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Background: Loss-of-function mutations in the ATP Binding Cassette A-1 (ABCA-1) transporter and the ApoE4 allele are associated with increased Alzheimer’s disease (AD) risk. We recently reported decreased ABCA-1 activity of cerebrospinal fluid (CSF) from participants with AD. Our aim is to study the interaction of ApoE4 with ABCA-1 activity in astrocytes, in human apoE4 (hApoE4) targeted replacement (TR) mice, and in CSF from humans grouped by APOE genotype. Methods: ABCA-1 expression and activity were assessed in astrocytes that express human ApoE isoforms and in hApoE mice. The ability of CSF to activate ABCA-1 was examined in cells. CSF from 59 older individuals with or without cognitive impairment was analyzed for ApoE particle size using native gel electrophoresis, and ABCA-1 cholesterol efflux activity. The effect of the ABCA-1 agonist CS-6253 on ABCA-1 activity and AD pathology was examined. Results: ApoE4 vs. apoE3 expressing astrocytes had decreased ABCA-1 expression that was associated with hypolipidated ApoE4. A similar discrepancy for ABCA-1 and apoE lipidation was found between hApoE4 and hApoE3 TR mice. CSF ApoE was resolved in four distinct bands by electrophoresis α 0 (>669 KDa), α 1 (600 KDa), α 2 (440 KDa) and α 3 (232-140 KDa). The amount of total ApoE present in α 0 size was reduced in ε4/ε4 vs ε3/ε3 individuals (3.208 % (SD 0.6156; N=3) vs 8.904 % (SD 0.6156; N=29), p2 size was increased in ε4/ε4 vs ε3/ε3 individuals (60.68 % (SD 8.207; N=3) vs 37.34 % (SD 16.80; N=31), p Conclusions: ApoE4 was associated with reduced ABCA-1 activity and hypolipidated ApoE4 in vivo , in vitro and in CSF . The ABCA-1 agonist CS6253 improved apoE lipidation in astrocytes, CSF and in hApoE4 TR mice. The findings stress the importance of ABCA-1 in apoE4 driven AD and ABCA-1 activity of CSF as a biomarker in AD.

Apabetalone (RVX-208, 200 mg/d) given orally to patients with diabetes mellitus (DM) and CVD leads to a 57% relative risk reduction in major adverse cardiovascular events (MACE). Potential actions of RVX-208, an inhibitor (BETi) of bromodomain extra-terminal (BET) proteins that are epigenetic readers of acetylated lysine residues within histones, in lowering MACE is explored by testing its effect on genes mediating monocyte adhesion to endothelial cells in response to high glucose (HG) and the dietary metabolite trimethyl-amine oxide (TMAO). Cultured THP-1 monocytes, HUVEC endothelial cells and primary human hepatocytes (PHH) were exposed to varying concentrations of glucose and TMAO. Results showed that HG (25.6 mM) induced Very Late Antigen-4 (VLA-4) mRNA, a gene mediating THP-1 adhesion by 1.3-fold and RVX-208 suppressed it >50%. Similarly, BETi blocked TMAO induction of VLA-4 mRNA by >50% in THP-1. In HUVECs RVX-208 abrogated HG induction of E-selectin and MYD88 mRNA by 2- and 1.3-fold, respectively and lowered TMAO induction of these mRNAs by >50%. Microbiome processing of dietary phospholipids followed by hepatic flavin mono-oxygenase-3 (FMO3) metabolism yields TMAO. In PHH exposed for 24 hrs to RVX-208, FMO3 mRNA was lower by 40% but it also suppressed a transcriptional regulator of FMO3, farnesoid X receptor (FXR). BETi suppressed both FXR mRNA and protein within 6 hrs by >80% suggesting a direct effect of BETi on the gene encoding FXR. Furthermore, ChiP data showed that BRD4, a BET protein, dissociated immediately from FXR gene upon exposure to RVX-208. Since BRD4 guides a complex containing RNA pol II along actively transcribed genes containing histones that are highly acetylated, the dissociation of BRD4 from FXR DNA would halt transcription of this gene. In summary, Apabetalone inhibits HG and TMAO enhanced adhesion of THP-1 to HUVECs a process that mimics a step in the pathogenesis of CVD. RVX-208 suppresses genes underlying cellular adhesion; VLA-4 in THP-1 and both E-selectin plus MYD88 in HUVECs. BETi blocks not only activity of TMAO but also its production by inhibiting FXR expression, a regulator of FMO3 gene transcription. The rapid actions of BETi in dissociating BRD4 from FXR DNA suggests a direct effect of RVX-208 on transcription of this gene.

Apabetalone (RVX-208) a bromodomain extra-terminal (BET) inhibitor selectively binds the 2nd ligand domain within a BET protein thus displacing it from acetylated lysine marks on histone tails. In clinical trials of ~1000 patients, many (n=499) of whom with CVD when given 200 mg/d RVX-208 had a 55% relative risk reduction in major adverse cardiac events (MACE) vs placebo. This benefit of RVX-208 may stem from ability of BET inhibition (BETi) to calm inflammation, metabolism, coagulation and complement pathways with well-known roles in CVD risks. These potential benefits of RVX-208 underlie BETonMACE a phase 3 CVD events trial. Of major interest is the anti-inflammatory (AI) effects seen in clinical data showing BETi lowers CRP by 28% in RVX-208 treated patients (n=331). RVX-208 lowered IL-6 and MCP-1 U937 cells exposed to LPS in a dose and time dependent manner by 90 and 85%, respectively within 24 hrs. RVX-208 displaced BET proteins BRD2-4 from chromatin. These AI effects underpin studies of RVX-297 a more potent cousin of RVX-208 that lowered IL-6 and MCP-1 in LPS stimulated U937 cells by 95 and 80% respectively within 24 hrs. In ChIP assays, RVX-297 also displaced BET protein BRD4 and pol II from promoters of cytokine genes IL-6 and IL-1β that mediate inflammation. Together, data from studying RVX-208 and -297 show both BET inhibitors displace transcription factors from promoter DNA that control expression of inflammatory genes (IGs) with key roles in CVD. Importance of BETi displacement of BRD4 and pol II from DNA becomes clear when added to the fact that cellular response to inflammation requires immediate and robust expression of defined set of IGs. For this rapid transcriptional response, the cell places chromatin structures upstream of IGs called super-enhancers (SE) or latent enhancers (LE) that act as molecular sinks for attracting BET proteins such as BRD4. The placement of a SE or LE adjacent to a promoter that controls a IG recruits this gene to the response against an inflammatory insult. Thus targeting BET proteins including BRD4 with a selective BETi may have broad effects on many genes or pathways by crippling SE or LE mediated cellular response to the inflammatory component of CVD. This mechanism may have potential implications to other inflammatory diseases.

Apabetalone is an inhibitor of the epigenetic readers bromodomain and extraterminal (BET) proteins, currently in a phase 3 outcomes trial in patients with cardiovascular disease (CVD) and diabetes mellitus. A post hoc analysis of phase 2b trials demonstrated a 55% relative risk reduction in major adverse cardiac events in CVD patients. Elevated inflammatory markers correlate with CVD. Inflammation also accompanies chronic kidney disease (CKD) and CKD patients are at risk of CVD. Previous research has shown that apabetalone modulates pathways that contribute to chronic inflammation, including the acute phase response (APR). Here, pathway analysis of gene microarrays showed downregulation of APR by apabetalone in primary human hepatocytes (PHH). Real-time PCR and ELISA analysis of RVX-208 treated PHHs confirmed that APR genes that correlate with CVD are suppressed by 20 to 95%, including CRP, ceruloplasmin (CP), serum amyloid P (SAP), PAI-1, alpha 2-macroglobulin (A2M), complement C2, C3 and C5, MBL2, serum amyloid A and interleukin 18. Apabetalone decreased IL-6-induced expression of CP, SAP and A2M, with most striking effects on CRP (-75%). Apabetalone also decreased LPS-induced expression of SAP in a mouse endotoxemia model. To assess effects of apabetalone on inflammatory mediators in CVD patients, SOMAscan™ 1.3K proteomic analysis was performed on plasma from phase 2b ASSERT (12 weeks; n=25) and ASSURE (26 weeks; n=47) clinical trials. This approach identified APR as the top downregulated pathway by apabetalone in both trials. APR biomarkers are elevated in CKD patients where they correlate with disease progression. To gain insight into the pharmacodynamics of the APR response to apabetalone, stage 4 CKD patients (n=8) received a single dose of the drug followed by plasma proteomics at several time points. At 12h post dose, APR was significantly downregulated by apabetalone. Of note, CRP was decreased in CKD patients after 12h of treatment (-7%, p=0.04) versus baseline, as well as in ASSERT (-43%, p=0.01) and ASSURE (-21%, p=0.02) trials versus placebo. Downregulation of the APR pathway by apabetalone may lead to reduced chronic inflammation in CVD and CKD patients and contribute to the reduction in MACE in patients with high residual CVD risk.

RVX-208 affects epigenetics by inhibiting bromodomain and extraterminal (BET) proteins from binding to their natural ligand, acetyl-lysine marks on histone tails and thereby modulates gene activity. In SUSTAIN and ASSURE phase IIb trials of CVD patients (n=499), giving 200 mg/d of RVX-208 orally lead a 55% relative risk reduction in major adverse cardiovascular events (MACE) vs. placebo. This marked reduction in MACE is unlikely due to RVX-208’s modest induction of ApoA-I/HDL, thus prompting studies of RVX-208 for its benefits beyond lipids. Methods included microarray surveys of human whole blood (WB) or primary hepatocytes (PH) exposed to RVX-208. Cytokines were assayed in U937 macrophage and peripheral blood mononuclear cells (PBMC) exposed to RVX-208. Plasma samples from phase IIb patients were measured using SOMAScan proteomic analysis. Results of the microarray studies using WB showed a potential anti-atherogenic effect of RVX-208 because it suppressed activity of 37/46 pro-atherogenic while inducing 8/18 anti-atherogenic genes. Additionally, RVX-208 had potential anti-thrombotic properties by affecting 18 genes related to platelet function (e.g. downregulation of CD64 and thrombospondin 1). RVX-208 had anti-inflammatory effects on the expression of >25 cytokines including downregulation of MCP-1, osteopontin and PARC genes. The suppression of these 3 genes by RVX-208 was evident in not only WB but also in the LPS stimulated U937 and/or PBMCs. Whether these in vitro findings extended into patients were examined by SOMAScan showing lower levels of osteopontin and PARC protein in plasma of treated patients. Furthermore, a key marker of inflammation RANTES was markedly lowered in treated patients. Why RVX-208 may affect genes connected to CVD was explored by exposing PH to RVX-208. These studies showed a 25% reduction in levels of mRNA encoding flavin mono-oxygenase-3 (FMO3), an enzyme that produces trimethylamine oxide (TMAO) a metabolite which predicts CVD risk. In summary, RVX-208 inhibits BET proteins to impact cellular epigenetics that in turn affects expression of genes with known roles in CVD. This activity may underlie RVX-208’s anti-atherogenic, -thrombotic and –inflammatory effects in reducing MACE observed in clinical trials.

The recently completed human trials SUSTAIN and ASSURE (n=499) showed a 55% reduction of major adverse cardiovascular events (MACE) in atherosclerotic patients treated with statins and oral RVX-208 (200 mg/day). RVX-208 increases ApoA-I, the dominant protein of HDL-c, by selectively inhibiting bromodomain extra-terminal proteins (BET) that are epigenetic readers which regulate chromatin function via binding to acetylated histones. Patients given RVX-208 had increased ApoA-I and HDL-c by 10.3% and 7.7%, respectively. Although significant, these increases may account, in part, for lower MACE. Thus, effects of RVX-208 on other biological processes that impacted atherosclerosis were examined. Specifically, biomarkers of vascular inflammation were measured and microarray techniques were used in studying human primary hepatocytes and whole blood. Results in human aortic endothelial cells (HAEC) exposed to RVX-208 showed suppressed VCAM-1 and MCP-1 with an IC50 of 1uM and 10uM, respectively. In U937 macrophages RVX-208 suppressed the pro-inflammatory cytokine IL-6 (IC50 1uM). Primary hepatocytes treated with RVX-208 lead to upregulation of gene sets within processes such as; transcription, translation and chromatin modeling, as previously seen with other BET inhibitors. More importantly RVX-208 down regulated gene sets within the; complement cascade, fibrin clotting, cholesterol and fatty acid synthesis, innate immune system and diabetes mellitus pathways. Any one of these changes, independently or cumulatively, may underlie the observed MACE reduction. Blood treated ex vivo with RVX-208 suppressed gene sets involved in pro-inflammatory signaling of monocytes and neutrophils, monocyte-endothelial cell interactions, extracellular matrix remodeling and Th1/Th2 cell responses. Moreover, RVX-208 affected genes with known roles in atherosclerosis and/or vascular inflammation yielding an overall anti-atherogenic profile. In summary, the orally active selective BET inhibitor RVX-208 significantly lowers MACE in patients with residual CVD risk receiving standard of care therapy including statins. The above results suggest that RVX-208 alters several biological pathways in a cardioprotective manner which may lead to lower MACE.

HDL mimetic peptides hold promise for the treatment of cardio-metabolic diseases. Transient increases in plasma TG and/or safety concerns may limit their therapeutic applications however, particularly in high risk patients with hypertriglyceridemia and diabetes. Presently, we sought to identify peptide structural features responsible for these adverse effects. The single helix peptide ATI-5261 was used as a model, since it forms a class A α-helix like many HDL mimetic peptides and has exceptional secondary structure and solubility. High dose ip injection (300 mg/kg) of ATI-5261 induced muscle toxicity in C57Bl/6 mice vs. vehicle (serum CPK@4 h = 32,314±2359 vs 143±44 IU/L), increased ALT and AST activities (233±10 & 4595±933 vs 26±6 & 84±21 IU/L) and elevated plasma TG (1951±77 vs 37±7 mg/dl). A majority (~90%) of the cytotoxicity and TG increase was eliminated by removing phenylalanine residues from the apolar face of ATI-5261. Similar results were obtained by removing cationic arginine residues from the lipid-water interface. Thus both features were necessary, but each not sufficient, to induce cytotoxicity and TG elevations. Based on this, a peptide (CS6253) was created that was non-toxic (no adverse effect level >500 mg/kg; t1/2 =7.0±0.6 h in rats), retained secondary structure (75±5% α-helicity), high solubility and ability to stimulate ABCA1-dependent cholesterol efflux efficiently (Km=0.80±0.40 vs. 0.86±0.25 μg/ml ATI-5261). In vivo CS6253 was TG neutral, promoted macrophage RCT, and reduced (32%) substantial atherosclerosis in apoE KO mice fed western diet 10 wks (lesion area = 22±4 vs. 15±2% with 30 mg CS6253/kg, ip injection every 48 h for 6 wks, p

Anti-diabetic activities of HDL apolipoprotein(apo)s have been described, complementing ability to reduce atherosclerosis. These effects have been attributed, in part, to apos stimulating cholesterol efflux from cells via ABCA1. This suggests apo mimetic peptides that promote ABCA1 cholesterol efflux may be useful to combat diabetes and its cardiovascular complications. Two such peptides (CS6253 and T6991-2) with optimized safety features (NOAEL = 500 mg/kg) and little TG elevating effects have been designed by us through structure-activity studies of a prototypic ABCA1 ligand peptide, ATI-5261, derived from the C-terminal (CT) domain of apoE. Glucose lowering and insulin sensitizing effects of these new peptides were presently evaluated in mouse models of obesity and diabetes. Peptides CS6253 and T6991-2 stimulated cholesterol efflux from macrophages via ABCA1 with high potency similar to the native apoE CT domain (Km= 0.33±0.14, 0.24±02, 0.21±0.02 μM). Administration of CS6253 at a dose of 30 mg/kg (SC) on alternate days for 6 weeks reduced atherosclerosis by 32% in apoE deficient mice fed high-fat, Western diet for 14 weeks (15±2 vs. 22±4% plaque lesions, CS6253 vs. control, p

Novel apolipoprotein (apo) mimetic peptides are potent mediators of ABCA1-mediated cholesterol efflux, mimicking native apoA-I. We investigated CS-6253 (CS) and ATI-5261 (ATI) in their ability to promote lipid transfer between nascent (n)HDL particles and plasma lipoproteins and examined cholesterol influx from remodelled HDL-like particles to hepatic cells through the SR-BI receptor. nHDL-like lipoproteins nHDL-CS and nHDL-ATI were generated by incubating CS or ATI in the presence of BHK cells expressing ABCA1 labelled with 3 [H]cholesterol and 3 [H] choline; native apoA-I was used as control. These nHDL particles were incubated with plasma at 37°C. Both CS and ATI increased LCAT activity significantly, although were approximately 50% less efficient than nHDL-apoA-I (fractional esterification rate (FER) =22.32±0.86%/h; vs nHDL-CS and nHDL-ATI; FER=11.40±0.045%/h; p3 [H]cholesterol from nHDL mimetics was esterified by LCAT, resulting in an increase in α1-migrating HDL-like particles in plasma (as shown by 2D-PAGGE). The ability of nHDL mimetics to transfer 3 [H]-phosphatidyl choline to plasma apoB-containing lipoproteins is (76±20%) in HDL-apoA-I, (38±0.5%) in nHDL-ATI and (54±13%) in nHDL-CS. These data show that nHDL mimetics are actively remodelled in the presence of plasma lipoproteins. We then investigated cholesterol delivery from HDL-CS and HDL-ATI mimetic to hepatic tissue via SR-BI using Fu5AH cells, having HDL-apoA-I as control. Kinetic parameters for SR-BI-mediated cholesterol influx are as follows: HDL-CS ( K m = 0.30±0.05 ug/ml; p K m = 0.37±0.13 ug/ml), while HDL-ATI was least efficient ( K m = 1.03±0.20 ug/ml). The inhibition of SR-BI selective uptake with BLT-1 affected the uptake of cholesterol from apoB precipitated plasma containing either HDL-CS, HDL-ATI or apoA-I. Here we present an in-vitro model of nHDL-CS and nHDL-ATI that actively undergo remodelling and maturation in plasma and replicate the function of apoA-I. These mature HDL mimetics generated from ABCA1 agonist peptides deliver cholesterol efficiently to hepatocytes specifically through the SR-BI receptor.

Novel approaches that target HDL biogenesis may increase nascent HDL-like particle formation that can be used for therapeutic purposes. Here, we evaluated two 26-amino acid apoA-I mimetic peptides designed from c-terminus of apoE, ATI-5261 and CS-6253 in their ability to promote cellular cholesterol efflux via the ABCA1 transporter. Using mifepristone-inducible ABCA1 BHK cells and cAMP-stimulated J774 macrophages, we examined ABCA1 oligomerization with dithiobis[succinimidyl propionate] (DSP) as a cross linker. Like native apoA-I, ATI-5261 and CS-6253 induced ABCA1 oligomerization at equimolar concentrations, and appear to interact with native oligomers of ABCA1, similar to apoA-I, as judged by crosslinking revealed by SDS-PAGE. By sucrose density gradient fractionation, we show that ABCA1 oligomerization occurs in non-raft domains. ATI-5261 and CS-6253 promoted cellular cholesterol efflux in a dose-dependent saturable manner. In BHK cells, the efficiency of cholesterol efflux (Km) on an equimolar basis was: apoA-I (0.16±0.02), ATI-5261 (0.45±0.08) and CS-6253 (0.68±0.15) and the efflux capacity (Vmax) (% in 24h) was: apoA-I (14.54±0.61), ATI-5261 (11.98±0.51) and CS-6253 (10.87±0.66), indicating a higher efflux efficiency for the apoA-I mimetics but with less capacity than native apoA-I. A similar trend was observed in J774 cells. Desorption of cellular cholesterol at 45 min occurred in both non-raft and raft-like membrane domains. We then examined by HPLC and 2-dimentional PAGGE the size and types of HDL-like particles formed. In 3[H]-cholesterol loaded BHK-ABCA1 cells, incubation with ATI-5261 and CS-6253 for 45 min and 12 hours yielded α-migrating particles with an apparent size ranging between 9 and 17 nm, as revealed by specific antibodies directed against ATI-5261 and CS-6253. Taken together, these observations suggest that ATI-5261 and CS-6253 induce cellular cholesterol efflux via ABCA1 oligomerization at molar concentrations similar to apoA-I and with higher efficiency. The particles generated by ATI-5261 and CS-6253 have α-migrating mobility with a size ranging between 9 and 17 nm and contain cholesterol, similar to nascent HDL particles.

Abstract The aim of the Probucol Quantitative Regression Swedish Trial (PQRST) (n=303) was to investigate whether probucol (0.5 g BID) added to diet and cholestyramine (8 g BID) could retard progression or induce regression of femoral atherosclerosis in hypercholesterolemic (>6.86 mmol/L) subjects. Probucol did not induce regression over the 3-year trial period as estimated by change in lumen volume on quantitative arteriography of a 20-cm segment of the femoral artery. In this report we studied in a representative subgroup (n=72) whether the reduction in HDL concentrations induced by probucol could explain the failure of the drug to be effective. We analyzed the effects of treatment on HDL particle size subclasses. Probucol lowered the relative level of HDL 2b , comprising the largest HDL particles, by 53% and the protein concentration of HDL 2b by 67%. The protein reduction in HDL was mainly confined to the apolipoprotein A-I moiety. The change in lumen volume correlated significantly with change in HDL, ie, HDL cholesterol ( r =.34, P 2 cholesterol ( r =.37, P 2b protein ( r =.44, P 2b value ( r =.51, P 2b distribution calculated on the active (n=35) and placebo (n=37) groups separately were also significant ( r =.39 and .32, respectively; both P 2b concentration and change in atherosclerosis was independent of the alteration in triglyceride concentration and could not be explained by treatment interaction. HDL 2b lowering was highly significantly correlated to probucol concentration. We suggest that the lowering effects of probucol on HDL and particularly on the HDL 2b fraction at least in part explain why regression of femoral atherosclerosis was not obtained by the drug.

Abstract The degree of atherosclerosis in the inner and outer curvature of the femoral artery was studied by using digitized angiography and edge-roughness calculations in 301 hyperlipidemic patients. When the two edges of the vessel were compared no significant difference was seen, but when the local curvature was taken into account, inner curves were found to be more atherosclerotic than outer curves, and both inner and outer curves were more affected than straight segments. The same pattern was encountered in subpopulations defined by clinical or blood lipid criteria. The suggested explanation is that flow disturbances such as low shear rates or separated flow, which tend to arise along the inner curvature, promote the development of atherosclerosis.