Liver-targeted Angptl4 silencing by antisense oligonucleotide treatment attenuates hyperlipidemia and atherosclerosis development in APOE*3-Leiden.CETP mice.

BACKGROUND AND AIMS: Angiopoietin-like 3 (ANGPTL3) and 4 (ANGPTL4) inhibit lipoprotein lipase to regulate tissue fatty acid uptake from triglyceride-rich lipoproteins such as VLDL. While pharmacological inhibition of ANGPTL3 is being evaluated as lipid-lowering strategy, systemic ANGPTL4 inhibition is not pursued due to adverse effects. This study aimed 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. METHODS AND RESULTS: Mice were subcutaneously injected twice-weekly 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 triglycerides (-48%) and total cholesterol (-56%), explained by higher VLDL-derived fatty acid 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 triglycerides in the refed and fasted state, respectively, combined Angptl3/4 silencing lowered plasma triglycerides independent of nutritional state. In cynomolgus monkeys, anti-ANGPTL4 ASO treatment was well tolerated without adverse effects. CONCLUSIONS: 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.

Doublecortin-like knockdown in mice attenuates obesity by stimulating energy expenditure in adipose tissue.

Crosstalk between peripheral metabolic organs and the central nervous system is essential for body weight control. At the base of the hypothalamus, ß-tanycytes surround the portal capillaries and function as gatekeepers to facilitate transfer of substances from the circulation into the cerebrospinal fluid and vice versa. Here, we investigated the role of the neuroplasticity gene doublecortin-like (DCL), highly expressed by ß-tanycytes, in body weight control and whole-body energy metabolism. We demonstrated that DCL-knockdown through a doxycycline-inducible shRNA expression system prevents body weight gain by reducing adiposity in mice. DCL-knockdown slightly increased whole-body energy expenditure possibly as a result of elevated circulating thyroid hormones. In white adipose tissue (WAT) triglyceride uptake was increased while the average adipocyte cell size was reduced. At histological level we observed clear signs of browning, and thus increased thermogenesis in WAT. We found no indications for stimulated thermogenesis in brown adipose tissue (BAT). Altogether, we demonstrate an important, though subtle, role of tanycytic DCL in body weight control through regulation of energy expenditure, and specifically WAT browning. Elucidating mechanisms underlying the role of DCL in regulating brain-peripheral crosstalk further might identify new treatment targets for obesity.

Time-restricted feeding attenuates hypercholesterolaemia and atherosclerosis development during circadian disturbance in APOE∗3-Leiden.CETP mice.

BACKGROUND: 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. METHODS: 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. FINDINGS: 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. INTERPRETATION: 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. FUNDING: 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.

Aging attenuates diurnal lipid uptake by brown adipose tissue.

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.

von Willebrand Factor: A Central Regulator of Arteriovenous Fistula Maturation Through Smooth Muscle Cell Proliferation and Outward Remodeling.

Background Arteriovenous fistula (AVF) maturation failure is a main limitation of vascular access. Maturation is determined by the intricate balance between outward remodeling and intimal hyperplasia, whereby endothelial cell dysfunction, platelet aggregation, and vascular smooth muscle cell (VSMC) proliferation play a crucial role. von Willebrand Factor (vWF) is an endothelial cell-derived protein involved in platelet aggregation and VSMC proliferation. We investigated AVF vascular remodeling in vWF-deficient mice and vWF expression in failed and matured human AVFs. Methods and Results Jugular-carotid AVFs were created in wild-type and vWF-/- mice. AVF flow was determined longitudinally using ultrasonography, whereupon AVFs were harvested 14 days after surgery. VSMCs were isolated from vena cavae to study the effect of vWF on VSMC proliferation. Patient-matched samples of the basilic vein were obtained before brachio-basilic AVF construction and during superficialization or salvage procedure 6 weeks after AVF creation. vWF deficiency reduced VSMC proliferation and macrophage infiltration in the intimal hyperplasia. vWF-/- mice showed reduced outward remodeling (1.5-fold, P=0.002) and intimal hyperplasia (10.2-fold, P<0.0001). AVF flow in wild-type mice was incremental over 2 weeks, whereas flow in vWF-/- mice did not increase, resulting in a two-fold lower flow at 14 days compared with wild-type mice (P=0.016). Outward remodeling in matured patient AVFs coincided with increased local vWF expression in the media of the venous outflow tract. Absence of vWF in the intimal layer correlated with an increase in the intima-media ratio. Conclusions vWF enhances AVF maturation because its positive effect on outward remodeling outweighs its stimulating effect on intimal hyperplasia.

Gene expression changes in the brain of a Cushing's syndrome mouse model.

Excess glucocorticoid exposure affects emotional and cognitive brain functions. The extreme form, Cushing's syndrome, is adequately modelled in the AdKO2.0 mouse, consequential to adrenocortical hypertrophy and hypercorticosteronemia. We previously reported that the AdKO2.0 mouse brain undergoes volumetric changes that resemble closely those of Cushing's syndrome human patients, as well as changes in expression of glial related marker proteins. In the present work, the expression of genes related to glial and neuronal cell populations and functions was assessed in regions of the anterior brain, hippocampus, amygdala and hypothalamus. Glucocorticoid target genes were consistently regulated, including CRH mRNA suppression in the hypothalamus and induction in amygdala and hippocampus, even if glucocorticoid receptor protein was downregulated. Expression of glial genes was also affected in the AdKO2.0 mouse brain, indicating a different activation status in glial cells. Generic markers for neuronal cell populations, and cellular integrity were only slightly affected. Our findings highlight the vulnerability of glial cell populations to chronic high levels of circulating glucocorticoids.

Liposomal Delivery Improves the Efficacy of Prednisolone to Attenuate Renal Inflammation in a Mouse Model of Acute Renal Allograft Rejection.

BACKGROUND: Systemic exposure to high-dose corticosteroids effectively combats acute rejection after kidney transplantation, but at the cost of substantial side effects. In this study, a murine acute renal allograft rejection model was used to investigate whether liposomal-encapsulated prednisolone (LP) facilitates local exposure to enhance its therapeutic effect. METHODS: Male BalbC recipients received renal allografts from male C57BL/6J donors. Recipients were injected daily with 5 mg/kg cyclosporine A and received either 10 mg/kg prednisolone (P), or LP intravenously on day 0, 3, and 6, or no additional treatment. Functional magnetic resonance imaging (fMRI) was performed on day 6 to study allograft perfusion and organs were retrieved on day 7 for further analysis. RESULTS: Staining of polyethylene-glycol-labeled liposomes and high performance liquid chromatography analysis revealed accumulation in the LP treated allograft. LP treatment induced the expression of glucocorticoid responsive gene Fkbp5 in the allograft. Flow-cytometry of allografts revealed liposome presence in CD45 cells, and reduced numbers of F4/80 macrophages, and CD3 T-lymphocytes upon LP treatment. Banff scoring showed reduced interstitial inflammation and tubulitis and fMRI analysis revealed improved allograft perfusion in LP versus NA mice. CONCLUSIONS: Liposomal delivery of prednisolone improved renal bio-availability, increased perfusion and reduced cellular infiltrate in the allograft, when compared with conventional prednisolone. Clinical studies should reveal if treatment with LP results in improved efficacy and reduced side effects in patients with renal allograft rejection.

Glomerular permeability is not affected by heparan sulfate glycosaminoglycan deficiency in zebrafish embryos.

Proteinuria develops when specific components in the glomerular filtration barrier have impaired function. Although the precise components involved in maintaining this barrier have not been fully identified, heparan sulfate proteoglycans are believed to play an essential role in maintaining glomerular filtration. Although in situ studies have shown that a loss of heparan sulfate glycosaminoglycans increases the permeability of the glomerular filtration barrier, recent studies using experimental models have shown that podocyte-specific deletion of heparan sulfate glycosaminoglycan assembly does not lead to proteinuria. However, tubular reabsorption of leaked proteins might have masked an increase in glomerular permeability in these models. Furthermore, not only podocytes but also glomerular endothelial cells are involved in heparan sulfate synthesis in the glomerular filtration barrier. Therefore, we investigated the effect of a global heparan sulfate glycosaminoglycan deficiency on glomerular permeability. We used a zebrafish embryo model carrying a homozygous germline mutation in the ext2 gene. Glomerular permeability was assessed with a quantitative dextran tracer injection method. In this model, we accounted for tubular reabsorption. Loss of anionic sites in the glomerular basement membrane was measured using polyethyleneimine staining. Although mutant animals had significantly fewer negatively charged areas in the glomerular basement membrane, glomerular permeability was unaffected. Moreover, heparan sulfate glycosaminoglycan-deficient embryos had morphologically intact podocyte foot processes. Glomerular filtration remains fully functional despite a global reduction of heparan sulfate.

Indirect Burst Pressure Measurements for the Mechanical Assessment of Biological Vessels.

IMPACT STATEMENT: Vascular tissue engineering (VTE) is a rapidly expanding field, with numerous approaches being explored both in preclinical and clinical settings. A pivotal factor in the development of VTE techniques is patient safety, notably with respect to the mechanical properties of the vessels. Of the mechanical properties, the bursting strength, representing the ability of a vessel to withstand the forces exerted on it by blood pressure, is the most important. The burst pressure is commonly assessed using one of three methods proposed by the ISO 7198. In this study, we evaluate the three burst pressure assessment methods exactly as they are presently in the field of VTE. We show that the indirect assessment methods, as they are presently used, provide inconsistent and therefore unreliable estimates of the true yield stress of a vessel.

Deficiency of TLR4 homologue RP105 aggravates outward remodeling in a murine model of arteriovenous fistula failure.

Arteriovenous access dysfunction is a major cause of morbidity for hemodialysis patients. The pathophysiology of arteriovenous fistula (AVF) maturation failure is associated with inflammation, impaired outward remodeling (OR) and intimal hyperplasia. RP105 is a critical physiologic regulator of TLR4 signaling in numerous cell types. In the present study, we investigated the impact of RP105 on AVF maturation, and defined cell-specific effects of RP105 on macrophages and vascular smooth muscle cells (VSMCs). Overall, RP105-/- mice displayed a 26% decrease in venous OR. The inflammatory response in RP105-/- mice was characterized by accumulation of anti-inflammatory macrophages, a 76% decrease in pro- inflammatory macrophages, a 70% reduction in T-cells and a 50% decrease in MMP-activity. In vitro, anti-inflammatory macrophages from RP105-/- mice displayed increased IL10 production, while MCP1 and IL6 levels secreted by pro-inflammatory macrophages were elevated. VSMC content in RP105-/- AVFs was markedly decreased. In vitro, RP105-/- venous VSMCs proliferation was 50% lower, whereas arterial VSMCs displayed a 50% decrease in migration, relative to WT. In conclusion, the impaired venous OR in RP105-/- mice could result from of a shift in both macrophages and VSMCs towards a regenerative phenotype, identifying a novel relationship between inflammation and VSMC function in AVF maturation.

Development and evaluation of in vivo tissue engineered blood vessels in a porcine model.

BACKGROUND: There's a large clinical need for novel vascular grafts. Tissue engineered blood vessels (TEBVs) have great potential to improve the outcome of vascular grafting procedures. Here, we present a novel approach to generate autologous TEBV in vivo. Polymer rods were engineered and implanted, evoking an inflammatory response that culminates in encapsulation by a fibrocellular capsule. We hypothesized that, after extrusion of the rod, the fibrocellular capsule differentiates into an adequate vascular conduit once grafted into the vasculature. METHODS AND RESULTS: Rods were implanted subcutaneously in pigs. After 4 weeks, rods with tissue capsules grown around it were harvested. Tissue capsules were grafted bilaterally as carotid artery interposition. One and 4-week patency were evaluated by angiography whereupon pigs were sacrificed. Tissue capsules before and after grafting were evaluated on tissue remodeling using immunohistochemistry, RNA profiling and mechanical testing. Rods were encapsulated by thick, well-vascularized tissue capsules, composed of circumferentially aligned fibroblasts, collagen and few leukocytes, with adequate mechanical strength. Patency was 100% after 1 week and 87.5% after 4 weeks. After grafting, tissue capsules remodeled towards a vascular phenotype. Gene profiles of TEBVs gained more similarity with carotid artery. Wall thickness and αSMA-positive area significantly increased. Interestingly, a substantial portion of (myo)fibroblasts present before grafting expressed smooth muscle cell markers. While leukocytes were hardly present anymore, the lumen was largely covered with endothelial cells. Burst pressure remained stable after grafting. CONCLUSIONS: Autologous TEBVs were created in vivo with sufficient mechanical strength enabling vascular grafting. Grafts differentiated towards a vascular phenotype upon grafting.