Susan Kühnast

Alirocumab inhibits atherosclerosis, improves the plaque morphology, and enhances the effects of a statin

Proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibition is a potential novel strategy for treatment of CVD. Alirocumab is a fully human PCSK9 monoclonal antibody in phase 3 clinical development. We evaluated the antiatherogenic potential of alirocumab in APOE*3Leiden.CETP mice. Mice received a Western-type diet and were treated with alirocumab (3 or 10 mg/kg, weekly subcutaneous dosing) alone and in combination with atorvastatin (3.6 mg/kg/d) for 18 weeks. Alirocumab alone dose-dependently decreased total cholesterol (−37%; −46%, P < 0.001) and TGs (−36%; −39%, P < 0.001) and further decreased cholesterol in combination with atorvastatin (−48%; −58%, P < 0.001). Alirocumab increased hepatic LDL receptor protein levels but did not affect hepatic cholesterol and TG content. Fecal output of bile acids and neutral sterols was not changed. Alirocumab dose-dependently decreased atherosclerotic lesion size (−71%; −88%, P < 0.001) and severity and enhanced these effects when added to atorvastatin (−89%; −98%, P < 0.001). Alirocumab reduced monocyte recruitment and improved the lesion composition by increasing the smooth muscle cell and collagen content and decreasing the macrophage and necrotic core content. Alirocumab dose-dependently decreases plasma lipids and, as a result, atherosclerosis development, and it enhances the beneficial effects of atorvastatin in APOE*3Leiden.CETP mice. In addition, alirocumab improves plaque morphology.

Anacetrapib reduces progression of atherosclerosis, mainly by reducing non-HDL-cholesterol, improves lesion stability and adds to the beneficial effects of atorvastatin

The present study is the first intervention study in a well-established, translational mouse model for hyperlipidaemia and atherosclerosis showing that anacetrapib dose-dependently reduces atherosclerosis development and adds to the anti-atherogenic effects of atorvastatin. This effect is mainly ascribed to the reduction in non-HDL-C despite a remarkable increase in HDL-C and without affecting HDL functionality. In addition, anacetrapib improves lesion stability.

Aliskiren inhibits atherosclerosis development and improves plaque stability in APOE*3Leiden.CETP transgenic mice with or without treatment with atorvastatin.

Objective Aliskiren is the first commercially available, orally active, direct renin inhibitor approved to treat hypertension. The renin–angiotensin system has been shown to be a significant contributor to the development of hypercholesterolemia-induced atherosclerosis. The aim of this study was to evaluate the antiatherosclerotic and plaque stabilization effects of aliskiren alone and in combination with atorvastatin. Methods APOE*3Leiden.CETP mice (n = 14–17/group) were fed a western-type diet (containing 0.25% cholesterol) alone or were treated with either aliskiren (15 mg/kg per day), atorvastatin (3.6 mg/kg per day) or a combination of aliskiren and atorvastatin. Effects on SBP, total cholesterol, inflammation markers and atherosclerotic size and composition were assessed. Results Aliskiren reduced SBP (−19%, P < 0.001) and atorvastatin reduced total cholesterol (−24%, P < 0.001). Atherosclerotic lesion area was reduced by aliskiren (−40%, P < 0.01), atorvastatin (−61%, P < 0.001) and the combination treatment (−69%, P < 0.001). Aliskiren alone and together with atorvastatin decreased the number of T cells in the aortic root area (−60%, P < 0.01; −41%, P < 0.05), as well as macrophage (−64%, P < 0.001; −72%, P < 0.001) and necrotic area (−52%, P = 0.071; −84%, P < 0.001) in the lesion. Atorvastatin alone and together with aliskiren decreased monocyte adherence (−43%, P < 0.05 and −51%, P < 0.01) and monocyte chemoattractant protein-1 (both −36%, P < 0.01). The combination treatment decreased the number of lesions (−17%, P < 0.05) and E-selectin (−17%, P < 0.05). Conclusion Aliskiren inhibited atherosclerosis development and improved plaque stability alone and in combination with atorvastatin, possibly via a mechanism involving T cells. These results suggest a potential benefit of using aliskiren in a clinical setting, particularly in combination with statin treatment.

Anacetrapib reduces (V)LDL cholesterol by inhibition of CETP activity and reduction of plasma PCSK9

Recently, we showed in APOE*3-Leiden cholesteryl ester transfer protein (E3L.CETP) mice that anacetrapib attenuated atherosclerosis development by reducing (V)LDL cholesterol [(V)LDL-C] rather than by raising HDL cholesterol. Here, we investigated the mechanism by which anacetrapib reduces (V)LDL-C and whether this effect was dependent on the inhibition of CETP. E3L.CETP mice were fed a Western-type diet alone or supplemented with anacetrapib (30 mg/kg body weight per day). Microarray analyses of livers revealed downregulation of the cholesterol biosynthesis pathway (P < 0.001) and predicted downregulation of pathways controlled by sterol regulatory element-binding proteins 1 and 2 (z-scores −2.56 and −2.90, respectively; both P < 0.001). These data suggest increased supply of cholesterol to the liver. We found that hepatic proprotein convertase subtilisin/kexin type 9 (Pcsk9) expression was decreased (−28%, P < 0.01), accompanied by decreased plasma PCSK9 levels (−47%, P < 0.001) and increased hepatic LDL receptor (LDLr) content (+64%, P < 0.01). Consistent with this, anacetrapib increased the clearance and hepatic uptake (+25%, P < 0.001) of [14C]cholesteryl oleate-labeled VLDL-mimicking particles. In E3L mice that do not express CETP, anacetrapib still decreased (V)LDL-C and plasma PCSK9 levels, indicating that these effects were independent of CETP inhibition. We conclude that anacetrapib reduces (V)LDL-C by two mechanisms: 1) inhibition of CETP activity, resulting in remodeled VLDL particles that are more susceptible to hepatic uptake; and 2) a CETP-independent reduction of plasma PCSK9 levels that has the potential to increase LDLr-mediated hepatic remnant clearance.

Niacin Reduces Atherosclerosis Development in APOE*3Leiden.CETP Mice Mainly by Reducing NonHDL-Cholesterol

Objective Niacin potently lowers triglycerides, mildly decreases LDL-cholesterol, and largely increases HDL-cholesterol. Despite evidence for an atheroprotective effect of niacin from previous small clinical studies, the large outcome trials, AIM-HIGH and HPS2-THRIVE did not reveal additional beneficial effects of niacin (alone or in combination with laropiprant) on top of statin treatment. We aimed to address this apparent discrepancy by investigating the effects of niacin without and with simvastatin on atherosclerosis development and determine the underlying mechanisms, in APOE*3Leiden.CETP mice, a model for familial dysbetalipoproteinemia (FD). Approach and Results Mice were fed a western-type diet containing cholesterol without or with niacin (120 mg/kg/day), simvastatin (36 mg/kg/day) or their combination for 18 weeks. Similarly as in FD patients, niacin reduced total cholesterol by -39% and triglycerides by −50%, (both P<0.001). Simvastatin and the combination reduced total cholesterol (−30%; −55%, P<0.001) where the combination revealed a greater reduction compared to simvastatin (−36%, P<0.001). Niacin decreased total cholesterol and triglycerides primarily by increasing VLDL clearance. Niacin increased HDL-cholesterol (+28%, P<0.01) and mildly increased reverse cholesterol transport. All treatments reduced monocyte adhesion to the endothelium (−46%; −47%, P<0.01; −53%, P<0.001), atherosclerotic lesion area (−78%; −49%, P<0.01; −87%, P<0.001) and severity. Compared to simvastatin, the combination increased plaque stability index [(SMC+collagen)/macrophages] (3-fold, P<0.01). Niacin and the combination reduced T cells in the aortic root (−71%, P<0.01; −81%, P<0.001). Lesion area was strongly predicted by nonHDL-cholesterol (R2 = 0.69, P<0.001) and to a much lesser extent by HDL-cholesterol (R2 = 0.20, P<0.001). Conclusion Niacin decreases atherosclerosis development mainly by reducing nonHDL-cholesterol with modest HDL-cholesterol-raising and additional anti-inflammatory effects. The additive effect of niacin on top of simvastatin is mostly dependent on its nonHDL-cholesterol-lowering capacities. These data suggest that clinical beneficial effects of niacin are largely dependent on its ability to lower LDL-cholesterol on top of concomitant lipid-lowering therapy.

Innovative pharmaceutical interventions in cardiovascular disease: Focusing on the contribution of non-HDL-C/LDL-C-lowering versus HDL-C-raisingA systematic review and meta-analysis of relevant preclinical studies and clinical trials

Non-HDL-cholesterol is well recognised as a primary causal risk factor in cardiovascular disease. However, despite consistent epidemiological evidence for an inverse association between HDL-C and coronary heart disease, clinical trials aimed at raising HDL-C (AIM-HIGH, HPS2-THRIVE, dal-OUTCOMES) failed to meet their primary goals. This systematic review and meta-analysis investigated the effects of established and novel treatment strategies, specifically targeting HDL, on inhibition of atherosclerosis in cholesteryl ester transfer protein-expressing animals, and the prevention of clinical events in randomised controlled trials. Linear regression analyses using data from preclinical studies revealed associations for TC and non-HDL-C and lesion area (R(2)=0.258, P=0.045; R(2)=0.760, P<0.001), but not for HDL-C (R(2)=0.030, P=0.556). In clinical trials, non-fatal myocardial infarction risk was significantly less in the treatment group with pooled odd ratios of 0.87 [0.81; 0.94] for all trials and 0.85 [0.78; 0.93] after excluding some trials due to off-target adverse events, whereas all-cause mortality was not affected (OR 1.05 [0.99-1.10]). Meta-regression analyses revealed a trend towards an association between between-group differences in absolute change from baseline in LDL-C and non-fatal myocardial infarction (P=0.066), whereas no correlation was found for HDL-C (P=0.955). We conclude that the protective role of lowering LDL-C and non-HDL-C is well-established. The contribution of raising HDL-C on inhibition of atherosclerosis and the prevention of cardiovascular disease remains undefined and may be dependent on the mode of action of HDL-C-modification. Nonetheless, treatment strategies aimed at improving HDL function and raising apolipoprotein A-I may be worth exploring.

Variable cartilage degradation in mice with diet-induced metabolic dysfunction : Food for thought

OBJECTIVE Human cohort studies have demonstrated a role for systemic metabolic dysfunction in osteoarthritis (OA) pathogenesis in obese patients. To explore the mechanisms underlying this metabolic phenotype of OA, we examined cartilage degradation in the knees of mice from different genetic backgrounds in which a metabolic phenotype was established by various dietary approaches. DESIGN Wild-type C57BL/6J mice and genetically modified mice (hCRP, LDLr-/-. Leiden and ApoE*3Leiden.CETP mice) based on C57BL/6J background were used to investigate the contribution of inflammation and altered lipoprotein handling on diet-induced cartilage degradation. High-caloric diets of different macronutrient composition (i.e., high-carbohydrate or high-fat) were given in regimens of varying duration to induce a metabolic phenotype with aggravated cartilage degradation relative to controls. RESULTS Metabolic phenotypes were confirmed in all studies as mice developed obesity, hypercholesteremia, glucose intolerance and/or insulin resistance. Aggravated cartilage degradation was only observed in two out of the twelve experimental setups, specifically in long-term studies in male hCRP and female ApoE*3Leiden.CETP mice. C57BL/6J and LDLr-/-. Leiden mice did not develop HFD-induced OA under the conditions studied. Osteophyte formation and synovitis scores showed variable results between studies, but also between strains and gender. CONCLUSIONS Long-term feeding of high-caloric diets consistently induced a metabolic phenotype in various C57BL/6J (-based) mouse strains. In contrast, the induction of articular cartilage degradation proved variable, which suggests that an additional trigger might be necessary to accelerate diet-induced OA progression. Gender and genetic modifications that result in a humanized pro-inflammatory state (human CRP) or lipoprotein metabolism (human-E3L.CETP) were identified as important contributing factors.