FoxO6-mediated ApoC3 upregulation promotes hepatic steatosis and hyperlipidemia in aged rats fed a high-fat diet.

FoxO6, an identified factor, induces hyperlipidemia and hepatic steatosis during aging by activating hepatic lipoprotein secretion and lipogenesis leading to increased ApoC3 concentrations in the bloodstream. However, the intricate mechanisms underlying hepatic steatosis induced by elevated FoxO6 under hyperglycemic conditions remain intricate and require further elucidation. In order to delineate the regulatory pathway involving ApoC3 controlled by FoxO6 and its resultant functional impacts, we employed a spectrum of models including liver cell cultures, aged rats subjected to HFD, transgenic mice overexpressing FoxO6 (FoxO6-Tg), and FoxO6 knockout mice (FoxO6-KO). Our findings indicate that FoxO6 triggered ApoC3-driven lipid accumulation in the livers of aged rats on an HFD and in FoxO6-Tg, consequently leading to hepatic steatosis and hyperglycemia. Conversely, the absence of FoxO6 attenuated the expression of genes involved in lipogenesis, resulting in diminished hepatic lipid accumulation and mitigated hyperlipidemia in murine models. Additionally, the upregulation of FoxO6 due to elevated glucose levels led to increased ApoC3 expression, consequently instigating cellular triglyceride mediated lipid accumulation. The transcriptional activation of FoxO6 induced by both the HFD and high glucose levels resulted in hepatic steatosis by upregulating ApoC3 and genes associated with gluconeogenesis in aged rats and liver cell cultures. Our conclusions indicate that the upregulation of ApoC3 by FoxO6 promotes the development of hyperlipidemia, hyperglycemia, and hepatic steatosis in vivo, and in vitro. Taken together, our findings underscore the significance of FoxO6 in driving hyperlipidemia and hepatic steatosis specifically under hyperglycemic states by enhancing the expression of ApoC3 in aged rats.

Exploring apolipoprotein C-III: pathophysiological and pharmacological relevance.

The availability of pharmacological approaches able to effectively reduce circulating LDL cholesterol (LDL-C) has led to a substantial reduction in the risk of atherosclerosis-related cardiovascular disease (CVD). However, a residual cardiovascular (CV) risk persists in treated individuals with optimal levels of LDL-C. Additional risk factors beyond LDL-C are involved, and among these, elevated levels of triglycerides (TGs) and TG-rich lipoproteins are causally associated with an increased CV risk. Apolipoprotein C-III (apoC-III) is a key regulator of TG metabolism and hence circulating levels through several mechanisms including the inhibition of lipoprotein lipase activity and alterations in the affinity of apoC-III-containing lipoproteins for both the hepatic receptors involved in their removal and extracellular matrix in the arterial wall. Genetic studies have clarified the role of apoC-III in humans, establishing a causal link with CVD and showing that loss-of-function mutations in the APOC3 gene are associated with reduced TG levels and reduced risk of coronary heart disease. Currently available hypolipidaemic drugs can reduce TG levels, although to a limited extent. Substantial reductions in TG levels can be obtained with new drugs that target specifically apoC-III; these include two antisense oligonucleotides, one small interfering RNA and an antibody.

Apolipoprotein C-III itself stimulates the Syk/cPLA2-induced inflammasome activation of macrophage to boost anti-tumor activity of CD8+ T cell.

Increased prevalence of cancer in obese individuals is involved with dyslipidemia- induced chronic inflammation and immune suppression. Although apolipoprotein C-III (ApoC3)-transgenic mice (ApoC3TG mice) or poloxamer 407 (P407)-treated mice had hyperlipidemia, CD8+ T cells with upregulated antitumor activities were observed in ApoC3TG mice, and decreased CD8+ T cell activities were observed in P407-treated mice. Increased ApoC3 expression in hepatocellular carcinoma was associated with increased infiltration of CD8+ T cells and predicted survival. Recombinant ApoC3 had no direct effects on CD8+ T cells. The upregulation of CD8+ T cells in ApoC3TG mice was due to cross-talk with context cells, as indicated by metabolic changes and RNA sequencing results. In contrast to dendritic cells, the macrophages of ApoC3TG mice (macrophagesTG) displayed an activated phenotype and increased IL-1ß, TNF-α, and IL-6 production. Coculture with macrophagesTG increased CD8+ T cell function, and the adoptive transfer of macrophagesTG suppressed tumor progression in vivo. Furthermore, spleen tyrosine kinase (Syk) activation induced by TLR2/TLR4 cross-linking after ApoC3 ligation promoted cellular phospholipase A2 (cPLA2) activation, which in turn activated NADPH oxidase 2 (NOX2) to promote an alternative mode of inflammasome activation. Meanwhile, mitochondrial ROS produced by increased oxidative phosphorylation of free fatty acids facilitated the classical inflammasome activation, which exerted an auxiliary effect on inflammasome activation of macrophagesTG. Collectively, the increased antitumor activity of CD8+ T cells was mediated by the ApoC3-stimulated inflammasome activation of macrophages, and the mimetic ApoC3 peptides that can bind TLR2/4 could be a future strategy to target liver cancer.

Unanticipated Enhancement of Intestinal TG Output by Apoc3 ASO Inhibition.

BACKGROUND: The objective of this study was to investigate whether apoC3 (apolipoprotein C3) inhibition with an antisense oligonucleotide (ASO) modulates intestinal triglyceride secretion. METHODS: Sprague-Dawley rats were treated with subcutaneous injections of apoC3 ASO 25 mg/kg twice weekly or inactive ASO for 4 weeks before the assessment of lymph flow, triglyceride and apoB48 (apolipoprotein B48) appearance in the lymph. Rats were surgically implanted with catheters in the mesenteric lymph duct and duodenum. Following an overnight fast, an intraduodenal lipid bolus (1.5-mL intralipid) was administered. Lymph fluid was collected for the following 4 hours to compare effects on lymph flow, lymph triglyceride and apoB48 concentration, and secretion. To assess suppression of apoC3 expression and protein abundance by apoC3 ASO compared with inactive ASO (placebo), intestinal and hepatic tissues were collected from a subset of animals before (fasting) and after an enteral lipid bolus (post-lipid). RESULTS: ApoC3 ASO significantly reduced apoC3 mRNA expression in the liver compared with inactive ASO (fasting: 42%, P=0.0048; post-lipid: 66%, P<0.001) and in the duodenum (fasting: 29%, P=0.0424; post-lipid: 53%, P=0.0120). As expected, plasma triglyceride also decreased significantly (fasting: 74%, P<0.001; post-lipid: 33%, P=0.0276). Lymph flow and cumulative lymph volume remained unchanged following apoC3 ASO therapy; however, lymph triglyceride, but not apoB48 output, increased by 38% (ANOVA, P<0.001). Last, no changes were observed in stool triglyceride, intestinal fat (quantified via oil red O staining), and expression of mRNAs involved in triglyceride synthesis, lipid droplet formation, and chylomicron transport and secretion. CONCLUSIONS: Despite the marked reduction in plasma triglyceride concentration that occurs with apoC3 ASO inhibition, intestinal triglyceride output surprisingly increased rather than decreased. These data demonstrate that the reduction of intestinal triglyceride output does not contribute to the potent plasma triglyceride-lowering observed with this novel therapy for hypertriglyceridemia. Further studies are required to explore the mechanism of this intestinal effect.

CD14+-Monocytes Exposed to Apolipoprotein CIII Express Tissue Factor.

Apolipoprotein CIII (ApoCIII) represents a key regulator of plasma lipid metabolism and a recognized risk factor for atherosclerosis and cardiovascular diseases. Beyond the regulation of lipoprotein trafficking, ApoCIII is also involved in endothelial dysfunction and monocyte recruitment related to atherothrombosis. With tissue factor (TF) being the primary initiator of the blood coagulation cascade, we hypothesized that ApoCIII-treated monocytes could express it. Hence, human CD14+-monocytes and autologous neutrophils were incubated with ApoCIII and sera from human subjects containing previously measured ApoCIII amounts. By RT-qPCR and ELISA, CD14+-monocytes, but not neutrophils, were found to show increased mRNA expression and production of TNFα, IL-1ß and IL-6 as well as TF mRNA once exposed to ultra-purified ApoCIII. By flow cytometry, CD14+-monocytes were found to rapidly express TF on their cell surface membrane when incubated with either ApoCIII or sera with known concentrations of ApoCIII. Finally, preincubation with specific ApoCIII-neutralizing antibodies significantly reduced the ability of most sera with known concentrations of ApoCIII to upregulate TF protein, other than partially inhibiting cytokine release, in CD14+-monocytes. In sum, herein we demonstrate that ApoCIII activates CD14+-monocytes to express TF. The data identify a potential mechanism which links circulating apolipoproteins with inflammation and atherothrombosis-related processes underlying cardiovascular risk.

APOC-III: a Gatekeeper in Controlling Triglyceride Metabolism.

PURPOSE OF REVIEW: Apolipoprotein C-III (ApoC-III) is a widely known player in triglyceride metabolism, and it has been recently recognized as a polyhedric factor which may regulate several pathways beyond lipid metabolism by influencing cardiovascular, metabolic, and neurological disease risk. This review summarizes the different functions of ApoC-III and underlines the recent findings related to its multifaceted pathophysiological role. RECENT FINDINGS: The role of ApoC-III has been implicated in HDL metabolism and in the development of atherosclerosis, inflammation, and ER stress in endothelial cells. ApoC-III has been recently considered an important player in insulin resistance mechanisms, lipodystrophy, diabetic dyslipidemia, and postprandial hypertriglyceridemia (PPT). The emerging evidence of the involvement of ApoC-III in the in the pathogenesis of Alzheimer's disease open the way to further study if modification of ApoC-III level slows disease progression. Furthermore, ApoC-III is clearly linked to cardiovascular disease (CVD) risk, and progression of coronary artery disease (CAD) as well as the calcification of aortic valve and recent clinical trials has pointed out the inhibition of ApoC-III as a promising approach to manage hypertriglyceridemia and prevent CVD. Several evidences highlight the role of ApoC-III not only in triglyceride metabolism but also in several cardio-metabolic pathways. Results from recent clinical trials underline that the inhibition of ApoC-III is a promising therapeutical strategy for the management of severe hypertriglyceridemia and in CVD prevention.

Treatment of the metabolic syndrome by siRNA targeting apolipoprotein CIII.

Apolipoprotein CIII (apoCIII) is increased in obesity-induced insulin resistance and type-2 diabetes. Emerging evidences support the advantages of small interfering RNAs (siRNAs) to target disease-causing genes. The aim of this study was to develop siRNAs for in vivo silencing of apoCIII and investigate if this results in metabolic improvements comparable to what we have seen using antisense oligonucelotides against apoCIII. Twenty-four siRNAs were synthesized and tested in a dual luciferase reporter assay. The eight best were selected, based on knockdown at 20 nM, and of these, two were selected based on IC50 values. In vivo experiments were performed in ob/ob mice, an obese animal model for diabetes. To determine the dose-dependency, efficacy, duration of effect and therapeutic dose we used a short protocol giving the apoCIII-siRNA mix for three days. To evaluate long-term metabolic effects mice were treated for three days, every second week for eight weeks. The siRNA mix effectively and selectively reduced expression of apoCIII in liver in vivo. Treatment had to be repeated every two weeks to maintain a suppression of apoCIII. The reduction of apoCIII resulted in increased LPL activity, lower triglycerides, reduced liver fat, ceased weight gain, enhanced insulin sensitivity, and improved glucose homeostasis. No off-target or side effects were observed during the eight-week treatment period. These results suggest that in vivo silencing of apoCIII with siRNA, is a promising approach with the potential to be used in the battle against obesity-induced metabolic disorders.

Simultaneous Quantification of Apolipoprotein C-III O-Glycoforms by Protein-MRM.

Apolipoprotein C-III (APOC-III) regulates triglyceride levels, associated with a risk of cardiovascular disease. One gene generates several proteoforms, each with a different molecular mass and a unique function. Unlike peptide multiple reaction monitoring (MRM), protein-MRM without digestion is required to analyze clinically relevant individual proteoforms. We developed a protein-MRM method without digestion to individually quantify APOC-III proteoforms in human serum. We optimized the protein-MRM method following 60% acetonitrile extraction with C18 filtration. Bovine serum and myoglobin served as supporting cushions and the internal standard during sample preparation, respectively. Furthermore, we evaluated the LOD, lower limit of quantification, linearity, accuracy, and precision. Good correlation compared with turbidimetric immunoassay (TIA) and peptide-MRM was observed using 30 clinical sera. Individual APOC-III O-glycoforms were identified by top-down proteomics and simultaneously quantified using the protein-MRM method. The sum abundance of APOC-III proteoforms was significantly correlated with TIA and peptide-MRM. Our protein-MRM method provides an affordable and rapid quantification of potential disease-specific proteoforms. Precise quantification of each proteoform allows investigators to identify novel biological roles potentially related to cardiovascular disease or novel biomarkers. We expect our protein-oriented method to be more clinically useful than antibody-based immunoassays and peptide-oriented MRM analysis, especially for quantification of a biomarker proteoform with certain post-translational modifications.

Recent Apolipoprotein CIII trials.

PURPOSE OF REVIEW: This review will briefly revise the evidence concerning the pharmacological inhibition of Apolipoprotein CIII (ApoCIII) in patients with hypertriglyceridemia. RECENT FINDINGS: ApoCIII is a plasma apolipoprotein playing a major role in the metabolism of triglyceride-rich lipoproteins, namely chylomicrons and very-low-density lipoproteins as well as in the pathological processes involved in atherosclerosis. Therefore, ApoCIII is a potential new target for reducing plasma levels of TRLs and, thereby, cardiovascular risk. In recent years, there have been extensive preclinical and clinical pharmacological studies aimed at testing drugs directed against ApoCIII. SUMMARY: In this review, firstly we will summarize the molecular function of ApoCIII in lipoprotein metabolism. Then, we will examine the lipid-lowering potential of the pharmacological inhibition of ApoCIII based on the results of clinical trial employing Volansesorsen, the first approved antisense therapeutic oligonucleotide against ApoCIII mRNA. The future perspectives for ApoCIII inhibition will be also revised.

Plasma high-density lipoprotein cargo is altered in Alzheimer's disease and is associated with regional brain volume.

Cholesterol levels have been repeatedly linked to Alzheimer's Disease (AD), suggesting that high levels could be detrimental, but this effect is likely attributed to Low-Density Lipoprotein (LDL) cholesterol. On the other hand, High-Density Lipoproteins (HDL) cholesterol levels have been associated with reduced brain amyloidosis and improved cognitive function. However, recent findings have suggested that HDL-functionality, which depends upon the HDL-cargo proteins associated with HDL, rather than HDL levels, appears to be the key factor, suggesting a quality over quantity status. In this report, we have assessed the HDL-cargo (Cholesterol, ApoA-I, ApoA-II, ApoC-I, ApoC-III, ApoD, ApoE, ApoH, ApoJ, CRP, and SAA) in stable healthy control (HC), healthy controls who will convert to MCI/AD (HC-Conv) and AD patients (AD). Compared to HC we observed an increased cholesterol/ApoA-I ratio in AD and HC-Conv, as well as an increased ApoD/ApoA-I ratio and a decreased ApoA-II/ApoA-I ratio in AD. Higher cholesterol/ApoA-I ratio was also associated with lower cortical grey matter volume and higher ventricular volume, while higher ApoA-II/ApoA-I and ApoJ/ApoA-I ratios were associated with greater cortical grey matter volume (and for ApoA-II also with greater hippocampal volume) and smaller ventricular volume. Additionally, in a clinical status-independent manner, the ApoE/ApoA-I ratio was significantly lower in APOE ε4 carriers and lowest in APOE ε4 homozygous. Together, these data indicate that in AD patients the composition of HDL is altered, which may affect HDL functionality, and such changes are associated with altered regional brain volumetric data.

Apolipoprotein C3 and necrotic core volume are correlated but also associated with future cardiovascular events.

We aimed to clarify the relationship between apolipoprotein C3 (apo-C3) and the vascular composition of lesion plaque in stable coronary disease (SCD) before percutaneous coronary intervention (PCI), and to investigate major adverse cardiovascular events (MACEs) within 4 years. Data of 98 consecutive patients with SCD who underwent PCI between November 1, 2012, and March 10, 2015, were analyzed. Laboratory and virtual histology-intravascular ultrasound (VH-IVUS) examinations of culprit lesions were conducted before PCI. Patients were divided according to median apo-C3 into low apo-C3 (≤ 8.5 mg/dL) and high apo-C3 (> 8.5 mg/dL) groups. VH-IVUS data indicated that the percentage of necrotic core volume (%NC) was significantly higher in the high apo-C3 group than in the low apo-C3 group. Moreover, the %NC significantly correlated with the apo-C3 level (R = 0.2109, P = 0.037). Kaplan-Meier curve analysis revealed that freedom from MACEs exhibited a greater decrease in the high apo-C3 group than in the low apo-C3 group, and in the high %NC group than in the low %NC group. Multivariate Cox hazards analysis showed that the %NC and high apo-C3 were independent predictors of 4 year MACEs. Apo-C3 may be a useful marker of future MACEs in patients with SCD after PCI and contribute to %NC growth.

Postprandial metabolism of apolipoproteins B48, B100, C-III, and E in humans with APOC3 loss-of-function mutations.

BackgroundApolipoprotein C-III (apoC-III) is a regulator of triglyceride (TG) metabolism, and due to its association with risk of cardiovascular disease, is an emergent target for pharmacological intervention. The impact of substantially lowering apoC-III on lipoprotein metabolism is not clear.MethodsWe investigated the kinetics of apolipoproteins B48 and B100 (apoB48 and apoB100) in chylomicrons, VLDL1, VLDL2, IDL, and LDL in patients heterozygous for a loss-of-function (LOF) mutation in the APOC3 gene. Studies were conducted in the postprandial state to provide a more comprehensive view of the influence of this protein on TG transport.ResultsCompared with non-LOF variant participants, a genetically determined decrease in apoC-III resulted in marked acceleration of lipolysis of TG-rich lipoproteins (TRLs), increased removal of VLDL remnants from the bloodstream, and substantial decrease in circulating levels of VLDL1, VLDL2, and IDL particles. Production rates for apoB48-containing chylomicrons and apoB100-containing VLDL1 and VLDL2 were not different between LOF carriers and noncarriers. Likewise, the rate of production of LDL was not affected by the lower apoC-III level, nor were the concentration and clearance rate of LDL-apoB100.ConclusionThese findings indicate that apoC-III lowering will have a marked effect on TRL and remnant metabolism, with possibly significant consequences for cardiovascular disease prevention.Trial registrationClinicalTrials.gov NCT04209816 and NCT01445730.FundingSwedish Heart-Lung Foundation, Swedish Research Council, ALF grant from the Sahlgrenska University Hospital, Novo Nordisk Foundation, Sigrid Juselius Foundation, Helsinki University Hospital Government Research funds, Finnish Heart Foundation, and Finnish Diabetes Research Foundation.

Key Molecules of Triglycerides Pathway Metabolism Are Disturbed in Patients With Systemic Lupus Erythematosus.

Background: Elevated triglycerides or triglyceride-rich lipoproteins are an additional cause of cardiovascular (CV) disease. Given that patients with systemic lupus erythematosus (SLE) have a high prevalence of premature CV disease and show an altered lipid profile, our objective was to study whether three molecules that play a central role in the triglyceride metabolism: apolipoprotein C-III (ApoC3), angiopoietin-like protein 4 (ANGPLT4), and lipoprotein lipase (LPL) differ between SLE patients and controls, and how they are related to disease characteristics, including disease damage. Methods: Cross-sectional study that included 347 women, 185 of them diagnosed with SLE and 162 age-matched controls. ANGPTL4, ApoC3 and LPL, and standard lipid profiles were analyzed in SLE patients and controls. A multivariable analysis was performed to assess whether ANGPTL4, ApoC3 and LPL molecules differ between patients and controls and to study their relationship with SLE disease damage. Results: After fully multivariable analysis that included classic CV risk factors, and the modifications that the disease itself produces over the lipid profile, it was found that ApoC3 was significantly lower (beta coef. -1.2 [95%CI -1.6- -0.8) mg/dl, <0.001), and ANGPTL4 (beta coef. 63 [95%CI 35-90] ng/ml, <0.001) and LPL (beta coef. 79 [95%CI 30-128] ng/ml, p=0.002) significantly higher in patients with SLE compared to controls. Disease damage score was significantly and independently associated with higher serum levels of LPL (beta coef. 23 [95%CI 10-35] ng/ml, p=0.001). Mediation analysis suggested that the relationship between disease damage and LPL was direct and not mediated by ApoC3 or ANGPLT4. Conclusion: The ApoC3, ANGPLT4 and LPL axis is disrupted in patients with SLE. Disease damage explains this disturbance.

Apolipoprotein C3 Is Downregulated in Patients With Inflammatory Bowel Disease.

INTRODUCTION: Inflammatory bowel disease (IBD) has been associated with an abnormal lipid profile. Apolipoprotein C-III (ApoC3) is a key molecule of triglyceride metabolism that is known to be related to inflammation and cardiovascular disease. In this study, we aim to study whether ApoC3 serum levels differ between patients with IBD and controls and whether the hypothetical disturbance of ApoC3 can be explained by IBD characteristics. METHODS: This is a cross-sectional study that included 405 individuals, 197 patients with IBD and 208 age-matched and sex-matched controls. ApoC3 and standard lipid profiles were assessed in patients and controls. A multivariable analysis was performed to analyze whether ApoC3 serum levels were altered in IBD and to study their relationship to IBD characteristics. RESULTS: After fully multivariable analysis including cardiovascular risk factors, use of statins, and changes in lipid profile caused by the disease itself, patients with IBD showed significant lower serum levels of ApoC3 (beta coef. -1.6 [95% confidence interval -2.5 to -0.7] mg/dL, P = 0.001). Despite this, inflammatory markers, disease phenotypes, or disease activity of IBD was not found to be responsible for this downregulation. DISCUSSION: Apolipoprotein C3 is downregulated in patients with IBD.

Efficacy and safety of the apolipoprotein C-III inhibitor Volanesorsen: a systematic evaluation and meta-analysis.

PURPOSE: Familial celiac disease syndrome (FCS) is a form of hypertriglyceridemia (HTG) caused by the accumulation of celiac particles. Currently, volanesorsen is considered to be used to treat patients with FCS and HTG to improve symptoms. To evaluate the effect of volanesorsen on lipid metabolism in patients with FCS, we performed a systematic evaluation and meta-analysis. METHODS: A systematic search of PubMed, Embase, ClinicalTrials.gov, and the Cochrane Library was conducted, and the bibliographies of original articles were checked manually. The quality of the studies was assessed using the Cochrane Risk of Bias tool. RESULTS: Four randomized, controlled trials involving 246 patients were analyzed in this study. Patients treated with volanesorsen showed (MD = -78.85%; 95% CI = -96.04 to -61.65, P = 0.67, I2 = 0%) decrease in TG and (MD = -80.08%; 95% CI = -90.02 to -71.54, P = 0.25, I2 = 29%) decrease in ApoC-III levels compared to patients in the placebo group showing a significant decrease. In addition, HDL-C increased (MD = 46.01% 95% CI = 41.03 to 50.99, P = 0.41, I2 = 0%), NHDL-C decreased (MD = -32.12%; 95% CI = -44.39 to -19.85, P = 0.11, I2 = 55%), VLDL-C decreased (MD = -65.88%; 95% CI = -83.97 to -47.79, P = 0.71, I2 = 0%), apo A1 increased (MD = 13.12%; 95% CI = 7.83 to 18.40, P = 0.72, I2 = 0%), and apoB increased (MD = 7.94 %; 95% CI = -1.90 to 17.78, P = 0.54, I2 = 0%) all suggest that volanesorsen has an overall FCS with a therapeutic effect. However, LDL-C increased (MD = 99.59%; 95% CI = 69.19 to 130.00, P = 0.61, I2 = 0%) and apo B48 decreased (MD = 82.89%; 95% CI = -100.88 to -64.91, P = 0.42, I2 = 0%), showing an inverse effect, suggesting that volanesorsen's did not target all proteins of lipid metabolism.

Addressing dyslipidemic risk beyond LDL-cholesterol.

Despite the success of LDL-lowering drugs in reducing cardiovascular disease (CVD), there remains a large burden of residual disease due in part to persistent dyslipidemia characterized by elevated levels of triglyceride-rich lipoproteins (TRLs) and reduced levels of HDL. This form of dyslipidemia is increasing globally as a result of the rising prevalence of obesity and metabolic syndrome. Accumulating evidence suggests that impaired hepatic clearance of cholesterol-rich TRL remnants leads to their accumulation in arteries, promoting foam cell formation and inflammation. Low levels of HDL may associate with reduced cholesterol efflux from foam cells, aggravating atherosclerosis. While fibrates and fish oils reduce TRL, they have not been uniformly successful in reducing CVD, and there is a large unmet need for new approaches to reduce remnants and CVD. Rare genetic variants that lower triglyceride levels via activation of lipolysis and associate with reduced CVD suggest new approaches to treating dyslipidemia. Apolipoprotein C3 (APOC3) and angiopoietin-like 3 (ANGPTL3) have emerged as targets for inhibition by antibody, antisense, or RNAi approaches. Inhibition of either molecule lowers TRL but respectively raises or lowers HDL levels. Large clinical trials of such agents in patients with high CVD risk and elevated levels of TRL will be required to demonstrate efficacy of these approaches.

CRISPR/Cas9-mediated knockout of APOC3 stabilizes plasma lipids and inhibits atherosclerosis in rabbits.

BACKGROUND: High levels of apolipoprotein C3 (APOC3) can lead to hypertriglyceridemia, which increases the risk of cardiovascular disease. We aim to create APOC3-knockout (KO) rabbits and explore the effects of APOC3 deletion on the occurrence and development of atherosclerosis. METHODS: An sgRNA anchored to exon 2 of APOC3 was designed to edit embryo genomes using the CRISPR/Cas9 system. The founder rabbits were sequenced, and their lipid profile, inflammatory cytokines, and atherosclerotic plaques were analyzed. RESULTS: When given a normal chow (NC) diet, all APOC3-KO rabbits had 50% lower triglyceride (TG) levels than those of the matched age control group. Additionally, their plasma lipoprotein lipase increased. When fed a high-fat diet, APOC3 deficiency was observed to be more conducive to the maintenance of plasma TG, total cholesterol, and low-density lipoprotein cholesterol levels, and the inhibition of the inflammatory response and the protection against atherosclerosis in rabbits. CONCLUSION: APOC3 deficiency can delay the formation of atherosclerosis-induced HFD in rabbits, indicating this is a novel therapeutic target to treat atherosclerosis.

Guanidinylated Apolipoprotein C3 (ApoC3) Associates with Kidney and Vascular Injury.

BACKGROUND: Coexistent CKD and cardiovascular diseases are highly prevalent in Western populations and account for substantial mortality. We recently found that apolipoprotein C-3 (ApoC3), a major constituent of triglyceride-rich lipoproteins, induces sterile systemic inflammation by activating the NOD-like receptor protein-3 (NLRP3) inflammasome in human monocytes via an alternative pathway. METHODS: To identify posttranslational modifications of ApoC3 in patients with CKD, we used mass spectrometry to analyze ApoC3 from such patients and from healthy individuals. We determined the effects of posttranslationally modified ApoC3 on monocyte inflammatory response in vitro, as well as in humanized mice subjected to unilateral ureter ligation (a kidney fibrosis model) and in a humanized mouse model for vascular injury and regeneration. Finally, we conducted a prospective observational trial of 543 patients with CKD to explore the association of posttranslationally modified ApoC3 with renal and cardiovascular events in such patients. RESULTS: We identified significant posttranslational guanidinylation of ApoC3 (gApoC3) in patients with CKD. We also found that mechanistically, guanidine and urea induce guanidinylation of ApoC3. A 2D-proteomic analysis revealed that gApoC3 accumulated in kidneys and plasma in a CKD mouse model (mice fed an adenine-rich diet). In addition, gApoC3 augmented the proinflammatory effects of ApoC3 in monocytes in vitro . In humanized mice, gApoC3 promoted kidney tissue fibrosis and impeded vascular regeneration. In CKD patients, higher gApoC3 plasma levels (as determined by mass spectrometry) were associated with increased mortality as well as with renal and cardiovascular events. CONCLUSIONS: Guanidinylation of ApoC3 represents a novel pathogenic mechanism in CKD and CKD-associated vascular injury, pointing to gApoC3 as a potential therapeutic target.

Apolipoprotein C-II and C-III preferably transfer to both high-density lipoprotein (HDL)2 and the larger HDL3 from very low-density lipoprotein (VLDL).

Triglyceride hydrolysis by lipoprotein lipase (LPL), regulated by apolipoproteins C-II (apoC-II) and C-III (apoC-III), is essential for maintaining normal lipid homeostasis. During triglyceride lipolysis, the apoCs are known to be transferred from very low-density lipoprotein (VLDL) to high-density lipoprotein (HDL), but the detailed mechanisms of this transfer remain unclear. In this study, we investigated the extent of the apoC transfers and their distribution in HDL subfractions, HDL2 and HDL3. Each HDL subfraction was incubated with VLDL or biotin-labeled VLDL, and apolipoproteins and lipids in the re-isolated HDL were quantified using western blotting and high-performance liquid chromatography (HPLC). In consequence, incubation with VLDL showed the increase of net amount of apoC-II and apoC-III in the HDL. HPLC analysis revealed that the biotin-labeled apolipoproteins, including apoCs and apolipoprotein E, were preferably transferred to the larger HDL3. No effect of cholesteryl ester transfer protein inhibitor on the apoC transfers was observed. Quantification of apoCs levels in HDL2 and HDL3 from healthy subjects (n = 8) showed large individual differences between apoC-II and apoC-III levels. These results suggest that both apoC-II and apoC-III transfer disproportionately from VLDL to HDL2 and the larger HDL3, and these transfers might be involved in individual triglyceride metabolism.

Reversal of hypertriglyceridemia in diabetic BTBR ob/ob mice does not prevent nephropathy.

The etiology of diabetic nephropathy in type 2 diabetes is multifactorial. Sustained hyperglycemia is a major contributor, but additional contributions come from the hypertension, obesity, and hyperlipidemia that are also commonly present in patients with type 2 diabetes and nephropathy. The leptin deficient BTBR ob/ob mouse is a model of type 2 diabetic nephropathy in which hyperglycemia, obesity, and hyperlipidemia, but not hypertension, are present. We have shown that reversal of the constellation of these metabolic abnormalities with leptin replacement can reverse the morphologic and functional manifestations of diabetic nephropathy. Here we tested the hypothesis that reversal specifically of the hypertriglyceridemia, using an antisense oligonucleotide directed against ApoC-III, an apolipoprotein that regulates the interactions of VLDL (very low density lipoproteins) with the LDL receptor, is sufficient to ameliorate the nephropathy of Type 2 diabetes. Antisense treatment resulted in reduction of circulating ApoC-III protein levels and resulted in substantial lowering of triglycerides to near-normal levels in diabetic mice versus controls. Antisense treatment did not ameliorate proteinuria or pathologic manifestations of diabetic nephropathy, including podocyte loss. These studies indicate that pathologic manifestations of diabetic nephropathy are unlikely to be reduced by lipid-lowering therapeutics alone, but does not preclude a role for such interventions to be used in conjunction with other therapeutics commonly employed in the treatment of diabetes and its complications.