterol transport by reducing fecal sterol excretion, possibly through the inhibition of cholesterol 7-alpha-hydroxylase expression. The current observation may provide a potential mechanism for the high incidence of atherosclerotic coronary artery disease following the immunosuppressant therapy in organ transplanted recipients.