Wnt5a Promotes Lysosomal Cholesterol Egress and Protects Against Atherosclerosis.

BACKGROUND: Impairment of cellular cholesterol trafficking is at the heart of atherosclerotic lesions formation. This involves egress of cholesterol from the lysosomes and 2 lysosomal proteins, the NPC1 (Niemann-Pick C1) and NPC2 that promotes cholesterol trafficking. However, movement of cholesterol out the lysosome and how disrupted cholesterol trafficking leads to atherosclerosis is unclear. As the Wnt ligand, Wnt5a inhibits the intracellular accumulation of cholesterol in multiple cell types, we tested whether Wnt5a interacts with the lysosomal cholesterol export machinery and studied its role in atherosclerotic lesions formation. METHODS: We generated mice deleted for the Wnt5a gene in vascular smooth muscle cells. To establish whether Wnt5a also protects against cholesterol accumulation in human vascular smooth muscle cells, we used a CRISPR/Cas9 guided nuclease approach to generate human vascular smooth muscle cells knockout for Wnt5a. RESULTS: We show that Wnt5a is a crucial component of the lysosomal cholesterol export machinery. By increasing lysosomal acid lipase expression, decreasing metabolic signaling by the mTORC1 (mechanistic target of rapamycin complex 1) kinase, and through binding to NPC1 and NPC2, Wnt5a senses changes in dietary cholesterol supply and promotes lysosomal cholesterol egress to the endoplasmic reticulum. Consequently, loss of Wnt5a decoupled mTORC1 from variations in lysosomal sterol levels, disrupted lysosomal function, decreased cholesterol content in the endoplasmic reticulum, and promoted atherosclerosis. CONCLUSIONS: These results reveal an unexpected function of the Wnt5a pathway as essential for maintaining cholesterol homeostasis in vivo.

ABCB1 (P-glycoprotein) regulates vitamin D absorption and contributes to its transintestinal efflux.

Efficient intestinal absorption of dietary vitamin D is required in most people to ensure an adequate status. Thus, we investigated the involvement of ATP binding cassette subfamily B member 1 (ABCB1) in vitamin D intestinal efflux. Both cholecalciferol (D3) and 25-hydroxycholecalciferol [25(OH)D3] apical effluxes were decreased by chemical inhibition of ABCB1 in Caco-2 cells and increased by ABCB1 overexpression in Griptites or Madin-Darby canine kidney type II cells. Mice deficient for the 2 murine ABCB1s encoded by Abcb1a and Abcb1b genes ( Abcb1-/-) displayed an accumulation of 25(OH)D3 in plasma, intestine, brain, liver, and kidneys, together with an increased D3 postprandial response after gavage compared with controls. 25(OH)D3 efflux through Abcb1-/- intestinal explants was markedly decreased compared with controls. This reduction of 25(OH)D3 transfer from plasma to lumen was further confirmed in vivo in intestine-perfused mice. Docking experiments established that both D3 and 25(OH)D3 could bind with high affinity to Caenorhabditis elegans P-glycoprotein, used as an ABCB1 model. Finally, in a group of 39 healthy male adults, a single-nucleotide polymorphism (SNP) in ABCB1 (rs17064) was significantly associated with the fasting plasma 25(OH)D3 concentration. Thus, we showed here for the first time that ABCB1 is involved in neo-absorbed vitamin D efflux by the enterocytes and that it also contributes to vitamin D transintestinal excretion and likely impacts vitamin D status.-Margier, M., Collet, X., le May, C., Desmarchelier, C., André, F., Lebrun, C., Defoort, C., Bluteau, A., Borel, P., Lespine, A., Reboul, E. ABCB1 (P-glycoprotein) regulates vitamin D absorption and contributes to its transintestinal efflux.

Intestinal scavenger receptors are involved in vitamin K1 absorption.

Vitamin K1 (phylloquinone) intestinal absorption is thought to be mediated by a carrier protein that still remains to be identified. Apical transport of vitamin K1 was examined using Caco-2 TC-7 cell monolayers as a model of human intestinal epithelium and in transfected HEK cells. Phylloquinone uptake was then measured ex vivo using mouse intestinal explants. Finally, vitamin K1 absorption was compared between wild-type mice and mice overexpressing scavenger receptor class B type I (SR-BI) in the intestine and mice deficient in cluster determinant 36 (CD36). Phylloquinone uptake by Caco-2 cells was saturable and was significantly impaired by co-incubation with α-tocopherol (and vice versa). Anti-human SR-BI antibodies and BLT1 (a chemical inhibitor of lipid transport via SR-BI) blocked up to 85% of vitamin K1 uptake. BLT1 also decreased phylloquinone apical efflux by ∼80%. Transfection of HEK cells with SR-BI and CD36 significantly enhanced vitamin K1 uptake, which was subsequently decreased by the addition of BLT1 or sulfo-N-succinimidyl oleate (CD36 inhibitor), respectively. Similar results were obtained in mouse intestinal explants. In vivo, the phylloquinone postprandial response was significantly higher, and the proximal intestine mucosa phylloquinone content 4 h after gavage was increased in mice overexpressing SR-BI compared with controls. Phylloquinone postprandial response was also significantly increased in CD36-deficient mice compared with wild-type mice, but their vitamin K1 intestinal content remained unchanged. Overall, the present data demonstrate for the first time that intestinal scavenger receptors participate in the absorption of dietary phylloquinone.

Cholesterol and Sphingomyelin-Containing Model Condensed Lipid Monolayers: Heterogeneities Involving Ordered Microdomains Assessed by Two Cholesterol Derivatives.

Lipid monolayers are often considered as model membranes, but they are also the physiologic lipid part of the peripheral envelope of lipoproteins and cytosolic lipid bodies. However, their structural organization is still rather elusive, in particular when both cholesterol and sphingomyelin are present. To investigate such structural organization of hemimembranes, we measured, using alternative current voltammetry, the differential capacitance of condensed phosphatidylcholine-based monolayers as a function of applied potential, which is sensitive to their lipid composition and molecular arrangement. Especially, monolayers containing both sphingomyelin and cholesterol, at 15% w/w, presented specific characteristics of the differential capacitance versus potential curves recorded, which was indicative of specific interactions between these two lipid components. We then compared the behavior of two cholesterol derivatives (at 15% w/w), 21-methylpyrenyl-cholesterol (Pyr-met-Chol) and 22-nitrobenzoxadiazole-cholesterol (NBD-Chol), with that of cholesterol when present in model monolayers. Indeed, these two probes were chosen because of previous findings reporting opposite behaviors within bilayer membranes regarding their interaction with ordered lipids, with only Pyr-met-Chol mimicking cholesterol well. Remarkably, in monolayers containing sphingomyelin or not, Pyr-met-Chol and NBD-Chol presented contrasting behaviors, and Pyr-met-Chol mimicked cholesterol only in the presence of sphingomyelin. These two observations (i.e., optimal amounts of sphingomyelin and cholesterol, and the ability to discriminate between Pyr-met-Chol and NBD-Chol) can be interpreted by the existence of heterogeneities including ordered patches in sphingomyelin- and cholesterol-containing monolayers. Since such monolayer lipid arrangement shares some properties with the raft-type lipid microdomains well-described in sphingomyelin- and cholesterol-containing bilayer membranes, our data thus strongly suggest the existence of compact and ordered microdomains in model lipid monolayers.

Transintestinal cholesterol excretion is an active metabolic process modulated by PCSK9 and statin involving ABCB1.

OBJECTIVE: Transintestinal cholesterol excretion (TICE) is an alternate pathway to hepatobiliary secretion. Our study aimed at identifying molecular mechanisms of TICE. APPROACH AND RESULTS: We studied TICE ex vivo in mouse and human intestinal explants, and in vivo after bile diversion and intestinal cannulation in mice. We provide the first evidence that both low-density lipoprotein (LDL) and high-density lipoprotein deliver cholesterol for TICE in human and mouse jejunal explants at the basolateral side. Proprotein convertase subtilisin kexin type 9 (PCSK9)(-/-) mice and intestinal explants show increased LDL-TICE, and acute injection of PCSK9 decreases TICE in vivo, suggesting that PCSK9 is a repressor of TICE. The acute repression was dependent on the LDL receptor (LDLR). Further, TICE was increased when mice were treated with lovastatin. These data point to an important role for LDLR in TICE. However, LDLR(-/-) mice showed increased intestinal LDL uptake, contrary to what is observed in the liver, and tended to have higher TICE. We interpret these data to suggest that there might be at least 2 mechanisms contributing to TICE; 1 involving LDL receptors and other unidentified mechanisms. Acute modulation of LDLR affects TICE, but chronic deficiency is compensated for most likely by the upregulation of the unknown mechanisms. Using mice deficient for apical multidrug active transporter ATP-binding cassette transporter B1 a and b, and its inhibitor, we show that these apical transporters contribute significantly to TICE. CONCLUSIONS: TICE is operative in human jejunal explants. It is a metabolically active process that can be acutely regulated, inversely related to cholesterolemia, and pharmacologically activated by statins.

Intestinal SR-BI does not impact cholesterol absorption or transintestinal cholesterol efflux in mice.

Reverse cholesterol transport (RCT) can proceed through the classic hepatobiliary route or through the nonbiliary transintestinal cholesterol efflux (TICE) pathway. Scavenger receptor class B type I (SR-BI) plays a critical role in the classic hepatobiliary route of RCT. However, the role of SR-BI in TICE has not been studied. To examine the role of intestinal SR-BI in TICE, sterol balance was measured in control mice and mice transgenically overexpressing SR-BI in the proximal small intestine (SR-BI(hApoCIII-ApoAIV-Tg)). SR-BI(hApoCIII-ApoAIV-Tg) mice had significantly lower plasma cholesterol levels compared with wild-type controls, yet SR-BI(hApoCIII-ApoAIV-Tg) mice had normal fractional cholesterol absorption and fecal neutral sterol excretion. Both in the absence or presence of ezetimibe, intestinal SR-BI overexpression had no impact on the amount of cholesterol excreted in the feces. To specifically study effects of intestinal SR-BI on TICE we crossed SR-BI(hApoCIII-ApoAIV-Tg) mice into a mouse model that preferentially utilized the TICE pathway for RCT (Niemann-Pick C1-like 1 liver transgenic), and likewise found no alterations in cholesterol absorption or fecal sterol excretion. Finally, mice lacking SR-BI in all tissues also exhibited normal cholesterol absorption and fecal cholesterol disposal. Collectively, these results suggest that SR-BI is not rate limiting for intestinal cholesterol absorption or for fecal neutral sterol loss through the TICE pathway.

21-Methylpyrenyl-cholesterol stably and specifically associates with lipoprotein peripheral hemi-membrane: a new labelling tool.

Lipoproteins are important biological components. However, they have few convenient fluorescent labelling probes currently reported, and their physiological reliability can be questioned. We compared the association of two fluorescent cholesterol derivatives, 22-nitrobenzoxadiazole-cholesterol (NBD-Chol) and 21-methylpyrenyl-cholesterol (Pyr-met-Chol), to serum lipoproteins and to purified HDL and LDL. Both lipoproteins could be stably labelled by Pyr-met-Chol, but virtually not by NBD-Chol. At variance with NBD-Chol, LCAT did not esterify Pyr-met-Chol. The labelling characteristics of lipoproteins by Pyr-met-Chol were well distinguishable between HDL and LDL, regarding dializability, associated probe amount and labelling kinetics. We took benefit of the pyrene labelling to approach the structural organization of LDL peripheral hemi-membrane, since Pyr-met-Chol-labelled LDL, but not HDL, presented a fluorescence emission of pyrene excimers, indicating that the probe was present in an ordered lipid micro-environment. Since the peripheral membrane of LDL contains more sphingomyelin (SM) than HDL, this excimer formation was consistent with the existence of cholesterol- and SM-enriched lipid microdomains in LDL, as already suggested in model membranes of similar composition and reminiscent to the well-described "lipid rafts" in bilayer membranes. Finally, we showed that Pyr-met-Chol could stain cultured PC-3 cells via lipoprotein-mediated delivery, with a staining pattern well different to that observed with NBD-Chol non-specifically delivered to the cells.

Respective contributions of intestinal Niemann-Pick C1-like 1 and scavenger receptor class B type I to cholesterol and tocopherol uptake: in vivo v. in vitro studies.

The intestinal absorption of cholesterol and lipid micronutrients such as vitamin E has been shown to share some common pathways. The present study aims to further compare the uptake of cholesterol ([3H]cholesterol v. 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3-ol (NBD-cholesterol)) and tocopherol in Caco-2 TC-7 cells and in mouse intestine, with special focus on the respective roles of scavenger receptor class B type I (SR-BI) and Niemann-Pick C1-like 1 (NPC1L1). Conversely to NBD-cholesterol, the uptakes of [3H]cholesterol and tocopherol by Caco-2 cells were impaired by both block lipid transport-1 and ezetimibe, which inhibit SR-BI and NPC1L1, respectively. These inhibitions occurred only when cholesterol or tocopherol was delivered to cells included in micelles that contained biliary acid and at least oleic acid as a lipid. In vivo, after 2 h of digestion in mice, the uptake of the two cholesterol analogues and of tocopherol all showed distinct patterns along the duodenum-jejunum axis. [3H]Cholesterol uptake, which correlated closely to NPC1L1 mRNA expression in wild-type (wt) mice, was strongly inhibited by ezetimibe. Intestinal SR-BI overexpression did not change NPC1L1 expression and led to a significant increase in [3H]cholesterol uptake in the distal jejunum. Conversely, neither ezetimibe treatment nor SR-BI overexpression had an effect on NBD-cholesterol uptake. However, in contrast with SR-BI mRNA expression, tocopherol absorption increased strongly up to the distal jejunum in wt mice where it was specifically inhibited by ezetimibe, and was increased in the proximal intestine of intestinal SR-BI-overexpressing mice. Thus, cholesterol and tocopherol uptakes share common pathways in cell culture models, but display different in vivo absorption patterns associated with distinct contributions of SR-BI and NPC1L1.

F1-adenosine triphosphatase displays properties characteristic of an antigen presentation molecule for Vgamma9Vdelta2 T cells.

Human Vgamma9Vdelta2 T lymphocytes are activated by phosphoantigens provided exogenously or produced by tumors and infected cells. Activation requires a contact between Vgamma9Vdelta2 cells and neighboring cells. We previously reported a role for cell surface F1-adenosine triphosphatase (ATPase) in T cell activation by tumors and specific interactions between Vgamma9Vdelta2 TCRs and purified F1-ATPase. 721.221 cells do not express surface F1-ATPase and do not support phosphoantigen responses unless they are rendered apoptotic by high doses of zoledronate, a treatment that promotes F1-expression as well as endogenous phosphoantigen production. By monitoring calcium flux in single cells, we show in this study that contact of T cells with F1-ATPase on polystyrene beads can partially replace the cell-cell contact stimulus during phosphoantigen responses. Triphosphoric acid 1-adenosin-5'-yl ester 3-(3-methylbut-3-enyl) ester, an adenylated derivative of isopentenyl pyrophosphate, can stably bind to F1-ATPase-coated beads and promotes TCR aggregation, lymphokine secretion, and activation of the cytolytic process provided that nucleotide pyrophosphatase activity is present. It also acts as an allosteric activator of F1-ATPase. In the absence of Vgamma9Vdelta2 cells, triphosphoric acid 1-adenosin-5'-yl ester 3-(3-methylbut-3-enyl) ester immobilized on F1-ATPase is protected from nucleotide pyrophosphatase activity, as is the antigenic activity of stimulatory target cells. Our experiments support the notion that Vgamma9Vdelta2 T cells are dedicated to the recognition of phosphoantigens on cell membranes in the form of nucleotide derivatives that can bind to F1-ATPase acting as a presentation molecule.

A specific tongue microbiota signature is found in patients displaying an improvement of orosensory lipid perception after a sleeve gastrectomy.

Introduction: A preferential consumption of low-fat foods is reported by most of the patients after a vertical sleeve gastrectomy (VSG). The fact that a recent study shed light on a relationship between oral microbiota and fat taste sensitivity in obese patients prompted us to explore whether such a connection also exists in the context of a VSG. Methods: Thirty-two adult female patients with a severe obesity (BMI = 43.1 ± 0.7 kg/m2) and candidates for a VSG were selected. Oral microbiota composition surrounding the gustatory circumvallate papillae (CVP) and the lipid perception thresholds were explored before and 6 months after surgery. Results: VSG was found to be associated both with a qualitative (compositional changes) and quantitative (lower gene richness) remodeling of the peri-CVP microbiota. Analysis of the lipid perception allowed us to distinguish two subgroups: patients with a post-operative improvement of the fat taste sensitivity (i.e., with a lower threshold, n = 14) and unimproved patients (n = 18). Specific peri-CVP microbiota signatures also discriminated these two subgroups, unimproved patient being characterized by higher levels of Porphyromonas, Fusobacterium, and Haemophilus genera associated with lower levels of Atopobium and Prevotella genera as compared to the lipid-improved patients. Conclusion: Collectively, these data raise the possibility that the microbial environment surrounding gustatory papillae might play a role in the positive changes of fat taste sensitivity observed in some patients after VSG.

A severe form of abetalipoproteinemia caused by new splicing mutations of microsomal triglyceride transfer protein (MTTP).

Abetalipoproteinemia is a rare autosomal recessive disease characterized by low lipid levels and by the absence of apoB-containing lipoproteins. It is the consequence of microsomal triglyceride transfer protein (MTTP) deficiency. We report two patients with new MTTP mutations. We studied their functional consequences on the triglyceride transfer function using duodenal biopsies. We transfected MTTP mutants in HepG2 and HeLa cells to investigate their association with protein disulfide isomerase (PDI) and their localization at the endoplasmic reticulum. These children have a severe abetalipoproteinemia. Both of them had also a mild hypogammaglobulinemia. They are compound heterozygotes with c.619G>T and c.1237-28A>G mutations within the MTTP gene. mRNA analysis revealed abnormal splicing with deletion of exon 6 and 10, respectively. Deletion of exon 6 (Δ6-MTTP) introduced a frame shift in the reading frame and a premature stop codon at position 234. Despite the fact that Δ6-MTTP and Δ10-MTTP mutants were not capable of binding PDI, both MTTP mutant proteins normally localize at the endoplasmic reticulum. However, these two mutations induce a loss of MTTP triglyceride transfer activity. These two mutations lead to abnormal truncated MTTP proteins, incapable of binding PDI and responsible for the loss of function of MTTP, thereby explaining the severe abetalipoproteinemia phenotype of these children.

P2Y13 receptor is critical for reverse cholesterol transport.

UNLABELLED: A major atheroprotective functionality of high-density lipoproteins (HDLs) is to promote "reverse cholesterol transport" (RCT). In this process, HDLs mediate the efflux and transport of cholesterol from peripheral cells and its subsequent transport to the liver for further metabolism and biliary excretion. We have previously demonstrated in cultured hepatocytes that P2Y(13) (purinergic receptor P2Y, G protein-coupled, 13) activation is essential for HDL uptake but the potential of P2Y(13) as a target to promote RCT has not been documented. Here, we show that P2Y(13)-deficient mice exhibited a decrease in hepatic HDL cholesterol uptake, hepatic cholesterol content, and biliary cholesterol output, although their plasma HDL and other lipid levels were normal. These changes translated into a substantial decrease in the rate of macrophage-to-feces RCT. Therefore, hallmark features of RCT are impaired in P2Y(13)-deficient mice. Furthermore, cangrelor, a partial agonist of P2Y(13), stimulated hepatic HDL uptake and biliary lipid secretions in normal mice and in mice with a targeted deletion of scavenger receptor class B type I (SR-BI) in liver (hypomSR-BI-knockout(liver)) but had no effect in P2Y(13) knockout mice, which indicate that P2Y(13)-mediated HDL uptake pathway is independent of SR-BI-mediated HDL selective cholesteryl ester uptake. CONCLUSION: These results establish P2Y(13) as an attractive novel target for modulating RCT and support the emerging view that steady-state plasma HDL levels do not necessarily reflect the capacity of HDL to promote RCT.

Specific requirements for Vgamma9Vdelta2 T cell stimulation by a natural adenylated phosphoantigen.

Human Vgamma9Vdelta2 T lymphocytes recognize phosphorylated alkyl Ags. Isopentenyl pyrophosphate (IPP) was previously proposed as the main Ag responsible for Vgamma9Vdelta2 T cell activation by cancer cells. However, triphosphoric acid 1-adenosin-5'-yl ester 3-(3-methylbut-3-enyl) ester (ApppI), a metabolite in which the isopentenyl moiety is linked to ATP, was reported in cells activated with aminobisphosphonates. The contribution of this compound to tumor-stimulatory activity was thus examined. ApppI induces selective expansion of Vgamma9Vdelta2 T cells from PBMCs. In the absence of APCs, however, ApppI has little stimulatory activity on Vgamma9Vdelta2 T cells, and optimal activation with ApppI requires addition of a nucleotide pyrophosphatase releasing IPP plus AMP. Thus, ApppI has no intrinsic stimulatory activity. Nevertheless, stimulation by ApppI is strengthened by the presence of APCs. Moreover, in contrast to IPP, ApppI can be efficiently pulsed on dendritic cells as well as on nonprofessional APCs. Pulsed APCs display stable and phosphatase-resistant stimulatory activity, indicative of Ag modification. HPLC analysis of tumor cell extracts indicates that latent phosphoantigenic activity is stored intracellularly in the Vgamma9Vdelta2 cell-sensitive tumor Daudi and can be activated by a nucleotide pyrophosphatase activity. The presence of ApppI in Daudi cell extracts was demonstrated by mass spectrometry. Nucleotidic Ags such as ApppI are thus a storage form of phosphoantigen which may represent a major source of phosphoantigenic activity in tumor cells. The unique properties of ApppI may be important for the design of Ags used in anticancer immunotherapeutic protocols using Vgamma9Vdelta2 cells.

Orosensory Perception of Fat/Sweet Stimuli and Appetite-Regulating Peptides before and after Sleeve Gastrectomy or Gastric Bypass in Adult Women with Obesity.

The aim of this study was to explore the impact of bariatric surgery on fat and sweet taste perceptions and to determine the possible correlations with gut appetite-regulating peptides and subjective food sensations. Women suffering from severe obesity (BMI > 35 kg/m2) were studied 2 weeks before and 6 months after a vertical sleeve gastrectomy (VSG, n = 32) or a Roux-en-Y gastric bypass (RYGB, n = 12). Linoleic acid (LA) and sucrose perception thresholds were determined using the three-alternative forced-choice procedure, gut hormones were assayed before and after a test meal and subjective changes in oral food sensations were self-reported using a standardized questionnaire. Despite a global positive effect of both surgeries on the reported gustatory sensations, a change in the taste sensitivity was only found after RYGB for LA. However, the fat and sweet taste perceptions were not homogenous between patients who underwent the same surgery procedure, suggesting the existence of two subgroups: patients with and without taste improvement. These gustatory changes were not correlated to the surgery-mediated modifications of the main gut appetite-regulating hormones. Collectively these data highlight the complexity of relationships between bariatric surgery and taste sensitivity and suggest that VSG and RYGB might impact the fatty taste perception differently.

Obesity of mice lacking VAP-1/SSAO by Aoc3 gene deletion is reproduced in mice expressing a mutated vascular adhesion protein-1 (VAP-1) devoid of amine oxidase activity.

The product of Aoc3 gene is known as vascular adhesion protein-1 (VAP-1), a glycoprotein contributing to leukocyte extravasation and exhibiting semicarbazide-sensitive amine oxidase activity (SSAO). Regarding the immune functions of VAP-1/SSAO, it is known that mice bearing Aoc3 gene knock-out (AOC3KO) exhibit defects in leukocyte migration similar to those of mice expressing a mutated VAP-1 lacking functional SSAO activity (knock-in, AOC3KI). However, it has not been reported whether these models differ regarding other disturbances. Thus, we further compared endocrine-metabolic phenotypes of AOC3KO and AOC3KI mice to their respective control. Special attention was paid on adiposity, glucose and lipid handling, since VAP-1/SSAO is highly expressed in adipose tissue (AT). In both mouse lines, no tissue SSAO activity was found, while Aoc3 mRNA was absent in AOC3KO only. Although food consumption was unchanged, both AOC3KO and AOC3KI mice were heavier and fatter than their respective controls. Other alterations commonly found in adipocytes from both lines were loss of benzylamine insulin-like action with unchanged insulin lipogenic responsiveness and adiponectin expression. A similar downregulation of inflammatory markers (CD45, IL6) was found in AT. Glucose handling and liver mass remained unchanged, while circulating lipid profile was distinctly altered, with increased cholesterol in AOC3KO only. These results suggest that the lack of oxidase activity found in AOC3KI is sufficient to reproduce the metabolic disturbances observed in AOC3KO mice, save those related with cholesterol transport. Modulation of SSAO activity therefore constitutes a potential target for the treatment of cardiometabolic diseases, especially obesity when complicated by low-grade inflammation.

Lipoprotein(a): Pathophysiology, measurement, indication and treatment in cardiovascular disease. A consensus statement from the Nouvelle Société Francophone d'Athérosclérose (NSFA).

Lipoprotein(a) is an apolipoprotein B100-containing low-density lipoprotein-like particle that is rich in cholesterol, and is associated with a second major protein, apolipoprotein(a). Apolipoprotein(a) possesses structural similarity to plasminogen but lacks fibrinolytic activity. As a consequence of its composite structure, lipoprotein(a) may: (1) elicit a prothrombotic/antifibrinolytic action favouring clot stability; and (2) enhance atherosclerosis progression via its propensity for retention in the arterial intima, with deposition of its cholesterol load at sites of plaque formation. Equally, lipoprotein(a) may induce inflammation and calcification in the aortic leaflet valve interstitium, leading to calcific aortic valve stenosis. Experimental, epidemiological and genetic evidence support the contention that elevated concentrations of lipoprotein(a) are causally related to atherothrombotic risk and equally to calcific aortic valve stenosis. The plasma concentration of lipoprotein(a) is principally determined by genetic factors, is not influenced by dietary habits, remains essentially constant over the lifetime of a given individual and is the most powerful variable for prediction of lipoprotein(a)-associated cardiovascular risk. However, major interindividual variations (up to 1000-fold) are characteristic of lipoprotein(a) concentrations. In this context, lipoprotein(a) assays, although currently insufficiently standardized, are of considerable interest, not only in stratifying cardiovascular risk, but equally in the clinical follow-up of patients treated with novel lipid-lowering therapies targeted at lipoprotein(a) (e.g. antiapolipoprotein(a) antisense oligonucleotides and small interfering ribonucleic acids) that markedly reduce circulating lipoprotein(a) concentrations. We recommend that lipoprotein(a) be measured once in subjects at high cardiovascular risk with premature coronary heart disease, in familial hypercholesterolaemia, in those with a family history of coronary heart disease and in those with recurrent coronary heart disease despite lipid-lowering treatment. Because of its clinical relevance, the cost of lipoprotein(a) testing should be covered by social security and health authorities.

Effects of human follicular fluid and high-density lipoproteins on early spermatozoa hyperactivation and cholesterol efflux.

The preovulatory human follicular fluid contains only HDLs as a lipoprotein class with a typically high proportion of prebeta HDL. We first examined the role of follicular fluid and HDL subfractions on human spermatozoa capacitation, a process characterized by a hyperactivation of the flagellar movement and a depletion of plasma membrane cholesterol. Whole follicular fluid and isolated HDL, used at constant free cholesterol concentration, were both able to promote an early flagellar hyperactivation. Moreover, incubation of [(3)H]cholesterol-labeled spermatozoa with follicular fluid induced a rapid cholesterol efflux from spermatozoa that was confirmed by mass measurements of cholesterol transfer. Using isolated HDL, the cholesterol efflux had a similar time course and represented 70% of that mediated by whole follicular fluid. We then analyzed the time course of radioactive labeling of HDL subfractions. In the first minute of incubation, we found that the prebeta HDL fraction incorporated the main part of the radioactivity (60%), with the rest being found in alpha-HDL, but strikingly, the labeling of alpha-HDL increased with time at the expense of prebeta HDL.Thus, our results indicate that HDLs are involved in both spermatozoa hyperactivation and cholesterol effl ux and suggest the role of prebeta-HDL particles as fi rst cellular cholesterol acceptors.

Role of P-glycoprotein in the disposition of macrocyclic lactones: A comparison between ivermectin, eprinomectin, and moxidectin in mice.

Macrocyclic lactones (MLs) are lipophilic anthelmintics and substrates for P-glycoprotein (P-gp), an ATP-binding cassette transporter involved in drug efflux out of both host and parasites. To evaluate the contribution of P-gp to the in vivo kinetic disposition of MLs, the plasma kinetics, brain concentration, and intestinal excretion of three structurally different MLs (ivermectin, eprinomectin, and moxidectin) were compared in wild-type and P-gp-deficient [mdr1ab(-/-)] mice. Each drug (0.2 mg/kg) was administered orally, intravenously, or subcutaneously to the mice. Plasma, brain, and intestinal tissue concentrations were measured by high-performance liquid chromatography. The intestinal excretion rate after intravenous administration was determined at different levels of the small intestine by using an in situ intestinal perfusion model. P-gp deficiency led to a significant increase in the area under the plasma concentration-time curve (AUC) of ivermectin (1.5-fold) and eprinomectin (3.3-fold), whereas the moxidectin AUC was unchanged. Ivermectin and to a greater extent eprinomectin were both excreted by the intestine via a P-gp-dependent pathway, whereas moxidectin excretion was weaker and mostly P-gp-independent. The three drugs accumulated in the brains of the mdr1ab(-/-) mice, but eprinomectin concentrations were significantly lower. We concluded that eprinomectin disposition in mice is controlled mainly by P-gp efflux, more so than that of ivermectin, whereas moxidectin disposition appears to be mostly P-gp-independent. Given that eprinomectin and ivermectin have higher affinity for P-gp than moxidectin, these findings demonstrated that the relative affinity of MLs for P-gp could be predictive of the in vivo kinetic behavior of these drugs.

Proprotein convertase subtilisin kexin type 9 null mice are protected from postprandial triglyceridemia.

OBJECTIVES: Proprotein convertase subtilisin kexin type 9 (PCSK9) is a natural inhibitor of the low-density lipoprotein receptor, and its deficiency in humans results in low plasma LDL-cholesterol and protection against cardiovascular disease. We explored whether PCSK9 expression impacts postprandial triglyceridemia, another important cardiovascular risk factor. METHODS AND RESULTS: Real-time PCR and confocal microscopy were used to show that PCSK9 is expressed throughout the entire small intestine and in human enterocytes. On olive oil gavage, PCSK9-deficient mice showed a dramatically decreased postprandial triglyceridemia compared with their wild-type littermates. Lymph analysis revealed that intestinal TG output is not quantitatively modified by PCSK9 deletion. However, PCSK9-/- mice present with a significant reduction of lymphatic apoB secretion compared to PCSK9+/+ mice. Modulating PCSK9 expression in polarized CaCo-2 cells confirmed the relationship between PCSK9 and apoB secretion; PCSK9-/- mice consistently secrete larger TG-rich lipoprotein than wild-type littermates. Finally, kinetic studies showed that PCSK9-deficient mice have an increased ability to clear chylomicrons compared to wild-type littermates. CONCLUSION: These findings indicate that in addition to its effect on LDL-cholesterol, PCSK9 deficiency might protect against cardiovascular disease by reducing postprandial triglyceridemia.

Stimulation of cell surface F1-ATPase activity by apolipoprotein A-I inhibits endothelial cell apoptosis and promotes proliferation.

OBJECTIVE: Several findings argue for a protective effect of high-density lipoproteins (HDLs) against endothelial dysfunction. The molecular mechanisms underlying this protective effect are not fully understood, although recent works suggest that the actions of HDL on the endothelium are initiated by multiple interactions between HDLs (lipid or protein moiety) and cell surface receptors. We previously showed that the mitochondrial related F(1)-ATPase is a cell surface receptor for HDLs and their main atheroprotective apolipoprotein (apoA-I). Herein we test the hypothesis that the cell surface F(1)-ATPase may contribute to the ability of apoA-I and HDLs to maintain endothelial cell survival. METHODS AND RESULTS: Cell imaging and binding assays confirmed the presence of the F(1)-ATPase at the surface of human umbilical vein endothelial cells (HUVECs) and its ability to bind apoA-I. Cell surface F(1)-ATPase activity (ATP hydrolysis into ADP) was stimulated by apoA-I and was inhibited by its specific inhibitor IF(1)-H49K. Furthermore the antiapoptotic and proliferative effects of apoA-I on HUVECs were totally blocked by the F(1)-ATPase ligands IF(1)-H49K, angiostatin and anti-betaF(1)-ATPase antibody, independently of the scavenger receptor SR-BI and ABCA1. CONCLUSIONS: This study suggests an important contribution of cell surface F(1)-ATPase to apoA-I-mediated endothelial cell survival, which may contribute to the atheroprotective functions of apoA-I.