Effects of repeated weight cycling on non-alcoholic steatohepatitis in diet-induced obese mice.

Lifestyle interventions remain the treatment of choice for patients with obesity and metabolic complications, yet are difficult to maintain and often lead to cycles of weight loss and regain (weight cycling). Literature on weight cycling remains controversial and we therefore investigated the association between weight cycling and metabolic complications using preexistent obese mice. Ldlr-/-.Leiden mice received a high-fat diet (HFD) for 20 weeks to induce obesity. Subsequently, weight-cycled mice were switched between the healthy chow diet and HFD for four 2-week periods and compared to mice that received HFD for the total study period. Repeated weight cycling tended to decrease body weight and significantly reduced fat mass, whereas adipose tissue inflammation was similar relative to HFD controls. Weight cycling did not significantly affect blood glucose or plasma insulin levels yet significantly reduced plasma free fatty acid and alanine transaminase/aspartate transaminase levels. Hepatic macrovesicular steatosis was similar and microvesicular steatosis tended to be increased upon weight cycling. Weight cycling resulted in a robust decrease in hepatic inflammation compared to HFD controls while hepatic fibrosis and atherosclerosis development were not affected. These results argue against the postulate that repeated weight cycling leads to unfavorable metabolic effects, when compared to a continuous unhealthy lifestyle, and in fact revealed beneficial effects on hepatic inflammation, an important hallmark of non-alcoholic steatohepatitis.

A systems toxicology approach for identification of disruptions in cholesterol homeostasis after aggregated exposure to mixtures of perfluorinated compounds in humans.

Per- and polyfluoroalkyl substances (PFAS) are used in various household and industrial products. In humans, positive associations were reported between PFAS, including perfluorsulfonic acid and perfluorooctanoic acid, and cholesterol, a cardiometabolic risk factor. Animal studies show the opposite. Human-centered approaches are needed to better understand the effects of PFAS mixtures on cholesterol. Here, a systems toxicology approach is described, using a gene-centered cholesterol biokinetic model. PFAS exposure-gene expression relations from published data were introduced into the model. An existing PFAS physiologically based kinetic model was augmented with lung and dermal compartments and integrated with the cholesterol model to enable exposure-effect modeling. The final model was populated with data reflecting lifetime mixture exposure from: tolerable weekly intake values; the environment; high occupational exposures (ski waxing, PFAS industry). Results indicate that low level exposures (tolerable weekly intake, environmental) did not change cholesterol. In contrast, occupational exposures clearly resulted in internal PFAS exposure and disruption of cholesterol homeostasis, largely in line with epidemiological observations. Despite model limitations (eg, dynamic range, directionality), changes in cholesterol homeostasis were predicted for ski waxers, hitherto unknown from epidemiological studies. Here, future studies involving lipid metabolism could improve risk assessment.

Semaglutide Has Beneficial Effects on Non-Alcoholic Steatohepatitis in Ldlr-/-.Leiden Mice.

Semaglutide, a glucagon-like peptide-1 receptor agonist, is an antidiabetic medication that has recently been approved for the treatment of obesity as well. Semaglutide is postulated to be a promising candidate for the treatment of non-alcoholic steatohepatitis (NASH). Here, Ldlr-/-.Leiden mice received a fast-food diet (FFD) for 25 weeks, followed by another 12 weeks on FFD with daily subcutaneous injections of semaglutide or vehicle (control). Plasma parameters were evaluated, livers and hearts were examined, and hepatic transcriptome analysis was performed. In the liver, semaglutide significantly reduced macrovesicular steatosis (-74%, p < 0.001) and inflammation (-73%, p < 0.001) and completely abolished microvesicular steatosis (-100%, p < 0.001). Histological and biochemical assessment of hepatic fibrosis showed no significant effects of semaglutide. However, digital pathology revealed significant improvements in the degree of collagen fiber reticulation (-12%, p < 0.001). Semaglutide did not affect atherosclerosis relative to controls. Additionally, we compared the transcriptome profile of FFD-fed Ldlr-/-.Leiden mice with a human gene set that differentiates human NASH patients with severe fibrosis from those with mild fibrosis. In FFD-fed Ldlr-/-.Leiden control mice, this gene set was upregulated as well, while semaglutide predominantly reversed this gene expression. Using a translational model with advanced NASH, we demonstrated that semaglutide is a promising candidate with particular potential for the treatment of hepatic steatosis and inflammation, while for the reversal of advanced fibrosis, combinations with other NASH agents may be necessary.

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.

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.

Common Variants Associated With OSMR Expression Contribute to Carotid Plaque Vulnerability, but Not to Cardiovascular Disease in Humans.

Background and Aims: Oncostatin M (OSM) signaling is implicated in atherosclerosis, however the mechanism remains unclear. We investigated the impact of common genetic variants in OSM and its receptors, OSMR and LIFR, on overall plaque vulnerability, plaque phenotype, intraplaque OSMR and LIFR expression, coronary artery calcification burden and cardiovascular disease susceptibility. Methods and Results: We queried Genotype-Tissue Expression data and found that rs13168867 (C allele) was associated with decreased OSMR expression and that rs10491509 (A allele) was associated with increased LIFR expression in arterial tissues. No variant was significantly associated with OSM expression. We associated these two variants with plaque characteristics from 1,443 genotyped carotid endarterectomy patients in the Athero-Express Biobank Study. After correction for multiple testing, rs13168867 was significantly associated with an increased overall plaque vulnerability (ß = 0.118 ± s.e. = 0.040, p = 3.00 × 10-3, C allele). Looking at individual plaque characteristics, rs13168867 showed strongest associations with intraplaque fat (ß = 0.248 ± s.e. = 0.088, p = 4.66 × 10-3, C allele) and collagen content (ß = -0.259 ± s.e. = 0.095, p = 6.22 × 10-3, C allele), but these associations were not significant after correction for multiple testing. rs13168867 was not associated with intraplaque OSMR expression. Neither was intraplaque OSMR expression associated with plaque vulnerability and no known OSMR eQTLs were associated with coronary artery calcification burden, or cardiovascular disease susceptibility. No associations were found for rs10491509 in the LIFR locus. Conclusions: Our study suggests that rs1316887 in the OSMR locus is associated with increased plaque vulnerability, but not with coronary calcification or cardiovascular disease risk. It remains unclear through which precise biological mechanisms OSM signaling exerts its effects on plaque morphology. However, the OSM-OSMR/LIFR pathway is unlikely to be causally involved in lifetime cardiovascular disease susceptibility.

Systemic PFOS and PFOA exposure and disturbed lipid homeostasis in humans: what do we know and what not?

Associations between per- and polyfluoroalkyl substances (PFASs) and increased blood lipids have been repeatedly observed in humans, but a causal relation has been debated. Rodent studies show reverse effects, i.e. decreased blood cholesterol and triglycerides, occurring however at PFAS serum levels at least 100-fold higher than those in humans. This paper aims to present the main issues regarding the modulation of lipid homeostasis by the two most common PFASs, PFOS and PFOA, with emphasis on the underlying mechanisms relevant for humans. Overall, the apparent contrast between human and animal data may be an artifact of dose, with different molecular pathways coming into play upon exposure to PFASs at very low versus high levels. Altogether, the interpretation of existing rodent data on PFOS/PFOA-induced lipid perturbations with respect to the human situation is complex. From a mechanistic perspective, research on human liver cells shows that PFOS/PFOA activate the PPARα pathway, whereas studies on the involvement of other nuclear receptors, like PXR, are less conclusive. Other data indicate that suppression of the nuclear receptor HNF4α signaling pathway, as well as perturbations of bile acid metabolism and transport might be important cellular events that require further investigation. Future studies with human-relevant test systems would help to obtain more insight into the mechanistic pathways pertinent for humans. These studies shall be designed with a careful consideration of appropriate dosing and toxicokinetics, so as to enable biologically plausible quantitative extrapolations. Such research will increase the understanding of possible perturbed lipid homeostasis related to PFOS/ PFOA exposure and the potential implications for human health.

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.

Beneficial effects of elafibranor on NASH in E3L.CETP mice and differences between mice and men.

Non-alcoholic steatohepatitis (NASH) is the most rapidly growing liver disease that is nevertheless without approved pharmacological treatment. Despite great effort in developing novel NASH therapeutics, many have failed in clinical trials. This has raised questions on the adequacy of preclinical models. Elafibranor is one of the drugs currently in late stage development which had mixed results for phase 2/interim phase 3 trials. In the current study we investigated the response of elafibranor in APOE*3Leiden.CETP mice, a translational animal model that displays histopathological characteristics of NASH in the context of obesity, insulin resistance and hyperlipidemia. To induce NASH, mice were fed a high fat and cholesterol (HFC) diet for 15 weeks (HFC reference group) or 25 weeks (HFC control group) or the HFC diet supplemented with elafibranor (15 mg/kg/d) from week 15-25 (elafibranor group). The effects on plasma parameters and NASH histopathology were assessed and hepatic transcriptome analysis was used to investigate the underlying pathways affected by elafibranor. Elafibranor treatment significantly reduced steatosis and hepatic inflammation and precluded the progression of fibrosis. The underlying disease pathways of the model were compared with those of NASH patients and illustrated substantial similarity with molecular pathways involved, with 87% recapitulation of human pathways in mice. We compared the response of elafibranor in the mice to the response in human patients and discuss potential pitfalls when translating preclinical results of novel NASH therapeutics to human patients. When taking into account that due to species differences the response to some targets, like PPAR-α, may be overrepresented in animal models, we conclude that elafibranor may be particularly useful to reduce hepatic inflammation and could be a pharmacologically useful agent for human NASH, but probably in combination with other agents.

No effects of PCSK9-inhibitor treatment on spatial learning, locomotor activity, and novel object recognition in mice.

Monoclonal anti-proprotein convertase subtilisin/kexin type 9 (PSCK9) neutralizing antibodies effectively lower plasma cholesterol levels and decrease cardiovascular events but also raised some concern that cognitive function could worsen as a side effect. Here, we performed experiments in mice to characterize the effect of anti-PCSK9 antibodies on behavior and cognitive function in detail. APOE*3Leiden.CETP mice and B6129SF1/J wildtype mice were fed a Western type diet and treated with the fully human anti-PCSK9 antibody CmAb1 (PL-45134; 10mg*kg-1 s.c.) or vehicle for 6 weeks. Locomotor activity, anxiety levels, recognition memory, and spatial learning were investigated using the open field, novel object recognition test, and Morris water maze, respectively. Serum cholesterol levels in APOE*3Leiden.CETP mice after treatment with anti-PCSK9 antibody were significantly lower compared to controls whereas cholesterol levels in B6129SF1/J wildtype mice remained unchanged at low levels. No apparent differences were found regarding locomotor activity, anxiety, recognition memory, and spatial learning between animals treated with anti-PCSK9 antibody or vehicle in APOE*3Leiden.CETP and B6129SF1/J wildtype mice. In this study, we found no evidence that treatment with anti-PCSK9 antibodies lead to differences in behavior or changes of cognition in mice.

Inhibition of macrophage proliferation dominates plaque regression in response to cholesterol lowering.

Statins induce plaque regression characterized by reduced macrophage content in humans, but the underlying mechanisms remain speculative. Studying the translational APOE*3-Leiden.CETP mouse model with a humanized lipoprotein metabolism, we find that systemic cholesterol lowering by oral atorvastatin or dietary restriction inhibits monocyte infiltration, and reverses macrophage accumulation in atherosclerotic plaques. Contrary to current believes, none of (1) reduced monocyte influx (studied by cell fate mapping in thorax-shielded irradiation bone marrow chimeras), (2) enhanced macrophage egress (studied by fluorescent bead labeling and transfer), or (3) atorvastatin accumulation in murine or human plaque (assessed by mass spectrometry) could adequately account for the observed loss in macrophage content in plaques that undergo phenotypic regression. Instead, suppression of local proliferation of macrophages dominates phenotypic plaque regression in response to cholesterol lowering: the lower the levels of serum LDL-cholesterol and lipid contents in murine aortic and human carotid artery plaques, the lower the rates of in situ macrophage proliferation. Our study identifies macrophage proliferation as the predominant turnover determinant and an attractive target for inducing plaque regression.

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.

Anti-PCSK9 antibodies inhibit pro-atherogenic mechanisms in APOE*3Leiden.CETP mice.

LDL-cholesterol (LDL-C) is a causal pathogenic factor in atherosclerosis. Monoclonal anti-proprotein convertase subtilisin/kexin type 9 (PCSK9) neutralizing antibodies are novel potent LDL-lowering drugs which reduce cardiovascular events. To characterize their effect on atherogenesis, APOE*3Leiden.CETP mice were fed a high cholesterol/high fat diet (WTD) or normal chow (NC) for 18 weeks. Mice on WTD were injected with the human anti-PCSK9 antibody mAb1 (PL-45134, 10 mg*kg-1 s.c.) or 0.9% saline every 10 days. PCSK9 inhibition decreased total cholesterol in serum of APOE*3Leiden.CETP mice and prevented the development of atherosclerosis. The plaque area in the aortic root was reduced by half and macrophage infiltration determined by Ly6c and Mac-3 staining was ameliorated. PCSK9 inhibition decreased markers of inflammation in mononuclear cells (Il-6, Tnfa mRNA), and in serum (CXCL-1,-10,-13; complement factor C5a) compared to control WTD fed animals. The number of circulating Sca-1/VEGF-R2 positive endothelial progenitor cells of the peripheral blood and spleen-derived diLDL/lectin double positive circulating angiogenic cells was increased. To conclude, the PCSK9-mediated anti-atherosclerotic effect involves the upregulation of pro-regeneratory endothelial progenitor cells, a reduction of inflammation and change of plaque composition.