Erratum: Proof-of-concept study for liver-directed miQURE technology in a dyslipidemic mouse model.

[This corrects the article DOI: 10.1016/j.omtn.2023.04.004.].

Differential effects of plant and animal fats on obesity-induced dyslipidemia and atherosclerosis in Ldlr-/-.Leiden mice.

Cardiovascular disease (CVD) is closely associated with obesity through risk factors such as dyslipidemia and chronic low-grade inflammation, which may be affected by diet. Dietary fats have been extensively studied in relation to CVD risk, however these studies have not always yielded consistent results, most likely due to lack in control of experimental conditions and confounding factors. Here we studied the effects of different plant and animal fats on dyslipidemia, inflammation, and atherosclerosis. Ldlr-/-.Leiden mice were fed isocaloric energy-dense diets with translational macronutrient composition for 28 weeks. The diets were identical apart from the type of fat they contained: either (1) a mixture of olive and rapeseed oil, (2) sunflower oil, (3) pork fat, (4) beef fat, or (5) milk fat. The fatty acid composition of the diets was determined and effects on circulating lipid and inflammatory risk factors and atherosclerosis were examined, complemented by adipose tissue histology and liver transcriptomics. While visceral fat mass, adipocyte size, and adipose tissue inflammation were not differentially affected by the diets, atherosclerotic lesion load and severity was more pronounced with increasing dietary saturated fatty acid content and decreasing monounsaturated and polyunsaturated fatty acid content, and hence most pronounced with beef and milk fat. These differential effects were accompanied by increases in pro-atherogenic plasma lipids/lipoproteins (e.g., triglycerides, apolipoprotein B), activation of pro-atherogenic cytokine/chemokine signaling pathways in liver, and with circulating pro-atherogenic mediators of inflammation altogether providing a rationale for the differential effects of plant and animal fats.

Atorvastatin Attenuates Diet-Induced Non-Alcoholic Steatohepatitis in APOE*3-Leiden Mice by Reducing Hepatic Inflammation.

Patients with metabolic syndrome are often prescribed statins to prevent the development of cardiovascular disease. Conversely, data on their effects on non-alcoholic steatohepatitis (NASH) are lacking. We evaluated these effects by feeding APOE*3-Leiden mice a Western-type diet (WTD) with or without atorvastatin to induce NASH and hepatic fibrosis. Besides the well-known plasma cholesterol lowering (-30%) and anti-atherogenic effects (severe lesion size -48%), atorvastatin significantly reduced hepatic steatosis (-22%), the number of aggregated inflammatory cells in the liver (-80%) and hepatic fibrosis (-92%) compared to WTD-fed mice. Furthermore, atorvastatin-treated mice showed less immunohistochemically stained areas of inflammation markers. Atorvastatin prevented accumulation of free cholesterol in the form of cholesterol crystals (-78%). Cholesterol crystals are potent inducers of the NLRP3 inflammasome pathway and atorvastatin prevented its activation, which resulted in reduced expression of the pro-inflammatory cytokines interleukin (IL)-1ß (-61%) and IL-18 (-26%). Transcriptome analysis confirmed strong reducing effects of atorvastatin on inflammatory mediators, including NLRP3, NFκB and TLR4. The present study demonstrates that atorvastatin reduces hepatic steatosis, inflammation and fibrosis and prevents cholesterol crystal formation, thereby precluding NLRP3 inflammasome activation. This may render atorvastatin treatment as an attractive approach to reduce NAFLD and prevent progression into NASH in dyslipidemic patients.

Proof-of-concept study for liver-directed miQURE technology in a dyslipidemic mouse model.

A gene-silencing platform (miQURE) has been developed and successfully used to deliver therapeutic microRNA (miRNA) to the brain, reducing levels of neurodegenerative disease-causing proteins/RNAs via RNA interference and improving the disease phenotype in animal models. This study evaluates the use of miQURE technology to deliver therapeutic miRNA for liver-specific indications. Angiopoietin-like 3 (ANGPTL3) was selected as the target mRNA because it is produced in the liver and because loss-of-function ANGPTL3 mutations and/or pharmacological inhibition of ANGPTL3 protein lowers lipid levels and reduces cardiovascular risk. Overall, 14 candidate miRNA constructs were tested in vitro, the most potent of which (miAngE) was further evaluated in mice. rAAV5-miAngE led to dose-dependent (≤-77%) decreases in Angptl3 mRNA in WT mice with ≤-90% reductions in plasma ANGPTL3 protein. In dyslipidemic APOE∗3-Leiden.CETP mice, AAV5-miAngE significantly reduced cholesterol and triglyceride levels vs. vehicle and scrambled (miSCR) controls when administrated alone, with greater reductions when co-administered with lipid-lowering therapy (atorvastatin). A significant decrease in total atherosclerotic lesion area (-58% vs. miSCR) was observed in AAV5-miAngE-treated dyslipidemic mice, which corresponded with the maintenance of a non-diseased plaque phenotype and reduced lesion severity. These results support the development of this technology for liver-directed indications.

A Novel, Orally Bioavailable, Small-Molecule Inhibitor of PCSK9 With Significant Cholesterol-Lowering Properties In Vivo.

Proprotein convertase subtilisin kexin type 9 (PCSK9) inhibits the clearance of low-density lipoprotein (LDL) cholesterol (LDL-C) from plasma by directly binding with the LDL receptor (LDLR) and sending the receptor for lysosomal degradation. As the interaction promotes elevated plasma LDL-C levels, and therefore a predisposition to cardiovascular disease, PCSK9 has attracted intense interest as a therapeutic target. Despite this interest, an orally bioavailable small-molecule inhibitor of PCSK9 with extensive lipid-lowering activity is yet to enter the clinic. We report herein the discovery of NYX-PCSK9i, an orally bioavailable small-molecule inhibitor of PCSK9 with significant cholesterol-lowering activity in hyperlipidemic APOE∗3-Leiden.CETP mice. NYX-PCSK9i emerged from a medicinal chemistry campaign demonstrating potent disruption of the PCSK9-LDLR interaction in vitro and functional protection of the LDLR of human lymphocytes from PCSK9-directed degradation ex vivo. APOE∗3-Leiden.CETP mice orally treated with NYX-PCSK9i demonstrated a dose-dependent decrease in plasma total cholesterol of up to 57%, while its combination with atorvastatin additively suppressed plasma total cholesterol levels. Importantly, the majority of cholesterol lowering by NYX-PCSK9i was in non-HDL fractions. A concomitant increase in total plasma PCSK9 levels and significant increase in hepatic LDLR protein expression strongly indicated on-target function by NYX-PCSK9i. Determinations of hepatic lipid and fecal cholesterol content demonstrated depletion of liver cholesteryl esters and promotion of fecal cholesterol elimination with NYX-PCSK9i treatment. All measured in vivo biomarkers of health indicate that NYX-PCSK9i has a good safety profile. NYX-PCSK9i is a potential new therapy for hypercholesterolemia with the capacity to further enhance the lipid-lowering activities of statins.

The tyrosine kinase inhibitor nilotinib targets the discoidin domain receptor DDR2 in calcific aortic valve stenosis.

BACKGROUND AND PURPOSE: Tyrosine kinase inhibitors (TKI) used to treat chronic myeloid leukaemia (CML) have been associated with cardiovascular side effects, including reports of calcific aortic valve stenosis. The aim of this study was to establish the effects of first and second generation TKIs in aortic valve stenosis and to determine the associated molecular mechanisms. EXPERIMENTAL APPROACH: Hyperlipidemic APOE*3Leiden.CETP transgenic mice were treated with nilotinib, imatinib or vehicle. Human valvular interstitial cells (VICs) were isolated and studied in vitro. Gene expression analysis was perfromed in aortic valves from 64 patients undergoing aortic valve replacement surgery. KEY RESULTS: Nilotinib increased murine aortic valve thickness. Nilotinib, but not imatinib, promoted calcification and osteogenic activation and decreased autophagy in human VICs. Differential tyrosine kinase expression was detected between healthy and calcified valve tissue. Transcriptomic target identification revealed that the discoidin domain receptor DDR2, which is preferentially inhibited by nilotinib, was predominantly expressed in human aortic valves but markedly downregulated in calcified valve tissue. Nilotinib and selective DDR2 targeting in VICs induced a similar osteogenic activation, which was blunted by increasing the DDR2 ligand, collagen. CONCLUSIONS AND IMPLICATIONS: These findings suggest that inhibition of DDR2 by nilotinib promoted aortic valve thickening and VIC calcification, with possible translational implications for cardiovascular surveillance and possible personalized medicine in CML patients.

Chronic Oral Administration of Mineral Oil Compared With Corn Oil: Effects on Gut Permeability and Plasma Inflammatory and Lipid Biomarkers.

We investigated the effects of chronic oral administration of mineral oil, versus corn oil as control, on intestinal permeability, inflammatory markers, and plasma lipids in APOE*3-Leiden.CETP mice. Mice received mineral oil or corn oil 15 or 30 µL/mouse/day for 16 weeks (15 mice/group). Intestinal permeability was increased with mineral versus corn oil 30 µL/day, shown by increased mean plasma FITC-dextran concentrations 2 h post-administration (11 weeks: 1.5 versus 1.1 µg/ml, p = 0.02; 15 weeks: 1.7 versus 1.3 µg/ml, p = 0.08). Mean plasma lipopolysaccharide-binding protein levels were raised with mineral versus corn oil 30 µL/day (12 weeks: 5.8 versus 4.4 µg/ml, p = 0.03; 16 weeks: 5.8 versus 4.5 µg/ml, p = 0.09), indicating increased intestinal bacterial endotoxin absorption and potential pro-inflammatory effects. Plasma cholesterol and triglyceride concentrations were decreased with mineral oil, without affecting liver lipids among treated groups. Fecal neutral sterol measurements indicated increased fecal cholesterol excretion with mineral oil 30 µL/day (+16%; p = 0.04). Chronic oral administration of mineral oil in APOE*3-Leiden.CETP mice increased intestinal permeability, with potential pro-inflammatory effects, and decreased plasma cholesterol and triglyceride levels. Our findings may raise concerns about the use of mineral oil as a placebo in clinical studies.

Effects of mineral oil administration on the pharmacokinetics, metabolism and pharmacodynamics of atorvastatin and pravastatin in mice and dogs.

We investigated the effects of mineral oil on statin pharmacokinetics and inflammatory markers in animal models. A new synthesis strategy produced regioisomers that facilitated the characterization of the main metabolite (M1) of atorvastatin, a lipophilic statin, in C57BL/6NCrl mice. The chemical structure of M1 in mice was confirmed as ortho-hydroxy ß-oxidized atorvastatin. Atorvastatin and M1 pharmacokinetics and inflammatory markers were assessed in C57BL6/J mice given atorvastatin 5 mg/kg/day or 10 mg/kg/day, as a single dose or for 21 days, with or without 10 µL or 30 µL mineral oil. No consistent differences in plasma exposure of atorvastatin or M1 were observed in mice after single or repeat dosing of atorvastatin with or without mineral oil. However, mice administered atorvastatin 10 mg/kg with 30 µL mineral oil for 21 days had significantly increased plasma levels of serum amyloid A (mean 9.6 µg/mL vs 7.9 µg/mL without mineral oil; p < 0.01) and significantly increased proportions of C62Lhigh B cells (mean 18% vs 12% without mineral oil; p = 0.04). There were no statistically significant differences for other inflammatory markers assessed. In dogs, pharmacokinetics of atorvastatin, its two hydroxy metabolites and pravastatin (a hydrophilic statin) were evaluated after single administration of atorvastatin 10 mg plus pravastatin 40 mg with or without 2 g mineral oil. Pharmacokinetics of atorvastatin, hydroxylated atorvastatin metabolites or pravastatin were not significantly different after single dosing with or without mineral oil in dogs. Collectively, the results in mice and dogs indicate that mineral oil does not affect atorvastatin or pravastatin pharmacokinetics, but could cause low-grade inflammation with chronic oral administration, which warrants further investigation.

In Vivo Magnetic Resonance Imaging-Based Detection of Heterogeneous Endothelial Response in Thoracic and Abdominal Aorta to Short-Term High-Fat Diet Ascribed to Differences in Perivascular Adipose Tissue in Mice.

Background Long-term feeding with a high-fat diet (HFD) induces endothelial dysfunction in mice, but early HFD-induced effects on endothelium have not been well characterized. Methods and Results Using an magnetic resonance imaging-based methodology that allows characterization of endothelial function in vivo, we demonstrated that short-term (2 weeks) feeding with a HFD to C57BL/6 mice or to E3L.CETP mice resulted in the impairment of acetylcholine-induced response in the abdominal aorta (AA), whereas, in the thoracic aorta (TA), the acetylcholine-induced response was largely preserved. Similarly, HFD resulted in arterial stiffness in the AA, but not in the TA. The difference in HFD-induced response was ascribed to distinct characteristics of perivascular adipose tissue in the TA and AA, related to brown- and white-like adipose tissue, respectively, as assessed by histology, immunohistochemistry, and Raman spectroscopy. In contrast, short-term HFD-induced endothelial dysfunction could not be linked to systemic insulin resistance, changes in plasma concentration of nitrite, or concentration of biomarkers of glycocalyx disruption (syndecan-1 and endocan), endothelial inflammation (soluble form of vascular cell adhesion molecule 1, soluble form of intercellular adhesion molecule 1 and soluble form of E-selectin), endothelial permeability (soluble form of fms-like tyrosine kinase 1 and angiopoietin 2), and hemostasis (tissue plasminogen activator and plasminogen activator inhibitor 1). Conclusions Short-term feeding with a HFD induces endothelial dysfunction in the AA but not in the TA, which could be ascribed to a differential response of perivascular adipose tissue to a HFD in the AA versus TA. Importantly, early endothelial dysfunction in the AA is not linked to elevation of classical systemic biomarkers of endothelial dysfunction.

Dual targeting of hepatic fibrosis and atherogenesis by icosabutate, an engineered eicosapentaenoic acid derivative.

BACKGROUND & AIMS: While fibrosis stage predicts liver-associated mortality, cardiovascular disease (CVD) is still the major overall cause of mortality in patients with NASH. Novel NASH drugs should thus ideally reduce both liver fibrosis and CVD. Icosabutate is a semi-synthetic, liver-targeted eicosapentaenoic acid (EPA) derivative in clinical development for NASH. The primary aims of the current studies were to establish both the anti-fibrotic and anti-atherogenic efficacy of icosabutate in conjunction with changes in lipotoxic and atherogenic lipids in liver and plasma respectively. METHODS: The effects of icosabutate on fibrosis progression and lipotoxicity were investigated in amylin liver NASH (AMLN) diet (high fat, cholesterol and fructose) fed ob/ob mice with biopsy-confirmed steatohepatitis and fibrosis and compared with the activity of obeticholic acid. APOE*3Leiden.CETP mice, a translational model for hyperlipidaemia and atherosclerosis, were used to evaluate the mechanisms underlying the lipid-lowering effect of icosabutate and its effect on atherosclerosis. RESULTS: In AMLN ob/ob mice, icosabutate significantly reduced hepatic fibrosis and myofibroblast content in association with downregulation of the arachidonic acid cascade and a reduction in both hepatic oxidised phospholipids and apoptosis. In APOE*3Leiden.CETP mice, icosabutate reduced plasma cholesterol and TAG levels via increased hepatic uptake, upregulated hepatic lipid metabolism and downregulated inflammation pathways, and effectively decreased atherosclerosis development. CONCLUSIONS: Icosabutate, a structurally engineered EPA derivative, effectively attenuates both hepatic fibrosis and atherogenesis and offers an attractive therapeutic approach to both liver- and CV-related morbidity and mortality in NASH patients.

Icosabutate Exerts Beneficial Effects Upon Insulin Sensitivity, Hepatic Inflammation, Lipotoxicity, and Fibrosis in Mice.

Icosabutate is a structurally engineered eicosapentaenoic acid derivative under development for nonalcoholic steatohepatitis (NASH). In this study, we investigated the absorption and distribution properties of icosabutate in relation to liver targeting and used rodents to evaluate the effects of icosabutate on glucose metabolism, insulin resistance, as well as hepatic steatosis, inflammation, lipotoxicity, and fibrosis. The absorption, tissue distribution, and excretion of icosabutate was investigated in rats along with its effects in mouse models of insulin resistance (ob/ob) and metabolic inflammation/NASH (high-fat/cholesterol-fed APOE*3Leiden.CETP mice) and efficacy was compared with synthetic peroxisome proliferator-activated receptor α (PPAR-α) (fenofibrate) and/or PPAR-γ/(α) (pioglitazone and rosiglitazone) agonists. Icosabutate was absorbed almost entirely through the portal vein, resulting in rapid hepatic accumulation. Icosabutate demonstrated potent insulin-sensitizing effects in ob/ob mice, and unlike fenofibrate or pioglitazone, it significantly reduced plasma alanine aminotransferase. In high-fat/cholesterol-fed APOE*3Leiden.CETP mice, icosabutate, but not rosiglitazone, reduced microvesicular steatosis and hepatocellular hypertrophy. Although both rosiglitazone and icosabutate reduced hepatic inflammation, only icosabutate elicited antifibrotic effects in association with decreased hepatic concentrations of multiple lipotoxic lipid species and an oxidative stress marker. Hepatic gene-expression analysis confirmed the changes in lipid metabolism, inflammatory and fibrogenic response, and energy metabolism, and revealed the involved upstream regulators. In conclusion, icosabutate selectively targets the liver through the portal vein and demonstrates broad beneficial effects following insulin sensitivity, hepatic microvesicular steatosis, inflammation, lipotoxicity, oxidative stress, and fibrosis. Icosabutate therefore offers a promising approach to the treatment of both dysregulated glucose/lipid metabolism and inflammatory disorders of the liver, including NASH.

Alirocumab, evinacumab, and atorvastatin triple therapy regresses plaque lesions and improves lesion composition in mice.

Atherosclerosis-related CVD causes nearly 20 million deaths annually. Most patients are treated after plaques develop, so therapies must regress existing lesions. Current therapies reduce plaque volume, but targeting all apoB-containing lipoproteins with intensive combinations that include alirocumab or evinacumab, monoclonal antibodies against cholesterol-regulating proprotein convertase subtilisin/kexin type 9 and angiopoietin-like protein 3, may provide more benefit. We investigated the effect of such lipid-lowering interventions on atherosclerosis in APOE*3-Leiden.CETP mice, a well-established model for hyperlipidemia. Mice were fed a Western-type diet for 13 weeks and thereafter matched into a baseline group (euthanized at 13 weeks) and five groups that received diet alone (control) or with treatment [atorvastatin; atorvastatin and alirocumab; atorvastatin and evinacumab; or atorvastatin, alirocumab, and evinacumab (triple therapy)] for 25 weeks. We measured effects on cholesterol levels, plaque composition and morphology, monocyte adherence, and macrophage proliferation. All interventions reduced plasma total cholesterol (37% with atorvastatin to 80% with triple treatment; all P < 0.001). Triple treatment decreased non-HDL-C to 1.0 mmol/l (91% difference from control; P < 0.001). Atorvastatin reduced atherosclerosis progression by 28% versus control (P < 0.001); double treatment completely blocked progression and diminished lesion severity. Triple treatment regressed lesion size versus baseline in the thoracic aorta by 50% and the aortic root by 36% (both P < 0.05 vs. baseline), decreased macrophage accumulation through reduced proliferation, and abated lesion severity. Thus, high-intensive cholesterol-lowering triple treatment targeting all apoB-containing lipoproteins regresses atherosclerotic lesion area and improves lesion composition in mice, making it a promising potential approach for treating atherosclerosis.

Oncostatin M reduces atherosclerosis development in APOE*3Leiden.CETP mice and is associated with increased survival probability in humans.

OBJECTIVE: Previous studies indicate a role for Oncostatin M (OSM) in atherosclerosis and other chronic inflammatory diseases for which inhibitory antibodies are in development. However, to date no intervention studies with OSM have been performed, and its relation to coronary heart disease (CHD) has not been studied. APPROACH AND RESULTS: Gene expression analysis on human normal arteries (n = 10) and late stage/advanced carotid atherosclerotic arteries (n = 127) and in situ hybridization on early human plaques (n = 9) showed that OSM, and its receptors, OSM receptor (OSMR) and Leukemia Inhibitory Factor Receptor (LIFR) are expressed in normal arteries and atherosclerotic plaques. Chronic OSM administration in APOE*3Leiden.CETP mice (n = 15/group) increased plasma E-selectin levels and monocyte adhesion to the activated endothelium independently of cholesterol but reduced the amount of inflammatory Ly-6CHigh monocytes and atherosclerotic lesion size and severity. Using aptamer-based proteomics profiling assays high circulating OSM levels were shown to correlate with post incident CHD survival probability in the AGES-Reykjavik study (n = 5457). CONCLUSIONS: Chronic OSM administration in APOE*3Leiden.CETP mice reduced atherosclerosis development. In line, higher serum OSM levels were correlated with improved post incident CHD survival probability in patients, suggesting a protective cardiovascular effect.

The APOE∗3-Leiden Heterozygous Glucokinase Knockout Mouse as Novel Translational Disease Model for Type 2 Diabetes, Dyslipidemia, and Diabetic Atherosclerosis.

BACKGROUND: There is a lack of predictive preclinical animal models combining atherosclerosis and type 2 diabetes. APOE∗3-Leiden (E3L) mice are a well-established model for diet-induced hyperlipidemia and atherosclerosis, and glucokinase+/- (GK+/-) mice are a translatable disease model for glucose control in type 2 diabetes. The respective mice respond similarly to lipid-lowering and antidiabetic drugs as humans. The objective of this study was to evaluate/characterize the APOE∗3-Leiden.glucokinase+/- (E3L.GK+/-) mouse as a novel disease model to study the metabolic syndrome and diabetic complications. METHODS: Female E3L.GK+/-, E3L, and GK+/- mice were fed fat- and cholesterol-containing diets for 37 weeks, and plasma parameters were measured throughout. Development of diabetic macro- and microvascular complications was evaluated. RESULTS: Cholesterol and triglyceride levels were significantly elevated in E3L and E3L.GK+/- mice compared to GK+/- mice, whereas fasting glucose was significantly increased in E3L.GK+/- and GK+/- mice compared to E3L. Atherosclerotic lesion size was increased 2.2-fold in E3L.GK+/- mice as compared to E3L (p = 0.037), which was predicted by glucose exposure (R 2 = 0.636, p = 0.001). E3L and E3L.GK+/- mice developed NASH with severe inflammation and fibrosis which, however, was not altered by introduction of the defective GK phenotype, whereas mild kidney pathology with tubular vacuolization was present in all three phenotypes. CONCLUSIONS: We conclude that the E3L.GK+/- mouse is a promising novel diet-inducible disease model for investigation of the etiology and evaluation of drug treatment on diabetic atherosclerosis.

Dose Effects of Ammonium Perfluorooctanoate on Lipoprotein Metabolism in APOE*3-Leiden.CETP Mice.

Epidemiological studies have reported positive associations between serum perfluorooctanoic acid (PFOA) and total and non-high-density lipoprotein cholesterol (non-HDL-C) although the magnitude of effect of PFOA on cholesterol lacks consistency. The objectives of this study were to evaluate the effect of PFOA on plasma cholesterol and triglyceride metabolism at various plasma PFOA concentrations relevant to humans, and to elucidate the mechanisms using APOE*3-Leiden.CETP mice, a model with a human-like lipoprotein metabolism. APOE*3-Leiden.CETP mice were fed a Western-type diet with PFOA (10, 300, 30 000 ng/g/d) for 4-6 weeks. PFOA exposure did not alter plasma lipids in the 10 and 300 ng/g/d dietary PFOA dose groups. At 30 000 ng/g/d, PFOA decreased plasma triglycerides (TG), total cholesterol (TC), and non-HDL-C, whereas HDL-C was increased. The plasma lipid alterations could be explained by decreased very low-density lipoprotein (VLDL) production and increased VLDL clearance by the liver through increased lipoprotein lipase activity. The concomitant increase in HDL-C was mediated by decreased cholesteryl ester transfer activity and changes in gene expression of proteins involved in HDL metabolism. Hepatic gene expression and pathway analysis confirmed the changes in lipoprotein metabolism that were mediated for a major part through activation of the peroxisome proliferator-activated receptor (PPAR)α. Our data confirmed the findings from a phase 1 clinical trial in humans that demonstrated high serum or plasma PFOA levels resulted in lower cholesterol levels. The study findings do not show an increase in cholesterol at environmental or occupational levels of PFOA exposure, thereby indicating these findings are associative rather than causal.

Obeticholic Acid Modulates Serum Metabolites and Gene Signatures Characteristic of Human NASH and Attenuates Inflammation and Fibrosis Progression in Ldlr-/-.Leiden Mice.

Concerns have been raised about whether preclinical models sufficiently mimic molecular disease processes observed in nonalcoholic steatohepatitis (NASH) patients, bringing into question their translational value in studies of therapeutic interventions in the process of NASH/fibrosis. We investigated the representation of molecular disease patterns characteristic for human NASH in high-fat diet (HFD)-fed Ldlr-/-.Leiden mice and studied the effects of obeticholic acid (OCA) on these disease profiles. Multiplatform serum metabolomic profiles and genome-wide liver transcriptome from HFD-fed Ldlr-/-.Leiden mice were compared with those of NASH patients. Mice were profiled at the stage of mild (24 weeks HFD) and severe (34 weeks HFD) fibrosis, and after OCA intervention (24-34 weeks; 10 mg/kg/day). Effects of OCA were analyzed histologically, biochemically, by immunohistochemistry, using deuterated water technology (de novo collagen formation), and by its effect on the human-based transcriptomics and metabolomics signatures. The transcriptomics and metabolomics profile of Ldlr-/-.Leiden mice largely reflected the molecular signature of NASH patients. OCA modulated the expression of these molecular profiles and quenched specific proinflammatory-profibrotic pathways. OCA attenuated specific facets of cellular inflammation in liver (F4/80-positive cells) and reduced crown-like structures in adipose tissue. OCA reduced de novo collagen formation and attenuated further progression of liver fibrosis, but did not reduce fibrosis below the level before intervention. Conclusion: HFD-fed Ldlr-/-.Leiden mice recapitulate molecular transcriptomic and metabolomic profiles of NASH patients, and these signatures are modulated by OCA. Intervention with OCA in developing fibrosis reduces collagen deposition and de novo synthesis but does not resolve already manifest fibrosis in the period studied. These data show that human molecular signatures can be used to evaluate the translational character of preclinical models for NASH.

The BCR-ABL1 Inhibitors Imatinib and Ponatinib Decrease Plasma Cholesterol and Atherosclerosis, and Nilotinib and Ponatinib Activate Coagulation in a Translational Mouse Model.

Treatment with the second and third generation BCR-ABL1 tyrosine kinase inhibitors (TKIs) increases cardiovascular risk in chronic myeloid leukemia (CML) patients. We investigated the vascular adverse effects of three generations of TKIs in a translational model for atherosclerosis, the APOE*3Leiden.CETP mouse. Mice were treated for sixteen weeks with imatinib (150 mg/kg BID), nilotinib (10 and 30 mg/kg QD) or ponatinib (3 and 10 mg/kg QD), giving similar drug exposures as in CML-patients. Cardiovascular risk factors were analyzed longitudinally, and histopathological analysis of atherosclerosis and transcriptome analysis of the liver was performed. Imatinib and ponatinib decreased plasma cholesterol (imatinib, -69%, p < 0.001; ponatinib 3 mg/kg, -37%, p < 0.001; ponatinib 10 mg/kg-44%, p < 0.001) and atherosclerotic lesion area (imatinib, -78%, p < 0.001; ponatinib 3 mg/kg, -52%, p = 0.002; ponatinib 10 mg/kg, -48%, p = 0.006), which were not affected by nilotinib. In addition, imatinib increased plaque stability. Gene expression and pathway analysis demonstrated that ponatinib enhanced the mRNA expression of coagulation factors of both the contact activation (intrinsic) and tissue factor (extrinsic) pathways. In line with this, ponatinib enhanced plasma levels of FVII, whereas nilotinib increased plasma FVIIa activity. While imatinib showed a beneficial cardiovascular risk profile, nilotinib and ponatinib increased the cardiovascular risk through induction of a pro-thrombotic state.

Atorvastatin accelerates clearance of lipoprotein remnants generated by activated brown fat to further reduce hypercholesterolemia and atherosclerosis.

BACKGROUND AND AIMS: Activation of brown adipose tissue (BAT) reduces both hyperlipidemia and atherosclerosis by increasing the uptake of triglyceride-derived fatty acids by BAT, accompanied by formation and clearance of lipoprotein remnants. We tested the hypothesis that the hepatic uptake of lipoprotein remnants generated by BAT activation would be accelerated by concomitant statin treatment, thereby further reducing hypercholesterolemia and atherosclerosis. METHODS: APOE*3-Leiden.CETP mice were fed a Western-type diet and treated without or with the selective ß3-adrenergic receptor (AR) agonist CL316,243 that activates BAT, atorvastatin (statin) or both. RESULTS: ß3-AR agonism increased energy expenditure as a result of an increased fat oxidation by activated BAT, which was not further enhanced by statin addition. Accordingly, statin treatment neither influenced the increased uptake of triglyceride-derived fatty acids from triglyceride-rich lipoprotein-like particles by BAT nor further lowered plasma triglyceride levels induced by ß3-AR agonism. Statin treatment increased the hepatic uptake of the formed cholesterol-enriched remnants generated by ß3-AR agonism. Consequently, statin treatment further lowered plasma cholesterol levels. Importantly, statin, in addition to ß3-AR agonism, also further reduced the atherosclerotic lesion size as compared to ß3-AR agonism alone, without altering lesion severity and composition. CONCLUSIONS: Statin treatment accelerates the hepatic uptake of remnants generated by BAT activation, thereby increasing the lipid-lowering and anti-atherogenic effects of BAT activation in an additive fashion. We postulate that, in clinical practice, combining statin treatment with BAT activation is a promising new avenue to combat hyperlipidemia and cardiovascular disease.

The AT04A vaccine against proprotein convertase subtilisin/kexin type 9 reduces total cholesterol, vascular inflammation, and atherosclerosis in APOE*3Leiden.CETP mice.

AIMS: Proprotein convertase subtilisin/kexin type 9 (PCSK9) has emerged as a promising therapeutic target for the treatment of hypercholesterolaemia and atherosclerosis. PCSK9 binds to the low density lipoprotein receptor and enhances its degradation, which leads to the reduced clearance of low density lipoprotein cholesterol (LDLc) and a higher risk of atherosclerosis. In this study, the AT04A anti-PCSK9 vaccine was evaluated for its therapeutic potential in ameliorating or even preventing coronary heart disease in the atherogenic APOE*3Leiden.CETP mouse model. METHODS AND RESULTS: Control and AT04A vaccine-treated mice were fed western-type diet for 18 weeks. Antibody titres, plasma lipids, and inflammatory markers were monitored by ELISA, FPLC, and multiplexed immunoassay, respectively. The progression of atherosclerosis was evaluated by histological analysis of serial cross-sections from the aortic sinus. The AT04A vaccine induced high and persistent antibody levels against PCSK9, causing a significant reduction in plasma total cholesterol (-53%, P < 0.001) and LDLc compared with controls. Plasma inflammatory markers such as serum amyloid A (SAA), macrophage inflammatory protein-1ß (MIP-1ß/CCL4), macrophage-derived chemokine (MDC/CCL22), cytokine stem cell factor (SCF), and vascular endothelial growth factor A (VEGF-A) were significantly diminished in AT04A-treated mice. As a consequence, treatment with the AT04A vaccine resulted in a decrease in atherosclerotic lesion area (-64%, P = 0.004) and aortic inflammation as well as in more lesion-free aortic segments (+119%, P = 0.026), compared with control. CONCLUSIONS: AT04A vaccine induces an effective immune response against PCSK9 in APOE*3Leiden.CETP mice, leading to a significant reduction of plasma lipids, systemic and vascular inflammation, and atherosclerotic lesions in the aorta.