Interventional hepatic ApoC-III knockdown improves atherosclerotic plaque stability and remodeling upon triglyceride lowering

Apolipoprotein C-III (apoC-III) is a critical regulator of triglyceride metabolism and correlates positively with hypertriglyceridemia and cardiovascular disease (CVD). It remains unclear if therapeutic apoC-III lowering reduces CVD risk and if the CVD correlation depends on the lipid-lowering or antiinflammatory properties. We determined the impact of interventional apoC-III lowering on atherogenesis using an apoC-III antisense oligonucleotide (ASO) in 2 hypertriglyceridemic mouse models where the intervention lowers plasma triglycerides and in a third lipid-refractory model. On a high-cholesterol Western diet apoC-III ASO treatment did not alter atherosclerotic lesion size but did attenuate advanced and unstable plaque development in the triglyceride-responsive mouse models. No lesion size or composition improvement was observed with apoC-III ASO in the lipid-refractory mice. To circumvent confounding effects of continuous high-cholesterol feeding, we tested the impact of interventional apoC-III lowering when switching to a cholesterol-poor diet after 12 weeks of Western diet. In this diet switch regimen, apoC-III ASO treatment significantly reduced plasma triglycerides, atherosclerotic lesion progression, and necrotic core area and increased fibrous cap thickness in lipid-responsive mice. Again, apoC-III ASO treatment did not alter triglyceride levels, lesion development, and lesion composition in lipid-refractory mice after the diet switch. Our findings suggest that interventional apoC-III lowering might be an effective strategy to reduce atherosclerosis lesion size and improve plaque stability when lipid lowering is achieved

The human milk oligosaccharide 3′sialyllactose reduces low-grade inflammation and atherosclerosis development in mice

Macrophages contribute to the induction and resolution of inflammation and play a central role in chronic low-grade inflammation in cardiovascular diseases caused by atherosclerosis. Human milk oligosaccharides (HMOs) are complex unconjugated glycans unique to human milk that benefit infant health and act as innate immune modulators. Here, we identify the HMO 3'sialyllactose (3'SL) as a natural inhibitor of TLR4-induced low-grade inflammation in macrophages and endothelium. Transcriptome analysis in macrophages revealed that 3'SL attenuates mRNA levels of a selected set of inflammatory genes and promotes the activity of liver X receptor (LXR) and sterol regulatory element binding protein-1 (SREBP1). These acute antiinflammatory effects of 3'SL were associated with reduced histone H3K27 acetylation at a subset of LPS-inducible enhancers distinguished by preferential enrichment for CCCTC-binding factor (CTCF), IFN regulatory factor 2 (IRF2), B cell lymphoma 6 (BCL6), and other transcription factor recognition motifs. In a murine atherosclerosis model, both s.c. and oral administration of 3'SL significantly reduced atherosclerosis development and the associated inflammation. This study provides evidence that 3'SL attenuates inflammation by a transcriptional mechanism to reduce atherosclerosis development in the context of cardiovascular disease

Abstract 13528: Interventional Hepatic ApoC-III Knockdown Improves Atherosclerotic Plaque Stability and Remodeling via Lowering Remnant Lipoproteins

Introduction: Apolipoprotein C-III (apoC-III) is a key regulator in triglyceride (TG) metabolism and correlates positively with hypertriglyceridemia and incidences of CVD. Recent studies also identified apoC-III as an inducer of sterile inflammation by activating the inflammasome, another CVD risk factor. It remains unclear if therapeutic apoC-III lowering can reduce CVD risk, nor is it clear if this is dependent on lipid-lowering or anti-inflammatory properties or both. Hypothesis: We set out to test the concept that interventional apoC-III lowering reduces atherosclerosis development only when TG-lowering is achieved. Methods: We determined the impact of interventional apoC-III lowering on atherogenesis using an apoC-III antisense oligonucleotides (ASOs) in hypertriglyceridemic mouse models where the intervention results in TG-lowering ( Apoe -/- Ndst1 f/f Alb-Cre + , Ldlr -/- Ndst1 f/f Alb-Cre + ) or not ( Ldlr -/- Lrp1 f/f Alb-Cre + ). Results: ApoC-III ASO treatment did not alter atherosclerotic lesion volume in the murine models mice simultaneously fed a Western diet. However, when TG-lowering was obtained, apoC-III ASO treatment significantly attenuated advanced and unstable plaque development. In contrast, no improvement in lesion composition and hyperlipidemia was observed in apoC-III ASO-treated Ldlr -/- Lrp1 f/f Alb-Cre + mice lacking hepatic receptors responsible for apoC-III inhibition of TRL clearance. To mimic an interventional setting, we tested the impact of therapeutic apoC-III lowering in combination with a switch to a lipid-poor chow diet intervention after 12-week Western diet feeding. We observed that apoC-III ASO treatment significantly reduced atherosclerotic lesion size progression when an additive TG-lowering was achieved. No differences in lesion development were observed in similarly treated Ldlr -/- Lrp1 f/f Alb-Cre + mice wherein no additive TG-lowering was achieved by apoC-III ASO intervention. Conclusions: Our data highlight that the impact of apoC-III on atherogenesis depends on its lipid-modifying properties and suggest that interventional apoC-III lowering in hypertriglyceridemia patients can prevent plaque rupture and reduce CVD-associated mortality. </jats:p

Abstract 023: Clearance of ApoC-III Glycoforms Associated With Triglyceride Metabolism

Background: We previously reported an association between higher abundance of disialylated (apoC-III 2 ) over monosialylated (apoC-III 1 ) apoC-III and lower plasma triglyceride (TG) concentrations in three independent studies. Methods: The goal of this study was to get mechanistic insights in the metabolism of apoC-III 1 and apoC-III 2 . First, the relative abundances of human triglyceride-rich lipoproteins (TRL)-associated apoC-III glycoforms in plasma were assessed by mass spectrometric immunoassay (MSIA) over time after injecting human TRL in wild-type mice, mice lacking hepatic heparan sulfate proteoglycans (HSPG), and mice deficient for both low-density lipoprotein receptor (LDLR) and LDLR-Related Protein 1 (LRP-1). Secondly, the relative abundance of plasma apoC-III glycoforms was assessed by MSIA in 11 participants randomized to volanesorsen (apoC-III antisense drug; n=11) or placebo (n=6) treatment for 13 weeks. Results: Kinetic studies revealed that half of TRL-associated apoC-III (total) was rapidly cleared via LDLR and LRP1 (t 1/2 = 16.5 min), and the remaining half via the much slower but higher capacity HSPGs (t 1/2 = 55.5 min). After injection, a significant increase in the relative abundance of apoC-III 2 was observed in HSPG-deficient mice , while no increase in mice lacking LDLR and LRP-1. Clinically, the relative abundance of apoC-III 2 significantly increased (42% increase compared to placebo, p=0.05) and that of apoC-III 1 significantly decreased (15% decrease compared to placebo, p=0.007) in participants randomized to volanesorsen. The decrease in the relative abundance of apoC-III 1 after volanesorsen treatment was strongly correlated with the decrease in TG levels (r=0.63, p=0.006). Conclusions: Our results indicate slower clearance of apoC-III-containing particles through HSPGs, which preferentially clear apoC-III 2. In contrast, apoC-III 2 is less effectively cleared by LDLR/LRP-1 compared to apoC-III 1 . Clinically, the increase in the apoC-III 2 /apoC-III 1 ratio upon antisense lowering of apoC-III might reflect faster clearance of apoC-III 1 as it associates with improved TG levels. </jats:p

Keratins as the main component for the mechanical integrity of keratinocytes

For decades, researchers have been trying to unravel one of the key questions in cell biology regarding keratin intermediate filament function in protecting epithelial cells against mechanical stress. For many different reasons, however, this fundamental hypothesis was still unproven. Here we answer this pivotal question by the use of keratin KO cells lacking complete keratin gene clusters to result in total loss of keratin filaments. This lack significantly softens cells, reduces cell viscosity, and elevates plastic cell deformation on force application. Reexpression of single keratin genes facilitates biomechanical complementation of complete cluster loss. Our manuscript therefore makes a very strong case for the crucial contribution of keratins to cell mechanics, with far-reaching implications for epithelial pathophysiology

ApoC-III inhibits clearance of triglyceride-rich lipoproteins through LDL family receptors.

Hypertriglyceridemia is an independent risk factor for cardiovascular disease, and plasma triglycerides (TGs) correlate strongly with plasma apolipoprotein C-III (ApoC-III) levels. Antisense oligonucleotides (ASOs) for ApoC-III reduce plasma TGs in primates and mice, but the underlying mechanism of action remains controversial. We determined that a murine-specific ApoC-III-targeting ASO reduces fasting TG levels through a mechanism that is dependent on low-density lipoprotein receptors (LDLRs) and LDLR-related protein 1 (LRP1). ApoC-III ASO treatment lowered plasma TGs in mice lacking lipoprotein lipase (LPL), hepatic heparan sulfate proteoglycan (HSPG) receptors, LDLR, or LRP1 and in animals with combined deletion of the genes encoding HSPG receptors and LDLRs or LRP1. However, the ApoC-III ASO did not lower TG levels in mice lacking both LDLR and LRP1. LDLR and LRP1 were also required for ApoC-III ASO-induced reduction of plasma TGs in mice fed a high-fat diet, in postprandial clearance studies, and when ApoC-III-rich or ApoC-III-depleted lipoproteins were injected into mice. ASO reduction of ApoC-III had no effect on VLDL secretion, heparin-induced TG reduction, or uptake of lipids into heart and skeletal muscle. Our data indicate that ApoC-III inhibits turnover of TG-rich lipoproteins primarily through a hepatic clearance mechanism mediated by the LDLR/LRP1 axis