The role of blood cholesterol quality in patients with advanced cancer receiving immune checkpoint inhibitors.

INTRODUCTION: Immune checkpoint inhibitors (ICIs) became the standard of care for several solid tumors. A limited fraction of patients (pts) achieves a long-term benefit. Plasmatic and intracellular cholesterol levels have emerged as promising biomarkers. The aim of the present study was to determine whether cholesterol efflux capacity (CEC), mediated by serum transporters (ABCA1 and ABCG1) and passive diffusion (PD), impacts on clinical outcome of advanced non-small cell lung cancer (NSCLC) and metastatic renal cell carcinoma (mRCC) pts treated with ICIs. MATERIAL AND METHODS: We retrospectively enrolled advanced NSCLC and mRCC pts consecutively treated with ICIs between October 2013 and October 2018. CEC and cholesterol loading capacity (CLC) were assessed by well-established specific cell models. As primary endpoint, CEC, PD and CLC were correlated with overall survival (OS) while the effects of these parameters on progression-free survival (PFS) and clinical benefit (CB), defined as complete/partial response or stable disease, represented secondary endpoints. RESULTS: NSCLC accounted for 94.2% of 70 enrolled cases, and serum sample suitable for CEC and PD determination was available in 68. Blood cholesterol and serum ABCA1, ABCG1, PD and CLC were associated with outcomes (OS, PFS and CB) at univariate analysis. At the multivariate analysis, only PD confirmed its positive prognostic value in terms of OS, PFS and CB. CONCLUSION: The favorable impact of cholesterol PD on clinical outcome might reflect its main conformation in mature HDL particles which potentially shape an inflamed context, ultimately promoting ICI efficacy. Further prospective studies are needed to support our findings and uncover targetable pathways.

Effects of a dietary intervention with Mediterranean vs lacto-ovo vegetarian diets on HDL function: Results from the CARDIVEG study.

BACKGROUND AND AIM: HDL-cholesterol efflux capacity (CEC) has been shown to be a better cardiovascular (CVD) risk marker than serum HDL concentration. Several foods and nutrients have been shown to improve HDL functions, however no effective dietetic nor pharmacological strategy is available to increase CEC. This study aims to evaluate the possible effect of Mediterranean diet (MD) and lacto-ovo-vegetarian diet (VD) on HDL function in a group of clinically healthy subjects at low-to-moderate CVD risk. METHODS AND RESULTS: Thirty apparently healthy subjects with a low-to-moderate cardiovascular risk profile (21 F; mean age: 51.3 ± 9.7 years) were randomly assigned to a 3-month MD or VD diet and then crossed. Participants on VD showed a reduction in total HDL CEC by 8.99% (p < 0.001) as well as a reduction in ABCA1 mediated-CEC by 18.62% (p < 0.001) compared to participants on MD. Regarding CEC mediated by aqueous diffusion, no significant changes were observed after treatment with either diet. Finally, a significant positive association between CEC mediated by the ABCA1 transporter and adiponectin was found (r = 0.462; p = 0.010). CONCLUSION: The results of this study suggest that HDL activity in promoting cholesterol efflux and thereby reducing the concentration of pro-atherogenic lipoproteins was more effective in participants undergoing MD than VD. Based on these findings, the MD could be considered a better therapeutic strategy for cardiovascular prevention than VD. CLINICAL TRIAL REGISTRATION URL: http://www. CLINICALTRIALS: gov. Unique identifier: NCT02641834.

Effect of a PCSK9 inhibitor and a statin on cholesterol efflux capacity: A limitation of current cholesterol-lowering treatments?

BACKGROUND: Cellular cholesterol efflux is a key step in reverse cholesterol transport that may impact on atherosclerotic cardiovascular risk. The process may be reliant on the availability of apolipoprotein (apo) B-100-containing lipoproteins to accept cholesterol from high-density lipoprotein. Evolocumab and atorvastatin are known to lower plasma apoB-100-containing lipoproteins that could impact on cholesterol efflux capacity (CEC). METHODS: We conducted a 2-by-2 factorial trial of the effects of subcutaneous evolocumab (420 mg every 2 weeks) and atorvastatin (80 mg daily) for 8 weeks on CEC in 81 healthy, normolipidaemic men. The capacity of whole plasma and apoB-depleted plasma, including ATP-binding cassette transporter A1 (ABCA1)-mediated and passive diffusion, to efflux cholesterol, was measured. RESULTS: Evolocumab and atorvastatin independently decreased whole plasma CEC (main effect p < .01 for both). However, there were no significant effects of evolocumab and atorvastatin on apoB-depleted plasma, ABCA1-mediated and passive diffusion-mediated CEC (p > .05 in all). In the three intervention groups combined, the reduction in whole plasma CEC was significantly correlated with the corresponding reduction in plasma apoB-100 concentration (r = .339, p < .01). In the evolocumab monotherapy group, the reduction in whole plasma CEC was also significantly correlated with the corresponding reduction in plasma lipoprotein(a) concentration (r = .487, p < .05). CONCLUSIONS: In normolipidaemic men, evolocumab and atorvastatin decrease the capacity of whole plasma to efflux cellular cholesterol. These effects may be chiefly owing to a fall in the availability of apoB-100-containing lipoproteins. Reduction in circulating lipoprotein(a) may also contribute to the decrease in whole plasma cholesterol efflux with evolocumab monotherapy.

Plasma HDL pattern, cholesterol efflux and cholesterol loading capacity of serum in carriers of a novel missense variant (Gly176Trp) of endothelial lipase.

BACKGROUND: Loss of function variants of LIPG gene encoding endothelial lipase (EL) are associated with primary hyperalphalipoproteinemia (HALP), a lipid disorder characterized by elevated plasma levels of high density lipoprotein cholesterol (HDL-C). OBJECTIVE: Aim of the study was the phenotypic and genotypic characterization of a family with primary HALP. METHODS: HDL subclasses distribution was determined by polyacrylamide gradient gel electrophoresis. Serum content of preß-HDL was assessed by (2D)-electrophoresis. Cholesterol efflux capacity (CEC) of serum mediated by ABCA1, ABCG1 or SR-BI was assessed using cells expressing these proteins. Cholesterol loading capacity (CLC) of serum was assayed using cultured human macrophages. Next generation sequencing was used for DNA analysis. Plasma EL mass was determined by ELISA. RESULTS: Three family members had elevated plasma HDL-C, apoA-I and total phospholipids, as well as a reduced content of preß-HDL. These subjects were heterozygous carriers of a novel variant of LIPG gene [c.526 G>T, p.(Gly176Trp)] found to be deleterious in silico. Plasma EL mass in carriers was lower than in controls. CEC of sera mediated by ABCA1 and ABCG1 transporters was substantially reduced in the carriers. This effect was maintained after correction for serum HDL concentration. The sera of carriers were found to have a higher CLC in cultured human macrophages than control sera. CONCLUSION: The novel p.(Gly176Trp) variant of endothelial lipase is associated with changes in HDL composition and subclass distribution as well as with functional changes affecting cholesterol efflux capacity of serum which suggest a defect in the early steps of revere cholesterol transport.

Probucol treatment is associated with an ABCA1-independent mechanism of cholesterol efflux to lipid poor apolipoproteins from foam cell macrophages.

Objective: Probucol is a cholesterol-lowering agent whose ability to prevent atherosclerosis is currently under study. Herein, we investigate the putative mechanism of probucol by observation of changes in cellular cholesterol efflux and lipid droplet morphology in macrophages. Results: The inhibitory activity of probucol was assessed in non-foam or foam cell macrophages expressing ABCA1 generated by treatment with fetal calf serum (FCS) alone or in combination with acetylated LDL, respectively. Probucol inhibited cholesterol efflux to apolipoprotein A-I (apoA-I) by 31.5±0.1% in THP-1 non-foam cells and by 18.5±0.2% in foam cells. In probucol-treated non-foam THP-1 cells, nascent high density lipoprotein (nHDL) particles with a diameter < 7 nm were generated, while in probucol-treated THP-1 foam cells nHDL particles of > 7 nm in diameter containing cholesterol were produced. Foam cells also displayed a significant accumulation of free cholesterol at the plasma membrane, as measured by percent cholestenone formed. Intracellularly, there was a significant decrease in lipid droplet number and an increase in size in probucol-treated THP-1 foam cells when compared to non-treated cells. Conclusions: We report for the first time that probucol is unable to completely inhibit cholesterol efflux in foam cells to the same extent as in non-foam cells. Indeed, functional nHDL is released from foam cells in the presence of probucol. This difference in inhibitory effect could potentially be explained by changes in the plasma membrane pool as well as intracellular cholesterol storage independently of ABCA1.

Efficacy of Nutraceutical Combination of Monacolin K, Berberine, and Silymarin on Lipid Profile and PCSK9 Plasma Level in a Cohort of Hypercholesterolemic Patients.

The guidelines for the treatment of dyslipidemias include the use of nutraceuticals (NUTs) in association with lifestyle modifications to achieve therapeutic goals. In NUT pill, different substances may be associated; in this study we investigated a combined NUT containing monacolin K (MonK)+KA (1:1), berberine (BBR), and silymarin. The aim of the study was to evaluate low-density lipoprotein cholesterol (LDL-C) reduction in 53 patients suffering from polygenic hypercholesterolemia, characterized by a low/intermediate cardiovascular risk calculated with SCORE algorithm. The effects on lipid profile of 2-month treatment with NUT containing MonK+KA (1:1), BBR, and sylimarin, were compared with Atorvastatin (ATO) 10 mg administrated in a matched control group. Serum proprotein convertase subtilisin/kexin type 9 (PCSK9) levels and the cholesterol loading capacity (CLC) were determined at baseline and at the end of the study in NUT-treated group; variations were assessed. NUT was effective as lipid-lowering agent with a wide interindividual response variability (mean LDL-C from 170.8 ± 19.9 to 123.8 ± 20.0 with a change of -47.0 ± 21.5 mg/dL; P < .001) and the effect was similar to that induced by ATO. The use of NUT significantly modified PCSK9 levels (P < .01) and CLC (P < .001), ultimately suppressing the serum-mediated foam cell generation directly measured on human macrophages. NUT reduces LDL-C levels with an effect similar to what is induced by 10 mg of ATO and ex vivo improves the functional profile of lipoproteins with antiatherogenic action.

Lipoprotein(a) concentration, genetic variants, apo(a) isoform size, and cellular cholesterol efflux in patients with elevated Lp(a) and coronary heart disease submitted or not to lipoprotein apheresis: An Italian case-control multicenter study on Lp(a).

BACKGROUND: Coronary artery disease (CAD) risk is greater with higher plasma lipoprotein(a)[Lp(a)] concentrations or smaller apoisoform size and putatively with increased cellular cholesterol loading capacity (CLC). The relationship between Lp(a) and CLC is not known. Information on Lp(a) polymorphisms in Italian patients is lacking. OBJECTIVE: The objective of this study was to determine relationships between Lp(a) and CLC, the impact of lipoprotein apheresis (LA), and describe the genetic profile of Lp(a). METHODS: We conducted a multicenter, observational study in Italian patients with hyperLp(a) and premature CAD with (n = 18)/without (n = 16) LA in which blood samples were analyzed for Lp(a) parameter and CLC. Genetic profiling of LPA was conducted in patient receiving LA. RESULTS: Mean macrophage CLC of the pre-LA serum was significantly higher than that of normolipidemic controls (19.7 ± 0.9 µg/mg vs 16.01 ± 0.98 µg/mg of protein, respectively). After LA, serum macrophage CLC was markedly lower relative to preapheresis (16.1 ± 0.8 µg/mg protein; P = .003) and comparable with CLC of the normolipidemic serum. LA did not significantly affect average apo(a) isoform size distribution. No anthropometric or lipid parameters studied were related to serum CLC, but there was a relationship between CLC and the Lp(a) plasma concentration (P = .035). DNA analysis revealed a range of common genetic variants. Two rare, new variants were identified: LPA exon 21, c.3269C>G, p.Pro1090Arg, and rs41259144 p.Arg990Gln, c.2969G>A CONCLUSIONS: LA reduces serum Lp(a) and also reduces macrophage CLC. Novel genetic variants of the LPA gene were identified, and geographic variations were noted. The complexity of these polymorphisms means that genetic assessment is not a predictor of CAD risk in hyperLp(a).

HDL-Mediated Cholesterol Efflux and Plasma Loading Capacities Are Altered in Subjects with Metabolically- but Not Genetically Driven Non-Alcoholic Fatty Liver Disease (NAFLD).

Background. Non-alcoholic fatty liver disease (NAFLD) increases the risk of atherosclerosis but this risk may differ between metabolically- vs. genetically-driven NAFLD. High-density lipoprotein (HDL)-mediated cholesterol efflux (CEC) and plasma loading capacity (CLC) are key factors in atherogenesis. Aims. To test whether CEC and CLC differ between metabolically- vs. genetically-determined NAFLD. Methods: CEC and CLC were measured in 19 patients with metabolic NAFLD and wild-type PNPLA3 genotype (Group M), 10 patients with genetic NAFLD carrying M148M PNPLA3 genotype (Group G), and 10 controls PNPLA3 wild-types and without NAFLD. CEC and CLC were measured ex vivo by isotopic and fluorimetric techniques using cellular models. Results: Compared with Group G, Group M showed reduced total CEC (-18.6%; p < 0.001) as well as that mediated by cholesterol transporters (-25.3% ABCA1; -16.3% ABCG1; -14.8% aqueous diffusion; all p < 0.04). No difference in CEC was found between Group G and controls. The presence of metabolic syndrome further impaired ABCG1-mediated CEC in Group M. Group M had higher plasma-induced CLC than Group G and controls (p < 0.001). Conclusions: Metabolically-, but not genetically-, driven NAFLD associates with dysfunctional HDL-meditated CEC and abnormal CLC. These data suggest that the mechanisms of anti-atherogenic protection in metabolic NAFLD are impaired.

Anti-ApoA-1 IgGs in Familial Hypercholesterolemia Display Paradoxical Associations with Lipid Profile and Promote Foam Cell Formation.

AIMS: Anti-Apolipoprotein A-1 autoantibodies (anti-ApoA-1 IgG) promote atherogenesis via innate immune receptors, and may impair cellular cholesterol homeostasis (CH). We explored the presence of anti-ApoA-1 IgG in children (5-15 years old) with or without familial hypercholesterolemia (FH), analyzing their association with lipid profiles, and studied their in vitro effects on foam cell formation, gene regulation, and their functional impact on cholesterol passive diffusion (PD). METHODS: Anti-ApoA-1 IgG and lipid profiles were measured on 29 FH and 25 healthy children. The impact of anti-ApoA-1 IgG on key CH regulators (SREBP2, HMGCR, LDL-R, ABCA1, and miR-33a) and foam cell formation detected by Oil Red O staining were assessed using human monocyte-derived macrophages. PD experiments were performed using a validated THP-1 macrophage model. RESULTS: Prevalence of high anti-ApoA-1 IgG levels (seropositivity) was about 38% in both study groups. FH children seropositive for anti-ApoA-1 IgG had significant lower total cholesterol LDL and miR-33a levels than those who were seronegative. On macrophages, anti-ApoA-1 IgG induced foam cell formation in a toll-like receptor (TLR) 2/4-dependent manner, accompanied by NF-kB- and AP1-dependent increases of SREBP-2, LDL-R, and HMGCR. Despite increased ABCA1 and decreased mature miR-33a expression, the increased ACAT activity decreased membrane free cholesterol, functionally culminating to PD inhibition. CONCLUSIONS: Anti-ApoA-1 IgG seropositivity is frequent in children, unrelated to FH, and paradoxically associated with a favorable lipid profile. In vitro, anti-ApoA-1 IgG induced foam cell formation through a complex interplay between innate immune receptors and key cholesterol homeostasis regulators, functionally impairing the PD cholesterol efflux capacity of macrophages.