👤 Trea C M Streefland

The chimeric cytokine IC7Fc conveys the metabolic signaling properties of the glycoprotein 130 receptor cytokines interleukin-6 and ciliary neurotrophic factor via membrane-bound signaling. IC7Fc was previously shown to slow the progression of type 2 diabetes mellitus, and here, we demonstrate its effect on atherosclerotic development. In APOE*3-Leiden.CETP mice, an atherosclerosis-prone model with a humanized lipoprotein metabolism, IC7Fc markedly lowered plasma triglyceride and total cholesterol levels. This was mechanistically explained by an inhibition of de novo lipogenesis in the liver, increased synthesis of bile acids from cholesterol, and down-regulated apolipoprotein B synthesis, which resulted in decreased cholesterol secretion in very low-density lipoprotein particles. As a consequence, IC7Fc treatment considerably reduced atherosclerotic lesion formation and vascular inflammation compared with current antihyperlipidemic therapy. In conclusion, IC7Fc is a promising pharmacological treatment for cardiometabolic diseases targeting hyperlipidemia and inflammation. Show less

LIPC encodes hepatic lipase (HL), a liver-bound protein with both phospholipase and triglyceride lipase activity, and involved in the catabolism of circulating lipoproteins. We recently identified the gain-of-function variant HL-E97G, with selectively increased phospholipase activity, as a new genetic cause of familial combined hypocholesterolaemia in humans. The role of HL in the development of atherosclerosis remains controversial. In this context, the action of HL-E97G on the development of atherosclerosis remains unknown. To evaluate the lipid-lowering and anti-atherogenic properties of HL-E97G vs. wildtype HL (HL-WT) in hypercholesterolaemic APOE*3-Leiden.CETP mice, a well-established model for human-like lipoprotein metabolism, and to assess dependence of these effects on the LDL receptor (LDLR) pathway in LDLR-deficient (Ldlr-/-) mice. APOE*3.Leiden.CETP mice or Ldlr-/- mice received an intravenous injection of AAV8 expressing either eGFP (control), HL-WT or HL-E97G (3 × 1011 GC/mouse) while being fed pro-atherogenic diets. Plasma cholesterol levels were measured monthly, and aortic atherosclerotic lesion sizes were assessed at termination. HL-E97G largely decreased plasma total cholesterol exposure in APOE*3-Leiden.CETP mice (-63% vs. control; -58% vs. HL-WT), resulting at least in part from increased uptake of (V)LDL by the liver, accompanied by a marked decrease in atherosclerotic lesion size (-98% vs. control; -97% vs. HL-WT) in the aortic root. Importantly, HL-E97G also strongly reduced plasma cholesterol exposure in Ldlr-/- mice (-80% vs. control; -77% vs. HL-WT), and decreased atherosclerotic lesion size in the aortic root (-54% vs. control; -41% vs. HL-WT) and the aortic arch (-73% vs. control; -70% vs. HL-WT). HL-E97G strongly reduces plasma cholesterol levels, by increasing the uptake of (V)LDL, to decrease atherosclerosis development in mice independently of the LDLR pathway. These data suggest that modulating HL function is a promising tool in patients with familial hypercholesterolaemia. Show less

[D APOE*3-Leiden.CETP mice were treated with saline, acyl-ExD3 or acyl-ExF1 via intraperitoneal injections for 6 weeks or intracerebroventricular infusion for 18 days. Body weight and composition were monitored at regular intervals, as were plasma glucose, triglyceride and cholesterol levels. At endpoint, mice were injected with very low-density lipoprotein (VLDL)-like particles containing glycerol tri[ Upon peripheral treatment, body weight gain was prevented and plasma glucose levels were reduced by acyl-ExF1, but circulating lipids were not affected by either acyl-ExF1 or acyl-ExD3. In contrast, central administration of either agonist strongly reduced plasma triglyceride and cholesterol levels, but did not affect glucose levels. Acyl-ExD3 and acyl-ExF1 increased [ The oppositely biased GLP-1 receptor agonists acyl-ExD3 and acyl-ExF1 do not differentially affect lipid metabolism in APOE*3-Leiden.CETP mice, while effects on glucose homeostasis and prevention of body weight gain are more pronounced upon peripheral acyl-ExF1 treatment. Show less

Angiopoietin-like 3 (ANGPTL3) and 4 (ANGPTL4) inhibit lipoprotein lipase to regulate tissue fatty acid (FA) uptake from triglyceride (TG)-rich lipoproteins such as very low density lipoproteins (VLDL). While pharmacological inhibition of ANGPTL3 is being evaluated as a lipid-lowering strategy, systemic ANGPTL4 inhibition is not pursued due to adverse effects. This study aims to compare the therapeutic potential of liver-specific Angptl3 and Angptl4 silencing to attenuate hyperlipidemia and atherosclerosis development in APOE*3-Leiden.CETP mice, a well-established humanized model for lipoprotein metabolism. Mice were subcutaneously injected twice per week with saline or liver-targeted antisense oligonucleotides against Angptl3, Angptl4, both, or a scrambled oligonucleotide. Plasma lipid levels, VLDL clearance, and hepatic VLDL production were determined, and atherosclerosis development was assessed. For toxicological evaluation, cynomolgus monkeys were treated with three dosages of liver-targeted ANGPTL4-silencing oligonucleotides. Liver-targeted Angptl4 silencing reduced plasma TGs (-48%) and total cholesterol (-56%), explained by higher VLDL-derived FA uptake by brown adipose tissue and lower VLDL production by the liver. Accordingly, Angptl4 silencing reduced atherosclerotic lesion size (-86%) and improved lesion stability. Hepatic Angptl3 silencing similarly attenuated hyperlipidemia and atherosclerosis development. While Angptl3 and Angptl4 silencing lowered plasma TGs in the refed and fasted state, respectively, combined Angptl3/4 silencing lowered plasma TGs independent of the nutritional state. In cynomolgus monkeys, anti-ANGPTL4 ASO treatment was well tolerated without adverse effects. Liver-targeted Angptl4 silencing potently attenuates hyperlipidemia and atherosclerosis development in APOE*3-Leiden.CETP mice, and liver-targeted ANGPTL4 silencing is well tolerated in non-human primates. These data warrant further clinical development of liver-targeted ANGPTL4 silencing. Show less

Circadian disturbance (CD) is the consequence of a mismatch between endogenous circadian rhythms, behaviour, and/or environmental cycles, and frequently occurs during shift work. Shift work has been associated with elevated risk for atherosclerotic cardiovascular disease (asCVD) in humans, but evidence for the effectiveness of prevention strategies is lacking. Here, we applied time-restricted feeding (TRF) as a strategy to counteract atherosclerosis development during CD in female APOE∗3-Leiden.CETP mice, a well-established model for humanized lipoprotein metabolism. Control groups were subjected to a fixed 12:12 h light-dark cycle, while CD groups were subjected to 6-h phase advancement every 3 days. Groups had either ad libitum (AL) access to food or were subjected to TRF with restricted food access to the dark phase. TRF did not prevent the increase in the relative abundance of circulating inflammatory monocytes and elevation of (postprandial) plasma triglycerides during CD. Nonetheless, TRF reduced atherosclerotic lesion size and prevented an elevation in macrophage content of atherosclerotic lesions during CD, while it increased the relative abundance of anti-inflammatory monocytes, prevented activation of T cells, and lowered plasma total cholesterol levels and markers of hepatic cholesterol synthesis. These effects were independent of total food intake. We propose that time restricted eating could be a promising strategy for the primary prevention of asCVD risk in shift workers, which warrants future study in humans. This work was funded by the Novo Nordisk Foundation, the Netherlands Ministry of Social Affairs and Employment, Amsterdam Cardiovascular Sciences, and the Dutch Heart Foundation. Show less

The metabolic and inflammatory processes that are implicated in the development of cardiovascular diseases are under control of the biological clock. While skeletal muscle function exhibits circadian rhythms, it is unclear to what extent the beneficial health effects of exercise are restricted to unique time windows. We aimed to study whether the timing of exercise training differentially modulates the development of atherosclerosis and elucidate underlying mechanisms. We endurance-trained atherosclerosis-prone female APOE*3-Leiden.CETP mice fed a Western-type diet, a well-established human-like model for cardiometabolic diseases, for 1 h five times a week for 4 weeks either in their early or in their late active phase on a treadmill. We monitored metabolic parameters, the development of atherosclerotic lesions in the aortic root and assessed the composition of the gut microbiota. Late, but not early, exercise training reduced fat mass by 19% and the size of early-stage atherosclerotic lesions by as much as 29% compared to sedentary animals. No correlation between cholesterol exposure and lesion size was evident, as no differences in plasma lipid levels were observed, but circulating levels of the pro-inflammatory markers ICAM-1 and VCAM-1 were reduced with late exercise. Strikingly, we observed a time-of-day-dependent effect of exercise training on the composition of the gut microbiota as only late training increased the abundance of gut bacteria producing short-chain fatty acids with proposed anti-inflammatory properties. Together, these findings indicate that timing is a critical factor to the beneficial anti-atherosclerotic effects of exercise with a great potential to further optimize training recommendations for patients. Show less

Brown adipose tissue (BAT) burns fatty acids (FAs) to produce heat, and shows diurnal oscillation in glucose and triglyceride (TG)-derived FA-uptake, peaking around wakening. Here we aimed to gain insight in the diurnal regulation of metabolic BAT activity. RNA-sequencing, chromatin immunoprecipitation (ChIP)-sequencing, and lipidomics analyses were performed on BAT samples of wild type C57BL/6J mice collected at 3-hour intervals throughout the day. Knockout and overexpression models were used to study causal relationships in diurnal lipid handling by BAT. We identified pronounced enrichment of oscillating genes involved in extracellular lipolysis in BAT, accompanied by oscillations of FA and monoacylglycerol content. This coincided with peak lipoprotein lipase (Lpl) expression, and was predicted to be driven by peroxisome proliferator-activated receptor gamma (PPARγ) activity. ChIP-sequencing for PPARγ confirmed oscillation in binding of PPARγ to Lpl. Of the known LPL-modulators, angiopoietin-like 4 (Angptl4) showed the largest diurnal amplitude opposite to Lpl, and both Angptl4 knockout and overexpression attenuated oscillations of LPL activity and TG-derived FA-uptake by BAT. Our findings highlight involvement of PPARγ and a crucial role of ANGPTL4 in mediating the diurnal oscillation of TG-derived FA-uptake by BAT, and imply that time of day is essential when targeting LPL activity in BAT to improve metabolic health. Show less

Brown adipose tissue (BAT) contributes to cardiometabolic health by taking up glucose and lipids for oxidation, a process that displays a strong diurnal rhythm. While aging has been shown to reduce thermogenic characteristics of BAT, it is as yet unknown whether this reduction is specific to the time of day. Therefore, we assessed whole-body and BAT energy metabolism in young and middle-aged male and female C57BL/6J mice and studied the consequences for lipid metabolism in humanized APOE*3-Leiden.CETP mice (also on a C57BL/6J background). We demonstrate that in middle-aged versus young mice body temperature is lower in both male and female mice, while uptake of triglyceride (TG)-derived fatty acids (FAs) by BAT, reflecting metabolic activity, is attenuated at its peak at the onset of the dark (wakeful) phase in female mice. This coincided with delayed plasma clearance of TG-rich lipoproteins and TG-depleted lipoprotein core remnants, and elevated plasma TGs at the same time point. Furthermore, middle-aged female mice showed increased adiposity, accompanied by lipid accumulation, increased expression of genes involved in lipogenesis, and reduced expression of genes involved in fat oxidation and the intracellular clock machinery in BAT. Peak abundance of lipoprotein lipase (LPL), a crucial regulator of FA uptake, was attenuated in BAT. Our findings suggest that LPL is a potential therapeutic target for restoring diurnal metabolic BAT activity, and that efficiency of strategies targeting BAT may be improved by including time of day as an important factor. Show less

Exercise has been shown to improve cardiometabolic health, yet neither the molecular connection nor the effects of exercise timing have been elucidated. The aim of this study was to investigate whether ad libitum or time-restricted mild exercise reduces atherosclerosis development in atherosclerosis-prone dyslipidemic APOE*3-Leiden.CETP mice and whether mild exercise training in men with obesity affects lipoprotein levels. Mice were group-housed and subjected to ad libitum or time-restricted (first or last 6 hours of the active phase) voluntary wheel running for 16 weeks while on a cholesterol-rich diet, after which atherosclerosis development was assessed in the aortic root. Furthermore, nine men with obesity followed a 12-week mild exercise training program. Lipoprotein levels were measured by nuclear magnetic resonance spectroscopy in plasma collected pre and post exercise training. Wheel running did not affect plasma lipid levels, uptake of triglyceride-derived fatty acids by tissues, and aortic atherosclerotic lesion size or severity. Markers of training status were unaltered. Exercise training in men with obesity did not alter lipoprotein levels. Mild exercise training does not reduce dyslipidemia or atherosclerosis development in APOE*3-Leiden.CETP mice or affect lipoprotein levels in humans. Future research on the effects of (time-restricted) exercise on atherosclerosis or lipid metabolism should consider more vigorous exercise protocols. Show less

Mannan oligosaccharides (MOS) have proven effective at improving growth performance, while also reducing hyperlipidemia and inflammation. As atherosclerosis is accelerated both by hyperlipidemia and inflammation, we aim to determine the effect of dietary MOS on atherosclerosis development in hyperlipidemic ApoE*3-Leiden.CETP (E3L.CETP) mice, a well-established model for human-like lipoprotein metabolism. Female E3L.CETP mice were fed a high-cholesterol diet, with or without 1% MOS for 14 weeks. MOS substantially decreased atherosclerotic lesions up to 54%, as assessed in the valve area of the aortic root. In blood, IL-1RA, monocyte subtypes, lipids, and bile acids (BAs) were not affected by MOS. Gut microbiota composition was determined using 16S rRNA gene sequencing and MOS increased the abundance of cecal Bacteroides ovatus. MOS did not affect fecal excretion of cholesterol, but increased fecal BAs as well as butyrate in cecum as determined by gas chromatography mass spectrometry. MOS decreased the onset of atherosclerosis development via lowering of plasma cholesterol levels. These effects were accompanied by increased cecal butyrate and fecal excretion of BAs, presumably mediated via interactions of MOS with the gut microbiota. Show less