ADCY9 (Adenylate Cyclase Type 9) Inactivation Protects From Atherosclerosis Only in the Absence of CETP (Cholesteryl Ester Transfer Protein).

BACKGROUND: Pharmacogenomic studies have shown that ADCY9 genotype determines the effects of the CETP (cholesteryl ester transfer protein) inhibitor dalcetrapib on cardiovascular events and atherosclerosis imaging. The underlying mechanisms responsible for the interactions between ADCY9 and CETP activity have not yet been determined. METHODS: Adcy9-inactivated ( Adcy9Gt/Gt) and wild-type (WT) mice, that were or not transgenic for the CETP gene (CETPtg Adcy9Gt/Gt and CETPtg Adcy9WT), were submitted to an atherogenic protocol (injection of an AAV8 [adeno-associated virus serotype 8] expressing a PCSK9 [proprotein convertase subtilisin/kexin type 9] gain-of-function variant and 0.75% cholesterol diet for 16 weeks). Atherosclerosis, vasorelaxation, telemetry, and adipose tissue magnetic resonance imaging were evaluated. RESULTS: Adcy9Gt/Gt mice had a 65% reduction in aortic atherosclerosis compared to WT ( P<0.01). CD68 (cluster of differentiation 68)-positive macrophage accumulation and proliferation in plaques were reduced in Adcy9Gt/Gt mice compared to WT animals ( P<0.05 for both). Femoral artery endothelial-dependent vasorelaxation was improved in Adcy9Gt/Gt mice (versus WT, P<0.01). Selective pharmacological blockade showed that the nitric oxide, cyclooxygenase, and endothelial-dependent hyperpolarization pathways were all responsible for the improvement of vasodilatation in Adcy9Gt/Gt ( P<0.01 for all). Aortic endothelium from Adcy9Gt/Gt mice allowed significantly less adhesion of splenocytes compared to WT ( P<0.05). Adcy9Gt/Gt mice gained more weight than WT with the atherogenic diet; this was associated with an increase in whole body adipose tissue volume ( P<0.01 for both). Feed efficiency was increased in Adcy9Gt/Gt compared to WT mice ( P<0.01), which was accompanied by prolonged cardiac RR interval ( P<0.05) and improved nocturnal heart rate variability ( P=0.0572). Adcy9 inactivation-induced effects on atherosclerosis, endothelial function, weight gain, adipose tissue volume, and feed efficiency were lost in CETPtg Adcy9Gt/Gt mice ( P>0.05 versus CETPtg Adcy9WT). CONCLUSIONS: Adcy9 inactivation protects against atherosclerosis, but only in the absence of CETP activity. This atheroprotection may be explained by decreased macrophage accumulation and proliferation in the arterial wall, and improved endothelial function and autonomic tone.

Dalcetrapib and anacetrapib differently impact HDL structure and function in rabbits and monkeys.

Inhibition of cholesteryl ester transfer protein (CETP) increases HDL cholesterol (HDL-C) levels. However, the circulating CETP level varies and the impact of its inhibition in species with high CETP levels on HDL structure and function remains poorly characterized. This study investigated the effects of dalcetrapib and anacetrapib, the two CETP inhibitors (CETPis) currently being tested in large clinical outcome trials, on HDL particle subclass distribution and cholesterol efflux capacity of serum in rabbits and monkeys. New Zealand White rabbits and vervet monkeys received dalcetrapib and anacetrapib. In rabbits, CETPis increased HDL-C, raised small and large α-migrating HDL, and increased ABCA1-induced cholesterol efflux. In vervet monkeys, although anacetrapib produced similar results, dalcetrapib caused opposite effects because the LDL-C level was increased by 42% and HDL-C decreased by 48% (P < 0.01). The levels of α- and preß-HDL were reduced by 16% (P < 0.001) and 69% (P < 0.01), resulting in a decrease of the serum cholesterol efflux capacity. CETPis modulate the plasma levels of mature and small HDL in vivo and consequently the cholesterol efflux capacity. The opposite effects of dalcetrapib in different species indicate that its impact on HDL metabolism could vary greatly according to the metabolic environment.

Development of a new bioactivatable fluorescent probe for quantification of apolipoprotein A-I proteolytic degradation in vitro and in vivo.

BACKGROUND AND AIMS: The potential benefits of high-density lipoproteins (HDL) against atherosclerosis are attributed to its major protein component, apolipoprotein A-I (apoA-I). Most of the apoA-I in the vascular wall appears to be in its lipid-poor form. The latter, however, is subjected to degradation by proteases localized in atherosclerotic plaques, which, in turn, has been shown to negatively impact its atheroprotective functions. Here, we report the development and in vivo use of a bioactivatable near-infrared full-length apoA-I-Cy5.5 fluorescent probe for the assessment of apoA-I-degrading proteolytic activities. METHODS: Fluorescence quenching was obtained by saturation of Cy5.5 fluorophore molecules on apoA-I protein. ApoA-I cleavage led to near-infrared fluorescence enhancement. In vitro proteolysis of the apoA-I probe by a variety of proteases including serine, cysteine, and metalloproteases resulted in an up to 11-fold increase in fluorescence (n = 5, p ≤ 0.05). RESULTS: We detected activation of the probe in atherosclerotic mice aorta sections using in situ zymography and showed that broad-spectrum protease inhibitors protected the probe from degradation, resulting in decreased fluorescence (-54%, n = 6 per group, p ≤ 0.0001). In vivo, the injected probe showed stronger fluorescence emission in the aorta of human apoB transgenic Ldlr-/- atherosclerotic mice (ATX) as compared to wild-type mice. In vivo observations were confirmed by ex vivo aorta imaging quantification where a 10-fold increase in fluorescent signal in ATX mice (p ≤ 0.05 vs. control mice) was observed. CONCLUSIONS: The use of this probe in different applications may help to assess new molecular mechanisms of atherosclerosis and may improve current HDL-based therapies by enhancing apoA-I functionality.

Genotype-Dependent Effects of Dalcetrapib on Cholesterol Efflux and Inflammation: Concordance With Clinical Outcomes.

BACKGROUND: Dalcetrapib effects on cardiovascular outcomes are determined by adenylate cyclase 9 gene polymorphisms. Our aim was to determine whether these clinical end point results are also associated with changes in reverse cholesterol transport and inflammation. METHODS AND RESULTS: Participants of the dal-OUTCOMES and dal-PLAQUE-2 trials were randomly assigned to receive dalcetrapib or placebo in addition to standard care. High-sensitivity C-reactive protein was measured at baseline and at end of study in 5243 patients from dal-OUTCOMES also genotyped for the rs1967309 polymorphism in adenylate cyclase 9. Cholesterol efflux capacity of high-density lipoproteins from J774 macrophages after cAMP stimulation was determined at baseline and 12 months in 171 genotyped patients from dal-PLAQUE-2. Treatment with dalcetrapib resulted in placebo-adjusted geometric mean percent increases in high-sensitivity C-reactive protein from baseline to end of trial of 18.1% (P=0.0009) and 18.7% (P=0.00001) in participants with the GG and AG genotypes, respectively, but the change was -1.0% (P=0.89) in those with the protective AA genotype. There was an interaction between the treatment arm and the genotype groups (P=0.02). Although the mean change in cholesterol efflux was similar among study arms in patients with GG genotype (mean: 7.8% and 7.4%), increases were 22.3% and 3.5% with dalcetrapib and placebo for those with AA genotype (P=0.005). There was a significant genetic effect for change in efflux for dalcetrapib (P=0.02), but not with placebo. CONCLUSIONS: Genotype-dependent effects on C-reactive protein and cholesterol efflux are supportive of dalcetrapib benefits on atherosclerotic cardiovascular outcomes in patients with the AA genotype at polymorphism rs1967309. CLINICAL TRIALS REGISTRATION: ClinicalTrials.gov; Unique Identifiers: NCT00658515 and NCT01059682.

HDL mimetic peptide CER-522 treatment regresses left ventricular diastolic dysfunction in cholesterol-fed rabbits.

OBJECTIVES: High-density lipoprotein (HDL) infusions induce rapid improvement of experimental atherosclerosis in rabbits but their effect on ventricular function remains unknown. We aimed to evaluate the effects of the HDL mimetic peptide CER-522 on left ventricular diastolic dysfunction (LVDD). METHODS: Rabbits were fed with a cholesterol- and vitamin D2-enriched diet until mild aortic valve stenosis and hypercholesterolemia-induced LV hypertrophy and LVDD developed. Animals then received saline or 10 or 30mg/kg CER-522 infusions 6 times over 2weeks. We performed serial echocardiograms and LV histology to evaluate the effects of CER-522 therapy on LVDD. RESULTS: LVDD was reduced by CER-522 as shown by multiple parameters including early filling mitral deceleration time, deceleration rate, Em/Am ratio, E/Em ratio, pulmonary venous velocities, and LVDD score. These findings were associated with reduced macrophages (RAM-11 positive cells) in the pericoronary area and LV, and decreased levels of apoptotic cardiomyocytes in CER-522-treated rabbits. CER-522 treatment also resulted in decreased atheromatous plaques and internal elastic lamina area in coronary arteries. CONCLUSIONS: CER-522 improves LVDD in rabbits, with reductions of LV macrophage accumulation, cardiomyocyte apoptosis, coronary atherosclerosis and remodelling.

Do reward-processing deficits in schizophrenia-spectrum disorders promote cannabis use? An investigation of physiological response to natural rewards and drug cues.

BACKGROUND: Dysfunctional reward processing is present in individuals with schizophrenia-spectrum disorders (SSD) and may confer vulnerability to addiction. Our objective was to identify a deficit in patients with SSD on response to rewarding stimuli and determine whether this deficit predicts cannabis use. METHODS: We divided a group of patients with SSD and nonpsychotic controls into cannabis users and nonusers. Response to emotional and cannabis-associated visual stimuli was assessed using self-report, event-related potentials (using the late positive potential [LPP]), facial electromyography and skin-conductance response. RESULTS: Our sample comprised 35 patients with SSD and 35 nonpsychotic controls. Compared with controls, the patients with SSD showed blunted LPP response to pleasant stimuli (p = 0.003). Across measures, cannabis-using controls showed greater response to pleasant stimuli than to cannabis stimuli whereas cannabis-using patients showed little bias toward pleasant stimuli. Reduced LPP response to pleasant stimuli was predictive of more frequent subsequent cannabis use (ß = -0.24, p = 0.034). LIMITATIONS: It is not clear if the deficit associated with cannabis use is specific to rewarding stimuli or nonspecific to any kind of emotionally salient stimuli. CONCLUSION: The LPP captures a reward-processing deficit in patients with SSD and shows potential as a biomarker for identifying patients at risk of heavy cannabis use.

Evaluation of links between high-density lipoprotein genetics, functionality, and aortic valve stenosis risk in humans.

OBJECTIVE: Studies have shown that high-density lipoprotein (HDL)-raising compounds induce regression of aortic valve stenosis (AVS) in animal models. However, whether patients with AVS have an impaired HDL metabolism is unknown. APPROACH AND RESULTS: A total of 1435 single nucleotide polymorphisms in genes associated with HDL cholesterol levels (in or around GALNT2, LPL, ABCA1, APOA5, SCARB1, LIPC, CETP, LCAT, LIPG, APOC4, and PLTP) were genotyped in 382 patients with echocardiography-confirmed AVS (aortic jet velocity ≥2.5 m/s) and 401 controls. After control for multiple testing, none of the genetic variants showed a positive association with case/control status (adjusted P≥0.05 for all single nucleotide polymorphisms tested). In a subsample of this cohort, HDL cholesterol levels, apolipoprotein AI levels, lecithin-cholesterol acyltransferase activity, pre-ß-HDL, HDL size, and 4 parameters of cholesterol efflux capacity were measured in apolipoprotein B-depleted serum samples from 86 patients with and 86 patients without AVS. Cholesterol efflux capacity was measured using J774 macrophages with and without stimulation of ATP-binding cassette A-1 expression by cAMP, and HepG2 hepatocytes for scavenger receptor class B type 1-mediated efflux. None of these parameters were different between cases and controls. However, compared with patients without coronary artery disease, sera from patients with coronary artery disease had lower HDL cholesterol levels, scavenger receptor class B type 1-mediated efflux, and HDL size (P≤0.003), independently of the presence or absence of AVS. CONCLUSIONS: Results of the present study suggest that, based on HDL genetics and HDL functionality, HDL metabolism does not seem to predict the risk of AVS. Because of our limited sample size, additional studies are needed to confirm these findings.

Palmitic acid increases medial calcification by inducing oxidative stress.

BACKGROUND: Aortic medial calcification is a cellular-regulated process leading to arterial stiffness. Although epidemiological studies have suggested an association between the saturation of fatty acids (FA) and arterial stiffness, there is no evidence that saturated FA can induce arterial calcification. This study investigated the capacity of palmitic acid (PA) to induce medial calcification and the signaling pathway(s) implicated in this process. METHODS: Rat aortic segments and vascular smooth muscle cells (VSMC) were exposed to calcification medium supplemented with PA. In vivo, rats were treated with warfarin to induce calcification and fed a PA-enriched diet. RESULTS: In vitro and ex vivo, palmitate increases calcification and ROS production. Palmitate increases extracellular-signal-regulated kinase (ERK1/2) phosphorylation and osteogenic gene expression. Inhibition of NADPH oxidase with apocynin or an siRNA prevents these effects. ERK1/2 inhibition attenuates the amplification of osteogenic gene expression and calcification induced by palmitate. In vivo, a PA-enriched diet amplified medial calcification and pulse wave velocity (PWV). These effects are mediated by ROS production as indicated by the inhibition of calcification and PWV normalization in rats concomitantly treated with apocynin. CONCLUSION: ROS induction by palmitate leads to ERK1/2 phosphorylation and subsequently induces the osteogenic differentiation of VSMC. © 2013 S. Karger AG, Basel.

Assessing the way people look to judge their intentions.

Faces of unknown persons are processed to infer the intentions of these persons not only when they depict full-blown emotions, but also at rest, or when these faces do not signal any strong feelings. We explored the brain processes involved in these inferences to test whether they are similar to those found when judging full-blown emotions. We recorded the event-related brain potentials (ERPs) elicited by faces of unknown persons who, when they were photographed, were not asked to adopt any particular expression. During the ERP recording, participants had to decide whether each face appeared to be that of a positively, negatively, ambiguously, or neutrally intentioned person. The early posterior negativity, the EPN, was found smaller for neutrally categorized faces than for the other faces, suggesting that the automatic processes it indexes are similar to those evoked by full-blown expressions and thus that these processes might be involved in the decoding of intentions. In contrast, in the same 200-400 ms time window, ERPs were not more negative at anterior sites for neutrally intentioned faces. Second, the peaks of the late positive potentials (LPPs) maximal at parietal sites around 700 ms postonset were not significantly smaller for neutrally intentioned faces. Third, the slow positive waves that followed the LPP were larger for faces that took more time to categorize, that is, for ambiguously intentioned faces. These three series of unexpected results may indicate processes similar to those triggered by full-blown emotions studies, but they question the characteristics of these processes.

SR-BI, CD36, and caveolin-1 contribute positively to cholesterol efflux in hepatic cells.

In non-hepatic cells, scavenger receptor class B type I (SR-BI), cluster of differentiation 36 (CD36), and caveolin-1 were described as mediators of cholesterol efflux, the first step of reverse cholesterol transport (RCT). Stable transformants of HepG2 cells overexpressing SR-BI, CD36, or caveolin-1 were generated, as well as cells overexpressing both caveolin-1 and SR-BI or caveolin-1 and CD36 in order to address the effect of caveolin-1 on both receptor activities. These cells were analyzed for their ability to efflux cholesterol to HDL(3). Our results show that overexpressing SR-BI, CD36, or caveolin-1 increases cholesterol efflux by 106, 92, and 48%, respectively. Moreover, the dual overexpressions of caveolin-1 and SR-BI or caveolin-1 and CD36 lead to a more prominent increase in cholesterol efflux. Studies were also conducted with primary cultures of SR-BI knockout (KO), CD36 KO, and SR-BI/CD36 double-KO (dKO) mice. SR-BI KO and SR-BI/CD36 dKO hepatic cells show 41 and 56% less cholesterol efflux, respectively, than normal hepatic cells. No significant difference was observed between the efflux of normal and CD36 KO cells. The difference between the role of human and murine CD36 correlated with the absence of CD36 dimers in mouse caveolae/rafts. Overall, our results show that SR-BI is clearly involved in cholesterol efflux in mouse and human hepatic cells, while CD36 plays a significant role in human cells.

Expression of caveolin-1 in hepatic cells increases oxidized LDL uptake and preserves the expression of lipoprotein receptors.

Oxidized LDL (OxLDL) that are positively associated with the risk of developing cardiovascular diseases are ligands of scavenger receptor-class B type I (SR-BI) and cluster of differentiation-36 (CD36) which can be found in caveolae. The contribution of these receptors in human hepatic cell is however unknown. The HepG2 cell, a human hepatic parenchymal cell model, expresses these receptors and is characterized by a very low level of caveolin-1. Our aim was to define the contribution of human CD36, SR-BI, and caveolin-1 in the metabolism of OxLDL in HepG2 cells and conversely the effects of OxLDL on the levels/localization of these receptors. By comparing mildly (M)- and heavily (H)-OxLDL metabolism between control HepG2 cells and HepG2 cells overexpressing CD36, SR-BI, or caveolin-1, we found that (1) CD36 increases M- and H-OxLDL-protein uptake; (2) SR-BI drives M-OxLDL through a degradation pathway at the expense of the cholesterol ester (CE) selective uptake pathway; (3) caveolin-1 increases M- and H-OxLDL-protein uptake and decreases CE selective uptake from M-OxLDL. Also, incubation with M- or H-OxLDL decreases the levels of SR-BI and LDL-receptor in control HepG2 cells which can be overcome by caveolin-1 expression. In addition, OxLDL move CD36 from low to high buoyant density membrane fractions, as well as caveolin-1 in cells overexpressing this protein. Thus, hepatic caveolin-1 expression has significant effects on OxLDL metabolism and on lipoprotein receptor levels.

Scavenger receptor of class B expressed by osteoblastic cells are implicated in the uptake of cholesteryl ester and estradiol from LDL and HDL3.

UNLABELLED: Lipoproteins transport many vitamins and hormones that have been shown to be necessary for bone formation. However, the metabolism of LDL and HDL3 by bone-forming osteoblastic cells remains unknown. Here we report that osteoblastic cells express scavenger receptors of class B that are implicated in the uptake of cholesterol and estradiol from LDL and HDL3. INTRODUCTION: The bone tissue is continuously remodeled, and its integrity requires a balance between osteoclastic bone resorption and osteoblastic bone formation. Recent studies have reported the importance of triglyceride-rich lipoproteins for the delivery of lipophilic vitamins necessary for normal bone metabolism. However, the ability of osteoblastic cells to process low- and high-density lipoproteins (LDL and HDL3) and the receptors involved remain unknown. MATERIALS AND METHODS: Binding, competition, degradation, and selective uptake assays with LDL and HDL3 radiolabeled in their protein and lipid moieties or with [3H]estradiol were conducted on human osteoblasts (MG-63 cell line and primary cultures of human osteoblasts [hOB cells]) and on mouse osteoblasts (MC3T3-E1 cell line and primary cultures of murine osteoblasts [mOB cells]). The expression of scavenger receptors (SRs) by osteoblastic cells was determined by RT-PCR and Western immunoblotting, and cellular localization was assessed by sucrose gradient fractionation. RESULTS: Osteoblastic cells were able to bind, internalize, and degrade HDL3 and LDL and are capable of selectively taking up cholesteryl esters (CEs) from these lipoproteins. Also, we provide evidence that osteoblastic cells express SR-BI, SR-BII, and CD36 (SR-Bs receptors) and that these receptors are localized in membrane lipid rafts or caveolin-rich membranes. The selective uptake of CE from LDL and HDL3 by osteoblastic cells was strongly inhibited by the known SR-B ligand oxidized LDL, indicating that SR-B receptors are responsible for the selective uptake. Finally, estradiol carried by LDL and HDL3 was selectively transferred to the osteoblastic cells also through SR-B receptors. CONCLUSIONS: Overall, our results suggest a novel mechanism for the routing of cholesterol and estradiol to osteoblasts involving the metabolism of LDL and HDL3 by SR-B receptors.

The role of human and mouse hepatic scavenger receptor class B type I (SR-BI) in the selective uptake of low-density lipoprotein-cholesteryl esters.

Low-density lipoprotein (LDL)-cholesteryl ester (CE) selective uptake has been demonstrated in nonhepatic cells overexpressing the scavenger receptor class B type I (SR-BI). The role of hepatic SR-BI toward LDL, the main carrier of plasma CE in humans, remains unclear. The aim of this study was to determine if SR-BI, expressed at its normal level, is implicated in LDL-CE selective uptake in human HepG2 hepatoma cells and mouse hepatic cells, to quantify its contribution and to determine if LDL-CE selective uptake is likely to occur in the presence of human HDL. First, antibody blocking experiments were conducted on normal HepG2 cells. SR-BI/BII antiserum inhibited (125)I-LDL and (125)I-HDL(3) binding (10 microg of protein/mL) by 45% (p < 0.05) and CE selective uptake by more than 85% (p < 0.01) for both ligands. Second, HepG2 cells were stably transfected with a eukaryotic vector expressing a 400-bp human SR-BI antisense cDNA fragment. Clone 17 (C17) has a 70% (p < 0.01) reduction in SR-BI expression. In this clone, (3)H-CE-LDL and (3)H-CE-HDL(3) association (10 microg of protein/mL) was 54 +/- 6% and 45 +/- 7% of control values, respectively, while (125)I-LDL and (125)I-HDL(3) protein association was 71 +/- 3% and 58 +/- 5% of controls, resulting in 46% and 55% (p < 0.01) decreases in LDL- and HDL(3)-CE selective uptake. Normalizing CE selective uptake for SR-BI expression reveals that SR-BI is responsible for 68% and 74% of LDL- and HDL(3)-CE selective uptake, respectively. Thus, both approaches show that, in HepG2 cells, SR-BI is responsible for 68-85% of CE selective uptake. Other pathways for selective uptake in HepG2 cells do not require CD36, as shown by anti-CD36 antibody blocking experiments, or class A scavenger receptors, as shown by the lack of competition by poly(inosinic acid). However, CD36 is a functional oxidized LDL receptor on HepG2 cells, as shown by antibody blocking experiments. Similar results for CE selective uptake were obtained with primary cultures of hepatic cells from normal (+/+), heterozygous (-/+), and homozygous (-/-) SR-BI knockout mice. Flow cytometry experiments show that SR-BI accounts for 75% of DiI-LDL uptake, the LDL receptor for 14%, and other pathways for 11%. CE selective uptake from LDL and HDL(3) is likely to occur in the liver, since unlabeled HDL (total and apoE-free HDL(3)) and LDL, when added in physiological proportions, only partially competed for LDL- and HDL(3)-CE selective uptake. In this setting, human hepatic SR-BI may be a crucial molecule in the turnover of both LDL- and HDL(3)-cholesterol.